RU2799785C2 - Modulation of the content of amino acids in the plant - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to dried plant material from Nicotiana tabacum for use in tobacco products. Also disclosed is a tobacco product containing said dried plant material from Nicotiana tabacum. Disclosed is a method for obtaining dried plant material from Nicotiana tabacum.

EFFECT: invention makes it possible to effectively obtain dried plant material from Nicotiana tabacum.

13 cl, 4 dwg, 2 tbl, 1 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention discloses polynucleotide sequences of genes encoding aspartate aminotransferase (AAT) from Nicotiana tabacum and their variants, homologues and fragments. Also disclosed are the polypeptide sequences encoded by them and their variants, homologues and fragments. Also disclosed is the modulation of the expression of one or more NtAAT genes, or the function or activity of the NtAAT polypeptide(s) they encode, to modulate the levels of one or more free amino acids, such as aspartate, and metabolites or by-products derived therefrom, such as ammonia, in a plant or part thereof.

BACKGROUND OF THE INVENTION

Acrylamide is a chemical compound of the formula C ₃ H ₅ NO (IUPAC name prop-2-enamide) and concerns have been raised about its potential toxicity. The source of acrylamide in the aerosol of smoking articles containing tobacco may be, at least in part, amino acids that are present in the tobacco material used to manufacture smoking articles. In cultivated types of tobacco, an increase in total free amino acids during drying is observed, in particular in air-cured types of tobacco and sun-cured types of tobacco. The change in amino acid content of dried tobacco material depends on the drying time at ambient temperature (air drying), which unlike flue drying (which is a fast drying process) allows the enzymatic reactions to remain active for 10-15 days after harvest. Additionally, air-cured tobacco material has a higher water content than other types of dried tobacco, which slows down the drying process at ambient temperature, and is therefore considered a second factor allowing some enzymatic reactions to remain active later in the drying phase. It is necessary to reduce the levels of amino acids and metabolites and by-products derived from them in plants, in particular in dried plant material and the smoke and aerosol obtained from them. Because ammonia is likely to be an amino acid by-product produced during drying, ammonia levels in dried, smoked, and aerosolized leaves may also be reduced. It is also necessary to reduce the level of formation of undesirable odors in the aerosol or smoke when tobacco is heated or burned.

The present invention addresses this need in the art.

BRIEF DESCRIPTION OF THE INVENTION

This document describes a number of polynucleotide sequences encoding AAT from Nicotiana tabacum that are involved in amino acid biosynthesis during early drying. Changes in NtATT genes that are not overexpressed during drying will not modulate the levels of amino acids and their derived metabolites. However, these genes are likely to be involved in other metabolic pathways and changes in their expression may result in a phenotype that is agronomically unfavorable (eg slow growth). Knowing which NtAAT genes are overexpressed during drying advantageously allows selection of plants with changes in the relevant genes only and reduces potential negative effects on other metabolic processes.

AAT catalyzes the reversible transfer of the α-amino group between aspartate and glutamate, and is therefore a key enzyme in amino acid metabolism through the generation of nitrogen flux from glutamate and aspartate. The synthesis of aspartate is important for the synthesis of other amino acids such as asparagine, threonine, isoleucine, cysteine, and methionine. During the drying process, aspartate is converted to asparagine by asparagine synthetase. Asparagine and glutamine are key compounds for N remobilization in aging leaves, asparagine is the main amino acid produced in dried Burley leaves. Asparagine is known to result in the formation of acrylamide when tobacco leaf is heated. Aspartate also plays a key role in the biosynthesis of other amino acids such as threonine, methionine, and cysteine, some of which, when heated, result in a sulfur odor. By modulating the expression and/or activity of the disclosed AAT, it is now possible to adjust the chemistry of plant parts such as leaves and the smoke or aerosols produced therefrom. Interestingly, some tobacco AAT genes can still be expressed for at least 8 days from the start of the air drying process, as described herein. For example, the amount of one or more amino acids such as aspartate and metabolites such as ammonia produced therefrom during and after drying can be modulated and hence the formation of acrylamide formed during heating of tobacco can be modulated, preferably reduced. As a further example, the formation of other amino acids that can result in a sulfur odor when tobacco is heated can also be modulated, preferably reduced. This document describes several AAT genomic polynucleotide sequences from Nicotiana tabacum , including NtAAT1-S (SEQ ID NO: 5), NtAAT1-T (SEQ ID NO: 7), NtAAT2-S (SEQ ID NO: 1), NtAAT2-T (SEQ ID NO: 3), NtAAT3-S (SEQ ID NO: 9), NtAAT3-T ( SEQ ID NO: 11 ), NtAAT4-S (SEQ ID NO: 13) and NtAAT4-T (SEQ ID NO: 15). Also shown are the respective predicted polypeptide sequences for NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), NtAAT2-T (SEQ ID NO: 4), NtAAT3-S (SEQ ID NO: 10), NtAAT3-T (SEQ ID NO: 12), NtAAT4-S (SEQ ID NO: 14) and NtAAT4-T (SEQ ID NO: 16). It has been shown that NtAAT2-S and NtAAT2-T, and to a lesser extent NtAAT1-S, NtAAT1-T, play a special role in aspartate biosynthesis during drying. On the contrary, the levels of NtAAT4-S and NtAAT4- T transcripts decrease mainly during the first two days of leaf drying, although NtAAT4-T remains expressed after eight days of air drying, so the participation of the NtAAT4-T protein during drying cannot be excluded. NtAAT3-S expression remains low during leaf drying and is not modulated during the yellowing phase. NtAAT3-T is sometimes slightly induced during drying, but the level of expression remains relatively low.

SOME BENEFITS

Advantageously, NtAAT polynucleotide sequences can be characterized by a high level of expression during drying, in particular from the beginning of drying. Modulating the expression of one or more NtAAT polynucleotide sequences can result in modulated levels of acrylamide in the aerosol because the level of aspartate can be modulated throughout the drying process. In particular, a decrease in expression of one or more NtAAT polynucleotide sequences may result in a decrease in the levels of acrylamide in the aerosol.

Because aspartate is key in the biosynthesis of other amino acids such as threonine, methionine, and cysteine, some of which result in a sulfur odor upon heating, modulation of the expression of NtAAT polynucleotide sequences can modulate this undesirable odor. Additionally, knowing that ammonia is likely to be a by-product of amino acids formed during drying, a reduction in the expression of NtAAT polynucleotide sequences and/or the activity of the protein they encode may provide a reduction in the level of ammonia in the dried plant material and the smoke and aerosol obtained from it. An increase in amino acid content occurs in air-cured tobacco and sun-cured tobacco because these drying processes result in an increased amino acid content in the dried tobacco material compared to tube-cured tobacco material. Thus, the present invention is particularly applicable to air-dried and fire-cured tobacco material.

Advantageously, there is a limited effect on nicotine levels in the modified plants described herein, which is necessary if the modified plants are to be used to produce tobacco plants and consumable tobacco products.

Advantageously, non-genetically modified plants can be created that may be more acceptable to consumers.

Advantageously, the present invention is not limited to the use of plants obtained by EMS mutagenesis. A plant produced by EMS mutagenesis may have less potential to transfer improved traits to the crop after selection. Once breeding has begun, the desired characteristic(s) of the plant obtained by EMS mutagenesis may be lost for various reasons. For example, multiple mutations may be required, a mutation may be dominant or recessive, and identification of a point mutation in a target gene may be difficult to achieve. On the contrary, the present invention introduces the use of NtAAT polynucleotides, which can in particular be manipulated to obtain plants with the desired phenotype. The present invention can be applied to various varieties of plants or crops.

In one aspect, a plant cell is described comprising: (i) a polynucleotide comprising, consisting of, or essentially consisting of a sequence that has at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; (ii) a polypeptide encoded by the polynucleotide specified in (i); (iii) a polypeptide comprising, consisting of, or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID No: 8, at least 93% sequence identity with SEQ ID NO: 2, or SEQ ID No: 4, or SEQ ID NO: 10, or SEQ ID No: 12, or at least 94% sequence identity with SEQ ID NO: 14 or SE Q ID NO: 16; or (iv) a construct, vector or expression vector comprising the isolated polynucleotide referred to in (i), wherein said plant cell contains at least one modification that modulates the expression or activity of the polynucleotide or polypeptide compared to that in a control plant cell in which the expression or activity of the polynucleotide or polypeptide has not been modified.

In another aspect, a plant cell is disclosed comprising: (i) a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; (ii) a polypeptide encoded by the polynucleotide specified in (i); (iii) a polypeptide comprising, consisting of, or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID No: 8, at least 93% sequence identity with SEQ ID NO: 2, or SEQ ID NO: 10, or SEQ ID No: 12, or at least 94% sequence identity with SEQ ID NO: 4, or SEQ ID NO: 14, or SEQ ID NO: 16; or (iv) a construct, vector or expression vector comprising the isolated polynucleotide referred to in (i), wherein said plant cell contains at least one modification that modulates the expression or activity of the polynucleotide or polypeptide compared to that in a control plant cell in which the expression or activity of the polynucleotide or polypeptide has not been modified. Preferably said plant cell contains a polynucleotide comprising, consisting of or essentially consisting of a sequence characterized by at least 80% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 1 or SEQ ID NO: 3, preferably, where the plant cell contains a polynucleotide containing, consisting of or essentially consisting of a sequence characterized by at least 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3.

Preferably, said plant cell comprises a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID No: 8, or at least 93% sequence identity with SEQ ID NO: 2 or SEQ ID No: 4, preferably wherein the plant cell comprises a polynucleotide containing, consisting of, or essentially consisting of a sequence having at least 93% sequence identity with SEQ ID NO: 2 or SEQ ID No: 4.

Preferably, said plant cell contains a polynucleotide comprising, consisting of or essentially consisting of a sequence characterized by at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID No: 8, or at least 93% sequence identity with SEQ ID NO: 2, or at least 94% sequence identity with SEQ ID No: 4, preferably, where the plant cell contains a polynucleotide containing, consisting of, or essentially consisting of a sequence characterized by at least 93% sequence identity to SEQ ID NO: 2 or at least 94% sequence identity to SEQ ID NO: 4. Preferably, expression and/or activity of one or more of NtAAT1-S (SEQ ID NO: 5 or SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 7 or SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 1 or SEQ ID NO: 2) and NtAAT2-T (SEQ ID NO: 3 or SEQ ID NO: 4) is modulated, while the expression and/or activity of one or more of NtAAT3-S (SEQ ID NO: 9 or SEQ ID NO: 10), NtAAT3-T (SEQ ID NO: 11 or SEQ ID NO: 12), NtAAT4-S (SEQ ID NO: 13 or SEQ ID NO: 14) and NtAAT 4-T (SEQ ID NO: 15 or SEQ ID NO: 16) is not modulated.

Preferably, at least one modification is a modification of the genome of a plant cell, or a modification of a construct, vector or expression vector, or a transgenic modification.

Preferably, the modification of the genome of the plant cell or the modification of the construct, vector or expression vector is a mutation or editing.

Preferably, such a modification provides a reduction in the expression or activity of the polynucleotide or polypeptide compared to those in the cell of the control plant.

Preferably, the plant cell contains an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 nucleotides of the RNA transcribed from the polynucleotide according to claim 1(i).

Preferably, the modulated expression or activity of the polynucleotide or polypeptide modulates the amino acid level in the dried or dried leaf derived from the plant cell compared to the amino acid level in the dried or dried leaf derived from the control plant, preferably where the amino acid is aspartate or a metabolite derived from it.

Preferably, the level of nicotine in the dried leaf or dried leaf obtained from the plant cell is substantially the same as the level of nicotine in the dried leaf or dried leaf obtained from the control plant cell; and/or where the level of acrylamide in the dried leaf or dried leaf obtained from the plant cell is reduced compared to the level of acrylamide in the dried leaf or dried leaf obtained from the control plant, and/or where the level of ammonia in the dried leaf or dried leaf obtained from the plant cell is reduced compared to the level of amino acids in the dried leaf or dried leaf obtained from from the control plant.

In an additional aspect, a plant or part thereof is described that contains a plant cell as described herein. In a further aspect, a plant or part of the plant described herein is described, wherein the amount of aspartate or metabolite derived therefrom is modified in at least part of the plant compared to that of a control plant or part thereof.

In a further aspect, a plant material, a dried plant material, or a homogenized plant material obtained from a plant or part thereof as described herein is described, preferably wherein the dried plant material is an air-dried plant material, a sun-dried plant material, or a fire-dried plant material.

In an additional aspect, the plant material described herein is described, including the biomass, seed, stem, flowers or leaves of a plant or parts thereof, as described herein.

In a further aspect, a tobacco product is described comprising a plant cell as described herein, a plant part as described herein, or plant material as described herein.

In an additional aspect, a method for producing a plant described herein is described, comprising the following steps: (a) obtaining a plant cell containing a polynucleotide containing, consisting of, or essentially consisting of a polynucleotide containing, consisting of, or essentially consisting of a sequence characterized by at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SE Q ID NO: 13 or SEQ ID NO: 15; (b) modifying a plant cell to modulate the expression of said polynucleotide as compared to that in a control plant cell; and (c) propagating the plant cell to obtain a plant.

Preferably, step (c) comprises cultivating a plant from a shoot or seedling containing a plant cell.

Preferably, the step of modifying a plant cell includes modifying the genome of the cell using genome editing or genomic engineering techniques.

Preferably, genome editing or genomic engineering techniques are selected from CRISPR/Cas technology, zinc finger nuclease mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide directed mutagenesis, and meganuclease mediated mutagenesis.

Preferably, the step of modifying a plant cell comprises transfecting the cell with a construct comprising a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 operably linked to the constitutive pro motor.

Preferably, the step of modifying the plant cell comprises introducing into the cell an interference polynucleotide containing a sequence that is at least 80% complementary to the RNA transcribed from the polynucleotide according to claim 1(i).

Preferably, the plant cell is transfected with a construct expressing an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 RNA nucleotides transcribed from the polynucleotide according to claim 1(i).

In an additional aspect, a method is described for obtaining a dried plant material with an adjusted amount of aspartate or a metabolite derived from it, compared to those in a control plant material, comprising the following steps: (a) obtaining a plant or part thereof or plant material described herein; (b) optionally collecting plant material from those; and (c) drying the plant material.

Preferably, the plant material comprises dried leaves, dried stems or dried flowers, or a mixture thereof.

Preferably, the drying method is selected from the group consisting of air drying, flame drying, flue drying and flue drying.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1 is a graph showing the content of total free amino acids after harvest (mature), two days after drying (48 hours) and at the end of drying in cultivated Virginia, Burley and Oriental tobacco.

Figure 2 is a graph showing the amounts of aspartate (asp) and asparagine (asn) after harvest in Swiss Burley tobacco leaf samples grown in the field (three replications of the leaf in bulk). The content of free amino acids was measured in leaf samples (located in the middle region of the stem), which were collected at regular intervals in the barn for air processing before completion of 50 days of drying.

Figure 3 is a graph showing the content of nicotine in the leaves located in the middle region of the stem, plants NtAAT2-S/T RNAi T0 (E324) and the corresponding control plants (CTE324).

Figure 4 is a graph showing the content of asparagine in the leaves located in the middle region of the stem, plants NtAAT2-S/T RNAi T0 (E324) and the corresponding control plants (CTE324).

DETAILED DESCRIPTION

The section headings used in this disclosure are for organizational purposes and are not intended to be limiting.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as usually understood by a person of ordinary skill in the art. In the event of conflict, this document, including the definitions, shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

The terms "include(includes)", "include(includes)," "characterized", "has", "may", "comprise(contains)", and variations thereof, as used herein, are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures.

The singular forms include references to the plural unless the context clearly dictates otherwise.

The term "and/or" means (a) or (b) or both (a) and (b).

The present invention contemplates other embodiments "comprising", "consisting of" and "essentially consisting of" the embodiments or elements set forth herein, whether explicitly stated or not.

When numeric ranges are set forth in this document, each intermediate number is provided explicitly with the same degree of precision. For example, in the case of the range 6-9, the numbers 7 and 8 are provided in addition to 6 and 9, and in the case of the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly provided.

The following terms, as used throughout this specification and claims, have the following meanings.

The terms "coding sequence" or "polynucleotide encoding" means the nucleotides (RNA or DNA molecules) that make up a polynucleotide that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of the individual or mammal receiving the polynucleotide. The coding sequence may be codon-optimized.

The terms "complementary sequence" or "complementary" may refer to Watson-Crick base pairing (e.g., A-T/U and C-G) or Hoogsteen between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared by two polynucleotides that when they are anti-parallel aligned with each other, the nucleotide bases at each position are complementary to each other.

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

The term "control" in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function, or activity of one or more of the same genes or polypeptides has not been modified (e.g., increased or decreased), and therefore may allow comparison with a plant in which the expression, function, or activity of one or more of the same genes or polypeptides has been modified. Used in this document, the expression "control plant" means a plant that is practically equivalent to the test plant or modified plant in all respects, with the exception of the tested parameters. For example, if we are talking about a plant in which the polynucleotide has been introduced, the control plant is an equivalent plant in which such a polynucleotide has not been introduced. The control plant may be an equivalent plant into which the control polynucleotide has been introduced. In such cases, a control polynucleotide is a polynucleotide for which its phenotypic effect on the plant is expected to be negligible or absent. The control plant may contain an empty vector. The control plant may correspond to the wild type plant. The control plant may be a null segregant, with the T1 segregant no longer containing the transgene.

The terms "donor DNA" or "donor template" refer to a double stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA may encode a fully functional polypeptide or a partially functional polypeptide.

The term "endogenous gene or polypeptide" refers to a gene or polypeptide that is derived from the genome of an organism and has not undergone a change, such as loss, gain or replacement of genetic material. An endogenous gene undergoes normal gene transfer and gene expression. The endogenous polypeptide undergoes normal expression.

The term "enhancer sequences" refers to sequences that can increase gene expression. These sequences may be located upstream, within the introns, or downstream of the transcribed region. The transcribed region consists of exons and introns located between them, from the promoter to the transcription termination site. The increase in gene expression can occur through various mechanisms, including increased transcriptional efficiency, stabilization of mature mRNA, and increased translation.

The term "expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment may refer to the transcription of a polynucleotide fragment (eg, transcription resulting in mRNA or functional RNA) and/or translation of the mRNA to produce a precursor or mature polypeptide. "Overexpression" refers to the production of a gene product in transgenic organisms at levels that exceed those in a null-segregant (or non-transgenic) organism from the same experiment.

The terms "functional" and "fully functional" describe a polypeptide that has a biological function or activity. The term "functional gene" refers to a gene that is transcribed to form an mRNA that is translated to form a functional or active polypeptide.

The term "genetic construct" refers to DNA or RNA molecules that contain a polynucleotide that encodes a polypeptide. The coding sequence may contain initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of driving expression.

The term "genome editing" refers to such a change in an endogenous gene that encodes an endogenous polypeptide, which achieves the expression of a polypeptide that is a truncated endogenous polypeptide or an endogenous polypeptide having an amino acid substitution. Genome editing may include replacing the portion of an endogenous gene to be targeted, or replacing the entire endogenous gene with a copy of the gene that is characterized by the presence of a truncation or amino acid substitution, using a repair mechanism such as HDR. Genome editing may also include creating an amino acid substitution in an endogenous gene by creating a double strand break in the endogenous gene, which is then repaired using NHEJ. NHEJ can be used to add or delete at least one base pair during repair, whereby an amino acid substitution can be obtained. Genome editing may also include deletion of a gene segment by simultaneous action of two nucleases on the same strand of DNA to effect truncation between two nuclease target sites, and repair of the DNA break by NHEJ.

The term "heterologous" in relation to a sequence means a sequence that is derived from a foreign species or, if derived from the same species, is substantially modified from its native form in composition and/or genomic locus due to intentional human interference.

The terms "repair by homologous recombination" or "HDR" refers to the mechanism of repair of double-stranded DNA damage in cells when a homologous DNA fragment is present in the nucleus, mainly in the G2 and S phases of the cell cycle. HDR uses donor DNA or a donor template to guide repair and can be used to create specific sequence changes in the genome, including targeted addition of entire genes. If the donor template is provided together with a site-specific nuclease, then the cellular apparatus will repair the break with homologous recombination, which is magnified by several orders of magnitude in the presence of DNA cleavage. If a homologous DNA fragment is not present, then NHEJ may occur instead.

The terms "homology" or "similarity" refers to the degree of sequence similarity between two polypeptides or two polynucleotide molecules compared by sequence alignment. The degree of homology between two individual compared polynucleotides is a function of the number of identical or matching nucleotides at the compared positions.

The terms "identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences are characterized by the presence of a certain percentage of residues that are the same within a certain area. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences within a given region, determining the number of positions where both sequences have identical residues to get the number of matching positions, dividing the number of matching positions by the total number of positions in the defined region, and multiplying the result by 100 to get the percent sequence identity. In cases where the two sequences are of different lengths, or the alignment creates one or more non-symmetrically spaced ends, and a certain comparison region includes only one sequence, then the residuals of the single sequence are included in the denominator and not in the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. The identity can be determined by yourself or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or the Smith-Waterman algorithm. 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=10.0, gap extension penalty=0.2 and matrix=Gonnet. For DNA and protein alignments: ENDGAP=-1 and GAPDIST=4. Those skilled in the art will appreciate that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, the percentage of identities is then calculated based on this alignment in the form (N/T), where N is the number of positions at which an identical residue is present in the sequences, and T is the total number of positions compared, including gaps but excluding overhangs.

The terms "increase" or "increased" refer to an increase of from about 10% to about 99%, or an increase of 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%, at least 99%, at least 100%, at least 150%, or at least 200%, or more, of an amount or function or activity, such as, without limitation, polypeptide function or activity, transcriptional function or activity, and/or protein expression. The term "increased" or the phrase "increased amount" may refer to an amount or function or activity of a modified plant or a product derived from a modified plant that is greater than would be found in a plant or product from the same plant variety treated in the same manner that has not been modified. Thus, in some cases, a wild-type plant of the same variety that has been processed in the same way is used as a control by which it is measured whether an increase in quantity has been achieved.

The term "reduction" or "reduced" as used herein refers to a reduction of from about 10% to about 99%, or a reduction of 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%, at least 99%, or at least 100%, or at least 150%, or at least 200%, or more, of an amount or function, such as polypeptide function, transcriptional function, or polypeptide expression. The term "increased" or the phrase "increased amount" may refer to an amount or function in a modified plant or product derived from a modified plant that is less than would be found in a plant or product from the same plant variety treated in the same manner that has not been modified. Thus, in some cases, a wild-type plant of the same variety that has been processed in the same way is used as a control to measure whether a reduction has been achieved.

The term "inhibit" or "inhibited" refers to a decrease of from about 98% to about 100%, or a decrease of at least 98%, at least 99%, and in particular 100%, of the amount or function or activity, such as, without limitation, the function or activity of a polypeptide, the transcriptional function or activity and/or expression of a polypeptide.

The term "introduced" means the delivery of a polynucleotide (eg, construct) or polypeptide into a cell. The expression "introduced" includes reference to the insertion of a polynucleotide into a eukaryotic cell, where the polynucleotide may be inserted into the cell's genome, and also includes a reference to the transient introduction of the polynucleotide or polypeptide into the cell. The expression "introduced" includes references to methods of stable or transient transformation, as well as to sexual interbreeding. Thus, the expression "introduced" in relation to the insertion of a polynucleotide (e.g., recombinant construct/expression construct) into a cell means "transfection" or "transformation" or "transduction" and includes reference to the insertion of the polynucleotide into a eukaryotic cell, where the polynucleotide can be inserted into the genome of the cell (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or expressed transient but (for example, transfected mRNA).

The terms "isolated" or "purified" refer to material that is substantially or essentially free of the components that normally accompany it, as found in its native state. Typically, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The polypeptide, which is the predominant molecule present in the formulation, is substantially purified. In particular, the isolated polynucleotide is separated from the open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" as used herein means that the polynucleotide or polypeptide yields substantially one band on an electrophoretic gel. In particular, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. Isolated polynucleotides can be purified from the host cell in which they naturally occur. Conventional nucleic acid purification methods known to those skilled in the art can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

The terms "modulate" or "modulating" refer to providing or facilitating a qualitative or quantitative change, adjustment or modification of a method, pathway, function or activity of interest. Without limitation, such a change, adjustment or modification may be an increase or decrease in the level of the corresponding process, pathway, function or activity of interest. For example, gene expression or polypeptide expression or the function or activity of a polypeptide can be modulated. Typically, relative change, adjustment or modification is determined by comparison with a control.

The term "non-homologous end-joining (NHEJ) pathway" as used herein refers to a pathway by which DNA double-strand breaks are repaired by direct ligation of the break ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process in which random microinsertions and microdeletions (inserts/deletions) are introduced at the DNA break point. This method can be used to deliberately destroy, delete or correct the reading frame in the sequences of target genes. Typically, NHEJ uses short homologous DNA sequences called microhomologues to guide repair. These microhomologues are often present in single strand protrusions at the ends of double strand breaks. If the overhangs are perfectly compatible, NHEJ will usually repair the gap accurately, although there may also be inaccurate repair resulting in loss of nucleotides, but it is much more common for the overhangs to be mismatched.

The term "non-naturally occurring" describes an entity such as a polynucleotide, genetic mutation, polypeptide, plant, plant cell, and plant material that is not naturally occurring or does not exist in nature. Such non-naturally occurring objects or artificial objects can be created, synthesized, initiated, modified, tampered with or manipulated in the ways described herein or as known in the art. Such non-naturally occurring objects or man-made objects can be created, synthesized, initiated, modified, tampered with or manipulated by humans. Thus, by way of example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material can be generated using conventional plant breeding techniques such as backcrossing, or gene manipulation techniques such as antisense RNA, interfering RNA, meganuclease, etc. As a further example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material can be generated by and or by transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell 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 does not occur naturally, or that does not exist 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 that of the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs naturally in a polynucleotide or polypeptide, such as a gene or polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, determining the presence or absence of genetic markers (eg microsatellite RNA markers).

The terms "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of an individual strand also defines the sequence of the complementary strand. Thus, the polynucleotide also spans a strand complementary to the single strand described. Many variants of the polynucleotide can be used for the same purpose as the specified polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and their complementary sequences. A single strand is a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, the polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single or double stranded, or may contain portions of both double and single stranded sequences. The polynucleotide may be DNA, either genomic or cDNA, RNA, or a hybrid molecule, where the polynucleotide may contain combinations of deoxyribo- and ribonucleotides, as well as combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be produced by chemical synthesis methods or by recombinant methods.

The specificity of single-stranded DNA for hybridization with complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al ., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). The rigidity of hybridization increases as the propensity to form DNA duplexes decreases. In polynucleotide hybridization reactions, stringency can be chosen to promote specificity (high stringency) hybridization reactions that can be used to identify, for example, full length clones from a library. Hybridization reactions with less specificity (low stringency) can be used to identify related but not exactly matching (homologous but not identical) DNA molecules or segments. DNA duplexes are stabilized by (1) a certain number of complementary base pairs; (2) a certain type of base pairs; (3) concentration of salts (ionic strength) in the reaction mixture; (4) the reaction temperature; and (5) the presence of certain organic solvents, such as formamide, which reduce the stability of the DNA duplex. Generally, the longer the probe, the higher the temperature required for proper annealing. The generally accepted approach is to change the temperature; higher relative temperatures lead to more severe reaction conditions. For "stringent conditions" hybridization, hybridization protocols have been described in which polynucleotides with at least 60% homology to one another remain hybridized. Generally, stringent conditions are chosen such that the temperature is approximately 5° C. lower than the melting point (Tm) temperature for a particular sequence at certain ionic strengths and pH. Tm is the temperature (at certain values of ionic strength, pH, and polynucleotide concentration) at which 50% of probes complementary to a given sequence hybridize to that sequence at equilibrium. Since these sequences are usually present in excess, at Tm 50% of the probes are occupied in the equilibrium state.

