RU2792235C2 - Plants with a shorter flowering time - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: transgenic plant having a reduced time period before flowering. A part of said plant is disclosed; an isolated polynucleotide or an isolated polypeptide, which the specified plant contains, RNAi construct, double-stranded RNA containing the specified polynucleotide. Also disclosed are the use of plant material in a tobacco product or smoking article; application of a herbal product in a tobacco product or smoking article; application of a tobacco product in a smoking article; a method for obtaining plant material from the specified plant; a method for identifying a molecule that provides modulation of the expression of a TFL1 polynucleotide or the activity of a TFL1 polypeptide in the specified plant.

EFFECT: effective reduction of the time period until flowering in a transgenic plant.

26 cl, 7 dwg, 2 tab, 6 ex

Description

FIELD OF THE INVENTION

The present invention discloses polynucleotide sequences of genes encoding Terminal Flower 1 (TFL1) from Nicotiana tabacum and variants, homologues and fragments thereof. Also disclosed are polypeptide sequences encoded in this manner and variants, homologues and fragments thereof. Also disclosed is the modification in a plant of the expression of one or more genes or the activity of the protein(s) so encoded in order to modulate the period of time prior to flowering. In one embodiment, the expression of one or more genes or the activity of the protein(s) so encoded(s) is reduced in order to shorten the period of time before flowering occurs. Plants, plant material, and the like with a modified period of time before flowering is also described.

BACKGROUND OF THE INVENTION

The time period before flowering is a tightly controlled mechanism in plants that has a direct impact on survival and reproduction. The transition to flowering is also directly related to crop yields. Plants have developed specialized signaling pathways that lead to the formation of reproductive structures instead of leaves. The T flowering locus (FT) and TFL1 are members of the phosphatidylethanolamine-binding proteins (PEBP) family, which are similar to mammalian PEBP and function as transcription factors. TFL1 acts antagonistically by delaying flowering recruitment. The FT protein interacts with the transcription factor bZIP of the flowering locus D (FD) in the shoot apical meristem to promote flowering. The TFL1 protein also binds to FD to suppress downstream genes such as LEAFY ( LFY) and APETALA1 (AP1) . After the transition to flowering , TFL1 is upregulated to balance FT activity.

There is a general need in the art to develop plants that have a reduced time to bloom, as this can lead to a number of advantages, especially those associated with commercial plant production. For example, this may provide a shorter period from seeding/planting to harvest, which may shorten the growing season. This may allow more rapid introduction of new traits through crossbreeding. This can lead to cost savings in the commercial production of plants. The present invention addresses this need.

BRIEF DESCRIPTION OF THE INVENTION

Seven TFL - 1 genes have been identified in Nicotiana tabacum , termed TFL1-1S (SEQ ID NO: 1 or 2 ), TFL1-1T (SEQ ID NO: 4 or 5), TFL1-2S (SEQ ID NO: 7 or 8) , TFL1 - 2T (SEQ ID NO: 10 or 11), TFL1 - 3T (SEQ ID NO: 13 or 14), TFL1 - 4S (SEQ ID NO: 16 or 17), and TFL1 - 4T (SEQ ID NO: 19 or 20). Surprisingly, the present inventors have found that when the expression of each of these genes is disrupted (eg, reduced), only TFL1 - 2S (SEQ ID NO: 7 or 8) and TFL1 - 2T (SEQ ID NO: 10 or 11) and TFL1 - 4T (SEQ ID NO: 19 or 20) affect the time to bloom by altering (eg, speeding up) flower development and thus changing (eg, shortening) the time to bloom. Unexpectedly, TFL1 - 1S (SEQ ID NO: 1 or 2) and TFL1 - 1T (SEQ ID NO: 4 or 5), TFL1 - 3T (SEQ ID NO: 13 or 14) and TFL1 - 4S (SEQ ID NO: 16 or 17) had almost no effect on the time period before flowering. Several motifs in TFL1-2S (SEQ ID NO: 7 or 8) and TFL1-2T (SEQ ID NO: 10 or 11) and TFL1-4T (SEQ ID NO: 19 or 20) have been identified that can be targeted . to destroy genes (eg, RNAi knockout, mutagenesis, etc., described herein) to change the time period before flowering (see, for example, example 4). Such motifs can be used as target regions to alter the expression of their respective genes in order to develop stable lines that bloom earlier or later. Without wishing to be bound by theory, it is believed that disrupting the expression of one or more TFL1 genes that are responsible for maintaining the vegetative state will facilitate the interaction of FT genes with promoters of flowering genes, thereby reducing the time period before flowering occurs.

ASPECTS AND EMBODIMENTS FOR CARRYING OUT THE INVENTION

Aspects and embodiments of the present invention are set forth in the appended claims.

In a first aspect, a mutant, non-naturally occurring, or transgenic plant, or portion thereof, is provided, characterized by reduced expression of a gene encoding Terminal Flower 1 ( TFL1 ), or reduced activity of a protein encoded by TFL1 , wherein said TFL1 contains, consists of, or essentially consists of (i) a polynucleotide sequence comprising, consisting of, or essentially consisting of a sequence having at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO: 11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide having at least 72% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO:21; where the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) or (iii) is reduced compared to a control plant in which the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) or (iii) ) were not reduced.

Accordingly, reduced expression of the polynucleotide or reduced activity of the polypeptide results in a shorter time to flowering compared to a control plant, respectively, wherein the time to flower is reduced by at least 8%, or at least 20%, or at least 28 %, or at least 30%.

Accordingly, the number of leaves is reduced by at least 16% or at least 22%.

Accordingly, plant height is reduced by at least 13%, or at least 23%, or remains approximately the same.

Accordingly, the plant contains at least one genetic alteration in the polynucleotide sequence encoding TFL1α .

Accordingly, the plant contains at least one mutation in the polynucleotide sequence encoding TFL1α .

Accordingly, at least one mutation is selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them.

Accordingly, the plant contains at least one mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S.

Accordingly, the plant contains at least one mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. Accordingly, the plant belongs to or is derived from the genus Nicotiana , respectively, where the plant is Nicotiana tabacum .

In a further aspect, a plant material is provided that is or can be obtained from a plant described herein.

In a further aspect, a herbal product is provided comprising at least a portion of the plant or plant material described herein.

In a further aspect, a method is provided for shortening the period of time before flowering in a plant, comprising modifying the plant by reducing the expression of at least one TFL1 gene or the activity of at least one protein encoded by it in said plant.

Accordingly, the method comprises (a) obtaining a plant or part thereof comprising (i) a polynucleotide sequence comprising, consisting of, or essentially consisting of a sequence having at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO: 8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide characterized by at least 72% sequence identity with SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:21; and (b) reducing TFL1 gene expression or TFL1 protein activity in the plant; and (c) obtaining a plant with a reduced time to flowering compared to a control plant in which TFL1 gene expression or TFL1 protein activity was not reduced.

In a further aspect, the use of reduced expression of at least one TFL1 gene, or activity of at least one protein encoded by it, is used to shorten the period of time before flowering in a plant.

Accordingly, TFL1 expression or TFL1 activity is reduced by a method selected from the group consisting of a) mutating the TFL1 gene in the plant; b) expression of the exogenous polynucleotide or polypeptide in the plant; and c) removing the TFL1 gene from the plant, or a combination of one or more of them.

Accordingly, at least one mutation is selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them.

Accordingly, at least one mutation is a mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S.

Accordingly, at least one mutation is a mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. Accordingly, the mutation is a mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S and a mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L.

In a further aspect, a method is provided for producing plant material with a reduced time to flowering compared to a control plant, said method comprising (a) obtaining a plant or plant material as described herein; (b) collecting plant material from the plant; (c) optionally drying or drying the plant material for a period of time; and (d) obtaining plant material that has a reduced time to flowering compared to a control plant.

In an additional aspect, a plant material is provided that is obtained or can be obtained using the method or application described herein.

In a further aspect, an isolated polynucleotide sequence is provided comprising, consisting of, or essentially consisting of a sequence having at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20.

In a further aspect, an isolated polypeptide encoded by the polynucleotide of claim 16 or a polypeptide having at least 72% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO:21 is provided.

Accordingly, in the isolated polypeptide, at least one mutation is selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them.

Accordingly, at least one mutation is a mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S.

Accordingly, at least one mutation is a mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. Accordingly, the mutation is a mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S and a mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L.

In a further aspect, an antibody is provided that specifically binds to the isolated polypeptide described herein.

In an additional aspect, a construct, vector, or expression vector is provided that contains the isolated polynucleotide described herein.

In a further aspect, a plant or plant material or plant cell is provided, comprising the construct, vector, or expression vector described herein.

In a further aspect, a plant cell is provided that is or can be obtained from the plant or plant material described herein.

In a further aspect, a plant material is provided that contains a cell as described herein.

In a further aspect, a tobacco product or smoking article is provided that contains the plant material described herein.

In an additional aspect, an RNAi construct is provided for suppressing TFL - 1 gene expression, comprising a sequence that hybridizes to a target sequence in the TFL - 1 mRNA and provides suppression of TFL - 1 gene expression through an RNA interference mechanism, where the specified target sequence is selected from the group, consisting of SEQ ID NO: 7, 8, 10, 11, 19 and/or 20.

In a further aspect, a double-stranded RNA is provided comprising at least two sequences that are at least partially complementary to each other, and wherein the sense strand comprises a first sequence and the antisense strand comprises a second sequence, and wherein at least one of the sequences comprises at least 10 contiguous nucleotides from TFL1 RNA, respectively, where at least one of the sequences contains 21-23 contiguous nucleotides from TFL1 RNA.

Accordingly, the double-stranded RNA contains a first sequence having at least 10 nucleotides from TFL1 , respectively 21-23 nucleotides from TFL-1; second sequence; and a third sequence containing a sequence inversely complementary to the first sequence located in the same orientation as the first sequence, where the second sequence is located between the first sequence and the third sequence, and the second sequence is operably linked to the first sequence and the third sequence.

Accordingly, the first sequence is selected from the group consisting of SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20 , and/or where the third sequence is a sequence reversely complementary to the sequence corresponding to SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:19, or SEQ ID NO:20.

Accordingly, the first sequence contains or consists of SEQ ID NO: 22, and the third sequence contains or consists of SEQ ID NO: 23; or the first sequence contains or consists of SEQ ID NO: 25, and the third sequence contains or consists of SEQ ID NO: 26; or the first sequence contains or consists of SEQ ID NO: 27, and the third sequence contains or consists of SEQ ID NO: 28; or the first sequence contains or consists of SEQ ID NO: 29, and the third sequence contains or consists of SEQ ID NO: 30; or the first sequence contains or consists of SEQ ID NO: 32, and the third sequence contains or consists of SEQ ID NO: 33; or the first sequence contains or consists of SEQ ID NO: 34, and the third sequence contains or consists of SEQ ID NO: 35; or the first sequence contains or consists of SEQ ID NO: 36, and the third sequence contains or consists of SEQ ID NO: 37; or the first sequence contains or consists of SEQ ID NO: 39 and the third sequence contains or consists of SEQ ID NO: 40.

Accordingly, the double-stranded RNA contains or consists of sequences selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 31, SEQ ID NO: 38, and SEQ ID NO: 41.

In a further aspect, an isolated polynucleotide sequence is provided comprising, consisting of, or essentially consisting of a sequence having at least 21 contiguous nucleotides from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:40, respectively, wherein the sequence contains, consists of, or essentially consists of at least 21 to 23 contiguous nucleotides.

In a further aspect, a method is provided for identifying a molecule that modulates the activity or expression of a TFL1 polynucleotide or TFL1 polypeptide, the method comprising (a) contacting the molecule with a plant containing the polynucleotide or polypeptide described herein, such as a polynucleotide sequence comprising , consisting or essentially consisting of a sequence characterized by at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO: 19, or SEQ ID NO:20, or a polypeptide encoded by a polynucleotide, or a polypeptide characterized by at least 72% sequence identity with SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:21; (b) monitoring one or more of (i) the level of expression of a TFL1 polynucleotide in a plant; (ii) the level of expression of the TFL1 polypeptide in the plant; (iii) modulating the activity of a TFL1 polypeptide in a plant; or (iv) modulating the activity of a TFL1 polynucleotide in a plant; and (c) identifying a molecule that modulates the activity or expression of a TFL1 polynucleotide or TFL1 polypeptide.

Combinations of one or more of the embodiments set forth are also disclosed.

SOME BENEFITS

The trait of fast flowering can provide for the selection of plant varieties with late flowering.

The fast flowering trait can produce commercial varieties of plants adapted to climatic conditions.

Controlling the time to bloom can provide increased seed or fruit production as well as flower extract production.

Controlling the period of time before flowering can ensure that there is no need for a treatment for maturation.

The fast flowering trait can provide a shorter time from seed/plant to harvest, which can shorten the growing season.

A fast flowering trait can allow more rapid introduction of new traits through crossbreeding.

A shorter plant life cycle could result in multiple crops of the plant per year, which could result in more sustainable production.

The fast flowering trait may result in earlier flower cutting, which may result in higher quality plant products.

It is advantageous to develop approaches based on non-genetically modified organisms (non-GMO) to reduce the time to flowering in plants through the use of gene inactivation. Due to the difficulties of growing and profiting from genetically modified crops in some countries, including European countries, it may be desirable to work with mutants with single nucleotide polymorphisms obtained by treatment with ethyl methanesulfonate (EMS) or the like, rather than through the use of genetic techniques. engineering. Mutants are not considered GMOs, even when the mutations are artificially induced. In the EU countries, for example, there are no special regulations for plants obtained as a result of mutation breeding. To date, the only known solution to shorten the time to flowering is to overexpress the FT genes, which may not be appropriate in a non-GMO environment. Knocking out the TFL1 genes using, for example, EMS/irradiation line selection or using selection based on natural TFL1 variants from different Nicotiana tabacum cultivars or an introgressive form from other Nicotiana species is an appropriate non-GMO solution. Alternatively, any gene editing technology could also be considered, but the regulation of such technical approaches is still unclear. The availability of TFL1 variants enables rapid selection by DNA testing without waiting for flowering and the need for tedious self-pollination steps.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1 . Expression of TFL1 and FT in the leaves of N. tabacum (TN90) flowering plants in the field. Transcript data are obtained by reading fragments per thousand bases of exon per million mapped fragments (FPKM) (see Nat Biotechnol. 2010 28(5):511-5). Expression of TFL1 and FT was determined by RNA sequence analyzes in immature flowers, lower stem leaves, mid stem leaves, upper stem leaves, petals, roots, sepals and stem.

Figure 2. Phenotypic analyzes of T0 RNAi TFL1 - 1S/T, TFL1 - 2S/T, TFL1 - 3T and TFL1 - 4T (20) and control (10) plants grown in a greenhouse under controlled conditions. The time to flowering (days after transplanting, DAT) of the four transgenic lines is shown in A and D, the leaf count values are shown in B and E, and the height of the four lines is shown in C and F. The means and standard deviations for each plant are shown.

Figure 3. Images of RNAi TFL1 - 2S/T plants in a greenhouse. Rapid flowering of RNAi TFL1 - 2S/T lines compared to control plants (Coltabaco 23RM), 117 days after potting (A) and after seed ball production, single plant comparison (B).

Figure 4 . Diagram illustrating the TFL1-2T-P131S mutation. Mutation of codon CCT > Pro to TCT > Ser.

Figure 5 . Bar graph illustrating that TFL1-2T-P131S mutant plants flower about 30% faster in both homozygous plants (mutant for both alleles) and heterozygous plants (mutant for one allele). The mutant plants have fewer leaves, but no effect on plant height was observed compared to the wild-type tobacco plant. WT= Nicotiana tabacum ; TFL1-2T-P131S wt=outer segregant of the mutated plant without mutation and is considered an additional control plant on the same phenotypic background as the mutant plants; TFL1-2T-P131S mut homo=homozygous mutant plant; TFL1-2T-P131S mut hetero = heterozygous mutant plant. N=4.

Figure 6 . Diagram illustrating the TFL1-4T-P110L mutation. Mutation of codon CCA > Pro to CTA > Leu.

Figure 7 . Bar graph illustrating that TFL1-4T-P110L mutant plants flower about 30% faster in heterozygous (single allele mutant) plants. The mutant plants have fewer leaves, but no effect on plant height was observed compared to the wild-type tobacco plant. WT= Nicotiana tabacum ; TFL1-4T-P110L mut hetero = heterozygous mutant plant. N=3.

DEFINITIONS

Technical terms and expressions used within the scope of this application should be given the meaning that is usually applied to them in this field of plant biology and molecular biology. All of the following definitions of terms apply to the entire content of this application. The word "comprising" does not exclude other elements or steps, and the singular forms do not exclude the plural. One stage can perform the functions of several features set forth in the claims. The terms "about", "substantially" and "about" in the context of a given numerical value or range refer to a value or range that is within 20%, within 10%, or within 5%, 4%, 3%, 2% or 1% of the set value or range.

The term "reduced time to bloom" or equivalents means a reduced time from sowing to first bloom compared to a control plant. The period can be shortened by at least about 5%, 6%, 7%, 8%, 9% 10%, 20%, 28%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more compared to the control plant.

The term "extended time to bloom" or equivalents means a longer period of time from sowing to the first flowers bloom compared to the control plant. The period can be extended by at least about 5%, 6%, 7%, 8%, 9% 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or more compared to the control plant.

The term "isolated" refers to any object that is taken from its natural environment, but this term does not imply any degree of purification.

An "expression vector" is a nucleic acid delivery vehicle that contains a combination of nucleic acid components to allow expression of the nucleic acid. Suitable expression vectors include episomes capable of extrachromosomal replication, such as double-stranded nucleic acid circular plasmids; linearized double-stranded nucleic acid plasmids; and other functionally equivalent expression vectors of any origin. The expression vector contains at least an upstream promoter operably linked to a nucleic acid, nucleic acid constructs, or a nucleic acid conjugate, as defined below.

The term "construct" refers to a double-stranded recombinant nucleic acid fragment containing one or more polynucleotides. The construction contains a "matrix thread", the bases of which are paired with a complementary "semantic or coding thread". This construct can be inserted into the vector in two possible orientations, either in the same (or sense) orientation, or in reverse (or antisense) orientation relative to the orientation of the promoter located in the vector, such as an expression vector.

"Vector" refers to a nucleic acid delivery vehicle that contains a combination of nucleic acid components to provide nucleic acid transfer, nucleic acid constructs and nucleic acid conjugates, and the like. Suitable vectors include episomes capable of extrachromosomal replication, such as double-stranded nucleic acid circular plasmids; linearized double-stranded nucleic acid plasmids; and other vectors of any origin.

"Promoter" refers to a nucleic acid element/sequence typically located upstream and operably linked to a double-stranded DNA fragment. Promoters may be derived entirely from regions near the native gene of interest, or may be composed of different elements derived from different native promoters or synthetic DNA segments.

The terms "homology, identity or similarity" refer to the degree of sequence similarity between two polypeptides or between two nucleic acid molecules compared by sequence alignment. Accordingly, the terms "homology, identity, or similarity" refer to the degree of sequence similarity between a complete sequence, such as a full sequence, of two polypeptides or between two nucleic acid molecules. The degree of identity between two distinct compared nucleic acid sequences is a function of the number of identical or matching nucleotides at comparable positions. Percent identity can be determined by visual inspection and mathematical calculations. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using a computer program such as ClustalW, BLAST, FASTA, or Smith-Waterman. The percent identity of two sequences can take on different values depending on (i) the method used to align the sequences, e.g., ClustalW, BLAST, FASTA, Smith-Waterman (used in different programs), or structural alignment from 3D comparison; and (ii) parameters used in the alignment method, e.g., local versus global alignment, pair substitution matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalty, e.g., functional form and constants . Once an alignment has been performed, there are different ways to calculate the percent identity between two sequences. For example, one can divide the number of identities by (i) the length of the shortest sequence; (ii) alignment length; (iii) average sequence length; (iv) number of positions without gaps; or (iv) the number of equivalent positions excluding protruding ends. In addition, it will be understood that the percent identity is also highly dependent on length. Therefore, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur randomly. The popular ClustalW multiple alignment program ( Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable method for generating multiple polypeptide or polynucleotide alignments. Suitable parameters for ClustalW may be as follows. For polynucleotide alignments: gap opening penalty=15.0, gap extension penalty=6.66 and template=identity. For polypeptide alignments: gap opening penalty=10. o, gap continuation penalty=0.2 and matrix=Gonnet. For DNA and protein alignments: ENDGAP=-1 and GAPDIST=4. One skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment. Accordingly, the percent identity is then calculated based on this alignment as (N/T), where N is the number of positions at which the identical residue is present in the sequences, and T is the total number of positions compared, including gaps but excluding overhangs. ends.

"Option" means almost the same sequence. A variant may have a similar function or substantially the same function relative to the wild-type sequence. In the case of TFL1, the same function is at least about 50%, 60%, 70%, 80% or 90% of the function of the wild-type protein under the same conditions. In the case of TFL1, a substantially similar function is at least about 90%, 95%, 96%, 97%, 98%, or 99% of the function of the wild-type protein under the same conditions. Variants may have one or more favorable mutations that result in a reduced level of TFL1 activity compared to the wild-type polypeptide. Variants may have one or more favorable mutations that result in TFL1 activity being knocked out ( ie, 100% downregulation and thus a non-functional polypeptide).

The term "plant" refers to any plant or part of a plant at any stage of its life cycle or development, as well as its offspring. In one embodiment, the plant is a "tobacco plant", which refers to a plant belonging to the genus Nicotiana. Preferred tobacco plant species are described herein. Accordingly, the plant is a mutant, non-naturally occurring or transgenic plant in which the expression of one or more genes or the activity of one or more proteins is modulated compared to a control plant. Accordingly, an alteration that renders a plant a mutant, non-naturally occurring, or transgenic plant results in modulation of the expression of one or more TFL1 genes or modulation of the activity of one or more TFL1 proteins. In certain embodiments, the alteration is a genetic alteration or genetic modification. Examples of mutations that can be introduced into plants to shorten the time to flowering are described herein.

"Plant parts" includes plant cells, plant protoplasts, tissue cultures of plant cells from which the whole plant can be regenerated, plant calluses, plant rhizomes, and plant cells that are intact in plants or plant parts such as germs, pollen, anthers, ovules. , seeds, leaves, flowers, stems, branches, fruit, roots, root tips, and the like. Progeny, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided they contain the introduced polynucleotides described herein. Plant leaves are particularly preferred for use in the present invention.

"Plant cell" refers to the structural and physiological unit of a plant. A plant cell may be in the form of a protoplast without a cell wall, an isolated single cell, or a cultured cell, or may be present as part of a more highly organized unit such as, but not limited to, plant tissue, a plant organ, or an entire plant.

The term "plant material" refers to any solid, liquid or gaseous composition, or combination thereof, derived from a plant, including biomass, leaves, stems, roots, flowers or flower parts, fruits, pollen, ova, zygotes, seeds, cuttings, secretions , extracts, cell or tissue cultures, or any other plant parts or products. In one embodiment, the plant material contains or consists of biomass, stems, seeds or leaves. In another embodiment, the plant material contains or consists of leaves.

The term "variety" refers to a population of plants that share permanent characteristics that separate them from other plants of the same species. While having one or more distinctive characters, a variety is further characterized by very little overall variation between individuals within that variety. The variety is often sold commercially.

The term "line" or "breeding line" as used herein refers to a group of plants that are used in plant breeding. A line differs from a variety in that it exhibits little inter-individual variability in one or more traits of interest, although there may be some inter-individual variability in other traits.

The term "non-naturally occurring" as used herein describes an entity (eg, polynucleotide, genetic mutation, polypeptide, plant and plant cell, and plant material) that is not naturally occurring or does not exist in nature. Such non-naturally occurring objects or man-made objects can be created, synthesized, manufactured, modified, tampered with or manipulated by the methods described herein or as known in the art. Such non-naturally occurring objects or man-made objects can be created, synthesized, manufactured, modified, tampered with or manipulated by humans. Thus, by way of example, a non-naturally occurring plant, a non-naturally occurring plant cell, or non-naturally occurring plant material can be generated using gene manipulation techniques such as using antisense RNA, interfering RNA, meganuclease, and the like. As a further example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material can be created using introgression or by transferring one or more genetic mutations (e.g., one or more polymorphisms) from the first plant or cell. plant to a second plant or plant cell (which may themselves be naturally occurring), such that the resulting plant, plant cell, or plant material, or progeny thereof, contains a genetic structure (e.g., genome, chromosome, or segment thereof) that is not formed naturally, or which is not exists in nature. The resulting plant, plant cell or plant material is thus artificial or not naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell can be created by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that contains a different genetic background from the first. plants or plant cells.

The term "modulation" may refer to a decrease, inhibition, increase, or other effect on the expression or activity of a polypeptide. The term may also refer to reducing, inhibiting, increasing or otherwise affecting the activity of a gene encoding a polypeptide, which may include, but is not limited to, modulating transcriptional activity. The term "modulating" can also refer to shortening or extending the period of time before flowering occurs.

The terms "reduction" or "reduced" or "reduction" or "reduced" as used herein refers to a reduction of from about 10% to about 99%, or a reduction of at least 10%, of at least 20%, at least 25% or 28%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of an amount or activity such as, but not limited to, polypeptide activity, transcriptional activity and protein expression.

The terms "inhibit" or "inhibited" as used herein refer to a reduction of from about 98% to about 100%, or a reduction of at least 98%, at least 99%, but in particular 100% of amount or activity, such as, but not limited to, polypeptide activity, transcriptional activity, and protein expression.

Cell transformation can be permanent or temporary. The terms "transiently transformed" or "transiently transformed" or variations of these terms refer to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the genome of the host cell. In contrast, the terms "stable transformation" or "stably transformed" refer to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. The term "stable transformant" refers to a cell into which one or more exogenous polynucleotides have been stably integrated into genomic or organelle DNA. It should be understood that an organism or cell thereof transformed with the nucleic acids, constructs and/or vectors of the present invention may be transiently as well as stably transformed. In certain embodiments, stable transformation is preferred.

The terms "magnification" or "enhanced" as used herein refers to an increase of from about 5% to about 99%, or an increase of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of an amount or activity such as, but not limited to, polypeptide activity, transcriptional activity, and protein expression.

The term "substantially" as used herein and when used in the context of an amount means that the amount is at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, or at least about 0.1% of the amount with which it is compared.

The term "control" in the context of a control plant or a control plant cell, etc., means a plant or plant cell in which the expression or activity of a gene or protein of interest has not been modulated, and therefore this control can provide a comparison or benchmark against the plant. or plant cells in which the expression or activity has been modified. Thus, in the context of the present invention, the control will not contain at least one modification or genetic alteration that reduces the expression or activity of TFL1. A control plant or plant cell may contain an empty vector. The control plant or plant cell may correspond to a wild-type plant or a wild-type plant cell, etc. In all such cases, the test plant and control plant are cultured and harvested using the same protocols for comparison purposes. Changes in the levels, ratios, activity, or distribution of the genes or polypeptides described herein, or changes in the phenotype of plants can be measured by comparing the test plant with a control plant, respectively, where the test plant and the control plant were cultivated and/or harvested using the same and the same protocols. The control plant may provide a reference point for measuring changes in the phenotype of the test plant. An indicator of change in phenotype can be measured at any point in time for a plant, including during plant development, senescence, or after drying. An indicator of change in phenotype can be measured for plants grown under any conditions, including plants grown in a grow room, greenhouse, or field.

DETAILED DESCRIPTION

In one embodiment, an isolated polynucleotide is provided, comprising a polynucleotide sequence consisting of, or consisting essentially of, that has at least 72% sequence identity with any of the sequences described herein, including any of the polynucleotides shown in the sequence listing. . Accordingly, an isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with it.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of, a polynucleotide sequence characterized by at least 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 20 or any of SEQ ID NO: 22-41.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of, a polynucleotide sequence characterized by at least 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2, or SEQ ID NO:5, or SEQ ID NO: 8 or SEQ ID NO: 11 or SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of, a polynucleotide sequence characterized by at least 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 19 or SEQ ID NO: 20.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of, a polynucleotide sequence characterized by at least 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 11, or SEQ ID NO: 20.

In another embodiment, polynucleotide variants are provided that are at least about 72%, 75%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% , 99.6%, 99.7%, 99.8% or 99.9% sequence identity with the sequence under SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 16 , or SEQ ID NO: 17, or SEQ ID NO: 19, or SEQ ID NO: 20, or any of SEQ ID NO: 22-41.

In another embodiment, polynucleotide variants are provided that are characterized by at least about 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99, 6%, 99.7%, 99.8% or 99.9% sequence identity with the sequence under SEQ ID NO:2, or SEQ ID NO:5, or SEQ ID NO:8, or SEQ ID NO: 11, or SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20.

In another embodiment, polynucleotide variants are provided that are characterized by at least about 72%, 73%, 74%, 75%, 78%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99, 6%, 99.7%, 99.8%, or 99.9% sequence identity with the sequence under SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20.

In another embodiment, fragments of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO : 20, or any of SEQ ID NOs: 22-41 with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them, which are characterized by at least about 72%, 73%, 74%, 75%, 80 %, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99, 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the corresponding fragments of SEQ ID NO:1 , or SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 20 or any from SEQ ID NO: 22-41. In certain embodiments, fragments may be 21-23 contiguous nucleotides in length. In certain embodiments, fragments may be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides in length. In certain embodiments, fragments may be at least about 10, 15, 20, 30, 40, 50, or 60 or more contiguous nucleotides in length.

In another embodiment, fragments of SEQ ID NO: 2 or SEQ ID NO: 5 or SEQ ID NO: 8 or SEQ ID NO: 11 or SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20 with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them, which are at least about 72%, 73%, 74%, 75%, 80%, 85%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the corresponding fragments of SEQ ID NO:2 or SEQ ID NO:5 or SEQ ID NO: 8 or SEQ ID NO: 11 or SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20.

In another embodiment, fragments of SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20 with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them are provided, which are characterized by at least about 72 %, 73%, 74%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98 %, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the corresponding fragments of SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20.

In another embodiment, polynucleotides are provided that provide a sufficient or substantial degree of identity or similarity to SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:7, or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19, or SEQ ID NO: 20, which encode a polypeptide that functions as a Terminal Flower 1 protein.

In another embodiment, polynucleotides are provided that provide a sufficient or substantial degree of identity or similarity to SEQ ID NO: 2, or SEQ ID NO: 5, or SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 14, or SEQ ID NO: 17 or SEQ ID NO: 20, which encode a polypeptide that functions as a Terminal Flower 1 protein.

In another embodiment, polynucleotides are provided that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20, which encode a polypeptide that functions as a Terminal Flower 1 protein.

In another embodiment, polynucleotide(s) are provided that encode(s) a protein with a Terminal Flower 1 protein activity that is at least about 72%, 73%, 74%, 75% , 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% or more of the activity of the protein shown under SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:14 , or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 19, or SEQ ID NO: 20.

In another embodiment, polynucleotide(s) are provided that encode(s) a protein with a Terminal Flower 1 protein activity that is at least about 72%, 73%, 74%, 75% , 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or more of the activity of the protein shown under SEQ ID NO: 3, or SEQ ID NO: 9, or SEQ ID NO: 12 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:21.

In another embodiment, there is(s) the polynucleotide(s) described herein that(s) encode(s) for a protein with a Terminal Flower 1 protein activity that is at least about 72% , 73%, 74%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% or more of the activity of the protein shown under SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:21.

To determine if a polypeptide is a functional Terminal Flower 1 protein, BLAST (Basic Local Alignment Search Tool) analyzes can be used to detect areas of similarity between biological sequences. The program can be used to compare nucleotide or protein sequences against sequence databases and calculate statistical significance. TFL1 transcription factor activity can be determined by the ability of TFL1 to have increased or decreased binding function - such as increased or decreased binding function to other proteins (eg, transcription factors) or increased or decreased binding to one or more nucleic acids. Transcriptional activity of TFL1 can be determined either biochemically by determining binding properties or by evaluating the outcome of transcription factor activity, such as increased or decreased expression of a target gene that responds to transcription factor activity. For example, in Arabidopsis thaliana, TFL1 acts by repressing the activity of the LFY and AP1 genes (Development (1998) 125: 1609-1615; Development (1999) 126: 1109-1120). The transcriptional activity of TFL1 can therefore be determined by measuring the activity of the LFY and/or AP1 genes in the presence and absence of TFL1. The end result is a reduced period of time before flowering, so that the main biological activity of TFL1 can be defined as inhibition of flowering. The biological role of TFL1/FT in flower development can be tested using gain-of-function and loss-of-function of the respective alleles (see The Plant Journal (2010) 63: 241-253). The polynucleotide described herein may include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragment(s) thereof. In addition, the polynucleotide may be single or double stranded DNA, DNA that is a mixture of single and double stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule with a mixture of single and double stranded regions or fragment(s) thereof. In addition, the polynucleotide may be composed of three-stranded sections containing DNA, RNA, or both, or fragment(s) thereof. The polynucleotide may contain one or more modified bases such as phosphorothioates and may be a peptidic nucleic acid. Typically, polynucleotides can be assembled from isolated or cloned cDNA fragments, genomic DNA, oligonucleotides, or single nucleotides, or a combination of the above. Although the polynucleotide sequences described herein are presented as DNA sequences, the sequences include their respective RNA sequences and their complementary (eg, fully complementary) DNA or RNA sequences, including their reverse complementary strands. The polynucleotides described herein may contain one or more substitution modifications. The polynucleotides described herein may contain one or more tags.

The polynucleotide described herein typically contains phosphodiester linkages, although in some cases polynucleotide analogs are included that may have alternative backbones containing, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages; and peptide polynucleotide backbones and bonds. Other polynucleotide analogs include polynucleotides with positively charged backbones, non-ionic backbones, and non-ribose backbones. Modifications to the ribose phosphate backbone can be made for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological media or as probes on a biochip. You can get a mixture of natural polynucleotides and analogues; alternatively, mixtures of different polynucleotide analogues and mixtures of naturally occurring polynucleotides and analogues can be prepared.

A variety of polynucleotide analogs are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide polynucleotide backbones and linkages. Other polynucleotide analogs include polynucleotides with positively charged backbones, non-ionic backbones, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.

Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs. These backbones are mostly non-ionic under neutral conditions in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may provide benefits. First, the peptide backbone may have improved hybridization kinetics. Peptide polynucleotides are characterized by greater melting point changes for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit melting temperature decreases of 2-4°C due to internal mismatch. In the case of a non-ionic peptide polynucleotide backbone, the drop is closer to 7-9°C. Similarly, due to their non-ionic nature, the hybridization of bases attached to these scaffolds is relatively insensitive to salt concentration. In addition, peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable.

Among the uses of the disclosed polynucleotides and fragments thereof is the use of the fragments as probes in nucleic acid hybridization assays or primers for use in nucleic acid amplification assays. Such fragments typically contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides from the DNA sequence. In other embodiments, the DNA fragment contains at least about 10, 15, 20, 30, 40, 50, or 60 or more contiguous nucleotides from the DNA sequence. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides from SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 20. In other embodiments, the DNA fragment contains at least about 10, 15, 20 , 30, 40, 50 or 60 or more contiguous nucleotides from SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO : 19, or SEQ ID NO: 20. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides from SEQ ID NO :2 or SEQ ID NO:5 or SEQ ID NO:8 or SEQ ID NO: 11, or SEQ ID NO: 14, or SEQ ID NO: 17, or SEQ ID NO: 20. In other embodiments, the DNA fragment contains at least about 10, 15, 20, 30, 40, 50, or 60 or more contiguous nucleotides from SEQ ID NO: 2 or SEQ ID NO: 5 or SEQ ID NO: 8 or SEQ ID NO: 11 or SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides from SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20. In other embodiments, the DNA fragment contains at least about 10, 15, 20, 30, 40, 50, or 60 or more contiguous nucleotides from SEQ ID NO: 8, or SEQ ID NO: 11, or SEQ ID NO: 20.

