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WO2010045679A1 - Eléments de contrôle transcriptionnel et leurs utilisations - Google Patents

Eléments de contrôle transcriptionnel et leurs utilisations Download PDF

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WO2010045679A1
WO2010045679A1 PCT/AU2009/001383 AU2009001383W WO2010045679A1 WO 2010045679 A1 WO2010045679 A1 WO 2010045679A1 AU 2009001383 W AU2009001383 W AU 2009001383W WO 2010045679 A1 WO2010045679 A1 WO 2010045679A1
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Prior art keywords
sequence
seq
nucleic acid
complement
set forth
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Robert George Birch
John M. Manners
Stephen R. Mudge
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University of Queensland UQ
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University of Queensland UQ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8226—Stem-specific, e.g. including tubers, beets
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters

Definitions

  • This invention relates generally to transcriptional control elements for use in plant genetic engineering. More particularly, the present invention relates to tissue-specific promoters for expression of heterologous nucleic acids in plants including monocotyledonous plants.
  • the invention also relates to chimeric nucleic acid constructs comprising a promoter of the invention operably linked to a foreign or endogenous polynucleotide that codes for a protein of interest or a transcript that is capable of modulating expression of a target gene.
  • the invention is further concerned with transformed plant cells, as well as differentiated plants and plant parts, containing the construct of the invention.
  • a primary goal of genetic engineering is to obtain plants having improved characteristics or traits. Many different types of characteristics or traits are considered advantageous, but those of particular importance include enhanced yield or quality of harvestable plant products, production of additional compounds in plants, enhanced stability or shelf life of the ultimate consumer product obtained from a plant, improvement in the nutritional value of edible portions of a plant, and plant resistance to diseases, insects, herbicides, cold stress, water stress or soil salinity.
  • a selected gene or genes
  • the selected gene (or genes) may be derived from a source different from the plant of interest or may be native to the desired plant, but engineered to have different or improved qualities. This new gene (or genes) may then be expressed in cells of the regenerated plant to exhibit the new trait or characteristic.
  • a promoter is a DNA sequence that directs the cellular machinery of a plant to produce (transcribe) RNA (transcript) from a contiguous transcribable region downstream (3') of the promoter.
  • the promoter region lies upstream (5') of the transcribable region.
  • DNA is typically comprised of two polynucleotide strands in anti- parallel orientation with complementary base pairing.
  • the terminology for the location and orientation of DNA sequence elements, including non-transcribed regulatory signals, uses as a reference the sequence of the corresponding transcript, which is a single-stranded polynucleotide with unambiguous 5' and 3' ends. Nucleotides are commonly counted from the point at which transcription commences, with positive (+) numbering in the downstream direction and negative (-) numbering in the upstream direction.
  • Promoters typically comprise sequence modules that function in concert to determine the overall promoter activity. conserved sequence motifs near the transcription start site are believed to function in the binding and orientation of RNA polymerase, whereas sequence motifs more distant from the transcription start site substantially modulate the level and developmental pattern of promoter activity.
  • many plant promoters include a motif with a TATA consensus in the vicinity of -30 nt , and a motif with a CAAT or AGGA consensus at about -70 nt upstream of the transcription start site (for reviews, see Messing, Geraghty, Heidecker, Hu, Kridl and Rubenstein, 1983, pp 211-21 in Genetic Engineering of Plants ed Kosuge, Meredith and Hollaender, Plenum; Waugh and Brown, 1991, pp 1-37 in Plant Genetic Engineering, ed Grierson, Blackie; Ferl and Paul, 2000, pp 312-57 in Biochemistry and Molecular Biology of Plants, ed Buchanan, Gruissem and Jones, ASPP.).
  • the promoter influences the rate at which the transcript of the gene is made. Assuming the transcript includes a coding region with appropriate translational signals, the promoter also influences the rate at which the resultant protein product of the gene is produced. Promoter activity also can depend on the presence of several other czs-acting regulatory elements which, in conjunction with cellular factors, determine strength, specificity, and transcription initiation site (for a review, see Zawel and Reinberg, 1992, Curr. Opin. Cell Biol. 4:488).
  • promoters are able to direct RNA synthesis at a higher rate relative to other promoters. These are called “strong promoters.” Certain other promoters have been shown to direct RNA production at higher levels only in particular types of cells or tissues and are often referred to as “tissue-specific promoters.” Promoters that are capable of directing RNA production in many or all tissues of a plant are called “constitutive promoters”. Thus, expression of a chimeric gene (or genes) introduced into a plant may potentially be controlled by identifying and using a promoter with the desired characteristics. The desired expression pattern depends on the nature of the gene product and the trait associated with expression of any particular gene.
  • segment ⁇ is more conserved across the class of promoter sequences than segment ⁇
  • segment ⁇ is more conserved than segment Z
  • segment Z is more conserved than segment ⁇
  • segment ⁇ is more conserved than segment ⁇
  • ⁇ is an optional spacer or intervening segment.
  • the present inventors have also determined that segment ⁇ is transcriptionally active in plant cells.
  • isolated nucleic acid molecules comprise a promoter sequence that is operable in plant cells, including monocotyledonous plant cells.
  • the promoter sequence generally comprises a nucleotide sequence (also referred to herein as segment ⁇ sequence ) selected from the group consisting of:
  • A, C, G and T represent the nucleic acid bases adenine, cytosine, guanine and thymine, respectively;
  • M is A or C;
  • R is A or G;
  • W is A or T
  • S is C or G
  • Y is C or T
  • K is G or T;
  • V is A or C or G;
  • H is A or C or T
  • D is A or G or T
  • B is C or G or T
  • N is G or A or C or T; and [0030] a is an integer from 0-3.
  • N ⁇ comprises 0 bases or is the sequence ACT.
  • sequences according to SEQ ID NO: 1 are suitably selected from the group consisting of:
  • the promoter sequence further comprises upstream of (a), (b) or (c) a nucleotide sequence (also referred to herein as segment ⁇ sequence ) selected from the group consisting of:
  • A, C, G, T, M, R, W, S, Y, K, V, H, D, B and N are as defined above;
  • each of b and c is an integer from 0-2; [0050] d is an integer from 0-4; and
  • e is an integer from 0- 1.
  • Nj is a sequence selected from GA or AA; N c is the sequence GT; N ⁇ is a sequence selected from ATA or CAAA; or N e is T. In other embodiments, any one or more of Nj, N c , N ⁇ or N e comprises 0 bases.
  • Representative examples of sequences according to SEQ ID NO: 12 are suitably selected from the group consisting of:
  • the promoter sequence further comprises upstream of (d), (e) or (f) a nucleotide sequence (also referred to herein as segment Z sequence ) selected from the group consisting of:
  • A, C, G, T 5 M, R, W, S, Y 5 K, V, H 5 D 5 B and N are as defined above;
  • N/ is a sequence selected from ATG, ATA or AAA; N g is a sequence selected from TA or TC; NA is the sequence CCCA; or N / is W. In other embodiments any one or more of N/, N g , N / ,, or N,- comprises 0 nucleotides.
  • sequences according to SEQ ID NO: 23 are suitably selected from the group consisting of:
  • the promoter sequence further comprises upstream of (g), (h) or (i) a nucleotide sequence (also referred to herein as segment ⁇ sequence ) selected from the group consisting of: [0087] Q) C YVYRKKN 7 WMDKSRYNVWRMMRN ⁇ CYMRDVBHBRN / YBRCN m HHMSWMRHHMABRVRYARVWN « RAVRMWRN 0 BRVDWGRRRBRRCN p RYTWYR GN ⁇ TSDDTRD WWMVDMRN.WARN ⁇ KGGRSWYTWRHMHMYHGRKN ⁇ VRRYVRSN ⁇ WRGK ⁇ RSDTGN v WRSCWAMYRWKHMYWTADCTTDHBBKN ⁇ RKYCWAN ⁇ AYCVCT MWTMHMN ⁇ YYHWVWGWKTCMYMAHRHKKYHMRWSRYYYRMMDSYCYMRH WN z MRDTSBCN ⁇ YTRRB
  • A, C, G, T 5 M, R, W 3 S, Y, K, V, H, D, B and N are as defined above;
  • each of r and w is an integer from 0 to 3; [0095] am is an integer from 0 to 5;
  • an is an integer from 0 to 7;
  • aa is an integer from 0 to 8.