"Stringent hybridization conditions" are conditions that allow a probe, primer, or oligonucleotide to hybridize only to its particular sequence. Stringent conditions are sequence dependent and will vary. Stringent conditions typically include: (1) low ionic strength and high temperature washes, eg 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) the presence of a denaturing agent during hybridization, e.g. 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5) at 42°C, or (3) the presence of 50% formamide. Typically, washes also include 5 x SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon milt DNA (50 µg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C. washing at 42° C. in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C. followed by a high stringency wash with 0.1 x SSC containing EDTA at 55° C. Preferably, conditions are such that sequences that are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

Under "moderate stringency conditions", wash solutions and hybridization conditions are used that are less stringent, such that the polynucleotide hybridizes to all of the polynucleotide in question, its fragments, derivatives, or analogs. One example involves hybridization in 6x SSC, 5x Denhardt's solution, 0.5% SDS and 100 μg/ml denatured DNA from salmon milt at 55°C followed by one or more washes in 1x SSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc. can be adjusted to match experimental factors such as probe length. Other conditions of moderate stringency have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, N.J. (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, N.Y. (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, N. Y. (1988)).

"Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than those for moderate stringency, such that the polynucleotide hybridizes to all of the polynucleotide in question, its fragments, derivatives, or analogs. A non-limiting example of low stringency hybridization conditions is hybridization in 35% formamide, 5 x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% ficolle, 0.2% BSA, 100 μg/ml denatured DNA from salmon milt, 10% (w/v) sulf those of dextran at 40°C followed by one or more washes in 2 x SSC, 25 mm Tris-HCl (pH 7.4), 5 mm EDTA and 0.1% SDS at 50°C. Other conditions of low stringency are well described, such as conditions for interspecific hybridization variants (see Ausubel et al., 1993; Kriegler, 1990).

The term "operably linked" means that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter may be located 5' (above) or 3' (below) from a gene that is under its control. The distance between a promoter and a gene may be about the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changing this distance can be done in concert without loss of promoter function. The term "operably linked" refers to the association of polynucleotide fragments in one fragment in such a way that the function of one is regulated by the other. For example, a promoter is operably linked to a polynucleotide fragment if it is capable of regulating transcription of that polynucleotide fragment.

The term "plant" refers to any plant at any stage of its life cycle or development and its descendants. In one embodiment, the plant is a tobacco plant that belongs to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant shoots, plant seeds, and plant cells and their progeny. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic sites, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plants are described herein.

The terms "polynucleotide", "polynucleotide sequence" or "polynucleotide fragment" are used interchangeably herein and refer to an RNA or DNA polymer that is single or double stranded and optionally contains synthetic, non-natural or adjusted nucleotide bases. Nucleotides (usually in the form of their 5'-monophosphate) are designated by their one-letter designations as follows: "A" for adenylate or deoxyadenylate (respectively for RNA or DNA), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for pur ines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. The 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, 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. Additionally, the polynucleotide may be composed of three-stranded sections containing DNA, RNA, or both, or fragment(s). The polynucleotide may contain one or more modified bases such as phosphothioates and may be a peptide nucleic acid (PNA). 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 polynucleotides described herein are presented as DNA sequences, the polynucleotides include their respective RNA sequences and their complementary (eg, fully complementary) DNA or RNA sequences, including their reverse complementary strands. The polynucleotides of the present invention are shown in the accompanying sequence listing.

The terms "polypeptide" or "polypeptide sequence" refer to a polymer of amino acids in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The terms also include modifications including, but not limited to, glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present invention are presented in the attached sequence listing.

The term "promoter" means a synthetic or naturally derived molecule that is capable of providing, activating or enhancing the expression of a polynucleotide in a cell. The term refers to an element/sequence of a polynucleotide located, as a rule, higher in sequence and functionally linked to a fragment of a double-stranded polynucleotide. 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 polynucleotide segments. The promoter may contain one or more specific transcription control sequences to further enhance expression and/or adjust expression spatially and/or temporally. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.

The terms "tissue-specific promoter" and "tissue-preferred promoter", used interchangeably herein, refer to a promoter that is predominantly, but not necessarily exclusively, expressed in one organ or tissue, but may also be expressed in one particular cell. The term "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. The expression "constitutive promoter" refers to a promoter that causes the expression of a gene in most cell types in most cases. An "inducible promoter" provides for selective expression of an operably linked DNA sequence in response to the presence of endogenous or exogenous stimuli, for example, via chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical and/or developmental cues. Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flood or drought, pathogens, phytohormones, wounds, or chemicals such as ethanol, jasmonate, salicylic acid, or antidotes.

The term "recombinant" as used herein refers to an artificial combination of two otherwise separated sequence segments, obtained, for example, by chemical synthesis or by manipulation of isolated polynucleotide segments using genetic engineering techniques. The term also includes reference to a cell or vector that has been modified by introducing a heterologous polynucleotide, or a cell derived from a cell so modified, but does not encompass modification of the cell or vector as a result of naturally occurring events (e.g., spontaneous mutation, natural transformation, or transduction, or transposition), such as those that occur without intentional human intervention.

The term "recombinant construct" refers to a combination of polynucleotides that do not normally occur together in nature. Accordingly, a recombinant construct may contain regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged differently than would normally occur in nature. The recombinant construct may be a recombinant DNA construct.

The expressions "regulatory sequences" and "regulatory elements", used interchangeably herein, refer to polynucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence that affect the transcription, processing, or stability of an RNA, or the translation of an associated coding sequence. Regulatory sequences include promoters, translational leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein.

The expression "site-specific nuclease" refers to an enzyme capable of specifically recognizing and cleaving DNA sequences. The site-specific nuclease may be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR/Cas9 based systems, and meganucleases.

The term "tobacco" is used collectively to refer to tobacco crops (e.g., a variety of field-grown and non-hydroponic tobacco plants), tobacco plants, and parts thereof, including but not limited to roots, stems, leaves, flowers, and seeds prepared and/or obtained as described herein. It is understood that the term "tobacco" refers to Nicotiana tabacum plants and products thereof.

The term "tobacco products" refers to consumer tobacco products, including, without limitation, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, chewing gum, and lozenges, and components, materials, and ingredients for the manufacture of consumer tobacco products. Preferably, these tobacco products are made from tobacco leaves and stems harvested from tobacco and cut, dried, dried and/or fermented in accordance with conventional tobacco production techniques.

The terms "transcriptional terminator", "termination sequences", or "terminator" refer to DNA sequences downstream of a coding sequence, including recognizable polyadenylation sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is typically characterized by the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

The term "transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including parental transgenic events, as well as those obtained by sexual crossing or asexual reproduction procedures from the original transgenic event. The term does not cover the adjustment of the genome (chromosomal or extrachromosomal) by conventional plant breeding techniques or by naturally occurring events such as accidental cross-pollination, infection by a non-recombinant virus, transformation by non-recombinant bacteria, non-recombinant transposition, or spontaneous mutation.

The expression "transgenic plant" refers to a plant which contains in its genome one or more heterologous polynucleotides, i.e. a plant which contains recombinant genetic material not normally found in it, and which has been introduced into the plant in question (or into the plant's ancestors) through human manipulation. For example, a heterologous polynucleotide may be stably integrated into the genome such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically improved germplasm has also progressed to the stage of introducing several traits into cultivated plants, which is often referred to as the gene pyramiding approach. In this approach, several genes can be introduced into a plant, conferring different characteristics of interest. Gene pyramiding can be accomplished in many ways, including, but not limited to, co-transformation, re-transformation, and crossing lines with different transgenes. Thus, a plant grown from a cell of a plant into which recombinant DNA is introduced by transformation is a transgenic plant, as is all progeny of that plant that contains the introduced transgene (either sexually or asexually). The term "transgenic plant" is understood to encompass the entire plant or tree and parts of the plant or tree, such as grains, seeds, flowers, leaves, roots, fruits, pollen, stems, and the like. Each heterologous polynucleotide can confer a distinct trait on the transgenic plant.

The term "transcriptional activator-like effector" or "TALE" refers to a polypeptide structure that recognizes and binds to a specific DNA sequence. The term "TALE DNA binding domain" refers to a DNA binding domain that contains an array of 33-35 amino acid tandem repeats, also known as RVD modules, each of which specifically recognizes one DNA base pair. RVD modules can be arranged in any order, gathering in an array that recognizes a certain sequence. The binding specificity of the TALE DNA binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids. The DNA binding domain of TALE can have 12 to 27 RVD modules, each containing an RVD and recognizing one DNA base pair. Specific RVDs have been identified that recognize each of the four possible DNA nucleotides (A, T, C and G). Since the DNA binding domains of TALE are modular, the repeats that recognize four different DNA nucleotides can be joined together to recognize any particular DNA sequence. These targeted DNA binding domains can then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

The terms "effector nucleases like transcription activators" or "TALEN", used interchangeably herein, refer to engineered fusion polypeptides based on the catalytic domain of a nuclease, such as Fok I endonuclease, and a designed TALE DNA-binding domain that can be targeted to a specifically synthesized DNA sequence.

The expression "TALEN monomer" refers to an engineered fusion polypeptide with a nuclease catalytic domain and a designed TALE DNA-binding domain. The two TALEN monomers can be designed to target and cleave the TALEN target site.

The term "transgene" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into another organism. This non-native DNA segment may retain the ability to produce an RNA or polypeptide in the transgenic organism, or it may provide an adjustment to the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to change the phenotype of the organism.

The term "variant" in relation to a polynucleotide means: (i) a portion or fragment of a polynucleotide; (ii) a sequence complementary to the polynucleotide or a portion thereof; (iii) a polynucleotide substantially identical to said polynucleotide or its complementary sequence; or (iv) a polynucleotide that hybridizes under stringent conditions to said polynucleotide, its complementary sequence, or its substantially identical polynucleotide.

The term "variant" in relation to a peptide or polypeptide means a peptide or polypeptide that differs in sequence by insertion, deletion or conservative substitution of amino acids, but retains at least one biological function or activity. A variant may also mean a polypeptide that retains at least one biological function or activity. Conservative amino acid substitution, i.e. substitution of an amino acid with another amino acid with similar properties (for example, degree of hydrophilicity and the distribution of charged sites) is understood in the art as generally including little change.

The term "variety" refers to a population of plants that have permanent characteristics that separate them from other plants of the same species. Although a variety has one or more distinguishing characteristics, it is additionally characterized by very little overall variation between individuals within that variety. The variety is often sold commercially.

The term "vector" refers to a polynucleotide delivery vehicle that contains a combination of polynucleotide components to transport polynucleotides, polynucleotide constructs and polynucleotide conjugates, etc. The vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as double-stranded circular plasmids; linearized plasmids from a double-stranded nucleotide sequence; and other vectors of any origin. An "expression vector" as used herein is a polynucleotide delivery vehicle that contains a combination of polynucleotide components to allow expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates, and the like. Suitable expression vectors include episomes capable of extrachromosomal replication, such as double-stranded circular plasmids; linearized plasmids from a double-stranded nucleotide sequence; and other functionally equivalent expression vectors of any origin. The expression vector contains at least a promoter upstream and operably linked to a polynucleotide, polynucleotide constructs, or polynucleotide conjugate, as defined below.

The term "zinc finger" refers to a polypeptide structure that recognizes and binds to DNA sequences. The zinc finger domain is the most common DNA-binding motif in the human proteome. One zinc finger domain contains approximately 30 amino acids and typically functions by binding to 3 consecutive base pairs of DNA through single amino acid side chain interactions with each base pair.

The term "zinc finger nuclease" or "ZFN" refers to a chimeric polypeptide molecule containing at least one zinc finger DNA-binding domain effectively associated with at least one nuclease or portion of a nuclease capable of cleaving DNA when fully assembled.

Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure will have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature systems and techniques used in connection with cell or tissue culture, molecular biology, immunology, microbiology, polypeptide and polynucleotide genetics and chemistry, and hybridization as described herein are well known and widely used in the art. The meaning and scope of the terms should be clear, but in the event of any implicit ambiguity, the definitions given in this document take precedence over any dictionary or non-document definition. In addition, unless otherwise required by context, terms in the singular will include the plural, and terms in the plural will include the singular.

Polynucleotides

In one embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of a sequence having at least 60% sequence identity with any of the sequences described herein, including any of the polynucleotides shown in the sequence listing. Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with them. Preferably, the polynucleotide(s) described herein encode(s) an active AAT polypeptide that has a level of function or activity of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of those of the polypeptide(s) shown(s). ) in the sequence listing.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of a polynucleotide having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7.

In certain embodiments, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3.

Preferably, the isolated polynucleotide(s) comprises, consists of, or essentially consists of a sequence characterized by at least about 85%, 86%, 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: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

Preferably, the isolated polynucleotide(s) comprises, consists of, or essentially consists of a sequence characterized by at least about 85%, 86%, 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: 5 or SEQ ID NO: 7.

Preferably, the isolated polynucleotide(s) comprises, consists of, or essentially consists of a sequence characterized by at least about 85%, 86%, 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: 1 or SEQ ID NO: 3.

In another embodiment, polynucleotides are provided comprising, consisting of, or essentially consisting of polynucleotides with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

In another embodiment, polynucleotides are provided that contain, consist of, or essentially consist of polynucleotides with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with SEQ ID NO: 5 or SEQ ID NO: 7.

In another embodiment, polynucleotides are provided that contain, consist of, or essentially consist of polynucleotides with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with SEQ ID NO: 1 or SEQ ID NO: 3.

In another embodiment, fragments of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 are provided with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them, characterized by at least about 80%, 85%, 86% 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15.

In another embodiment, fragments of SEQ ID NO: 5 or SEQ ID NO: 7 are provided with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them, characterized by at least about 80%, 85%, 86% 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: 5 or SEQ ID NO: 7.

In another embodiment, fragments of SEQ ID NO: 1 or SEQ ID NO: 3 are provided with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with them, characterized by at least about 80%, 85%, 86% 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: 3.

In another embodiment, polynucleotides are provided that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 5 or SEQ ID NO: 7 that encode a polypeptide that functions as an AAT.

In another embodiment, polynucleotides are provided that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 that encode a polypeptide that performs the function of an AAT.

In another embodiment, polynucleotides are provided that provide a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 that encode a polypeptide that functions as an AAT.

In another embodiment, polynucleotides are provided that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 5 or SEQ ID NO: 7 that encode a polypeptide that functions as an AAT.

In another embodiment, polynucleotides are provided that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 that encode a polypeptide that performs the function of an AAT.

In another embodiment, a polymer is provided that is a polynucleotide that comprises, consists of, or essentially consists of, the polynucleotide referred to herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

In another embodiment, a polymer is provided that is a polynucleotide that contains, consists of, or essentially consists of, a polynucleotide referred to herein as SEQ ID NO: 5 or SEQ ID NO: 7.

In another embodiment, a polymer is provided that is a polynucleotide that contains, consists of, or essentially consists of, a polynucleotide referred to herein as SEQ ID NO: 1 or SEQ ID NO: 3.

Preferably, the polynucleotides described herein encode an AAT polypeptide.

As described herein, NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3) are the genes expressed at the highest level after 48 hours of drying compared to green tobacco leaves. The expression level can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 times that of green tobacco leaves. The use of NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3) is preferred in certain embodiments of the present invention. The polynucleotide may include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide may be, without limitation, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragment(s) thereof. In addition, the polynucleotide can 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. Additionally, the polynucleotide may be composed of three-stranded sections containing DNA, RNA, or both, or fragment(s). 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 polynucleotides described herein are presented as DNA sequences, they include their respective RNA sequences and their complementary (eg, fully complementary) DNA or RNA sequences, including their reverse complementary sequences.

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

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

Other analogs include peptide polynucleotides, which are those based on peptide analogs of polynucleotides. These backbones are substantially 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 of a polynucleotide may have improved hybridization kinetics. Peptide polynucleotides are characterized by greater changes in melting point in the presence of mismatched base pairs compared to perfectly matched base pairs. DNA and RNA, as a rule, are characterized by a decrease in the melting point by 2-4 C in the presence of an internal mismatch. In the case of a non-ionic peptide backbone of a polynucleotide, the drop is closer to 7-9C. Similarly, due to their non-ionic nature, the hybridization of bases attached to these backbones is relatively insensitive to salt concentration. Additionally, 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 hybridization based assays or as primers for use in amplification based 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. Thus, according to one aspect, a method for detecting a polynucleotide is also provided, comprising the use of probes or primers, or both. Illustrative primers are described in this document.

The main parameters influencing the choice of hybridization conditions 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 Harbor, N.Y.). Using the genetic code data in combination with the polypeptide 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 include 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.

Also potentially useful are polynucleotides and oligonucleotides (e.g., primers or probes) that hybridize under conditions of reduced stringency, typically medium stringency, and typically high stringency, to the polynucleotide(s) described herein. The main parameters influencing the selection of hybridization conditions and guidance for developing suitable conditions are set forth in Sambrook J., E. F. Fritsch and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and can be readily determined by those of ordinary skill in the art based on, for example, the length or base composition of the polynucleotide.

This document defines one way to achieve conditions of moderate and high stringency. It should be understood that the temperature of flushing and the concentration of salts during washing with need can be adjusted to achieve the necessary degree of stiffness by applying the basic principles that control the hybridization reactions and stability of the duplex, as specialists know in this field of technology and are additionally described below (see, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Maniatis (1989, Maniatis Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y). When hybridization of polynucleotide with polynucleotide with an unknown sequence, it is assumed that the length of the hybrid will be the same as a hybridized polynucleotide. hybridized polynucleotides with known sequences, the length of the hybrid can be determined by aligning the sequences of polynucleotides and identifying the site or sites with optimal complementarity of the sequence. The temperature of hybridization for hybrids, the length of which is allegedly less than 50 pairs of base, should be less than the melting temperature of the hybrid, where the melting temperature is determined in accordance with the following. equations. For hybrids less than 18 bp in length, melting point (°C) = 2(number of bases A+T)+4(number of bases G+C). 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=0.165 M). 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 has at least 60% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 7%, 98%, 99% or 100%) with the 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 first 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 first 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 with respect to the first 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 first 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 the first sequence (the 5'-3' direction) and the sequence inversely complementary to the first sequence (the first sequence located 5'-3') are located within the same polynucleotide molecule/strand. The sequences set forth herein are shown in the 5'-3' direction.

At least one modification (eg, mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 5), NtAAT1-T (SEQ ID NO: 7), NtAAT2-S (SEQ ID NO: 1), NtAAT2-T (SEQ ID NO: 3), NtAAT3-S (SEQ ID NO: 9), NtAAT3-T ( SEQ ID NO: 11), NtA AT4-S (SEQ ID NO: 13) and NtAAT4-T (SEQ ID NO: 15).

In certain embodiments, at least one modification (eg, mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 5) and NtAAT1-T (SEQ ID NO: 7).

In certain embodiments, at least one modification (eg, mutation) may be included in one or more of NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3).

In certain embodiments, at least one modification (e.g., a mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 5), NtAAT1-T (SEQ ID NO: 7), NtAAT2-S (SEQ ID NO: 1), and NtAAT2-T (SEQ ID NO: 3).

In certain embodiments, at least one modification (e.g., a mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 5), NtAAT1-T (SEQ ID NO: 7), NtAAT2-S (SEQ ID NO: 1), and NtAAT2-T (SEQ ID NO: 3), wherein one or more of NtAAT3-S (SEQ ID NO: 9), NtAAT3 -T (SE Q ID NO: 11), NtAAT4-S (SEQ ID NO: 13), and NtAAT4-T (SEQ ID NO: 15) do not include modification(s) (eg, mutation(s)).

In certain embodiments, at least one modification (e.g., a mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 5), NtAAT1-T (SEQ ID NO: 7), NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3), while NtAAT3-S (SEQ ID NO: 9), NtAAT3 -T (SEQ ID NO: 11), NtAAT4-S (SEQ ID NO: 13) and NtAAT4-T (SEQ ID NO: 15) do not include modification(s) (eg, mutation(s)).

Polypeptides

In another aspect, an isolated polypeptide is provided comprising, consisting of, or essentially consisting of, a polypeptide having at least 60% sequence identity with any of the polypeptides described herein, including any of the polypeptides shown in the sequence listing. Preferably, the isolated polypeptide contains, consists of, or essentially consists of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 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 them.

In one embodiment, a polypeptide is encoded by any of the polynucleotides described herein, including a polynucleotide having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

In another embodiment, an isolated polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 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 SEQ ID NO: 6, SEQ ID No: 8, SEQ ID NO: 2, SEQ ID No: 4, SEQ ID NO: 10 or SEQ ID No: 12.

In another embodiment, an isolated polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 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 SEQ ID NO: 2 or SEQ ID No: 4.

In another embodiment, an isolated polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 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 SEQ ID NO: 6 or SEQ ID No: 8.

In another embodiment, an isolated polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 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% identity to sequences with SEQ ID NO: 6 or SEQ ID No: 8; at least 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: 2 or SEQ ID No: 4; or at least 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: 14 or SEQ ID NO: 16.

The polypeptide may contain sequences that have a sufficient or significant degree of identity or similarity with SEQ ID NO: SEQ ID NO: 6, SEQ ID No: 8, SEQ ID NO: 2, SEQ ID No: 4, SEQ ID NO: 10 or SEQ ID No: 12, functioning as an AAT. The polypeptide may contain sequences that have a sufficient or significant degree of identity or similarity with SEQ ID NO: 6 or SEQ ID No: 8, functioning as an AAT. The polypeptide may contain sequences that have a sufficient or significant degree of identity or similarity with SEQ ID NO: 2 or SEQ ID No: 4, functioning as an AAT. Fragments of the polypeptide(s) typically retain some or all of the functions of the AAT or the activity of the full length sequence.

As described herein, NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3) are the genes expressed at the highest level after 48 hours of drying compared to green tobacco leaves. The expression level can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 times that of green tobacco leaves. The use of NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3) is preferred in certain embodiments of the present invention. As discussed herein, polypeptides also include mutant variants obtained by introducing any type of adjustment (e.g., insertion, deletion, or substitution of amino acids; changes in glycosylation state; changes that affect refolding or isomerization, three-dimensional structures, or self-association states) that can be intentionally engineered or naturally isolated, provided that they still retain some or all of their functionality or activity. Preferably, the function or activity is modulated.

A deletion refers to the removal of one or more amino acids from a polypeptide. An insert refers to one or more amino acid residues introduced at a predetermined location in a polypeptide. Insertions may include insertions into the sequence of one or more amino acids. Substitution refers to the substitution of amino acids in a polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions typically include a single residue, but may be grouped depending on the functional limitations imposed on the polypeptide, and may range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions and/or insertions can be performed using peptide synthesis techniques, such as solid phase peptide synthesis, or by manipulating recombinant DNA. Methods for manipulating DNA sequences to produce substitution, insertion, or deletion variants of a polypeptide are well known in the art. A variant may be characterized by the presence of adjustments that cause a "silent" change to occur and result in a functionally equivalent polypeptide. Intentional amino acid substitutions can be made based on the similarity of residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity and amphipathic nature, provided that the binding that causes the secondary structure 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 polar uncharged amino acids having 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 one another:

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 Trp Tyr

The polypeptide may be a mature polypeptide, or an immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptides may be in linear form or be cyclized using known methods. Polypeptides typically contain at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.

At least one modification (eg, mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), NtAAT2-T (SEQ ID NO: 4), NtAAT3-S (SEQ ID NO: 10), NtAAT3-T (SEQ ID NO: 12), Nt AAT4-S (SEQ ID NO: 14) and NtAAT4-T (SEQ ID NO: 16).

In certain embodiments, at least one modification (e.g., mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), and NtAAT2-T (SEQ ID NO: 4).

In certain embodiments, at least one modification (eg, mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 6) and NtAAT1-T T (SEQ ID NO: 8).

In certain embodiments, at least one modification (eg, mutation) may be included in one or more of NtAAT2-S (SEQ ID NO: 2) and NtAAT2-T (SEQ ID NO: 4).

In certain embodiments, at least one modification (e.g., mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), and NtAAT2-T (SEQ ID NO: 4), wherein one or more of NtAAT3-S (SEQ ID NO: 10), NtAAT3-T ( SEQ ID NO: 12), NtAAT4-S (SEQ ID NO: 14) and NtAAT4-T (SEQ ID NO: 16) do not include modifications (eg, mutation(s)).

In certain embodiments, at least one modification (e.g., a mutation) may be included in one or more of NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), and NtAAT2-T (SEQ ID NO: 4), wherein NtAAT3-S (SEQ ID NO: 10), NtAAT3-T (SEQ ID NO: 10), : 12), NtAAT4-S (SEQ ID NO: 14) and NtAAT4-T (SEQ ID NO: 16) do not include modifications (eg mutation(s)).

Plant modification

Transformation

Recombinant constructs can be used to transform plants or plant cells in order to modulate the expression, function, or activity of a polypeptide. 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 the expression, function, or activity of a polypeptide is modulated may include mutant, non-naturally occurring, transgenic, human-created, or genetically engineered plants or plant cells. Preferably, the transgenic plant or plant cell contains a genome that has been corrected by stable integration of recombinant DNA. Recombinant DNA contains DNA that is genetically engineered and engineered outside of a cell, and contains DNA containing naturally occurring DNA, or cDNA, or synthetic DNA. A transgenic plant may include a plant regenerated from an originally transformed plant cell and transgenic plants that are the offspring of later generations or hybrids of the transformed plant. Preferably, the transgenic modification provides for an adjustment in the expression or function or activity of a polynucleotide or polypeptide described herein compared to that in 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 backbones 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, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.

The vectors may contain, 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 resistance to biocidal agents, such as resistance to an antibiotic (eg, kanamycin, G418, bleomycin, or hygromycin) or herbicide (eg, glyphosate, chlorsulfuron, or phosphinothricin). Additionally, the expression vector may contain 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 polypeptide, glutathione-S-transferase, polyhistidine, c-myc, or hemagglutinin sequences are typically expressed as a fusion fragment with 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 the recombinant polynucleotide into its genome to ensure its stable transformation. The plant or plant cell described herein may be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. The plant or plant cell may 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 such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.

A number of methods are available in the art for transforming a plant cell, including biolistics; gene gun techniques; transformation mediated by Agrobacterium; transformation mediated by a viral vector; freeze-thaw method; microparticle bombardment; direct DNA uptake; ultrasonic treatment; microinjection; plant virus-mediated transfer; and electroporation. An Agrobacterium-based 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 polypeptide 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 polypeptide 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, if necessary, plants can be regenerated from the transformed cultures using methods known to those skilled in the art.