The main parameters influencing the choice of hybridization conditions for polynucleotides and guidance for developing suitable conditions are described in Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbour, N.Y.). Using the genetic code data in combination with the amino acid sequences described herein, sets of degenerate oligonucleotides can be obtained. Such oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR) by which DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for genetic libraries. Such libraries would include, without limitation, cDNA libraries, genomic libraries, and even electronic label libraries for expressed sequences or DNA. Homologous sequences identified by this method will then be used as probes to identify homologues of the sequences specified herein.

In addition, polynucleotides and oligonucleotides (e.g., primers or probes) that hybridize under reduced stringency conditions, typically medium stringency conditions, and typically high stringency conditions, with the polynucleotide(s) described(s) have potential use. ) in this document. The main parameters influencing the choice of hybridization conditions and guidance for developing suitable conditions can be easily determined by those of ordinary skill in the art based on, for example, the length or composition of the bases of the polynucleotide. One way to achieve moderately stringent conditions involves using a pre-wash solution containing 5x standard sodium citrate, 0.5% sodium dodecyl sulfate, 1.0 mM ethylenediaminetetraacetic acid (pH 8.0), hybridization buffer with approximately 50% formamide, 6x standard sodium citrate and a hybridization temperature of approximately 55°C (or other similar hybridization solutions such as those containing approximately 50% formamide with a hybridization temperature of approximately 42°C) and washing conditions at approximately 60°C in 0.5x standard sodium citrate , 0.1% sodium dodecyl sulfate. Typically, high stringency conditions are defined as hybridization conditions as described above, but with a wash at about 68° C., 0.2x standard sodium citrate, 0.1% sodium dodecyl sulfate. SSPE (1x SSPE is 0.15 M sodium chloride, 10 mM sodium phosphate, 1.25 mM EDTA, pH 7.4) can be replaced with standard sodium citrate (1x standard sodium citrate is 0.15 M sodium chloride and 15 mM sodium citrate) in hybridization buffers and wash buffers; washes are performed within 15 minutes of completion of hybridization. It should be understood that the wash temperature and wash salt concentration can be adjusted as needed to achieve the desired degree of stringency using the basic principles that govern hybridization reactions and duplex stability as is known to those skilled in the art and further described below (see e.g. e.g. , Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) When a polynucleotide is hybridized to a target polynucleotide of unknown sequence, the length of the hybrid is assumed to be as for the polynucleotide to be hybridized When polynucleotides are hybridized to known sequences, the length of the hybrid can be determined by aligning the polynucleotide sequences and identifying the site or sites with optimal sequence complementarity. yat less than 50 base pairs in length, should be 5-10 C less than the melting point of the hybrid, where the melting point is determined in accordance with the following equations. For hybrids less than 18 bp in length, melting point (°C) = 2(number of A+T bases)+4(number of G+C bases). For hybrids longer than 18 bp, melting point (°C) = 81.5+16.6(log10 [Na+])+0.41(% G+C)-(600/N) where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1x standard sodium citrate=0.165M). Typically, each such hybridizing polynucleotide has a length that is at least 25% (typically at least 50%, 60%, or 70%, and most often at least 80%) of the length of the polynucleotide to which it hybridizes and is characterized at least 60% sequence identity (e.g., at least 70%, 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) c polynucleotide with which it hybridizes.

As will be appreciated by one of skill in the art, linear DNA has two possible orientations: the 5'-3' direction and the 3'-5' direction. For example, if the reference sequence is located in the 5'-3' direction, and if the second sequence is located in the 5'-3' direction in the same polynucleotide molecule/strand, then the reference sequence and the second sequence are oriented in the same direction or have the same orientation. Typically, the promoter sequence and the gene of interest under the control of that promoter are in the same orientation. However, if relative to the reference sequence located in the 5'-3' direction, the second sequence is located in the 3'-5' direction in the same polynucleotide molecule/strand, then the reference sequence and the second sequence are oriented in the antisense direction or have an antisense orientation. Two sequences having antisense orientations with respect to each other can alternatively be described as having the same orientation if a reference sequence (the 5'-3' direction) and a sequence inversely complementary to the reference sequence (the reference sequence located 5'-3' ) are located within a single polynucleotide molecule/strand. The sequences set forth herein are shown in the 5'-3' direction.

The recombinant constructs provided herein can be used to transform plants or plant cells to modulate protein expression levels and/or activity. The recombinant polynucleotide construct may comprise a polynucleotide encoding one or more of the polynucleotides described herein operably linked to a regulatory region suitable for expression of the polypeptide. Thus, a polynucleotide may contain a coding sequence that encodes for a polypeptide as described herein. Plants or plant cells in which protein expression and/or activity levels are modulated may include mutant, non-naturally occurring, transgenic, human-created or genetically engineered plants or plant cells. Accordingly, the plant or plant cell contains a genome that has been modified by stable integration of recombinant DNA. Recombinant DNA includes DNA that is genetically engineered and engineered outside of a cell, and includes DNA containing naturally occurring DNA, or cDNA, or synthetic DNA. The plant may include a plant regenerated from an originally transformed plant cell and progeny plants from later generations or crosses of the transformed plant. Accordingly, the modification alters the expression or activity of a polynucleotide or polypeptide described herein as compared to a control plant.

The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide that modulates expression operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.

Also provided are vectors containing recombinant polynucleotide constructs such as those described herein. Suitable vector bases include, for example, those commonly used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses and retroviruses. Numerous vectors and expression systems are commercially available. Vectors may include, for example, origins of replication, nuclear matrix binding sites, or markers. The marker gene may confer a selectable phenotype to a plant cell. For example, the marker can confer biocidal resistance, such as resistance to an antibiotic (eg, kanamycin, G418, bleomycin, or hygromycin), or to a herbicide (eg, glyphosate, chlorsulfuron, or phosphinothricin). In addition, the expression vector may include a tag sequence designed to facilitate manipulation or detection (eg, purification or localization) of the expressed polypeptide. Label sequences such as luciferase, beta-glucuronidase, green fluorescent protein, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences are typically expressed as a fusion to the encoded polypeptide. Such labels can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

A plant or plant cell can be transformed by integrating a recombinant polynucleotide into its genome to obtain a stable transformation. The plant or plant cell described herein can be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. A plant or plant cell can also be transiently transformed such that the recombinant polynucleotide does not integrate into its genome. Transiently transformed cells typically lose all or some of the introduced recombinant polynucleotide with each cell division, so that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions. This document also contemplates the use of genome editing.

There are a number of methods available in the art for transforming a plant cell, all of which are covered herein, including biolistics, gene gun methods, Agrobacterium-mediated transformation, viral vector-mediated transformation, and electroporation. Agrobacterium's system for integrating foreign DNA into plant chromosomes has been extensively studied, modified and used for plant genetic engineering. Deproteinized recombinant DNA molecules containing DNA sequences corresponding to the purified protein of interest, operably linked in sense or antisense orientation to regulatory sequences, are coupled to the corresponding T-DNA sequences using conventional methods. They are introduced into protoplasts using a polyethylene glycol technique or an electroporation technique, both of which are standard. Alternatively, such vectors containing recombinant DNA molecules encoding the purified protein of interest are introduced into living Agrobacterium cells, which then transfer the DNA into plant cells. Transformation with deproteinized DNA without accompanying T-DNA vector sequences can be performed by protoplast fusion with DNA-containing liposomes or by electroporation. Deproteinized DNA without accompanying T-DNA vector sequences can also be used to transform cells with inert, high speed microparticles.

If a cell or cultured tissue is used as a recipient tissue for transformation, plants can be regenerated from the transformed cultures, if necessary, using techniques known to those skilled in the art.

The choice of regulatory regions to include in a recombinant construct depends on several factors, including, but not limited to, potency, selectivity, inducibility, desired level of expression, and preferred expression in certain cells or tissues. It is routine for one skilled in the art to modulate the expression of a coding sequence by appropriate selection and placement of regulatory regions relative to the coding sequence. Transcription of a polynucleotide can be modulated in a similar manner. Some suitable regulatory sites initiate transcription exclusively or predominantly in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are well known in the art.

Examples of promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissue or cell types (eg, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present at different developmental stages, or present in response to different conditions. environment. Examples of promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of promoters for directing RNAi polypeptide production include the cauliflower mosaic virus (CaMV/35S) 35S promoter, SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin gene promoters. Those of skill in the art can create many variants of recombinant promoters.

Tissue-specific promoters are transcriptional controls that are active only in certain cells or tissues at certain times during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression may be preferential, for example, when the expression of polynucleotides in certain tissues is preferred. Examples of tissue-specific developmental promoters include promoters that can initiate transcription only (or mostly only) in certain tissues, such as vegetative tissues, such as roots or leaves, or reproductive tissues, such as fruits, ovules, seeds, pollen. , stamens, flowers or any embryonic tissue. Promoters specific for reproductive tissues can be, for example, anther specific, ovule specific, germ specific, endosperm specific, integument specific, seed and seed coat specific, pollen specific, petal specific, sepal specific. or their combinations.

Examples of leaf-specific promoters include the pyruvatortophosphate dikinase (PPDK) gene promoter from the C4 plant (maize), the cab-m1Ca+2 promoter from maize, the myb-related gene promoter from Arabidopsis thaliana (Atmyb5), the ribulose bisphosphate carboxylase (RBCS) gene promoters (e.g., genes RBCS1, RBCS2 and RBCS3A expressed in leaves and seedlings grown in the light, RBCS1 and RBCS2 expressed in developing tomato fruits, or the promoter of the ribulose bisphosphate carboxylase gene, which is expressed at high levels almost exclusively in mesophilic cells of leaf blades and leaf axils).

Examples of senescent tissue-specific promoters include the tomato promoter active during fruit ripening, senescence and leaf abscission, promoter of the gene encoding maize cysteine protease, promoter of the 82E4 gene, and promoter of the SAG genes.

Anther-specific promoters are further examples. Root-preferred promoters known to those skilled in the art can be selected. Seed-preferred promoters include both seed-specific promoters (promoters active during seed development, such as seed storage protein promoters) and germinating seed promoters (promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, the Cim1 (cytokinin-induced signal) gene promoter; cZ19B1 (19 kDa maize zein); milps (myoinositol-1-phosphate synthase); mZE40-2, also known as Zm-40; nuclc and celA (cellulose synthase). The gamma-zein gene promoter is an endosperm-specific promoter. The Glob-1 gene promoter is a germ-specific promoter. For dicotyledonous plants, seed-specific promoters include, but are not limited to, the promoter of the bean bean phaseolin gene, the napin gene, the β-conglycinin gene, the soybean lectin gene, the cruciferin gene, and the like. For monocots, seed-specific promoters include, but are not limited to, the maize 15 kD zein gene promoter, the 22 kD zein gene promoter, the 27 kD zein gene promoter, the g-zein gene promoter, the 27 kD gamma-zein gene promoter (such as the gzw64A promoter, see Genbank accession number S78780), waxy gene promoter, shrunken 1 gene promoter, shrunken 2 gene promoter, globulin 1 gene promoter (see Genbank accession number L22344), Itp2 gene promoter, cim1 gene promoter, maize end1 and end2 gene promoters, nuc1 gene promoter, Zm40 gene promoter, eep1 and eep2 gene promoter; lec1 gene promoter, thioredoxin H gene promoter; mlip15 gene promoter, PCNA2 gene promoter, and shrunken-2 gene promoter.

Examples of inducible promoters include those responsive to pathogens, anaerobic conditions, heat, light, drought, low temperature, or high salt concentration. Pathogen-inducible promoters include promoters of pathogenesis-associated proteins (PR proteins) that are induced after infection with a pathogen (eg, PR proteins, SAR proteins, beta-1,3-glucanase, chitinase).

In addition to plant promoters, other suitable promoters of bacterial origin (e.g. , the octopine synthase gene promoter, the nopaline synthase gene promoter, and other promoters derived from Ti plasmids) can be derived, or they can be derived from viral promoters (e.g., the 35S and 19S RNA promoters of the mosaic virus cauliflower mosaic virus (CaMV), constitutive tobacco mosaic virus promoters, 19S and 35S promoters of cauliflower mosaic virus (CaMV), or 35S promoter of boletus mosaic virus). In certain embodiments, the mirabilis mosaic virus (MMV) promoter is preferred. In certain embodiments, the 35S promoter is preferred.

In another aspect, an isolated polypeptide is provided, comprising a polypeptide sequence consisting of or essentially consisting of it, which has at least 72% sequence identity with any of the polypeptide sequences described herein, including any of the polypeptides shown in the sequence listing. Accordingly, an isolated polypeptide contains, consists of, or essentially consists of a sequence characterized by at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99, 5% 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with it.

In one embodiment, the isolated polypeptide contains, consists of, or essentially consists of a sequence characterized by at least 72%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4 %, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 3, or SEQ ID NO: 9, or SEQ ID NO: 12 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:21.

In another embodiment, the isolated polypeptide contains, consists of, or essentially consists of a sequence characterized by at least 72%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4 %, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO: 21.

In certain embodiments, the activity of a polypeptide comprising, consisting of, or essentially consisting of a sequence encoding a Terminal Flower 1 protein and having at least 72% sequence identity with SEQ ID NO: 3, or SEQ ID NO: 9, or SEQ ID NO: 12 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:21 was modulated. In another embodiment, the activity of a polypeptide comprising, consisting of, or essentially consisting of a sequence encoding a Terminal Flower 1 protein and having at least 72% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO: 21, modulated.

The polypeptide may contain fragments of sequences that share a sufficient or substantial degree of identity or similarity to function as a Terminal Flower 1 protein. 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8 %, 99.9% or 100% activity.

As discussed herein, polypeptides also include mutants obtained by introducing alterations of any type (e.g., one or more insertions, deletions, or substitutions of amino acids; changes in glycosylation patterns, changes that affect refolding or isomerization, three-dimensional structure, or self-association states) when provided that they still retain some or all of their function or activity as a Terminal Flower 1 protein. Accordingly, function or activity as a Terminal Flower 1 protein is modulated, reduced or suppressed. Accordingly, the function or activity as a Terminal Flower 1 protein is suppressed such that the activity of the Terminal Flower 1 protein is not detectable. Illustrative mutants are described in this document.

Polypeptides include variants obtained by introducing any type of alteration (eg, insertions, deletions or substitutions of amino acids, changes in glycosylation patterns, changes that affect refolding or isomerization, three-dimensional structures or self-association states) that can be intentionally engineered or easily isolated. The alteration may be one or more stop codons. A variant may have alterations that produce a silent change and result in a functionally equivalent protein. Intentional amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and the amphipathic nature of the residues, provided that the secondary binding activity of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar end groups with similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine. Conservative substitutions can be made, for example, in accordance with the table below. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for each other.

ALIPHATIC non-polar Gly Ala Pro Ile Leu Val Polar - uncharged Cys Ser Thr Met Asn Gly Polar - charged AspGlu Lys Arg AROMATIC His Phe TrpTyr

The polypeptide may be a mature protein or an immature protein, or a protein derived from an immature protein. The polypeptides may be linear or cyclized using known methods. Polypeptides typically contain at least 10, at least 20, at least 30, or at least 40, or at least 50, or at least 100, or at least 200, or at least 300 , or at least 400, or at least 500, or at least 600 contiguous amino acids.

Also disclosed is a polypeptide encoded by SEQ ID NO: 3, or SEQ ID NO: 9, or SEQ ID NO: 12, or SEQ ID NO: 15, or SEQ ID NO: 18, or SEQ ID NO: 21, which is characterized by 100% sequence identity with it, or a polypeptide containing, consisting or essentially consisting of the sequence presented under SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:15, or SEQ ID NO:21.

The polypeptide can be obtained by culturing the transformed or recombinant host cells under culture conditions suitable for expression of the polypeptide. The resulting expressed polypeptide can then be purified from such a culture using known purification methods. Purification of a polypeptide may include the use of an affinity column containing agents that bind to the polypeptide; one or more stages using a column with such affinity resins; one or more steps involving hydrophobic interaction chromatography or immunoaffinity chromatography. Alternatively, the polypeptide may be expressed in a form that will facilitate purification. For example, it can be expressed as a fusion polypeptide, for example, with a maltose-binding polypeptide, glutathione-5-transferase, his-tag or thioredoxin. Kits for expression and purification of fusion polypeptides are commercially available. A polypeptide can be labeled with an epitope and then purified using a specific antibody for that epitope. One or more liquid chromatography steps, such as reverse phase high performance liquid chromatography, can be used to further purify the polypeptide. Some or all of the above purification steps in various combinations can be used to obtain a substantially homogeneous recombinant polypeptide. A polypeptide thus purified may be substantially free of other polypeptides and is defined herein as "substantially purified polypeptide"; such purified polypeptides include polypeptides, fragments, variants, and the like. Expression, isolation, and purification of polypeptides and fragments can be performed using any suitable technique, including, without limitation, the methods described herein.

In addition, an affinity column, for example with a monoclonal antibody designed against polypeptides, can be used to affinity purify the expressed polypeptides. These polypeptides can be removed from the affinity column using conventional techniques, for example, in a high salt elution buffer followed by dialysis in a low salt buffer for use, or by changing the pH or other components depending on the affinity matrix used, or by competitive removal with a natural substrate of the affinity fragment.

Isolated or substantially purified polynucleotides or protein compositions are described. An "isolated" or "purified" polynucleotide or protein, or a biologically active portion thereof, contains substantially or essentially no components that would normally complement or interact with the polynucleotide or protein found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when prepared by recombination techniques, or substantially free of precursors or other substances when prepared by chemical synthesis. Optimally, an "isolated" polynucleotide does not contain sequences (optimally protein-encoding sequences) that naturally flank the polynucleotide (e.g., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, an isolated polynucleotide may comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb ., 0.5 kb, or 0.1 kb a nucleotide sequence that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Protein that is substantially free of cellular material includes preparations of a protein having a contaminating protein content of less than about 30%, 20%, 10%, 5%, or 1% (based on dry weight).

The polypeptide can also be obtained using known conventional methods of chemical synthesis. Methods for constructing polypeptides or fragments thereof by synthetic means are known to those skilled in the art. Synthetically engineered polypeptide sequences, due to common primary, secondary or tertiary structural or conformational characteristics with native polypeptides, may share biological properties with them, including biological activity.

Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as nucleic acid sequencing, determining the presence or absence of genetic markers (eg, microsatellite RNA markers).

Also provided are antibodies that are immunoreactive with respect to the polypeptides described herein. The polypeptides, fragments, variants, fusion polypeptides, and the like set forth herein can be used as "immunogens" in the production of antibodies immunoreactive thereto. Such antibodies can specifically bind to the polypeptide through the antigen-binding sites of the antibody. Specific binding antibodies are those that will specifically recognize and bind to polypeptide, homologues and variants, and not to other molecules. In one embodiment, the antibodies are specific for polypeptides having the amino acid sequence set forth herein and do not cross-react with other polypeptides.

More specifically, the polypeptides, fragment, variants, fusion polypeptides, and the like contain antigenic determinants or epitopes that cause the formation of antibodies. These antigenic determinants or epitopes may be linear or conformational (discontinuous). Linear epitopes consist of a single amino acid stretch of a polypeptide, while conformational or discontinuous epitopes consist of amino acid portions from different regions of the polypeptide chain that have been brought into close proximity by folding the polypeptide. Epitopes can be identified by any means known in the art. In addition, epitopes from polypeptides can be used as research reagents, in assays, and for the purification of specifically binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes, or variants thereof, can be generated using techniques known in the art, for example, solid phase synthesis, chemical or enzymatic cleavage of a polypeptide, or recombinant DNA technology.

Both polyclonal and monoclonal antibodies to polypeptides can be obtained using conventional techniques. Hybridoma cell lines that produce monoclonal antibodies specific for polypeptides are also discussed in this document. Such hybridomas can be obtained and identified using well-known techniques. To produce antibodies, various host animals can be immunized by introducing a polypeptide, fragment, variant or mutant thereof. For example, such animal hosts may include, but are not limited to, rabbits, mice, and rats. Various adjuvants can be used to enhance the immunological response. Depending on the type of host, such adjuvants include, but are not limited to, Freund's adjuvants (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole hemocyanin , dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum . Monoclonal antibodies can be isolated using conventional techniques. Such monoclonal antibodies may be any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

Antibodies can also be used in assays to detect the presence of polypeptides or fragments, either in vitro or in vivo . Antibodies can also be used to purify polypeptides or fragments using immunoaffinity chromatography.

Also disclosed are fragments of the polynucleotides described herein and polypeptides encoded in this manner. Polynucleotide fragments may encode protein fragments that retain the biological activity of the native protein. Alternatively, polynucleotide fragments useful as hybridization probes or PCR primers generally do not encode a protein fragment that retains biological activity. In addition, fragments of the disclosed nucleotide sequences include those fragments that can be assembled into recombinant constructs as described herein. Polynucleotide sequence fragments can range from at least about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides , approximately 300 nucleotides, approximately 400 nucleotides, approximately 500 nucleotides, approximately 600 nucleotides, approximately 700 nucleotides, approximately 800 nucleotides, approximately 900 nucleotides, approximately 1000 nucleotides, approximately 1100 nucleotides, approximately 1200 nucleotides, approximately 1300 nucleotides or approximately 1500 nucleotides, approximately 2000 nucleotides, approximately 3000 nucleotides, approximately 4000 nucleotides, approximately 5000 nucleotides, approximately 6000 nucleotides, approximately 7000 nucleotides, with about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 15,000 nucleotides, about 20,000 nucleotides, and up to a full-length polynucleotide encoding the polypeptides described herein.

Polypeptide sequence fragments can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, approximately 600 amino acids or up to the full length polypeptide described herein.

Modulating the expression or activity of one or more TFL1 proteins or one more TFL1 nucleic acid sequences is advantageous for the reasons described herein. The expression of TFL1-2S (SEQ ID NO: 7 or 8 ) or TFL1-2T (SEQ ID NO: 10 or 11) or TFL1-4T (SEQ ID NO : 19 or 20) can be modulated individually in a plant such that that modulate the expression of only one of TFL1-2S (SEQ ID NO: 7 or 8), or TFL1-2T (SEQ ID NO: 10 or 11), or TFL1-4T (SEQ ID NO: 19 or 20). The expression of two or more of TFL1-2S (SEQ ID NO: 7 or 8 ) or TFL1-2T (SEQ ID NO: 10 or 11) or TFL1-4T (SEQ ID NO: 19 or 20 ) can be modulated in a plant such in a manner that modulates the expression of two or more of TFL1-2S (SEQ ID NO: 7 or 8 ) or TFL1-2T ( SEQ ID NO: 10 or 11) or TFL1-4T (SEQ ID NO: 19 or 20). For example, expression of TFL1-2S (SEQ ID NO: 7 or 8) and TFL1-2T (SEQ ID NO: 10 or 11 ) can be modulated. For example , expression of TFL1-2S (SEQ ID NO: 7 or 8) and TFL1-4T (SEQ ID NO: 19 or 20 ) can be modulated. For example , expression of TFL1-2T (SEQ ID NO: 10 or 11) and TFL1-4T (SEQ ID NO: 19 or 20 ) can be modulated. For example , expression of TFL1-2S (SEQ ID NO: 7 or 8) and TFL1-4T (SEQ ID NO: 19 or 20 ) can be modulated.

The activity of TFL1-2S (SEQ ID NO: 9) or TFL1-2T (SEQ ID NO: 12) or TFL1-4T (SEQ ID NO: 21) can be modulated individually in a plant such that the expression of only one of the TFL1-2S (SEQ ID NO: 9) or TFL1-2T (SEQ ID NO: 12) or TFL1-4T (SEQ ID NO: 21). The expression of two or more of TFL1-2S (SEQ ID NO: 9) or TFL1-2T (SEQ ID NO: 12) or TFL1-4T (SEQ ID NO: 21) can be modulated in a plant such that the expression of two or more of TFL1-2S (SEQ ID NO: 9) or TFL1-2T (SEQ ID NO: 12) or TFL1-4T (SEQ ID NO: 21). For example, expression of TFL1-2S (SEQ ID NO: 9) and TFL1-2T (SEQ ID NO: 12) can be modulated. For example, expression of TFL1-2S (SEQ ID NO: 9) and TFL1-4T (SEQ ID NO: 21) can be modulated. For example, expression of TFL1-2T (SEQ ID NO: 12) and TFL1-4T (SEQ ID NO: 21) can be modulated. For example, expression of TFL1-2S (SEQ ID NO: 21) and TFL1-4T (SEQ ID NO: 21) can be modulated.

In certain embodiments, modulation (e.g., reduction) of Terminal Flower 1 protein expression can be accomplished at the genomic and/or transcript level using a variety of molecules that disrupt transcription and/or translation, including, but not limited to, antisense molecules, siRNA molecules, ribozyme or deoxyribozyme. Insertion of one or more mutations in at least one gene can also be used, including deletions, insertions, site-specific mutations, zinc finger nucleases, and the like. In other embodiments, expression can be suppressed at the protein level using antagonists or enzymes that cleave the polypeptide, etc.

In one aspect, a mutant plant, or portion thereof, is described comprising at least one mutation in (i) a polynucleotide sequence comprising, consisting of, or essentially consisting of a sequence having at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide having at least 72% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO:21; or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide shown in (i), wherein at least one mutation results in a reduction in the expression or activity of the Terminal Flower 1 protein compared to a control plant that does not contain at least one mutation . A plant or plant cell may therefore contain one or more mutations in TFL1-2S (SEQ ID NO: 7 or 8) and/ or TFL1-2T ( SEQ ID NO: 10 or 11) and/or TFL1-4T ( SEQ ID NO: 19 or 20), wherein said mutation results in reduced expression or reduced function of said gene or protein encoded by it.

The expression or function of the mutant(s) can be modulated, suppressed or reduced. A mutant plant or plant cell may have one or more additional mutations in one or more other genes or polypeptides. In certain embodiments, the implementation of the mutants may have one or more additional mutations in one or more other genes or polypeptides.

Said mutant plant or plant cell may be heterozygous or homozygous for the mutation(s). Said mutant plant or plant cell may be heterozygous for at least one mutation and homozygous for at least one different mutation. Accordingly, the mutant plant or plant cell is homozygous for the mutation(s).

Illustrative mutants and mutations are described in this document.

In one embodiment, at least one mutation is selected from the group consisting of a mutation at position T143 and/or G129 in SEQ ID NO: 9; or mutations at position R120 and/or G129 and/or P131 in SEQ ID NO: 12; or a mutation at position P110 or H86 in SEQ ID NO: 21, or a combination of two or more of them.

In one embodiment, at least one mutation in SEQ ID NO: 9 is selected from mutations at positions {T143,G129} {T143,H84} {G129,H84}{T143,G129,H84}.

In one embodiment, at least one mutation in SEQ ID NO: 12 is selected from mutations at positions {R120,G129} {R120,P131} {G129,P131} {R120,G129,P131} {R120,P131,D142}

In one embodiment, at least one mutation in SEQ ID NO: 12 is selected from mutations at positions {P110,H86}.

In one embodiment, at least one mutation is a mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S.

In one embodiment, at least one mutation is a mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L.

In one embodiment, the mutations are a mutation at position P131 of SEQ ID NO: 12, respectively, where the mutation is P131S, and a mutation at position P110 of SEQ ID NO: 21, respectively, where the mutation is P110L.

All possible combinations of such mutations are disclosed which include any 2, 3, 4, 5, 6 or 7 mutations selected from positions T143 and G129 in SEQ ID NO: 9 and positions R120 and/or G129 and/or P131 in SEQ ID NO: 12, and positions P110 or H86 in SEQ ID NO: 21.

In one embodiment, the mutation is T143I in SEQ ID NO: 9. In one embodiment, the mutation is G129R in SEQ ID NO: 9. In one embodiment, the mutation is G129E in SEQ ID NO: 9. In one embodiment, the mutation is is H84STOP in SEQ ID NO: 9. In one embodiment, the mutation is R120C in SEQ ID NO: 12. In one embodiment, the mutation is G129E in SEQ ID NO: 12. In one embodiment, the mutation is P131S in SEQ ID NO: 12. In one embodiment, the mutation is P110L in SEQ ID NO: 21. In one embodiment, the mutation is H86STOP in SEQ ID NO: 21.

All possible combinations of such mutations are disclosed which include any 2, 3, 4, 5, 6, 7, 8 or 9 mutations selected from T143I and/or G129R and/or G129E and/or H84STOP in SEQ ID NO: 9 and/or R120C and/or G129E and/or P131S in SEQ ID NO: 12 and/or P110L and/or H86STOP in SEQ ID NO: 21.

In another aspect, a method is provided for shortening the period of time before flowering in a plant or plant material derived from a plant, said method comprising introducing into the genome of said plant one or more mutations that reduce the expression of at least one TFL - 1 gene, where the specified at least one gene TFL - 1 encodes TFL1 - 2S (SEQ ID NO: 7 or 8), TFL1 - 2T (SEQ ID NO: 10 or 11) and TFL1 - 4T (SEQ ID NO: 19 or 20).

Also provided is a method for identifying a plant with a reduced period of time before flowering, said method comprising screening a nucleic acid sample from a plant of interest for the presence of one or more mutations in TFL1-2S (SEQ ID NO: 7 or 8), TFL1 - 2T (SEQ ID NO: 10 or 11) and TFL1 - 4T (SEQ ID NO: 19 or 20).

Also disclosed is a plant or plant cell that is heterozygous or homozygous for one or more mutations in the gene encoding TFL1 - 2S (SEQ ID NO: 7 or 8), TFL1 - 2T (SEQ ID NO: 10 or 11) and TFL1 - 4T (SEQ ID NO: 19 or 20), where the indicated(s) mutation(s) lead to reduced gene expression or reduced function of the protein encoded by it.

In some embodiments, an advantageous mutation(s) is introduced into a plant or plant cell using a mutagenesis approach, and the introduced mutation is identified or selected using methods known to one of skill in the art, such as Southern blotting, DNA sequencing, PCR analysis or phenotypic analysis. Mutations that affect gene expression, or that disrupt the function of the encoded protein, can be determined using methods well known in the art. Mutations of the type of insertion into the exon of the gene, as a rule, lead to the formation of non-functional mutants. Mutations in conserved residues can be particularly effective in suppressing or reducing the metabolic function of the encoded protein.

Methods for producing mutant polynucleotides and polypeptides are also disclosed. Any plant of interest, including a plant cell or plant material, can be genetically modified in a variety of ways known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemical compound-induced mutagenesis, ionizing agent-induced mutagenesis. radiation mutagenesis, mutagenesis using modified bases, mutagenesis using nicked double-stranded DNA, double-strand break mutagenesis, mutagenesis using repair-disrupted host strains, mutagenesis by whole gene synthesis, DNA shuffling, and other equivalent methods.

Mutant variants of a polypeptide can be used to create mutant, non-naturally occurring or transgenic plants (e.g., mutant, non-naturally occurring, transgenic, human-made or genetically engineered) or plant cells containing one or more mutant polypeptide variants. Accordingly, mutant variants of the polypeptide retain the activity of the wild-type polypeptide. The activity of a mutant polypeptide variant may be higher, lower, or approximately the same as that of a wild-type polypeptide.

Mutations in the nucleotide sequences and polypeptides described herein may include human-made mutations or synthetic mutations, or mutations created using genetic engineering techniques. Mutations in the nucleotide sequences and polypeptides described herein may be mutations that are made or that can be made by a method that includes an in vitro or in vivo manipulation step. Mutations in the nucleotide sequences and polypeptides described herein may be mutations that are made or that can be made by a method that involves human intervention. As an example, the method may include mutagenesis using exogenously added substances, such as mutagenic, teratogenic or carcinogenic organic compounds, such as ethyl methanesulfonate (EMS), which lead to random mutations in the genetic material. As a further example, the method may include one or more genetically engineered steps, such as one or more of the genetically engineered steps described herein, or combinations thereof. As a further example, the method may include one or more plant crossing steps.

The activity of one or more Terminal Flower 1 polypeptides in a plant is reduced or suppressed according to the present invention if the conversion activity is statistically lower than the conversion activity of the same Terminal Flower 1 polypeptide(s) in a plant that has not been modified with inhibition of the conversion activity of this Terminal Flower 1 polypeptide and which was cultured and harvested using the same protocols. The activity of the Terminal Flower 1 polypeptide in a plant is considered abolished if the activity is not detectable using the analytical methods described herein. Methods for determining the activity of a Terminal Flower 1 polypeptide are described herein.

In addition to mutagenesis products, compositions that can modulate the expression or activity of one or more of the polynucleotides or polypeptides described herein include, without limitation, sequence-specific polynucleotides that can disrupt the transcription of one or more endogenous genes; sequence-specific polynucleotides that can disrupt translation of RNA transcripts (eg, double-stranded RNA, siRNA, ribozymes); sequence-specific polynucleotides that can disrupt the stability of one or more proteins; sequence-specific polynucleotides that can interfere with the enzymatic activity of one or more proteins or the binding activity of one or more proteins with respect to substrates or regulatory proteins; antibodies that show specificity for one or more proteins; small molecule compounds that can interfere with the stability of one or more proteins or the enzymatic activity of one or more proteins or the binding activity of one or more proteins; zinc finger proteins that bind one or more polynucleotides; and meganucleases that have activity on one or more polynucleotides. Gene editing technologies, genetic editing technologies and genome editing technologies are well known in the art.

One way to edit a gene involves the use of transcription activator-like effector nucleases (TALEN), which cause double-strand breaks, to which the cell can respond with the involvement of repair mechanisms. Non-homologous end joining relinks DNA on both sides of the double strand break, where there is little or no sequence overlap for complementary binding. This repair mechanism induces errors in the genome through insertion or deletion or chromosomal rearrangement. Any such errors may render the gene products encoded at that location non-functional. Another gene editing method involves the use of the bacterial CRISPR/Cas system. Bacteria and archaebacteria are characterized by chromosomal elements called regularly spaced short palindromic repeats (CRISPR) that are part of the adaptive immune system that protects against viral and plasmid DNA entry. In type II CRISPR systems, CRISPR RNA (crRNA) functions with transactivating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-strand breaks into the target DNA. Targeted cleavage with Cas9 requires crRNA and tracrRNA base pairing, as well as crRNA and target DNA base pairing. Target recognition is facilitated by the presence of a short motif, called the protospacer-adjacent motif (PAM), which corresponds to the NGG sequence. This system can be used for genome editing. Cas9 is usually programmed with a double RNA consisting of crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid "guide RNA" to target Cas9. The use of non-coding guide RNA targeting DNA for site-specific cleavage promises to be significantly simpler than existing technologies such as TALEN. The use of the CRISPR/Cas strategy with nuclease complex retargeting only requires the introduction of a new RNA sequence and there is no need to redesign the specificity of protein transcription factors.

Antisense technology is another known method that can be used to modulate the expression of a polypeptide. The polynucleotide of the gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence such that the antisense RNA strand is transcribed. Then, a plant cell is transformed with a recombinant construct and an antisense RNA strand is obtained. The polynucleotide is not necessarily the complete sequence of the gene to be repressed, but will generally be substantially complementary to at least a portion of the sense strand of the gene to be repressed.

The polynucleotide can be transcribed into a ribozyme or a catalytic RNA that affects the expression of the mRNA. Ribozymes can be engineered to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous polynucleotides may encode ribozymes designed to cleave specific mRNA transcripts, thus preventing expression of the polypeptide. Hammerhead-type ribozymes are useful for degrading specific mRNAs, although various ribozymes that cleave mRNAs at site-specific recognition sequences can be used. Hammerhead ribozymes cleave the mRNA at sites defined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target RNA must contain the nucleotide sequence 5'-UG-3'. The construction and production of hammerhead ribozymes is known in the art. Hammerhead ribozyme sequences can be inserted into stable RNA such as transfer RNA (tRNA) to increase the efficiency of in vivo cleavage.