  • n is an integer from 0 to 9;
  • j is an integer from 0 to 16;
  • u is an integer from 0 to 42.
  • N 7 comprises C or a sequence selected from: TCTTAAGGTTGAATA [SEQ ID NO: 35] or CAATCCGGTCG [SEQ ID NO: 36]; N* is K; N / is B; N n , is a sequence selected from TG, TT or GA N ra is a sequence selected from GA, GGA, AGA, GGAGAAAGA or ACAAGATAA; N 0 is a sequence selected from AG, TT or CT; N p is T or A; N ? is A; N 7 . is a sequence selected from CTT or CTC; N 15 .
  • N is the sequence GT; N; is C or a sequence selected from CC or AA; N M is C or a sequence selected from TAGTGACTAAAGTTTAGTCCCTGAACTGAACTTTAATCC [SEQ ID NO: 37], TAGTGACTAAAGTTTAGTCCTTGAACTGAACTTTAATCC [SEQ ID NO: 38], TAGTGGACTGAACTTTAATCC [SEQ ID NO: 39]
  • Representative sequences according to SEQ ID NO: 34 may be selected from the group consisting of: [0104] CTCTAGTCAAGGGACTAAACCAGCTAAAAGACATCCGCTGCCCC
  • the promoter sequence further comprises upstream of Q), (k) or (1) a nucleotide sequence (also referred to herein as segment ⁇ sequence ) selected from the group consisting of:
  • nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 51 or a complement thereof;
  • each of at and av is an integer from 0 to 1 ;
  • each of aw and ax is an integer from 0 to 2;
  • each of au, ay and az is an integer from 0 to 3;
  • N ⁇ comprises C, G or A; N ⁇ M comprises a sequence selected from GGA, GGC or GAT; N ⁇ v comprises C or T; N ⁇ w comprises a sequence selected from AC, CC or AG; H z* comprises the sequence AG; Hy comprises a sequence selected from TGC or ACC; and N ⁇ z comprises A or a sequence selected from CGC or CCT.
  • any one or more of H 7/ , H n ,, H n ,, N w , N 0 *, H 0 , or H E comprises 0 nucleotides.
  • sequences according to SEQ ID NO: 51 include:
  • an intervening sequence (also referred to herein as "segment ⁇ sequence") is interposed between the nucleotide sequence according to any one of (d), (e) or (f) and the nucleotide sequence according to the nucleotide sequence according to any one of (g), (h) or (i).
  • the intervening sequence comprises from about 100 to about 150 nucleotides, typically from about 110 to about 140 nucleotides and suitably from about 115 to about 120 nucleotides.
  • the intervening sequence is selected from the group consisting of:
  • GGTGTGCTCACTTCGCGAAAAGTAGGCGAAAAGTTACTGTAGCA TTTTTCGTTGTTATTTGGCAAAATTTGTCCAATCATGGACTAACTAGGCTCAAAAG CTTCGTCCCAACATTTACAGACAAACTATGCAATTAGTTATCTTTTACCTACAT TTAATGCTCCATGCATACGTCGAAAGATTTGATGTGATGGGTGTAAGAGGAAATA TTTTGGGTTGGGATGGGGAAGTGAACAAGGCCTCGG
  • SEQ ID NO: 60 or a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 60 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 60 or a complement thereof, under at least medium or high stringency conditions;
  • the promoter sequence has a structure represented by formula (I):
  • ⁇ is any one of (m), (n) or (o); [0140] ⁇ is any one of G) 5 (k) or (1);
  • Z is any one of (g), (h) or (i); [0142] ⁇ is an optional spacer or intervening sequence; [0143] ⁇ is any one of (d), (e) or (f); and [0144] ⁇ is any one of (a), (b) or (c).
  • the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 72 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 72 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 72 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 72 or a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 72 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 72 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 72 or a complement thereof,
  • the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
  • the present invention provides isolated nucleic acid molecules, which comprise a promoter sequence that is operable in plant cells, including monocotyledonous plant cells.
  • the promoter sequence generally comprises a nucleotide sequence selected from the group consisting of:
  • promoter elements positioned upstream or 5' to the TATA box in segment ⁇ of the exemplified promoter sequences set forth in SEQ ID NO: 63-82 influence the transcription initiation rate and/or tissue (e.g., sink tissue) expression of operably connected nucleic acid sequences, and could be fused, therefore, to heterologous core promoter sequences to produce chimeric promoters with similar expression patterns to the exemplified promoter sequences.
  • the present invention provides isolated nucleic acid molecules comprising chimeric promoter sequences that generally comprise at least one segment selected from the group consisting of a heterologous core promoter sequence in operable connection with a nucleotide sequence selected from the group consisting of :
  • nucleotide sequence represented by (1) comprises segment ⁇ , and optionally ⁇ . In other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ and Z, and optionally ⁇ . In yet other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , Z and ⁇ , and optionally ⁇ . In still other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , Z 3 ⁇ and ⁇ , and optionally ⁇ .
  • a promoter of the invention can be fused to a transcribable sequence to create a chimeric construct.
  • This construct can then be introduced into a host cell, typically a plant cell or plant or plant part, by any method of choice.
  • chimeric nucleic acid constructs comprising an isolated nucleic acid comprising a promoter sequence as broadly described above, in operable connection with a heterologous (e.g., foreign or endogenous) nucleic acid sequence to be transcribed.
  • the chimeric construct further comprises a 3 ' non- translated sequence that is operably linked to the heterologous nucleic acid sequence and that functions in plant cells to terminate transcription and/or to cause addition of a polyadenylated nucleotide sequence to the 3 ' end of a transcribed RNA sequence.
  • the heterologous nucleic acid sequence is heterologous with respect to the plant cell in which it is or will be introduced.
  • the heterologous nucleic acid sequence encodes a structural or regulatory protein, or alternatively, a transcript capable of modulating expression of a corresponding target gene.
  • the transcript comprises an antisense RNA or a ribozyme or other transcribed region aimed at downregulation of expression of the corresponding target gene.
  • the other transcribed region may comprise a sense transcript aimed at sense suppression (co- suppression) of the corresponding target gene or a hairpin transcript aimed at RNAi-mediated downregulation of the target gene.
  • the chimeric construct in some embodiments may be further characterized in that the promoter sequence is capable of conferring transcription of the heterologous nucleic acid sequence in a specific tissue (e.g., stem tissue) of the plant.
  • the promoter sequence is capable of regulating transcription of the heterologous nucleic acid preferentially in stem tissue of a plant.
  • the chimeric construct may be further characterized in that the promoter sequence is capable of regulating transcription of the heterologous nucleic acid sequence in mature stem tissue of a plant.
  • Plants falling within the scope of the present invention encompass any taxonomic grouping, including angiosperms, gymnosperms, monocotyledons and dicotyledons.
  • the plant is selected from monocotyledonous plants such as cereals, sugarcane, bananas and pineapples.
  • the plant is a sucrose- accumulating plant such as sugarcane, sugarbeet or sweet sorghum.
  • the present invention provides methods for expression of a heterologous nucleic acid sequence. These methods generally comprise introducing into a plant cell a chimeric construct as broadly described above.
  • the present invention contemplates methods for producing a transformed plant cell, wherein the methods generally comprise introducing into a plant cell a chimeric construct as broadly described above.
  • the present invention provides methods for producing transformed plant cells. These methods generally comprise introducing into regenerable plant cells a chimeric construct as broadly described above so as to yield transformed plant cells and identifying or selecting the transformed plant cells.
  • the present invention provides methods for selecting stable genetic transformants from transformed plant cells, wherein the methods generally comprise introducing into regenerable plant cells a chimeric construct as broadly described above so as to yield transformed plant cells and identifying or selecting a transformed plant cell line from the transformed plant cells.
  • the regenerable cells are regenerable dicotyledonous plant cells. In other embodiments, the regenerable cells are monocotyledonous plant cells such as regenerable graminaceous or non-graminaceous monocotyledonous plant cells.