The choice of regulatory regions to be included in a recombinant construct depends on several factors, including, without limitation, potency, selectivity, inducibility, desired level of expression, and preferential expression in certain cells or tissues. It is a common task for a person skilled in the art to modulate the expression of a coding sequence through the correct selection of regulatory regions and their placement in relation 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.

Suitable promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present at different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of suitable promoters for directing RNAi polynucleotide production include the cauliflower mosaic virus (CaMV/35S) 35S promoter, SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin gene or phaseolin gene promoters. Many variants of recombinant promoters can be created by those skilled in the art.

Tissue-specific promoters are transcriptional controls that are active only in certain cells or tissues at certain points in time during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression may be preferential, for example, if expression of polynucleotides in certain tissues is preferred. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or mostly only) in certain tissues, such as vegetative tissues, e.g. 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, embryo specific, endosperm specific, integument specific, seed and seed coat specific, pollen specific, petal specific, sepal specific, or combinations thereof.

Suitable leaf-specific promoters include the pyruvatortophosphate dikinase (PPDK) gene promoter from a C4 plant (maize), the cab-m1Ca+2 promoter from maize, the myb-related genes promoter from Arabidopsis thaliana (Atmyb5), ribulose bisphosphate carboxylase (RBCS) promoters (e.g., tomato leaf-expressed genes RBCS 1, RBCS2, and RBCS3A). and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits, or the promoter of the ribulose bisphosphate carboxylase gene, expressed at high levels almost exclusively in mesophilic cells of leaf blades and leaf axils).

Suitable aging tissue-specific promoters include the promoter from tomato, active during fruit ripening, senescence and leaf abscission, the promoter of the gene encoding maize cysteine protease, the 82E4 promoter, and the promoter of the SAG genes. Suitable anther-specific promoters can be used. Suitable root-preferred promoters known to those skilled in the art can be selected. Suitable promoters for expression preferably in seeds include both seed-specific promoters (promoters active during seed development, such as seed storage polypeptide promoters) and germinating seed promoters (promoters active during seed germination). Such seed-preferred promoters include Cim1 (cytokinin-induced signal); 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 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. 27 kDa zein gene promoter, g-zein gene promoter, 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 accession number L223 44 in Genbank), 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 polypeptides (PR polypeptides) that are inducible after infection with a pathogen (eg, PR polypeptides, SAR polypeptides, beta-1,3-glucanase, chitinase).

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

Suitable methods for introducing polynucleotides into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., Biotechniques 4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986)), Agrobacterium-mediated transformation (U.S. Patent No. 5,981,840 and 5,563,055), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)) and ballistic particle acceleration (see, for example, US Pat. Nos. 4,945,050; 5,879,918; 5,886,244; Tissue, and Organ Culture: Fundamental Methods, ed Gamborg and Phillips (Springer-Verlag, Berlin) (1995) and McCabe et al., Biotechnology 6:923-926 (1988)).

Mutation

Disclosed is a plant or plant cell containing a mutation in one or more of the polynucleotides or polypeptides described herein, where said mutation results in modulation of AAT function or activity. In addition to the mutations described, the mutant plant or plant cells may be characterized by the presence of one or more additional mutations either in the same polynucleotides or polypeptides as described herein or in one or more other polynucleotides or polypeptides in the genome.

Also provided is a method for modulating the level of an AAT polypeptide described herein in a (dried) plant or in a (dried) plant material, said method comprising introducing into the genome of said plant one or more mutations that modulate the expression of at least one gene, wherein said at least one gene is selected from sequences according to the present invention.

Also provided is a method for identifying a plant with modulated AAT levels, said method comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in sequences in accordance with the present invention, and optionally correlating the identified mutation(s) with mutation(s) known to modulate levels of one or AAT.

Also disclosed is a plant or cell that is heterozygous or homozygous for one or more mutations in a gene of the present invention, said mutation resulting in modulated expression of the gene or the function or activity of the polypeptide encoded by it.

A number of approaches can be used to combine mutations in a single plant, including sexual crossing. A plant characterized by the presence of one or more favorable heterozygous or homozygous mutations in a gene according to the present invention that modulate the expression of a gene or the function or activity of the polypeptide encoded by such can be crossed with a plant characterized by the presence of one or more favorable heterozygous or homozygous mutations in one or more genes that modulate their expression or function or activity of the polypeptide that encodes oh those. In one embodiment, crosses are carried out to introduce one or more favorable mutations in a heterozygous or homozygous state in a gene in accordance with the present invention in the same plant.

The function or activity of one or more polypeptides of the present invention in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to suppress the function or activity of that polypeptide and that has been cultured, harvested and dried using the same protocols.

In some embodiments, the mutation(s) are introduced into a plant or plant cell using a mutagenesis enabling approach, and the introduced mutation is identified or selected using methods known to those skilled in the art, such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or that disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in exons of a gene, as a rule, result in the formation of non-functional mutant variants. Mutations at conserved residues may be particularly effective in suppressing the metabolic function of the encoded polypeptide. For example, it is understood that a mutation in one or more of the highly conserved regions will likely provide an adjustment to the function of the polypeptide, while a mutation outside of these highly conserved regions will likely have little or no effect on the function of the polypeptide. Additionally, a single nucleotide mutation can result in a stop codon, the presence of which will result in a truncated polypeptide and, depending on the degree of truncation, loss of function.

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 radiation-induced mutagenesis, modified base mutagenesis, DNA duplex mutagenesis gap mutagenesis, double-strand break mutagenesis, mutagenesis using repair-defective host lines, mutagenesis by whole gene synthesis, DNA shuffling, and other equivalent methods.

Also disclosed are fragments of polynucleotides and polypeptides. Polynucleotide fragments may encode polypeptide fragments that retain the biological function of the native polypeptide and are therefore involved in the plant's metabolite transport network. Alternatively, polynucleotide fragments that are useful as hybridization probes or PCR primers generally do not encode polypeptide fragments that retain biological function. In addition, fragments of the disclosed polynucleotides include those fragments that can be assembled into recombinant constructs, as discussed herein. Polynucleotide fragments can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, up to a full length polynucleotide encoding the polypeptides described herein. Polypeptide 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, and up to the full length polypeptide described herein. 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-generated, or genetically engineered plants) or plant cells containing one or more mutant polypeptide variants. Preferably, mutant variants of the polypeptide retain the function of the unmutated polypeptide. The function of a mutant polypeptide variant may be higher, lower, or approximately the same as that of an unmutated polypeptide.

Mutations in the polynucleotides and polypeptides described herein may include human-made mutations, or artificial mutations, or genetically engineered mutations. Mutations in the polynucleotides and polypeptides described herein may be mutations that are made or can be made by a method that includes an in vitro or in vivo manipulation step. Mutations in the polynucleotides and polypeptides described herein may be mutations that are made or that can be made by a method that involves human intervention.

Methods by which a mutation is randomly introduced into a polynucleotide may include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals such as mutagenic, teratogenic, or carcinogenic organic compounds to induce mutations. To create mutations, mutagens can be used that create primarily point mutations and short deletions, insertions, missense mutations, simple repeat sequences, transversions and/or transitions, including chemical mutagens and radiation. Mutagens include 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, nitro zoguanidine, 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. The dose of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue so as to obtain a mutation rate that is below a threshold level characterized by lethality or reproductive sterility. Any method for obtaining a plant polynucleotide known to those skilled in the art can be used to obtain a plant polynucleotide for mutation screening.

The mutation method may include one or more plant crossing steps.

Once a mutation has been introduced, screening can be performed to identify mutations that give rise to genes with premature stop codons or otherwise non-functional genes. Once a mutation has been introduced, screening can be performed to identify mutations that give rise to functional genes that are capable of being expressed at increased or decreased levels. Screening for mutant variants can be performed by sequencing, or by using one or more probes or primers specific for a given gene or polypeptide. Polynucleotides can also be engineered to generate specific mutations that can result in modulation of gene expression, modulation of mRNA stability, or modulation of polypeptide stability. In this document, such plants are referred to as "non-naturally occurring" or "mutant" plants. Typically, mutant or non-naturally occurring plants contain at least a portion of the foreign or synthetic or human-made nucleotide (eg, DNA or RNA) that was not present in the plant prior to being manipulated. The foreign nucleotide 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.

Transgenics and editing

In addition to mutagenesis, compositions that can modulate the expression or function or activity of one or more of the polynucleotides or polypeptides described herein include 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 polypeptides; sequence-specific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to substrates or regulatory polypeptides; antibodies that show specificity for one or more polypeptides; small molecule compounds that can interfere with the stability of one or more polypeptides, or the enzymatic function of one or more polypeptides, or the binding function of one or more polypeptides; zinc finger polypeptides that bind one or more polynucleotides; and meganucleases that have a function on one or more polynucleotides. Gene editing technologies, genetic editing technologies and genome editing technologies are well known in the art.

Zinc finger nucleases

Zinc finger polypeptides can be used to modulate the expression or function 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 search is made for a unique site for binding the polypeptide to zinc fingers. Alternatively, the genomic DNA sequence is searched for two unique zinc finger binding sites for the polypeptide, with both sites on opposite strands and close to each other, e.g. 1, 2, 3, 4, 5, 6 or more base pairs apart. Accordingly, zinc finger polypeptides that bind to polynucleotides are provided.

A zinc finger polypeptide can be designed to recognize a selected target site in a gene. The zinc finger polypeptide may comprise any combination of motifs derived from natural zinc finger DNA binding domains and non-natural zinc finger DNA binding domains by truncation or extension, or by a site-directed mutagenesis method in combination with a selection method such as, but not limited to, phage display selection, bacterial two-hybrid system selection, or bacterial one-hybrid system selection. 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 polynucleotide and that does not occur in a cell or organism containing the polynucleotide to be modified. Methods for designing a zinc finger polypeptide that binds specific polynucleotides that are unique to a target gene are known in the art.

In other embodiments, a zinc finger polypeptide can be selected for binding to a regulatory sequence of a polynucleotide. More specifically, the regulatory sequence may contain a transcription initiation site, a start codon, an exon region, an exon-intron interface, a terminator, or a stop codon. Accordingly, the present invention provides mutant, non-naturally occurring or transgenic plant or plant cells produced 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 cells by zinc finger nuclease mediated mutagenesis. The methods for delivering the zinc finger polypeptide and the zinc finger nuclease to the plant are similar to those described below for delivery of the meganuclease.

Meganucleases

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, in particular, to effect 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 adjusted DNA recognition properties. Meganuclease polypeptides can be delivered to plant cells by a number of different mechanisms known in the art.

The present invention encompasses the use of meganucleases to inactivate the polynucleotide(s) described herein (or any combination thereof described herein) in a plant cell or 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) causing the polynucleotide(s) to be substantially inactivated by the meganuclease.

Meganucleases can be used to cleave at meganuclease recognition sites within 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 using a suitable transformation method. To achieve maximum efficiency, it is necessary to link the meganuclease expression cassette to a selectable marker and 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 be undesirable if the plant may require authorization. In such cases, the meganuclease expression cassette (and associated selectable marker gene) can be segregated in subsequent generations of the plant using conventional breeding techniques.

Following delivery of the meganuclease expression cassette, the plant cells are grown initially under conditions that are typical of the particular transformation procedure that was used. This may mean growing the transformed cells in a medium below 26°C, often in the dark. Such standard conditions can be applied over 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 can be increased to stimulate the function of the engineered meganuclease to cleave and mutate at the meganuclease recognition site.

TALEN

One method of gene editing involves the use of effector nucleases like transcription activator (TALEN) that induce the formation of double-strand breaks to which the cell can respond through repair mechanisms. NHEJ reconnects DNA on both sides of the double strand break where there is very little or no sequence overlap to allow for annealing. 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. For certain applications, precise removal of the polynucleotide may be necessary. 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 TALENs, which are able to recognize and bind to a 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.

CRISPR/Cas

Another method of gene editing involves the use of the bacterial CRISPR/Cas system. Bacteria and archaebacteria are characterized by the presence of chromosomal elements called regularly spaced short palindromic repeats (CRISPR), which are part of the adaptive immune system that protects against viral and plasmid DNA entry. In the case of type II CRISPR systems, CRISPR RNAs (crRNA) function with transactivating crRNA (tracrRNA) and CRISPR-associated (Cas) polypeptides to introduce double-strand breaks into the target DNA. Cleavage of the target by 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 to target DNA for site-specific cleavage promises to be significantly simpler than existing technologies such as TALEN. With the CRISPR/Cas strategy, retargeting of the nuclease complex only requires the introduction of a new RNA sequence, and there is no need to reverse engineer the specificity of the transcription factors based on the polypeptide. CRISPR/Cas technology has been implemented in plants in the method of international patent application WO 2015/189693, which discloses a virus-mediated genome editing platform that is widely applicable to plant species. Tobacco rattle virus (TRV) RNA2 genome was designed to carry and deliver guide RNA to Nicotiana benthamiana plants overexpressing Cas9 endonuclease. In the context of the present invention, a guide RNA can be obtained from any of the sequences disclosed herein and the ideas from WO 2015/189693 used to edit the genome of a plant cell and obtain the desired mutant plant. The rapid pace of technology development has resulted in a wide variety of protocols with broad applications in plants that have been well categorized in a number of recent scientific reviews (e.g., Plant Methods (2016) 12:8; and Front Plant Sci. (2016) 7:506). An overview of CRISPR/Cas systems with particular emphasis on their application is described in Biotechnology Advances (2015) 33, 1, 41-52). More recent developments regarding the use of CRISPR/Cas to manipulate plant genomes are described in Acta Pharmaceutica Sinica B (2017) 7, 3, 292-302 and Curr. Op. in Plant Biol. (2017) 36, 1-8. CRISPR/Cas9 based plasmids for use in plants are listed at "addgene", a non-commercial plasmid repository (addgene.org), and CRISPR/Cas9 based plasmids are commercially available.

Antisense modification

Antisense oligonucleotide technology is another well known technique that can be used to modulate polypeptide expression. The polynucleotide of the gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence such that an 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 is generally 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 catalytic RNA that affects the expression of the mRNA. Ribozymes can be designed 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 a 5'-UG-3' polynucleotide. 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 translation of the RNA transcript(s) is an interfering RNA. RNA interference or RNA silencing is an evolutionarily conserved process by which specific mRNAs can be targeted for enzymatic degradation. Double-stranded RNA (double-stranded RNA) is introduced into the cell or produced in the cell (for example, double-stranded RNA virus or interfering RNA polynucleotides) to initiate the interfering RNA pathway. Double-stranded RNA can be converted into several 21-24 bp small interfering RNA (siRNA) duplexes. using RNase III, which are endonucleases specific for double-stranded RNA. Subsequently, siRNAs can be recognized by RNA-induced silencing complexes that promote siRNA unwinding in an ATP-dependent process. The untwisted siRNA antisense strand directs activated RNA-induced silencing complexes to a targeted mRNA that contains a sequence complementary to the siRNA antisense strand. 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 by activated RNA-induced silencing complexes at a single site defined by the binding site of the 5'end of the siRNA strand. Activated RNA-induced silencing complexes can be reused to catalyze another cleavage event.

Interfering RNA expression vectors may contain interfering RNA constructs encoding interfering RNA polynucleotides that mediate RNA interference by downregulating 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 other sequences known to those skilled in the art, such as various selectable markers.

Double-stranded RNA molecules can include siRNA molecules assembled from a single oligonucleotide in a stem-loop structure, where the self-complementary sense and antisense portions of the siRNA molecule are connected by polynucleotide-based or non-polynucleotide-based linker(s), as well as circular 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 siRNA molecule capable of mediating the interfering RNA.

The use of small hairpin RNA molecules 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 from the 3' or 5' end of one or both strands). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form 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). The spacer sequence is typically an unrelated polynucleotide that resides between two complementary stretches of polynucleotides that, after annealing to a double-stranded polynucleotide, form a small hairpin RNA. The spacer sequence typically contains from about 3 to about 100 nucleotides.

Any RNA polynucleotide of interest can be obtained by selecting an appropriate sequence composition, loop size and stem length to produce a hairpin duplex. When designing, a suitable stem length range for a hairpin duplex includes stem lengths of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, such as about 14-30 nucleotides, about 30-50 nucleotides, about 50-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, about 200-300 nucleotides, about 300-400 nucleotides, about 400-500 nucleotides, about 500-600 nucleotides, and about 600-700 nucleotides. When designing, a suitable range of loop lengths for the hairpin duplex includes a loop length of about 4-25 nucleotides, about 25-50 nucleotides, or more if the stem length of the hairpin duplex 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 siRNA molecule is a double-stranded RNA or ssRNA molecule from about 15 to about 35 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 17 to about 30 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 19 to about 25 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 21 to about 23 nucleotides in length. In certain embodiments, hairpin structures with duplex regions longer than 21 nucleotides can facilitate efficient siRNA-driven silencing regardless of loop sequence and length. Exemplary sequences for performing RNAi are described herein.

The target mRNA sequence is typically about 14 to about 50 nucleotides in length. Therefore, the target mRNA can be screened for regions of about 14 to about 50 nucleotides in length that preferably meet one or more of the following criteria: an A+T/G+C ratio of about 2:1 to about 1:2; the presence of an AA dinucleotide or a CA dinucleotide at the 5' end; the presence of a sequence of at least 10 consecutive nucleotides that is unique to the target mRNA (ie, the sequence is not present in other mRNA sequences from the same plant); and the absence of "strings" of more than three consecutive nucleotides representing guanine (G) or more than three consecutive nucleotides representing cytosine (C). Evaluation against these criteria can be performed 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 sequence is unique to a target mRNA. Alternatively, the sequence can be selected (and the siRNA sequence designed) using commercially available computer software (eg, OligoEngine, Target Finder and Design Tool for siRNA, which are commercially available).

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

In an exemplary embodiment, the siRNA 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 polynucleotides. described in this document.

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

One method for inducing double-stranded RNA silencing in plants is transformation with a gene construct producing hairpin RNA (see Nature (2000) 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 by generating hairpin RNA based on intron splicing ( Plant J. (2001), 27, 581-590). Preferably, the length of the stem is from about 50 nucleotides to about 1 thousand nucleotides in length. Methods for producing hairpin RNA based on intron splicing are well described in the art (see, for example, Bioscience, Biotechnology, and Biochemistry (2008) 72, 2, 615-617).

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

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

Interfering RNA molecules may contain one or more 5' or 3' cap structures. 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 interfering RNA molecules is the chemical coupling to the interfering RNA molecule of one or more fragments or conjugates that enhance the function, 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 include 2' modifications, introduction of non-natural bases, covalent attachment of a ligand, and replacement of phosphate bonds with thiophosphate bonds. In this embodiment, the strength of the duplex structure is strengthened by at least one and typically two chemical bonds.

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.

The interfering RNA molecule can be conjugated to ligands, for example, to enhance its absorption into the cell. In certain embodiments, the hydrophobic ligand is conjugated to a molecule to facilitate direct penetration across the cell membrane. In certain instances, conjugation of the cationic ligand to oligonucleotides often results in improved nuclease resistance.

"Targeted induced local damage in the genome" (TILLING) is another mutagenesis technology that can be used to create and/or identify polynucleotides encoding polypeptides with modified expression, function and/or activity. TILLING also enables the selection of plants carrying such mutant variants. TILLING combines high density mutagenesis with high throughput screening methods. TILLING methods 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).

Various embodiments are directed to expression vectors containing one or more polynucleotides or one or more interfering RNA constructs that contain one or more of the polynucleotides described herein.

Various embodiments are directed to expression vectors containing one or more polynucleotides or one or more interfering RNA constructs described herein.

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 a DNA fragment of the first strand by operably linking to a second promoter. Each polynucleotide strand can be transcribed from a single expression vector, or from different expression vectors. An RNA duplex having RNA interference properties can be enzymatically converted to siRNA to modulate RNA levels.

Thus, various embodiments are directed to expression vectors comprising 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; and (b) a second sequence containing a sequence that is complementary (eg, inversely complementary) to 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.

Various compositions and methods are provided for modulating the expression level of an endogenous gene by modulating mRNA translation. A host plant (tobacco) 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 expression of RNA polynucleotides having a sequence complementary to the mRNA portion.

Various expression vectors for modulating translation of an mRNA may contain a promoter operably linked to a polynucleotide wherein the sequence is located in an antisense orientation with respect to the promoter. The length of antisense RNA polynucleotides can vary and can be 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 about 200-300 nucleotides.

Mobile genetic elements

Alternatively, targeting for gene inactivation can be accomplished 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 polypeptide function. 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 can 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.

Ribozymes

Alternatively, gene inactivation targeting can be accomplished by introducing ribozymes derived from a range of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either on their own (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.

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 the expression or function or activity of those genes or their products. 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 polypeptide or a portion thereof. As a further example, mutant or non-naturally occurring plants or plant cells may have one or more mutations in a region outside one or more of the gene(s), such as a region upstream or downstream of the gene it regulates, provided that they modulate the function or expression of the gene(s). Upstream elements may include promoters, enhancers, or transcription factors. Some elements, such as enhancers, may be upstream or downstream of the gene they regulate. The element(s) are not necessarily located adjacent to the gene it regulates, as some elements have been found to be several thousand base pairs upstream or downstream from 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 (genes), in the first 700 nucleotides from the gene(s), in the first 800 nucleotides from the gene(s), in the first 900 nucleotides from the gene(s), in the first 1000 nucleotides from the gene(s), in the first 1100 nucleotides from the gene(s), in the first 1200 nucleotides from the gene(s), in the first 1300 nucleotides from the gene (genes), in the first 1400 nucleotides from the gene(s) or in the first 1500 nucleotides from 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 a combination thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (eg, mutant, non-naturally occurring or transgenic plants or plant cells, etc., 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 tested 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 tissue, or plant cells, can be mutated to create mutant plants. However, the type of plant material subjected to mutagenesis may be of importance when a plant polynucleotide is screened for mutations. For example, if pollen is subjected to mutagenesis prior to pollination of an unmutated plant, first generation plants are grown from the seeds obtained from this pollination. Each cell of the first generation plants will contain mutations that originated in the pollen; thus, these first generation plants can then be screened for mutations instead of waiting for the second generation to appear.

Obtaining modified plants, screening and crossing

A polynucleotide derived from individual plants, plant cells, or plant material can optionally be combined to expedite screening for mutations in a plant population derived from mutated tissue, plant cells, or plant material. One or more next generations of plants, plant cells, or plant material can be tested. The size of the optionally pooled group depends on the sensitivity of the screening method used.

After optionally pooling the samples, they can be analyzed using polynucleotide-specific amplification techniques such as PCR. 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 sample. Preferably, 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 the 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 designed 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 some embodiments, a plant can be regenerated or grown from a plant, plant tissue, or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue can be used, such as, without limitation, tissue culture or regeneration from protoplasts. Preferably, plants can be regenerated by growing the transformed plant cells on callus induction medium, shoot induction medium and/or root induction medium. See, for example, Plant Cell Reports (1986) 5:81-84. These plants can then be grown and either pollinated with the same transformed line or other lines and the resulting hybrid identified as expressing the desired phenotypic characteristic. You can grow two or more generations to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited, and then collect seeds to ensure that the expression of the desired phenotypic characteristic has been achieved. Thus, the expression "transformed seeds" as used herein refers to seeds that contain a nucleotide construct stably integrated into the plant genome.

Accordingly, 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 function of the polynucleotide(s) described herein. At least one mutant plant cell is then propagated to produce a mutant plant, wherein the mutant plant has a modulated level of the described polypeptide(s) (or any combination thereof described herein) compared to that of 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 that of the control plant, preferably 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 inserted (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 originates) to provide the trait. Thus, as an example, a mutation that occurs naturally in the 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. Thus, a person skilled in the art can 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. The naturally occurring mutant allele(s) 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 of the mutations in the genes described herein. The same technique can also be applied to introgression of one or more unnatural mutation(s) from a first plant into a second plant. Plants displaying the desired trait can be selected from a pool of mutant plants. Preferably, selection is made using the polynucleotide data described herein. Therefore, it is possible to carry out selection on a genetic basis in comparison with the control. Such a screening approach may include the use of conventional amplification and/or hybridization techniques as discussed herein. Thus, a further aspect of the present invention relates to a method for identifying a mutant plant comprising the steps of: (a) obtaining a sample containing a polynucleotide from a plant; and (b) determining the sequence of a polynucleotide, wherein a difference in the sequence of the polynucleotide compared to that of a control plant indicates that said plant is a mutant plant. In another aspect, a method is provided for identifying a mutant plant that accumulates one or more amino acids at an increased or decreased level compared to a control plant, comprising the steps of: (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 level of at least one amino acid in said plant. In another aspect, a method is provided for producing a mutant plant that is characterized by increased or decreased levels of at least one amino acid compared to a control plant, comprising the steps of: (a) obtaining a sample from a first plant; (b) determining if said sample contains one or more mutations in one or more of the polynucleotides described herein that result in modulation of at least one amino acid levels, and (c) transferring the one or more mutations to a second plant. The mutation(s) can be transferred to the second plant using various methods that are 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 is characterized by increased or decreased levels of at least one amino acid 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 that result in modulation of at least one amino acid levels; and (c) introgression of one or more mutations from the first plant into the second plant. In one embodiment, the introgression step includes 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 that is a cultivar or elite cultivar) obtained or obtained using the methods described herein. In certain embodiments, "mutant plants" may have one or more mutations located only in a particular region of the plant, for example, in the sequence of one or more of the polynucleotide(s) 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 some embodiments, mutant plants may have one or more mutations located in more than one region of the plant genome, 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 herein; or may lack one or more mutations in the 3'-untranslated region of the polynucleotide(s) described herein; or may lack one or more mutations in the 5'-untranslated region of the polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the polynucleotide(s) described herein; or may not have one or more mutations in the non-coding region of the polynucleotide(s) described 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 on the plant, plant cell, or plant material; (b) obtaining a sample from said plant, plant cell or plant material or their descendants; and (c) determining the polynucleotide sequence of a gene, or variant, or fragment, where a difference in said sequence indicates the presence of one or more mutations therein. The method also selects for plants having a mutation(s) that occurs at regions of the genome that affect gene expression in a plant cell, such as a transcription initiation site, start codon, intron region, exon-intron interface, terminator, or stop codon.

Plant families, species, varieties, seeds and tissue culture

Plants suitable for genetic modification to be applied to them include 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, Cucur bitaceae, 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, L actuca, 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, Vitis and Zea.

Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (Gerard's bearded vulture), Pennisetum purpureum (elephantgrass), Phalaris arundinacea (reedweed), 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 escul enta (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), Coffeycliseca (cocoa) fe), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot and sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (giant gourd), Cucurbita moschata (nutmeg gourd), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmo schus 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), Sorghuycliseor (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 plant), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet) or Pennisetum glaucum (pearl millet).

Various embodiments are directed to mutant tobacco, non-naturally occurring tobacco, or transgenic tobacco plants or plant cells modified to modulate gene expression levels, resulting in plants or plant cells, such as a tobacco plant or plant cell, in which the expression level of the polypeptide is modulated in the tissues of interest compared to a 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). Другие виды включают 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. Preferably, the tobacco plant is N. tabacum.

The use of tobacco varieties and elite varieties of tobacco 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 naturally occur in a particular tobacco variety or tobacco variety (e.g., elite tobacco variety), or may be a genetic mutation(s) that(s) exist(s) in nature, provided that the mutation does not naturally occur in a particular tobacco variety or tobacco variety (e.g., elite tobacco variety).