In one embodiment, the sequence-specific polynucleotide that can disrupt the translation of the RNA transcript(s) is an interfering RNA. RNA interference or RNA silencing is an evolutionarily conserved process in which specific mRNAs can be targeted for enzymatic degradation. Double-stranded RNA (double-stranded RNA) is introduced into or produced in the cell (eg, double-stranded RNA virus or interfering RNA polynucleotides) to initiate the interfering RNA pathway. Double-stranded RNA can be converted into several 21-23 bp small interfering RNA duplexes. using RNase III, which are endonucleases specific for double-stranded RNA. Small interfering RNAs can then be recognized by RNA-induced silencing complexes that promote the unwinding of small interfering RNAs by an ATP-dependent process. The untwisted antisense strand of the small interfering RNA directs activated RNA-induced silencing complexes to a targeted mRNA containing a sequence complementary to the antisense strand of the small interfering RNA. The targeted mRNA and the antisense strand can form an A-coil, and the major groove of the A-coil can be recognized by activated RNA-induced silencing complexes. The target mRNA can be cleaved with activated RNA-induced silencing complexes at a single site defined by the binding site of the 5' end of the small interfering RNA strand. Activated RNA-induced silencing complexes can be reused to catalyze another cleavage event.

An exemplary sense RNAi target sequence for TFL1-1S/T is shown under SEQ ID NO: 22. An example antisense RNAi target sequence for TFL1-1S/T is shown under SEQ ID NO: 23. An exemplary TFL1-1S/T RNAi construct is shown under SEQ ID NO: 24.

An exemplary sense RNAi target sequence for TFL1-1S is provided under SEQ ID NO: 25. An example antisense RNAi target sequence for TFL1-1S is provided under SEQ ID NO: 26.

An exemplary sense RNAi target sequence for TFL1-1T is provided under SEQ ID NO: 27. An example antisense RNAi target sequence for TFL1-1T is provided under SEQ ID NO: 28.

An exemplary sense RNAi target sequence for TFL1-2S/T is shown under SEQ ID NO: 29. An example antisense RNAi target sequence for TFL1-2S/T is shown under SEQ ID NO: 30. An exemplary TFL1-2S/T RNAi construct is shown under SEQ ID NO: 31.

An exemplary sense RNAi target sequence for TFL1-2S is provided under SEQ ID NO: 32. An exemplary antisense RNAi target sequence for TFL1-2S is provided under SEQ ID NO: 33.

An exemplary sense RNAi target sequence for TFL1-2T is provided under SEQ ID NO: 34. An example antisense RNAi target sequence for TFL1-2T is provided under SEQ ID NO: 35.

An exemplary sense RNAi target sequence for TFL1-3T is provided under SEQ ID NO: 36. An exemplary antisense RNAi target sequence for TFL1-3T is provided under SEQ ID NO: 37. An exemplary TFL1-3T RNAi construct is provided under SEQ ID NO: 38.

An exemplary sense RNAi target sequence for TFL1-4T is provided under SEQ ID NO: 39. An exemplary antisense RNAi target sequence for TFL1-4T is provided under SEQ ID NO: 40. An exemplary TFL1-4T RNAi construct is provided under SEQ ID NO: 41.

Sequences of about 21 to 23 nucleotides in length are disclosed for any of SEQ ID NOs: 22, 23, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 39, 40, or 41.

Methods for gene silencing using such coding sequences and ways of using them are also discussed.

Interfering RNA expression vectors may contain interfering RNA constructs encoding interfering RNA polynucleotides that exhibit activity against RNA interference by reducing the expression level of mRNA, pre-mRNA, or related RNA variants. Expression vectors may contain an upstream promoter operably linked to an interfering RNA construct, as further described herein. Interfering RNA expression vectors may contain a suitable minimal core promoter, an interfering RNA construct of interest, an upstream (5') regulatory region, a downstream (3') regulatory region, including transcription termination and polyadenylation signals, and others. sequences known to those skilled in the art, such as various selectable markers.

Examples of interfering RNA constructs are provided under SEQ ID NOs: 24, 31, 38 and 41.

In one embodiment, the expression vector contains a promoter, such as a strong constitutive MMV (Mirabilis Mosaic Virus) promoter located upstream and operably linked to an interfering RNA construct, and a downstream (3') regulatory region, such as a 3'-terminator. the nos sequence of the Agrobacterium tumefaciens nopaline synthase gene.

Polynucleotides can be obtained in a variety of forms, including double-stranded structures (i.e., a double-stranded RNA molecule containing an antisense strand and a complementary sense strand), double-stranded hairpin structures, or single-stranded structures (i.e., an ssRNA molecule containing only an antisense strand) . The structures may contain a double helix, asymmetric double helix, hairpin, or asymmetric hairpin secondary structure having a self-complementary sense and antisense strands. Double-stranded interfering RNA can be enzymatically converted into double-stranded small interfering RNAs. One strand of the small interfering RNA double helix can be annealed with a complementary sequence in the target mRNA and related RNA variants. SIRNA/mRNA double helixes are recognized by RNA-induced silencing complexes, which can cleave RNA at multiple sites in a sequence-dependent manner, resulting in the destruction of the target mRNA and related RNA variants.

Double-stranded RNA molecules may include small interfering RNA molecules assembled from a single oligonucleotide in a stem-loop structure, where the self-complementary sense and antisense regions of the small interfering RNA molecule are connected by polynucleotide-based or non-polynucleotide-based linker(s), as well as a circular a single-stranded RNA with two or more loop structures and a stem containing self-complementary sense and antisense strands, where the circular RNA can be processed either in vivo or in vitro to form an active small interfering RNA molecule capable of mediating RNA interferences.

Double-stranded RNA may contain at least two sequences that are at least partially complementary to each other. The sense strand may contain the first sequence, and the antisense strand may contain the second sequence. At least one of the sequences may contain at least 10 contiguous nucleotides from the TFL1 RNA. At least one of the sequences may contain approximately 21-23 contiguous nucleotides from the TFL1 RNA.

In one embodiment, the double-stranded RNA has a first sequence containing at least about 10 contiguous nucleotides from TFL1 , respectively, about 21-23 contiguous nucleotides from TFL-1. The double-stranded RNA may have a second sequence. The double-stranded RNA may have a third sequence containing a sequence inversely complementary to the first sequence in the same orientation as the first sequence. The second sequence may be located between the first sequence and the third sequence. The second sequence may be operably linked to the first sequence and the third sequence.

The first sequence may be selected from the group consisting of SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20 , and/or where the third sequence is a sequence reversely complementary to the sequence corresponding to SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:19, or SEQ ID NO:20.

The first sequence may contain or consist of SEQ ID NO: 22, and the third sequence may contain or consist of SEQ ID NO: 23; or the first sequence may contain or consist of SEQ ID NO: 25, and the third sequence may contain or consist of SEQ ID NO: 26; or the first sequence may contain or consist of SEQ ID NO: 27, and the third sequence may contain or consist of SEQ ID NO: 28; or the first sequence may contain or consist of SEQ ID NO: 29, and the third sequence may contain or consist of SEQ ID NO: 30; or the first sequence may contain or consist of SEQ ID NO: 32, and the third sequence may contain or consist of SEQ ID NO: 33; or the first sequence may contain or consist of SEQ ID NO: 34, and the third sequence may contain or consist of SEQ ID NO: 35; or the first sequence may contain or consist of SEQ ID NO: 36, and the third sequence may contain or consist of SEQ ID NO: 37; or the first sequence may contain or consist of SEQ ID NO: 39 and the third sequence may contain or consist of SEQ ID NO: 40.

The double-stranded RNA may contain or consist of a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 31, SEQ ID NO: 38, and SEQ ID NO: 41.

The use of small RNA molecules forming hairpins is also contemplated. They contain a specific antisense sequence in addition to a reverse complementary (sense) sequence, usually separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complementary strand so that they can be annealed to form a double-stranded RNA molecule (optionally with additional processing steps that may result in the addition or deletion of one, two, three or more nucleotides 3 '-end or 5'-end of one or both strands). The spacer may be of sufficient length to allow complementary binding of the antisense and sense sequences and formation of a double strand structure (or stem) prior to cleavage of the spacer (and optionally subsequent processing steps that may result in the addition or deletion of one, two, three, four or more nucleotides from the 3' end or from the 5' end of one or both strands). A spacer sequence is generally an unrelated nucleotide sequence that is located between two complementary portions of a nucleotide sequence that, when complementary bound into a double-stranded polynucleotide, contains a small hairpin RNA. The spacer sequence typically contains from about 3 to about 100 nucleotides.

Any RNA polynucleotide of interest can be prepared by selecting an appropriate sequence composition, loop size and stem length to produce a double helix hairpin. An exemplary DNA sequence of antisense and sense RNAi target sequences and constructs is shown under SEQ ID Nos: 22-41. A suitable range for constructing the stem length of a hairpin double helix includes a stem length of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, e.g., about 14-30 nucleotides, about 30-50 nucleotides, approximately 50-100 nucleotides, approximately 100-150 nucleotides, approximately 150-200 nucleotides, approximately 200-300 nucleotides, approximately 300-400 nucleotides, approximately 400-500 nucleotides, approximately 500-600 nucleotides, and approximately 600- 700 nucleotides. A suitable range for constructing the loop length of the hairpin double helix includes a loop length of about 4-25 nucleotides, about 25-50 nucleotides, or longer if the stem length of the hairpin double helix is significant. In certain embodiments, the double-stranded RNA or ssRNA molecule is about 15 to about 40 nucleotides in length. In another embodiment, the small interfering RNA molecule is a double-stranded RNA or ssRNA molecule about 15 to about 35 nucleotides in length. In another embodiment, the small interfering RNA molecule is a double-stranded RNA or ssRNA molecule from about 17 to about 30 nucleotides in length. In another embodiment, the small interfering RNA molecule is a double-stranded RNA or ssRNA molecule from about 19 to about 25 nucleotides in length. In another embodiment, the small interfering RNA molecule is a double-stranded RNA or ssRNA molecule about 21 to about 23 nucleotides in length. In certain embodiments, hairpin structures with double helix regions longer than 21 nucleotides can facilitate efficient small interfering RNA driven silencing regardless of loop sequence and length. Illustrative sequences used for RNA interference are set forth under SEQ ID NOs: 22-33.

The target mRNA sequence is typically about 14 to about 50 nucleotides in length. The target mRNA can thus be screened for regions of about 14 to about 50 nucleotides in length that preferably meet one or more of the following criteria for the target sequence: an A+T/G+C ratio of about 2:1 to about 1:2; an AA dinucleotide or a CA dinucleotide at the 5' end of the target sequence; a sequence of at least 10 contiguous nucleotides unique to the target mRNA (i.e., the sequence is not present in other mRNA sequences from the same plant); and no "bands" of more than three contiguous guanine (G) nucleotides or more than three contiguous cytosine (C) nucleotides. These criteria can be evaluated using various techniques known in the art, for example, computer programs such as BLAST can be used to search public databases to determine if a selected target sequence is unique to the target mRNA. Alternatively, one can select a target sequence (and design a small interfering RNA sequence) using commercially available computer software (eg, OligoEngine, Target Finder and Design Tool for small interfering RNAs, which are commercially available).

In one embodiment, target mRNA sequences are selected that are from about 14 to about 30 nucleotides in length and meet one or more of the criteria above. In another embodiment, target sequences are selected that are from about 16 to about 30 nucleotides in length and meet one or more of the criteria above. In a further embodiment, target sequences are selected that are from about 19 to about 30 nucleotides in length and meet one or more of the criteria above. In another embodiment, target sequences are selected that are from about 19 to about 25 nucleotides in length and meet one or more of the criteria above.

In an exemplary embodiment, small interfering RNA molecules contain a specific antisense sequence that is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides from any of the polynucleotide sequences described herein.

The specific antisense sequence contained in a small interfering RNA molecule may be identical or substantially identical to a sequence complementary to the target sequence. In one embodiment, the specific antisense sequence contained in the small interfering RNA molecule is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence complementary to the sequence of the target mRNA. Methods for determining sequence identity are known in the art and can be determined, for example, using the BLASTN program of the University of Wisconsin Computer Group (GCG) software, or provided on the NCBI website.

The specific antisense sequence of small interfering RNA molecules may exhibit variability in terms of differences (eg, nucleotide substitution, including transition or transversion) at one, two, three, four or more nucleotides of the target mRNA sequence. If such nucleotide substitutions are in the antisense strand of the double-stranded RNA molecule, then the complementary nucleotide in the sense strand with which the substituted nucleotide would normally form hydrogen bonds upon base pairing may or may not be suitably substituted. Double-stranded RNA molecules in which one or more nucleotide substitutions occur in the sense sequence but not in the antisense strand are also contemplated. If the antisense sequence of a small interfering RNA molecule contains one or more mismatches between the small interfering RNA nucleotide sequence and the target nucleotide sequence, as described above, these mismatches can be detected at the 3' end, 5' end, or in the central part of the antisense sequence.

In another embodiment, the small interfering RNA molecules contain a specific antisense sequence that is capable of selectively hybridizing under stringent conditions to a portion of a naturally occurring target gene or target mRNA. As is known to those skilled in the art, variations in the stringency of the hybridization conditions can be achieved by varying the time, temperature, or concentration of the solutions used for the hybridization and washing steps. Suitable conditions may also depend in part on the specific nucleotide sequences used, for example, the sequence of the target mRNA or gene.

One method for inducing double-stranded RNA silencing in plants is transformation with a gene construct producing hairpin RNA (see Smith et al . (2000) Nature , 407, 319-320). Such constructs contain inverted portions of the target gene sequence separated by an appropriate spacer. Insertion of a functional plant intron region as a spacer fragment further enhances the efficiency of gene silencing induction due to the production of hairpin RNA spliced to the intron (Wesley et al . (2001) Plant J. , 27, 581-590). Accordingly, the length of the stem is from about 50 nucleotides to about 1 thousand nucleotides in length. Methods for producing hairpin RNA spliced to an intron are well described in the art (see, for example, Bioscience, Biotechnology, and Biochemistry (2008) 72, 2, 615-617).

Interfering RNA molecules having a double-stranded or double-stranded structure, such as double-stranded RNA or small hairpin RNA, may have blunt ends, or may have sticky 3' or 5' ends. As used herein, the term "sticky end" refers to an unpaired nucleotide or nucleotides that protrude from the double strand if the 3' end of one strand of RNA extends beyond the 5' end of the other strand (3' sticky end), or vice versa ( sticky 5'-end). Sticky-end nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. In one embodiment, at least one strand of the interfering RNA molecule has a sticky 3' end from about 1 to about 6 nucleotides in length. In other embodiments, the 3' sticky end is about 1 to about 5 nucleotides, about 1 to about 3 nucleotides, and about 2 to about 4 nucleotides in length.

If the interfering RNA molecule contains a sticky 3'-end at one end of the molecule, the other end may be blunt-ended, or also have a sticky end (5' or 3'). If an interfering RNA molecule contains a sticky end at both ends of the molecule, the length of the sticky ends may be the same or different. In one embodiment, the interfering RNA molecule contains sticky 3' ends from about 1 to about 3 nucleotides in length at both ends of the molecule. In a further embodiment, the interfering RNA molecule is a double-stranded RNA having a sticky 3' end of 2 nucleotides at both ends of the molecule. In yet another embodiment, the sticky end nucleotides of the interfering RNA are TT dinucleotides or UU dinucleotides.

When determining the percent identity of an interfering RNA molecule containing one or more sticky ends with the target mRNA sequence, the sticky end(s) may or may not be taken into account. For example, nucleotides from the sticky 3' end and up to 2 nucleotides from the 5' or 3' end of the double strand can be modified without significant loss of activity of the small interfering RNA molecule.

Interfering RNA molecules may contain one or more 5' or 3' cap structures. The interfering RNA molecule may contain a cap at the 3' end of the sense strand, the antisense strand, or both the sense and antisense strands; or at the 5' end of the sense strand, the antisense strand, or both the sense and antisense strands of the interfering RNA molecule. Alternatively, the interfering RNA molecule may contain a cap at both the 3' end and the 5' end of the interfering RNA molecule. The term "cap structure" refers to a chemical modification included at either end of an oligonucleotide that protects the molecule from degradation by endonucleases and may also facilitate delivery or localization within a cell.

Another modification applied to the interfering RNA molecules is the chemical coupling to the interfering RNA molecule of one or more fragments or conjugates that increase the activity, cellular distribution, cellular uptake, bioavailability, or stability of the interfering RNA molecule. Polynucleotides can be synthesized or modified using methods conventional in the art. Chemical modifications may include, but are not limited to, 2' modifications, introduction of non-natural bases, covalent attachment of a ligand, and replacement of phosphate bonds with thiophosphate bonds. In this embodiment, the integrity of the double helix structure is enhanced by at least one and typically two chemical bonds. Chemical bonding can be achieved using any of a variety of well known techniques, for example, by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; through metal ion coordination or through the use of purine analogues.

The nucleotides of one or both of the two single strands can be modified to modulate the activation of cellular enzymes such as, for example, but not limited to, certain nucleases. Techniques for reducing or inhibiting cellular enzyme activation are known in the art and include, without limitation, 2'-amino modifications, 2'-fluoro modifications, 2'-alkyl modifications, uncharged backbone modifications, morpholine modifications, 2'-O-methyl modifications, and phosphoramidate. Thus, at least one 2'-hydroxyl group of the double-stranded RNA nucleotides is replaced by a chemical group. Also, at least one nucleotide can be modified to form a closed nucleotide. Such a closed nucleotide contains a methylene or ethylene bridge that connects the 2'-oxygen of the ribose to the 4'-carbon of the ribose. The introduction of a closed nucleotide into the oligonucleotide improves the affinity for complementary sequences and increases the melting point by several degrees.

It is possible to conjugate ligands to an interfering RNA molecule, for example, to increase cellular absorption towards it. In certain embodiments, the hydrophobic ligand is conjugated to a molecule to facilitate direct penetration across the cell membrane. These approaches are used to facilitate the penetration of antisense oligonucleotides into the cell. In certain instances, conjugation of the cationic ligand to oligonucleotides often results in improved nuclease resistance. Representative examples of cationic ligands include propylammonium and dimethylpropylammonium. Antisense oligonucleotides can retain their high binding affinity for mRNA when the cationic ligand is distributed throughout the oligonucleotide.

The molecules and polynucleotides described herein can be prepared using well known solid phase synthesis techniques. Any other means for such synthesis known in the art may be used additionally or alternatively.

Various embodiments are directed to expression vectors containing one or more polynucleotides or one or more interfering RNA constructs described herein. Illustrative designs are shown in Figure 21.

Various embodiments are directed to expression vectors containing one or more polynucleotides or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides described herein that are capable of self-annealing to form a hairpin structure, wherein the construct contains ( a) one or more of the polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence containing a sequence inversely complementary to the first sequence located in the same orientation as the first sequence, where the second sequence is located between the first sequence and the third sequence, and the third sequence is operably linked to the first sequence and the third sequence.

The disclosed sequences can be used to construct various polynucleotides that do not form hairpin structures. For example, a double-stranded RNA can be formed by (1) transcribing a first strand of DNA by operably linking to a first promoter and (2) transcribing a sequence reversely complementary to the sequence of the first strand DNA fragment by operably linking to a second promoter. Each strand of a polynucleotide can be transcribed from a single expression vector, or from different expression vectors. An RNA double helix having RNA interference activity can be enzymatically converted into small interfering RNAs to modulate RNA levels.

Thus, various embodiments are directed to expression vectors containing one or more of the interfering RNA polynucleotides or constructs described herein, encoding interfering RNA polynucleotides capable of self-annealing, wherein the construct comprises (a) one or more of the polynucleotides described herein. document; and (b) a second sequence containing a complementary (eg, reverse complementary) sequence of the first sequence in the same orientation as the first sequence.

Various compositions and methods are provided for modulating endogenous expression levels of one or more of the polypeptides described herein (or any combination thereof described herein) by promoting co-suppression of gene expression. The phenomenon of co-suppression occurs as a result of the introduction of multiple copies of the transgene into the cell of the host plant. Integration of multiple copies of a transgene can lead to modulation of the expression of the transgene and the targeted endogenous gene. The degree of co-suppression depends on the degree of sequence identity between the transgene and the targeted endogenous gene. Silencing of both the endogenous gene and the transgene can occur due to extensive methylation of repressed loci (ie, the endogenous promoter and the endogenous gene of interest), which can disrupt transcription. Alternatively, in some cases co-suppression of the endogenous gene and the transgene may occur through post-transcriptional gene silencing, in which transcripts can be generated, but increased levels of disruption prevent transcript accumulation. The mechanism of co-suppression through post-transcriptional gene silencing is thought to be similar to RNA interference in that RNA appears to be important as an initiator and target in these processes, and may be mediated at least in part by the same molecular mechanism. possibly through RNA-directed mRNA degradation.

Cosuppression of nucleic acids can be achieved by introducing multiple copies of the nucleic acid or fragments thereof as transgenes into the genome of the plant of interest. The host plant can be transformed with an expression vector containing a promoter operably linked to such nucleic acid or fragments thereof. Various embodiments are directed to expression vectors for promoting co-suppression of endogenous genes containing a promoter operably linked to a polynucleotide.

Various embodiments are directed to methods for modulating the expression level of one or more of the polynucleotides described herein (or any combination thereof, as described herein) by integrating multiple copies of the polynucleotide(s) into the plant genome, comprising transforming a host plant cell using an expression vector that contains a promoter operably linked to the polynucleotide.

Various compositions and methods are provided for modulating the expression level of an endogenous gene by modulating mRNA translation. The host plant cell can be transformed with an expression vector containing a promoter operably linked to a polynucleotide located in an antisense orientation with respect to the promoter to allow the expression of RNA polynucleotides characterized by a sequence complementary to the mRNA portion.

Various expression vectors for modulating mRNA translation may contain a promoter operably linked to the polynucleotide in an antisense orientation with respect to the promoter. The length of antisense RNA polynucleotides can vary and range from about 15-20 nucleotides, about 20-30 nucleotides, about 30-50 nucleotides, about 50-75 nucleotides, about 75-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides and approximately 200-300 nucleotides.

Also, genes can be targeted for inactivation by introducing transposons (eg, IS elements) into the genomes of plants of interest. These transposable genetic elements can be introduced by sexual cross-pollination and insertion mutants can be screened for loss of protein activity. The disrupted gene of the parent plant can be introduced into other plants by crossing the parent plant with a plant that has not undergone transposon-induced mutagenesis, such as by sexual cross-pollination. Any standard selection techniques known to those skilled in the art may be used. In one embodiment, one or more genes can be inactivated by insertion of one or more transposons. Mutations can lead to homozygous destruction of one or more genes, to heterozygous destruction of one or more genes, or to a combination of homozygous and heterozygous destruction if more than one gene is destroyed. Suitable transposable elements include retrotransposons, retroposons, and SINE-like elements. Such methods are known to those skilled in the art.

Alternatively, genes can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either independently (viroid RNA) or with the help of a helper virus (satellite RNA). Examples of suitable RNAs include avocado sunspot viroid-derived and satellite RNAs derived from tobacco ring spot virus, alfalfa temporary banding virus, tobacco velvet spot virus, Solanum nodiflorum spot virus, and subterranean clover spot virus. Various target RNA-specific ribozymes are known to those skilled in the art.

As discussed herein, the expression of one or more polypeptides can be modulated in non-transgenic ways, such as by creating one or more mutations in one or more genes, as discussed herein. Methods by which a random mutation is introduced into a gene sequence may include chemical mutagenesis, EMS mutagenesis, and radiation mutagenesis. Methods by which one or more targeted mutations are introduced into a cell include, but are not limited to, genome editing techniques such as zinc finger nuclease mediated mutagenesis, TILLING, homologous recombination, oligonucleotide-directed mutagenesis and mutagenesis mediated by meganuclease. In one embodiment, TILLING is used. It is a technology that can be used to create and/or identify polynucleotides encoding polypeptides with modified expression and/or activity. TILLING also allows selection of plants carrying such mutations. TILLING combines high density mutagenesis with high throughput screening methods. Methods for performing TILLING are well known in the art (see McCallum et al ., (2000) Nat Biotechnol 18: 455-457 and Stemple (2004) Nat Rev Genet 5(2): 145-50).

Some non-limiting examples of mutations are deletions, insertions, and missense mutations of at least one nucleotide, single nucleotide polymorphisms, and a simple repeat sequence. Once a mutation has been introduced, screening can be performed to identify mutations that create early stop codons or otherwise non-functional genes. Once a mutation has been introduced, screening can be performed to identify mutations that create functional genes capable of being expressed at elevated levels. Mutant screening can be done by sequencing, or using one or more probes or primers specific for a given gene or protein. It is also possible to design specific mutations in polynucleotides that result in modulation of gene expression, modulation of mRNA stability, or modulation of protein stability. Such plants are referred to herein as "non-natural" or "mutant" plants. Typically, mutant or non-naturally occurring plants will contain at least a portion of the foreign or synthetic or human-created nucleic acid (eg, DNA or RNA) that is not present in the plant prior to being manipulated. The foreign nucleic acid may be one nucleotide, two or more nucleotides, two or more contiguous nucleotides, or two or more non-contiguous nucleotides such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600 , 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.

Mutant or non-naturally occurring plants or plant cells may have any combination of one or more mutations in one or more genes that results in modulation of protein levels. For example, mutant or non-naturally occurring plants or plant cells may have one mutation in one gene; multiple mutations in the same gene; one mutation in two or more, or three or more, or four or more genes; or multiple mutations in two or more, or three or more, or four or more genes. Examples of such mutations are described in this document. As a further example, mutant or non-naturally occurring plants or plant cells may have one or more mutations in a particular portion of the gene(s), for example, in the region of a gene that encodes the active site of a protein or portion thereof. As a further example, mutated or non-naturally occurring plants or plant cells may have one or more mutations in a region outside of one or more genes, such as upstream or downstream of the gene it regulates, provided that they modulate the activity or expression of the gene(s). Upstream elements may include promoters, enhancers, or transcription factors. Some elements, such as enhancers, can be located upstream or downstream of the gene they regulate. The element(s) are not necessarily located near the gene it(s) regulates, as some elements have been found to be several thousand base pairs upstream or downstream. chains relative to the gene they regulate. Mutant or non-naturally occurring plants or plant cells may have one or more mutations located in the first 100 nucleotides of the gene(s), in the first 200 nucleotides of the gene(s), in the first 300 nucleotides of the gene(s), in the first 400 nucleotides of the gene(s), in the first 500 nucleotides of the gene(s), in the first 600 nucleotides of the gene(s), in the first 700 nucleotides of the gene(s), in the first 800 nucleotides of the gene(s), in the first 900 nucleotides of the gene(s), in the first 1000 nucleotides of the gene(s), in the first 1100 nucleotides of the gene(s), in the first 1200 nucleotides of the gene(s), in the first 1300 nucleotides of the gene(s), in the first 1400 nucleotides of the gene(s) or in the first 1500 nucleotides of the gene(s). Mutant or non-naturally occurring plants or plant cells may have one or more mutations located in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of the gene(s) or combinations thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (eg, mutant, non-naturally occurring or transgenic plants or plant cells, and the like, as described herein) containing variants of the mutant polypeptide.

In one embodiment, plant seeds are subjected to mutagenesis and then grown into first generation mutant plants. The first generation plants are then allowed to self-pollinate, and the seeds from the first generation plants are grown into second generation plants, which are then screened for mutations at their loci. Although mutated plant material can be screened for mutations, the advantage of screening second generation plants is that all somatic mutations correspond to mutations in the germline. One of skill in the art will appreciate that various plant material, including, without limitation, seeds, pollen, plant tissues, or plant cells, can be mutated to create mutant plants. However, the type of plant material subjected to mutagenesis may be of importance when the plant nucleic acid is screened for mutations. For example, if pollen is subjected to mutagenesis prior to pollination of a non-mutagenetic plant, first generation plants are grown from seeds resulting from such pollination. Every cell of the first generation plants will contain the mutations created in the pollen; thus, these first generation plants can then be twitched to be screened for mutations instead of waiting for the second generation.

Mutagens that produce primarily point mutations and short deletions, insertions, transversions and/or transitions, including chemical mutagens and irradiation, can be used to create mutations. Mutagens include, without limitation, ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl- N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro -9[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

Also contemplated are spontaneous mutations at the locus, which may not be directly caused by the mutagen, provided that they result in the desired phenotype. Suitable mutagenic agents may also include, for example, ionizing radiation such as x-rays, gamma rays, fast neutron radiation and ultraviolet radiation. Any method for obtaining plant nucleic acid known to those skilled in the art can be used to obtain plant nucleic acid for mutation screening.

The derived nucleic acid from individual plants, plant cells, or plant material can optionally be pooled in order to facilitate screening for mutations in a plant population derived from mutated plant tissues, cells, or material. One or more successive generations of plants, plant cells, or plant material may be screened. The size of the optionally pooled group depends on the sensitivity of the screening method used.

Once the nucleic acid samples have optionally been pooled, they can be subjected to polynucleotide-specific amplification techniques such as polymerase chain reaction. Any one or more primers or probes specific for a gene or sequences immediately adjacent to a gene can be used to amplify sequences within an optionally pooled nucleic acid sample. Accordingly, one or more primers or probes are designed to amplify regions of the locus where beneficial mutations are most likely to occur. Most preferably, the primer is designed to detect mutations in regions of the polynucleotide. Additionally, it would be preferred for primer(s) and probe(s) to avoid known polymorphic sites to facilitate screening for point mutations. To facilitate identification of amplification products, one or more primers or probes can be labeled using any conventional labeling method. Primer(s) or probe(s) can be designed based on the sequences described herein using methods that are well known in the art.

To facilitate identification of amplification products, the primer(s) or probe(s) may be labeled using any conventional labeling method. They can be constructed based on the sequences described herein using methods that are well known in the art. Polymorphisms can be identified by means known in the art and some of those described in the literature.

In a further aspect, a method for producing a mutant plant is provided. The method includes obtaining at least one plant cell containing a gene encoding a functional polynucleotide described in this document (or any combination thereof, described in this document). This at least one plant cell is then treated under conditions effective to modulate the activity of the polynucleotide(s) described herein. At least one mutant plant cell is then propagated into a mutant plant, wherein the mutant plant has a modulated level of the described polypeptide(s) (or any combination thereof described herein) compared to a control plant. In one embodiment of this method for producing a mutant plant, the step of treating includes exposing at least one cell to a chemical mutagenic agent as described above and under conditions effective to produce at least one mutant plant cell. In another embodiment of this method, the treatment step comprises exposing at least one cell to a source of ionizing radiation under conditions effective to produce at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype is modified from a control plant, respectively, by methods other than genetic engineering and genetic modification.

In certain embodiments, the mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that occur naturally in another plant, plant cell, or plant material and provide the desired trait. The mutation can be introduced (eg, by introgression) into another plant, plant cell, or plant material (eg, a plant, plant cell, or plant material with a different genetic background than the plant from which the mutation is derived) to create a mutation that is not occurs naturally in a given plant and to provide them with a given trait. Thus, by way of example, a mutation that occurs naturally in a first plant can be introduced into a second plant, such as a second plant with a different genetic background from that of the first plant. One skilled in the art can thus search for and identify a plant that naturally carries in its genome one or more mutant alleles of the genes described herein that provide the desired trait. In certain embodiments, one or more mutations in one allele is sufficient to shorten the period of time before flowering occurs. The mutant allele(s) that occur(s) in nature can be transferred to a second plant in a variety of ways, including selection, backcrossing, and introgression to produce lines, varieties, or hybrids that have one or more mutations in the genes described in this document. Plants showing the desired trait can be selected from a pool of mutant plants. Accordingly, selection is made using the nucleotide sequence data described herein. Therefore, it is possible to screen for a genetic trait as compared to a control. Such a screening approach may include the use of conventional nucleic acid amplification and/or hybridization techniques, as discussed herein. Thus, a further aspect relates to a method for identifying a mutant plant with a reduced time to flowering compared to a control plant, comprising (a) obtaining a sample from the plant to be screened; (b) determining whether said sample contains one or more mutations in one or more of the polynucleotides described herein; and (c) determining the rate of onset of flowering of said plant.

In another aspect, a method is provided for producing a mutant plant that has a reduced time to flowering compared to a control plant, comprising the steps of (a) obtaining a sample from a first plant; (b) determining whether said sample contains one or more mutations in one or more of the polynucleotides described herein, resulting in a shortened period of time before flowering occurs; and (c) transferring one or more mutations to a second plant. The mutation(s) can be transferred to a second plant using various methods known in the art, such as genetic engineering, gene manipulation, introgression, plant breeding, backcrossing, and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant.

In another aspect, a method is provided for producing a mutant plant that has a reduced time to flowering compared to a control plant, comprising the steps of (a) obtaining a sample from a first plant; (b) determining whether said sample contains one or more mutations in one or more of the polynucleotides described herein, resulting in a shortened period of time before flowering occurs; and (c) introgression of one or more mutations from the first plant into the second plant. In one embodiment, the introgression step involves plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar.

An additional aspect relates to a mutant plant (including a mutant plant of a cultivar or elite cultivar) obtained or obtainable using the methods described herein. In certain embodiments, a "mutant plant" may contain one or more mutations located only in a particular region of the plant, such as in the sequence of one or more of the polynucleotides described herein. In this embodiment, the rest of the genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to mutagenesis.

In certain embodiments, mutant plants may have one or more mutations located in more than one region of the plant, such as in the sequence of one or more of the polynucleotides described herein and in one or more additional regions of the genome. In this embodiment, the rest of the genomic sequence of the mutant plant will not be the same or substantially the same as the plant prior to mutagenesis. In certain embodiments, mutant plants may lack one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of the polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of the polynucleotide(s) described herein; or may not have one or more mutations in the promoter of the polynucleotide(s) described(s) herein; or may not have one or more mutations in the 3'-untranslated region of the polynucleotide(s) described(s) herein; or may not have one or more mutations in the 5'-untranslated region of the polynucleotide(s) described(s) herein; or may not have one or more mutations in the coding region of the polynucleotide(s) described(s) herein; or may not have one or more mutations in the non-coding region of the polynucleotide(s) described(s) herein; or any combination of two or more, three or more, four or more, five or more, or six or more of their parts.

In a further aspect, a method is provided for identifying a plant, plant cell, or plant material containing a mutation in a gene encoding a polynucleotide described herein, comprising (a) performing mutagenesis of the plant, plant cell, or plant material; (b) obtaining a nucleic acid sample from said plant, plant cell or plant material or their descendants; and (c) determining the nucleic acid sequence of a gene encoding a polynucleotide described herein, or variants or fragments thereof, where a difference in said sequence is indicative of one or more mutations therein.

Zinc finger proteins can also be used to modulate the expression or activity of one or more of the polynucleotides described herein. In various embodiments, a genomic DNA sequence containing part or all of the coding sequence of a polynucleotide is modified by zinc finger nuclease-mediated mutagenesis. In the genomic DNA sequence, a unique binding site for the zinc finger protein is searched for. Alternatively, two unique zinc finger binding sites are searched in the genomic DNA sequence, with both sites on opposite strands and close to each other, e.g., 1, 2, 3, 4, 5, 6 apart. or more base pairs apart. Accordingly, zinc finger proteins are provided that bind to polynucleotides.

The zinc finger protein can be engineered to recognize a selected target site in a gene. The zinc finger protein may contain any combination of motifs derived from natural zinc finger DNA binding domains and non-natural zinc finger DNA binding domains obtained by truncation or extension or a site-directed mutagenesis method in combination with a selection method. such as, but not limited to, selection by phage display, selection by a bacterial dihybrid system, or selection by a bacterial single hybrid system. The term "non-natural zinc finger DNA binding domain" refers to a zinc finger DNA binding domain that binds a sequence of three base pairs in a target nucleic acid and that does not occur in the cell or organism containing the nucleic acid to be modified. Methods for constructing a zinc finger protein that binds specific nucleotide sequences that are unique to a target gene are known in the art.