  • the expression of the chimeric construct in the transformed cells imparts a phenotypic characteristic to the transformed cells. Suitably, the imparted phenotype results from expression of the heterologous nucleic acid sequence.
  • transformed plant cells are provided, containing a chimeric construct as broadly described above.
  • the present invention contemplates methods for producing a differentiated transgenic plant. These methods generally comprise introducing a chimeric construct as broadly described above into regenerable plant cells so as to yield regenerable transformed cells, identifying or selecting a population of transformed cells, and regenerating a differentiated transgenic plant from the population.
  • the expression of the chimeric construct renders the differentiated transgenic plant identifiable over the corresponding non-transgenic plant.
  • the invention provides differentiated transgenic plants comprising plant cells containing a chimeric construct as broadly described above.
  • the chimeric construct is transmitted through a complete cycle of the differentiated transgenic plant to its progeny so that it is expressed by the progeny plants.
  • the present invention also provides cells, tissues, leaves, fruit, flowers, seeds and other reproductive material, material used for vegetative propagation, progeny plants including Fl hybrids, male-sterile plants and all other plants and plant products derivable from the differentiated transgenic plant.
  • the 942-nt sequence set forth in SEQ ID NO: 83 is a transcribable nucleic acid sequence comprising an ORF, as set forth in SEQ ID NO: 83, which codes for a 313- amino acid sequence, as set forth in SEQ ID NO: 84.
  • This ORF is transcribed at high levels in stem tissues, including mature stem tissues, of sugarcane (Saccharum sp.).
  • nucleotide sequences that correspond or are complementary to at least a portion of the sequence set forth in SEQ ID NO: 83 may be useful as probes for isolating homologous transcribable sequences from other plants, especially from other sugarcane plants and more broadly other monocotyledonous plants such as cereals, bananas and pineapples to, in turn, permit the isolation of promoter sequences with analogous qualities to those described herein.
  • the present invention contemplates isolated nucleic acid molecules comprising a promoter sequence or biologically active fragment thereof or variant of these, wherein the promoter sequence is located upstream of a transcribable nucleic acid sequence that hybridizes to a nucleic acid probe derived from the polynucleotide sequence set forth in SEQ ID NO: 83.
  • the isolated promoter sequence is of sufficient length such that it is capable of initiating and/or regulating transcription of a DNA sequence to which it is coupled.
  • the promoter sequence may be between about 150 nts and 1200 nts in length and usually greater than 250 nts in length.
  • analogous promoter sequences may be obtained from plants, especially from monocotyledonous plants such as cereals, sugarcane, bananas and pineapples, which contain a nucleotide sequence that is capable of hybridizing to a nucleic acid probe derived from the sequence set forth in SEQ ID NO: 83 under at least medium stringency conditions, and especially under high stringency conditions.
  • Figure 1 is a photographic representation of various Northern analyses showing expression of ScRlMYBl in different sugarcane tissues, (a) Northern analysis using total RNA isolated from meristems (M), internodes 1 to 3 (1-3), internode 5 (5), internode 12 (12), internode 20 (20), expanding leaf (EL) and mature leaf (ML) from field-grown Ql 17 plants, plus root (R) from glasshouse-grown Ql 17 plants, probed with the 838bp Hin ⁇ lll fragment of the ScRlMYBl cDNA.
  • Figure 2 is a photographic representation of a Southern analysis showing genetic complexity of ScRlMYBl in sugarcane. Genomic DNA from cultivar Ql 17 was digested with the restriction enzymes BamBI (B), EcoRI (E), HmdIII (H), Kpnl (K), Sad (S) zn ⁇ Xbal (X), and probed with the 838bp Hin ⁇ lll fragment of the ScRlMYBl cDNA.
  • Figure 3 is a diagrammatic representation illustrating an alignment of the proteins encoded by eight alleles of ScRlMYBl identified in sugarcane cultivar Ql 17.
  • the MYB DNA binding domain is marked by a solid bar above
  • the CCHC-type zinc finger is marked by a dashed line
  • the positions of introns 1 and 2 are marked by vertical arrows.
  • "Zl us” represents the sequence encoded by the partially spliced Zl transcript.
  • Figure 4 is a graphical representation showing allelic expression patterns in mature stem (internode 12) of sugarcane cultivars Ql 17 and Q200, as revealed by cloning and sequencing RT-PCR products.
  • Figure 5 is a diagrammatic representation of a sequence alignment of the promoter sequences corresponding to alleles Al, A2, A3, A4, A5, A6, A7, A8, A9 and B of the ScRlMYBl gene. The nucleotide sequences delineating segments ⁇ , ⁇ , Z, ⁇ , ⁇ and ⁇ are shown. TABLE A
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • amplicon refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an “amplicon” may include the sequence of probes or primers used in amplification.
  • biologically active fragment refers to a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30% of the activity of a reference promoter sequence.
  • biologically active fragment refers to a part of an indicated DNA sequence that modulates RNA transcription or that, when fused to a particular gene and introduced into a plant cell, causes expression of the gene at a level higher than is possible in the absence of such part of the indicated DNA sequence.
  • biologically active fragments encompass a portion of a promoter sequence that when added to a sequence including one or more 'core' promoter elements or motifs such as a TATA motif, promotes transcription in at least one tissue type to a greater extent than the 'core' sequence without the addition of the portion.
  • biologically active fragments of at least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 ,60 , 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides in length, or almost up to the number of nucleotides present in a foil-length promoter sequence.
  • chimeric construct chimeric nucleic acid
  • chimeric DNA chimeric DNA
  • a polynucleotide (a) having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or (b) encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein.
  • This phrase also includes within its scope a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.
  • growing or “regeneration” as used herein mean growing a whole, differentiated plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).
  • heterologous refers to a nucleic acid sequence linked to a nucleic acid sequence to which it is not naturally linked.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • RNA U pairs with A and C pairs with G.
  • the terms "match” and “mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an "isolated polynucleotide”, as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an isolated polynucleotide refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • isolated polynucleotide is free of sequences (e.g., protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide was derived.
  • an isolated promoter polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide was derived.
  • marker gene is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker.
  • a selectable marker gene confers a trait for which one can 'select' based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells).
  • a screenable marker gene confers a trait that one can identify through observation or testing, i.e., by 'screening' (e.g., ⁇ -glucuronidase, neomycin phosphotransferase II, luciferase, or other enzyme activity not present in untransformed cells).
  • a "naturally-occurring" nucleic acid molecule refers to a
  • RNA or DNA molecule having a nucleotide sequence that occurs in nature For example a naturally-occurring nucleic acid molecule can encode a protein that occurs in nature.
  • nucleic acid extract obtained from a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source.
  • the nucleic acid extract may be obtained from tissue isolated directly from a host plant.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof, including nucleotides with modified or substituted sugar groups and the like) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring
  • the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like.
  • PNAs peptide nucleic acids
  • phosphorothioate phosphorodithioate
  • phophoroselenoate phosphorodiselenoate
  • phosphoroanilothioate phosphoraniladate
  • phosphoroamidate methyl phosphonates
  • 2-O-methyl ribonucleic acids 2-O-methyl rib
  • Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a variant nucleic acid sequence. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.
  • operably connected means placing a transcribable nucleic acid sequence as defined herein under the regulatory control of a promoter sequence, which then controls the transcription and optionally translation of the gene.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous nucleic acid sequence to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.
  • plant and “differentiated plant” refer to a whole plant or plant part containing differentiated plant cell types, tissues and/or organ systems. Plantlets and seeds are also included within the meaning of the foregoing terms. Plants included in the invention are any plants amenable to transformation techniques, including angiosperms, gymnosperms, monocotyledons and dicotyledons. In specific embodiments, the plant is a monocotyledonous plant, illustrative examples of which include turf, turf grass, cereal, maize, rice, oat, wheat, barley, orchid, iris, lily, onion, banana, pineapple, sugarcane, sorghum, and palm.
  • plant cell refers to any plant cell or cell line including protoplasts, gamete-producing cells, and cells which regenerate into whole plants. Plant cells also include cells in plants as well as protoplasts in culture.