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 26 3, 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 94 4, msKY 14xL8 Narrow Leaf Madole Narrow Leaf Madole LC NBH 98 N-126 N-777LC N-7371LC 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 ilep 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 a low level of conversion of nicotine to nornicotine are also contemplated, even though they are not specifically mentioned herein.

Embodiments are also directed to compositions and methods for producing plant mutants, non-naturally occurring plants, hybrid plants, and transgenic plants that have been modified to modulate the expression or function of the polynucleotide(s) described herein (or any combination thereof, as described herein). Advantageously, the resulting mutant plants, non-naturally occurring plants, hybrid plants or transgenic plants may be similar in general appearance to control plants or substantially the same. Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stem height, leaf ingrowth angle, leaf size (width and length), internode distance, and lamina-to-vein ratio can be assessed by field observations.

One aspect relates to the seed of a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant described herein. Preferably, the seed is tobacco seed. An additional aspect relates to the pollen or ovule of a mutant plant, non-naturally occurring plant, hybrid plant or transgenic plant described herein. Furthermore, a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant as described herein is provided, which further comprises a male sterility polynucleotide.

Also provided is tissue culture from 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 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. The plant material described herein may be dried tobacco material, such as air-dried or sun-dried tobacco material. Examples of air-cured and sun-cured tobacco varieties are those of the Burley type and the dark type. The plant material described herein may be flue-cured tobacco material such as that of the Virginia type.

The CORESTA recommendation for tobacco drying is described in CORESTA Handbook #17, April 2016, Sustainability in Leaf Tobacco Production.

One object is to provide mutant, transgenic or non-naturally occurring plants or parts thereof that exhibit modulated levels of NtAAT, resulting in modulated levels of at least one amino acid, such as aspartate, in plant material, such as dried leaves. Since aspartate is known to result in acrylamide when tobacco leaf is heated, modulating aspartate levels can also result in modulating acrylamide levels. The synthesis of aspartate is also important for the synthesis of other amino acids such as asparagine, threonine, isoleucine, cysteine, and methionine. Accordingly, the levels of one or more of these other amino acids may be modulated if the levels of aspartate are modulated. The presence of certain amino acids, such as threonine, methionine, and cysteine, can result in a sulfur odor in smoke or aerosols that are produced by heating. Therefore, modulating the levels of these amino acids also modulates this sulfur odor.

In some embodiments, the activity and/or expression of one or more of NtAAT1-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S, and NtAAT4-T are modulated.

In some embodiments, the activity and/or expression of one or more of NtAAT1-S and NtAAT1-T are modulated.

In some embodiments, the activity and/or expression of one or more of NtAAT2-S and NtAAT2-T are modulated.

In some embodiments, the activity and/or expression of one or more of NtAAT1-S, NtAAT1-T, NtAAT2-S, and NtAAT2-T are modulated.

In some embodiments, the expression and/or activity of one or more of NtAAT1-S, NtAAT1-T, NtAAT2-S, and NtAAT2-T are modulated, while the expression and/or activity of one or more of NtAAT3-S, NtAAT3-T, NtAAT4-S, and NtAAT4-T are not modulated.

In some embodiments, the expression and/or activity of one or more of NtAAT1-S, NtAAT1-T, NtAAT2-S, and NtAAT2-T are modulated, while the expression and/or activity of NtAAT3-S NtAAT3-T, NtAAT4-S, and NtAAT4-T are not modulated.

Preferably, the mutant, transgenic, or non-naturally occurring plants, or parts thereof, have substantially the same appearance as the control plant.

Accordingly, this document describes mutant, transgenic or non-naturally occurring plants or plant parts or cells that are characterized by modulated levels of at least one amino acid compared to those in control cells or control plants. Mutant, transgenic or non-naturally occurring plants or plant cells have been modified to modulate the synthesis or function of one or more of the polypeptides described herein by modulating the expression of one or more of the corresponding polynucleotides described herein. Preferably, modulated levels of at least one amino acid are observed in at least green leaves, preferably dried leaves.

A further aspect relates to a mutant, non-naturally occurring, or transgenic plant or cell, wherein the expression or function of one or more of the AAT polypeptides described herein is modulated (e.g., reduced) and the plant part (e.g., green leaves, preferably dried leaves, or dried tobacco) has modulated (e.g., reduced) levels of at least one amino acid, by at least 5% compared to a control plant in which the expression or the function of said polypeptide(s) has not been modulated (eg reduced). In some embodiments, the level of at least one amino acid in a plant, such as green leaves, preferably dried leaves or dried tobacco, can be modulated (e.g., reduced), such as increased by 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 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200%, in amount or function. The level of at least one amino acid may be reduced to undetectable amounts.

Another additional aspect relates to a dried plant material, such as a dried leaf or dried tobacco, obtained or derived from a mutant, non-naturally occurring or transgenic plant or cell, wherein the expression of one or more of the polynucleotides described herein, or the function of the polypeptide encoded by them, is modulated (e.g., reduced), and where the level of at least one amino acid is modulated (e.g., reduced) by at least 5%, at 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%, at least 99%, or at least 100%, or at least 150%, or at least 200% compared to the control plant.

Preferably, the appearance of said plant or part (eg, leaf) is substantially the same as that of a control plant. Preferably, the plant is a tobacco plant or a coffee plant.

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 function of one or more of the polynucleotides or polypeptides described herein, which may result in plants, or plant components (e.g., leaves, such as green leaves or dried leaves, or tobacco), or plant cells with a modulated content of at least one amino acid.

Mutant, non-naturally occurring or transgenic plants may be similar in appearance to control plants or substantially the same. In one embodiment, the leaf weight of the mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant. In one embodiment, the number of leaves in the mutant, non-naturally occurring, or transgenic plant is substantially the same as the control plant. In one embodiment, the leaf weight and number of leaves of the mutant, non-naturally occurring, or transgenic plant is substantially the same as the control plant. In one embodiment, the stem height of the mutant, non-naturally occurring, or transgenic plants is substantially the same as the control plants at, for example, one, two, or three or more months after transplanting into the field, or 10, 20, 30, or 36 or more days after topping. For example, the stem height of the mutant, non-naturally occurring or transgenic plants is no less than the stem height of the control plants. In another embodiment, the chlorophyll content of the mutant, non-naturally occurring, or transgenic plants is substantially the same as that of control plants. In another embodiment, the stem height of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants, and the chlorophyll content of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants. In other embodiments, the size or shape or number or color of the leaves of the mutant, non-naturally occurring, or transgenic plants are substantially the same as control plants. Preferably, the plant is a tobacco plant or a coffee plant.

In another aspect, a method is provided for modulating the amount of at least one amino acid in at least part of a plant (e.g., in leaves, such as dried leaves or in tobacco), comprising the steps of: (i) modulating the expression or function of one or more of the polypeptides described herein (or any combination thereof, as described herein), preferably, wherein the polypeptide(s) are encoded by the corresponding polynucleotides described herein; (ii) measuring the level of at least one amino acid in at least a portion (eg, in leaves, such as dried leaves, or in tobacco, or in smoke) of the mutant, non-naturally occurring, or transgenic plant obtained in step (i); and (iii) identifying a mutant, non-naturally occurring or transgenic plant in which the level of at least one amino acid has been modulated compared to a control plant. Preferably, the appearance of said mutant, non-naturally occurring or transgenic plant is substantially the same as that of the control plant. Preferably the plant is a tobacco plant.

In another aspect, a method is provided for modulating the amount of at least one amino acid in at least a portion of dried plant material, such as dried leaves, comprising the steps of: (i) modulating the expression or function of one or more polypeptides (or any combination thereof, as described herein), preferably, wherein the polypeptide(s) are encoded by the corresponding polynucleotides described herein; (ii) collecting plant material, such as one or more leaves, and drying for a specified period of time; (iii) measuring the level of at least one amino acid in at least a portion of the dried plant material obtained in step (ii) or during step (ii); and (iv) identifying the dried plant material in which the level of at least one amino acid has been modulated compared to a control plant.

The increase in expression level compared to the control can be from about 5% to about 100%, or the increase is 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% or more, such as 200%, 300%, 500%, 1000% or more, which includes an increase in transcriptional function, or polynucleotide expression level, or polypeptide expression level, or combinations thereof.

The increase in function or activity compared to control can be from about 5% to about 100%, or the increase is 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% or more, such as 200%, 300%, 500%, 1000% or more.

The decrease in expression level compared to the control can be from about 5% to about 100%, or the decrease is 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 9 5%, at least 98%, or 100%, which includes a reduction in transcriptional function, or polynucleotide expression level, or polypeptide expression level, or combinations thereof. Preferably, the expression is reduced.

The decrease in function or activity compared to control can be from about 5% to about 100%, or the decrease is 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 9 5%, at least 98% or 100%. Preferably, the function or activity is reduced.

The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the polynucleotides or polypeptides described herein in a plant species of interest, preferably in tobacco.

A variety of polynucleotide-based methods can be used to increase the level of gene expression in plants and 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 causing the gene to be overexpressed in the plant or plant cell. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, a promoter can drive expression to modulate the levels of a given enzyme in a plant or in a particular tissue thereof. In one exemplary embodiment, a vector is created carrying one or more of the polynucleotides described herein (or any combination thereof, as described herein) to allow overexpression of the gene in a plant or plant cell. The vector carries a suitable promoter, such as the cauliflower mosaic virus CaMV 35S promoter upstream of the transgene, which triggers its constitutive expression in all plant tissues. The vector also carries an antibiotic resistance gene to allow selection of transformed calluses and cell lines.

Expression of sequences from promoters can be enhanced by including expression control sequences, including enhancers, chromatin activating elements, transcription factor responsive elements, and the like. Such control sequences can be constitutive and increase transcription in a versatile manner; or they may be facultative and increase the level of transcription in response to specific signals. Separately, the signals associated with aging and the signals that are active during the drying procedure should be specified.

Various embodiments are thus directed to methods of modulating the level of expression of one or more of the polynucleotides described herein (or any combination thereof, as described herein) by integrating multiple copies of the polynucleotide into the plant genome, which methods comprise transforming a host plant cell with an expression vector that contains a promoter operably linked to one or more of the polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

In one embodiment, the plant used in the present invention is an air-dried plant because such plants are high in amino acids (more than about 27.4 mg/g free amino acids dry weight when grown in the field) and high in ammonia (greater than about 0.18% dry weight when grown in the field) at the end of drying. Air-dried mutant, transgenic, or non-naturally occurring plants or parts thereof may have an amino acid content that corresponds to a field grown free amino acid content of less than about 27.4 mg/g dry weight at the end of drying, such as a field grown free amino acid content of less than about 20 mg/g dry weight at the end of drying, or a field grown free amino acid content of less than about 1 5 mg/g based on dry weight at the end of drying, or free amino acids when grown in the field less than about 10 mg/g based on dry weight at the end of drying, or free amino acids when grown in the field less than about 5 mg/g based on dry weight at the end of drying. Air-dried mutant, transgenic, or non-naturally occurring plants or parts thereof may have an ammonia content that, when grown in the field, is about 0.18% dry weight at the end of drying, or less than about 0.15% dry weight when grown in the field at the end of drying, or less than about 0.10% dry weight when grown in the field at the end of drying, or less than about 0.05% based on dry weight when grown in the field at the end of drying.

In another embodiment, the plant used in the present invention is a sun-dried plant because such plants are high in amino acids (when grown in the field, free amino acids are greater than about 26.5 mg/g dry weight at the end of drying) and high in ammonia (when grown in the field, more than about 0.14% dry weight at the end of drying). Mutant, transgenic, or non-naturally occurring, sun-dried plants or parts thereof may have an amino acid content that corresponds to a field grown free amino acid content of less than about 26.5 mg/g dry weight at the end of drying, such as a field grown free amino acid content of about 20 mg/g dry weight at the end of drying, or a field grown free amino acid content of less than about 20 mg/g. g based on dry weight at the end of drying, or a free amino acid content when grown in the field of less than about 15 mg/g based on dry weight at the end of drying, or a content of free amino acids when grown in the field is less than about 10 mg/g based on dry weight at the end of drying, or a content of free amino acids when grown in the field is less than about 5 mg/g based on dry weight at the end of drying. Mutant, transgenic, or non-naturally occurring, sun-dried plants or parts thereof may have an ammonia content that when grown in the field is less than about 0.14% dry weight at the end of drying, or when grown in the field is less than about 0.10% dry weight at the end of drying, or when grown in the field is less than about 0.05% dry weight at the end of drying.

In another embodiment, the plant used in the present invention is a plant that has been fire-dried. Such plants are characterized by an amino acid content that corresponds to a field-grown free amino acid content of greater than about 3 mg/g dry weight at the end of drying, and an ammonia content that, when field grown, is greater than about 0.02% dry weight at the end of drying. Flame-dried mutant, transgenic, or non-naturally occurring plants or parts thereof can have an amino acid content that corresponds to a field-grown free amino acid content of less than about 3 mg/g dry weight at the end of drying, such as a field-grown free amino acid content of about 2.5 mg/g dry weight at the end of drying, or a field-grown free amino acid content of less than about 2, 0 mg/g dry weight at the end of drying, or free amino acids when grown in the field less than about 1.5 mg/g dry weight at the end of drying, or free amino acids when grown in the field less than about 1.0 mg/g based on dry weight at the end of drying, or free amino acids when grown in the field less than about 0.5 mg/g based on dry weight at the end of drying. Mutant, transgenic, or non-naturally occurring, sun-dried plants or parts thereof may have an ammonia content that when grown in the field is less than about 0.02% dry weight at the end of drying, or when grown in the field is less than about 0.10% dry weight at the end of drying, or when grown in the field is less than about 0.05% dry weight at the end of drying.

In some embodiments, air-dried or sun-dried plants are preferred.

The content of amino acids can be measured using various methods known in the art. One such method is MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker Srl, NutriSciences Company. In order to detect amino acids in the dried leaves of the plant, after removal of the midrib, the dried lamellae are, if necessary, dried at 40° C. for 2-3 days. The tobacco material is then ground into a fine powder (~100 μM) prior to analysis for amino acid content. Another method for measuring the amino acid content of plant material is described in UNI EN ISO 13903:2005. In one embodiment, a method used to measure the amino acid content of plant material is described in UNI EN ISO 13903:2005.

Ammonia content can be determined by Skalar: MT24-Nitrate, Total Alkaloids, Ammonia, Chloride, TKN. To detect ammonia in the dried leaves of the plant, after removal of the midrib, the dried plates are dried, if necessary, at 40° C. for 2-3 days. The tobacco material is then ground into a fine powder (~100 μM) prior to analysis for ammonia content. Other methods for measuring ammonia are known in the art and include those described in Health Canada (1999) Determination of ammonia in whole tobacco. Tobacco Control Program. Health Canada Official Method T-302; and Tobacco Manufacturers Organization (2002) UK smoke constituent study. Annex A Part 5 Method: determination of ammonia yields in mainstream cigarette smoke using the Dionex DX-500 ion chromatograph, Report Nr GC15/M24/02.

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 different genetic characteristics. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desired genetic traits or genetic background is obtained. One purpose for which such breeding methods are suitable is to introduce the desired genetic trait into other varieties, breeding lines, hybrids or varieties, especially those of commercial interest. Another purpose is to facilitate the accumulation of genetic modifications of various 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 seed from a progeny tobacco plant; (b) growing the seed of the progeny tobacco plant under conditions suitable for plant growth 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 seed of a progeny tobacco plant; (d) growing the seed of the progeny tobacco plant of step (c) under conditions suitable for plant growth to produce additional non-naturally occurring plants; and (e) repeating the crossing and rearing steps (c) and (d) several times to produce further generations of non-naturally occurring plants. The method may optionally include before step (a) the step of obtaining a parent plant that contains the characterized genetic features 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 produce generations of non-naturally occurring plants. Backcrossing is an example of such a method in which a progeny is 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 genetic characteristics more closely related 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 for the presence of variant genes are evaluated in the field using standard field procedures. Control genotypes are provided, including the genotype of the original non-mutated parent, and planting sites are located in the field according to a randomized block plan or other appropriate field planning. For tobacco, standard agronomic practices are used, for example, tobacco is harvested, weighed and sampled for chemical and other conventional testing before and during drying. Statistical data analysis is performed to confirm the similarity of the selected lines with the parent line. Cytogenetic analyzes of selected plants are optionally performed to confirm the relationships of chromosome set and chromosome conjugation.

DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies can be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant gene alleles in other tobacco 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 backcross generations can be screened with a marker derived from the 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 mode of inheritance or the applied MAS technology, it may be necessary for the selected plants to self-pollinate before each backcrossing cycle to facilitate the discovery of the individual plants needed. Backcrossing or other selection procedure can be repeated until the desired phenotype of the recurrent parent is restored.

According to the present invention, in a breeding program, successful crosses produce F1 plants that are fertile. Selected F1 plants can be crossed with one of the parents, and the first generation plants resulting from backcrossing 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 produces 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, a population of F2 generation plants is screened for variant gene expression, e.g., a plant that does not express a polypeptide due to the absence of a gene is identified according to standard methods, e.g., using a PCR technique using primers designed based on the polynucleotide sequence information for the polynucleotide(s) described herein (or any combination thereof described herein).

Hybrid tobacco varieties can be produced by preventing the female parent plants (i.e. parent forms) of the first variety from self-pollinating, allowing pollen from the male parent plant of the second variety to fertilize the female parent plant, and producing F1 hybrid seeds on the female plant. Self-pollination of female plants can be prevented by castrating the flowers early in flower development. Alternatively, pollen production on the female parent plants can be prevented using some form of male sterility. For example, male sterility can be achieved through cytoplasmic male sterility (CMS) or transgenic male sterility, where the transgene suppresses microsporogenesis and/or pollen production or causes self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments in which 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 F1 tobacco simple hybrids. In such embodiments, plants of parental varieties can be grown in substantially uniform contiguous populations to facilitate natural cross-pollination of female parent plants by male parent plants. F1 seeds produced on female parent plants are selectively harvested by conventional means. It is also possible to grow two varieties of the parent plant en masse and collect a mixture of F1 hybrid seeds produced on the female and seeds produced on the male by self-pollination. Alternatively, a trilinear cross can be made where a single F1 hybrid is used as the female and crossed with another male. 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. For example, a population from the progeny of a single transformation line may be screened for those plants that have the desired level of expression or function of the polypeptide(s) encoded therewith. Physical and biochemical methods can be used to detect levels of expression or activity. These include Southern analysis or PCR amplification to detect the polynucleotide; Northern blot, RNase S1 protection assay, primer extension assay, or RT-PCR amplification to detect RNA transcripts; enzyme assays to detect the enzymatic or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzymatic staining and immunostaining, and enzymatic analysis can also be used to detect the presence or expression or function 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 and parts thereof described herein can be modified either before or after modulating the expression, function, or activity of one or more polynucleotides and/or polypeptides in accordance with the present invention.

One or more of the following additional genetic modifications may be present in mutant, non-naturally occurring or transgenic plants or parts thereof.

One or more genes that are involved in the conversion of nitrogen metabolism intermediates can be modified, resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include genes encoding nicotine demethylase such as CYP82E4, CYP82E5 and CYP82E10 described in WO2006/091194, WO2008/070274, WO2009/064771 and WO2011/088180 and nitrate reductase as described in WO2016046288.

One or more genes that are involved in heavy metal uptake or heavy metal transport may be modified, resulting in lower levels of heavy metals. Non-limiting examples include polypeptide families associated with multidrug resistance, cation diffusion mediator (CDF) families, Zrt-, Irt-like polypeptide (ZIP) family, cation exchanger (CAX) family, copper transporter (COPT) family, heavy metal ATPase family (e.g. HMA as described in WO2009/074325 and WO2017/129739), a family of macrophage polypeptide homologues associated with natural resistance (NRAMP) and other members of the ATP-binding cassette (ABC) transporter family (e.g. MRP) as described in WO2012/028309 that are involved in the transport of heavy metals such as cadmium.

Other illustrative modifications can result in plants with modulated isopropyl malate synthase expression or function, resulting in a change in sucrose ester composition that can be used to change the aroma profile (see WO2013/029799).

Other exemplary modifications can result in plants with modulated threonine synthase expression or function, where methional levels can be modulated (see WO2013/029800).

Other exemplary modifications may result in plants with modulated expression or function of one or more of neoxanthine synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase to modulate beta-damascenone content to alter the aroma profile (see WO2013/064499).

Other exemplary modifications may result in plants with modulated expression or function of chloride channel members of the CLC family to modulate their nitrate levels (see WO2014/096283 and WO2015/197727).

Other illustrative modifications can result in plants with modulated expression or function of one or more AATs to modulate the levels of one or more amino acids such as aspartate in the leaf and modulate the levels of acrylamide in the aerosol produced by heating or burning the leaf (see WO2017042162).

Examples of other modifications include modulating herbicide tolerance, for example, glyphosate is an 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 polypeptide 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 examples of modifications result in plants that are resistant to insects. toxinsBacillus thuringiensis(Bt) can provide an efficient path delaying the emergence of Bt-resistant pests, as recently shown on broccoli, where the Bt genescry1AcAnd cry1C as part of the "pyramid" provided control of the cabbage moth, resistant to any individual polypeptide, and significantly delayed evolutionary 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 a fertility adjustment, 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 enzyme acylglycerol phosphate acyltransferase from Arabidopsis; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in the synthesis of mannitol and sorbitol, improve drought tolerance.

Another exemplary modification results in plants in which the activity of one or more endogenous glycosyltransferases such as N-acetylglucosaminyltransferase, β(1,2)-xylosyltransferase and a(1,3)-fucosyltransferase is modulated (see WO/2011/117249).

Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and nornicotine metabolites that form during drying can be modulated (see WO2015169927).

Other illustrative modifications can result in plants with improved storage polypeptides and oils, plants with increased photosynthetic efficiency, plants with a long shelf life, plants with increased carbohydrate content, and plants resistant to fungi. 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 genes that are involved in the nicotine synthesis pathway can be modified to produce plants or plant parts that produce modulated levels of nicotine when dried. The nicotine synthesis genes may be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MPO1, MPO2, MYC2a, MYC2b , NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT, or a combination of one or more of them.

One or more genes that are involved in regulating the amount of one or more alkaloids can be modified to produce plants or plant parts that produce modulated alkaloid levels when dried. Alkaloid control genes may be selected from the group consisting of BBLa , BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2 , or a combination of two or more of these.

One or more of these traits can be introgressed into, or directly transformed into, mutant, non-naturally occurring, or transgenic plants of another variety.

In various embodiments, mutant plants, non-naturally occurring plants or transgenic plants, as well as biomass, are provided in which the expression level of one or more polynucleotides of the present invention is modulated to thereby modulate the level of the polypeptide(s) encoded by them.

Parts of the plants described herein, in particular the lamina and midrib of these plants, can be incorporated into or used in the manufacture of a variety of consumer products, including, without limitation, aerosol generating materials, aerosol generating devices, smoking articles, smoking articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol forming materials include tobacco compositions, tobacco species, tobacco extract, cut tobacco, cut filler, dried tobacco, exploded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco species. Smoking articles and smoking articles are generally aerosol generating devices. Examples of smoking or smoking articles include cigarettes, cigarillos and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol generating devices, instead of combustion, the tobacco composition or other aerosol generating material is heated by one or more electrical heating elements to produce an aerosol. In another type of heated aerosol generating device, the aerosol is produced 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 tobacco-containing aerosol forming materials may contain tobacco in any form, including dried particles, lumps, granules, powders, or suspension coated on, mixed with, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tablets, foams, or pellets. As used herein, the term "smoke" is used to describe the type of aerosol that is produced by smoking articles such as cigarettes or by burning an aerosol-forming material.

In one embodiment, dried plant material is also provided from the mutant, transgenic, and non-naturally occurring plants described herein. Methods for drying green tobacco leaves are known to those skilled in the art and include, without limitation, air drying, fire drying, fire tube drying, and sun drying, as described herein.

In another embodiment, tobacco products are described, including aerosolized tobacco-containing materials containing plant material such as leaves, preferably dried leaves, from the mutant tobacco plants, transgenic tobacco plants, or non-naturally occurring tobacco plants described herein. The tobacco products described herein may be blended tobacco products, which may additionally contain unmodified tobacco.

Products and methods for crop management and agriculture.

For mutant, non-natural or transgenic plants, there may be other uses in, for example, agriculture. For example, the mutant, non-naturally occurring, or transgenic plants described herein can be used to make animal feed and human food.

The present invention also provides methods for producing seeds, comprising cultivating a mutant plant, a non-naturally occurring plant, or a transgenic plant as 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 using 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 label 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 means for 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 nucleotides that hybridize (eg, specifically hybridize) to the polynucleotide(s) described herein. 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 methods for gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or plaques of bacterio phage) or gene detection (eg, as one or more amplification 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 a polynucleotide(s). As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer that is derived from a polynucleotide sequence and a second primer that hybridizes to a sequence above or below the polynucleotide sequence, such as a promoter sequence, the 3' end of a precursor mRNA, or a sequence derived 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 originate from or be obtained from a plant, plant cell, or plant material, or a tobacco product made 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 steps of: (a) obtaining a sample containing or suspected to contain a polynucleotide; (b) bringing said sample into contact 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, wherein the presence of an amplification product is indicative of the presence of a 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 a polynucleotide(s) is also contemplated. 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 a polynucleotide(s). The kit may contain polynucleotide amplification reagents, such as PCR reagents, 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 reagents for DNA sequencing. The kit may contain reagents and instructions for using the kit.

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. Methods based on the use of molecular 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 may be used in a particular genotyping method, including those based on amplified fragment length polymorphism (AFLP). AFLP is the result of allelic differences between amplified fragments caused by polynucleotide variability. Thus, the present invention further provides methods for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically linked to those genes or polynucleotides, using techniques such as AFLP analysis.

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 out the preferred mode currently provided for the implementation of the present invention, are intended to illustrate and not to limit the present invention.

EXAMPLES

Example 1 Identification of Major Genes whose Activation Correlates with Amino Acid Levels after Drying in Burley, Virginia and Oriental Tobacco Leaf

To identify key functions contributing to changes in free amino acid levels during the initial drying time of Burley, Virginia and Oriental tobacco leaf, an analysis was made of the dominance of functions of genes activated in dried leaves after 48 hours of drying compared to mature leaves at harvest (log2>2-fold change, adjusted p-value <0.05) in Burley, Virginia and Oriental tobacco. Identified genes involved in the production of free amino acids, which are active after 48 hours of drying, regardless of the types of drying and varieties of tobacco. We studied tobacco genes that affect the production of aspartate during early drying and belong to the AAT family.

Complete set of polynucleotides identified in tobacco genomeNtAAT, which representNtAAT1-S (SEQ ID NO: 5),NtAAT1-T (SEQ ID NO: 7),NtAAT2-S (SEQ ID NO: 1),NtAAT2-T (SEQ ID NO: 3),NtAAT3-S (SEQ ID NO: 9),NtAAT3-T (SEQ ID NO: 11),NtAAT4-S (SEQ ID NO: 13) andNtAAT4-T (SEQ ID NO: 15), and whose predicted polypeptide sequences are NtAAT1-S (SEQ ID NO: 6), NtAAT1-T (SEQ ID NO: 8), NtAAT2-S (SEQ ID NO: 2), NtAAT2-T (SEQ ID NO: 4, NtAAT3-S (SEQ ID NO: 10), NtAAT3-T (SEQ ID NO: 12), NtA AT4-S (SEQ ID NO: 14) and NtAAT4-T (SEQ ID NO: 16).