A zinc finger motif nuclease can be constructed by fusing a first polynucleotide encoding a zinc finger protein that binds to the polynucleotide and a second polynucleotide encoding a non-specific endonuclease such as, but not limited to, type IIS endonuclease. The fusion protein between the zinc finger protein and the nuclease may comprise a spacer consisting of two base pairs, or alternatively a spacer which may consist of three, four, five, six, seven or more base pairs. In various embodiments, a zinc finger nuclease introduces a double-strand break in a regulatory region, a coding region, or a non-coding region of a polynucleotide's genomic DNA sequence and results in a decrease in the expression level of the polynucleotide or a decrease in the activity of the protein encoded by it. Cleavage with zinc finger nucleases often results in the deletion of DNA at the cleavage site, followed by DNA repair by joining the non-homologous ends.

In other embodiments, the implementation of the protein "zinc finger" can be selected for binding to the regulatory sequence of the polynucleotide. More specifically, the regulatory sequence may comprise a transcription start site, a start codon, an exon region, an exon-intron interface, a terminator, or a stop codon. Accordingly, the present invention provides a mutant, non-naturally occurring, or transgenic plant or plant cell obtained by zinc finger nuclease-mediated mutagenesis near or within one or more of the polynucleotides described herein, and methods for producing such plant or plant cell by zinc finger nuclease-mediated mutagenesis. Methods for delivering the zinc finger protein and the zinc finger motif nuclease into the plant are similar to those described below for delivery of the meganuclease.

In another aspect, methods are described for producing mutant, non-naturally occurring or transgenic or otherwise genetically modified plants using meganucleases such as I-CreI. Naturally occurring meganucleases, as well as recombinant meganucleases, can be used to specifically cause a double-strand break at one site, or at a relatively small number of sites, in plant genomic DNA to degrade one or more of the polynucleotides described herein. The meganuclease may be an engineered meganuclease with altered DNA recognition properties. Meganuclease proteins can be delivered to plant cells by a number of different mechanisms known in the art.

The present invention also encompasses the use of meganucleases to inactivate the polynucleotide(s) described herein (or any combination thereof described herein) in a plant cell or in a plant. In particular, the present invention provides a method for inactivating a polynucleotide in a plant using a meganuclease, comprising (a) obtaining a plant cell containing a polynucleotide described herein; (b) introducing a meganuclease or a meganuclease-encoding construct into said plant cell; and (c) allowing the meganuclease to substantially inactivate the polynucleotide(s)

Meganucleases can be used to cleave meganuclease recognition sites in the coding regions of a polynucleotide. Such cleavage often results in deletion of the DNA at the meganuclease recognition site, followed by repair of the mutagenic DNA by joining the non-homologous ends. Such mutations in the coding sequence of a gene are usually sufficient to inactivate the gene. This method for modifying a plant cell comprises, firstly, delivering a meganuclease expression cassette into the plant cell by a suitable transformation method. To achieve maximum efficiency, it is required to link the meganuclease expression cassette to a selectable marker and to select successfully transformed cells in the presence of a selective agent. This approach will result in the integration of the meganuclease expression cassette into the genome, which, however, may not be desirable if the plant is likely to require regulatory approval. In such cases, the meganuclease expression cassette (and associated selectable marker gene) can be isolated from subsequent generations of the plant using conventional breeding techniques. Alternatively, plant cells can be initially transformed with a meganuclease expression cassette without a selectable marker and can be grown on media without a selective agent. Under such conditions, a portion of the treated cells will acquire a meganuclease expression cassette and express the engineered meganuclease transiently without integration of the meganuclease expression cassette into the genome. Since this does not provide transformation efficiency, this last transformation procedure requires the screening of more treated cells to obtain the desired genome modification. The approach described above can also be applied to modify a plant cell using a zinc finger protein or a zinc finger motif nuclease.

Following delivery of the meganuclease expression cassette, plant cells are grown initially under conditions that are typical of the particular transformation procedure used. This may mean that the transformed cells grow on media at temperatures below 26°C, often in the dark. Such standard conditions can be used for a period of time, preferably 1-4 days, to ensure the recovery of the plant cell after the transformation process. At any time after this initial recovery period, the growth temperature may be increased to stimulate the activity of the engineered meganuclease to cleave and mutate the meganuclease recognition site.

Certain applications may require precise removal of a polynucleotide from the plant genome. Such applications are possible using a pair of engineered meganucleases, each of which cleaves a meganuclease recognition site on either side of the intended deletion. It is also possible to use TAL effector nucleases (TALEN), which are able to recognize and bind to the gene and introduce a double-strand break into the genome. Thus, in another aspect, methods are provided for producing mutant, non-naturally occurring or transgenic or otherwise genetically modified plants described herein using TAL effector nucleases.

Plants suitable for use in the present invention include, without limitation, monocot and dicot plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vit is and Zea.

Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (bearded vulture), Pennisetum purpureum (elephantgrass), Phalaris arundinacea (reed grass), Cynodon dactylon (pig finger), Festuca arundinacea (cane fescue), Spartina pectinata (comb spartina), Medicago sativa (alfalfa), Arundo donax (cane arundo), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticale), bamboo, Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor bean), Elaeis guineensis (oil palm), Linum usitatissimum (flax), Brassica juncea, Beta vulgaris (sugar beet), Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musyclise alca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, Brussels sprouts) , Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea ycliseca (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot and sweet pepper), Allium cepa (onion ), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (giant squash), Cucurbita moschata (nutmeg squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant ), Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Lupinus albus (lupine), Uniola paniculata (oats), bent grass (Agrostis spp.), Populus tremuloides (aspen poplar), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (chaff) and Phleum pratense (timothy), Panicum virgatum (millet), Sorghu52yclise52or (sorghum, Sudan grass), Miscanthus giganteus (miscanthus), Saccharum sp. (sugarcane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus ( sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet) or Pennisetum glaucum (pearl millet).

Various embodiments are directed to mutant, non-naturally occurring transgenic plants or plant cells modified to modulate gene expression levels, resulting in plants or plant cells, such as a tobacco plant or a tobacco plant cell, in which the expression level of the polypeptide is modulated in tissues. of interest compared to control. The disclosed compositions and methods can be applied to any species of the genus Nicotiana , including N. rustica and N. tabacum (eg, LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico). Other species include N. acaulis, N. acuminata , N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii , N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis , N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae.

The use of tobacco varieties and elite tobacco varieties is also contemplated in this document. A transgenic, non-naturally occurring or mutant plant, therefore, may be a variety of tobacco or an elite variety of tobacco that contains one or more transgenes or one or more genetic mutations, or a combination thereof. The genetic mutation(s) (e.g., one or more polymorphisms) may be mutations that do not occur naturally in a particular tobacco variety or tobacco variety (e.g., premium tobacco), or may -gut) is a genetic mutation(s) that exists(-s) in nature, provided that the mutation does not occur naturally in a particular variety of tobacco or variety of tobacco (for example, in an elite variety tobacco).

Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1, Prilep P12-2/1, Yaka JK-48, Yaka JB 125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Также предполагаются подразновидности вышеуказанного с низким уровнем превращения никотина в норникотин, даже если они специально не указаны в данном документе.Particularly useful varieties of Nicotiana tabacum include Burley-type tobacco, dark-type tobacco, fire-cured tobacco, and oriental-type tobacco. Non-limiting examples of varieties or varieties are BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC tobacco Galpao, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, hybrid 403LC, hybrid 404LC, hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC , PD 7312 LC, Perique tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51 , RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 hybrid 49 Burley 21 KY8959 KY9 MD 609 PG01 PG04 PO1 PO2 PO3 RG11 RG 8 VA509 AS44 Banket A1 Basma Drama B84/31 Basma I Zichna ZP4/B Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405,

Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1, Prilep P12-2/1, Yaka JK-48, Yaka JB 125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Sub-varieties of the above with low levels of nicotine to nornicotine conversion are also contemplated, even though they are not specifically listed here.

Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, and transgenic plants that have been modified to modulate the expression or activity of the polynucleotide(s) described herein (or any of them). combinations as described in this document). Various phenotypic characteristics, such as degree of maturity, number of leaves per plant, stem height, leaf angle, leaf size (width and length), internode length, and lamina-vein ratio, can be assessed by field observations.

One aspect relates to the seeds of a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant described herein. Preferably, the seeds are tobacco seeds. An additional aspect relates to the pollen or ovule of a mutant plant, non-naturally occurring plant, hybrid plant or transgenic plant described herein. In addition, a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant as described herein, which further comprises a male sterile nucleic acid, is provided.

Also provided is tissue culture of regenerated cells of a mutant plant, non-naturally occurring plant, hybrid plant, or transgenic plant or part thereof, as described herein, wherein plants are regenerated from the culture capable of expressing all of the morphological and physiological characteristics of the parent. Regenerated cells include, without limitation, cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, petioles, pith and bolls, or callus or protoplasts derived therefrom.

Yet another further aspect relates to dried plant material, such as dried leaf or dried tobacco, obtained or obtainable from a mutant, non-naturally occurring or transgenic plant or cell.

Embodiments are also directed to compositions and methods for producing mutant, non-naturally occurring, or transgenic plants or plant cells that have been modified to modulate the expression or activity of one or more of the polynucleotides or polypeptides described herein, which can result in plants with reduced period of time before flowering.

In another aspect, a method is provided for shortening the period of time until a plant blooms, comprising (i) modulating (e.g., decreased) expression or activity of one or more of the polypeptides or polynucleotides described herein; (ii) measuring the flowering rate of the mutant, non-naturally occurring or transgenic plant obtained in step (i); and (iii) identifying a mutant, non-naturally occurring, or transgenic plant that has a reduced time to flowering compared to a control plant. Accordingly, the plant is a tobacco plant.

The reduction in expression level compared to the control may be from about 5% to about 100%, or the reduction may be at least 10%, at least 20%, at least 25%, at least 30%, at least 40 %, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100%, which includes a decrease in transcriptional activity or the level of expression of the polynucleotide or the level of expression of the polypeptide, or a combination thereof.

The decrease in activity compared to the control may be from about 5% to about 100%, or the decrease may be at least 10%, at least 20%, at least 25%, at least 30%, at least 40% , at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100 %.

The polynucleotides and recombinant constructs described herein can be used to modulate expression in plant species of interest, respectively, in tobacco.

A variety of polynucleotide-based methods can be used, for example, to increase the level of gene expression in plants and in plant cells. By way of example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared that contains the gene of interest together with an upstream promoter capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein. After transformation and subject to growth under suitable conditions, the promoter can drive expression. In one exemplary embodiment, a vector carrying one or more of the polynucleotides described herein (or any combination thereof, as described herein) is created to overexpress a gene in a plant or in a plant cell. The vector carries a suitable promoter, such as the cauliflower mosaic virus CaMV 35S promoter, upstream of the transgene that controls its constitutive expression in all plant tissues. The vector also carries an antibiotic resistance gene to allow selection of transformed calluses and cell lines.

Various embodiments are directed to methods for reducing the expression level of one or more of the polynucleotides described herein by integrating multiple copies of the polynucleotide into the plant genome, comprising transforming a host plant cell with an expression vector that contains a promoter operably linked to one or more of the polynucleotides. described in this document. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

A plant carrying a mutant allele of one or more of the polynucleotides described herein (or any combination thereof as described herein) can be used in a plant breeding program to create usable lines, varieties and hybrids. In particular, the mutant allele is introgressed into the commercially important varieties described above. Thus, plant breeding methods are provided which involve crossing a mutant plant, a non-naturally occurring plant, or a transgenic plant, as described herein, with a plant having a different genetic identity. The method may further comprise crossing the progeny plant with another plant and optionally re-crossing until a progeny with the desired genetic traits or genetic background is obtained. One purpose such breeding methods serve is to introduce the desired genetic trait into other varieties, breeding lines, hybrids or varieties, especially those of commercial interest. Another goal is to facilitate the accumulation of genetic modifications of different genes in individual varieties, lines, hybrids or plant varieties. Intraspecific as well as interspecific crosses are provided. The progeny plants that result from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the present invention.

In one embodiment, a method for producing a non-naturally occurring plant is provided, comprising (a) crossing a mutant or transgenic plant with a second plant to produce tobacco progeny seed; (b) growing the progeny seed under plant growth conditions to produce a non-naturally occurring plant. The method may further comprise (c) crossing a previous generation of a non-naturally occurring plant with itself or with another plant to produce a progeny seed; (d) growing the progeny seed from step (c) under plant growth conditions to produce additional non-naturally occurring plants; and (e) repeating the crossing and rearing steps (c) and (d) many times to produce next generations of non-naturally occurring plants. The method may optionally include, prior to step (a), the step of obtaining a parent plant that has a genetic identity that is characteristic of, and is not identical to, the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and rearing steps are repeated 0 to 2 times, 0 to 3 times, 0 to 4 times, 0 to 5 times, 0 to 6 times, 0 to 7 times, 0 to 8 times, 0 to 9 times, or 0 to 10 times to obtain generations of non-naturally occurring plants. Backcrossing is an example of such a method where the offspring are crossed with one of its parents or with another plant genetically similar to its parent to produce a progeny plant in the next generation that has a genetic identity closer to one of the parents. Plant breeding techniques, in particular plant breeding, are well known and can be used in the methods of the present invention. The present invention further provides non-naturally occurring plants obtained using these methods. Certain embodiments omit the plant selection step.

In some embodiments of the methods described herein, lines resulting from selection and screening of variant genes are evaluated in the field using standard field procedures. Control genotypes are included, including the original unmutated parents, and planting sites are located in the field according to a randomized block plan or other appropriate field planning. For tobacco, standard agricultural practices are used, for example, tobacco is harvested, weighed and sampled for chemical and other conventional testing before and during drying or drying. Statistical analyzes of the data are performed to confirm the similarity of the selected lines with the parent line. Cytogenetic analyzes of selected plants are optionally performed to confirm chromosome set and chromosome conjugation relationships.

DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies can be used in a marker-assisted selection (MAS) breeding program to transfer or propagate mutant alleles of a gene in other plants, as described herein. For example, a breeder can create segregated populations by hybridizing a genotype containing a mutant allele with an agronomically desired genotype. Plants from F2 or backcrossed generations can be screened with a marker derived from a genomic sequence or fragment thereof using one of the techniques listed herein. Plants identified as having the mutant allele can be backcrossed or selfed to create a second population to be screened. Depending on the intended inheritance pattern or the MAS technology used, it may be necessary to self-pollinate the selected plants before each backcrossing cycle to facilitate identification of the individual plants needed. Backcrossing or other selection procedure can be repeated until the desired phenotype of the recurrent parent is restored.

In a breeding program, successful crosses produce F1 plants that are fertile. Selected F1 plants can be crossed with one of the parents, and first generation backcross plants can be self-pollinated to produce a population that is again screened for expression of a variant gene (eg, a null version of the gene). The process of backcrossing, selfing and screening is repeated, for example, at least 4 times until the final screening results in a plant that is fertile and sufficiently similar to the recurrent parent. This plant is self-pollinated, if necessary, and then the progeny are screened again to confirm that the plant is expressing the variant gene. In some embodiments, the F2 generation plant population is screened for expression of the variant gene, e.g., a plant that does not express the polypeptide due to the absence of the gene is identified according to standard methods, e.g., using a PCR technique using primers designed from the information the nucleotide sequence for the polynucleotide(s) described herein (or any combination thereof described herein).

Hybrid varieties can be produced by preventing the female parent plants (i.e., maternal parents) of the first variety from self-pollinating, allowing pollen from the male parent plants of the second variety to fertilize the female parent plants, and producing F1 hybrid seeds on the female plants. Self-pollination of female plants can be prevented by castrating the flowers early in flower development. Alternatively, the production of pollen on the female parent plants can be prevented by using a male sterility type. For example, male sterility can be obtained using cytoplasmic male sterility (CMS) or transgenic male sterility, where the transgene suppresses microsporogenesis and/or pollen production, or causes self-incompatibility. Female parent plants characterized by CMS are particularly useful. In embodiments where the female parent plants are characterized by CMS, pollen is harvested from the male fertile plants and applied by hand to the stigmas of the CMS female parent plants and the resulting F1 seeds are harvested.

The varieties and lines described herein can be used to form simple F1 hybrids. In such embodiments, the plants of the parental varieties can be grown in substantially homogeneous populations to facilitate natural cross-pollination by the male parent plants of the female parent plants. F1 seeds produced on female parent plants are selectively harvested by conventional means. It is also possible to grow two varieties of parent plants in a mass and collect a mixture of F1 hybrid seeds produced on the female parent and seeds produced on the male parent as a result of self-pollination. Alternatively, a trilinear cross can be made where a single F1 hybrid is used as the female parent and crossed to a different male parent. As another alternative, double cross hybrids can be created, where the F1 offspring of two different simple hybrids are crossed with themselves.

A population of mutant, non-naturally occurring, or transgenic plants can be screened or selected for those members of the population that have the desired trait or phenotype. The desired trait or phenotype may be a shorter time to flower (e.g., at least 30% shorter time to flower compared to the wild type plant) and optionally fewer leaves than the wild type plant, and optionally the same height that of a wild-type plant.

For example, a population of progeny of a line with one transformation can be screened for those plants that have the desired level of expression or activity of the polypeptide(s) encoded(s) with it. Physical and biochemical methods can be used to determine levels of expression or activity. These include Southern analysis or PCR amplification to detect the polynucleotide; Northern blotting, RNase S1 protection, primer extension or real-time PCR amplification to detect RNA transcripts; enzyme assays to detect enzymatic or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blotting, immunoprecipitation and enzyme immunoassays for the detection of polypeptides. Other techniques such as in situ hybridization, enzymatic staining and immunostaining, and enzymatic assays can also be used to detect the presence or expression or activity of polypeptides or polynucleotides.

This document describes mutant, non-naturally occurring or transgenic plant and plant cells containing one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates, or one or more vectors/expression vectors.

Without limitation, the plants described herein can be modified for other purposes either before or after the expression or activity has been modulated according to the present invention. One or more of the following genetic modifications may be present in mutant, non-naturally occurring or transgenic plants. In one embodiment, one or more genes that are involved in the conversion of nitrogen metabolism intermediates are modified to produce plants (such as leaves) that, when dried, produce lower levels of at least one tobacco-specific nitrosamine than control plants. Non-limiting examples of genes that can be modified include genes encoding asparagine synthetase, such as CYP82E4 , CYP82E5 and CYP82E10 , which are involved in the conversion of nicotine to nornicotine and are described in publications WO2006091194, WO2008070274, WO200906470271 and PCT/US20118 document. In another embodiment, one or more genes that are involved in heavy metal uptake or heavy metal transport are modified to result in plants or plant parts (such as leaves) having a lower heavy metal content than control plants or parts thereof. without modification(s). Non-limiting examples include the genes of the multidrug resistance protein family, the cation diffusion mediator (CDF) family, the Zrt-, Irt-like protein (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPase family P-type (e.g., HMA as described in WO2009074325), the macrophage protein homologs associated with natural resistance (NRAMP) families, and the ATP-binding cassette (ABC) transporter family (e.g., MRPs as described in WO2012/028309, which involved in the transport of heavy metals such as cadmium). The term "heavy metal" as used herein includes transition metals. Examples of other modifications include herbicide tolerance, for example, glyphosate is the active ingredient in many broad spectrum herbicides. Glyphosate-resistant transgenic plants have been developed by gene transfer of aroA (glyphosate EPSP synthetase from Salmonella typhimurium and E. coli ). Sulfonylurea-resistant plants were obtained by transformation of the mutant ALS (acetolactate synthetase) gene from Arabidopsis. The photosystem II OB protein from the mutant Amaranthus hybridus was transferred into plants to produce atrazine-resistant transgenic plants; and bromoxynil-resistant transgenic plants were obtained by inserting the bxn gene from the bacterium Klebsiella pneumoniae. Other exemplary modifications result in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins may provide an effective way of delaying the emergence of Bt-resistant pests, as recently shown in broccoli, where the Bt cry1Ac and cry1C pyramidal genes controlled cabbage moths resistant to any one protein and significantly delayed the development of resistant insects. . Another exemplary modification results in plants that are resistant to diseases caused by pathogens (eg, viruses, bacteria, fungi). Plants expressing the Xa21 gene (bacterial necrosis resistance) have been developed, with plants expressing both the Bt fusion gene and the chitinase gene (yellow grass moth resistance and rhizoctonia tolerance). Another exemplary modification results in an altered reproductive capacity, such as male sterility. Another exemplary modification results in plants that are tolerant of abiotic stress (eg, drought, temperature change, salinity), and tolerant transgenic plants were obtained by transferring the glycerol phosphate acyltransferase enzyme from Arabidopsis; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in the synthesis of mannitol and sorbitol, which improve drought tolerance. Other exemplary modifications may result in plants with improved accumulation of proteins and oils, plants with increased photosynthetic efficiency, plants with a long shelf life, plants with increased carbohydrate content, and plants resistant to fungi; plants encoding an enzyme involved in the biosynthesis of alkaloids. Also contemplated are transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) and/or cystathionine gamma synthase (CGS) has been modulated.

One or more of these traits can be introgressed into, or directly transformed into, mutant, non-naturally occurring, or transgenic plants of another variety. Introgression of the trait(s) into mutant, non-naturally occurring or transgenic plants can be achieved by any plant breeding method known in the art, such as pedigree selection, backcrossing, double haploid selection, etc. (see Wernsman, EA and Rufty, RC 1987. Chapter Seventeen Tobacco pages 669-698 in Cultivar Development Crop Species WH Fehr (ed.), MacMillan Publishing Co, Inc., New York, NY page 761). The molecular biology based techniques described above, in particular using RFLP and microsatellite markers, can be used in such backcrosses to identify offspring that have the highest degree of genetic identity with the recurrent parent. This makes it possible to expedite the production of varieties having at least 90%, preferably at least 95%, more preferably at least 99% genetic identity with the recurrent parent, even more preferably genetically identical to the recurrent parent and additionally characterized by the trait(s) introgressed(s) from a donor parent. Such determination of genetic identity may be based on molecular markers known in the art.

The last generation of backcrosses can be self-pollinated to produce pure breeding offspring for the nucleic acid(s) to be transferred. The resulting plants typically have substantially all of the morphological and physiological characteristics of the mutant, non-naturally occurring, or transgenic plants in addition to the trait(s) transferred (eg, one or more traits defined by a single gene). The exact backcrossing protocol will depend on the trait being changed to determine the appropriate test protocol. Although backcrossing methods are simplified if the trait being transferred is the dominant allele, the recessive allele can also be transferred. In this case, it may be necessary to use progeny testing to determine whether the desired trait has been successfully transferred.

In various embodiments, mutant plants, non-naturally occurring plants, or transgenic plants, as well as biomass, are provided in which the expression level of a polynucleotide (or any combination thereof, as described herein) is reduced to reduce the period of time before flowering occurs.

In various embodiments, mutant plants, non-naturally occurring plants, or transgenic plants, as well as biomass, are provided in which the activity of the polypeptide (or any combination thereof, as described herein) is reduced to reduce the time to flowering.

Parts of such plants, in particular tobacco plants, and more particularly the lamina and midrib of tobacco plants, can be incorporated into or used in the manufacture of various consumer products, including, without limitation, aerosol generating materials, aerosol generating devices, smoking articles, for smoking, smokeless products and tobacco products. Examples of aerosol forming materials include, without limitation, tobacco compositions, tobacco species, tobacco extract, cut tobacco, cut filler, cured or dried tobacco, exploded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco species. Smoking articles and smoking articles are types of aerosol generating devices. Examples of smoking articles and smoking articles include, without limitation, cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobaccos and snuffs. In certain aerosol generating devices, instead of combusting (or burning), the tobacco composition or other aerosol generating material is subjected to heat, such as with one or more electrical heating elements or a carbon heat source, to form an aerosol. Typically, in such heated smoking articles, the aerosol is generated by the transfer of heat from the heat source to a physically separated aerosol-forming substrate or material, which may be located within, around, or downstream of the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate as a result of heat transfer from the heat source and are trapped in the air drawn through the smoking article. As the released compounds cool, they condense to form an aerosol that is inhaled by the user. Such devices include, for example, electrically heated aerosol generating devices in which the aerosol is formed by transferring heat from the aerosol generating device to the aerosol generating substrate of the heated smoking article. Accordingly, during the heating of the aerosol-forming substrate, no combustion or burning of tobacco occurs. A suitable aerosol generating article is described in WO2013/098405 and comprises an aerosol generating substrate for generating an inhalable aerosol when heated by an internal heating element of the aerosol generating device. It may comprise an electrically heated aerosol generating device comprising an internal heating element. It may further comprise, in a linear sequential arrangement, an aerosol generating substrate, a support element located immediately downstream of the aerosol generating substrate, a cooling aerosol element located downstream of the support element, and an outer wrapper surrounding the aerosol generating substrate, a support element and an element cooling the aerosol. The support member may be adjacent to the aerosol generating substrate. The aerosol generating substrate is penetrated by the heating element of the aerosol generating device.

As used herein, the term "combustion" refers to a redox chemical reaction where the reacting molecules, namely fuel and oxidant, mix and rearrange to become product molecules while releasing heat. Combustion can be positively noted by the presence of appropriate amounts of oxides of nitrogen in the gaseous products, not formed as a result of the decomposition of nitrates present in the original reacting substrate, and by clear evidence of a simultaneous general exothermic process. The formation of corresponding amounts of nitrogen oxides in combustion gases can be determined by comparing the total amounts of nitrogen oxides formed under the conditions of interest (for example, in air) and nitrogen oxides formed under the same conditions but in the absence of oxygen (for example, in an atmosphere of pure nitrogen or helium). In another type of heated aerosol generating device, the aerosol is formed by transferring heat from a combustible fuel cell or heat source to a physically separated aerosol generating material that may be located in, around, or downstream of the heat source. Smokeless tobacco products and various aerosol-forming tobacco-containing materials may contain tobacco in any form, including in the form of dried particles, trimmings, granules, powders or suspension, applied to, mixed with, surrounded by, or otherwise combined with other ingredients. them in any format such as flakes, films, tablets, foams or balls. As used herein, the term "smoke" is used to describe the type of aerosol produced by smoking articles, such as combustible cigarettes, or by burning an aerosol-forming material.

In certain embodiments, heating without combusting or burning plant material is preferred.

One embodiment also provides dried plant material from the mutant, transgenic, and non-naturally occurring tobacco plants described herein. Methods for drying green tobacco leaves are known to those of ordinary skill in the art and include, without limitation, air drying, fire drying, smoke drying, and sun drying. The method of drying green tobacco leaves depends on the type of tobacco harvested. For example, Burley and certain dark varieties are typically air-dried, while pipe tobacco, chewing tobacco, and snuff are fire-dried.

In another embodiment, dried plant material from the mutant, transgenic, and non-naturally occurring plants described herein is also provided. Methods for drying leaves are known to those of ordinary skill in the art and include, but are not limited to, air drying and sun drying. The appropriate method of drying the leaves depends on the type of plant being harvested. Accordingly, the plant material is dried after collection. Thus, the use of dried material and dried after collection material is discussed in this document.

In another embodiment, tobacco products are described, including aerosol-forming tobacco-containing materials containing plant material such as leaves, preferably dried or dried leaves, from mutant tobacco plants, transgenic tobacco plants, or non-naturally occurring tobacco plants described herein. document. The tobacco products described herein may be a blended tobacco product, which may further comprise unmodified tobacco.

The present invention also provides methods for producing seeds, comprising cultivating a mutant plant, a non-naturally occurring plant, or a transgenic plant described herein, and collecting the seeds of the cultivated plants. The seeds of the plants described herein can be conditioned and packaged in a packaging material by means known in the art to form an article of manufacture. Packaging material such as paper or cloth is well known in the art. The seed package may have a label, such as a tag or label attached to the packaging material, a label printed on the package, which describes the origin of the seeds it contains.

Compositions, methods, and kits for plant genotyping for identification, selection, or selection may include a means of detecting the presence of a polynucleotide (or any combination thereof, as described herein) in a polynucleotide sample. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more polynucleotides and optionally one or more probes and optionally one or more amplification or detection reagents.

Accordingly, gene-specific oligonucleotide primers or probes containing about 10 or more contiguous polynucleotides corresponding to the polynucleotide(s) described herein are disclosed. Said primers or probes may contain or consist of about 15, 20, 25, 30, 40, 45, or 50 or more contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the polynucleotide(s) described herein. document. In some embodiments, primers or probes may comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides, which can be used in sequence-dependent gene identification methods. (eg, Southern hybridization) or isolation (eg, in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (eg, as one or more amplifying primers in nucleic acid amplification and detection). One or more specific primers or probes can be designed and used to amplify and detect part or all of the polynucleotide(s). As a specific example, two primers can be used in a polymerase chain reaction protocol to amplify a nucleic acid fragment encoding a nucleic acid, such as DNA or RNA. The polymerase chain reaction can also be carried out using one primer that is derived from a nucleic acid sequence and a second primer that hybridizes to a sequence upstream or downstream of the nucleic acid sequence, such as a promoter sequence, the 3' end of the mRNA precursor or a sequence obtained from a vector. Examples of thermal and isothermal techniques applicable to in vitro amplification of polynucleotides are well known in the art. The sample may be a plant, a plant cell, or plant material, or a tobacco product, or may be derived from them, wherein the tobacco product is made or obtained from a plant, plant cell, or plant material, as described herein.

In a further aspect, a method is also provided for detecting a polynucleotide(s) described herein (or any combination thereof described herein) in a sample, comprising the step of (a) obtaining a sample containing, or suspected to contain, a polynucleotide; (b) contacting said sample with one or more primers or one or more probes to specifically detect at least a portion of the polynucleotide(s); and (c) detecting the presence of an amplification product, where the presence of an amplification product is indicative of the presence of the polynucleotide(s). ) in the sample. In a further aspect, the use of one or more primers or probes to specifically detect at least a portion of the polynucleotide(s) is also provided. Also provided are kits for detecting at least a portion of a polynucleotide(s) that contain one or more primers or probes for specifically detecting at least a portion of the polynucleotide(s). The kit may contain reagents for polynucleotide amplification, such as PCR, or reagents for probe hybridization detection technology, such as Southern blot, Northern blot, in situ hybridization, or microarray. The kit may contain reagents for antibody binding detection technology such as Western blotting, ELISA, SELDI mass spectrometry, or test strips. The kit may contain DNA sequencing reagents.

In some embodiments, the kit may contain instructions for one or more of the methods described. The disclosed kits may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding, in particular for quantifying codominant traits.

The present invention also provides a method for genotyping a plant, plant cell or plant material containing the polynucleotide described herein. Genotyping provides a means of distinguishing chromosome pair homologues and can be used to distinguish between segregants in a plant population. Molecular methods using markers can be used for phylogenetic studies characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, localizing chromosomal segments that affect monogenic traits, cloning based on genetic maps, and studying quantitative inheritance. Any number of molecular marker assays can be used in a particular genotyping method, including amplified fragment length polymorphisms (AFLPs). AFLPs are the product of allelic differences between amplification fragments caused by nucleotide sequence variation. Thus, the present invention further provides methods for tracking the segregation of one or more genes or nucleic acids, as well as chromosomal sequences genetically related to those genes or nucleic acids, using techniques such as AFLP analysis.

One embodiment also provides dried or desiccated plant material from the mutant, transgenic, and non-naturally occurring plants described herein. For example, methods for drying or drying tobacco leaves are known to those skilled in the art and include, without limitation, air drying, fire drying, smoke drying, and sun drying. The method of drying green tobacco leaves depends on the type of harvested tobacco, as described herein.

In another embodiment, tobacco products are described, including tobacco products containing plant material, such as leaves, respectively, dried plant material, such as dried or dried leaves, from the mutant, transgenic, and non-naturally occurring plants described herein. , or which are obtained through the methods described in this document. The tobacco products described herein may additionally contain unmodified tobacco.

In another embodiment, tobacco products are described comprising plant material, preferably leaves, such as dried or desiccated leaves, from the mutant, transgenic, and non-naturally occurring plants described herein. For example, the plant material can be added to the inside or outside of the tobacco product so that the desired flavor is released upon combustion. The tobacco product according to this embodiment may even be unmodified tobacco or modified tobacco. The tobacco product according to this embodiment may even be obtained from a mutant, transgenic, or non-naturally occurring plant that has modifications in one or more genes other than those disclosed herein.

In a further aspect, a method is provided for identifying a molecule that modulates the activity or expression of a TFL1 polynucleotide or TFL1 polypeptide. Exemplary molecules that can be used in such a method are described herein, including antisense molecules, siRNA, ribozyme molecules, deoxyribozyme molecules, TALEN, zinc finger nucleases, and the like. The use of chemical compounds is also contemplated.

A mutant, non-naturally occurring or transgenic plant or part thereof is also described having modulated (eg, decreased or increased) expression of the gene encoding Terminal Flower 1 ( TFL1 ), or modulated (eg, decreased or increased) activity of the protein encoded by TFL1 . wherein said TFL1 contains, consists of, or essentially consists of (i) a polynucleotide sequence containing, consisting of, or essentially consisting of a sequence characterized by at least 72% sequence identity with SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide having at least 72% sequence identity with SEQ ID NO:9 or SEQ ID NO:12 or SEQ ID NO:21; where the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) or (iii) is modulated (for example, reduced or increased) compared to a control plant in which the expression or activity of the polynucleotide or polypeptide presented in (i) , (ii) or (iii) was not modulated (eg reduced or increased).

Modulated expression of the polynucleotide or modulated activity of the polypeptide provided modulation of the period of time before the onset of flowering compared with the control plant, respectively, where the period of time before flowering is modulated by at least 8% or at least 20%.

Increased expression of the polynucleotide or increased activity of the polypeptide provides an extension of the time to flowering compared to the control plant, respectively, where the time period before flowering is extended by at least 8% or at least 20%.

Also disclosed is a method for modulating (eg, increasing or decreasing) the period of time before the onset of flowering in a plant, including modifying the plant by modulating the expression of at least one TFL1 gene or the activity of at least one protein encoded by it in the specified plant; respectively, where the method includes: (a) obtaining a plant or part thereof, containing (i) a polynucleotide sequence containing, consisting of, or essentially consisting of a sequence characterized by at least 72% sequence identity with SEQ ID NO: 7, or SEQ ID NO :8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide characterized by at least 72% sequence identity with SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:21; and (b) modulating (eg, increasing or decreasing) TFL1 gene expression or TFL1 protein activity in a plant; and (c) obtaining a plant with a modulated period of time to flowering compared to a control plant in which TFL1 gene expression or TFL1 protein activity has not been modulated.

Accordingly, TFL1 expression or TFL1 activity is modulated by a method selected from the group consisting of a) mutating the TFL1 gene in a plant; b) expression of the exogenous polynucleotide or polypeptide in the plant; and c) removing the TFL1 gene from the plant, or a combination of one or more of them.

Also disclosed is a method for increasing the period of time before the onset of flowering in a plant, including modifying the plant by increasing the expression of at least one TFL1 gene or the activity of at least one protein encoded by it in the specified plant; respectively, where the method includes (a) obtaining a plant or part thereof, containing (i) a polynucleotide sequence containing, consisting of, or essentially consisting of a sequence characterized by at least 72% sequence identity with SEQ ID NO: 7, or SEQ ID NO: 8 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:19 or SEQ ID NO:20; or (ii) a polypeptide encoded by a polynucleotide shown in (i); or (iii) a polypeptide characterized by at least 72% sequence identity with SEQ ID NO:9, or SEQ ID NO:12, or SEQ ID NO:21; and (b) increasing TFL1 gene expression or TFL1 protein activity in the plant; and (c) obtaining a plant with an extended time to flowering compared to a control plant in which TFL1 gene expression or TFL1 protein activity has not been modulated.