  • plant tissue is meant differentiated and undifferentiated tissue derived from roots, shoots, pollen, seeds, tumour tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as embryos and calluses.
  • polynucleotide or "nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • primer an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent.
  • the primer is usually single-stranded for maximum efficiency in amplification but can alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers.
  • the primer may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3 r end of the template sequence.
  • primers can be large polynucleotides, such as from about 35 nucleotides to several kb or more.
  • Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis.
  • substantially complementary it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide.
  • the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential.
  • non-complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template.
  • non-complementary nucleotide residues or a stretch of non- complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
  • Probe refers to a molecule that binds to a specific sequence or sub- sequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.
  • promoter refers to a nucleic acid which directs expression of another nucleic acid to which it is operably linked, by initiating, regulating or otherwise controlling transcription of the nucleic acid. Promoters usually comprise a TATA box and often a "CAAT” box, capable of directing RNA polymerase to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. They may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which may influence the transcription initiation rate, tissue expression and/or temporal expression of an operably connected nucleic acid sequence.
  • a "core promoter” is intended to mean a promoter sequence consisting only of all basal elements needed for transcription initiation, e.g. , a TATA box and/or an initiator, without ancillary (e.g., upstream) promoter elements.
  • “Constitutive promoter” refers to a promoter that directs transcription in many or all tissues of a plant.
  • stem-specific promoter is meant a promoter that preferentially directs transcription in stem tissue of a plant.
  • Preferentially directs transcription means that the rate of transcription of an operably linked nucleic acid is higher in the nominated tissue or developmental stage than in another tissue or developmental stage used for comparison.
  • the term "recombinant” as used herein refers to a nucleic acid or polypeptide resulting from in vitro manipulation into a form not normally found in nature. As used in the art, “recombinant” usually refers to the products of recombinant DNA technology.
  • sequence comparison programs such as BESTFIT (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. Sequences of a similar or substantially different length may be aligned and compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by BESTFIT.
  • regulatory element refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which includes sequences that control the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site.
  • An example of a complex regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter.
  • a promoter generally comprises a core promoter region, responsible for the initiation of transcription, and optionally other regulatory elements that modify gene expression.
  • nucleotide sequences, located within introns, or 3' of the coding region sequence may also contribute to the regulation of expression of a coding region of interest.
  • suitable introns include, but are not limited to, the maize IVS6 intron, or the maize actin intron.
  • a regulatory element may also include those elements located downstream (3') to the site of transcription initiation, or within transcribed regions, or both.
  • a post-transcriptional regulatory element may include elements that are active following transcription initiation, for example translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability determinants.
  • a "reference sequence” is at least 6 but frequently 15 to 18 and often at least
  • two polynucleotides may each comprise (1) a sequence of nucleotide bases that is similar between the two polynucleotides, and (2) a sequence of nucleotide bases that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for comparison may be conducted by computerized implementations of algorithms (such as EMBOSS programs NEEDLE and WATER accessible on the EMBL-EBI website http://www.ebi.ac.ulc/Tools/emboss/align/index.html.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a comparison window.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity can be determined using the EMBOSS programs NEEDLE or WATER or other algorithms such as those described above.
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco,
  • sequence similarity refers to the extent that optimally aligned sequences show the same amino acid or conservatively substituted amino acids according to a matrix or algorithm specified in the sequence comparison program, for example the Blossum or PAM matrices used with multiple sequence alignment program Clustal.
  • stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between immobilized nucleotide sequences and the labeled polynucleotide sequences that remain bound to them following the hybridization procedure.
  • Stringent conditions refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize.
  • the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization and subsequent washes, and the time allowed for these processes.
  • non- stringent hybridization conditions are selected; about 20 to 25° C lower than the thermal melting point (T m ).
  • T m is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH.
  • highly stringent washing conditions are selected to be about 5 to 15° C lower than the T m .
  • moderately stringent washing conditions are selected to be about 15 to 30° C lower than the T m .
  • Highly permissive (low stringency) washing conditions may be as low as 50° C below the T m , allowing a high level of mis-matching between hybridized sequences.
  • transcribable DNA sequence or "transcribed DNA sequence”, excludes the non-transcribed regulatory sequence that drives transcription.
  • the transcribable sequence may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA.
  • a transcribable sequence may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control.
  • transcribable sequence may contain an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.
  • the transcribable sequence may also encode a fusion protein. It is contemplated that introduction into plant tissue of chimeric nucleic acid constructs of the invention will include constructions wherein the transcribable sequence and its promoter are each derived from different species. [0232]
  • transformation means alteration of genotype by introduction of genetic material (e.g., the chimeric construct of the present invention) into an organism.
  • transgenic or “transformed” with respect to a plant cell, plant part (including seed), plant tissue or plant means a plant cell, plant part, plant tissue or plant which comprises an isolated chimeric DNA construct according to the invention which has been introduced into the genome of a plant cell, plant part, plant tissue or plant.
  • vector is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast, virus, mammal, avian, reptile or fish into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art.
  • wild type refers to an untransformed plant cell, plant part, plant tissue or plant, i.e., one where the genome has not been altered by the presence of a chimeric nucleic acid construct as defined herein.
  • transcribable DNA sequences useful for isolating promoter sequences were first isolated through their linkage to a transcribable sequence, which comprises an ORF whose sequence is set forth in SEQ ID NO: 83, and which was found to be transcribed at high levels in mature stem tissues of sugarcane (Sacchamm sp.).
  • nucleotide sequences that correspond or are complementary to at least a portion of the sequence set forth in SEQ ID NO: 83 may be useful as probes for isolating homologous transcribable sequences from other plants, especially from other sugarcane plants and more broadly other monocotyledonous plants such as cereals, turf, turf grass, rice, orchid, iris, lily, onion, banana, pineapples, sugarcane, sorghum, and palm and to, in turn, permit the isolation of promoter sequences with analogous qualities to those described herein.
  • the probes may be used in any suitable screening procedure. For example, a microarray screening procedure, as described below, may be used to identify genes expressed differentially in various tissues.
  • the present invention provides promoter sequences useful for expression of transcribable sequences in plants.
  • stem-specific promoters for expression of chimeric or heterologous nucleic acid sequences in plants, especially monocotyledonous plants are provided.
  • Representative examples of such promoter sequences may be selected from the sequences set forth in SEQ ID NOS: 63 to 82. Sequence analysis has revealed that these promoter sequences share a common segmental architecture, which can be represented by the following formula:
  • segment ⁇ is more conserved across the class of promoter sequences than segment ⁇ , segment ⁇ is more conserved than segment Z, segment Z is more conserved than segment ⁇ , segment ⁇ is more conserved than segment ⁇ , and ⁇ is an optional spacer or intervening segment, as illustrated in Figure 5.
  • Segment ⁇ appears to comprise a core promoter region containing a TATA box and (AGGA)CAAT box and it has been experimentally determined that this segment is transcriptionally active in plant cells.
  • segments ⁇ , Z, ⁇ and ⁇ comprise promoter elements, which preferentially direct transcription in plant sink tissue, including stem tissue.
  • transcription is directed in stem tissue of monocotyledonous plants, illustrative examples of which include turf, turf grass, cereal, maize, rice, oat, wheat, barley, orchid, iris, lily, onion, banana, pineapple, sugarcane, sorghum, and palm.
  • the promoter sequences of the present invention can be used to prepare biologically active fragments that have promoter activity, to isolate corresponding sequences from other organisms, particularly other plants and more particularly other monocotyledonous plants, or to synthesize synthetic sequences. They can also be used in combination with native or heterologous core promoter regions, control elements or other regulatory sequences to modulate transcription and/or translation.
  • the present invention contemplates that biologically-active fragments of any one of SEQ ID NOS: 63 to 82, which comprise less than the entire promoter sequences disclosed herein, may be utilized to drive expression of an operably linked nucleotide sequence of interest, such as a nucleotide sequence encoding a heterologous protein. It is within skill in the art to determine whether such fragments decrease or increase expression levels or alter the nature of expression, i.e., constitutive or inducible expression. Such fragments should retain promoter activity, or the ability to modulate the activity of a coupled core promoter region, particularly the ability to control expression of operably linked nucleotide sequences.