Gene expression analyzes showed that NtAAT2-S (SEQ ID NO: 1) and NtAAT2-T (SEQ ID NO: 3) are the genes expressed at the highest level (>11x) after 48 hours of drying in Burley, Virginia and Oriental type tobacco compared to green tobacco leaves (see Table 1). Interestingly, NtAAT1-T (SEQ ID NO: 7) was also activated after 48 hours of drying, but to a lesser extent (>2.5). In mature leaves, only NtAAT2-S and NtAAT2-T are initially activated, indicating that these genes are the main triggers for aspartate production for asparagine synthesis.

The NtAAT2-S and NtAAT2-T genes are characterized by a high level of expression not only during early leaf drying, when chlorophyll is destroyed, but also in the petals (see Table 2). Their expression is very low in roots and leaves (see Table 2) and other tissues, indicating that the function of NtAAT2-S and NtAAT2-T is associated with localization in aerial organs that do not contain chlorophyll. The same observation seems to be true for NtAAT1-S and NtAAT1-T , but to a lesser extent. Therefore, the present inventors cannot rule out that NtAAT1-S and NtAAT1-T also promote aspartate synthesis in dried leaves. In contrast, NtAAT3-S / NtAAT3-T and NtAAT4-S / NtAAT4-T appear to be more constitutively expressed in all plant tissues (see Table 2).

Co-expression analysis confirmed that NtAAT2-S , NtAAT2-T , NtASN1-S and NtASN1-T are co-regulated during the early drying phase. To do this, we used a database of transcriptomes obtained by drying Burley tobacco, consisting of transcriptomes from 34 non-dried and early-dried samples of Burley tobacco. 168 genes were found to be co-expressed with NtASN1-S and NtASN1-T and 12 genes with NtAAT2-S and NtAAT2-T (cut-off >0.9). From this set of transcriptomes, all NtAAT2-S and NtAAT2-T and NtASN1-S and NtASN1-T transcriptomes were present in two sets of RNA sequences (9 total sequences) associated with 5 other transcripts. This co-expression, associated with the time-dependence experiment during drying (see Figure 2), and co-expression in petals and leaves subjected to early drying (see Table 2 and WO2017/042162), indicates that all, NtAAT2-S , and NtAAT2-T , and NtASN1-S , and NtASN1-T , in concert contribute to the assimilation of neither spending in amino acids and asparagine.

The silencing of NtAAT2-S and NtAAT2-T in Burley tobacco plants was investigated to determine whether both genes contribute to the reduction of aspartate levels in dried Burley tobacco leaves. A specific DNA fragment (SEQ ID NO: 17) within the coding sequence for both NtAAT2-S and NtAAT2-T was cloned with the mirabilis mosaic virus (MMV) strong promoter in the GATEWAY vector. This NtAAT2-S and NtAAT2-T gene fragment was flanked between MMV and the 3'-nos terminator sequence of the nopaline synthase gene from Agrobacterium tumefaciens . Burley tobacco line TN90e4e5e10 (Zyvert) was transformed using standard Agrobacterium mediated transformation protocols. TN90e4e5e10 (Zyvert) is a selection from an ethyl methanesulfonate (EMS) mutagenesis Burley population that contains knockout mutations in CYP82E4, CYP82E5v2 and CYP82E10 (see Phytochemistry (2010) 71: 17-18) to prevent nornicotine production. The use of such a baseline may avoid possible difficulties in interpreting future TSNA data.

To ensure selection of plants with low levels of aspartate, leaves of 16 individual T0 plants (E324) and 4 corresponding control lines (CTE324) were analyzed after 60 hours of drying to determine the effect on nicotine, as a control (see figure 3) and aspartate (see figure 4). No significant difference in nicotine content between leaves of T0 plants (E324) and control lines (CTE324) was observed. The best T0 lines showing the lowest levels of aspartate were lines 3, 8, 13, 16, 17 and 20, in which the amount of aspartate was either found at very low levels (75 μg/g) or at undetectable levels. Seeds were harvested from these top T0 lines showing the lowest levels of aspartate. T1 progeny were analyzed by qPCR to determine the potency of NtAAT2-S and NtAAT2-T silencing events for both aspartate and asparagine content.

Because aspartate plays a key role in the synthesis of other amino acids such as asparagine, threonine, isoleucine, cysteine, and methionine, manipulation of the NtAAT genes (for example, via a constitutive promoter or an aging-specific promoter such as SAG12 or E4) can alter the chemical composition of dried tobacco leaves. Similarly, knocking out the NtAAT genes using a gene editing strategy such as CRISPR-Cas or mutant selection can alter the amino acid chemistry of the leaves as well as the smoke and aerosol chemistry of major commercial tobacco varieties.

Any publication cited or described in this document provides relevant information disclosed prior to the filing date of this application. The statements made in this document should not be construed as an admission that the authors of the present invention have no reason to contrast it with earlier such disclosures. 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 present invention, as stated, 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 of NtAAT (FPKM) during early drying in Burley (BU), Virginia (FC) and Oriental (OR) tobacco

BU FC OR Greens mature 48 hours drying Greens mature 48 hours drying Greens mature 48 hours drying NtAAT1-S 7.8 6.7 9.2 11.4 8.1 15.8 12.9 12.9 13.0 NtAAT1-T 10.5 13.4 28.5 11.0 5.5 28.1 12.4 10.0 38.9 NtAAT2-S 9.8 21.9 234.1 1.9 6.0 65.1 18.5 49.3 665.6 NtAAT2-T 2.7 9.4 105.8 1.1 2.3 12.0 5.1 11.7 80.4 NtAAT3-S 14.5 10.7 17.7 21.2 13.2 11.1 24.5 18.1 22.6 NtAAT3-T 14.7 13.0 21.0 18.2 16.6 9.7 29.4 29.0 22.7 NtAAT4-S 44.5 39.8 29.0 45.6 41.7 28.9 6.4 7.0 3.2 NtAAT4-T 60.7 47.0 21.6 57.3 55.9 19.2 44.9 47.3 25.8

TABLE 2

Expression of NtAAT genes in leaves, petals, and roots of Burley (BU) and Virginia (FC) tobacco plants grown in the field

Sheet Petal Root BU FC BU FC BU FC NtAAT1-S 5.2 9.8 17.3 27.6 15.0 11.1 NtAAT1-T 6.6 6.9 52.4 33.5 3.8 4.2 NtAAT2-S 7.6 4.1 152.7 213.0 7.1 6.1 NtAAT2-T 2.1 1.0 68.2 128.7 3.4 4.5 NtAAT3-S 20.5 19.5 25.4 26.0 30.9 18.9 NtAAT3-T 20.9 19.9 37.4 36.9 31.3 24.4 NtAAT4-S 69.0 44.8 42.3 49.8 29.1 24.9 NtAAT4-T 81.5 49.1 43.2 46.9 18.2 18.8

--->

SEQUENCE LIST

<110> Philip Morris Products S.A.

<120> MODULATION OF PLANT AMINO ACID CONTENT

<130> P10499EP

<140>EP18164766.0

<141> 2018-03-28

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 6933

<212> DNADNA

<213> Nicotiana tabacum

<400> 1

atgaacatgt cacaacaatc accgtcaccg tccgctgacc ggaggttgag tgttctggcg 60

agacaccttg aactgtcgtc ctccgccacc gtcgaatcct ctatcgtcgc tgctcctacc 120

tctggaaatg ctggaaccaa ctctgtcttc tctcacatcg ttcgcgctcc cgaagatcct 180

attctcggcg taactctctc tctctctctc tctctctctc ttcatccaca cacacacgca 240

ctcactcaca taacatatta agtatatgcg tgctcaaatg ttctgtatgt attcatttgt 300

tccgtatcaa atgttctctt gttataagct gaattttaga ggaattgtag tgctatttgc 360

taatcgaaag agcttgatac tcattctctt cctattgaat taaatattcc ttttttctta 420

tggatgatga atttaagact tttttttagt ccgatcacta cgaaatttcg atttcaagtt 480

gatagaagtg aaaaatgatg gggttaacat atcaattgag cgaataaaaa gagaaattcg 540

600

660

tatcttggtg ttatgttttg gttattaggt cactattgct tacaataaag atagtagccc 720

catgaagttg aatttgggag ttggtgcata tcgcacagag gtgatcatcc tttttggatt 780

ttgtatttgc gctattatgg tcaatggagc actattatca gttgctggat aatcatcctt 840

tttgatattt ccttgattga aatctaaaaa cacgaataaa aagatattta ctgatggatc 900

tgtgttttgg tttcttcaga ttgacgcatt tctgttaatt gaaaagaatt gtgattgttt 960

tggtgattgt ggtgttattt tagcttcata cagttaatcc gacgccgtag tgtactagtg 1020

tttggctgat gtgctgccaa gagataatgt ttaagattat ggtttgccat aattgataaa 1080

atttaatatt aaaagtactt ggctggatgt tctgcgtttg cataacttgt aatgcatatg 1140

aaaaagttac ctttgatttt cataattagt gagaaaactc aagtagcttc cgcattcctg 1200

tcattgcact atcaaacaca ttaaacggtt tccgacatat ctacctagtt tggaacttca 1260

1320

tcaaaagatc acagagaaga ttgcatttat tttttgtagt ctagttggct cagagtctgt 1380

caaacacaac ttgttacatc gcattttacc tgttagttaa gaaacttggg tcatcaacaa 1440

atttgtcatg aggtggttat ttcttggggc tttgtgaatt gctctcagca atctgctagc 1500

tttcttatgt ggactcaaaa caatgaagct cttgagttga tgtgttgatt tttcaatcag 1560

agtaaaacaa gttctatatt tggctgtgag agtaaagtgg gagctattaa aattcctagc 1620

1680

ttgatgaagg gctagtagtt gtgtatactt agttcttttt caaatttcat aagtatctct 1740

tgatggtttt tctcgctgac tgttgaatat ggggctccac agtttgtgtt gctatattga 1800

aatgtttcag ctaataaaac taacgtgttt ctttttcttt ctcccttttt tggggttatc 1860

aggaaggaaa acctcttgtt ttgaatgttg taagacaagc agagcagcta ctagtaaatg 1920

acaggtactt gcattgccat ttcatggagt atgaataaaa tgtttcctta attctatgtg 1980

attaaacttc aagatttctg caggtctcgc gttaaagagt acctatctat tacgggactg 2040

gcagacttca ataaattgag tgctaagctg atacttggcg ccgacaggta taaaagttcc 2100

2160

tgcataggat gatatcttca gataataagg ctccattcca agtgtttgat ggcttggtag 2220

atctttgtga agcatctatt aacatttggt cacatttttt taaaaccaac ttcccatccc 2280

atccatgcca ttccacgtgt cagttattca tgaaaatgct gttcacttgc atacatgtta 2340

ctgccgttgt gttgatttcc tcaactctac tcataatttc tctgtgtggt cgcattctgg 2400

2460

ttagcgtgta aagcaagcaa ctcttgatgc gtgtgatcaa gtgtattgct gtctagagct 2520

gacagatgtt aaatttatct tatgcgtttc caagatcttc cagatgttct atgtaatctt 2580

tttaggccag cttaaacttt gacttgcttc atatacattt atgttaaagg agagttgtta 2640

atatacttca atttttcaca tttttaattc ctctttttac ctgtggtcct cacgagctct 2700

tactttcttt gcttggtaca gccccgctat tcaagagaac agagtaacaa ctgtgcagtg 2760

cttgtctggc acaggctcat tgaggggttgg agctgaattt ttggctcgac attatcatca 2820

agtaaactgc tacatcttcc taacctacct ttcattttcc ttcgttttct tagccttcgt 2880

gggtaaacaa tcttcaaagt tgaattaacc ttgatgtaac cattcctgca gcgcacaatt 2940

tatattcccc aaccaacatg gggaaaccac ccaaaagttt tcactttagc tggattatcg 3000

gtaaagagtt accgctacta tgatccagca actcgtggac tcaattttca aggtatgaaa 3060

cacttcccta caatataatg atgtaacagg atattgtccc

3180 ctgtttacta ttactctctt ccaggatgat ggatgttctt

gattacaaat tatcacaagt ctgaatcaag ttgtggatgg atggtttcac ttgtttgatt 3240

3300

ttctgcgagg aaataagcga agagagatgg agatataact tgataataat ggaatgcaac 3360

aaacgcctaa tttaacatat tagggaccaa ctaacgtcta catttgacat tagctcttaa 3420

cattttgact ttttaatacc ttaccaaaaa taaaaaagat tgacattcta atgtcgcacg 3480

3540

gtcaaaaaag aaattcagtt gtcgaaatgt tcttgaaaaa agttactgca accgcaatgg 3600

3660

aggccattgg agaggggccg ggaaacacca tctgaaagag gtacagtggt accagaagga 3720

3780

tgatgttgtg acagcagtca caataaagaa aagtggtgca atcagaatga tcactggaaa 3840

ggctagaatt gtagaactat cataagaaag tgaattgtgg agggaaatct ctgtgaaaag 3900

acaaaatcta tttaggtcca caagatcata gaggctctaa tgccatgtga gaaactgaga 3960

gagtggacgg aaataaatag attacttgat aaaatacaat ccatacgttt aaatccgaat 4020

gactaacttt aattttaaca caacttttac atctaaaagt atgacacgtg acattctaac 4080

ctttcgtgct tgtgttcaca actttgcata tcgccgactt gtttacaaga actttctttt 4140

tcgtacatga caggtttgtt ggaagacctt ggatctgctc catcgggagc ggtagtgcta 4200

cttcacgctt gtgcccataa ccccactggt gttgatccaa ccattgatca gtgggagcaa 4260

attaggagat tgatgagatc aagaggattg ttgcccttct ttgatagtgc atatcaggta 4320

agagatcatc aacagatgtg cagagcactt tggctgttgg agttgttgct gtgtgagcat 4380

ttaaaagtga tgtggtttgt tcagtatatg tcaattaacc ttgatattca aactttgata 4440

ttctagggct ttgccagtgg aagcctagat acagatgcac agtctgttcg catgtttgtg 4500

gcagatggag gtgaagtact tgttgctcaa agttatgcaa agaatatggg gctttatggt 4560

gaacgtgttg gagctctaag cattgtacgt cttaaaggac aatggacaac tgtgccttat 4620

ttctgaaaat ttatatctcc agttggtcat ttgttgcatt acctttattt ttctcagatt 4680

gattctcatg atgcataaac tgtcttactg ttttcatagt ggccttcttt tgtgatgtta 4740

aaatttggta gttatgaact gtttaaagct tatatagctt acttccaaat aaataactgt 4800

gagccttgga catcacatat aaattatttt atatcacgga ttcgagccgt ggaaacaacc 4860

tcttgcagaa atgtagggta aggttgcgta taatagaccc ttgtcatccg gcccttcccc 4920

ggacccctgc gcatagcggg agcttagtgc aacgggttgc ctttttttca tcctgaggca 4980

taaaaagttt gtaatttctc aagaatgaat aaagagcctg ttataacagg caatttgcat 5040

atcatatggt gttgtttgtc gcacagtgat gacatattta tcacacaaat gaaagaaaaa 5100

tgaaggatat agttctgaac cctcagttaa actctgctga cagttataat tcttcaaatt 5160

ttctcaaatc tgtaggtctg caggaatgct gatgtggcga gcagagttga gagccagctg 5220

aagttggtga ttaggccaat gtattccaat ccgcccatcc atggtgcgtc aatagttgcc 5280

acaatcctta aagacaggta atatatcaac catcaggaaa ttgcttcttg ggaccctaaa 5340

aagccatttc ctttctttct atatgataga atccagtgta tgttcaaaaa ttatgtttag 5400

tcattgttct gcaaaataaa tcactaattt tctgcagaaa catgtaccgt gaatggaccc 5460

ttgagctgaa agcaatggct gatagaatca tcagaatgcg tcagcaatta tttgatgctt 5520

tacgtgctag aggtaaattt gctgcattat tttcacgtat gtgtgctctt attacatgtt 5580

tcttgttgca tcgacttcgg atatttttct catttttgat aatttcggtt caagtgtcat 5640

tataaatgct acatgttcgt ggcatatact tctacccata aaatatgctg caacttgttc 5700

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atgaaagctt aatttaggaa aaagattaag caaacaaaac atggaattcg acaaattcaa 5820

acatttctcc aaatcttaat agatctcgat cctccttagt gctttcatca acttcttagg 5880

taattcacct cttaactttg gctctgactg ggttctctac ctttggaaaa ccatccccaa 5940

agatgtccct tgcacgatcc ttttggggac atgaactgtt ggatcaggca agaaactatc 6000

ccccaataaa gaaaaattgt tggatcagat ttttttcctga taggttgcat tctttcagca 6060

ttccccttaa agtttgtgat ttggacgttg tcctcatttt ggtataaaaa atgtcattgg 6120

aaactttcca ttttggcaca tcaggtgtta gaatcatcat gtcttcataa attggctata 6180

gacaaagtct catgtcgtca gctcctttct tcagtatcag gcattcttta atcaatgtaa 6240

gtgtcgagca ttgcatgagt aggatactta tttctattta catgaattga tgggcaagtc 6300

6360

gaattcaatt gattggcagg tacacctggt gactggagtc acattatcaa gcagattgga 6420

6480

atctacatga catcagatgg gtaatatgtc atttctcagc aaaaagtact gtatatcata 6540

tcagactacc atgtctcctc cacatctgat atgtgatttt attacctcgt aagaatttct 6600

accctcggat ggtaaaacag aaagagggaa gggagttaaa atcttttcag ccatcagtta 6660

gttcttttct tgcagtattc ttgctacctt agctttgatg aacgctaaga gaaatgtggc 6720

6780

6840

ttttgcagac gcatcagtat ggcaggtctg agctccagga cagttccaca tctagcagat 6900

gccatacatg ctgctgtcgc tcgggctcgt tga 6933

<210> 2

<211> 450

<212> PROTEIN

<213> Nicotiana tabacum

<400> 2

Met Asn Met Ser Gln Gln Ser Pro Ser Pro Ser Ala Asp Arg Arg Leu

1 5 10 15

Ser Val Leu Ala Arg His Leu Glu Leu Ser Ser Ser Ala Thr Val Glu

20 25 30

Ser Ser Ile Val Ala Ala Pro Thr Ser Gly Asn Ala Gly Thr Asn Ser

35 40 45

Val Phe Ser His Ile Val Arg Ala Pro Glu Asp Pro Ile Leu Gly Val

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Thr Ile Ala Tyr Asn Lys Asp Ser Ser Pro Met Lys Leu Asn Leu Gly