Accordingly, TFL1 expression or TFL1 activity is increased by a method selected from the group consisting of a) mutating the TFL1 gene in a plant; b) expression of the exogenous polynucleotide or polypeptide in the plant; and c) removing the TFL1 gene from the plant, or a combination of one or more of them.

Also described is a method for obtaining plant material with a modulated period of time before the onset of flowering compared with a control plant, while this method includes (a) obtaining a plant or plant material, as described in this document; (b) collecting plant material from the plant; (c) optionally drying or drying the plant material for a period of time; and (d) obtaining plant material that has a modulated period of time before flowering compared to a control plant.

Also described is a method for obtaining plant material with an extended period of time before the onset of flowering compared with a control plant, while this method includes (a) obtaining a plant or plant material, as described in this document; (b) collecting plant material from the plant; (c) optionally drying or drying the plant material for a period of time; and (d) obtaining plant material that has an extended time to flowering compared to a control plant.

The present invention is further described in the examples below, which are provided to describe the present invention in more detail. These examples, which set forth the preferred mode currently contemplated for carrying out the present invention, are intended to illustrate and not limit the present invention.

EXAMPLES

Example 1 Identification of Tobacco TFL1 Genes

The tobacco TFL1 gene family was identified and the identity of 7 tobacco TFL1 genes was determined. They are called TFL1-1S, TFL1-1T, TFL1-2S, TFL1-2T, TFL1-3T, TFL1-4T and TFL1-3S. The sequences of these genes are described in this document.

Example 2 Analysis of TFL1 Expression Levels

To identify active forms of TFL1 in the vegetative tissues of leaves and roots, N. tabacum plants of three different varieties (Burley-TN90, Virginia-K326, and Oriental type - BX_Basma Xanthi) were grown in Magenta boxes with bactoagar-MS and expression data (FPKM) were calculated after isolation. RNA and sequencing of the corresponding cDNA libraries ( Nat Commun . (2014) 5:3833). The data are shown in Table 1. TFL1 was mainly expressed in root tissues. The main TFL1 genes expressed in roots under these conditions ( vegetative state) are TFL1-2S , TFL1-2T , TFL1-3T and TFL1-4T . It can be seen that no TFL1-4S transcripts have been identified in Virginia (K326), Burley (TN90) or Dark tobacco, with only a very small number of RNA copies found in the oriental-type Basma Xanthi ( BX) roots. The reason why transcripts were not detected in K326 and TN90 is not clear, but this may be due to promoter disruption, since the 5'-end of the 2 kb. the promoter at K326 and TN90 could not be identified.

To determine whether gene expression profiles differ between flowering plants, N. tabacum (variety TN90) was grown in the field and RNA was isolated from immature flowers, leaves at the bottom of the stem, leaves at the middle part of the stem, leaves at the top of the stem, petals, roots, sepals and stem immediately after the appearance of the first flowers. The results are shown in figure 1. In these plants, the level of expression of TFL1 - 2T and TFL1 - 4T remained low in the roots, stem and immature flowers (FPKM values do not exceed 1.1).

Example 3 - Genetic manipulation of TFL1 : Tobacco plants with TFL1 - RNAi

Four specific DNA fragments were designed to silence the six expressed copies of TFL1 (see SEQ ID NOS: 24, 27, 30 and 33) and cloned between the strong constitutive MMV (Mirabilis Mosaic Virus) promoter and the nos 3' terminator sequence of the Agrobacterium nopaline synthase gene. tumefaciens ( Plant Mol Biol . (1999) 40: 771-82): (1) C100S3-MMVp-GW-pDONR221-TFL1-1 S/T; (2) C100S3-MMVp-GW-pDONR221 - TFL1-2 S/T; (3) C100S3-MMVp-GW-pDONR221 - TFL1-3T; (4) C100S3-MMVp-GW-pDONR221 - TFL1-4T.

A dark tobacco variety was transformed with each of the four constructs using standard Agrobacterium mediated transformation protocols ( Cold Spring Harb Symp Quant Biol. (1985) 50: 433-437). For each construct, 20 independent T0 plants were selected on kanamycin and then transferred to soil in a greenhouse section set to 9 hours light (24°C +/- 3°C) and 15 hours dark (18°C +/- 3°C). C) during the first three months, then 15 hours of light and 9 hours of darkness. Control plants were subjected to the same in vitro procedure (no transformation step) and were also simultaneously transferred to the greenhouse. Seedlings were first cultivated in trays and transplanted into 5 liter pots after 25 days. The plants were watered every day with a fertilizer solution (Yara, The Netherlands). Each plant for the four constructs and their respective controls were analyzed phenotypically and data was recorded as flower appearance after transplant (DAT), height and number of leaves (see figure 2 and table 2). The TFL1-1S/T and TFL1-4T RNAi plant data and their respective controls (Figures 2A,B,C) needed to be compared separately from the TFL1-2S/T and TFL1-3T group (Figures 2D,E,F) because the first the group of plants were transplanted and cultivated in the greenhouse 3 weeks after the second group of plants. All plants were grown under similar conditions in a greenhouse. The data showed that the TFL1-2S/T RNAi transgenic lines flowered significantly faster than control plants, showing a 20% reduction in time to flowering. In addition, the RNAi line TFL1-2S/T also showed a significant decrease in height and number of leaves, which correlates with a reduced time period before flowering (figures 2A, B, C). Data are statistically significant for all measured parameters (P<0.001) and phenotypes were clearly visible in the greenhouse (figure 3). In addition, another TFL1 gene product, TFL1-4T , appears to play a role in regulating the time period before flowering. Indeed, an 8% reduction in time to flowering was observed, but not significantly compared to control, with the other two parameters measured, height (16% reduction versus control) and number of leaves (13% reduction versus control) are statistically significant.

The data collected on RNAi plants, combined with expression data, suggest that TFL1-1S and TFL1-1T make a minimal contribution to regulating the time to flowering and maintaining the vegetative state of plants.

In conclusion, the data indicate that the most efficient TFL1 genes for maintaining a vegetative state in tobacco under the described growth conditions are TFL1-2S , TFL1-2T > TFL1-4T > TFL1-3T >TFL1-1S , TFL1-1T . Therefore, silencing or knocking out TFL1 - 2S and/or TFL1 - 2T and/or TFL1 - 4 - T is likely to be a solution to ensure a shortened time to flowering.

Example 4 - TFL1 Mutations to Shorten Time to Bloom

For tobacco breeding, the EMS-treated tobacco variety population was screened for mutations in TFL1α . All amino acid substitutions identified in TFL1-2S , TFL1-2T and TFL - 4T were analyzed for possible effect on protein function using the SIFT program ( Nucleic Acids Res. (2003) 1;31(13):3812-4). A small SIFT score (<0.05) implies that the amino acid residue is probably not functionally acceptable. In TFL1-2S , TFL1-2T, and TFL - 4T, seven mutations were identified with a SIFT score ratio below 0.05, as shown below:

TFL1-2S_G129E 0.0002

TFL1-2S_G129R 0.0002

TFL1-2S_T143I 0.0011

TFL1-2T_R120C 0

TFL1-2T_P131S 0.0007

TFL1-2T_G129E 0.0002

TFL1-4T_P110L 0

The SIFT score is used as a tool to facilitate mutation selection. In a greenhouse experiment, mutations and combinations of mutations were tested and the rate of onset of flowering was measured.

In TFL1-2S, TFL1-2T, and TFL1-4T, the His88 motif is conserved at positions 84, 88, and 86, respectively. A mutation (eg, a missense mutation) at this amino acid position can disrupt the three-dimensional structure of the transcription factor, thus affecting the binding properties at the promoter. Accordingly, the mutation may be a stop mutation.

The Asp144 motif is conserved in TFL1-2S and TFL1-2T at positions 138 and 142, respectively. It is replaced by glutamate in TFL1-4T at position 139. Three mutations close to TFL1-2S (D138), TFL1-2T (D142), respectively, have been identified near this region and may affect the three-dimensional structure of the transcription factor and, thereby altering binding to the promoter.

Example 5 - Mutation TFL1 - 2T - P131S

A tobacco plant containing the TFL1-2T-P131S mutation (as shown in Figure 4) was grown under greenhouse conditions. Time to flowering, number of leaves, and height of mutant plants were monitored relative to the wild-type plant. Tobacco plants containing this mutation flowered 30% faster and had fewer leaves (6-7) if the TFL1-2T copy contained the P131S mutation in both homozygotes and heterozygotes. No effect on plant height was observed. The mutant tobacco plant had a shorter time to flowering compared to the wild type plant.

Example 6 - TFL1-4T - P110L Mutation A tobacco plant containing the TFL1-4T-P110L mutation (as shown in Figure 6) was grown under greenhouse conditions. Time to flowering, number of leaves, and height of mutant plants were monitored relative to the wild-type plant. No homozygous plants were available for this mutant, but as observed for TFL1-2T-P131S, tobacco plants flower about 30% faster (eg, about 28% faster) and have fewer leaves (8-9) if TFL1- 4T contains the heterozygous P110L mutation. No effect on height was observed. The mutant tobacco plant has a shorter time before flowering compared to the wild type plant. The data suggest that only one allele is sufficient to accelerate flowering for both TFL1-2T-P131S and TFL1-4T-P110L mutations.

Any publication cited or described in this document provides relevant information disclosed prior to the filing date of this application. The statements made herein are not to be construed as an admission that the inventors of the present invention have no basis for setting it apart from such disclosures as before. All publications mentioned in the above description are incorporated into this document by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods of carrying out the invention, which are obvious to those skilled in cellular, molecular and plant biology or related fields, are intended to be within the scope of the appended claims.

TABLE 1

Expression data (FPKM) of TFL1 in vegetative (non-flowering) plants grown on bactoagar - MS. No transcripts were detected in TN90 and K326 (none).

table 2

Statistical analyzes of the data presented in figure 2 were performed using a modified two-sample Welch test ( Biometrika (1947) 34 (1-2): 28-35; BioMed Central Medical Research Methodology (2012) 12: 78)

SEQUENCES

SEQ ID NO: 1

The genomic sequence of the TFL1 - 1S N. tabacum gene, containing 2 kb. before ATG (in bold )

atg

SEQ ID NO: 2

Genomic sequence of the TFL1 gene - 1S N. tabacum

atggcaaggaatgtagagcctctagtagtagggagagtagtaggggatgttcttgattcatttagtcccacagttaaaatgacagtcacttacaacaataaacaagtttgcaatggccaagagctcttcccttctgcagtcactattagacctagggttgaagttcaaggtggtgatatgagaactttcttcacattggtaacttttctaattttccttcaggttattaacttttcattgctatatagacatctttcaaacctgtctagaagattcttttcttgagctgagggttcggagacagcctctctatcccacacaagataaggttaacgtctgcatataccttgccgtcccctgacaccacatatgggattatactgcgtatgttgttgttgttgtattgctatatagacatctttgataaatcactaatcatgcatgatattattcttcttattgtaggtcatcacagatcctgatgtacctggccctagtgatccgtatctgagagaacatgttcactggtatgtactacattcaagaaaatctatggaaaaaaaataactagaagagatgagtccaaaagaaaggagattaattattgtttgatttgtattttttatttgcattagattaagtcatgcaattacctagtaataaattcgttataaggacatacacaaaaggtatgctcaacataattaaagaaagaaaagcataagcattatattcttatatgcaagctcccaatctgattgtggcgaagctaagaatttcttcgggtgtttaaatttaaaagaagtgaaaaaaaatccgataaaaaatttatgtgttatatacctctaaaacttaatattgtacctatatacatattgtaatttttcgacgaatgaccactcttattgcccatgctgccaatgccaatatgtaaaatacttcgccaatagacaaatatgtatcacattaattagttacctaaataggtaattatccataa taagtgggactcgtaacttgaaaaatatggttagtaatttgctacaacatgttaagatacagtgagaatgtaaatgtatatatgtcctttcacgccgtcgtaagtttatataagttaaatctggttaacaatgtaggtcatcataacactcacaattgggaataataactcttaatcatacttctttacattcatatttgcaggatagtgactgatattccaggaactacagatgccacctttggtaaattggctattttggtttcttttattaagctggtgtttgacatgctagaattaatgtactttaattttgagcaggaaaagagttggttagctatgagatcccaaggcctaatattggaatacataggtttgtgtttgttctctttaagcagaaatgcagacaatcagtcagcccacctacttcaagggatcatttcaacactcgcaactttgccaacgtaaatgaccttggtccgcctgtcgccgccgtcttcttcaatgcacaacgagagaccgccgccaggaggcgctaa

SEQ ID NO: 3

N. tabacum TFL1-1S gene amino acid sequence obtained from SEQ ID NO: 1 or SEQ ID NO : 2.

MARNVEPLVVGRVVGDVLDSFSPTVKMTVTYNNKQVCNGQELFPSAVTIRPRVEVQGGDMRTFFTLVITDPDVPGPSDPYLREHVHWIVTDIPGTTDATFGKELVSYEIPRPNIGIHRFVFVLFKQKCRQSVSPPTSRDHFNTRNFANVNDLGPPVAAVFFNAQRETAARRR

SEQ ID NO: 4

The genomic sequence of the TFL1 gene - 1T N. tabacum , containing 2 kb. before ATG (in bold )

atg

SEQ ID NO: 5

Genomic sequence of the TFL1 gene - 1T N. tabacum

atggcaaggaatgtagagcctctagttgtagggagagtagtaggggatgttcttgattcattcagtcccaaagttaaaatgacagtcacttacaacaataaacaagtttgcaatggccaagagctcttcccttctgcggtcaccattagacctagggttgaggttcaaggtggtgatatgagaactttcttcacattggtaacttttctaaattctccttaaggttattaacttttcatttctatatagacacatcgagggtcaacagagacaactctatctcacacaaggtaggggtaaggtattcgtataccctacccggcccacatgtgagatcacactgaatatgttgttgttgtcgcatttaggggtgtacaaatgaaactgacaaactgcaccaatctgataatccgagtcaaatcgagaaaaaatccgattatggtttggtttgatttggtttggtgatggaaaaaaccccgacatatttggttttgtttggttttaactaaaaaaagtcaaaccgaaaccaaaccaaccagacattatatgtgtagaaattttaaatatatttaatacataaaaatatttatggtagtgtaatttataaatatttcttaagatttttcatagtttatcttttaacgtattatttcaaacttgggcttataatttttggatgctccaataagttttatagtccataaatgttagtaactcaaataaatcctaaaccaaaatcaaatcaatactaatgctaataaaagacattcaattcaattgtactatgaatgaaaatagtgttggatatatatttttatagtttttccacggtttagataaaatgtataacttatttttctttgagtatggttagtcatgtaaataatcttattaatcataattttaaattatgtttatttttattatggcttattaataatatttaattttttgtgcaattttattatctttattgttgaatattttagtacaatgccac gactcatctcatatttatgttattttattgaaaaacacctcatatagttctgcctcattaggattaaaaaaatatttggagcacaaattttactttttgtgttatgaagactttatgaaaaaaaaataaaataaaaacccgaaaacccgaaacctcgagaaaaatcgagattaaaaatccgacttttattggtttggtttggtatttagatttaataacccgatacaattagtttggtttggtaattagaaaatccgaatcaaacccctaaccgtgtacacccctagtcgtattggtatatagacatctttgataaatcattaatcatgcatgatattcttcgatctccttattgtaggtcatgacagaccctgatgttcctggccctagtgatccgtatctgagagaacatcttcactggtatgcactatattcaagaaaagctatggaaaaaaataactagatgagataggtaaaaaagaaaggagtttaatgattgtttgatttgcattaattatattaagtcatgccattatctagtaataaactggttataaggacatacacaaaaggtatggtcaacatataatcaaagaaagaaaagcataagcatccttatgcaagctgccaatgtcatgtaaaatacttcgctaatagacaaatatatattacattagttacctaaaagataggtatataatcagtgggactcctaacttaaaaaataaggttagtaatctgctataacgatacactgagaatcgtcgtcgtcagtttataagttaaaaattaatgtaggtcatcacaacactcacaaagagtgcctcaattgggaagagtatgttatatagttagaatttatgttacatatggaaccacagtactacagaaggataactcttaaacatacttctttaccatcatatttgcaggatagtgactgatattccaggaacaacagatgccacctttggtaagctcactattttggcattttcat ttttccttcaatttcttttagtatatagctaggttggtttttgacatgctaattttgagcaggaaaagagttggttagctatgagatcccacggcctaatattggaatacataggtttgtgtttgttctgtttaagcagaaatgcagacagtcagttagtccacatgatgtttccagagatcacttcaacactcgcaactttgccaacgtaaacgatcttggcccgcctgtcgccgccgtcttcttcaatgcacaacgagagaccgccgccaggagacgctaa

SEQ ID NO: 6

N. tabacum TFL1-1T gene amino acid sequence obtained from SEQ ID NO: 4 or SEQ ID NO : 5.

MARNVEPLVVGRVVGDVLDSFSPKVKMTVTYNNKQVCNGQELFPSAVTIRPRVEVQGGDMRTFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGKELVSYEIPRPNIGIHRFVFVLFKQKCRQSVSPHDVSRDHFNTRNFANVNDLGPPVAAVFFNAQRETAARRR

SEQ ID NO: 7

The genomic sequence of the TFL1 - 2S N. tabacum gene, containing 2 kb. before ATG (in bold )

atg

SEQ ID NO: 8

Genomic sequence of the TFL1 - 2S gene of N. tabacum

atgtctgttacttataacagcagcaagcatgtttataatgggcatgaactctttccttcctcagtcacctctaaacctagggttgaagttcatggaggtgatttgagatctttctttacaatggtacatactgcttccttcgattttcaatacttttattaggggtggagcttagcggcggagccaagattttaactaaggggagtcaaaatataaataagtaagcacacaaaaaaatcaagggggtcaacgtatagtatatacacataaaattaagaatttaacatatttataccgtgtaattttccagcgaaggggtgtcaattgactctccttgccaatgagtggctccgccactggcggagctagagttctagttacggttcgttgtattgtgttaagaagtccacttatactgtcttttctagaatttagaattcataaattcaaaattatggctctgcccttaaatttatttttatacatttctattatatagtaaatcgtttatattgaccccttatttttcttttttaccttaattgacagatcatgatagacccagatgttcctggtcctagtgatccatatctcagggaacacctacactggtaaagaaataagttttttaattactaactcattcaattttatcgtcccttcttttccttgtttacttggagggaaaataatacgatctcatcgaaaagataaaaattcttcaggcttgttatctaaaaacttgttaaaaaataccgtaatgaaaagacatatgagtttgttattaggtatttgactaaatatgatcgatcatatggtgttcggacaagaaatattttgtgaaaaggtccgcatacttttaaaaaaagaaaatctgccttgactcttgagtttgtgcttctcgggaaaacaatttcttccttctttttttttttttttttggtttattgacctttacatattaaagacaccactgagacacatatctagaaaaattgta tttgggaacgcaaaagcaaagaaaacatgtgttattaatcttatgtcaatgccaccagcagctcaggaaaaatatggtcgatatattgtgatttgcttgcaaaaggagcaaagaagaaatcttttgataatgtttgttatgacgatgtacttaaagcaaataagttagaggtcgtttggtacatgggataaggataataattttgggataaagtttaggattaactttatcttatatttggtttggagtattagctaaccgcgaggtatttttcaaactaaaatagtgggattagctatcccatataaaaagtaggatagctaatcccatgggatatcccaccctatcggatagtaatagtccaataggagacaactctaatttgtacagacataatgtccagtcacaccttgtttttttgtcatgacacatattaagcatgaataataatatttcgacaatcttgtagcatttgattagacttagcaaattataaatatgtccaataattggtcacattgttctaataattacttgtttcccttatcattatatatagtgcttcattcactaaacagaacccaaaaaaaaaaaaaaaaaaactgcaaaatggtcatatcatgtagtaacggaataaaaacgtactcagttttatgataaaatcaaagtgacatatttgtacgctttgatagttgacaaatacctgaaaaaagaatttgaccatctttacaggattgtcacagacattccaggcactacagattgctcgtttggtatgtatctttaacccaaatttcaagcttcgaaatagtaacagcttttgtttttaatattttatttgtcttaaatacatattttccttattataaatttcttcgcctagtggtaacgggatcaggtattgattcgtatttattttttattgatcaacaaaaaaagagtacaaaagaaagaattgtttttctacacttagatttatatatatgcaatgtctagaaat taatgagtttacaaattcattgatgtgtatatctcacaatcaaatccaaaatactgatccaaaaattttgatcagggaaagaaatagttggctatgaaatgccaaggccaaatattggaattcacaggtttgtatttctgctgttcaagcagaagaagaggcaaacagtattgactgcacctctctccagggatcgatttaatacgcgtaaattcgcagaagaaaatgagcttgggtctcctgttgcagcagttttcttcaattgccagagggaaactgctgccagaaggcgttga

SEQ ID NO: 9: N. tabacum TFL1-2S gene amino acid sequence derived from SEQ ID NO: 7 or SEQ ID NO : 8

MSVTYNSSKHVYNGHELFPSSVTSKPRVEVHGGDLRSFFTMIMIDPDVPGPSDPYLREHLHWIVTDIPGTTDCSFGKEIVGYEMPRPNIGIHRFVFLLFKQKKRQTVLTAPLSRDRFNTRKFAEENELGSPVAAVFFNCQRETAARRR

SEQ ID NO: 10: Genomic sequence of the N. tabacum TFL1-2T gene containing 2 kb . before ATG (in bold)

atg

SEQ ID NO: 11: Genomic sequence of the N. tabacum TFL1-2T gene

atgtctgttacttataacagcagcaagcatgtctataatggacatgaactctttccttcctcagtcacctctaaacctagggttgaagttcatggaggtgatttgagatctttctttacactggtacatactccttcgattttcactacttttaatttattaggggcgaagctagagttctagctacgggttcgttgtattaattgtgttaagaagtccacttaagctgtcttttttagaatttagaatccataaactcaaaatagtgactttgcttctaaattaatttttatgcatttctcttatatcgtgtatgtgaatattgaccccttattttttcttttttaccttaattgacagatcatgatagacccagatgttcctggtcctagtgatccatatctcagggaacatctacactggtaaagacatacgttttttaattactaactcattcaattttatcgccccttcttttccttgtttacttggagggaaaataatacgatctcgtcaagaagatcaaaaatcttcaggcttgttatttaggaacttgttcaaaaataccgttttgaaaagaacatatgagtttgttattaggtatttgactaaataggaacgatcatatggtgttcggacaagaaaatttttgtgaaaaggtccgcatactttaaaaaaaaaaaaaaaaaaaaaaaatccgccttgactcttgagtttctgcttcttggaaaaaacatttcttctttttttttttgggttttttgacctttatatattaattaaagacaccactgagacacttaattaaaaaattgtatatgggaacgcaaaaagaaaaaaaaacatgtgttattaatcttatgtcaatgccatcagcaactcaggaaaatacggtcgatatactgtgatttgcttgcgaaaggagcaaagaagaaatcttttgataatgtttgttatgacgatgcacttaacctaaaataagttaggggccgtttggtaa atgaaataaggataataatctcggaacaaagtttaggattaactttatcccatatttgatttggagtattagttaattgcgggataactttcaaattaaaatagtaggattagttatctcatatataaagtaaaatacctaatcccaataatataataggagacaactctaatttgcgtagacataatgtccagtctcactttgtatatttgtcatgacgcatattaagcatgaatgataatatttcgacaatcttgtggcatttgattacactcagcaaattataaatatgtccaataattgcattaataattacttgttcctcttatcattatagtgcctcattcactaaaccgaacccaaaagaacactgcaaaatggtcatatcatgtagtaacagaaaaaaaaaacgtactcgattttatgataaaatcaaagtgacatatgtgtcgctttgataattgacaaatacctgaaaaaagaatttgaccatctttacaggattgtcacagacattccaggcactacagattgctcgtttggtatgtatctttaacccaaagttcaagctatgaaatagtaacagcttttctttttaatattttatttgtcttaaatacatattttccttattataaatttattcgcctagtggtaacgggatcaggtattgattcgtatttaatttttattgttcaacaaaaaagagtacaaaaagaaagaattgattttctacacttagatttatatgcaatatctagaaatcagaagatcagcaatgagtttactaattcatcgatgtgtatatcgcacaatcaaatccaattactaataatactgatctaaaaatttcgatcagggagagaaatagttgggtatgaaatgccaaggccaaatattggaatccacaggtttgtatttctgctgttcaagcagaagaagaggcaaacattattgagtgcacctctctccagggatcgatttaatacgcgcaaattct cagaagaaaatgagcttgggtctcctgttgcagcagctttcttcaattgccagagggaaaccgctgccagaaggcgttga

SEQ ID NO: 12: N. tabacum TFL1-2T gene amino acid sequence derived from SEQ ID NO: 10 or SEQ ID NO: 11

MSVTYNSSKHVYNGHELFPSSVTSKPRVEVHGGDLRSFFTLIMIDPDVPGPSDPYLREHLHWIVTDIPGTTDCSFGREIVGYEMPRPNIGIHRFVFLLFKQKKRQTLLSAPLSRDRFNTRKFSEENELGSPVAAAFFNCQRETAARRR

SEQ ID NO: 13: Genomic sequence of the N. tabacum TFL1-3T gene containing 2 kb. before ATG

atg

SEQ ID NO: 14: Genomic sequence of N. tabacum TFL1-3T gene

atggctcaaatgacagatccccttgtgattagtagggtggttggagatgttgttgattatttctctccaagtgttaagatgtgtgttatttataaccccagtaagcatgtctataatgggcatgaactctttccatcccttgttacctctaaacctaaggttgaagttcatggaggtgacatgagatccttctttacactggtaattaattcacactacttcaatagttttcttgttcttatattttattatctatctatatatatataataaaggagcggcaaagccaccatataaatgacaaatgtaaacttttaggacaaaactccaaaaaagttggagttttaaaattattttatatataaaataaataaataaataaataaataaactatcaattcaaattggggagtagtttcttactaatatgatagctatatctatatctatatctatctatatatgtaaaacatttatatgatgccaagtggcataaccactgataagatttttaaatttgaatatgaatgaattttaaatgaagttctaacttcttaaaaataaaccctaatataggttactatttttagtaatgattgaaattattattaaaatattttgttgaaaacaacatagagataaaatttgattattaaatttatgtattacaacaataataattattgaaaatattgctaaaattttcatgaaaggattcacccataattattagtataatagaaaactaaaaaattattaagtctaaagttctagatctctatatttataaacgtataaactgttattttattttctgaaaaaagcaaaaatactgaagagaaaaatgataaaaatattttaaaatatgtaagtcatgtgcaaataataaagtgaacaaatgatgtagtagtatactgaataagatatgtttttttgtcataaaataagtatatgcataactcatctcaataatttgctgactccatctgagtcaaaatat cttctaaattcaagcgaagataattatctatcgcattatttttttatcattaatataaggcaagacgaatctatatctcatatgggactttttaaatagatacatctttataaatgaaccactttatgagttttatcacgaattacaagtaagaataacttgaagattgaaagaatttttggatttatttaattataatatatttttattcattttaaattaatttatattttcaaattatttgtagcaacctatataattatgatatttgagtattatcttataagttatttgatgattgtcgtttgatttaattattgaactattactacaggggacataagatgataattataattttgtagaaacatatgatctaatgtgctcaaataaattactatcatactttgatatgactaatattctttaataatttttgcgcatcgggcgggtactaatactagttcttaaaaaaagggtagcgcgatgcacaaagcattccgcattcacacaggatcctaggaattgggtcgcaccccacagtctaccctaatgcaaacattagcgactactttcacggctcgaactcgtcacttatagatcatacagagacaaatttactgttgctccaagttccctttcttattttattattcttataatttctattcttatattgttataaattatttttttctttttgatagatcatgactgaccctgatgttcctggtcctagcgatccatatcttagggagcacttacattggtatgtatcatactatcatcaactttgaaagcttaaaacactgtaaagttgatgattcacaccaaagattttaatcgtcgtcgtgttacttccatataaatcagtatcgagaagtatgtggccatcactccatcaacgacaccaaaatgaaataaagagtccctatatcaatacaatataaattaatcttaaacatgaagttgactttaaattggataaattgtttccact actaagcttagcgtataaattagtcctttgactttcaattttgtataataatgcgaagctttttttcttgtaaatgcaatttttgtccttgagggtttgcaacttcttttttaaggaaaaaaaaaagactaaagttgtgtgacactaaaaccaagagttagcttaatacttcatggacacacgttagcataaaacatataaccgatattcaaaattacaaaaatgatagaatcataatttttgtttctatttaaaaaggaagtaagccaaattactactaacatagtggacttaaagggtattaattttttgttattttaatgatatctgttcatgacttcttgactacttctactcctttatatcaatcaaattataatttacttcgtttgactatctaatttacagggtaattacagacattccaggcactacagattcctcgtttggtatggaataatattgtattccttttttacttttctgcctagcatttctaaatagagtagtccgatacacgaaatatttcactttacgcaggatccagaaataaaggaccataccccaattgggtgtaatataagtagtcatgggcgcatgcagtattttagtgacgggtttaattgcactcataattttggacgcttagcataaagtagtagatatgtatccataacttcaaaaatataataggttcaatgttaaaaatttcaaaagagatgaactcatagagtttaaatcatgatccgcctctgtaggcagtctaccctaatgaaagaatcagtggctgatttcacagttcaaaaccgtaacctatgaatcacataaagccaactttaccatcgctccaagactcgccttcttctgcctaacattactactgctaataaagagaattttaataaaactactaatgctaattattattctttgctaaaatcttcatcaggaaaagaagtggtgggctatgaaatgccaatgcctaacattggaatccata ggtttgtgtttctgctcttcaagcagaagaagaggcaaacagtgagcgcaccattatccagggaccgattcaatacgcggaaatacgcagaagaaaatgagcttggctctccagttgctgctgttttcttcaactgccaaagggaaaccgcggccagaaagcgttga

SEQ ID NO: 15: N. tabacum TFL1-3T gene amino acid sequence derived from SEQ ID NO: 13 or SEQ ID NO: 14

MAQMTDPLVISRVVGDVVDYFSPSVKMCVIYNPSKHVYNGHELFPSLVTSKPKVEVHGGDMRSFFTLIMTDPDVPGPSDPYLREHLHWVITDIPGTTDSSFGKEVVGYEMPMPNIGIHRFVFLLFKQKKRQTVSAPLSRDRFNTRKYAEENELGSPVAAVFFNCQRETAARKR

SEQ ID NO: 16: Genomic sequence of the N. tabacum TFL1-4S gene containing 2 kb. before ATG (in bold )

atg

SEQ ID NO: 17: Genomic sequence of N. tabacum TFL1-4S gene

atggcaagaagtttagagcctctaattgttgggagagtagtaggagatgttcttgattcatttagtcctataatgaaaatgacaatatcatataacaacaaattagtgtgcaatggccatgaactccttccttctgttgtcactgctagacctaaagttgaagttcaagggggagatttgagaactttcttcacattggtatttttttcttgatttctacttaatttccaagatcatcaagttcccattatttctttaaaaaaaaaaaaagcagttcggtgcactaaactcccgctatgcgcggggttcggtgaagcaccgaaccataagggtctattgtacgcaaccttaccctgcatttatgcaagaggcttgctcaccattacaagttatattaatttaacatgttatatataaccacaaaggctgtcgtgggatggtaaatatccttctatccttaatcagaagtttcgggttcaagttatagccctaggaatatagtcgtctttggtagggatcctttacccccaaaactttccgccgtgaatccagattagtaaacctcaaagcgggtatcgggcattggatgacaaaccaaaaaaacttcaacgtgttatagcatgttataacttattacagttaatttagttttccagtcgatactatattaaatagagtgcctgtaatttactttggagtgatttgattgttattttttcgcatcgtcagtacataaaacttatattaattttcgaatatgtaggtcatgacagaccctgatgttcctggccctagtgatccttatctaagagagcatctccactggtatgccctaaactcaatttttttttaaaaaaaaaaaatagaaaatgagaaaaaatatgtaaaaatctacaaatatgagaagatcatgattaattggaactatttttactgactatttgacaggatagtaactgacattccaggtaccactgatgctacttttggtaagttctct gtatcttctgcaaaattacaagcacatgtgaagataaaagaagtttttctattattcacttattttgtctagctagttatatagaataattataagatcaacaattttgtatagtagtgaatgttggacttctaaagtcgaacatgtccacttgatgagtgtcacaaaaatgtagaaactaaacaatcgtttggacataaaaaaaaaagtaagtttttttgagttaaattgaaaaagaaaatatttagaatttgaaattgtggatatacatttaaattgaaaagcattgcagttttgtaaggaaaataaactttcatatacataaaaaagtgattttttggaaactcatcttcaagaatatttttaaaaatttccgtccaatgtataaccaaacattattttgaaaaagattaaaaaaaggaaaaactttaggaacaacgggtcccaagataaatgtgtctagtcatataagattagataaattaggattttattatatttggtagaaggtgcaagaagcatatgtaaataataaattgagaagtcacttaagatattttgatcatgtcccacatcgataacaagaggtaccattctatatatgttaaatcatggtaagttaaagtattatatcacatattaaatggtgatataatagacctaaatcacatgaaacgaaattgtcccgaaaggtctataaatttttgaaattcatgtagacgaagctaaaagtaggatacaataaaaaaaaaattaaagatctatattggcgatactatttagttgggattgcattttagttattctagtacatttactttaatctaatttttgctagctaggagtcttttaatcttattagaaatttacataccaaaaaatttagagaacttgctaggacaattggtatttctttatataatattgtggaagttgtattagagtatgttgtttacattacactctttgagtgcgttccttctccgaactagctaatgca tgaacacgagatgccttctgcaccgtgctaccctattaatatataaaaaaatggtagcccggtgcattaagctcccgctatgcgcgggttccgaaaaaggatcagaccacaagggtctatgtttgcaaccttacttgtatttctgcaagaaactgtttccacggctcgaacccatgatcttctggtcacatgacaataactttaccggttacaccaaggttccccttcacgcgctgcccttttaatattgtctattaatatttcctactagagttatacacccctttgttattactcactcttagggtgattattaacatataatatgtttaatatttatactaaaaacaggacgagaattggttagctatgagattccaatgccaaatattggaatccataggtttgtatttgtacttttcaagcaaaaacgaagacaatcagttagctctcctacttcaagggatcacttcaacactagaaattttgctgaagaaaatgatcttggccaacctgttgctgctgttttcttcaatgcacagcgagaaaccgccgcacgaagacgctaa

SEQ ID NO: 18: N. tabacum TFL1-4S gene amino acid sequence derived from SEQ ID NO: 16 or SEQ ID NO: 17 .