  • the 5' portion of a promoter up to the TATA box near the transcription start site can be deleted without abolishing promoter activity, as described by Opsahl-Sorteberg, H-G. et al. (2004, Gene 341:49-58).
  • Biologically active fragments of promoters can be readily identified by randomly preparing and assaying deletion mutants of the promoter sequences of the invention (e.g., SEQ ID NOS: 63 to 82). With this strategy, a series of constructs is prepared, wherein each construct contains a different portion of the clone (a subclone), and these constructs are then screened for activity.
  • the activity of a promoter can be determined by standard methods known in the art, as disclosed for example in Medberry et al. (1992, Plant Cell 4:185; 1993, The Plant J. 3:619), Sambrook et al. (1989, supra) and McPherson et al. (U.S. Patent No. 5, 164,316).
  • a suitable means for screening for activity is to operably link a deleted promoter construction to a selectable or screenable marker, and to isolate only those cells or tissues or plants which express the marker gene.
  • a number of different, deleted promoter constructs are identified which still retain the desired, or even enhanced, activity.
  • the smallest segment which is required for activity is thereby identified through comparison of the selected constructs.
  • This segment may then be used for the construction of vectors for the expression of heterologous genes.
  • biologically active fragments may be identified by fusion to a core promoter region that is coupled to a reporter gene, and screening transformed plant cells for reporter gene expression at developmental stages of interest, as disclosed for example in Puente et al. (1996, EMBOJ. 15: 3732).
  • promoter elements positioned upstream or 5' to the TATA box in segment ⁇ influence the transcription initiation rate and/or tissue (e.g., stem-specific) expression of operably connected nucleic acid sequences.
  • tissue e.g., stem-specific expression of operably connected nucleic acid sequences.
  • the present invention also contemplates fusing any one or more of segments ⁇ , ⁇ , Z, ⁇ and ⁇ to a heterologous core promoter (e.g., a core promoter region of any promoter operative in the host organism into which it is desired to express a transcribable sequence of interest) to thereby produce a chimeric promoter sequence.
  • a heterologous core promoter e.g., a core promoter region of any promoter operative in the host organism into which it is desired to express a transcribable sequence of interest
  • Suitable core promoter sequences are well known in the art and are generally derived from plant-operative promoters, such as but not limited to the CaMV35S, nopaline synthase, ferrodoxin-RolD, maize ubiquitin and rice actin promoters. Reference also may be made to Puente et al. (1996, EMBO J. 15: 3732) and Klimyuk et al. (1995 Molecular & General Genetics 249: 357), who disclose illustrative plant core promoters. Alternatively the core promoter may correspond to a consensus core promoter sequence. Non-limiting examples of core promoter sequences are set forth in SEQ ID NO: 85- 87.
  • Representative biologically-active fragments of the promoter sequences set forth in SEQ ID NOS: 63 to 82 may comprise at least about 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides, or almost up to the number of nucleotides present in a full-length promoter sequence.
  • Non-limiting examples of such biologically active fragments are set forth in SEQ ID NO: 2 to 11, which correspond to segment ⁇ , respectively, of alleles A4, A7, A3, A6, Al, A2, A9, A8, A5 and B, as described herein.
  • Longer fragments can be produced by adding corresponding upstream sequences such as but not limited to ⁇ , ⁇ , Z, ⁇ and ⁇ .
  • biologically active fragments may include any one or more of segments ⁇ , ⁇ , Z, ⁇ and ⁇ , which can be fused to core promoter sequences, to create chimeric promoter sequences with expression patterns, in some embodiments, reflecting the activity of the promoter sequences set forth in SEQ ID NO: 62-83.
  • Illustrative examples of biologically active fragments according to the present invention are listed in Table 1 below. TABLE l
  • the present invention also encompasses promoter sequence variants that are substantially complementary to a reference sequence.
  • Such variants are identified by blotting techniques that include a step whereby nucleic acids are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), followed by a hybridization step, and a detection step.
  • Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence.
  • Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences.
  • Such techniques are well known by those skilled in the art, and have been described in Ausubel et al. (1994-1998, supra) at pages 2.9.1 through 2.9.20.
  • Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane-bound DNA to a complementary nucleotide sequence labeled radioactively, enzymatically or fluorochromatically.
  • dot blotting and slot blotting DNA samples are directly applied to a synthetic membrane prior to hybridization as above.
  • An alternative blotting step is used when identifying complementary polynucleotides in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridization.
  • a typical example of this procedure is described in Sambrook et al. ("Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989) Chapters 8- 12.
  • polynucleotides are blotted/transferred to a synthetic membrane, as described above.
  • a reference polynucleotide such as a polynucleotide of the invention is labeled as described above, and the ability of this labeled polynucleotide to hybridize with an immobilized polynucleotide is analyzed.
  • radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10 8 dpm/mg to provide a detectable signal.
  • a radiolabeled nucleotide sequence of specific activity 10 8 to 10 9 dpm/mg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA, usually 10 ⁇ g. Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization can also increase the sensitivity of hybridization (see Ausubel supra at 2.10.10).
  • polynucleotide sequence variants according to the invention will hybridize to a reference polynucleotide under at least low stringency conditions.
  • Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA) 5 1 mM EDTA, 0.5 MNaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • the polynucleotide variants hybridize to a reference polynucleotide under at least medium stringency conditions.
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42°C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55°C.
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 60-65°C.
  • BSA Bovine Serum Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C
  • 2 x SSC 0.1% SDS
  • BSA Bovine Serum Albumin
  • BSA Bovine Serum Albumin
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42°C, and about 0.01 M to about 0.02 M salt for washing at 55 0 C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C.
  • Other stringent conditions are well known in the art.
  • T m 81.5 + 16.6 (log 10 M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length)
  • T m of a duplex DNA decreases by approximately 1 °C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at T m - 15 °C for high stringency, or T m - 30 °C for moderate stringency.
  • a membrane ⁇ e.g. , a nitrocellulose membrane or a nylon membrane
  • immobilized DNA is hybridized overnight at 42 0 C in a hybridization buffer (50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe.
  • a hybridization buffer 50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA
  • the membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1% SDS for 15 min at 45°C, followed by 2 x SSC, 0.1% SDS for 15 min at 5O 0 C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55 0 C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68 0 C).
  • 2 x SSC 0.1% SDS for 15 min at 45°C
  • 2 x SSC 0.1% SDS for 15 min at 5O 0 C
  • two sequential higher stringency washes i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55 0 C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68 0 C.
  • Methods for detecting a labeled polynucleotide hybridized to an immobilized polynucleotide are well known to practitioners in the art. Such methods include autoradiography, phosphorimaging, chemiluminescent, fluorescent and colorimetric detection.
  • variants will comprise regions that show at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity over a reference promoter sequence of identical size ("comparison window") or when compared to an aligned sequence in which the alignment is performed by a computer program known in the art, as described in the definitions section on sequence relationships. What constitutes suitable variants may be determined by conventional techniques.
  • polynucleotides according to any one of SEQ ID NO: 62-82 can be mutated using random mutagenesis (e.g., transposon mutagenesis), oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlier prepared variant or non- variant version of an isolated natural promoter according to the invention.
  • random mutagenesis e.g., transposon mutagenesis
  • oligonucleotide-mediated (or site-directed) mutagenesis e.g., oligonucleotide-mediated (or site-directed) mutagenesis
  • PCR mutagenesis e.g., PCR mutagenesis
  • cassette mutagenesis e.g., cassette mutagenesis of an earlier prepared variant or non- variant version of an isolated natural promoter according to the invention.
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing nucleotide substitution variants of a promoter of the invention. This technique is well known in the art as, for example, described by Adelman et al. (1983, DNA 2:183). Briefly, promoter DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a template DNA, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the promoter of interest.
  • a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the promoter of interest.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the DNA template can be generated by those vectors that are either derived from bacteriophage Ml 3 vectors, or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. (1987, Methods Enzymol. 153:3).