65 70 75 80

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

85 90 95

Val Arg Gln Ala Glu Gln Leu Leu Val Asn Asp Arg Ser Arg Val Lys

100 105 110

Glu Tyr Leu Ser Ile Thr Gly Leu Ala Asp Phe Asn Lys Leu Ser Ala

115 120 125

Lys Leu Ile Leu Gly Ala Asp Ser Pro Ala Ile Gln Glu Asn Arg Val

130 135 140

Thr Thr Val Gln Cys Leu Ser Gly Thr Gly Ser Leu Arg Val Gly Ala

145 150 155 160

Glu Phe Leu Ala Arg His Tyr His Gln Arg Thr Ile Tyr Ile Pro Gln

165 170 175

Pro Thr Trp Gly Asn His Pro Lys Val Phe Thr Leu Ala Gly Leu Ser

180 185 190

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

195 200 205

Gln Gly Leu Leu Glu Asp Leu Gly Ser Ala Pro Ser Gly Ala Val Val

210 215 220

Leu Leu His Ala Cys Ala His Asn Pro Thr Gly Val Asp Pro Thr Ile

225 230 235 240

Asp Gln Trp Glu Gln Ile Arg Arg Leu Met Arg Ser Arg Gly Leu Leu

245 250 255

Pro Phe Phe Asp Ser Ala Tyr Gln Gly Phe Ala Ser Gly Ser Leu Asp

260 265 270

Thr Asp Ala Gln Ser Val Arg Met Phe Val Ala Asp Gly Gly Glu Val

275 280 285

Leu Val Ala Gln Ser Tyr Ala Lys Asn Met Gly Leu Tyr Gly Glu Arg

290 295 300

Val Gly Ala Leu Ser Ile Val Cys Arg Asn Ala Asp Val Ala Ser Arg

305 310 315 320

Val Glu Ser Gln Leu Lys Leu Val Ile Arg Pro Met Tyr Ser Asn Pro

325 330 335

Pro Ile His Gly Ala Ser Ile Val Ala Thr Ile Leu Lys Asp Arg Asn

340 345 350

Met Tyr Arg Glu Trp Thr Leu Glu Leu Lys Ala Met Ala Asp Arg Ile

355 360 365

Ile Arg Met Arg Gln Gln Leu Phe Asp Ala Leu Arg Ala Arg Gly Thr

370 375 380

Pro Gly Asp Trp Ser His Ile Ile Lys Gln Ile Gly Met Phe Thr Phe

385 390 395 400

Thr Gly Leu Asn Ser Glu Gln Val Ala Phe Met Thr Lys Glu Tyr His

405 410 415

Ile Tyr Met Thr Ser Asp Gly Arg Ile Ser Met Ala Gly Leu Ser Ser

420 425 430

Arg Thr Val Pro His Leu Ala Asp Ala Ile His Ala Ala Val Ala Arg

435 440 445

Ala Arg

450

<210> 3

<211> 6935

<212> DNA

<213> Nicotiana tabacum

<400> 3

atgaacatgt cacaacaatc accgtccgct gaccggaggt tgagtgtttt ggcgaggcac 60

cttgaaccgt cgtcctccgc caccgtcgaa acctccatcg tcgctgctcc tacctctgga 120

aatgctggaa ccaactctgt cttctctcac atcgttcgtg ctcccgaaga tcctattctc 180

ggggtaactt tctctctctc tctctctctc tctcttcatc cacacgcact cactcacata 240

acatatgtat aagtatttaa gtatatgcgt gctcaaatgt tctgtatata ttcatttgtt 300

ccgtatcaaa tgttctcttg ttataagctg aattttagag gaattgtagt gttatttgct 360

aatcgcaaga gcttgcatac tcattctctt cgtattgaat taaatattcc ttttttctta 420

tggatgacga atttaagcag ttttttgagt ccgatcacta cgaaatttcg atttcaagtt 480

gatagaagtg aaaaatgatg gtgtttacat attaattgag cgaataaaaa gagaaattcg 540

agtgttgata tcttcaaaaa tgttgttaaa tgtagagata tactgtgatt tagtttctgt 600

tataatcttt gccttttttc ttttgaaatt tgaatattgt ttgttgaagt cttcgtgaca 660

tattggtgtt atgttttggt tattaggtta ctattgcata caataaagat agcagcccca 720

tgaagttgaa tttgggagtt ggtgcatatc gcacagaggt gatcatcctt tttggctttt 780

gtatttgcgc tattatcgtc gatggagcac tattatcagt agctggataa tcatcctttt 840

tgatatttcc ttgattgaaa tccaaaaaca cgaataaaaa gaaatttact gatggatctg 900

tgttttggtt tcttcagatt tacgcatttc tgttaattga aaaaatatta tgattgtctt 960

ggtgattgtg gtgttgtttt ggcttcatat agttaatccg acgccgtagt gtactaatgt 1020

ttggctgatg tgctgccaag agaaatgttt aagattatgg tctgccataa ctgataaaat 1080

ttaatattaa aagtacttgg ctggatgttc tgcgtttgca taacttgtaa cgcatatgaa 1140

aaaattacct ttgattttca taattagtga gaaaattaag tagcttccgc attcctgtca 1200

ttgcactatc aaacacatac ggtttatgat atatctacct agtttggaac tttgtgattt 1260

ctatttttca caccttgtaa taaatgataa ttcttggatc tgtggtgtct ttgttcaaaa 1320

gatcacagag aagattgcac ttatgttttg tagtctagtt ggctcagact ctgtcaaaca 1380

catcgcattt tacctgttag ttaagaaact tgggtcatca acaaatttgt 1440

catgaggtgg ttatttcttg gggttttgtg aattgctctc agcaatctgc tagctttctt 1500

1560

caagttctat atttggctgt gagagtaaag tgggagctat taaaattcct agctgaattt 1620

atgtttctta atatcttaaa tccttaaagg tagagggaga ggaaggaggc ttattgatga 1680

aggactagta gttgtgtata cttagttctt tctcaaattt cataagaatc tcttgagggt 1740

ttttctcgct gactgttgaa tatggggctc cacagtttgt gttgctatat tgaaatgttt 1800

cagctaataa tactaacgtg tttctttttc tttctccctt ttgtggggtt atcaggaagg 1860

aaaaccgctt gttttgaatg ttgtaagaca agcagagcag ctactagtaa atgacaggta 1920

cttgtgttgc catttcatgg agtatgaata aaatgtttcc ttaattctat gtgattaaac 1980

ttcaagattt ctgcaggtct cgcgttaaag agtacctatc tattactgga ctggcagact 2040

tcaataaatt gagtgctaag ctgatacttg gtgccgacag gtatacaagt tcctgttctc 2100

tgtatagtgt tgctgataag atatgcaggg agataaagca tgtattttcc tgttgcatag 2160

gatgatatct tccgataata aggctccgtt ccaagtgttt gatggcttgg tagatctttg 2220

tgaagcatct attaacattt gctcacgttt ttttaaaacc aacttcccat cccatccatg 2280

ccattccacg tgtcagttat tcatgaaaat gctgttcact tgcatacatg ttactgccgt 2340

agtgttggtt tctctcaact ctactcataa tttctgtgtg gtcgcattct ggtgatctga 2400

tttatgtgat aatatctgta catgttgttt tgaaatttgg gtagtgtctc tttgattagc 2460

gtgtaaagca ggcaactctt gatgcatgtg gtgaagtgta tgattgctgt ctagagctgt 2520

2580

taggccagct taaactttga cttgcttcat atacatttat gttaaaggag agttgttaat 2640

atacttcaat tttcacattt ttaattcctc ttttacctgt ggtccttacg agctcttact 2700

ttctttgctt ggtacagccc tgctattcaa gagaacagag taacaactgt gcagtgcttg 2760

tctggcacag gctcattgag ggttggagct gaatttttgg ctcgacatta tcatcaagta 2820

aactgctacg tcttcttaac ctacctttca ctctccttcg ttttcttagc cttcgtggggt 2880

aaacaatctt caaagttgaa ttgaccttga tgtaaccatt cctgcagcgc actatttata 2940

ttccccaacc aacatgggga aaccacccaa aagttttcac tttagctggg ttatcagtaa 3000

agagttaccg ctactatgat ccagcaactc gtggactcaa ttttcaaggt atgaaacact 3060

tcccttcaat ataatgatgt aacgggatat tgtcccatta gatatctatg gccatgctgt 3120

ttcctattac tctcttccag gatgatggat gttcttttag tcttattctg gtatttgatt 3180

acaaattatc acaagtctga atcaagttgt ggatagatgg tttcacttga ttgattgcat 3240

tgtaatccag caaacttgta aatccgtcat catctatgct ttttctttat accttttttc 3300

3360

cgcctaattt aacatattag ggaccaacta acatcagcat ttgacattag ctcttaacat 3420

tttgactttt taacacctta tcaaaaaaaa gaaggaaaaa gattgacatt ctaatgtcgc 3480

acggaaccaa aggtgggaat agctgataac atagaaaagt aaccaaacaa gtcctggaat 3540

cttgtcaaaa aaaaattcag ttgccaaaat gttcttggaa aaaattactg caaccacaat 3600

ggtcggaaga ataggaggaa gaaattcaat aatgcgggtc aaatagaggga ggtgccacta 3660

aaaggccatt ggagaggggc cgggaaacac catctgaaag aggcacagtg gtaccagaag 3720

gattatcgaa tgctgatgca tagaaacgag tcagagattg aaacagtcac tggaaagaag 3780

3840

ctggaaaggc tcgaattgta gaactatcat aagaaagtga attgtggctg ggagactctt 3900

tgaaagacaa aatctattta ggtctccaca agatcatgga tgctctaatg ctatgtgaga 3960

aactaagaga gtggacgaaa ataaatggat tacttgataa aatacaatcc atacgtttaa 4020

atccgaatga ctaactttaa ttttaacaca acttttacat ctaaaagtat gacacgtgac 4080

acttaacctt tcatgcttgt gttcacaact tcgcatgtcg ccgacttgtt tacaagaact 4140

ttatttttca tacatgacag gtttgttgga agaccttgga tctgctccat cgggagcgat 4200

agtgctactt catgcttgtg cccataaccc cactggtgtt gatccaacca ttgatcagtg 4260

ggagcaaatt aggagattga tgagatcaag aggattgttg cccttctttg atagtgcata 4320

tcaggtaaga aatcatcaac agatgttcag agcactttgg ctgttggagt tgttgctgta 4380

tgagcattta aaagtgaggc ggtttattca gtatatgtca attaaccttg atattcaaac 4440

tttgatattc tagggctttg ccagtggaag cctagataca gatgcacagt ctgttcgcat 4500

gtttgtcgca gatggaggtg aagtacttgt tgctcaaagt tatgcaaaga atatggggct 4560

ttatggtgaa cgtgtcggag ctctaagcat cgtatgtctc aaaggacaaa ggacaactgt 4620

4680

tcagattgat tctcatgatg catgaactgt cttactgttt tcatagtgtt cttttgtgat 4740

cttaaaattt ggtagttatg aactgttaaa gcttatatag cttacttcca aatatataac 4800

tgtgagcctt ggacatcaca tataaattat tttatatcac ggggtcgaga tgtggaaaca 4860

acctcttgca gaaatgtagg gtaaggttgc gtacaataga cccttgtggt ccggcccttc 4920

ctcggacccc tgcacatagc gggagcttag tgcactgggt tgcccttgtt ttcatcctga 4980

ggcataaaaa gtttttaatt tctcaagaat gaataaagag cctgttataa caggcacttt 5040

gcatgtcata tggtgttgtt tgtcacacag ttatgcatat agtgtagttt atcacacaaa 5100

tgaaaaaaat tgaaggatat agttctgaac cctcagttaa actctgctga cagatataat 5160

tcttcaaaat tttctcgaat ctgtaggtct gcagaaatgc tgatgtggcg agcagagttg 5220

agagccagct gaagttggtg attaggccaa tgtattccaa tccgcccatc catggtgcgt 5280

caatagttgc cacaatcctt aaagacaggt aatatatcaa ccatcaggaa attgcttctt 5340

gggaccccaa aaaagccatt tccttttcttt ctatatgata gaatccagtg tatgatcaaa 5400

aattatgttt agtcattgtt ctgcaaaata aatcactaaa tttctgcaga aacatgtacc 5460

atgaatggac ccttgagctg aaagcaatgg ctgatagaat catcagaatg cgtcagcaat 5520

tatttgatgc tttacgtgct agaggtaaat ttgctgcatt attttcacgt atgcgtgctc 5580

ttattacatg tttcttgctg catcgacttc ggatattttt ctcatttttg ataatttcgg 5640

ttcaagtgtc attataaatg ctacatgttc gtggcgtata cttctacata taaaatatgc 5700

tgcaactcgt tccagctcat ttgtctagat aattgatgaa aaggaccaat cttcacagct 5760

gactctcctg aatgaaagct taacgaagga aaaagattaa gcaaacaaaa catggaattc 5820

gacaaattca aacatttctc caaatcttaa tacatctcga tccttcttag tgctttcatc 5880

aacttcttag gtaattcacc tcttaacttt ggctctgact ggattctcta cctttggaaa 5940

accatcccca aagatgtccc ttgcacgatc cttttgggga catgaactgt tggatcaggc 6000

aagaaactat cccccaataa agaaaaattg ttggatcaga ttttttcctg ataggttgca 6060

6120

aaatgtcatt ggaaacttcc attttggcac atcaggtgtt agaatcatca tgtcttcata 6180

aattggcaat agacaaagtc tcatgtcgtc aactcctttc ttcagtgtca ggcattcttt 6240

aatcaatgca agtgtcgagc attgcatgaa taggatacct attactattt acatgaattg 6300

atgggcaagt cgggcatttt ttagtcggct taaaggtcaa gcattgcatg aacaagatat 6360

ctatttctat tgacttcaat tgattggcag gtacacctgg tgactggagt cacattatca 6420

agcagattgg aatgtttact ttcacgggac ttaactcaga gcaagttgcc ttcatgacca 6480

aagagtacca catctacatg acatcagatg ggtaatgtgt catttcttag cacaaagttc 6540

tgtatatgtc atatcagact accatgtccc ccctacatct gatatgtgat tttattacct 6600

6660

tttgttcttt tcttgtagta ttcttgctac cttagctttg atgttcgcta agagaaatgt 6720

ggcggtacta atgaacattt ctagagcatg gttctttcta agtttgtatt taattgtggc 6780

aacttcaatt 6840

cattttgcag acgcatcagt atggcaggtc tgagctccag gacagttcca catctagcag 6900

atgccataca tgctgctgtt gctcgagctc gttga 6935

<210> 4

<211> 448

<212> PROTEIN

<213> Nicotiana tabacum

<400> 4

Met Asn Met Ser Gln Gln Ser Pro Ser Ala Asp Arg Arg Leu Ser Val

1 5 10 15

Leu Ala Arg His Leu Glu Pro Ser Ser Ser Ala Thr Val Glu Thr Ser

20 25 30

Ile Val Ala Ala Pro Thr Ser Gly Asn Ala Gly Thr Asn Ser Val Phe

35 40 45

Ser His Ile Val Arg Ala Pro Glu Asp Pro Ile Leu Gly Val Thr Ile

50 55 60

Ala Tyr Asn Lys Asp Ser Ser Pro Met Lys Leu Asn Leu Gly Val Gly

65 70 75 80

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

85 90 95

Gln Ala Glu Gln Leu Leu Val Asn Asp Arg Ser Arg Val Lys Glu Tyr

100 105 110

Leu Ser Ile Thr Gly Leu Ala Asp Phe Asn Lys Leu Ser Ala Lys Leu

115 120 125

Ile Leu Gly Ala Asp Ser Pro Ala Ile Gln Glu Asn Arg Val Thr Thr

130 135 140

Val Gln Cys Leu Ser Gly Thr Gly Ser Leu Arg Val Gly Ala Glu Phe

145 150 155 160

Leu Ala Arg His Tyr His Gln Arg Thr Ile Tyr Ile Pro Gln Pro Thr

165 170 175

Trp Gly Asn His Pro Lys Val Phe Thr Leu Ala Gly Leu Ser Val Lys

180 185 190

Ser Tyr Arg Tyr Tyr Asp Pro Ala Thr Arg Gly Leu Asn Phe Gln Gly

195 200 205

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

210 215 220

His Ala Cys Ala His Asn Pro Thr Gly Val Asp Pro Thr Ile Asp Gln

225 230 235 240

Trp Glu Gln Ile Arg Arg Leu Met Arg Ser Arg Gly Leu Leu Pro Phe

245 250 255

Phe Asp Ser Ala Tyr Gln Gly Phe Ala Ser Gly Ser Leu Asp Thr Asp

260 265 270

Ala Gln Ser Val Arg Met Phe Val Ala Asp Gly Gly Glu Val Leu Val

275 280 285

Ala Gln Ser Tyr Ala Lys Asn Met Gly Leu Tyr Gly Glu Arg Val Gly

290 295 300

Ala Leu Ser Ile Val Cys Arg Asn Ala Asp Val Ala Ser Arg Val Glu

305 310 315 320

Ser Gln Leu Lys Leu Val Ile Arg Pro Met Tyr Ser Asn Pro Pro Ile

325 330 335

His Gly Ala Ser Ile Val Ala Thr Ile Leu Lys Asp Arg Asn Met Tyr

340 345 350

His Glu Trp Thr Leu Glu Leu Lys Ala Met Ala Asp Arg Ile Ile Arg

355 360 365

Met Arg Gln Gln Leu Phe Asp Ala Leu Arg Ala Arg Gly Thr Pro Gly

370 375 380

Asp Trp Ser His Ile Ile Lys Gln Ile Gly Met Phe Thr Phe Thr Gly

385 390 395 400

Leu Asn Ser Glu Gln Val Ala Phe Met Thr Lys Glu Tyr His Ile Tyr

405 410 415

Met Thr Ser Asp Gly Arg Ile Ser Met Ala Gly Leu Ser Ser Arg Thr

420 425 430

Val Pro His Leu Ala Asp Ala Ile His Ala Ala Val Ala Arg Ala Arg

435 440 445

<210> 5

<211> 6388

<212> DNA

<213> Nicotiana tabacum

<400> 5

atggcgatcc gagccgcgat ttccggtcgt cccctcaagt ttagctcgtc ggtcggagcg 60

cgatctttgt cgtcgttgtg gcgaaacgtc gagccggctc ctaaagatcc tatcctcggc 120

gttaccgaag ctttcctcgc cgatcctact cctcataaag tcaatgttgg tgttgtaagt 180

ttttttttct ctttgctttg tttgattttc cacttcattt cgtgtaagct aggatttagc 240

ttacttgacc atttcgctat tcttcatagg ccatagctgt aaaaatggtt ttactgtgac 300

360

tatgcattcc acttttttca acttgatcta tttaagaaaa aaattgaaaa agatttgacc 420

ttttttctta aattatttct tttaaatttt ttatttttgt gattatattata tagggagctt 480

acagagacaa caatggaaaa cccgtggtac tggagtgtgt tagagaagca gagcggagga 540

600

atttgtaatt aattacgatt acgttaactt tgatctatta gaaaatagaa acttcagcca 660

gtaagattac tttttttctt cgaggagtgt gagatgtaaa cccaggtcgg atgcactgga 720

attcttcact agtttgtgca aatatattct taatattttt caaaaacttc ctgtgtacac 780

acacacatac atgtaattaa ttaagtaatt acctactgat ctaggatttt taggtagttc 840

ggaaaaatat attttcaata tcgtttaaga attttctggg tatgcgtagt ttgagtatga 900

taaaatgggt gcatgtgcac cactgcttac gctagggaac agcctctaat tagctgttgg 960

1020

1080

aggtctacag aaatgggtgg tacgatattt tccagccgtc agctcacta accagtttga 1140

ttctttggga ctttttcttt gtattctcac gtttacttct agtggatggt gcagatggat 1200

ttctttacta attctttctt ctgcgtttgc agagctttct cccaagataa attattaata 1260

1320

1380

tttattcatt gtggttggag aaagtattaa aataacaagt gaaaagtctg ttttacgtca 1440

agtttgaatt tgaatttttg aattgatttg cttctttaag tttatgaaac catctatgag 1500

aatattattg gcactccatt gtctcatttt atgtgaaggc atttgacgtt gcacaaaatt 1560

taaaaatata aagaaactta tgacgtgtaa gttagatata tgcagagtac catagttatc 1620

cttttaagta agtgatagtg agagtttcac atttcttttt gttctctctt cttataaaag 1680

aatgaatttt tgtatcccaa caagtcatat cattaatgtt aacacgggga tactaagatt 1740

aactaatgtc taaaaagaga aagatttcat tcttcttgga cgggctaaaa aggaaagtat 1800

gtcacataaa atgagacaga gatatgttag gatttgtcct gaatttctaa tcaattagga 1860

ctctctttct tgaaggaatg gaaaaagact cctaaaagga ttaaatctcc ttaatatgtc 1920

ttatcctttt cttggaagac aagttttgca catctataaa ttaagggatct ctgcttttca 1980

2040

tttttctctc gatagttttt cttcttttat attagtttta tcatatgtag atcaattgac 2100

2160

cgtccatcaa gttttgtatt gttagttttc gcatgcacca tgttatttcg aacccaacaa 2220

gtggtatcag agccaatggt tcagagtcaa cggttgaacg aggttgaaga aagattcaat 2280

gcgtgtttag atcaagacga taatgaagat ttattcaaca tgctacatgt ggagataaat 2340

ttacaattac aattgtattc aacacgcctg ctgttgcatc tttattggaa taaaaactct 2400

ttctaaccca tattttggcc actttaaacc aatgccaatt ttaagtcatg cctacttgca 2460

acacatgagt tccagtgcca gttttaactc acgcttcttt ttaatgcatg agcttctttt 2520

atcccatgtt tattcttaac acgcgggctc cttttaaccc atacttggtt tttttttaac 2580

caataccaag attttcaatt tttaatcatg gcaagcagca gtgatgaaga ttatgtgaag 2640

aaggtgaatc aactttgtca agaaaattca ggccaagggg agattgttag gatttgtcct 2700

gaatttctaa tcaattagga ctctctttct tgaaggaatg gaaaaagact cctaaaagga 2760

ttaaatctct ttactatgtc ttatcatttt cttggaagac aagttttgca catctataaa 2820

ttaaggatct ctgcttctca caagagcaca aaaaaaaaaa tatccacaat gtagttatta 2880

aagagttttg tttagggggga gatttttctt tcaatagttt ttcttctttt atattagttt 2940

3000

3060

3120

gatgggggag aactacgcta tctgatctca aaaaaagtgt gcatcatgtg atactatgtg 3180

3240

gtagtgtcaa catgatcgag gagtcactga agttagccta tggggagaac tcagacttga 3300

taaaagataa gcgcattgca gcaattcaag ctttatccgg gactggagca tgccgaattt 3360

ttgcagactt ccaaaggcgc ttttgtcctg attcacagat ttatattcct gttcctacat 3420

ggtctaagta agtgtattct tctgcttctc ggcatctcta cagcatccta attgatcttc 3480

ctcaattggt ttttgcactt taaaacatga gtatgcaaat accttcaaaa ttttctaatt 3540

tcctgtcatt actaatataa agttcttggc agtcatcata acatttggag agatgctcat 3600

gtccctcaga aaatgtatca ttattatcat cctgaaacaa aggggttgga cttcgctgca 3660

ctgatggatg atataaaggt aagaaaacat atatttgagg ttgtttgcca tgatggttgg 3720

ttctcctgtt tgatgatata gtgtccctcc tcaagtggca attatgtgtt ctatcctgac 3780

gtatttcaat ttttcattgac atagaatgcc ccaaatggat cattctttct gcttcatgct 3840

tgcgctcaca atcctactgg ggtggatcct acagaggaac aatggaggga gatctcacac 3900

3960

aatttattac actggttcca ggtgaaggga cattttgctt tctttgacat ggcctatcaa 4020

ggatttgcta gtgggaatcc agagaaggat gctaaggcta tcaggatatt tcttgaagat 4080

ggtcatccga taggatgtgc ccaatcatat gcaaaaaata tgggactata tggccagaga 4140

gttggttgcc taaggtaaac tactactccc accatcatat cttatttgcc ctagttacaa 4200

tctggagagt caaacaaact ttttattaga ccatagttgg tctatttttc aaatgatcta 4260

attccaaaag cagttactac tatttcatgc aaattctaga ttaattaacc ttttgctata 4320

cctcattatc ttcatttagt aggcagtagc taattttacc atatatcata tttttcatat 4380

4440

gttctttccg gtaatttcag ttcatttcca cctaaaagtc caactcgaag agaaaaacag 4500

aattttgcta gtcaaaactt agatccaatg aaaagcacca aaattttggt tttaaattat 4560

aaaacatgtc tactctaggt ttttttccta ggcaagcgat gtgattttac aatcttacaa 4620

taaggcatca atcaatccca aactagtttg gttcaacaat ataaatcttt gttcctattt 4680

catggcattg ggcccattgc attccaataa tggtaaataa gacaaattag aggttatcta 4740

agattagaga ttccttatta aaatctcata cttatatcta cattgtcacg caagtctctg 4800

accttaaaag aagacttcta gcagacacca gacttaacgt aagtgtaaca acaacaacta 4860

agttttaatc caaactagtt agtatcgctt atatgaatcc cttgcttcca ttgtgtagca 4920

ctaaacaaca acaacaacat accgagtata atctcacata gtggggtctg gggagggtag 4980

tgtgtacgca gactttaccc ctgccttgtg gagaaagaga ggctatttcc aatagaccct 5040

cggctcaaaa ccattgtgta gcaatgggagg catgaaatag aaaacttgtc ttctgattaa 5100

aatctgttat taaattaatg aggagaaaaa attggattac ttgttggtaa atcttatttg 5160

ttgttttaaa cacacacaaa gccgaaagac ctctgatact ttttcctgaga tgcttccgca 5220

attcactgca gtgtggtttg tgaggatgaa aaacaagcag tggcagtgaa aagtcagttg 5280

cagcagcttg ctaggcccat gtatagtaat ccacctgttc atggcgcgct cgttgtttct 5340

accatccttg gagatccaaa cttgaaaaag ctatggcttg gggaagtgaa ggtaatatgg 5400

ttagaacaag aaaagattta tgattatata actatcattg gtattttgac aaaaggtagg 5460

aactaggaac cttgctatta aagatatttt cttcccttta ttttggaaaa aaaggtattt 5520

tcttgctttc ttcaaatgtt tgagatttgg atagagccgt tacatggaaa tgctgtgcaa 5580

ttttctgcta ctcacatgga aaagatcttt ttcttttgct gatctgttta agcacctatt 5640

tgctaaagcc tactatgtca gtatgttgtt caatcttttc agccacagaa acaggtctaa 5700

accagttcca aactttaaat aatcttacca tggtagtttc agcaaagata aattggtccg 5760

tgcagccatt aactgttttc tttgtcgggc ttcttaaact tgttttctcc aaggtctagt 5820

tgttggtgct gtggctgctt tttagctttg tgctcattat cagagcatca tatgtttaag 5880

tgtaaaggtt caatgactaa gttctttttc cagggcatgg ctgatcgcat tatcgggatg 5940

agaactgctt taagagaaaa ccttgagaag ttggggtcac ctctatcctg ggagcacata 6000

accaatcagg tattgaaatc aacaacttct gttgttttct atgctactag tatataacta 6060

ttaagaaaat tactgtggtt cacctactgc gccattaata ctcgatacca ccaacagatt 6120

ggcatgttct gttatagtgg gatgacaccc gaacaagtcg accgtttgac aaaagagtat 6180

cacatctaca tgactcgtaa tggtcgtatc aggtataatc attaggtcac caatttctgc 6240

6300

6360

tattcatgag gccaccaaat cagcttaa 6388

<210> 6

<211> 424

<212> PROTEIN

<213> Nicotiana tabacum

<400> 6

Met Ala Ile Arg Ala Ala Ile Ser Gly Arg Pro Leu Lys Phe Ser Ser

1 5 10 15

Ser Val Gly Ala Arg Ser Leu Ser Ser Leu Trp Arg Asn Val Glu Pro

20 25 30

Ala Pro Lys Asp Pro Ile Leu Gly Val Thr Glu Ala Phe Leu Ala Asp

35 40 45

Pro Thr Pro His Lys Val Asn Val Gly Val Gly Ala Tyr Arg Asp Asn

50 55 60

Asn Gly Lys Pro Val Val Leu Glu Cys Val Arg Glu Ala Glu Arg Arg

65 70 75 80

Ile Ala Gly Ser Phe Asn Met Glu Tyr Leu Pro Met Gly Gly Ser Val

85 90 95

Asn Met Ile Glu Glu Ser Leu Lys Leu Ala Tyr Gly Glu Asn Ser Asp

100 105 110

Leu Ile Lys Asp Lys Arg Ile Ala Ala Ile Gln Ala Leu Ser Gly Thr

115 120 125

Gly Ala Cys Arg Ile Phe Ala Asp Phe Gln Arg Arg Phe Cys Pro Asp

130 135 140

Ser Gln Ile Tyr Ile Pro Val Pro Thr Trp Ser Asn His His Asn Ile

145 150 155 160

Trp Arg Asp Ala His Val Pro Gln Lys Met Tyr His Tyr Tyr His Pro

165 170 175

Glu Thr Lys Gly Leu Asp Phe Ala Ala Leu Met Asp Asp Ile Lys Asn

180 185 190

Ala Pro Asn Gly Ser Phe Phe Leu Leu His Ala Cys Ala His Asn Pro

195 200 205

Thr Gly Val Asp Pro Thr Glu Glu Gln Trp Arg Glu Ile Ser His Gln

210 215 220

Phe Lys Val Lys Gly His Phe Ala Phe Phe Asp Met Ala Tyr Gln Gly

225 230 235 240

Phe Ala Ser Gly Asn Pro Glu Lys Asp Ala Lys Ala Ile Arg Ile Phe

245 250 255

Leu Glu Asp Gly His Pro Ile Gly Cys Ala Gln Ser Tyr Ala Lys Asn

260 265 270