MARSLEPLIVGRVVGDVLDSFSPIMKMTISYNNKLVCNGHELLPSVVTARPKVEVQGGDLRTFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGRELVSYEIPMPNIGIHRFVFVLFKQKRRQSVSSPTSRDHFNTRNFAEENDLGQPVAAVFFNAQRETAARRR

SEQ ID NO: 19: Genomic sequence of the N. tabacum TFL1-4T gene containing 2 kb. before ATG (in bold )

atg

SEQ ID NO: 20: Genomic sequence of N. tabacum TFL1-4T gene

atggcaagaagtttggagcctctaatagttgggagagtagtaggagatgttcttgattcatttagtcctatagtgaaaatgacaattacttataacaacaaattagtgtgcaatggtcatgaattctttccttctattgtcacttctagacctaaggttgaagttcaaggaggagatttgagaactttcttcacactggtaatttttcttgattttttccttaattccaagatcatcaagttccatttatttctttacaagttatattaatttaaccctttataatcaccaaaggctggcgtgggttgctaagtaaccttccatcgttaatcagatgtttcgggttcgagctagccctgggattacaatcgttttttgtaggaagcgctttaacccccaaaatttttcagcacgaacccggattagtaaacctcaaaactcgtgccaaatactagatgacaaaccaaaagagttttcaacctgttataacatatgttacttgttacaattattagttttccggtcaatagtatattatgtaattttctttgaagtgacttgattgttattttttcacattatcagtgcataaaacttatactattattttttaatatgtaggtcatgacagaccctgatgttcccggccctagtgatccttatctacgagagcatctccactggtacactctctataatagtttcatttgttccgaattttcttggctgttatataaaaaatatattataacatagcatgaaaattggttccacaaaaacttaatttttatagtgaataattgttatatattgatattgttatagagaggtctgtctatatgccctaaactcaatgaaaaaaaatagaaaatgagaaaaaatatgtaaaatctacaaatatgagaagatcatttttagttgaaactatccttatatactactgaatatttagctggcaaataaaattgacagtgttttactgattgtttgacaggatagt aactgacattccaggtaccactgatgctacttttggtaagttttattagtttcttctgcaagattacaagcacatgtgaagaagatacaagatgtttttccattactcacttattttgtcttgctaataattatatagaacaattgtaagatcaacagtgttatataatagtgaatgttggacttctaaagtcgaacatgtccacatgatgagcgtcacaaaaatgcagatacgagctcgtttggattgacttaaaaaatgtggtttttcagcaaaaataacttttaagccaaaaaacaataagttagggttgtccacctttttgcttttggcttaatttaagcattttaaaatttattttaagcaatttttgacttagccaaacaccgaaaaaagctaaaagaaacttaaaagctgatttgactagcttaaaagtaaatccaaacaccctctaactaagcatttggacataaaaaaaatatgtcatttttgaaaaaagtagttcttttgagttaagtcaaaaaagaatatataaaatttgaaattgtatttagacatgcatttcacttgaaaattattagagttttatgagaaaaatgaacttttagatgaaaaagtggtttttggaaactcatcttcaagaatttttccaaaacttcagtccaatcgtataaccaaacattattttgataaaaacatcgaaaataaaaataaatctatggagaaacgggtcccaagataaatgtgtctagtcatataagattattcaaaattaagaatttatcacatttgtaaaagatgtaagtagcatatgtaaatgataaaatgagaagtcacttgagatgttttgatcatgtcctacgtcgatcttcagaggtaccattccgtatacgtgattggtaagtaaaggtattaaaaagagacataatggacctaaattacgtgaaacgaaattgtcttgaaaagtctttcaaatttttgaaatccatgtagacgaatc gaaaagtagggcacaatgaaatatgatcaaaggtttataatggtgatacaagttagttgggattacgttttagttatgccagtatatttactttaatctaatattttcttggagttttttaatcttattagaaatttacttaccaaaaatttagagaacttgctagaacaatataattgataattcttcatatatattgtcttcgagctgtagaaacagccactaatgtttgcattaggatatgttgtctacatcacacttattgtgtgttgccctcaccggaccctgcatgaacgtatgatgccttatgcaccgcgccccttttaatattatttattaattaatatttcctgctagagttatactcctttgttattactcattcttaggttgatgattaacttataatatgcttaatctttatactaaaaataggaagagaattggttagctatgagattccaaggccaaatattggaatccataggtttgtatttgtacttttcaagcaaagacgaagacaatcagttagccctcctacttcaagggaaaacttcaacactagaaattttgccgaagaaaatgatcttagccaacctgttgctgctgttttcttcaatgcacagcgagaaaccgccgcgcgaagacgctaa

SEQ ID NO: 21 : N. tabacum TFL1-4T gene amino acid sequence derived from SEQ ID NO: 19 or SEQ ID NO: 20.

MARSLEPLIVGRVVGDVLDSFSPIVKMTITYNNKLVCNGHEFFPSIVTSRPKVEVQGGDLRTFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGRELVSYEIPRPNIGIHRFVFVLFKQRRRQSVSPPTSRENFNTRNFAEENDLSQPVAAVFFNAQRETAARRR

SEQ ID NO: 22: DNA sequence of target sense RNAi TFL1-1S/T N. tabacum sequence

agttaaaatgacagtcacttacaacaataaacaagtttgcaatggccaagagctcttc

SEQ ID NO: 23: DNA sequence of target antisense RNAi TFL1 - 1S/T N. tabacum

Gaagagctcttggccattgcaaacttgtttattgttgtaagtgactgtcattttaact

SEQ ID NO: 24: DNA sequence of the N. tabacum RNAi TFL1-1S/T construct

Ggtaccacaagtttgtacaaaaaagcaggct aagctt gtcga ccatgg agttaaaatgacagtcacttacaacaataaacaagtttgcaatggccaagagctcttctggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgttagtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaactaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagcatattttttacttttatttaagtatactatattccaacaggtaagttaacgaagagctcttggccattgcaaacttgtttattgttgtaagtgactgtcattttaact ggcgcgcccgggcaattg acccagctttcttgtacaaagtggtgagctc

SEQ ID NO: 25: DNA sequence of target N. tabacum TFL1-1S sense RNAi sequence

tgcagtcactattagacctagggttgaagttcaaggtggtgatatgagaactttcttcacattggtcatcacagatcctgatgtacct

SEQ ID NO: 26: DNA sequence of N. tabacum antisense RNAi TFL1-1S target DNA sequence

aggtacatcaggatctgtgatgaccaatgtgaagaaagttctcatatcaccaccttgaacttcaaccctaggtctaatagtgactgca

SEQ ID NO: 27: DNA sequence of target N. tabacum sense RNAi TFL1-1T sequence

tgcggtcaccattagacctagggttgaggttcaaggtggtgatatgagaactttcttcacattggtcatgacagaccctgatgttcct

SEQ ID NO: 28: DNA sequence of N. tabacum antisense RNAi TFL1-1T target DNA sequence

aggaacatcagggtctgtcatgaccaatgtgaagaaagttctcatatcaccaccttgaacctcaaccctaggtctaatggtgaccgca

SEQ ID NO: 29: DNA sequence of target sense RNAi TFL1-2S/T N. tabacum sequence

catgaactctttccttcctcagtcacctctaaacctagggttgaagttcatggaggtgatttgagatctttctttaca

SEQ ID NO: 30: DNA sequence of N. tabacum antisense RNAi TFL1-2S/T target DNA sequence

tgtaaagaaagatctcaaatcacctccatgaacttcaaccctaggtttagaggtgactgaggaaggaaagagttcatg

SEQ ID NO: 31: DNA sequence of the N. tabacum RNAi TFL1-2S/T construct

ggtaccacaagtttgtacaaaaaagcaggctaagcttgtcgaccatggcatgaactctttccttcctcagtcacctctaaacctagggttgaagttcatggaggtgatttgagatctttctttacatggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgttagtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaactaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagcatattttttacttttatttaagtatactatattccaacaggtaagttaactgtaaagaaagatctcaaatcacctccatgaacttcaaccctaggtttagaggtgactgaggaaggaaagagttcatgggcgcgcccgggcaattgacccagctttcttgtacaaagtggtgagctc

SEQ ID NO: 32: DNA sequence of target N. tabacum sense RNAi TFL1-2S sequence

gaaagaaatagttggctatgaaatgccaaggccaaatattggaattcacaggtttgtatttctgctgttcaagcagaagaagaggcaaacagtattgactgcacctctctccagggatcga

SEQ ID NO: 33: DNA sequence of N. tabacum antisense RNAi TFL1-2S target DNA sequence

tcgatccctggagagaggtgcagtcaatactgtttgcctcttcttctgcttgaacagcagaaatacaaacctgtgaattccaatatttggccttggcatttcatagccaactatttctttc

SEQ ID NO: 34: DNA sequence of target N. tabacum TFL1-2T sense RNAi sequence

gagagaaatagttgggtatgaaatgccaaggccaaatattggaatccacagcagctttcttcaattgccagagggaaaccgctgccagaaggcgttgaagaagatgttta

SEQ ID NO: 35: DNA sequence of N. tabacum antisense RNAi TFL1-2T target DNA sequence

taaacatcttcttcaacgccttctggcagcggtttccctctggcaattgaagaaagctgctgtggattccaatatttggccttggcatttcatacccaactatttctctc

SEQ ID NO: 36: DNA sequence of N. tabacum target sense RNAi TFL1-3T sequence

atggctcaaatgacagatccccttgtgattagtagggtggttggagatgttgttgattatttctctccaagtgttaagatgtgtgttatttataaccccagtaagcatgtctataatgggcatgaactctttccatcc

SEQ ID NO: 37: DNA sequence of N. tabacum antisense RNAi TFL1-3T target DNA sequence

ggatggaaagagttcatgcccattatagacatgcttactggggttataaataacacacatcttaacacttggagagaaataatcaacaacatctccaaccaccctactaatcacaaggggatctgtcatttgagccat

SEQ ID NO: 38: DNA sequence of the N. tabacum RNAi TFL1-3T construct

ggtaccacaagtttgtacaaaaaagcaggctaagcttgtcgaccatggatggctcaaatgacagatccccttgtgattagtagggtggttggagatgttgttgattatttctctccaagtgttaagatgtgtgttatttataaccccagtaagcatgtctataatgggcatgaactctttccatcctggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgttagtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaactaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagcatattttttacttttatttaagtatactatattccaacaggtaagttaacggatggaaagagttcatgcccattatagacatgcttactggggttataaataacacacatcttaacacttggagagaaataatcaacaacatctccaaccaccctactaatcacaaggggatctgtcatttgagccatggcgcgcccgggcaattgacccagctttcttgtacaaagtggtgagctc

SEQ ID NO: 39: DNA sequence of target N. tabacum TFL1-4T sense RNAi sequence

tagtcctatagtgaaaatgacaattacttataacaacaaattagtgtgcaatggtcatgaattctttccttctattgtcacttctagacctaa

SEQ ID NO: 40: DNA sequence of N. tabacum antisense RNAi TFL1-4T target DNA sequence

ttaggtctagaagtgacaatagaaggaaagaattcatgaccattgcacactaatttgttgttataagtaattgtcattttcactataggacta

SEQ ID NO: 41: DNA sequence of the N. tabacum RNAi TFL1-4T construct

Ggtaccacaagtttgtacaaaaaagcaggctaagcttgtcgaccatggtagtcctatagtgaaaatgacaattacttataacaacaaattagtgtgcaatggtcatgaattctttccttctattgtcacttctagacctaatggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgttagtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaactaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagcatattttttacttttatttaagtatactatattccaacaggtaagttaacttaggtctagaagtgacaatagaaggaaagaattcatgaccattgcacactaatttgttgttataagtaattgtcattttcactataggactaggcgcgcccgggcaattgacccagctttcttgtacaaagtggtgagctc

--->

SEQUENCE LIST

<110> Philip Morris Products S.A.