  • the DNA that is to be mutated may be inserted into one of the vectors to generate single-stranded template. Production of single-stranded template is described, for example, in Sections 4.21- 4.41 of Sambrook et al. (1989, supra).
  • the single-stranded template may be generated by denaturing double-stranded plasmid (or other DNA) using standard techniques.
  • the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
  • a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of the promoter under test, and the other strand (the original template) encodes the native unaltered sequence of the promoter under test.
  • This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli.
  • the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer having a detectable label to identify the bacterial colonies having the mutated DNA.
  • the resultant mutated DNA fragments are then cloned into suitable expression hosts such as E. coli using conventional technology and clones that retain the desired promoter activity are detected. Where the clones have been derived using random mutagenesis techniques, positive clones would have to be sequenced in order to detect the mutation.
  • linker-scanning mutagenesis of DNA may be used to introduce clusters of point mutations throughout a sequence of interest that has been cloned into a plasmid vector.
  • the linker sequence actually provides the desired clusters of point mutations as it is moved or "scanned” across the region by its position at the varied endpoints of the deletion mutation series.
  • An alternate protocol is also described by Ausubel et ah, supra, which makes use of site directed mutagenesis procedures to introduce small clusters of point mutations throughout the target region. Briefly, mutations are introduced into a sequence by annealing a synthetic oligonucleotide containing one or more mismatches to the sequence of interest cloned into a single-stranded M 13 vector. This template is grown in an Escherichia coli duf ung ⁇ strain, which allows the incorporation of uracil into the template strand.
  • the oligonucleotide is annealed to the template and extended with T4 DNA polymerase to create a double-stranded heteroduplex. Finally, the heteroduplex is introduced into a wild-type E. coli strain, which will prevent replication of the template strand due to the presence of apurinic sites (generated where uracil is incorporated), thereby resulting in plaques containing only mutated DNA.
  • Region-specific mutagenesis and directed mutagenesis using PCR may also be employed to construct promoter variants according to the invention.
  • reference may be made, for example, to Ausubel et at, supra, in particular Chapters 8.2 A and 8.5.
  • An isolated nucleic acid promoter sequence or variant according to the invention can be fused to a heterologous nucleic acid to form a chimeric construct.
  • the heterologous nucleic acid may be a foreign or endogenous DNA sequence.
  • the chimeric construct includes regulatory sequences which influence expression of the heterologous nucleic acid in plants.
  • the chimeric construct is present in an expression vector which includes regulatory sequences that enable selective propagation in bacteria.
  • a 3' non-translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
  • the polyadenylation signal is characterized by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
  • the 3' non-translated regulatory DNA sequence typically includes from about 50 to 1,000 base pairs and contains plant transcriptional and translational termination sequences.
  • suitable 3' non-translated sequences are the 3' transcribed non- translated regions containing a polyadenylation signal from the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al, 1983, Nucl. Acid Res., 11:369) and the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens.
  • suitable 3' non-translated sequences may be derived from plant genes such as the 3' end of the protease inhibitor I or II genes from potato or tomato, the soybean storage protein genes and the pea E9 small subunit of the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed.
  • 3' non-translated regulatory sequences can be obtained de novo as, for example, described by An (1987, Methods in Enzymology, 153:292), which is incorporated herein by reference.
  • the chimeric construct of the present invention can further include enhancers, either translation or transcription enhancers, as may be required.
  • enhancer * regions are well known to persons skilled in the art, and can include an ATG initiation codon and adjacent sequences.
  • the initiation codon must be in phase with the reading frame of the coding sequence relating to the foreign or endogenous DNA sequence to ensure translation of the entire sequence.
  • the translation control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the foreign or endogenous DNA sequence.
  • the sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.
  • transcriptional enhancers include, but are not restricted to, elements from the CaMV 35S promoter and octopine synthase genes as for example described by Last et al (U.S. Patent No. 5,290,924, which is incorporated herein by reference). It is proposed that the use of an enhancer element such as the ocs element, and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation.
  • leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence.
  • leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as for example described by Joshi (1987, Nucl. Acid Res., 15:6643), which is incorporated herein by reference.
  • other leader sequences e.g., the leader sequence of RTBV, have a high degree of secondary structure that is expected to decrease mRNA stability and/or decrease translation of the mRNA.
  • leader sequences that do not have a high degree of secondary structure, (ii) that have a high degree of secondary structure where the secondary structure does not inhibit mRNA stability and/or decrease translation, or (iii) that are derived from genes that are highly expressed in plants, will be most preferred.
  • sucrose synthase intron as, for example, described by Vasil et al. (1989, Plant Physiol, 91:5175), the Adh intron I as, for example, described by Callis et al. (1987, Genes Develop., II), or the TMV omega element as, for example, described by Gallie et al. (1989, The Plant Cell, 1:301)
  • Adh intron I as, for example, described by Callis et al. (1987, Genes Develop., II
  • TMV omega element as, for example, described by Gallie et al. (1989, The Plant Cell, 1:301
  • Other such regulatory elements useful in the practice of the invention are known to those of skill in the art.
  • targeting sequences may be employed to target a protein product of the foreign or endogenous DNA sequence to an intracellular compartment within plant cells or to the extracellular environment.
  • a DNA sequence encoding a transit or signal peptide sequence may be operably linked to a sequence encoding a desired protein such that, when translated, the transit or signal peptide can transport the protein to a particular intracellular or extracellular destination, respectively, and can then be post- translationally removed.
  • Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.
  • the transit or signal peptide can direct a desired protein to a particular organelle such as a plastid (e.g., a chloroplast), rather than to the cytoplasm.
  • a plastid e.g., a chloroplast
  • the chimeric DNA construct can further comprise a plastid transit peptide encoding DNA sequence operably linked between a promoter region or promoter variant according to the invention and the foreign or endogenous DNA sequence.
  • a promoter region or promoter variant for example, reference may be made to Heijne et al. (1989, Eur. J. Biochem., 180:535) and Keegstra et al. (1989, Ann. Rev. Plant Physiol. Plant MoI. Biol, 40:471), which are incorporated herein by reference.
  • the chimeric construct can also be introduced into a vector, such as a plasmid.
  • Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.
  • Additional DNA sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the chimeric construct, and sequences that enhance transformation of prokaryotic and eukaryotic cells.
  • the vector suitably contains an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • the vector may be integrated into the host cell genome when introduced into a host cell. For integration, the vector may rely on the foreign or endogenous DNA sequence or any other element of the vector for stable integration of the vector into the genome by homologous recombination.
  • the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell.
  • the additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location in the chromosome.
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleic acid sequences.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUBl 10, pE194, pTA1060, and pAM.beta.l permitting replication in Bacillus.
  • the origin of replication may be one having a mutation to make its function temperature-sensitive in a Bacillus cell (see, e.g. , Ehrlich, 1978, Proc. Natl. Acad. Sd. USA 75:1433).
  • the chimeric nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the expressible foreign or endogenous DNA sequence.
  • a selectable or screenable marker gene as, or in addition to, the expressible foreign or endogenous DNA sequence.
  • the actual choice of a marker is not crucial as long as it is functional ⁇ i.e., selective) in combination with the plant cells of choice.
  • the marker gene and the foreign or endogenous DNA sequence of interest do not have to be linked, since co-transformation of unlinked genes as, for example, described in U.S. Pat. No. 4,399,216 is also an efficient process in plant transformation.
  • selectable or screenable marker genes include genes that encode a "secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells.
  • markers that encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity.
  • Secretable proteins include, but are not restricted to, proteins that are inserted or trapped in the cell wall ⁇ e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S); small, diffusible proteins detectable, e.g. by ELISA; and small active enzymes detectable in extracellular solution ⁇ e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin acety transferase).
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, erythromycin, chloramphenicol or tetracycline resistance.
  • exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (ne ⁇ ) gene conferring resistance to kanamycin, paromomycin, G418 and the like as, for example, described by Potrykus et al. (1985, MoI. Gen. Genet.
  • EPSPS
  • acetolactate synthase gene which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
  • EP-A- 154 204 a mutant acetolactate synthase gene that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
  • a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan
  • dalapon dehalogenase gene that confers resistance to the herbicide.