Met Gly Leu Tyr Gly Gln Arg Val Gly Cys Leu Ser Val Val Cys Glu

275 280 285

Asp Glu Lys Gln Ala Val Ala Val Lys Ser Gln Leu Gln Gln Leu Ala

290 295 300

Arg Pro Met Tyr Ser Asn Pro Val His Gly Ala Leu Val Val Ser

305 310 315 320

Thr Ile Leu Gly Asp Pro Asn Leu Lys Lys Leu Trp Leu Gly Glu Val

325 330 335

Lys Gly Met Ala Asp Arg Ile Ile Gly Met Arg Thr Ala Leu Arg Glu

340 345 350

Asn Leu Glu Lys Leu Gly Ser Pro Leu Ser Trp Glu His Ile Thr Asn

355 360 365

Gln Ile Gly Met Phe Cys Tyr Ser Gly Met Thr Pro Glu Gln Val Asp

370 375 380

Arg Leu Thr Lys Glu Tyr His Ile Tyr Met Thr Arg Asn Gly Arg Ile

385 390 395 400

Ser Met Ala Gly Val Thr Thr Gly Asn Val Gly Tyr Leu Ala Asn Ala

405 410 415

Ile His Glu Ala Thr Lys Ser Ala

420

<210> 7

<211> 4789

<212> DNA

<213> Nicotiana tabacum

<400> 7

atggcgattc gagccgcgat ttccggtcgt tccctcaagc atattagctc gtcggtcgga 60

gcgcgatctt tgtcgtcgtt gtggcgaaac gtcgagccgg ctcctaaaga tcctatcctt 120

ggcgttaccg aagctttcct cgccgatcct actccccata aagtcaatgt tggcgttgtg 180

agtttttttt tcctctttgt tttgcttcat tttccacctc atttcgtgta tgcaaggatt 240

tagcttactt gaccattttcg ctatacttcc cttggtaggc catagctgta aaaaatagtt 300

ttactgtgac gaatcatcga catatggata cagagtattc taatggagta gtcaacaaca 360

taagtcgatc tcaatcgctt tgggattgag aaagagttta ttgatttaat ttttgtatgc 420

gttccacttt tttcaacttg atctatttaa gaaaaaaatt gaaaaagatt tgaccttttt 480

tcttaaatta tttcttttat aaaatttgct tttgtgatta ttatacaggg agcttacagg 540

gacgacaacg gaaaacccgt ggtactggag tgtgtcagag aagcagagcg gaggatcgct 600

ggcagtttca acatgtgagt gcttctcctg atttattcat ttttttctgt tatttatttg 660

taattaatta cgattacgtt aaatttgatc tattagaaaa tataaacttc agccagtaag 720

attacttttt ttcttcgagg agtttgagat gtaaaaccca ggtcggatgc actgggattc 780

ttaagtagtt tgtacaaata tattcttaat agttttgtaa aatttgctgt atacacacac 840

atgtaattaa ttacctactg atctaggatt tataggtagt tcggaaaaat atattttcaa 900

tatcgtttaa gaatttcctg ggtgtgtata gtttgagtat gagaaaatgg gtgcatgtgc 960

accactgctt acgctagggga tcagcttcta aatagctggt ggtgagtctg gtgagtggtg 1020

actgttcttt attttcagtt actgtagcca caaattgttg gttattgatt aagtataaat 1080

1140

gggtggtccg atattttcca cccgtcagtt cctctaacta gtttgattct ttgggacttt 1200

ttctttgtat tctcacgttt acgcctagtg gatggtgcag atggatttct ttactaattc 1260

tttcttctgc gcttgcagag ctttctccca agataaatta ttaatatcaa attgaccttt 1320

cgatagttca atggtgttta actttttcaa atattgcccc acatcccatt ttatattatg 1380

1440

ttcattgtgg taggagaaac tagaaagtat taaaataaca agtgaaaagt ctgttttatg 1500

1560

ttatgaaacc atctatgaga atattattag cactccattt gtctcatttt atgtgaaggc 1620

atctgacttt gcacaaagtt aaaaaatata aagatactta cgacgtgaaa gtttgatata 1680

tgccaagtac cataattatc cttttaagca agtgatagtg agagtttaac atttcttttt 1740

gttctctctt cttataaaag aatgaatttt gtatcaagtg ggtcccaaca agtcattcat 1800

taagggtaaa acggggatgc taagattaac taatttccaa aaagagaaag atttaattct 1860

tctaggacag gctaaaaatg gaaagtgttt cacataaaat gagacataga caatataagt 1920

ttgtcaaaca ttttgtgacg tcctaaaata gaaagtgtcc tgagatggag gagaactacg 1980

ttatctgttc tcaaaaaagt gtgtatcatg tgatactatg tggatagttt agttcacatg 2040

ttaacaattc atcatttatc agggaatatc ttcctatggg aggtagtgtc aacatgatcg 2100

aggagtcact gaagttagcc tatggggaga actcagactt gataaaagat aagcgcattg 2160

cagcaattca agctttatct gggactggag catgccgaat ttttgcagac ttccaaaggc 2220

2280

cttctgcttc tcggcatctc tacagcatcc taattgatct tcctcaattg gtttttgcac 2340

attaaaacat gagtatgcaa ataccttcaa aatttttctaa tttcctgtca ttactaatat 2400

aaaattcttg gcagtcatca taacatttgg agagatgctc atgtccctca gaaaacgtat 2460

cattattatc atcctgaaac aaaggggttg gacttcactg cactgatgga tgatataaag 2520

gtaagaaaac atatatttga ggttgttttc catgatggtt tgttctcctg tttgatgata 2580

tagcgtccct cctcaagtgg caattatgtg ttctatcctg acgtatttca attttcattg 2640

acatagaatg ccccaaatgg atcattcttt ctgcttcatg cttgtgctca caatcctact 2700

ggggtggatc ctacagagga acaatggagg gagatctcgc accacttcaa ggtaatgatt 2760

ttgtatattt tgtctctcct ttttcttgta ccaagtcata ctaaatttat tacactggtt 2820

ccaggtgaag ggacattttg ctttctttga catggcctat caaggatttg ctagtgggaa 2880

tccagagaag gatgctaagg caatcaggat atttcttgaa gatggtcatc cgataggatg 2940

tgcccaatca tatgcaaaaa atatgggact atatggccag agagttggtt gcctaaggta 3000

3060

actttttgtt agaccttagt cggtctattt ttcaaatgtt ctaattccaa aagcagttac 3120

tactatttcc tgtaaattct agattaatta actttttatt atacctcatt atcttcattt 3180

3240

3300

tttccacctc aaagtccaac tcgacgtgaa aagcagaatt ttgctagtca aaacttggat 3360

ccaacaatta tttagaataa attgagttct ttccgataac ttcagttcat ttccacctca 3420

aagtccaact cgacgagaaa aacagaattt tgctagtcaa aacttggatc caatgaaaag 3480

caccaaaatt ttggttttaa attacaaaat aatgtatact ctaggttttt gtcctatgca 3540

agtgatttta cggtcttaaa ataaagcatc aatcaatccc ttaaacacac acaaagccct 3600

ctaatacatt tgctgagatg cttccgcaat tcactgcagt gtggtttgtg aggatgaaaa 3660

gcaagcagtg gcagtgaaaa gtcagttgca gcaacttgct aggcccatgt acagtaatcc 3720

acctgttcat ggtgcgctcg ttgtttctac catccttgga gatccaaact tgaaaaagct 3780

3840

ctatcattgg tattttgacg acagatagga actaggaacc ttgctattaa agatattttc 3900

3960

agaactatca catggaaatg ctgtaccatt ttctgctact cacatggaaa agatcctttt 4020

cttttgctga tctgtttaag caccaatttg ccatagcttt gttgtcctat attttcagcc 4080

acagaaataa gtctaaacca gtcccaagct ttaataagct ttcattgcgt ggtagtgtca 4140

gccgcataaa ttggtcagtg cagccattaa ctgttttctt catggggcct gttaaccttg 4200

tatttctcca aggtcaagtt gttggtgttg tggctgcttt ttagctttgt actcattatc 4260

agagcatcat atgttaaacg taaaggttca atgactaaga gttttttttc cagggcatgg 4320

ctgatcgcat catcgggatg agaactgctt taagagaaaa ccttgagaag aagggctcac 4380

ctctatcgtg ggagcacata accaatcagg tattgaaatc aatgacttct gttgcgttct 4440

atactagtat ataactatta gaacactatg gctcacctat tgccccatta atactcgata 4500

ctgcctacag attggcatgt tctgctatag tgggatgaca cccgaacaag ttgaccgttt 4560

gacaaaagag tatcacatct acatgactcg taatggtcgt atcaggtata atcactcatt 4620

cacgaatttc tgcttaatgc tccggtgttc ttgtacgagt taatatctca ttaatttttc 4680

cactatgtta tactgtgtgt tttgtatgtt gtgcagtatg gcaggagtta ctactggaaa 4740

tgttggttac ttggcaaacg ctattcatga ggttaccaaa tcagcttaa 4789

<210> 8

<211> 425

<212> PROTEIN

<213> Nicotiana tabacum

<400> 8

Met Ala Ile Arg Ala Ala Ile Ser Gly Arg Ser Leu Lys His Ile Ser

1 5 10 15

Ser Ser Val Gly Ala Arg Ser Leu Ser Ser Ser Leu Trp Arg Asn Val Glu

20 25 30

Pro Ala Pro Lys Asp Pro Ile Leu Gly Val Thr Glu Ala Phe Leu Ala

35 40 45

Asp Pro Thr Pro His Lys Val Asn Val Gly Val Gly Ala Tyr Arg Asp

50 55 60

Asp Asn Gly Lys Pro Val Val Leu Glu Cys Val Arg Glu Ala Glu Arg

65 70 75 80

Arg Ile Ala Gly Ser Phe Asn Met Glu Tyr Leu Pro Met Gly Gly Ser

85 90 95

Val Asn Met Ile Glu Glu Ser Leu Lys Leu Ala Tyr Gly Glu Asn Ser

100 105 110

Asp Leu Ile Lys Asp Lys Arg Ile Ala Ala Ile Gln Ala Leu Ser Gly

115 120 125

Thr Gly Ala Cys Arg Ile Phe Ala Asp Phe Gln Arg Arg Phe Cys Pro

130 135 140

Asp Ser Gln Ile Tyr Ile Pro Val Pro Thr Trp Ser Asn His His Asn

145 150 155 160

Ile Trp Arg Asp Ala His Val Pro Gln Lys Thr Tyr His Tyr Tyr His

165 170 175

Pro Glu Thr Lys Gly Leu Asp Phe Thr Ala Leu Met Asp Asp Ile Lys

180 185 190

Asn Ala Pro Asn Gly Ser Phe Phe Leu Leu His Ala Cys Ala His Asn

195 200 205

Pro Thr Gly Val Asp Pro Thr Glu Glu Gln Trp Arg Glu Ile Ser His

210 215 220

His Phe Lys Val Lys Gly His Phe Ala Phe Phe Asp Met Ala Tyr Gln

225 230 235 240

Gly Phe Ala Ser Gly Asn Pro Glu Lys Asp Ala Lys Ala Ile Arg Ile

245 250 255

Phe Leu Glu Asp Gly His Pro Ile Gly Cys Ala Gln Ser Tyr Ala Lys

260 265 270

Asn Met Gly Leu Tyr Gly Gln Arg Val Gly Cys Leu Ser Val Val Cys

275 280 285

Glu Asp Glu Lys Gln Ala Val Ala Val Lys Ser Gln Leu Gln Gln Leu

290 295 300

Ala Arg Pro Met Tyr Ser Asn Pro Val His Gly Ala Leu Val Val

305 310 315 320

Ser Thr Ile Leu Gly Asp Pro Asn Leu Lys Lys Leu Trp Leu Gly Glu

325 330 335

Val Lys Gly Met Ala Asp Arg Ile Ile Gly Met Arg Thr Ala Leu Arg

340 345 350

Glu Asn Leu Glu Lys Lys Gly Ser Pro Leu Ser Trp Glu His Ile Thr

355 360 365

Asn Gln Ile Gly Met Phe Cys Tyr Ser Gly Met Thr Pro Glu Gln Val

370 375 380

Asp Arg Leu Thr Lys Glu Tyr His Ile Tyr Met Thr Arg Asn Gly Arg

385 390 395 400

Ile Ser Met Ala Gly Val Thr Thr Gly Asn Val Gly Tyr Leu Ala Asn

405 410 415

Ala Ile His Glu Val Thr Lys Ser Ala

420 425

<210> 9

<211> 4551

<212> DNA

<213> Nicotiana tabacum

<400> 9

atggcaaatt cctccaattc tgtttttgcg catgttgttc gtgctcctga agatcccatc 60

ttaggagtac gtccctttcc actctttcta ttttacattt ccactgaata tgtttcttct 120

gtggctcctt taataatctt ccgtaaatat actattagtg gatttgataa gctacttctc 180

tctccctctc tctttttttt tcttattttg ggttagatta aaatgaacat taattaatga 240

tcagatgatt tggttaaaga tgatatctag gagatcggca taaataagtt gattggaatg 300

360

tagtacttct ttctttttac tgtgatctgg caattcctta tttttattcct ggtgtagttg 420

atgaaaggtg tagatttgat tctttaactt gctctattga gaaggtaatt tgtgcttctc 480

aagtgtttat taatgttgtt ttcttctgtt gtgttacttc attaaaacag gtcacagttg 540

cttataacaa agataccagc ccagtgaagt tgaatttggg tgttggcgca tatcgcactg 600

aggtctgcca cttctacttt gtctcgttgt tctttattat tattattttt ttattatagc 660

caaaaaaagt tgccccttga atggatttgg tcctgctatg tgttgaatcc ttggttaagt 720

ttttctttaa taggctcctt cacaaggata gaaaattgta gacactgatg cttacacatt 780

agtaatattt tttcccctga tgcataatga agtgaaacca cttgtgcttc taaaaaatca 840

tactttgggg caaggtgaag tacacatttt tataagtggt tgtttttttc ttcaatcttg 900

agttgaatgt tagtgttaag taggagccgc aaacgggcgg gtcgggtcgg atttggttca 960

gatcgaaaat gggtaatgaa aaaacaggta aattatctga ctcgacccat atttaatacg 1020

gtaaaaaca ggttaaccgg cggataatat gggtaaccat attattcatg tcttcttgca 1080

tatgatcaat tatgggagaa ttcttagcct caaatgggaa cccccaattt gaggctttac 1140

aaatttaaaa gttagaccca ttggttaacc attttctaaa tggataatat ggttcttatc 1200

catatttgac ccatttttaa aaagttcatt atccaaccca ttttttagtg gataatatgg 1260

gtgtttaact gatttctttt aaccattttg acacccctag tgttaagctt gaaaacgact 1320

aatgcatagt ctgatgacaa cttgcaggaa ggaaagcccc ttgttcttaa tgtggtgaga 1380

cgggctgaac aaatgctcgt caatgacacg taacttgcca aattagaaac tagcttacag 1440

attttctttt gagatatgat cacctgatac caagattgga atctaaggct gctatgatgc 1500

aggtctcggg tgaaggagta tctctcaatt actggactag cggattttaa caaactgagt 1560

1620

aatgcttgag tcaatttttt ttaaaaaaaa tgctcactat ccatgtcgct ctatttaaac 1680

ctatcttgcc aaaccacttg tataaatgaa aatgagccgt cgatattctt ccttccatct 1740

agtttgatat ttgaattaga gattgttgct aaaagggaat gctttatctc tacagcgtag 1800

agtaactgaa tacctgttaa acatgttcct ccgtatttca tcttattatg atgccttgca 1860

tctgaagaaa attgttctag agttaacttt ctctcctctt tgttgtactg attatctgtg 1920

tgtggtgaac gcgatatcag gaaatatgtg tcttctgtca ctattactcc ttgttaagtc 1980

atatgtaatt gacttgttat gatatcaaca gatttactta tgtttagatg tagtttaaat 2040

gctttttgtg ctgttttgtt gcttatacag ccctgccatt caagagaaca gggtgactac 2100

tgttcagtgc ttgtcgggca caggttcttt gagggttggg gctgaatttc tggctaagca 2160

ttatcatgaa gttagtattc cttgctctct ttccctttat atgtctaaat caaatggaca 2220

cttgtataag cttctactgt ttgttttgtt gccagcatac catatatata ccacagccaa 2280

catggggaaa ccatccgaag gttttcactt tagccgggct ttcagtaaaa tattaccgtt 2340

actacgaccc agcaacacga ggcctggatt tccaaggtac tactgtaatc actgttctta 2400

aagttctaca gttgtaagta agagccgatt tctctttttt atggacaagt gaactttctc 2460

ctggtcgtgt ctagaaagat ctatatttta tgtgtagcta gcacaggatc tttatttatt 2520

taattttgta ttctcttggt aaagatataa gcatagttta tctgtggctt ttcctgtatt 2580

tgggtgttgc atatcaaatt taatcatgaa ccctgtagga cttttggatg atcttgctgc 2640

tgcacccgct ggagcaatag ttcttctcca tgcatgtgct cataacccaa ctggcgttga 2700

tccaacaaat gaccagtgg agaaaatcag gcagttgatg aggtccaagg gcctgttgcc 2760

tttctttgac agtgcttacc aggtaaagct tatgatggga ttttgaattc aagtgatact 2820

tcgttaagaa tgattaccaa ataatttgaa gcgccaaact atgtattaat gggctgccca 2880

acggaccctt actataatga atatttttga tattgcaggg ttttgccagt ggcaacctag 2940

atgcagatgc acaatctgtt cgcatgtttg tggctgatgg tggtgaatgt cttgcagctc 3000

agagttatgc caaaaacatg ggactgtatg gggagcgtgt tggtgccctt agcattgtaa 3060

gtccttttgt cggttgtaat tgctttccct ttttagtaag cgataaaatt ggtggctgaa 3120

gaactatcca tggctatatc atgctatcta tgtctaaaga tgattttcct tgaaagcata 3180

attcaggtta tattccctag aaggctaaaa agaagttgtt ctgatggtac aatgaacaca 3240

gtctctagag atattgaaag ccaaattttt gaatatggct tcccctttga ttgtaattgg 3300

3360

3420

gtgttgtgtt ataggtttgc aaagacgcag atgttgcaag cagagtcgaa agccagctaa 3480

agctggttat caggccaatg tactctaatc caccaattca tggtgcgtct attgttgcta 3540

ctatactcaa ggacaggttt gtgcaactat ttacaagatt ctgttttgct gttagtagat 3600

gctatacctt ctacattttg atgtggtttc tcatctaatg gtgatagaca aatgtacgat 3660

gaatggacaa ttgagctgaa agcaatggcc gacaggatta ttagcatgcg ccaacaactc 3720

tttgatgcct tgcaagctcg aggtatttga tcttcatatt tgttctttct ggggaagcat 3780

actgtattct gtatgatggg tttgactgct actgcaatag gagctttttc ctgaaaagta 3840

ccatggtgaa acaaccacgg caactaaatc ttttgacttc attgttcagt ttagtgctaa 3900

tgtaagtttt attctgttat gcaggtacga caggtgattg gagtcatatc atcaagcaaa 3960

ttggaatgtt tactttcaca ggattaaata ctgagcaagt ttcattcatg actagagagc 4020

4080

gtttagtttg ttactttgggg ttgctttttt ctcagtagaa acttaaataa ttggaactta 4140

gaagtccttc gttgattatt tcggcttgaa ttctttaata aggagaattt cagatttata 4200

4260

aaagtgcagt tctgttttcc cccctcccag attagactaa ttcccaaaag aacttacctt 4320

caatctatgg aacatttagt attctggtat cagttgaaac atctctttgt tgaagttaag 4380

attttggtta aaaagatctt catctctagt aacattttct acattccatt tttgaagga 4440

atgattttct cctttctcat ttgcaggaga attagcatgg caggccttag ttctcgcaca 4500

attcctcatc ttgccgatgc catacatgct gctgttacca aagcggccta a 4551

<210> 10

<211> 446

<212> PROTEIN

<213> Nicotiana tabacum

<400> 10

Met Ala Asn Ser Ser Asn Ser Val Phe Ala His Val Val Arg Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Lys Pro Leu Val Leu Asn Val Val Arg Arg Ala Glu Gln Met Leu Val

50 55 60

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

65 70 75 80

Asp Phe Asn Lys Leu Ser Ala Lys Leu Ile Phe Gly Ala Asp Ser Pro

85 90 95

Ala Ile Gln Glu Asn Arg Val Thr Thr Val Gln Cys Leu Ser Gly Thr

100 105 110

Gly Ser Leu Arg Val Gly Ala Glu Phe Leu Ala Lys His Tyr His Glu

115 120 125

His Thr Ile Tyr Ile Pro Gln Pro Thr Trp Gly Asn His Pro Lys Val

130 135 140

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

145 150 155 160

Ala Thr Arg Gly Leu Asp Phe Gln Gly Thr Thr Val Ile Thr Val Leu

165 170 175

Lys Val Leu Gln Leu Tyr Lys His Ser Leu Ser Val Ala Phe Pro Val

180 185 190

Phe Gly Cys Cys Ile Ser Asn Leu Ile Met Asn Pro Val Gly Leu Leu

195 200 205

Asp Asp Leu Ala Ala Ala Pro Ala Gly Ala Ile Val Leu Leu His Ala

210 215 220

Cys Ala His Asn Pro Thr Gly Val Asp Pro Thr Asn Asp Gln Trp Glu

225 230 235 240

Lys Ile Arg Gln Leu Met Arg Ser Lys Gly Leu Leu Pro Phe Phe Asp

245 250 255

Ser Ala Tyr Gln Gly Phe Ala Ser Gly Asn Leu Asp Ala Asp Ala Gln

260 265 270

Ser Val Arg Met Phe Val Ala Asp Gly Gly Glu Cys Leu Ala Ala Gln

275 280 285

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

290 295 300

Ser Ile Val Cys Lys Asp Ala Asp Val Ala Ser Arg Val Glu Ser Gln

305 310 315 320

Leu Lys Leu Val Ile Arg Pro Met Tyr Ser Asn Pro Pro Ile His Gly

325 330 335

Ala Ser Ile Val Ala Thr Ile Leu Lys Asp Arg Gln Met Tyr Asp Glu

340 345 350

Trp Thr Ile Glu Leu Lys Ala Met Ala Asp Arg Ile Ile Ser Met Arg

355 360 365

Gln Gln Leu Phe Asp Ala Leu Gln Ala Arg Gly Thr Thr Gly Asp Trp

370 375 380

Ser His Ile Ile Lys Gln Ile Gly Met Phe Thr Phe Thr Gly Leu Asn

385 390 395 400

Thr Glu Gln Val Ser Phe Met Thr Arg Glu His His Ile Tyr Met Thr

405 410 415

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

420 425 430

His Leu Ala Asp Ala Ile His Ala Ala Val Thr Lys Ala Ala

435 440 445

<210> 11

<211> 4176

<212> DNA

<213> Nicotiana tabacum

<400> 11

atggcaaatt cctccaattc tgtttttgcc catgttgttc gtgctcctga agatcccatc 60

ttaggagtac ctccctttcc actctttcta ttttacattt ccactgaata tgtttcttct 120

gtggctcctt taataatctt ccgtaaatat attattagtg gatttgataa gctacttctc 180

tctctctctc tctctctctc tctctctctc tctctctctc tctctctctt ttatttttctt 240

300

atcttggaga tcggcataaa taagttgatt ggaatgatcg ctatagggtt acctattgta 360

tgcattggat catggatgtg tttactaatt atttaatacc tctttctttt tactgtgatc 420

tggcaattcc ttattttatt cctggtgtgg ttgatggaag ggtgtagatt tgattcttta 480

acttgctcta ttgagaagat aatttgttct tctcaagtgt ttagtaatgg tttttttcct 540

gttgtgctac ttcattaaaa caggtcacag ttgcttataa caaagatacc agcccggtga 600

agttgaattt gggtgttggc gcatatcgca ctgaggtctg ccacttctac tttgtctcgt 660

tattctttat tttttatttt ttattataac caaaataagt tgccccttga atggatttgg 720

tcctgctatg ttttgttgaa tccttggtta agttttttctt taataggctc cttcacaagg 780

atacaaaatt gtagacactg atgcatacac attaatattt ttttttccctg atgcataatg 840

aagtgaaacc acttgatttt ataagtggtt gttttttttct tcaatcttga gttggatgtt 900

agtgttaagc ttgaaaatta tgttctacta atgcatagtc cgatgacaac ttgcaggaag 960

gaaagcccct tgttcttaat gtggtgagac gagctgaaca aatgctcgtc aatgacacgt 1020

aacttgccaa attagaaact agcttacaga ttttcttttg agatatgatc acctgatgcc 1080

1140

ctggactagc ggattttaac aaactgagtg caaagcttat atttggatct gacaggtttg 1200

gagaattttt ggtgcagttg ctcttgataa atgcttgaat caaaaatata aaaaaatgct 1260

cactatccat gtcgctccag ttaaacctat cttgccaaac cacttgtata aaagaaaatg 1320

1380

gggaatgctt tatctctaca aagtagagta actgaatacc tgttaaaaca tattcctccg 1440

tatttcatct tattatgatg ccttgcatca gaagaaaatt gttctagagt taactttctc 1500

tcctctttgt tgtactgact ttctgtgtaa ggtgaacgtg atatcaggaa atatgtgtct 1560

1620

agatgtagtt taaatgcttt ttgtgctgtt ttgttgctga tacagccctg ccattcaaga 1680

gaacagggtg actactgttc agtgcttgtc gggcacaggt tctttgaggg ttggggctga 1740

gtttctggct aagcattatc atgaagttag tattccttgc tctctttccc tttatatgtc 1800

taaatcaaat ggacacttct ataagcttct actgtttgtt ttgttgccag catactatat 1860

atataccaca gccaacatgg ggaaaccatc cgaaggtttt cactttagct gggctttcag 1920

taaaatatta tcgttactac gacccagcaa cacgaggcct ggatttccaa ggtactactg 1980

taatcaatgt tcttaaagtt ctacagttgt aagtaagaac cgatttctct ttttcatgga 2040

caagtgaact tgctcctggt cgtgtctaga aagatctata tattatgtgt agctagcaca 2100

2160

ggcttctcct gtatttggggt gttgcgtatc aaatttaatc atgaaccctg taggactttt 2220

ggatgatctt gctgctgcac ccgctggagt aatagttctt ctccatgcat gtgctcataa 2280

cccaactggc gttgatccaa caaatgacca gtgggagaaa atcaggcagt tgatgaggtc 2340

caaggggctg ttacctttct ttgacagtgc ttaccaggta aagcttatga tgggattttg 2400

aattcaagtg atacttcgtt aagaatgatt accaaataat ttgaagcccc aaactatgta 2460

2520

ccactggcaa cctagatgca gatgcacaat ctgttcgcat gtttgtggct gatggtggtg 2580

aatgtcttgc agctcagagt tatgccaaaa acatgggact gtatggggag cgtgttggtg 2640

cccttagcat tgtaagtcct tttgtcggtt gtaattgctt tcccttttta ataagcaata 2700

aaattgcttt ccctttttaa taagcaatat agcatgatat ccatggctat atcatgctat 2760

ttatgtctaa agatgatttt ttctttggaa gcataattca ggttatattc cctaaaaggc 2820

taaaaagagg ttgttctgtt ggtacaatga acacagtctc tagagatatt gaaagccaat 2880

tttttgaaga tggcttccac ttagattgta attggaaaag aaagagaagg acaaagtgga 2940

attagtaccg gattgtatgt ttaggaaaaa gtgtcgtttt ttttgagttt tatcagacag 3000

3060

tgcagatgtt gcaagcagag tcgaaagcca gctaaagctg gttatcaggc caatgtactc 3120

taatccacca attcatggtg cgtctattgt tgctactata ctcaaggaca ggtttgtaca 3180

actatataca agattctgtt ttgttgttag tagatgctat accttctaca ttttgatgtg 3240

gttgctcatc taatggtgat agacaaatgt acgatgaatg gacaattgag ctgaaagcaa 3300

tggccgacag gattattagc atgcgccaac aactctttga tgccttgcaa gctcgaggta 3360

3420

ctgctactgc aataggaact ttttctggaa aagtgccagg gtgaaagaac cacggcaact 3480

aaatcttctg acttcattgt tcagtttagt gctaatgtaa gttttattct gttatgcagg 3540

tacagcaggt gattggagtc atatcatcaa acaaattggc atgtttactt tcacaggatt 3600

3660

gtaaggacat ctgactgttg atattttttt ttatttgttt agtttgttac tttgggttgc 3720

ttttttctca gtagaaactt aaataattgg aacttagaag cccttatcat tgattatttc 3780

ggcttgaatt ctttaataag gagaatttca gacttatagc ttcagttttg agaggaagca 3840

taaacaagtc cagctctgtc attcatactt aaaatttaca gaagaaagtg cagttctgtt 3900

tttcccccct cccaaattat attgattctc aaaagaactt accttcaatc tatggcacat 3960

4020

atcatcatct ctagtgacat tttctacttt ccatttttag aaggaatgat tttctccttt 4080

ctcatttgca ggagaattag catggcaggc cttagttctc gcacaattcc tcatcttgcc 4140

gatgccatac atgctgctgt taccaaagcg gcctaa 4176

<210> 12

<211> 446

<212> PROTEIN

<213> Nicotiana tabacum

<400> 12

Met Ala Asn Ser Ser Asn Ser Val Phe Ala His Val Val Arg Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Lys Pro Leu Val Leu Asn Val Val Arg Arg Ala Glu Gln Met Leu Val