<120> Plants with reduced time to bloom

<130> P10400EP

<140> EP16205377.1

<141> 2017-01-05

<160> 41

<170> PatentIn version 3.5

<210> 1

<211> 3539

<212> DNA

<213> Nicotiana tabacum

<400> 1

tgtttattgt acattttgaa ctgcatcgag ctgcacgatt gacagatcta gtctgcaaca 60

gagtactttg gtttctaatt atagtaatcc taacacctca gaaatttcca aaattggaat 120

tatgggtgca aatcatcttt atatatactc tcaaatgact tctcagatct tacggtcact 180

aattgtctgt aaattaagta tggtggagtt cactttatat actctgagcg gtgtgcccat 240

cctcctccat cagaatatca cttaaaacaa ttaacgtact ttataatctt tccctattgt 300

360

taggattacc ctaaatgaaa aaagcaatag attccctggc aatatgttta cttatatttt 420

gaatttttgc aaaaaaaata aataaatgtg tgtcaacttg cttagttgggg accacatgca 480

aaaactaatt caaggatttc atctgataat tttatagtgg aggaaaag gcttggatta 540

600

660

gtatatatat atatatatat atatatatat atatataaga aatataaaaa aaattggcta 720

ttatttttca gagcagctat acaatatcat tttccatatc aggtaagctg aacaacaaat 780

catggaccaa ttcgaagagc accagtcagg tgtgaaagag aactgacatg atagcataaa 840

atacatatca ctaactcctc cacatctaag agcatacatt taatttccta tatggaagat 900

aagattaatt agaaaagtgt ttgataaagc taagatatgt tagtaccagt ttgtagtatc 960

1020

1080

agtcatatat agtacatatg acccctccta aaggaaaaga aagtagggaa agtaccaagc 1140

tagggcatat ttaaaggaaa ataaaatggc aattttaaga tgttagagta agggcagggg 1200

taggccaaag aagtattgggg gagaagtgat tagacaagac atgccactgc ttcacctaac 1260

cgcggacata actagcgata ggaagatgtg gaggtcgaga attaaggttg tgagttgaca 1320

1380

tttattactt tgttatgtca ctcgcttcca ttactagtta tctgttgtta ttgcttgttg 1440

ctttattttt accatttttt tagccgaggg tctatcaaaa acagtctctc tgcctttata 1500

ggataggggt aaggctgcgt acacactacc ctttccagac cccacgtatg ggattaatcc 1560

1620

aagtacatgt attaagattt cattattttc ggggaagaat ttcgaatgta tttaaaccat 1680

aaaacgtcat tctcctgcaa gtatttttgg tgatcaagat aagtatagtt aaaaattgag 1740

1800

ttcaggggta gggttacttt tttttttaaaa aatattctat ttgaagttcc ataaaatccc 1860

attttttatt tgcaactata ttgagtagat gttccgagac agcaactata agtgaggctc 1920

tatttcactg ttcaaccaaa atctctcaca agctaagttc cttgcaatcc aaaagatctc 1980

tctctctctc tctctctcta atggcaagga atgtagagcc tctagtagta gggagtag 2040

taggggatgt tcttgattca tttagtccca cagttaaaat gacagtcact tacaacaata 2100

aacaagtttg caatggccaa gagctcttcc cttctgcagt cactattaga cctagggttg 2160

aagttcaagg tggtgatatg agaactttct tcacattggt aacttttcta attttccttc 2220

2280

cttgagctga gggttcggag acagcctctc tatcccacac aagataaggt taacgtctgc 2340

atataccttg ccgtcccctg acaccacata tgggattata ctgcgtatgt tgttgttgtt 2400

gtattgctat atagacatct ttgataaatc actaatcatg catgatatta ttcttcttat 2460

tgtaggtcat cacagatcct gatgtacctg gccctagtga tccgtatctg agagaacatg 2520

ttcactggta tgtactacat tcaagaaaat ctatggaaaa aaaataacta gaagagatga 2580

2640

gtcatgcaat tacctagtaa taaattcgtt ataaggacat acacaaaagg tatgctcaac 2700

ataattaaag aaagaaaagc ataagcatta tattcttata tgcaagctcc caatctgatt 2760

2820

2880

atttttcgac gaatgaccac tcttattgcc catgctgcca atgccaatat gtaaaatact 2940

tcgccaatag acaaatatgt atcacattaa ttagttacct aaataggtaa ttatccataa 3000

taagtgggac tcgtaacttg aaaaatatgg ttagtaattt gctacaacat gttaagatac 3060

3120

ctggttaaca atgtaggtca tcataacact cacaattggg aataataact cttaatcata 3180

cttctttaca ttcatatttg caggatagtg actgatattc caggaactac agatgccacc 3240

tttggtaaat tggctatttt ggtttctttt attaagctgg tgtttgacat gctagaatta 3300

3360

ttggaataca taggtttgtg tttgttctct ttaagcagaa atgcagacaa tcagtcagcc 3420

cacctacttc aagggatcat ttcaacactc gcaactttgc caacgtaaat gaccttggtc 3480

cgcctgtcgc cgccgtcttc ttcaatgcac aacgagagac cgccgccagg aggcgctaa 3539

<210> 2

<211> 1539

<212> DNA

<213> Nicotiana tabacum

<400> 2

atggcaagga atgtagagcc tctagtagta gggagagtag taggggatgt tcttgattca 60

120

gagctcttcc cttctgcagt cactattaga cctagggttg aagttcaagg tggtgatatg 180

agaactttct tcacattggt aacttttcta attttccttc aggttattaa cttttcattg 240

ctatatagac atctttcaaa cctgtctaga agattctttt cttgagctga gggttcggag 300

acagcctctc tatcccacac aagataaggt taacgtctgc atataccttg ccgtcccctg 360

acaccacata tgggattata ctgcgtatgt tgttgttgtt gtattgctat atagacatct 420

ttgataaatc actaatcatg catgatatta ttcttcttat tgtaggtcat cacagatcct 480

gatgtacctg gccctagtga tccgtatctg agagaacatg ttcactggta tgtactacat 540

tcaagaaaat ctatggaaaa aaaataacta gaagagatga gtccaaaaga aaggaatta 600

attattgttt gatttgtatt ttttatttgc attagattaa gtcatgcaat tacctagtaa 660

taaattcgtt ataaggacat acacaaaagg tatgctcaac ataattaaag aaagaaaagc 720

ataagcatta tattcttata tgcaagctcc caatctgatt gtggcgaagc taagaatttc 780

ttcgggtgtt taaatttaaa agaagtgaaa aaaaatccga taaaaaattt atgtgttata 840

tacctctaaa acttaatatt gtacctatat acatattgta atttttcgac gaatgaccac 900

tcttattgcc catgctgcca atgccaatat gtaaaatact tcgccaatag acaaatatgt 960

atcacattaa ttagttacct aaataggtaa ttatccataa taagtgggac tcgtaacttg 1020

1080

tatgtccttt cacgccgtcg taagtttata taagttaaat ctggttaaca atgtaggtca 1140

tcataacact cacaattggg aataataact cttaatcata cttctttaca ttcatatttg 1200

1260

ggtttctttt attaagctgg tgtttgacat gctagaatta atgtacttta attttgagca 1320

1380

tttgttctct ttaagcagaa atgcagacaa tcagtcagcc cacctacttc aagggatcat 1440

ttcaacactc gcaactttgc caacgtaaat gaccttggtc cgcctgtcgc cgccgtcttc 1500

ttcaatgcac aacgagagac cgccgccagg aggcgctaa 1539

<210> 3

<211> 172

<212> PROTEIN

<213> Nicotiana tabacum

<400> 3

Met Ala Arg Asn Val Glu Pro Leu Val Val Gly Arg Val Val Gly Asp

1.5 10 15

Val Leu Asp Ser Phe Ser Pro Thr Val Lys Met Thr Val Thr Tyr Asn

20 25 30

Asn Lys Gln Val Cys Asn Gly Gln Glu Leu Phe Pro Ser Ala Val Thr

35 40 45

Ile Arg Pro Arg Val Glu Val Gln Gly Gly Asp Met Arg Thr Phe Phe

50 55 60

Thr Leu Val Ile Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro Tyr

65 70 75 80

Leu Arg Glu His Val His Trp Ile Val Thr Asp Ile Pro Gly Thr Thr

85 90 95

Asp Ala Thr Phe Gly Lys Glu Leu Val Ser Tyr Glu Ile Pro Arg Pro

100 105 110

Asn Ile Gly Ile His Arg Phe Val Phe Val Leu Phe Lys Gln Lys Cys

115 120 125

Arg Gln Ser Val Ser Pro Pro Thr Ser Arg Asp His Phe Asn Thr Arg

130 135 140

Asn Phe Ala Asn Val Asn Asp Leu Gly Pro Pro Val Ala Ala Val Phe

145 150 155 160

Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg

165 170

<210> 4

<211> 4283

<212> DNA

<213> Nicotiana tabacum

<400> 4

ttcgagtcgt gataagtgtt ttgcaaaaat ataagataag actgcatgcg tacaatagac 60

tcttttggtc cggccctttt ctggaccctg cgcataacgg aagcttagtg cacccggcaa 120

ccgtttttca acgttccaga tgcaagaata ttataaggag gctttttgac acttttaaat 180

ttaatctaat agagtttaag tttcatgcat cgactgtcaa aaaaatattt atataatcac 240

taatacgata attacaagtg aaatgctata ctaaacatta agtagtaacc taataaaacg 300

gtagctagct aatctactat atatcacgta gtattaaaat tacacttatg taaaaatctt 360

tacggtgtta agaataactt aaaagcaatt tagtaataca ttaatgagtg aaaaaggtga 420

agaggaaacg tggtgtgttt tgaactgcat cgagctgcac gattgacaga gctagtctgg 480

aacagagtac tttggtttct aattatagta atcctaacat ctcagaaatt tccataatag 540

gaattatggg tgcaaatcat ctttatatat actctcaaat taatgacttc tcagcaagta 600

ctgtcattgt ctgtaaatta agtatggtgg agttcacttt atactctgag cagtgtgccc 660

atcctcctcc atcacttaaa acaattaatg tactttataa tctttcccta ttgtttacac 720

acacaattta ggaaagtttt taacaaagac aagcccatgt ttcaagattt gtttcttagg 780

attaccctaa atgaaaaaag caatagattc cctggcaatt tacttatatt ttgaattttt 840

gcaaaaacaa aaagaaaaag tgacaacttg cttagttggg accacatgca aaaactaatt 900

taaggaattc atctgatatt tttatagtgg agaggaaaag ggctggatta atttaaatat 960

tccttatttg ccaaaattat ttatattcga tagctgtaaa aatatataaa atttgtatat 1020

ttgtttttgt atagtataca cggaaatgta tatatataca agaaattaaa aaaaaactat 1080

tattttcaga gcagttatac aatattattt tccctatcat gtaagcttag cttatcaaca 1140

aatcatggac caattctaag agctccattc aggtgtgaaa gagagctgac atgatagtat 1200

aaaatacaca tcactaactc ctccacattt gagagctata gattaatttc ctatatggaa 1260

1320

atcaagctaa aattttagtg tcaatttaag gataatttta ctatgattag aagacaagtt 1380

atcattcaca aatttctgaa aggaacttat aagacttttt atttttattt tttattgtac 1440

ttacatttgt caattaagta catgtgaccc ctcctaaagg aaaagaaagt agggaaatta 1500

aagtaccaag ttagcgcata cttaaagcca aggtaagcaa aatggcaatt ttaaggtgtt 1560

1620

1680

ataagtatag ttaacgttga acaatcttct ttgcttacta tgtcttgtat aaaactaaca 1740

aagaaaaatt cagtttattt ttcaggggta gggttacttt tttaaaaaaa aatattctat 1800

ttgaagttcc ataagatccc atttttaatt tgtaactata ttgagtagat attccaagac 1860

agcaactata aatgaggatc tatttcaccg ttcaatcaaa atctctcaca agctaagttc 1920

ctagcaatcc aaaaaagatc tctctctctc tctctctctc tctctctctc tctctctctc 1980

tctctctctc tctctctcta atggcaagga atgtagagcc tctagttgta gggagtag 2040

taggggatgt tcttgattca ttcagtccca aagttaaaat gacagtcact tacaacaata 2100

aacaagtttg caatggccaa gagctcttcc cttctgcggt caccattaga cctagggttg 2160

aggttcaagg tggtgatatg agaactttct tcacattggt aacttttcta aattctcctt 2220

aaggttatta acttttcatt tctatataga cacatcgagg gtcaacagag acaactctat 2280

ctcacacaag gtaggggtaa ggtattcgta taccctaccc ggcccacatg tgagatcaca 2340

ctgaatatgt tgttgttgtc gcatttaggg gtgtacaaat gaaactgaca aactgcacca 2400

Atctgataat ccgagtcaaa tcgagaaaaa

ggtgatggaa aaaaccccga catatttggt tttgtttggt tttaactaaa aaaagtcaaa 2520

accaaccaga cattatatgt gtagaaattt taaatatatt taatacataa 2580

2640

aacgtattat ttcaaacttg ggcttataat ttttggatgc tccaataagt tttatagtcc 2700

ataaatgtta gtaactcaaa taaatcctaa accaaaatca aatcaatact aatgctaata 2760

aaagacattc aattcaattg tactatgaat gaaaatagtg ttggatatat atttttatag 2820

tttttccacg gtttagataa aatgtataac ttatttttct ttgagtatgg ttagtcatgt 2880

aaataatctt attaatcata attttaaatt atgtttattt ttattatggc ttattaataa 2940

tatttaattt tttgtgcaat ttttattatct ttattgttga atattttagt acaatgccac 3000

gactcatctc atatttatgt tattttattg aaaaacacct catatagttc tgcctcatta 3060

ggattaaaaa aatatttgga gcacaaattt tactttttgt gttatgaaga ctttatgaaa 3120

3180

3240

ggtaattaga aaatccgaat caaaccccta accgtgtaca cccctagtcg tattggtata 3300

tagacatctt tgataaatca ttaatcatgc atgatattct tcgatctcct tattgtaggt 3360

3420

gtatgcacta tattcaagaa aagctatgga aaaaaataac tagatgagat aggtaaaaaa 3480

3540

3600

aaagcataag catccttatg caagctgcca atgtcatgta aaatacttcg ctaatagaca 3660

3720

3780

agttaaaaat taatgtaggt catcacaaca ctcacaaaga gtgcctcaat tgggaagagt 3840

atgttatata gttagaattt atgttacata tggaaccaca gtactacaga aggataactc 3900

3960

gatgccacct ttggtaagct cactattttg gcattttcat ttttccttca atttctttta 4020

gtatatagct aggttggttt ttgacatgct aattttgagc aggaaaagag ttggttagct 4080

atgagatccc acggcctaat attggaatac ataggtttgt gtttgttctg tttaagcaga 4140

aatgcagaca gtcagttagt ccacatgatg tttccagaga tcacttcaac actcgcaact 4200

ttgccaacgt aaacgatctt ggcccgcctg tcgccgccgt cttcttcaat gcacaacgag 4260

agaccgccgc caggagacgc taa 4283

<210> 5

<211> 2283

<212> DNA

<213> Nicotiana tabacum

<400> 5

atggcaagga atgtagagcc tctagttgta gggagagtag taggggatgt tcttgattca 60

ttcagtccca aagttaaaat gacagtcact tacaacaata aacaagtttg caatggccaa 120

gagctcttcc cttctgcggt caccattaga cctagggttg aggttcaagg tggtgatatg 180

agaactttct tcacattggt aacttttcta aattctcctt aaggttatta acttttcatt 240

tctatataga cacatcgagg gtcaacagag acaactctat ctcacacaag gtaggggtaa 300

ggtattcgta taccctaccc ggcccacatg tgagatcaca ctgaatatgt tgttgttgtc 360

gcatttaggg gtgtacaaat gaaactgaca aactgcacca atctgataat ccgagtcaaa 420

tcgagaaaaa atccgattat ggtttggttt gatttggttt ggtgatggaa aaaaccccga 480

catatttggt tttgtttggt tttaactaaa aaaagtcaaa ccgaaaccaa accaaccaga 540

600

tttataaata tttcttaaga tttttcatag tttatctttt aacgtattat ttcaaacttg 660

ggcttataat ttttggatgc tccaataagt tttatagtcc ataaatgtta gtaactcaaa 720

taaatcctaa accaaaatca aatcaatact aatgctaata aaagacattc aattcaattg 780

840

aatgtataac ttatttttct ttgagtatgg ttagtcatgt aaataatctt attaatcata 900

attttaaatt atgtttattt ttattatggc ttattaataa tatttaattt tttgtgcaat 960

tttattatct ttattgttga atattttagt acaatgccac gactcatctc atatttatgt 1020

tattttattg aaaaacacct catatagttc tgcctcatta ggattaaaaa aatatttgga 1080

gcacaaattt tactttttgt gttatgaaga ctttatgaaa aaaaaataaa ataaaaaccc 1140

gaaaacccga aacctcgaga aaaatcgaga ttaaaaatcc gacttttatt ggtttggttt 1200

ggtatttaga tttaataacc cgatacaatt agtttggttt ggtaattaga aaatccgaat 1260

caaaccccta accgtgtaca cccctagtcg tattggtata tagacatctt tgataaatca 1320

ttaatcatgc atgatattct tcgatctcct tattgtaggt catgacagac cctgatgttc 1380

1440

1500

ttgatttgca ttaattatat taagtcatgc cattatctag taataaactg gttataagga 1560

catacacaaa aggtatggtc aacatataat caaagaaaga aaagcataag catccttatg 1620

caagctgcca atgtcatgta aaatacttcg ctaatagaca aatatatatt acattagtta 1680

cctaaaagat aggtatataa tcagtgggac tcctaactta aaaaataagg ttagtaatct 1740

gctataacga tacactgaga atcgtcgtcg tcagtttata agttaaaaat taatgtaggt 1800

catcacaaca ctcacaaaga gtgcctcaat tgggaagagt atgttatata gttagaattt 1860

atgttacata tggaaccaca gtactacaga aggataactc ttaaacatac ttctttacca 1920

tcatatttgc aggatagtga ctgatattcc aggaacaaca gatgccacct ttggtaagct 1980

cactattttg gcattttcat ttttccttca atttctttta gtatatagct aggttggttt 2040

ttgacatgct aattttgagc aggaaaagag ttggttagct atgagatccc acggcctaat 2100

2160

ccacatgatg tttccagaga tcacttcaac actcgcaact ttgccaacgt aaacgatctt 2220

ggcccgcctg tcgccgccgt cttcttcaat gcacaacgag agaccgccgc caggagacgc 2280

taa 2283

<210> 6

<211> 173

<212> PROTEIN

<213> Nicotiana tabacum

<400> 6

Met Ala Arg Asn Val Glu Pro Leu Val Val Gly Arg Val Val Gly Asp

1.5 10 15

Val Leu Asp Ser Phe Ser Pro Lys Val Lys Met Thr Val Thr Tyr Asn

20 25 30

Asn Lys Gln Val Cys Asn Gly Gln Glu Leu Phe Pro Ser Ala Val Thr

35 40 45

Ile Arg Pro Arg Val Glu Val Gln Gly Gly Asp Met Arg Thr Phe Phe

50 55 60

Thr Leu Val Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro Tyr

65 70 75 80

Leu Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro Gly Thr Thr

85 90 95

Asp Ala Thr Phe Gly Lys Glu Leu Val Ser Tyr Glu Ile Pro Arg Pro

100 105 110

Asn Ile Gly Ile His Arg Phe Val Phe Val Leu Phe Lys Gln Lys Cys

115 120 125

Arg Gln Ser Val Ser Pro His Asp Val Ser Arg Asp His Phe Asn Thr

130 135 140

Arg Asn Phe Ala Asn Val Asn Asp Leu Gly Pro Pro Val Ala Ala Val

145 150 155 160

Phe Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg

165 170

<210> 7

<211> 4296

<212> DNA

<213> Nicotiana tabacum

<400> 7

catgaccttt tagctactct taactcttct gattgttctg ctgtaacttg tccccttgag 60

ttaaatgtaa agttaaaggc taaagaaggg gatcctctcc ctaatcctga aaattataga 120

ggcctcgttg gtaagctaaa tttcctcact cacactaggc ctgacataag ttttgttgtg 180

caacatctta gtcagttcat gcaacagccc tgctttcctc acatgaaggc agctttgcac 240

ctgttgaggt atctcagaga cacttctaat tttggcctct tatactcgaa ttctactgat 300

ctctctttgc aggcttattg tgatagtgat tggggatcct gccctgataa ctggagattt 360

gtttctgatt tctgtttatt ctttggtggc agtctcattg ggtggaaatc taagaaacat 420

gcagtggtct ctttatcttc ggctgaagtt gagtatagat ctatgagcaa ggctgtggct 480

gaaattactt gggtgtgtag gcgtctatct gatcttgggg tctcttctgc ttctcttgtt 540

cctctccatt gtgacagtat ctctgccatt cacattgcct acaatcctgt cttctatgag 600

660

atcagtttat ctcacatttc cagtgcttct cagctcgcga atgtcttcat caaacccctg 720

tgtgggcctt ctcaccatct tcatattcga aagttgggag ttctctcacc ctcctacttg 780

agggggggct gttgagatag gctgaaatca gtgtggctca gacccaatta ttatttattt 840

atgtacatca gattaggccc attagttagt ctttagttag tcttttattt ctttacatat 900

attggggccat gtatacatac atagagaccc gattttgtaa tagttagatg attcattttt 960

cggttcttaa tcaataagaa atatctcgaa ctttctctct atctctcttt aaccctaaat 1020

1080

1140

1200

tatatatggt tgaaacatga 1260

1320

1380

ttttccataa aatggcttaa atctataagc aaaagatatt tgaaaataat ttttttttta 1440

aaaaagctct caaattttac agccaaacgg aagcttagga taaaaaaggg ggaaatgggg 1500

gagatgggtg ggcaggttgg gctgaagaga aatagacaac agtgcattaa catgtcaaat 1560

catctttatc cctctttcta aaccttacaa ggagtacttt ttatttttctt ttttcttttt 1620

ttggccctaa taaaaattaa aacacatatt ctctagctgc taagctataa ctttaactca 1680

ttggcaccac gacgagtagg agaataacct ttttgggctt ttcttttctt ttctttggtc 1740

cccttttttt gaactatcaa tattttagtc caaacacacc tgactctaca gtgatctgat 1800

ggccactata aatattggct ttttgcaact ctcttctcac caaaatacaa atcggttgaa 1860

ctcttcatat ataatattcc cactactatt actcttaact ttaaatagat ttcttatata 1920

tgggttcaaa aatgtctgat ccccttgtga ttggtagagt gattggggaa gttgttgatt 1980

atttcactcc aagtgttaag atgtctgtta cttataacag cagcaagcat gtttataatg 2040

ggcatgaact ctttccttcc tcagtcacct ctaaacctag ggttgaagtt catggaggtg 2100

atttgagatc tttctttaca atggtacata ctgcttcctt cgattttcaa tacttttatt 2160

aggggtggag cttagcggcg gagccaagat tttaactaag gggagtcaaa atataaataa 2220

2280

ttaacatatt tataccgtgt aattttccag cgaaggggtg tcaattgact ctccttgcca 2340

atgagtggct ccgccactgg cggagctaga gttctagtta cggttcgttg tattgtgtta 2400

agaagtccac ttatactgtc ttttctagaa tttagaattc ataaattcaa aattatggct 2460

ctgcccttaa atttattttt atacatttct attatatagt aaatcgttta tattgacccc 2520

ttatttttct tttttacctt aattgacaga tcatgataga cccagatgtt cctggtccta 2580

gtgatccata tctcagggaa cacctacact ggtaaagaaa taagtttttt aattactaac 2640

tcattcaatt ttatcgtccc ttcttttcct tgtttacttg gagggaaaat aatacgatct 2700

catcgaaaag ataaaaattc ttcaggcttg ttatctaaaa acttgttaaa aaataccgta 2760

atgaaaagac atatgagttt gttattaggt atttgactaa atatgatcga tcatatggtg 2820

ttcggacaag aaatttttg tgaaaaggtc cgcatacttt taaaaaaaga aaatctgcct 2880

2940

tggtttattg acctttacat attaaagaca ccactgagac acatatctag aaaaattgta 3000

tttgggaacg caaaagcaaa gaaaacatgt gttattaatc ttatgtcaat gccaccagca 3060

gctcaggaaa aatatggtcg atatattgtg atttgcttgc aaaaggagca aagaagaaat 3120

3180

acatgggata aggataataa ttttgggata aagtttagga ttaactttat cttatatttg 3240

gtttggagta ttagctaacc gcgaggtatt tttcaaacta aaatagtggg attagctatc 3300

ccatataaaa agtaggatag ctaatcccat gggatatccc accctatcgg atagtaatag 3360

3420

3480

acttagcaaa ttataaatat gtccaataat tggtcacatt gttctaataa ttacttgttt 3540

cccttatcat tatatatagt gcttcattca ctaaacagaa cccaaaaaaa aaaaaaaaaa 3600

aactgcaaaa tggtcatatc atgtagtaac ggaataaaaa cgtactcagt tttatgataa 3660

aatcaaagtg acatatttgt acgctttgat agttgacaaa tacctgaaaa aagaatttga 3720

ccatctttac aggattgtca cagacattcc aggcactaca gattgctcgt ttggtatgta 3780

tctttaaccc aaatttcaag cttcgaaata gtaacagctt ttgtttttaa tattttattt 3840

gtcttaaata catattttcc ttattataaa ttttcttcgcc tagtggtaac gggatcaggt 3900

attgattcgt atttattttt tattgatcaa gtacaaaaga aagaattgtt 3960

tttctacact tagatttata tatatgcaat gtctagaaat taatgagttt acaaattcat 4020

tgatgtgtat atctcacaat caaatccaaa atactgatcc aaaaattttg atcagggaaa 4080

4140

ctgttcaagc agaagaagag gcaaacagta ttgactgcac ctctctccag ggatcgattt 4200

aatacgcgta aattcgcaga agaaaatgag cttgggtctc ctgttgcagc agttttcttc 4260

aattgccaga gggaaactgc tgccagaagg cgttga 4296

<210> 8

<211> 2296

<212> DNA

<213> Nicotiana tabacum

<400> 8

atgtctgtta cttataacag cagcaagcat gtttataatg ggcatgaact ctttccttcc 60

tcagtcacct ctaaacctag ggttgaagtt catggaggtg atttgagatc tttctttaca 120

atggtacata ctgcttcctt cgattttcaa tacttttatt aggggtggag cttagcggcg 180

gagccaagat tttaactaag gggagtcaaa atataaataa gtaagcacac aaaaaaatca 240

agggggtcaa cgtatagtat atacacataa aattaagaat ttaacatatt tataccgtgt 300

aattttccag cgaaggggtg tcaattgact ctccttgcca atgagtggct ccgccactgg 360

cggagctaga gttctagtta cggttcgttg tattgtgtta agaagtccac ttatactgtc 420

ttttctagaa tttagaattc ataaattcaa aattatggct ctgcccttaa atttattttt 480

atacatttct attatatagt aaatcgttta tattgacccc ttatttttct tttttacctt 540

aattgacaga tcatgataga cccagatgtt cctggtccta gtgatccata tctcagggaa 600

660

ttcttttcct tgtttacttg gagggaaaat aatacgatct catcgaaaag ataaaaattc 720

ttcaggcttg ttatctaaaa acttgttaaa aaataccgta atgaaaagac atatgagttt 780

gttattaggt atttgactaa atatgatcga tcatatggtg ttcggacaag aaatattttg 840

tgaaaaggtc cgcatacttt taaaaaaaga aaatctgcct tgactcttga gtttgtgctt 900

ctcgggaaaa caatttcttc cttctttttt tttttttttt tggtttattg acctttacat 960

attaaagaca ccactgagac acatatctag aaaaattgta tttgggaacg caaaagcaaa 1020

gaaaacatgt gttattaatc ttatgtcaat gccaccagca gctcaggaaa aatatggtcg 1080

1140

gacgatgtac ttaaagcaaa taagttagag gtcgtttggt acatgggata aggataataa 1200

ttttgggata aagtttagga ttaactttat cttatatttg gtttggagta ttagctaacc 1260

gcgaggtatt tttcaaacta aaatagtggg attagctatc ccatataaaa agtaggatag 1320

ctaatcccat gggatatccc accctatcgg atagtaatag tccaatagga gacaactcta 1380

atttgtacag acataatgtc cagtcacacc ttgttttttt gtcatgacac atattaagca 1440

tgaataataa tatttcgaca atcttgtagc atttgattag acttagcaaa ttataaatat 1500

1560

gcttcattca ctaaacagaa cccaaaaaaa aaaaaaaaaa aactgcaaaa tggtcatatc 1620

1680

acgctttgat agttgacaaa tacctgaaaa aagaatttga ccatctttac aggattgtca 1740

cagacattcc aggcactaca gattgctcgt ttggtatgta tctttaaccc aaatttcaag 1800

cttcgaaata gtaacagctt ttgtttttaa tattttattt gtcttaaata catatttttcc 1860

ttattataaa ttttcttcgcc tagtggtaac gggatcaggt attgattcgt atttattttt 1920

tattgatcaa caaaaaaaga gtacaaaaga aagaattgtt tttctacact tagatttata 1980

tatatgcaat gtctagaaat taatgagttt acaaattcat tgatgtgtat atctcacaat 2040

caaatccaaa atactgatcc aaaaattttg atcagggaaa gaaatagttg gctatgaaat 2100

gccaaggcca aatattggaa ttcacaggtt tgtatttctg ctgttcaagc agaagaagag 2160

gcaaacagta ttgactgcac ctctctccag ggatcgattt aatacgcgta aattcgcaga 2220

agaaaatgag cttgggtctc ctgttgcagc agttttcttc aattgccaga gggaaactgc 2280

tgccagaagg cgttga 2296

<210> 9

<211> 148

<212> PROTEIN

<213> Nicotiana tabacum

<400> 9

Met Ser Val Thr Tyr Asn Ser Ser Lys His Val Tyr Asn Gly His Glu

1.5 10 15

Leu Phe Pro Ser Ser Val Thr Ser Lys Pro Arg Val Glu Val His Gly

20 25 30

Gly Asp Leu Arg Ser Phe Phe Thr Met Ile Met Ile Asp Pro Asp Val

35 40 45

Pro Gly Pro Ser Asp Pro Tyr Leu Arg Glu His Leu His Trp Ile Val

50 55 60

Thr Asp Ile Pro Gly Thr Thr Asp Cys Ser Phe Gly Lys Glu Ile Val

65 70 75 80

Gly Tyr Glu Met Pro Arg Pro Asn Ile Gly Ile His Arg Phe Val Phe

85 90 95

Leu Leu Phe Lys Gln Lys Lys Arg Gln Thr Val Leu Thr Ala Pro Leu

100 105 110

Ser Arg Asp Arg Phe Asn Thr Arg Lys Phe Ala Glu Glu Asn Glu Leu

115 120 125

Gly Ser Pro Val Ala Ala Val Phe Phe Asn Cys Gln Arg Glu Thr Ala

130 135 140

Ala Arg Arg Arg

145

<210> 10

<211> 4080

<212> DNA

<213> Nicotiana tabacum

<400> 10

agggcacgac cctaagacct tcttcatagc tataaatagt gagctcaggt ttcattgtaa 60

120

tcttctgatt tctagattct ttttgttttt gtgcccgaaa accttgttcc tggaattgtt 180

gcttctgttg tttcgtccat atcttaaggc taagtgttat ataattcttc aattatttat 240

ttatttattt caggttcaaa ttaattcact tatctaaaaa tcatgtataa atttaattgt 300

accattttac gggtgaacag tttggcgccc atcgtggggc ctagataacc gtgtaactaa 360

aggacaaacg tcttttcggg aacttttcta ttttcaagaa ctcaaacccg agatttagac 420

ctctgaggga tctgatcatc tcactacatc gctgagtggt agttgattcc atatacgatt 480

aacctagttt acaactaaat taaattatgt gcattaatcc aagcaacttt tgatgatcag 540

ctgatcaacc taacgtaaga aagcaattaa tttagatgca tatattctac aaatggaaat 600

tagtaggagc aagcaagtta tgcaaaagaa aggaaaagag aaaacattag aagtaggcca 660

aagaaagaag aaggaagagg aagcaatcag ccactgttct agaatggaat atggagaaaa 720

ataataaatt aaattcagat ttctataagt agtaatcctc ttctttctat taccggttaa 780

agctgcagaa attttctttt tcttgacatg acctgaccat agcttccacc attgtttgca 840

ggctggtggt ggagtccctt tataccctca tctctcctac ctaagaacca taggattagg 900

tgattcaagt ttttattttt aacaaaaaat gaaaaattta tgaaggaagt tcaacttttt 960

attaccttaa ataaaaaaga ccttgatgct ttaagtagct ccaagacggt agctgcaaat 1020

tccatctgct tttccttttt aataaaataa tgtactacct actatctgaa agtttaactt 1080

ctatgattct gtaggttttg taaaacactt gggggtattt atattttata ggggattgca 1140

attagaggca gatacaattt ggtttagtta accaccgata ttactcaaac aatttggctt 1200

taaatctggt tagtgtttgg atatagattt ggttgaaatt tgaagaaaaa aaatgagttt 1260

ttaaaaatga gatgaaaaat aatttttgaa agttaaaatt gtatttggac atgcatttta 1320

1380

tgagatttga agattttatt ttcaaaattt gcattataat ctataaacaa atagatacta 1440

tttgagaaca aaatttaaaa aataaagctt tcaaacttat gacgaaaggg aagcttagga 1500

taaaaagggg ggaaatggcc tgggagatgg gtgggcaggt tgggctgaag agaaatagac 1560

aacagtgcat taacatgtca aatcatcttt atccctcttt aaaaacatta ttaggagtac 1620

ttcttttttt cttggggtgc aaaagcctaa tacaagttaa aacacatatt ctctagctgc 1680

taagctataa ctttaactca ttggtaccac gacgagtagg agaataaact ttttgggctt 1740

ttcttttctt ttctttggtt cccatttttt gaactatcaa tattttagtc caaacacacc 1800

tgactctaca gtgatctgat ggccactata aatattggct ttttgcagct ccaaaataca 1860

aatcggtcga actcttcata tatattactc ttaactttaa ataaatagat ttcttatata 1920

tgggttcaaa aatgtctgat ccccttgtga ttggtagagt gataggggaa gttgttgatt 1980

atttcactcc aagtgttaag atgtctgtta cttataacag cagcaagcat gtctataatg 2040

gacatgaact ctttccttcc tcagtcacct ctaaacctag ggttgaagtt catggaggtg 2100

2160

taggggcgaa gctagagttc tagctacggg ttcgttgtat taattgtgtt aagaagtcca 2220

2280

aattaatttt tatgcatttc tcttatatcg tgtatgtgaa tattgacccc ttattttttc 2340

ttttttacct taattgacag atcatgatag acccagatgt tcctggtcct agtgatccat 2400

atctcaggga acatctacac tggtaaagac atacgttttt taattactaa ctcattcaat 2460

tttatcgccc cttcttttcc ttgtttactt ggagggaaaa taatacgatc tcgtcaagaa 2520

2580

acatatgagt ttgttattag gtatttgact aaataggaac gatcatatgg tgttcggaca 2640

agaaaatttt tgtgaaaagg tccgcatact ttaaaaaaaa aaaaaaaaaa aaaaaatccg 2700

2760

tttgaccttt atatattaat taaagacacc actgagacac ttaattaaaa aattgtatat 2820

gggaacgcaa aaagaaaaaa aaacatgtgt tattaatctt atgtcaatgc catcagcaac 2880

tcaggaaaat acggtcgata tactgtgatt tgcttgcgaa aggagcaaag aagaaatctt 2940

ttgataatgt ttgttatgac gatgcactta acctaaaata agttaggggc cgtttggtaa 3000

3060

ttggagtatt agttaattgc gggataactt tcaaattaaa atagtaggat tagttatctc 3120

atatataaag taaaatacct aatcccaata atataatagg agacaactct aatttgcgta 3180

gacataatgt ccagtctcac tttgtatatt tgtcatgacg catattaagc atgaatgata 3240

atatttcgac aatcttgtgg catttgatta cactcagcaa attataaata tgtccaataa 3300

ttgcattaat aattacttgt tcctcttatc attatagtgc ctcattcact aaaccgaacc 3360

caaaagaaca ctgcaaaatg gtcatatcat gtagtaacag aaaaaaaaaa cgtactcgat 3420

tttatgataa aatcaaagtg acatatgtgt cgctttgata attgacaaat acctgaaaaa 3480

agaatttgac catctttaca ggattgtcac agacattcca ggcactacag attgctcgtt 3540

tggtatgtat ctttaaccca aagttcaagc tatgaaatag taacagcttt tctttttaat 3600

attttatttg tcttaaatac atattttcct tattataaat ttattcgcct agtggtaacg 3660

ggatcaggta ttgattcgta tttaattttt attgttcaac aaaaaagagt acaaaaagaa 3720

3780

gagtttacta attcatcgat gtgtatatcg cacaatcaaa tccaattact aataatactg 3840

atctaaaaat ttcgatcagg gagagaaata gttgggtatg aaatgccaag gccaaatatt 3900

3960

gcacctctct ccagggatcg atttaatacg cgcaaattct cagaagaaaa tgagcttggg 4020

tctcctgttg cagcagcttt cttcaattgc cagagggaaa ccgctgccag aaggcgttga 4080

<210> 11

<211> 2080

<212> DNA

<213> Nicotiana tabacum

<400> 11

atgtctgtta cttataacag cagcaagcat gtctataatg gacatgaact ctttccttcc 60

tcagtcacct ctaaacctag ggttgaagtt catggaggtg atttgagatc tttctttaca 120

ctggtacata ctccttcgat tttcactact tttaatttat taggggcgaa gctagagttc 180

tagctacggg ttcgttgtat taattgtgtt aagaagtcca cttaagctgt cttttttaga 240

atttagaatc cataaactca aaatagtgac tttgcttcta aattaatttt tatgcatttc 300

360

atcatgatag acccagatgt tcctggtcct agtgatccat atctcaggga acatctacac 420

tggtaaagac atacgttttt taattactaa ctcattcaat tttatcgccc cttcttttcc 480

ttgtttactt ggagggaaaa taatacgatc tcgtcaagaa gatcaaaaat cttcaggctt 540

gttatttagg aacttgttca aaaataccgt tttgaaaaga acatatgagt ttgttattag 600

660

tccgcatact ttaaaaaaaa aaaaaaaaaa aaaaaatccg ccttgactct tgagtttctg 720

cttcttggaa aaaacatttc ttcttttttt ttttgggttt tttgacctttt atatattaat 780

taaagacacc actgagacac ttaattaaaa aattgtatat gggaacgcaa aaagaaaaaa 840

aaacatgtgt tattaatctt atgtcaatgc catcagcaac tcaggaaaat acggtcgata 900

tactgtgatt tgcttgcgaa aggagcaaag aagaaatctt ttgataatgt ttgttatgac 960

gatgcactta acctaaaata agttaggggc cgtttggtaa atgaaataag gataataatc 1020

tcggaacaaa gtttaggatt aactttatcc catatttgat ttggagtatt agttaattgc 1080

gggataactt tcaaattaaa atagtaggat tagttatctc atatataaag taaaatacct 1140

aatcccaata atataatagg agacaactct aatttgcgta gacataatgt ccagtctcac 1200

tttgtatatt tgtcatgacg catattaagc atgaatgata atatttcgac aatcttgtgg 1260

catttgatta cactcagcaa attataaata tgtccaataa ttgcattaat aattacttgt 1320

tcctcttatc attatagtgc ctcattcact aaaccgaacc caaaagaaca ctgcaaaatg 1380

gtcatatcat gtagtaacag aaaaaaaaaa cgtactcgat tttatgataa aatcaaagtg 1440

acatatgtgt cgctttgata attgacaaat acctgaaaaa agaatttgac catctttaca 1500

ggattgtcac agacattcca ggcactacag attgctcgtt tggtatgtat ctttaaccca 1560

1620

1680

tttaattttt attgttcaac aaaaaagagt acaaaaagaa agaattgatt ttctacactt 1740

agatttatat gcaatatcta gaaatcagaa gatcagcaat gagtttacta attcatcgat 1800

gtgtatatcg cacaatcaaa tccaattact aataatactg atctaaaaat ttcgatcagg 1860

gagagaaata gttgggtatg aaatgccaag gccaaatatt ggaatccaca ggtttgtatt 1920

tctgctgttc aagcagaaga agaggcaaac attattgagt gcacctctct ccagggatcg 1980

atttaatacg cgcaaattct cagaagaaaa tgagcttggg tctcctgttg cagcagcttt 2040

cttcaattgc cagagggaaa ccgctgccag aaggcgttga 2080

<210> 12

<211> 148

<212> PROTEIN

<213> Nicotiana tabacum

<400> 12

Met Ser Val Thr Tyr Asn Ser Ser Lys His Val Tyr Asn Gly His Glu

1.5 10 15

Leu Phe Pro Ser Ser Val Thr Ser Lys Pro Arg Val Glu Val His Gly

20 25 30

Gly Asp Leu Arg Ser Phe Phe Thr Leu Ile Met Ile Asp Pro Asp Val

35 40 45

Pro Gly Pro Ser Asp Pro Tyr Leu Arg Glu His Leu His Trp Ile Val

50 55 60

Thr Asp Ile Pro Gly Thr Thr Asp Cys Ser Phe Gly Arg Glu Ile Val

65 70 75 80

Gly Tyr Glu Met Pro Arg Pro Asn Ile Gly Ile His Arg Phe Val Phe

85 90 95

Leu Leu Phe Lys Gln Lys Lys Arg Gln Thr Leu Leu Ser Ala Pro Leu

100 105 110

Ser Arg Asp Arg Phe Asn Thr Arg Lys Phe Ser Glu Glu Asn Glu Leu

115 120 125

Gly Ser Pro Val Ala Ala Ala Phe Phe Asn Cys Gln Arg Glu Thr Ala

130 135 140

Ala Arg Arg Arg

145

<210> 13

<211> 5167

<212> DNA

<213> Nicotiana tabacum

<400> 13

tgaagttgtg tttggacatg cgttgtattt gagaaaaaat tgaagttttg tgagaggaat 60

ttttttgacc ccaaaactac ataatttgaa ttattattta aaaaaaatga tcatattaca 120

tgaacaaaca gtgttttcaa tttatttttg aaaaaaacag ccaaaatcta gccaaatggg 180

agctaagtgt atgatcaaga ttcatgtccc aaatggaaaa gaatattaac aaaaaaaagg 240

gcagtaaaga agaggtggct acaataggat cgcgcaaaag aaagatggaa aaaagaggaa 300

caggaggggg gaataagcag cacaagaagt tattataagt cagctcttcc agaaaggaat 360

atggagaaaa gttaacctca ggtttctata aataggaatg tccaactttc atttactagt 420

ttaagctgca gaaattctct ttttcttgac atgacctttt caccatcttt aatttggtgg 480

gctttgtggt ggagtccctt tataccatag gctctcctag aggatccata acattagatt 540

ggtaaggttc taagtggact cacgatatga aatttgtgat cgaacctata actcgtctga 600

660

gaatctaaac aaaaactatt gagttcatcc gtaccgatac ctaatactct agctccaacc 720

ctgattctaa tataatgaaa ataaaaccac atctaggaag ttcatgacct gttcttacct 780

taaatgccaa aggccttaaa cctttgatag cttgagaata gccaaacaag tatagattcc 840

atctacttta attttctttc tgattaagat atattgcaac tcctgtaaat gcgcaaggag 900

tcagctggtt cttcccccat ttctatattt tttagtatca ctttcttttc ttaattattc 960

cttcttacat ttgaatcttt ttccatcagc tagctgtttt gatagtagta aaaatgcgaa 1020

ggctcttctt actaatattt caatgaccaa tgaatttaga tggagaagca agttctatta 1080

1140

gtcttttctt gtctatttaa acaagaatca atttaactct cttaatgaca tgctctttag 1200

tcacagaaaa attataacaa attgaagaca ttagttatta atgttctttt cgcactaaaa 1260

gttttttaaa aattttttat cttaaatgtt tgtgactaat caaatatcat catataaaat 1320

taatctggga atatgacatt tttcaatata actaatatgg tgcaaattgc atacactacg 1380

caataaattt gtggttaggg tcatatcatt agcctgtcaa atcatcttta tccctctttc 1440

tctaaacgac ttcttttctc cctttttttt cgcccctcaa acaaagcaaa tagactattc 1500

tctagctgct aattagctaa acaatgactt taactcgttg tgcccagagg agaataacct 1560

ttatctctct tctcttttct ttgtttccat ctttaattta gacttctttt tttggttttt 1620

1680

1740

gaaagaatat cattcaacca caacttttgt tggtcccatc tttatatcta tatgttctta 1800

ctatatttta atccatttcc cacttccaat gatttaaaga agctatagga taggtgcatt 1860

tggaccacta taaatatagg ttttgcagtt ctatgctcca tacaaatatc cagcaagaaa 1920

ctaaactata tattactga gttactacta atagttttca ctcaatctat ttccactctt 1980

tctcctcttc attatattat atggctcaaa tgacagatcc ccttgtgatt agtagggtgg 2040

ttggagatgt tgttgattat ttctctccaa gtgttaagat gtgtgttatt tataacccca 2100

gtaagcatgt ctataatggg catgaactct ttccatccct tgttacctct aaacctaagg 2160

ttgaagttca tggaggtgac atgagatcct tctttacact ggtaattaat tcacactact 2220

2280

gcaaagccac catataaatg acaaatgtaa acttttagga caaaactcca aaaaagttgg 2340

agttttaaaa ttattttata tataaaataa ataaataaat aaataaataa actatcaatt 2400

caaattgggg agtagtttct tactaatatg atagctatat ctatatctat atctatctat 2460

atatgtaaaa catttatatg atgccaagtg gcataaccac tgataagatt tttaaatttg 2520

aatatgaatg aattttaaat gaagttctaa cttcttaaaa ataaacccta atataggtta 2580

2640

aaaatttgat tattaaattt atgtattaca acaataataa ttattgaaaa tattgctaaa 2700

attttcatga aaggattcac ccataattat tagtataata gaaaactaaa aaattattaa 2760

gtctaaagtt ctagatctct atatttataa acgtataaac tgttatttta ttttctgaaa 2820

aaagcaaaaa tactgaagag aaaaatgata aaaatatttt aaaatatgta agtcatgtgc 2880

aaataataaa gtgaacaaat gatgtagtag tatactgaat aagatatgtt ttttgtcat 2940

aaaataagta tatgcataac tcatctcaat aatttgctga ctccatctga gtcaaaatat 3000

3060

caagacgaat ctatatctca tatgggactt tttaaataga tacatcttta taaatgaacc 3120

3180

ggatttattt aattataata tatttttatt cattttaaat taatttattt tttcaaatta 3240

tttgtagcaa cctatataat tatgatattt gagtattatc ttataagtta tttgatgatt 3300

3360

tgtagaaaca tgtgctcaaa taaattacta tcatactttg atatgactaa 3420

tattctttaa taatttttgc gcatcgggcg ggtactaata ctagttctta aaaaaagggt 3480

agcgcgatgc acaaagcatt ccgcattcac acaggatcct aggaattggg tcgcacccca 3540

cagtctaccc taatgcaaac attagcgact actttcacgg ctcgaactcg tcacttatag 3600

3660

aatttctatt cttatattgt tataaattat ttttttcttt ttgatagatc atgactgacc 3720

ctgatgttcc tggtcctagc gatccatatc ttagggagca cttacattgg tatgtatcat 3780

actatcatca actttgaaag cttaaaacac tgtaaagttg atgattcaca ccaaagattt 3840

taatcgtcgt cgtgttactt ccatataaat cagtatcgag aagtatgtgg ccatcactcc 3900

3960

aacatgaagt tgactttaaa ttggataaat tgtttccact actaagctta gcgtataaat 4020

tagtcctttg actttcaatt ttgtataata atgcgaagct ttttttcttg taaatgcaat 4080

ttttgtcctt gagggtttgc aacttctttt ttaaggaaaa aaaaaagact aaagttgtgt 4140

gacactaaaa ccaagagtta gcttaatact tcatggacac acgttagcat aaaacatata 4200

accgatattc aaaattacaa aaatgataga atcataattt ttgtttctat ttaaaaagga 4260

agtaagccaa attactacta acatagtgga cttaaagggt attaattttt tgttatttta 4320

atgatatctg ttcatgactt cttgactact tctactcctt tatatcaatc aaattataat 4380

ttacttcgtt tgactatcta atttacaggg taattacaga cattccaggc actacagatt 4440

cctcgtttgg tatggaataa tattgtattc cttttttact tttctgccta gcatttctaa 4500

atagagtagt ccgatacacg aaatatttca ctttacgcag gatccagaaa taaaggacca 4560

taccccaatt gggtgtaata taagtagtca tgggcgcatg cagtatttta gtgacgggtt 4620

taattgcact cataattttg gacgcttagc ataaagtagt agatatgtat ccataacttc 4680

aaaaatataa taggttcaat gttaaaaatt tcaaaagaga tgaactcata gagtttaaat 4740

catgatccgc ctctgtaggc agtctaccct aatgaaagaa tcagtggctg atttcacagt 4800

tcaaaaccgt aacctatgaa tcacataaag ccaactttac catcgctcca agactcgcct 4860

tcttctgcct aacattacta ctgctaataa agagaatttt aataaaacta ctaatgctaa 4920

ttattattct ttgctaaaat cttcatcagg aaaagaagtg gtgggctatg aaatgccaat 4980

gcctaacatt ggaatccata ggtttgtgtt tctgctcttc aagcagaaga agaggcaaac 5040

agtgagcgca ccattatcca gggaccgatt caatacgcgg aaatacgcag aagaaaatga 5100

gcttggctct ccagttgctg ctgttttctt caactgccaa agggaaaccg cggccagaaa 5160

gcgttga 5167

<210> 14

<211> 3167

<212> DNA

<213> Nicotiana tabacum

<400> 14

atggctcaaa tgacagatcc ccttgtgatt agtagggtgg ttggagatgt tgttgattat 60

ttctctccaa gtgttaagat gtgtgttatt tataacccca gtaagcatgt ctataatggg 120

catgaactct ttccatccct tgttacctct aaacctaagg ttgaagttca tggaggtgac 180

atgagatcct tctttacact ggtaattaat tcacactact tcaatagttt tcttgttctt 240

atattttatt atctatctat atatatataa taaaggagcg gcaaagccac catataaatg 300

360

tataaaataa ataaataaat aaataaataa actatcaatt caaattgggg agtagtttct 420

tactaatatg atagctatat ctatatctat atctatctat atatgtaaaa catttatatg 480

atgccaagtg gcataaccac tgataagatt tttaaatttg aatatgaatg aattttaaat 540

gaagttctaa cttcttaaaa ataaacccta atataggtta ctatttttag taatgattga 600

660

atgtattaca acaataataa ttattgaaaa tattgctaaa attttcatga aaggattcac 720

ccataattat tagtataata gaaaactaaa aaattattaa gtctaaagtt ctagatctct 780

atatttataa acgtataaac tgttatttta ttttctgaaa aaagcaaaaa tactgaagag 840

aaaaatgata aaaattttt aaaatatgta agtcatgtgc aaataataaa gtgaacaaat 900

gatgtagtag tatactgaat aagatatgtt tttttgtcat aaaataagta tatgcataac 960

tcatctcaat aatttgctga ctccatctga gtcaaaatat cttctaaatt caagcgaaga 1020

taattatcta tcgcattatt tttttatcat taatataagg caagacgaat ctatatctca 1080

1140

aattacaagt aagaataact tgaagattga aagaattttt ggatttattt aattataata 1200

tatttttatt cattttaaat taatttatat tttcaaatta tttgtagcaa cctatataat 1260

1320

aactattact acaggggaca taagatgata attataattt tgtagaaaca tatgatctaa 1380

1440

gcatcgggcg ggtactaata ctagttctta aaaaaagggt agcgcgatgc acaaagcatt 1500

ccgcattcac acaggatcct aggaattggg tcgcacccca cagtctaccc taatgcaaac 1560

attagcgact actttcacgg ctcgaactcg tcacttatag atcatacaga gacaaattta 1620

1680

tataaattat ttttttcttt ttgatagatc atgactgacc ctgatgttcc tggtcctagc 1740

gatccatatc ttagggagca cttacattgg tatgtatcat actatcatca actttgaaag 1800

cttaaaacac tgtaaagttg atgattcaca ccaaagattt taatcgtcgt cgtgttactt 1860

ccatataaat cagtatcgag aagtatgtgg ccatcactcc atcaacgaca ccaaaatgaa 1920

ataaagagtc cctatatcaa tacaatataa attaatctta aacatgaagt tgactttaaa 1980

ttggataaat tgtttccact actaagctta gcgtataaat tagtcctttg actttcaatt 2040

ttgtataata atgcgaagct ttttttcttg taaatgcaat ttttgtcctt gagggtttgc 2100

2160

gcttaatact tcatggacac acgttagcat aaaacatata accgatattc aaaattacaa 2220

aaatgataga atcataattt ttgtttctat ttaaaaagga agtaagccaa attactacta 2280

acatagtgga cttaaagggt attaattttt tgttatttta atgatatctg ttcatgactt 2340

cttgactact tctactcctt tatatcaatc aaattataat ttacttcgtt tgactatcta 2400

atttacaggg taattacaga cattccaggc actacagatt cctcgtttgg tatggaataa 2460

tattgtattc cttttttact tttctgccta gcatttctaa atagagtagt ccgatacacg 2520

aaatatttca ctttacgcag gatccagaaa taaaggacca taccccaatt gggtgtaata 2580

2640

gacgcttagc ataaagtagt agatatgtat ccataacttc aaaaatataa taggttcaat 2700

gttaaaaatt tcaaaagaga tgaactcata gagtttaaat catgatccgc ctctgtaggc 2760

agtctaccct aatgaaagaa tcagtggctg atttcacagt tcaaaaccgt aacctatgaa 2820

tcacataaag ccaactttac catcgctcca agactcgcct tcttctgcct aacattacta 2880

ctgctaataa agagaattttt aataaaacta ctaatgctaa ttattattct ttgctaaaat 2940

cttcatcagg aaaagaagtg gtgggctatg aaatgccaat gcctaacatt ggaatccata 3000

ggtttgtgtt tctgctcttc aagcagaaga agaggcaaac agtgagcgca ccattatcca 3060

gggaccgatt caatacgcgg aaatacgcag aagaaaatga gcttggctct ccagttgctg 3120

ctgttttctt caactgccaa agggaaaccg cggccagaaa gcgttga 3167

<210> 15

<211> 173

<212> PROTEIN

<213> Nicotiana tabacum

<400> 15

Met Ala Gln Met Thr Asp Pro Leu Val Ile Ser Arg Val Val Gly Asp

1.5 10 15

Val Val Asp Tyr Phe Ser Pro Ser Val Lys Met Cys Val Ile Tyr Asn

20 25 30

Pro Ser Lys His Val Tyr Asn Gly His Glu Leu Phe Pro Ser Leu Val

35 40 45

Thr Ser Lys Pro Lys Val Glu Val His Gly Gly Asp Met Arg Ser Phe

50 55 60

Phe Thr Leu Ile Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro

65 70 75 80

Tyr Leu Arg Glu His Leu His Trp Val Ile Thr Asp Ile Pro Gly Thr

85 90 95

Thr Asp Ser Ser Phe Gly Lys Glu Val Val Gly Tyr Glu Met Pro Met

100 105 110

Pro Asn Ile Gly Ile His Arg Phe Val Phe Leu Leu Phe Lys Gln Lys

115 120 125

Lys Arg Gln Thr Val Ser Ala Pro Leu Ser Arg Asp Arg Phe Asn Thr

130 135 140

Arg Lys Tyr Ala Glu Glu Asn Glu Leu Gly Ser Pro Val Ala Ala Val

145 150 155 160

Phe Phe Asn Cys Gln Arg Glu Thr Ala Ala Arg Lys Arg

165 170

<210> 16

<211> 4569

<212> DNA

<213> Nicotiana tabacum

<220>

<221> other signs

<222> (1896)..(1896)