  • Screenable markers include, but are not limited to, a uidA gene encoding a ⁇ -glucuronidase (GUS) enzyme for which various chromogenic substrates are known; a ⁇ -galactosidase gene encoding an enzyme for which chromogenic substrates are known; an aequorin gene (Prasher et al., 1985, Biochem. Biophys. Res.
  • Microbiol, 129:2703 which encodes an enzyme capable of oxidizing tyrosine to dopa and dopaquinone which in turn condenses to form the easily detectable compound melanin; or axylE gene (Zukowsky et ⁇ l, 1983, Proc. N ⁇ tl Ac ⁇ d. ScL USA 80:1101), which encodes a catechol dioxygenase that can convert chromogenic catechols.
  • the isolated promoters sequences of the invention may be used, inter ⁇ li ⁇ , to drive expression of a foreign or endogenous nucleic acid sequence.
  • Illustrative agronomic properties encoded by the foreign or endogenous sequence include, but are not limited to, increased yield of a desired endogenous plant component, production of additional compounds by reactions involving endogenous plant components, decreased production of an undesired plant component, insect resistance or tolerance, herbicide resistance or tolerance, disease resistance or tolerance, tolerance to other stresses (e.g. drought, salinity, cold).
  • the foreign or endogenous nucleic acid sequence may comprise a region transcribed into a molecule that modulates the expression of a corresponding target gene.
  • the molecule may be an antisense RNA or a ribozyme or other transcript aimed at downregulation of expression of the corresponding target gene.
  • Anti-sense regulation, co-suppression and the use of ribozymes and hairpin may comprise a region transcribed into a molecule that modulates the expression of a corresponding target gene.
  • the molecule may be an antisense RNA or a ribozyme or other transcript aimed at downregulation of expression of the corresponding target gene.
  • RNA in plants are well known in the art.
  • the skilled person is referred to United States Patent 5,759,829 for an example of antisense technology; and to U. S. Patent 5,283,184, U. S. Patent 5,686,649, and WIPO PCT specification WO9853083 for examples of co-suppression technology; and to U.S. patent 5,707,835, U.S. patent 5,747,335 and U.S. patent 5,840,874 which each provide examples of ribozyme technology; and to USA patent application 2008/0104732 for examples of hairpin RNA technology for RNAi induction.
  • Each of these patent documents is incorporated herein by reference.
  • the foreign or endogenous nucleic acid sequence may encode a molecule which is readily detectable or measurable, e.g. ⁇ -glucuronidase or luciferase; a selectable product, e.g. , neomycin phosphotransferase (nptll) conferring resistance to aminoglycosidic antibiotics such as geneticin and paramomycin; a product conferring herbicide tolerance, e.g. glyphosate resistance or glufosinate resistance; a product affecting starch biosynthesis or modification e.g. starch branching enzyme, starch synthases, ADP- glucose pyrophosphorylase; a product involved in fatty acid biosynthesis, e.g.
  • a selectable product e.g. , neomycin phosphotransferase (nptll) conferring resistance to aminoglycosidic antibiotics such as geneticin and paramomycin
  • a product conferring herbicide tolerance e.g.
  • a product conferring insect resistance e.g. crystal toxin protein of Bacillus thuringiensis
  • a product conferring viral resistance e.g. viral coat protein
  • a product conferring fungal resistance e.g. chitinase, ⁇ -l,3-glucanase or phytoalexin
  • a product altering sucrose metabolism e.g. invertase or sucrose synthase
  • a gene encoding valuable pharmaceuticals e.g. antibiotics, secondary metabolites, pharmaceutical peptides or vaccines.
  • both dicotyledonous and monocotyledonous plants that are amenable to transformation can be modified by introducing a chimeric DNA construct according to the invention into a recipient cell and growing a new plant that harbors and expresses the foreign or endogenous DNA sequence.
  • a construct of the invention may be introduced into a plant cell utilizing A. tumefaciens containing the Ti plasmid. In using an A.
  • the Agrobacterium harbors a binary Ti plasmid system.
  • a binary system comprises (1) a first Ti plasmid having a virulence region essential for the introduction of transfer DNA (T-DNA) into plants, and (2) a chimeric plasmid.
  • the chimeric plasmid contains at least one border region of the T-DNA region of a wild-type Ti plasmid flanking the nucleic acid to be transferred.
  • Binary Ti plasmid systems have been shown effective to transform plant cells as, for example, described by De Framond (1983, Biotechnology, 1:262) and Hoekema et al. (1983, Nature, 303:179). Such a binary system is preferred inter alia because it does not require integration into the Ti plasmid in Agrobacterium. [0293] Methods involving the use of Agrobacterium include, but are not limited to:
  • Ti plasmid may be manipulated in the future to act as a vector for these other monocot plants. Additionally, using the Ti plasmid as a model system, it may be possible to artificially construct transformation vectors for these plants. Ti plasmids might also be introduced into monocot plants by artificial methods such as microinjection, or fusion between monocot protoplasts and bacterial spheroplasts containing the T-region, which can then be integrated into the plant nuclear DNA.
  • gene transfer can be accomplished by in situ transformation by Agrobacterium, as described by Bechtold et al. (1993, CR. Acad. Sci. Paris, 316:1194). This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells.
  • nucleic acids may be introduced using root-inducing (Ri) plasmids of Agrobacterium as vectors.
  • Cauliflower mosaic virus may also be used as a vector for introducing of exogenous nucleic acids into plant cells (U.S. Pat. No. 4,407,956).
  • CaMV DNA genome is inserted into a parent bacterial plasmid creating a recombinant DNA molecule that can be propagated in bacteria.
  • the recombinant plasmid again may be cloned and further modified by introduction of the desired nucleic acid sequence.
  • the modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.
  • Nucleic acids can also be introduced into plant cells by electroporation as, for example, described by Fromm et al. (1985, Proc. Natl. Acad. Sci, U.S.A, 82:5824) and Shimamoto et al. (1989, Nature 338:274-276).
  • plant protoplasts are electroporated in the presence of vectors or nucleic acids containing the relevant nucleic acid sequences. Electrical impulses of high field strength reversibly permeabilize membranes allowing the introduction of nucleic acids. Electroporated plant protoplasts reform the cell wall, divide and form a plant callus.
  • nucleic acids can be introduced into a plant cell by contacting the plant cell using mechanical or chemical means.
  • a nucleic acid can be mechanically transferred by microinjection directly into plant cells by use of micropipettes.
  • a nucleic acid may be transferred into the plant cell by using polyethylene glycol which forms a precipitation complex with genetic material that is taken up by the cell.
  • polyethylene glycol which forms a precipitation complex with genetic material that is taken up by the cell.
  • silicon carbide or tungsten whiskers for example as described in United States Patent No. 5,302,523.
  • Transgenes driven by various promoter sequences can be efficiently silenced during the regeneration and growth of mature plants.
  • constructs are usually designed and transferred so as to minimize insofar as possible features likely to contribute to the production of aberrant transcripts and associated sequence-specific gene silencing responses in plants.
  • Simple integrations at a known favorable site for stable transgene expression may be obtained by homologous recombination in plants.
  • Accessory components such as site-specific recombinases, transposases or rare cutting nucleases can be used to facilitate simple and/or targeted integration as described below.
  • Site-specific recombinase systems such as CXQ-IOX from bacteriophage Pl, can be employed for resolution of complex integration events into simple patterns (Srivastava 1999, 2001; De Buck 2001a, 2001b, 2007).
  • Site-specific recombination systems can also be used to bring about targeted integration of transgenes into predetermined genomic loci through a two-round transformation procedure.
  • the first round of transformation introduces a construct containing a lox site.
  • Single-copy lines are selected as recipients for the next step, in which Cre recombinase activity integrates a /ox-flanked gene of interest specifically into the single-copy lox 'landing pad'.
  • the three necessary components can be brought together either by crossing transgenic plant lines with separate components, or by a second round of transformation into the selected recipient lines.
  • Several site-specific recombinase systems including FLP-FRT and R-RS have shown promise in plants, but Cre-lox is the best characterized (Ow 2002; Ow 2007).