50 55 60

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

65 70 75 80

Asp Phe Asn Lys Leu Ser Ala Lys Leu Ile Phe Gly Ser Asp Ser Pro

85 90 95

Ala Ile Gln Glu Asn Arg Val Thr Thr Val Gln Cys Leu Ser Gly Thr

100 105 110

Gly Ser Leu Arg Val Gly Ala Glu Phe Leu Ala Lys His Tyr His Glu

115 120 125

His Thr Ile Tyr Ile Pro Gln Pro Thr Trp Gly Asn His Pro Lys Val

130 135 140

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

145 150 155 160

Ala Thr Arg Gly Leu Asp Phe Gln Gly Thr Thr Val Ile Asn Val Leu

165 170 175

Lys Val Leu Gln Leu Tyr Lys His Ser Leu Ser Val Ala Ser Pro Val

180 185 190

Phe Gly Cys Cys Val Ser Asn Leu Ile Met Asn Pro Val Gly Leu Leu

195 200 205

Asp Asp Leu Ala Ala Ala Pro Ala Gly Val Ile Val Leu Leu His Ala

210 215 220

Cys Ala His Asn Pro Thr Gly Val Asp Pro Thr Asn Asp Gln Trp Glu

225 230 235 240

Lys Ile Arg Gln Leu Met Arg Ser Lys Gly Leu Leu Pro Phe Phe Asp

245 250 255

Ser Ala Tyr Gln Gly Phe Ala Thr Gly Asn Leu Asp Ala Asp Ala Gln

260 265 270

Ser Val Arg Met Phe Val Ala Asp Gly Gly Glu Cys Leu Ala Ala Gln

275 280 285

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

290 295 300

Ser Ile Val Cys Lys Asp Ala Asp Val Ala Ser Arg Val Glu Ser Gln

305 310 315 320

Leu Lys Leu Val Ile Arg Pro Met Tyr Ser Asn Pro Pro Ile His Gly

325 330 335

Ala Ser Ile Val Ala Thr Ile Leu Lys Asp Arg Gln Met Tyr Asp Glu

340 345 350

Trp Thr Ile Glu Leu Lys Ala Met Ala Asp Arg Ile Ile Ser Met Arg

355 360 365

Gln Gln Leu Phe Asp Ala Leu Gln Ala Arg Gly Thr Ala Gly Asp Trp

370 375 380

Ser His Ile Ile Lys Gln Ile Gly Met Phe Thr Phe Thr Gly Leu Asn

385 390 395 400

Thr Glu Gln Val Ser Phe Met Thr Arg Glu His His Ile Tyr Met Thr

405 410 415

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

420 425 430

His Leu Ala Asp Ala Ile His Ala Ala Val Thr Lys Ala Ala

435 440 445

<210> 13

<211> 4857

<212> DNA

<213> Nicotiana tabacum

<400> 13

atggtttcca caatgttctc tctagcttct gccactccgt cagcttcatt ttccttgcaa 60

gataatctca aggtaatttc atcgtcaatt acattatttg gaaatttgcc ttatcttaga 120

ctattcctaa tgaggtggat tcatgctgtt gtttgtgttt gaacagtcaa agctaaagct 180

ggggactact agccaaagtg cctttttcgg gaaagacttc gtgaaggcaa aggtaggatt 240

tttgtgttgt ttgtgtacat ttggtgagag gtaatagctc tactgctata gagaaactcc 300

ctgtaggttc tgtcctttag agtatagaag agaaggaaag agtttaattg ggaataatgg 360

tggggatggg atgatttgca tacaattgaa catgtgtttc ttgctttggt atattatgat 420

ataggatgat ccaatcatgc tccgtaaatc aactccagaa cttattattc tttcggcact 480

tactaattat aaaaatcggg ttggagtcct gaaaataagt gattgcctaa ccaacttaca 540

gaactaattt tattatccgt atactcaaat caaaacgaca ttatgccagt actggtttct 600

tgagagggat gatattagtg tagaattatt tataaagttg cagtttaacg tagggtgttt 660

tactaaccag aaaggtgtag atgattccat tcagtttatt agatgctaag aagtataaca 720

gtgaggcctg tgaaacttct ggtagtacca acgattgggg ttttatggcg tttaggaatt 780

tagacattaa ttggcacatt ttagaacgaa aaatatgaca tttaacttac aacagttctt 840

ttctgaataa aatattacta gtaactaatt tgtttgaact ttgccattgc taaaatgtgg 900

ctcaagatct tcttggtact tctatttgta atatcagagt tataggggtc taattctagc 960

tcttgagtcg aaattgttat tagtaaagat aattctttct tgtccccctt cagtgctaac 1020

tcacttatgg tattggttta taaaaaattg tgattcagat tataaagtaa 1080

aaaattatgc ctcagtttgt acagcatttt gggttatctg acgttcaatt caacagggtt 1140

ctttaatatc tatttttcta tcttttgtaa tcattgcaac accgagctgt ttaatgtgct 1200

caaaggctat tattagtcct cccactcacc agatccttag aaaaaagccc agaagagaaa 1260

ggcaaagaat acaagcccag accgattgct cgttttataa attttgggaa ttgggatctc 1320

ttttctcata ttcttacttt ttttctctttc ttttttttcca gtcaaatggc cgtactacta 1380

tgactgttgc tgtgaacgtc tctcgatttg agggaataac tatggctcct cctgacccca 1440

ttcttggagt ttctgaagca ttcaaggctg atacaaatga actgaagctt aaccttggtg 1500

ttggagctta ccgcacggag gagcttcaac catatgtcct caatgttgtt aagaaagtaa 1560

1620

cccatgggtg gatacccttg tccaagggga gtcaatttat tacactctgt aaataggtta 1680

1740

gttgaatcgc ccctggcttc ttctgtttac ttctatatat tttgtatcca atgggtgaaa 1800

tcctaagcgt tcatcattca ttaactgttt taataacctt 1860

ctataatttt gcatctgaat gatgaggaaa ttgcttttct gtaggcagaa aaccttatgc 1920

tagaaagagg agataacaaa gaggtacttg atttactaaa ttcatctttt ggccttgact 1980

agtgtcactt ggtgccaatt cttacttatt ttttaatcta tggatatata gtatcttcca 2040

atagaaggtt tggctgcatt caacaaagtc acagcagagt tattgtttgg agcagataac 2100

ccagtgattc agcaacaaag ggtaagtatt tttgttttta actcttagga aaatatatcc 2160

2220

gtgatcaggt ggctactatt caaggtctgt caggaactgg gtcattgcgt attgctgcag 2280

cactgataga gcgttacttc cctggctcta aggttttaat atcatctcca acctggggta 2340

2400

gttttgcagg aaatcataag aacattttca atgatgccag ggtgccttgg tctgaatatc 2460

gatattatga tcccaaaaca gttggcctgg attttgctgg gatgatagaa gatattaagg 2520

ttattatcgt cctcgcattt gtaatctttg tggttgaaat tgtaaagcag cagtgagcac 2580

2640

actttcagta attgaaggag agatgcgttg ttcattctag aatagcactg tatctcccaa 2700

ttgcattttc tgtttcctgt tcttcctccc tatgtttgca ttgatccatg tctctgctaa 2760

acatggacaa tttgcgccct tggcaatgac atgtgtgttg cttgcttttc ttctctttct 2820

atttcttggt aggagtgact tggttctttc aatgtgagca gtcatatttc tgaaaatgaa 2880

2940

cttgtgagga tggaatatgc aaaaataggc tgcacgcttg cctttttagat ctttggttcc 3000

tatgtcggtt gtgaatgtag atttctattt ttcaacattg tctcgcaagg aaaataggat 3060

tatccagtat tggatgtctt tcctatgttt

3180

tcttgtgcaa gtcattgaca tagttatgg cattacttgt ttataggctg ctcctgaagg 3240

atcatttatc ttgctccatg gctgtgcgca caacccaact ggtattgatc ccacaattga 3300

3360

cgcctaccag gtaatctgtg ctaaacccaa ttatttcatt tggtgaagct gtaaaatttc 3420

3480

ttggttttga aatagtgaaa gatctctcgt atttcatttg ttcttttggt gtgaggagag 3540

3600

3660

tcagtaaagt tattagaaca agataatctg aagtcatttc tattcagaga attgcattga 3720

atagctgtat actataataa tcgagatgcc tcatctgtct acacgctgcc ctacagggat 3780

ttgcaagcgg cagccttgac gaagatgcct catctgtgag attgtttgct gcacgtggca 3840

3900

gagctattaa tgttctttgc tcatccgctg atgcagcgac aaggtacagt cacccgcact 3960

agcaactaca taattgtcct ctgtatagga aaaatgatgc actggaaaac aatggttcca 4020

4080

atctcccatc acctgagcct atggcttgat tggattttat gtgggcgaac caatagaatt 4140

atttgcttaa ttttctcaac taatggatgc atctctgcta actcacaggg tgaaaagcca 4200

gctaaaaagg cttgctcgac caatgtactc aaatccccca attcacggtg ctagaattgt 4260

tgccaatgtc gttggaattc ctgagttctt tgatgaatgg aaacaagaga tggaaatgat 4320

ggcaggaagg ataaagagtg tgagacagaa gctatacgat agcctctcca ccaaggataa 4380

gagtggaaag gactggtcat acattttgaa gcagattgga atgttctcct tcacaggcct 4440

caacaaagct caggtaaatc cccgtgattt aagctattgc ttcatcacaa tatgcttaaa 4500

ttcaatttga tcattcatcg caaagcacat tctgaactca gcacatattt tcattaacac 4560

4620

gagtctctta ctggagtata actagattat cgacaatgca tacatttctt tccctgtacc 4680

tgcacttctg gtgctcatat ttgatctctc ttcttggcca cgcagagcga gaacatgacc 4740

aacaagtggc atgtgtacat gacaaaagac gggaggatat cgttggctgg attatcagct 4800

gctaaatgcg aatatcttgc agatgccata attgactcgt actacaatgt cagctaa 4857

<210> 14

<211> 462

<212> PROTEIN

<213> Nicotiana tabacum

<400> 14

Met Val Ser Thr Met Phe Ser Leu Ala Ser Ala Thr Pro Ser Ala Ser

1 5 10 15

Phe Ser Leu Gln Asp Asn Leu Lys Ser Lys Leu Lys Leu Gly Thr Thr

20 25 30

Ser Gln Ser Ala Phe Phe Gly Lys Asp Phe Val Lys Ala Lys Ser Asn

35 40 45

Gly Arg Thr Thr Met Thr Val Ala Val Asn Val Ser Arg Phe Glu Gly

50 55 60

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

65 70 75 80

Lys Ala Asp Thr Asn Glu Leu Lys Leu Asn Leu Gly Val Gly Ala Tyr

85 90 95

Arg Thr Glu Glu Leu Gln Pro Tyr Val Leu Asn Val Val Lys Lys Ala

100 105 110

Glu Asn Leu Met Leu Glu Arg Gly Asp Asn Lys Glu Tyr Leu Pro Ile

115 120 125

Glu Gly Leu Ala Ala Phe Asn Lys Val Thr Ala Glu Leu Leu Phe Gly

130 135 140

Ala Asp Asn Pro Val Ile Gln Gln Gln Arg Val Ala Thr Ile Gln Gly

145 150 155 160

Leu Ser Gly Thr Gly Ser Leu Arg Ile Ala Ala Ala Leu Ile Glu Arg

165 170 175

Tyr Phe Pro Gly Ser Lys Val Leu Ile Ser Ser Pro Thr Trp Gly Asn

180 185 190

His Lys Asn Ile Phe Asn Asp Ala Arg Val Pro Trp Ser Glu Tyr Arg

195 200 205

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

210 215 220

Asp Ile Lys Ala Ala Pro Glu Gly Ser Phe Ile Leu Leu His Gly Cys

225 230 235 240

Ala His Asn Pro Thr Gly Ile Asp Pro Thr Ile Glu Gln Trp Glu Lys

245 250 255

Ile Ala Asp Val Ile Gln Glu Lys Asn His Ile Pro Phe Phe Asp Val

260 265 270

Ala Tyr Gln Gly Phe Ala Ser Gly Ser Leu Asp Glu Asp Ala Ser Ser

275 280 285

Val Arg Leu Phe Ala Ala Arg Gly Met Glu Leu Leu Val Ala Gln Ser

290 295 300

Tyr Ser Lys Asn Leu Gly Leu Tyr Gly Glu Arg Ile Gly Ala Ile Asn

305 310 315 320

Val Leu Cys Ser Ser Ala Asp Ala Ala Thr Arg Val Lys Ser Gln Leu

325 330 335

Lys Arg Leu Ala Arg Pro Met Tyr Ser Asn Pro Pro Ile His Gly Ala

340 345 350

Arg Ile Val Ala Asn Val Val Gly Ile Pro Glu Phe Phe Asp Glu Trp

355 360 365

Lys Gln Glu Met Glu Met Met Ala Gly Arg Ile Lys Ser Val Arg Gln

370 375 380

Lys Leu Tyr Asp Ser Leu Ser Thr Lys Asp Lys Ser Gly Lys Asp Trp

385 390 395 400

Ser Tyr Ile Leu Lys Gln Ile Gly Met Phe Ser Phe Thr Gly Leu Asn

405 410 415

Lys Ala Gln Ser Glu Asn Met Thr Asn Lys Trp His Val Tyr Met Thr

420 425 430

Lys Asp Gly Arg Ile Ser Leu Ala Gly Leu Ser Ala Ala Lys Cys Glu

435 440 445

Tyr Leu Ala Asp Ala Ile Ile Asp Ser Tyr Tyr Asn Val Ser

450 455 460

<210> 15

<211> 5206

<212> DNA

<213> Nicotiana tabacum

<220>

<221> Other sign

<222> (4436)..(4436)

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

<400> 15

atggcttcca caatgttctc tctagcttct gccgctccat cagcttcatt ttccttgcaa 60

gataatctca aggtaatttc attgtgaatt acattatttg gaaatttgcc ctatcttaga 120

ctgttcctaa tgaggtggat tcatgctgtt gtttgtgttt gaacagtcaa agctaaagct 180

ggggactact agccaaagtg cctttttcgg gaaagacttc gcgaaggcaa aggtaggatt 240

tttgtgttgt ttgtgtacat ttggtgagag gtaatagctc tactgatata gagcaactcc 300

ctgtaggttc tgtcctttag agtatagaag agaagagaag agtttaattg ggaataatgg 360

tggggatgga atgatttgca tacaaatgaa catgtgtttc ttgcttttgg tgtatgatat 420

aggatgatcc aatcatgctc cgtaaatcaa ctccagaact tattattctt tcggcacttc 480

taattataaa aatctggttg gagtaatgaa tataagtgat tacctaacca acttacagaa 540

ttgattttat tatccatata ctgaaattca aaaacggcgt tttgccagta ctggtttctt 600

gagagggatg atattaatat agaattattt tataaagttg cagtttaacg tagggtattt 660

tactaactag aaaggtgata gatggttccg ttcagtttat tagaagtata acagtgaggc 720

ctgttaaact tttgctagta tcaatgattg gggttttatg gcgtttagga atttagacat 780

caattggcac attttagaac gaaaaacatg acatttaagt tacatcagtt cttttctgaa 840

taaaatagta ctagtaaata acttgtttga actttgccat ttgctaaaat gtggctcaag 900

atcttcttgg tacttctatt tgtaatatca gagttatagg ggtctaattc taccactgtt 960

ttgagtcaaa atgttattag taaagataat tctttcttgt cccccttcag tgctaacatt 1020

aaaaattgtg cttcagatca ctttataaag 1080

1140

ctctttaata tctatgtttc tactttttgt aatctacatc gagctgttta atgtgctcaa 1200

aggctttaat tagtcctcct actcaccaga tccttagaaa aaagcccaga agagaaaggc 1260

1320

ggagggagga gcatgaacca aagatgatac attgatatta ttttctaaat ttgggaattg 1380

tgatcttatc ttaaattttt acttttttct ctttttcttt ttttatagtc aaatggtcgg 1440

actactatgg ctgtttctgt gaacgtctct cgatttgagg gaataacaat ggctcctcct 1500

gaccccattc ttggagtttc tgaagcattc aaggctgata caaatgaact gaagcttaac 1560

cttggagttg gagcttaccg cacagaagat cttcaaccct atgtcctcaa tgttgttaaa 1620

1680

accttgttcc catgtgttcg tcattaaaca tagtaataac tttctatagt tttgcatctg 1740

aatgatgagg aaattacttt tctgtaggca gaaaacctta tgctagagag aggtgacaac 1800

aaagaggtac ttgatatact aaattcatct tttggcctat tagtgtctct tggtgccatt 1860

tcttacttat tttttgtcca tgaatatata gtatcttcca atagaaggtt tggctgcatt 1920

caacaaagtc acagcagagt tattgtttgg agcagataat ccagtgattc agcaacaaag 1980

ggtaagtatt ttggttttta actcttagca aaaaagtatc ctggaacaaa cttgtagatt 2040

cagtttccac ggattgaatg gcattgtatg tttcttgatc aggtggctac tattcaaggt 2100

ctatcaggaa ctgggtcatt gcgtattgct gcagcactga tagagcgtta cttccctggc 2160

tctaaggttt tgatatcatc tccaacctgg ggtacgtata tagtgctttg gattaatttg 2220

gttgaatctc ataatactga tttttgcagt tatgttttgc aggaaatcat aagaacattt 2280

tcaatgatgc cagggtgcct tggtctgaat atcgatatta tgatcccaaa acagttggtc 2340

tagattttgc tgggatgata gaagatataa aggttattat cttcctcact tttgtaatct 2400

ttgtggttga aattgtaaag cagcagtgag cagtgtcttt ttcctttctc cacaagtcca 2460

ttgatggtgc ctttgcatgt gggacatgct ttgactttca gtcgttgaag gagagatgcg 2520

ttattcattc taggatagca ttgtatctcc caaatgcttt ttctgtttcc tgctcttcct 2580

tcccattttt gcatcgatcc tgtctctgct aaacatggac aatttgcgcc cttggcaaat 2640

ggcaatgact tgtgtgttgc ttttcttctc tttctatttt ttggtaggag tgacttggtt 2700

ctttcagtgt gagcagtcat atttctgaaa atgaaaatca gaggaacttg gtgctcacac 2760

ttagagaaag tttgttatgt tttgggatgt gaaaggaatt gacagaacaa gtttgataat 2820

atattttttc ttgtgaggat ggaatatgct aaaaataggc tgcactcttt ccttttagat 2880

ctttagttcc tatgtcggtt gtgaatgtcg atttctattt tcaacatttt ctcacgaaga 2940

aaataggatt atccagtact ggatgtctct cctatgtctg atatatgtgt atgtgcagtc 3000

3060

ttccttccag cttttgtgta agtcattgac atagtttaat gaaactactt gtttataggc 3120

tgctcctgaa ggatcattca tcttgctcca tggctgtgca cacaacccaa ctggtattga 3180

tcccacaatt gaacaatggg aaaagattgc tgatgtaatt caggagaaga accacattcc 3240

attttttgat gttgcctacc aggtaatctg tgctaaaccc aattattttc atttggtgaa 3300

3360

3420

tggtgtgtgg agactataca ttgttgtatt gatagatgag cgaatttgat tgatgttggt 3480

ggttaagcca catgtgttac tttgtccata tttttttaca ccgtcttggt ttttatcaat 3540

gaaatttact gatttttcag tgaaattatt agaacaagat catctgaagt catttctgtt 3600

cagagaattg gattgaatag ctgtatacta taataatcga gatgcctcat ctgtctacac 3660

gctgcactgc agggattcgc aagcggcagc cttgatgaag atgcctcatc tgtgagattg 3720

tttgctgcac gtggcatgga gcttttggtt gctcaatcat atagtaaaaa tctgggtctg 3780

tatggagaaa ggattggagc tattaatgtt ctttgctcat ctgctgatgc agcgacaagg 3840

tacaacggcc agcactaata atctacatat ttctcctctg tattggtaaa atgatgttgc 3900

3960

4020

atgtggttga accaatataa cttttcttct gttaatggat gcatatctac taacttacag 4080

ggtgaaaagc cagctaaaaa ggcttgctcg accaatgtac tcaaatcccc ccattcacgg 4140

tgctagaatt gttgccaatg tcgttggaat tcctgagttc tttgatgaat ggaaacaaga 4200

gatggaaatg atggcaggaa ggataaagag tgtgagacag aagctatatg atagcctctc 4260

cgccaaggat aaaagtggaa aggactggtc atacattctg aagcagattg gaatgttctc 4320

cttcacaggc ctcaacaaag ctcaggtaaa accccgtgaa ttaagttatt gctgttgcgg 4380

aagccaaata tatagagagt gattaaatca caactactat atctaaaggt agctangtaa 4440

atgagacaat aataaaatga acaccagaaa ttaatgaggt tcggcaaaat ttgatttttt 4500

gcctagttct cggacacaat caactcaaat ttatttcact ccaaaaatac aaatgaaata 4560

ctacaagaga gaaagaagat tcaaatgcct taggaaataa gaaggcaagt gagagatgtt 4620

tacaaatgaa caaaatcctt gctatttata gaagagaaat ggccttaata atgtcatgca 4680

tgacatcata ttaagtgtga acatgtaatg taaatgcacg aaaaatgcat ctaccaattt 4740

cttaaggctt caaatgttca cactagttca cattaatctt gtcaaaattc aacaattgct 4800

gcatcacaat atgcttaaat tcaatttgat ttggttgaca actttctagc tttgatcatt 4860

catcacaaag cgcattcttc actcagcacg tatttttatt aagacattct ttccttccat 4920

tctgaccgat ttcataagtt aaatatgcaa aagatagtgc agtgagagtc tccttactgg 4980

attataacta tggactaaag ttaaatgcat acatttcttt ccctgtactt gcacttctcg 5040

tgctcatatt tgatatctct tcttggctac acagagcgag aacatgacca acaagtggca 5100

tgtgtacatg acaaaagacg ggaggatatc gttggctgga ttatctgctg ccaaatgtga 5160

atatcttgca gatgccataa ttgactcata ctacaatgtc agctaa 5206

<210> 16

<211> 462

<212> PROTEIN

<213> Nicotiana tabacum

<400> 16

Met Ala Ser Thr Met Phe Ser Leu Ala Ser Ala Ala Pro Ser Ala Ser

1 5 10 15

Phe Ser Leu Gln Asp Asn Leu Lys Ser Lys Leu Lys Leu Gly Thr Thr

20 25 30

Ser Gln Ser Ala Phe Phe Gly Lys Asp Phe Ala Lys Ala Lys Ser Asn

35 40 45

Gly Arg Thr Thr Met Ala Val Ser Val Asn Val Ser Arg Phe Glu Gly

50 55 60

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

65 70 75 80

Lys Ala Asp Thr Asn Glu Leu Lys Leu Asn Leu Gly Val Gly Ala Tyr

85 90 95

Arg Thr Glu Asp Leu Gln Pro Tyr Val Leu Asn Val Val Lys Lys Ala

100 105 110

Glu Asn Leu Met Leu Glu Arg Gly Asp Asn Lys Glu Tyr Leu Pro Ile

115 120 125

Glu Gly Leu Ala Ala Phe Asn Lys Val Thr Ala Glu Leu Leu Phe Gly

130 135 140

Ala Asp Asn Pro Val Ile Gln Gln Gln Arg Val Ala Thr Ile Gln Gly

145 150 155 160

Leu Ser Gly Thr Gly Ser Leu Arg Ile Ala Ala Ala Leu Ile Glu Arg

165 170 175

Tyr Phe Pro Gly Ser Lys Val Leu Ile Ser Ser Pro Thr Trp Gly Asn

180 185 190

His Lys Asn Ile Phe Asn Asp Ala Arg Val Pro Trp Ser Glu Tyr Arg

195 200 205

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

210 215 220

Asp Ile Lys Ala Ala Pro Glu Gly Ser Phe Ile Leu Leu His Gly Cys

225 230 235 240

Ala His Asn Pro Thr Gly Ile Asp Pro Thr Ile Glu Gln Trp Glu Lys

245 250 255

Ile Ala Asp Val Ile Gln Glu Lys Asn His Ile Pro Phe Phe Asp Val

260 265 270

Ala Tyr Gln Gly Phe Ala Ser Gly Ser Leu Asp Glu Asp Ala Ser Ser

275 280 285

Val Arg Leu Phe Ala Ala Arg Gly Met Glu Leu Leu Val Ala Gln Ser

290 295 300

Tyr Ser Lys Asn Leu Gly Leu Tyr Gly Glu Arg Ile Gly Ala Ile Asn

305 310 315 320

Val Leu Cys Ser Ser Ala Asp Ala Ala Thr Arg Val Lys Ser Gln Leu

325 330 335

Lys Arg Leu Ala Arg Pro Met Tyr Ser Asn Pro Pro Ile His Gly Ala

340 345 350

Arg Ile Val Ala Asn Val Val Gly Ile Pro Glu Phe Phe Asp Glu Trp

355 360 365

Lys Gln Glu Met Glu Met Met Ala Gly Arg Ile Lys Ser Val Arg Gln

370 375 380

Lys Leu Tyr Asp Ser Leu Ser Ala Lys Asp Lys Ser Gly Lys Asp Trp

385 390 395 400

Ser Tyr Ile Leu Lys Gln Ile Gly Met Phe Ser Phe Thr Gly Leu Asn

405 410 415

Lys Ala Gln Ser Glu Asn Met Thr Asn Lys Trp His Val Tyr Met Thr

420 425 430

Lys Asp Gly Arg Ile Ser Leu Ala Gly Leu Ser Ala Ala Lys Cys Glu

435 440 445

Tyr Leu Ala Asp Ala Ile Ile Asp Ser Tyr Tyr Asn Val Ser

450 455 460

<210> 17

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence used to generate plants with AAT2S/T RNAi

<400> 17

gctattcaag agaacagagt aacaactgtg cagtgcttgt ctggcacagg ctcattgagg 60

gttggagctg aatttttggc tcgacattat catcaacgca c 101

<---

Claims (32)

1. Dried plant material from Nicotiana tabacum for use in tobacco products, comprising: (i) a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 15; (ii) a polypeptide encoded by the polynucleotide specified in (i); (iii) a polypeptide comprising, consisting of or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID No: 8, at least 93% sequence identity with SEQ ID NO: 2, or at least 94% sequence identity with SEQ ID NO: 4, or SEQ ID NO: 14, or SEQ ID NO: 16; or (iv) a construct, vector or expression vector containing the isolated polynucleotide referred to in (i), wherein the dried plant material from Nicotiana tabacum contains at least one modification that modulates the expression or activity of a polynucleotide or polypeptide compared to that of a control dried plant material without modifying the expression or activity of the polynucleotide or polypeptide. 2. The dried Nicotiana tabacum plant material according to claim 1, wherein at least one modification is a genome modification of the dried Nicotiana tabacum plant material, or a construct, vector or expression vector modification, or a transgenic modification; preferably, where the modification of the genome of the dried plant material from Nicotiana tabacum or the modification of the construct, vector or expression vector is a mutation or editing. 3. The desiccated Nicotiana tabacum plant material according to any one of the preceding claims, wherein the modification results in a decrease in the expression or activity of the polynucleotide or polypeptide compared to that in the control plant material; preferably, where the plant material contains an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 nucleotides of the RNA transcribed from the polynucleotide (i) of claim 1. 4. The dried Nicotiana tabacum plant material according to any one of the preceding claims, wherein the dried Nicotiana tabacum plant material is a leaf, wherein the modulated expression or activity of the polynucleotide or polypeptide modulates the amino acid level in the dried Nicotiana tabacum leaf or the dried Nicotiana tabacum leaf compared to the amino acid level in the dried control Nicotiana tabacum leaf, or you dried leaf of Nicotiana tabacum , preferably wherein the amino acid is aspartate or a metabolite derived therefrom; and/or wherein the level of nicotine in a dried or dried Nicotiana tabacum leaf is essentially the same as the level of nicotine in a control dried or dried Nicotiana tabacum leaf; and/or where the level of acrylamide in the dried or dried Nicotiana tabacum leaf is reduced compared to the level of acrylamide in the control dried or dried Nicotiana tabacum leaf; and/or wherein the ammonia level in the dried or dried Nicotiana tabacum leaf is reduced compared to the amino acid level in the control dried or dried Nicotiana tabacum leaf. 5. Dried Nicotiana tabacum plant material according to any one of the preceding claims, preferably wherein the plant material contains biomass, seed, stem, flowers or leaves. 6. The dried Nicotiana tabacum plant material according to any one of the preceding claims, wherein the dried plant material is an air-dried plant material, a sun-dried plant material, or a fire-dried plant material. 7. The dried Nicotiana tabacum plant material according to any one of the preceding claims, wherein the plant material is a homogenized plant material. 8. Tobacco product containing the dried plant material from Nicotiana tabacum according to any one of the preceding claims. 9. The method of obtaining dried plant material from Nicotiana tabacum according to any one of paragraphs. 1-8, including stages: (a) obtaining a Nicotiana tabacum plant cell comprising a polynucleotide comprising, consisting of, or essentially consisting of a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 15; (b) modifying a plant cell to modulate the expression of said polynucleotide as compared to that in a control cell of the Nicotiana tabacum plant; And (c) regenerating a plant cell into a Nicotiana tabacum plant; (d) collection of plant material from it; And (c) drying the plant material. 10. The method according to p. 9, where stage (c) includes cultivating a plant from a shoot or seedling containing a plant cell; and/or where the step of modifying a plant cell includes modifying the genome of the cell using genome editing or genomic engineering techniques; preferably, wherein the genome editing or genomic engineering techniques are selected from CRISPR/Cas technology, zinc finger nuclease mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide directed mutagenesis, and meganuclease mediated mutagenesis. 11. The method according to claim 9 or 10, wherein the step of modifying a plant cell includes transfecting the cell with a construct containing a polynucleotide containing, consisting of, or essentially consisting of a sequence characterized by at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 15, operably linked to a constitutive promoter; and/or where the step of modifying a plant cell includes introducing into the cell an interference polynucleotide containing a sequence that is at least 80% complementary to the RNA transcribed from the polynucleotide of claim 1(i); preferably, wherein the plant cell is transfected with a construct expressing an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 RNA nucleotides transcribed from the polynucleotide of claim 1(i). 12. The method according to any one of paragraphs. 9-11, in which the plant material contains leaves, stems or flowers. 13. The method according to any one of paragraphs. 9-12, in which the drying is selected from air drying, fire drying, pipe drying and solar drying.