<223> n is a, c, g, or t

<400> 16

60

aatgttggcc ggaaagatgc ctgtgggtgc tgtttgggggg aaaaaaacaa aagctcgggt 120

aaattatcaa tacccgagtt ggttcctttc gtttgccact ggaccaactc ctgattttgc 180

ttatatatgg gttccaaact aaaaatactt atatatttaa taaatttatc aatacaaata 240

caaggctcgg gtaaaagtta ttaggttctc ggaaacccat acccgatact atggatccgc 300

360

aaagaataag tttaatgtga caatttgaat ggttgagaca acatgccaaa agctaattta 420

agggatttta tttgacattt atatatggga gagaagaaaa agtattgccc agtatattat 480

tttaagctct acaaccaatc aagaatcaat tcctagaatc cattcaggtg tcaaagaata 540

ctgacatgat ataataaaat acaatttata tcacatcagt atttgctttt tcttgggaga 600

ttagataaaa agagatcaga aatggagttt tatggtacta ggagaattca aggatttact 660

acttttgtgg cacaacataa gctcccaatt tttttaagga atttataaaa gttggttttc 720

taagtactta caattgtcaa atttacaagt catttagtac ataaaaagaa acccaatgat 780

gaggttcagg aaaaaaaaaa atcctatact gtgatttcct agttggcgtt cggacataaa 840

aattatgaaa ttccgaaaaa aaaaattgtt ttaagttgaa aatggtatgt gaaaattaaa 900

gttatatatg gacataaata taatttggag ctgtttttga atttttgtga gtgctttgaa 960

gtgaaatttt ctaaaaacag ctttttggag tttttcaaat tccggagttc aacttcaagc 1020

1080

1140

agaattgaaa gaattttaaa ttaaagtatt tttagatata taaaaataat gtacttttta 1200

1260

ttcataacta acattaacca aaagcattta ttgatcctat tttgacacca ttttatttta 1320

atacaattca taaatttcaa gaatttgaat acattagctt aatctcactt aaattttgag 1380

gtgatgcctg ttctctttct agtcacaact ttaatgtaca ttttatgt caaattaata 1440

cctgaatttg taacccatca aatatcgcca cataatatga aacagtgaaa atatcttata 1500

ttcctgtatt ttatgactaa gacattaagt agctaacaac gatcgaaaaa cattcctaat 1560

aacaagcgaa ttacaactct gtcggataat cgtctgaaac cctaaaaagc tactgaaatg 1620

1680

1740

gagggttggg gagtaggacc tcaaaaacaa agaattttaa tactttggag tttccttagg 1800

1860

tatatatata tatatatata tatatatata tatatnatat aaggtgtcca tctgatcaag 1920

tatccaatac aaaccattct taagtctttg aaaatttctc ttttttttcct tatctctatc 1980

tctgtctaat tttctttatt atggcaagaa gtttagagcc tctaattgtt gggagtag 2040

taggagatgt tcttgattca tttagtccta taatgaaaat gacaatatca tataacaaca 2100

aattagtgtg caatggccat gaactccttc cttctgttgt cactgctaga cctaaagttg 2160

aagttcaagg gggagatttg agaactttct tcacattggt atttttttct tgatttctac 2220

ttaatttcca agatcatcaa gttcccatta tttctttaaa aaaaaaaaaa gcagttcggt 2280

gcactaaact cccgctatgc gcggggttcg gtgaagcacc gaaccataag ggtctattgt 2340

acgcaacctt accctgcatt tatgcaagag gcttgctcac cattacaagt tatattaatt 2400

taacatgtta tatataacca caaaggctgt cgtgggatgg taaatatcct tctatcctta 2460

atcagaagtt tcgggttcaa gttatagccc taggaatata gtcgtctttg gtagggatcc 2520

tttaccccca aaactttccg ccgtgaatcc agattagtaa acctcaaagc gggtatcggg 2580

cattggatga caaaccaaaa aaacttcaac gtgttatagc atgttataac ttattacagt 2640

taatttagtt ttccagtcga tactatatta aatagagtgc ctgtaattta ctttggagtg 2700

2760

taggtcatga cagaccctga tgttcctggc cctagtgatc cttatctaag agagcatctc 2820

2880

tatgtaaaaa tctacaaata tgagaagatc atgattaatt ggaactattt ttactgacta 2940

tttgacagga tagtaactga cattccaggt accactgatg ctacttttgg taagttctct 3000

3060

tattttgtct agctagttat atagaataat tataagatca acaattttgt atagtagtga 3120

atgttggact tctaaagtcg aacatgtcca cttgatgagt gtcacaaaaa tgtagaaact 3180

3240

3300

ggaaaataaa ctttcatata cataaaaaag tgattttttg gaaactcatc ttcaagaata 3360

3420

3480

aattaggatt ttattatatt tggtagaagg tgcaagaagc atatgtaaat aataaattga 3540

gaagtcactt aagatatttt gatcatgtcc cacatcgata acaagaggta ccattctata 3600

3660

3720

cgaagctaaa agtaggatac aataaaaaaa aaattaaaga tctatattgg cgatactatt 3780

3840

aggagtcttt taatcttatt agaaatttac ataccaaaaa atttagagaa cttgctagga 3900

3960

cactctttga gtgcgttcct tctccgaact agctaatgca tgaacacgag atgccttctg 4020

caccgtgcta ccctattaat atataaaaaa atggtagccc ggtgcattaa gctcccgcta 4080

tgcgcgggtt ccgaaaaagg atcagaccac aagggtctat gtttgcaacc ttacttgtat 4140

ttctgcaaga aactgtttcc acggctcgaa cccatgatct tctggtcaca tgacaataac 4200

tttaccggtt acaccaaggt tccccttcac gcgctgccct tttaatattg tctattaata 4260

tttcctacta gagttataca cccctttgtt attactcact cttagggtga ttattaacat 4320

ataatatgtt taatatttat actaaaaaca ggacgagaat tggttagcta tgagattcca 4380

atgccaaata ttggaatcca taggtttgta tttgtacttt tcaagcaaaa acgaagacaa 4440

tcagttagct ctcctacttc aagggatcac ttcaacacta gaaattttgc tgaagaaaat 4500

gatcttggcc aacctgttgc tgctgttttc ttcaatgcac agcgagaaac cgccgcacga 4560

agacgctaa 4569

<210> 17

<211> 2569

<212> DNA

<213> Nicotiana tabacum

<400> 17

atggcaagaa gtttagagcc tctaattgtt gggagagtag taggagatgt tcttgattca 60

tttagtccta taatgaaaat gacaatatca tataacaaca aattagtgtg caatggccat 120

gaactccttc cttctgttgt cactgctaga cctaaagttg aagttcaagg gggagatttg 180

agaactttct tcacattggt atttttttct tgatttctac ttaatttcca agatcatcaa 240

gttcccatta tttctttaaa aaaaaaaaaa gcagttcggt gcactaaact cccgctatgc 300

gcggggttcg gtgaagcacc gaaccataag ggtctattgt acgcaacctt accctgcatt 360

tatgcaagag gcttgctcac cattacaagt tatattaatt taacatgtta tatataacca 420

caaaggctgt cgtgggatgg taaatatcct tctatcctta atcagaagtt tcgggttcaa 480

gttatagccc taggaatata gtcgtctttg gtagggatcc tttaccccca aaactttccg 540

ccgtgaatcc agattagtaa acctcaaagc gggtatcggg cattggatga caaaccaaaa 600

aaacttcaac gtgttatagc atgttataac ttattacagt taatttagtt ttccagtcga 660

tactatatta aatagagtgc ctgtaattta ctttggagtg atttgattgt tattttttcg 720

catcgtcagt acataaaact tatattaatt ttcgaatatg taggtcatga cagaccctga 780

tgttcctggc cctagtgatc cttatctaag agagcatctc cactggtatg ccctaaactc 840

aatttttttt taaaaaaaaa aaatagaaaa tgagaaaaaa tatgtaaaaa tctacaaata 900

tgagaagatc atgattaatt ggaactattt ttactgacta tttgacagga tagtaactga 960

cattccaggt accactgatg ctacttttgg taagttctct gtatcttctg caaaattaca 1020

1080

atagaataat tataagatca acaattttgt atagtagtga atgttggact tctaaagtcg 1140

aacatgtcca cttgatgagt gtcacaaaaa tgtagaaact aaacaatcgt ttggacataa 1200

1260

tggatataca tttaaattga aaagcattgc agttttgtaa ggaaaataaa ctttcatata 1320

cataaaaaag tgattttttg gaaactcatc ttcaagaata tttttaaaaa tttccgtcca 1380

atgtataacc aaacattatt ttgaaaaaga ttaaaaaaag gaaaaacttt aggaacaacg 1440

ggtcccaaga taaatgtgtc tagtcatata agattagata aattaggatt ttattatatt 1500

tggtagaagg tgcaagaagc atatgtaaat aataaattga gaagtcactt aagatatttt 1560

gatcatgtcc cacatcgata acaagaggta ccattctata tatgttaaat catggtaagt 1620

taaagtatta tatcacatat taaatggtga tataatagac ctaaatcaca tgaaacgaaa 1680

ttgtcccgaa aggtctataa atttttgaaa ttcatgtaga cgaagctaaa agtaggatac 1740

aataaaaaaa aaattaaaga tctatattgg cgatactatt tagttgggat tgcattttag 1800

ttattctagt acatttactt taatctaatt tttgctagct aggagtcttt taatcttatt 1860

agaaatttac ataccaaaaa atttagagaa cttgctagga caattggtat ttctttatat 1920

aatattgtgg aagttgtatt agagtatgtt gtttacatta cactctttga gtgcgttcct 1980

tctccgaact agctaatgca tgaacacgag atgccttctg caccgtgcta ccctattaat 2040

atataaaaaa atggtagccc ggtgcattaa gctcccgcta tgcgcgggtt ccgaaaaagg 2100

atcagaccac aagggtctat gtttgcaacc ttacttgtat ttctgcaaga aactgtttcc 2160

acggctcgaa cccatgatct tctggtcaca tgacaataac tttaccggtt acaccaaggt 2220

2280

2340

actaaaaaca ggacgagaat tggttagcta tgagattcca atgccaaata ttggaatcca 2400

taggtttgta tttgtacttt tcaagcaaaa acgaagacaa tcagttagct ctcctacttc 2460

aagggatcac ttcaacacta gaaattttgc tgaagaaaat gatcttggcc aacctgttgc 2520

tgctgttttc ttcaatgcac agcgagaaac cgccgcacga agacgctaa 2569

<210> 18

<211> 172

<212> PROTEIN

<213> Nicotiana tabacum

<400> 18

Met Ala Arg Ser Leu Glu Pro Leu Ile Val Gly Arg Val Val Gly Asp

1.5 10 15

Val Leu Asp Ser Phe Ser Pro Ile Met Lys Met Thr Ile Ser Tyr Asn

20 25 30

Asn Lys Leu Val Cys Asn Gly His Glu Leu Leu Pro Ser Val Val Thr

35 40 45

Ala Arg Pro Lys Val Glu Val Gln Gly Gly Asp Leu Arg Thr Phe Phe

50 55 60

Thr Leu Val Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro Tyr

65 70 75 80

Leu Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro Gly Thr Thr

85 90 95

Asp Ala Thr Phe Gly Arg Glu Leu Val Ser Tyr Glu Ile Pro Met Pro

100 105 110

Asn Ile Gly Ile His Arg Phe Val Phe Val Leu Phe Lys Gln Lys Arg

115 120 125

Arg Gln Ser Val Ser Ser Pro Thr Ser Arg Asp His Phe Asn Thr Arg

130 135 140

Asn Phe Ala Glu Glu Asn Asp Leu Gly Gln Pro Val Ala Ala Val Phe

145 150 155 160

Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg

165 170

<210> 19

<211> 4652

<212> DNA

<213> Nicotiana tabacum

<400> 19

atccccagag gcggatctag gatttgaacc ttatgggttc tcagcatttg ggcaaaagtg 60

atactttaag caagtgagga agttttttaa tgttggccgg aaagatgcct gtgggtgctg 120

tttgggggaa aaaaacaaaa gctcgggtaa attatcaata cccgagttgg ttcctttcgt 180

ttgccactgg accaactcct gattttgctt atatatggggt tccaaactaa aaatacttat 240

300

aaacccatac ccgatactat ggatccgccc ctgcttatcc ctaccttgtg tgaggtagaa 360

420

ttgagacaac atgccaaaag ctaatttaag ggattttatt tgacatttat atatgggaga 480

gaagaaaaag tattgcccag tatattattt taagctctac aaccaatcaa gaatcaattc 540

ctagaatcca ttcaggtgtc aaagaatact gacatgatat aataaaatac aatttatatc 600

660

tggtactagg agaattcaag gatttactac ttttgtggca caacataagc tcccaatttt 720

tttaaggaat ttataaaagt tggttttcta agtacttaca attgtcaaat ttacaagtca 780

tttagtacat aaaaagaaac ccaatgatga ggttcaggaa aaaaaaaaaa tcctatactg 840

tgatttccta gttggcgttc ggacataaaa attatgaaat tccgaaaaaa aaaattgttt 900

taagttgaaa atggtatgtg aaaattaaag ttatatatgg acataaatat aatttggagc 960

1020

ttttcaaatt ccggagttca acttcaagcg aaaaattaaa attttcatga tcaaatgttg 1080

1140

taactagttt cattttagtc attaagggta gaattgaaag aattttaaat taaagtattt 1200

aaaaataatg tactttttaa aacacacaaa aaaaggagtg ccatatatta 1260

atttaatata aggatatata gtggatgcat tcataactaa cattaaccaa aagcatttat 1320

1380

cattagctta atctcactta aattttgagg tgatgcctgt tctctttcta gtcacaactt 1440

taatgtacat tttatatgtc aaattaatac ctgaatttgt aacccatcaa atatcgccac 1500

tatcttatat tcctgtattt tatgactaag acattaagta 1560

gctaacaacg atcgaaaaac attcctaata acaagcgaat tacaactctg tcggataatc 1620

gtctgaaacc ctaaaaagct actgaaatga ttttcctacta gtataattcc gatgaaattt 1680

tgttcgaaaa ttctataaga aatacacgta tttttagtag tgaaaaaaga tttgttgtaa 1740

tttttttaggg tggggtgggg tgatttgggg agggttgggg agtaggacct caaaaacaaa 1800

gaattttaat actttggagt ttccttaggt cccatgtttt atactttctt ttattctcct 1860

tcaccattat agctataact tagtacatat atatatatgg tggccctctg atccaatgta 1920

aaatgcaaac cattcttaag atctttgaaa tttctctctt ttttttcttt atctctatct 1980

ctgtctaatt ctctctatta tggcaagaag tttggagcct ctaatagttgggagagtagt 2040

aggagatgtt cttgattcat ttagtcctat agtgaaaatg acaattactt ataacaacaa 2100

attagtgtgc aatggtcatg aattctttcc ttctattgtc acttctagac ctaaggttga 2160

agttcaagga ggagatttga gaactttctt cacactggta atttttcttg attttttcct 2220

taattccaag atcatcaagt tccatttatt tctttacaag ttatattaat ttaacccttt 2280

ataatcacca aaggctggcg tgggttgcta agtaaccttc catcgttaat cagatgtttc 2340

gggttcgagc tagccctgggg attacaatcg ttttttgtag gaagcgcttt aacccccaaa 2400

atttttcagc acgaacccgg attagtaaac ctcaaaactc gtgccaaata ctagatgaca 2460

aaccaaaaga gttttcaacc tgttataaca tatgttactt gttacaatta ttagttttcc 2520

ggtcaatagt atattatgta attttctttg aagtgacttg attgttattt tttcacatta 2580

tcagtgcata aaacttatac tattattttt taatatgtag gtcatgacag accctgatgt 2640

tcccggccct agtgatcctt atctacgaga gcatctccac tggtacactc tctataatag 2700

tttcatttgt tccgaatttt cttggctgtt atataaaaaa tatattataa catagcatga 2760

2820

atagagaggt ctgtctatat gccctaaact caatgaaaaa aaatagaaaa tgagaaaaaa 2880

tatgtaaaat ctacaaatat gagaagatca tttttagttg aaactatcct tatatactac 2940

tgaatattta gctggcaaat aaaattgaca gtgttttact gattgtttga caggatagta 3000

3060

gattacaagc acatgtgaag aagatacaag atgtttttcc attactcact tattttgtct 3120

3180

acttctaaag tcgaacatgt ccacatgatg agcgtcacaa aaatgcagat acgagctcgt 3240

ttggattgac ttaaaaaatg tggtttttca gcaaaaataa cttttaagcc aaaaaacaat 3300

3360

3420

gactagctta aaagtaaatc caaacaccct ctaactaagc atttggacat aaaaaaaata 3480

3540

3600

3660

3720

gagaaacggg tcccaagata aatgtgtcta gtcatataag attattcaaa attaagaatt 3780

3840

gatgttttga tcatgtccta cgtcgatctt cagaggtacc attccgtata cgtgattggt 3900

aagtaaaggt attaaaaaga gacataatgg acctaaatta cgtgaaacga aattgtcttg 3960

aaaagtcttt caaatttttg aaatccatgt agacgaatcg aaaagtaggg cacaatgaaa 4020

tatgatcaaa ggtttataat ggtgatacaa gttagttggg

gtatatttac tttaatctaa tattttcttg gagttttttta atcttattag aaatttactt 4140

accaaaaatt tagagaactt gctagaacaa tataattgat aattcttcat atatattgtc 4200

ttcgagctgt agaaacagcc actaatgttt gcattaggat atgttgtcta catcacactt 4260

attgtgtgtt gccctcaccg gaccctgcat gaacgtatga tgccttatgc accgcgcccc 4320

ttttaatatt atttattaat taatatttcc tgctagagtt atactccttt gttattactc 4380

attcttaggt tgatgattaa cttataatat gcttaatctt tatactaaaa ataggaagag 4440

aattggttag ctatgagatt ccaaggccaa atattggaat ccataggttt gtatttgtac 4500

ttttcaagca aagacgaaga caatcagtta gccctcctac ttcaagggaa aacttcaaca 4560

ctagaaattt tgccgaagaa aatgatctta gccaacctgt tgctgctgtt ttcttcaatg 4620

cacagcgaga aaccgccgcg cgaagacgct aa 4652

<210> 20

<211> 2653

<212> DNA

<213> Nicotiana tabacum

<400> 20

atggcaagaa gtttggagcc tctaatagtt gggagagtag taggagatgt tcttgattca 60

tttagtccta tagtgaaaat gacaattact tataacaaca aattagtgtg caatggtcat 120

gaattctttc cttctattgt cacttctaga cctaaggttg aagttcaagg aggagatttg 180

agaactttct tcacactggt aatttttctt gattttttcc ttaattccaa gatcatcaag 240

ttccatttat ttctttacaa gttatattaa tttaaccctt tataatcacc aaaggctggc 300

gtgggttgct aagtaacctt ccatcgttaa tcagatgttt cgggttcgag ctagccctgg 360

gattacaatc gttttttgta ggaagcgctt taacccccaa aatttttcag cacgaacccg 420

gattagtaaa cctcaaaact cgtgccaaat actagatgac aaaccaaaag agttttcaac 480

ctgttataac atatgttact tgttacaatt attagttttc cggtcaatag tatattatgt 540

aattttcttt gaagtgactt gattgtttatt ttttcacatt atcagtgcat aaaacttata 600

ctattatttt ttaatatgta ggtcatgaca gaccctgatg ttcccggccc tagtgatcct 660

tatctacgag agcatctcca ctggtacact ctctataata gtttcatttg ttccgaattt 720

tcttggctgt tatataaaaa atatattata acatagcatg aaaattggtt ccacaaaaac 780

ttaattttta tagtgaataa ttgttatata ttgatattgt tatagagagg tctgtctata 840

tgccctaaac tcaatgaaaa aaaatagaaa atgagaaaaa atatgtaaaa tctacaaata 900

tgagaagatc atttttagtt gaaactatcc ttatatacta ctgaatattt agctggcaaa 960

taaaattgac agtgttttac tgattgtttg acaggatagt aactgacatt ccaggtacca 1020

ctgatgctac ttttggtaag ttttattagt ttcttctgca agattacaag cacatgtgaa 1080

gaagatacaa gatgtttttc cattactcac ttatttttgtc ttgctaataa ttatatagaa 1140

caattgtaag atcaacagtg ttatataata gtgaatgttg gacttctaaa gtcgaacatg 1200

tccacatgat gagcgtcaca aaaatgcaga tacgagctcg tttggattga cttaaaaaat 1260

gtggtttttc agcaaaaata acttttaagc caaaaaacaa taagttaggg ttgtccacct 1320

ttttgctttt ggcttaattt aagcatttta aaatttattt taagcaattt ttgacttagc 1380

caaacaccga aaaaagctaa aagaaactta aaagctgatt tgactagctt aaaagtaaat 1440

ccaaacaccc tctaactaag catttggaca taaaaaaaat atgtcatttt tgaaaaaagt 1500

agttcttttg agttaagtca aaaaagaata tataaaattt gaaattgtat ttagacatgc 1560

1620

tttttggaaa ctcatcttca agaatttttc caaaacttca gtccaatcgt ataaccaaac 1680

attattttga taaaaacatc gaaaataaaa ataaatctat ggagaaacgg gtcccaagat 1740

aaatgtgtct agtcatataa gattattcaa aattaagaat ttatcacatt tgtaaaagat 1800

gtaagtagca tatgtaaatg ataaaatgag aagtcacttg agatgttttg atcatgtcct 1860

acgtcgatct tcagaggtac cattccgtat acgtgattgg taagtaaagg tattaaaaag 1920

agacataatg gacctaaatt acgtgaaacg aaattgtctt gaaaagtctt tcaaattttt 1980

gaaatccatg tagacgaatc gaaaagtagg gcacaatgaa atatgatcaa aggtttataa 2040

tggtgataca agttagttgg gattacgttt tagttatgcc agtatattta ctttaatcta 2100

2160

tgctagaaca atataattga taattcttca tatatattgt cttcgagctg tagaaacagc 2220

cactaatgtt tgcattagga tatgttgtct acatcacact tattgtgtgt tgccctcacc 2280

ggaccctgca tgaacgtatg atgccttatg caccgcgccc cttttaatat tatttattaa 2340

ttaatatttc ctgctagagt tatactcctt tgttattact cattcttagg ttgatgatta 2400

acttataata tgcttaatct ttatactaaa aataggaaga gaattggtta gctatgagat 2460

2520

acaatcagtt agccctccta cttcaaggga aaacttcaac actagaaatt ttgccgaaga 2580

aaatgatctt agccaacctg ttgctgctgt tttcttcaat gcacagcgag aaaccgccgc 2640

gcgaagacgc taa 2653

<210> 21

<211> 172

<212> PROTEIN

<213> Nicotiana tabacum

<400> 21

Met Ala Arg Ser Leu Glu Pro Leu Ile Val Gly Arg Val Val Gly Asp

1.5 10 15

Val Leu Asp Ser Phe Ser Pro Ile Val Lys Met Thr Ile Thr Tyr Asn

20 25 30

Asn Lys Leu Val Cys Asn Gly His Glu Phe Phe Pro Ser Ile Val Thr

35 40 45

Ser Arg Pro Lys Val Glu Val Gln Gly Gly Asp Leu Arg Thr Phe Phe

50 55 60

Thr Leu Val Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro Tyr

65 70 75 80

Leu Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro Gly Thr Thr

85 90 95

Asp Ala Thr Phe Gly Arg Glu Leu Val Ser Tyr Glu Ile Pro Arg Pro

100 105 110

Asn Ile Gly Ile His Arg Phe Val Phe Val Leu Phe Lys Gln Arg Arg

115 120 125

Arg Gln Ser Val Ser Pro Pro Thr Ser Arg Glu Asn Phe Asn Thr Arg

130 135 140

Asn Phe Ala Glu Glu Asn Asp Leu Ser Gln Pro Val Ala Ala Val Phe

145 150 155 160

Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg

165 170

<210> 22

<211> 58

<212> DNA

<213> Nicotiana tabacum

<400> 22

agttaaaatg acagtcactt acaacaataa acaagtttgc aatggccaag agctcttc 58

<210> 23

<211> 58

<212> DNA

<213> Nicotiana tabacum

<400> 23

gaagagctct tggccattgc aaacttgttt attgttgtaa gtgactgtca ttttaact 58

<210> 24

<211> 482

<212> DNA

<213> Nicotiana tabacum

<400> 24

ggtaccacaa gtttgtacaa aaaagcaggc taagcttgtc gaccatggag ttaaaatgac 60

agtcacttac aacaataaac aagtttgcaa tggccaagag ctcttctggt aacctttaat 120

180

cattcttttt atgaaccaaa catgcatgca aagatatttt tagatatttg gacggcgagt 240

gagatttgaa actaggaccg tttgcctgat acaatattaa aatatgtaac cattttatgt 300

acaagtttaa actgttgata gtagcatatt ttttactttt atttaagtat actatattcc 360

aacaggtaag ttaacgaaga gctcttggcc attgcaaact tgtttattgt tgtaagtgac 420

tgtcatttta actggcgcgc ccgggcaatt gacccagctt tcttgtacaa agtggtgagc 480

tc 482

<210> 25

<211> 88

<212> DNA

<213> Nicotiana tabacum

<400> 25

tgcagtcact attagaccta gggttgaagt tcaaggtggt gatatgagaa ctttcttcac 60

attggtcatc acagatcctg atgtacct 88

<210> 26

<211> 88

<212> DNA

<213> Nicotiana tabacum

<400> 26

aggtacatca ggatctgtga tgaccaatgt gaagaaagtt ctcatatcac caccttgaac 60

ttcaacccta ggtctaatag tgactgca 88

<210> 27

<211> 88

<212> DNA

<213> Nicotiana tabacum

<400> 27

tgcggtcacc attagaccta gggttgaggt tcaaggtggt gatatgagaa ctttcttcac 60

attggtcatg acagaccctg atgttcct 88

<210> 28

<211> 88

<212> DNA

<213> Nicotiana tabacum

<400> 28

aggaacatca gggtctgtca tgaccaatgt gaagaaagtt ctcatatcac caccttgaac 60

ctcaacccta ggtctaatgg tgaccgca 88

<210> 29

<211> 78

<212> DNA

<213> Nicotiana tabacum

<400> 29

catgaactct ttccttcctc agtcacctct aaacctaggg ttgaagttca tggaggtgat 60

ttgagatctt tctttaca 78

<210> 30

<211> 78

<212> DNA

<213> Nicotiana tabacum

<400> 30

tgtaaagaaa gatctcaaat cacctccatg aacttcaacc ctaggtttag aggtgactga 60

ggaaggaaag agttcatg 78

<210> 31

<211> 522

<212> DNA

<213> Nicotiana tabacum

<400> 31

ggtaccacaa gtttgtacaa aaaagcaggc taagcttgtc gaccatggca tgaactcttt 60

ccttcctcag tcacctctaa acctagggtt gaagttcatg gaggtgattt gagatctttc 120

tttacatggt aacctttaat gtttaaccgt tcacatttct aatatttact tatttgtaac 180

atgtcgtcac gtgttagttt cattcttttt atgaaccaaa catgcatgca aagatatttt 240

tagatatttg gacggcgagt gagatttgaa actaggaccg tttgcctgat acaatattaa 300

aatatgtaac cattttatgt acaagtttaa actgttgata gtagcatatt ttttactttt 360

atttaagtat actatattcc aacaggtaag ttaactgtaa agaaagatct caaatcacct 420

ccatgaactt caaccctagg tttagaggtg actgaggaag gaaagagttc atgggcgcgc 480

ccgggcaatt gacccagctt tcttgtacaa agtggtgagc tc 522

<210> 32

<211> 121

<212> DNA

<213> Nicotiana tabacum

<400> 32

gaaagaaata gttggctatg aaatgccaag gccaaatatt ggaattcaca ggtttgtatt 60

tctgctgttc aagcagaaga agaggcaaac agtattgact gcacctctct ccagggatcg 120

a 121

<210> 33

<211> 121

<212> DNA

<213> Nicotiana tabacum

<400> 33

tcgatccctg gagagaggtg cagtcaatac tgtttgcctc ttcttctgct tgaacagcag 60

aaatacaaac ctgtgaattc caatatttgg ccttggcatt tcatagccaa ctatttcttt 120

c 121

<210> 34

<211> 110

<212> DNA

<213> Nicotiana tabacum

<400> 34

gagagaaata gttgggtatg aaatgccaag gccaaatatt ggaatccaca gcagctttct 60

tcaattgcca gagggaaacc gctgccagaa ggcgttgaag aagatgttta 110

<210> 35

<211> 110

<212> DNA

<213> Nicotiana tabacum

<400> 35

taaacatctt cttcaacgcc ttctggcagc ggtttccctc tggcaattga agaaagctgc 60

tgtggattcc aatatttggc cttggcattt catacccaac tatttctctc 110

<210> 36

<211> 138

<212> DNA

<213> Nicotiana tabacum

<400> 36

atggctcaaa tgacagatcc ccttgtgatt agtagggtgg ttggagatgt tgttgattat 60

ttctctccaa gtgttaagat gtgtgttatt tataacccca gtaagcatgt ctataatggg 120

catgaactct ttccatcc 138

<210> 37

<211> 138

<212> DNA

<213> Nicotiana tabacum

<400> 37

ggatggaaag agttcatgcc cattatagac atgcttactg gggttataaa taacacacat 60

cttaacactt ggagagaaat aatcaacaac atctccaacc accctactaa tcacaagggg 120

atctgtcatt tgagccat 138

<210> 38

<211> 642

<212> DNA

<213> Nicotiana tabacum

<400> 38

ggtaccacaa gtttgtacaa aaaagcaggc taagcttgtc gaccatggat ggctcaaatg 60

acagatcccc ttgtgattag tagggtggtt ggagatgttg ttgattattt ctctccaagt 120

gttaagatgt gtgttattta taaccccagt aagcatgtct ataatgggca tgaactcttt 180

ccatcctggt aacctttaat gtttaaccgt tcacatttct aatatttact tatttgtaac 240

atgtcgtcac gtgttagttt cattcttttt atgaaccaaa catgcatgca aagatatttt 300

tagatatttg gacggcgagt gagatttgaa actaggaccg tttgcctgat acaatattaa 360

aatatgtaac cattttatgt acaagtttaa actgttgata gtagcatatt ttttactttt 420

atttaagtat actatattcc aacaggtaag ttaacggatg gaaagagttc atgcccatta 480

tagacatgct tactggggtt ataaataaca cacatcttaa cacttggaga gaaataatca 540

acaacatctc caaccaccct actaatcaca aggggatctg tcatttgagc catggcgcgc 600

ccgggcaatt gacccagctt tcttgtacaa agtggtgagc tc 642

<210> 39

<211> 93

<212> DNA

<213> Nicotiana tabacum

<400> 39

tagtcctata gtgaaaatga caattactta taacaacaaa ttagtgtgca atggtcatga 60

attctttcct tctattgtca cttctagacc taa 93

<210> 40

<211> 93

<212> DNA

<213> Nicotiana tabacum

<400> 40

ttaggtctag aagtgacaat agaaggaaag aattcatgac cattgcacac taatttgttg 60

ttataagtaa ttgtcatttt cactatagga cta 93

<210> 41

<211> 552

<212> DNA

<213> Nicotiana tabacum

<400> 41

ggtaccacaa gtttgtacaa aaaagcaggc taagcttgtc gaccatggta gtcctatagt 60

gaaaatgaca attacttata acaacaaatt agtgtgcaat ggtcatgaat tctttccttc 120

tattgtcact tctagaccta atggtaacct ttaatgttta accgttcaca tttctaatat 180

ttacttatt gtaacatgtc gtcacgtgtt agtttcattc tttttatgaa ccaaacatgc 240

atgcaaagat atttttagat atttggacgg cgagtgagat ttgaaactag gaccgtttgc 300

ctgatacaat attaaaatat gtaaccattt tatgtacaag tttaaactgt tgatagtagc 360

atatttttta cttttattta agtatactat attccaacag gtaagttaac ttaggtctag 420

aagtgacaat agaaggaaag aattcatgac cattgcacac taatttgttg ttataagtaa 480

ttgtcatttt cactatagga ctaggcgcgc ccgggcaatt gacccagctt tcttgtacaa 540

agtggtgagc tc 552

<---

Claims (54)

1. A transgenic plant having a reduced period of time before flowering compared to a control plant, wherein said transgenic plant is characterized by reduced expression of the gene encoding Terminal Flower 1 ( TFL1 ) or reduced activity of a protein encoded by TFL1 , wherein said plant contains: (i) a polynucleotide sequence comprising SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 19 or SEQ ID NO: 20; or (ii) a polypeptide encoded by the polynucleotide shown in (i); where the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) is reduced compared to a control plant in which the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) was not reduced. 2. The transgenic plant according to claim 1, wherein the reduced expression of the polynucleotide or the reduced activity of the polypeptide shortens the time to bloom compared to the control plant, respectively, where the time to bloom is reduced by at least 8% or at least 20 %; and/or where the plant contains at least one genetic alteration in the polynucleotide sequence encoding TFL1. 3. Transgenic plant according to claim 1 or 2, where the plant contains at least one mutation in the polynucleotide sequence encoding TFL1; respectively, where at least one mutation is selected from the group consisting of: mutations at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them. 4. A transgenic plant according to claim 1 or 2, wherein the plant contains at least one mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S. 5. A transgenic plant according to claim 1 or 2 or 4, wherein the plant contains at least one mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. 6. A transgenic plant according to any one of the preceding claims, wherein the plant belongs to or is derived from the genus Nicotiana, respectively, wherein the plant is Nicotiana tabacum. 7. Part of the transgenic plant according to claim 1, characterized by reduced expression of the gene encoding Terminal Flower 1 ( TFL1 ), or reduced activity of the protein encoded by TFL1 , where the specified part contains: (i) a polynucleotide sequence comprising SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 19 or SEQ ID NO: 20; or (ii) a polypeptide encoded by the polynucleotide shown in (i); where the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) is reduced compared to a control plant in which the expression or activity of the polynucleotide or polypeptide presented in (i), (ii) was not reduced. 8. The part of claim 7 wherein the reduced expression of the polynucleotide or the reduced activity of the polypeptide results in a shorter time to flowering compared to a control plant, respectively, wherein the time to flower is reduced by at least 8% or at least 20% ; and/or where the plant contains at least one genetic alteration in the polynucleotide sequence encoding TFL1 . 9. Part according to claim 7 or 8, where the part contains at least one mutation in the polynucleotide sequence encoding TFL1 ; respectively, where at least one mutation is selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them. 10. The part according to claim 7 or 8, where the part contains at least one mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S. 11. The part according to claim 7 or 8 or 10, where the part contains at least one mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. 12. Part according to paragraphs. 7-11, where the plant belongs to or is derived from the genus Nicotiana , respectively, where the plant is Nicotiana tabacum . 13. The use of plant material in a tobacco product or smoking article, where the specified plant material is obtained or can be obtained from a plant according to any one of paragraphs. 1–6; where the claimed plant material is a leaf. 14. The use of a herbal product in a tobacco product or smoking article, where said herbal product contains at least the plant material according to claim 13. 15. The use of a tobacco product in a smoking article, where said tobacco product contains plant material according to claim 13. 16. A method for reducing the period of time before the onset of flowering in a plant, including modifying the plant by reducing the expression of at least one TFL1 gene or the activity of at least one protein encoded by it, in the specified plant; respectively, where the method includes (a) obtaining a plant containing (i) the polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 19 or SEQ ID NO: 20; or (ii) a polypeptide encoded by the polynucleotide shown in (i); And (b) reducing TFL1 gene expression or TFL1 protein activity in the plant; And (c) obtaining a plant with a reduced time to flowering compared to a control plant in which TFL1 gene expression or TFL1 protein activity was not reduced. 17. The method according to p. 16, where the expression of TFL1 or TFL1 activity is reduced using a method selected from the group consisting of a) mutating the TFL1 gene in a plant; b) expression of the exogenous polynucleotide or polypeptide in the plant; and c) removing the TFL1 gene from the plant, or a combination of one or more of them, respectively, where the plant is mutated at a position selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them. 18. A method for obtaining plant material from a plant according to claim 1 with a reduced period of time before flowering compared to a control plant, while this method includes (a) obtaining a plant according to any one of paragraphs. 1-6 or plant material according to claim 13; (b) collecting plant material from the plant; (c) optionally drying or drying the plant material for a period of time; And (d) obtaining plant material that has a reduced time to flowering compared to a control plant. 19. The use of plant material in a tobacco product or smoking article, where the specified plant material is obtained or can be obtained using the method according to any one of paragraphs. 16–18. 20. An isolated polynucleotide for reducing the expression or activity of certain TFL1 polynucleotides, wherein said polynucleotide comprises SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 19 , or SEQ ID NO: 20. 21. A selected polypeptide for inhibiting the expression of TFL-1, where the selected polypeptide contains at least one mutation selected from the group consisting of a mutation at position T143 or G129 in SEQ ID NO: 9; or mutations at position R120 or G129 or P131 in SEQ ID NO: 12; or mutations at position P110 or H86 in SEQ ID NO: 21, or combinations of two or more of them; respectively, where the mutation is T143I, or G129R, or G129E, or H84STOP in SEQ ID NO: 9; or where the mutation is R120C or G129E or P131S in SEQ ID NO: 12; or where the mutation is P110L or H86STOP in SEQ ID NO: 21 or a combination of two or more of them. 22. The isolated polypeptide of claim 21, wherein the isolated polypeptide contains at least one mutation at position P131 in SEQ ID NO: 12, respectively, where the mutation is P131S. 23. An isolated polypeptide according to claim 21 or 22, wherein the plant contains at least one mutation at position P110 in SEQ ID NO: 21, respectively, where the mutation is P110L. 24. An RNAi construct to suppress TFL-1 expression, containing a sequence that hybridizes with a target sequence in the mRNA of the TFL-1 gene and provides suppression of TFL-1 gene expression through the mechanism of RNA interference, where the specified target sequence is selected from the group consisting of SEQ ID NO: 7, 8, 10, 11, 19 and/or 20. 25. Double-stranded RNA for inhibiting TFL-1 expression, where said RNA contains at least two sequences that are at least partially complementary to each other, and where the sense strand contains the first sequence, and the antisense strand contains the second sequence, and where at least one of the sequences contains at least 10 contiguous nucleotides from TFL1 RNA, respectively, where at least one of the sequences contains 21-23 contiguous nucleotides from TFL1 RNA; respectively, where the double-stranded RNA contains the first sequence containing at least 10 contiguous nucleotides from TFL1 , respectively, 21-23 contiguous nucleotides from TFL-1; second sequence; And a third sequence containing a sequence inversely complementary to the first sequence, located in the same orientation as the first sequence, where the second sequence is located between the first sequence and the third sequence and the second sequence is operably linked to the first sequence and the third sequence; respectively, where the first sequence is selected from the group consisting of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 19, or SEQ ID NO: 20, and/or where the third sequence is a sequence reversely complementary to the sequence corresponding to SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 19, or SEQ ID NO: 20; respectively, where the first sequence contains or consists of SEQ ID NO: 22 and the third sequence contains or consists of SEQ ID NO: 23; or the first sequence contains or consists of SEQ ID NO: 25 and the third sequence contains or consists of SEQ ID NO: 26; or the first sequence contains or consists of SEQ ID NO: 27 and the third sequence contains or consists of SEQ ID NO: 28; or the first sequence contains or consists of SEQ ID NO: 29 and the third sequence contains or consists of SEQ ID NO: 30; or the first sequence contains or consists of SEQ ID NO: 32 and the third sequence contains or consists of SEQ ID NO: 33; or the first sequence contains or consists of SEQ ID NO: 34 and the third sequence contains or consists of SEQ ID NO: 35; or the first sequence contains or consists of SEQ ID NO: 36 and the third sequence contains or consists of SEQ ID NO: 37; or the first sequence contains or consists of SEQ ID NO: 39 and the third sequence contains or consists of SEQ ID NO: 40; respectively, where the double-stranded RNA contains or consists of a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 31, SEQ ID NO: 38 and SEQ ID NO: 41. 26. A method for identifying a molecule that modulates the expression of a TFL1 polynucleotide or the activity of a TFL1 polypeptide, the method comprising (a) contacting the molecule with a plant containing a polynucleotide sequence comprising SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 19, or SEQ ID NO: 20, or a polypeptide encoded by a polynucleotide; (b) monitoring one or more of (i) the level of expression of a TFL1 polynucleotide in a plant; (ii) the level of expression of the TFL1 polypeptide in the plant; (iii) modulating the activity of a TFL1 polypeptide in a plant; or (iv) modulating the activity of a TFL1 polynucleotide in a plant; And (c) identifying a molecule that modulates the expression of a TFL1 polynucleotide or the activity of a TFL1 polypeptide.

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