  • Conditions (ii) and (iii) can be accomplished together if the landing pad is a promoter-fox-cre cassette, so that targeted integration abolishes Cre production (provided the recipient line was hemizygous for the landing pad) and commences integrated marker gene expression.
  • Counter- selection has been essential to eliminate lines with non-targeted integration events in most attempts at homologous recombination in plants, but not when using an efficient recombinase strategy. With an inducible cre gene and appropriate vector design it should be possible to achieve both single-copy site-specific integration and removal of unwanted selectable marker genes following selection of transformed cells (Ow 2007).
  • Agrobacterium-mediaXed gene transfer can be used with a recombinase- mediated cassette exchange (RMCE) strategy in which (i) the landing pad and replacement gene are each flanked by a wild-type and incompatible mutant lox site, (ii) Cre is provided by transient or integrative transformation using a separate T-DNA, and (ii ⁇ ) stringent selection for site-specific integration is based on expression of a replacement selectable marker gene from a promoter in the landing pad (Nanto 2005; Louwerse 2007).
  • RMCE recombinase- mediated cassette exchange
  • Nuclear scaffold or matrix attachment regions are sequences of about 300 base pairs to several kilobases that play a structural role in anchoring chromatin to the framework of the nuclear scaffold (Allen 2000, Chernov 2004).
  • the inclusion of flanking S/MARs in constructs has been reported to substantially increase transgene expression in plants (Allen 1996, Ulker 1999), Vain 1999,,Brouwer 2002, Mankin 2003).
  • Duplicated T-DNA borders can be used to minimise the incidence of integrated sequences from the flanking binary or Ti vector (Thole 2007).
  • Constructs employing tandem terminators can be used to reduce read-through (Luo 2007).
  • the transgene expression cassette may be isolated without vector backbone by either restriction digestion or PCR amplification (Agrawal 2005 ; Kumar 2006).
  • the genetic code is universal, the frequency of use of particular codons in the redundant set encoding any amino acid varies between organisms and even between protein classes (for example those expressed at high or low levels). This is thought to be related to the abundance of corresponding tRNAs in cells, which might in turn limit translation of transgenes containing rarely used codons. Synonymous mutations may be introduced into transgene sequences, either to use the most abundant codons in recipient cell genes or to more closely match the overall codon usage frequencies of the recipient (De Rocher 1998). Potential poly(A) addition signals can be removed simultaneously (Diehn 1998, Misztal 2004). Recently, Li (2007) reported that replacement of uncommon codons while maintaining codon diversity gave superior expression relative to use of the most abundant codon throughout a synthetic gene.
  • Heterologous genes may simultaneously be modified to eliminate signals for other processing events that limit transgene expression, for example intron splice signals, sequence context around the start codon, or mRNA destabilizing elements (Gutierrez 1999, Haseloff 1997, Holmberg 2001, Khanna 2006).
  • RNAi RNAi protein
  • HC-Pro potyvirus helper component-protease
  • Co-transformation with silencing suppressors or crossing to combine the transgene with a silencing suppressor may be used to inhibit silencing (Anandalakshmi 1998, Johansen 2001, Lim 2005, Lewsey 2007).
  • the methods used to regenerate transformed cells into differentiated plants are not critical to this invention, and any method suitable for a target plant can be employed. Normally, a plant cell is regenerated to obtain a whole plant following a transformation process.
  • Regeneration from protoplasts varies from species to species of plants, but generally a suspension of protoplasts is first made, hi certain species, embryo formation can then be induced from the protoplast suspension, to the stage of ripening and germination as natural embryos.
  • the culture media will generally contain various amino acids and hormones, necessary for growth and regeneration. Examples of hormones utilized include auxins and cytokinins. It is sometimes advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.
  • Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these variables are controlled, regeneration is reproducible. Regeneration also occurs from plant callus, explants, organs or parts. Transformation can be performed in the context of organ or plant part regeneration as, for example, described in Methods in Enzymology, Vol. 118 and Klee et al. (1987, Annual Review of Plant Physiology, 38:467), which are incorporated herein by reference. Utilizing the leaf disk-transformation-regeneration method of Horsch et al. (1985, Science, 227:1229, incorporated herein by reference), disks are cultured on selective media, followed by shoot formation in about 2-4 weeks. Shoots that develop are excised from calli and transplanted to appropriate root-inducing selective medium. Rooted plantlets are transplanted to soil as soon as possible after roots appear. The plantlets can be repotted as required, until reaching maturity.
  • the mature transgenic plants are propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.
  • the mature transgenic plants can be self-crossed to produce a homozygous inbred plant.
  • the inbred plant produces seed containing the newly introduced foreign gene(s). These seeds can be grown to produce plants that would produce the selected phenotype, e.g., early flowering.
  • Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells that have been transformed as described. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
  • the literature describes numerous techniques for regenerating specific plant types and more are continually becoming known. Those of ordinary skill in the art can refer to the literature for details and select suitable techniques without undue experimentation.
  • assays include, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting and PCR; a protein expressed by the heterologous DNA may be analysed by western blotting, high performance liquid chromatography or ELISA (e.g., nptll) as is well known in the art.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting and PCR
  • a protein expressed by the heterologous DNA may be analysed by western blotting, high performance liquid chromatography or ELISA (e.g., nptll) as is well known in the art.
  • cDNA clone MCSA063B04 was identified as putatively up- regulated in mature vs. immature stem based on microarray data (Casu et al 2004). EST sequencing of this clone revealed a putative methionine sulfoxide reductase (GenBank Accession number CF572917). However, the present inventors found by Northern analysis that this gene is not up-regulated in mature stems. Surprisingly, further sequence determination and analysis of the full insert in clone MCSA063B04 revealed the presence of likely concatenated cDNAs. A previously unknown region within this chimeric cDNA clone comprised a full open reading frame with a predicted translation product similar to SHAQKYF RlMYB transcription factors. The sequence of this region is provided as SEQ ID NO: 83.
  • Transcripts hybridising to a probe comprising SEQ ID NO: 83 were shown, by northern analysis, to be strongly up-regulated in mature vs. immature stem (Figure Ia), and also detectable in mature roots ( Figure Ib), but not in leaf tissue.
  • SNPs single nucleotide polymorphisms
  • intron 1 results in a smaller predicted protein (152 and 124 amino acids, respectively), that does not include any of the MYB domain but does include the N-terminal zinc finger and serine-rich domains ( Figure 3).
  • the presence of both spliced and unspliced transcript explains the double band observed on the northern blot probed with the ScRlMYBl probe ( Figure 1).
  • the Al promoter :Z1 coding sequence was identified 5 times among the 13 BAC clones, suggesting that the high proportion of Zl transcripts corresponds with a high genomic copy number of this allele in Q200. It can be concluded that the Al promoter is functional for ScRlMYBl expression in both cultivars. The analysis also indicates that the Xl transcribed sequence is linked to both the A4 and B promoter sequences in Ql 17, so these promoters are likely to be functional in their native state.
  • the Al promoter [SEQ ID NO.77] was fused to the coding region for a vacuole-targeted sucrose isomerase that converts sucrose into isomers isomaltulose and trehalulose that are not normally present in sugarcane.
  • Transgenic plants obtained after bombardment using this construct were grown in the field for 8 months, and then juice was extracted from mature stem internodes. Among 21 analysed plants, 15 produced detectable levels of isomaltulose and trehalulose.
  • the novel sucrose isomers made up three quarters of the total sugar in these basal internodes, showing that the Al promoter drives strong transgene expression in mature stem internodes of sugarcane.
  • DNA sequencing of plasmid templates was done using a BigDye Terminator 3.1 DNA sequencing kit (Applied Biosystems), and separations by the Australian Genomic Research Facility (Brisbane, Queensland, Australia).
  • the degree of allelic variation for ScRlMYBl was assessed by cloning and sequencing multiple PCR products amplified from Ql 17 using a forward primer beginning at the start codon (MYB F: S'-ATGGCTAGGAAATGCTCCAG-S') and a reverse primer beginning at the stop codon (MYB R: 5'-TTATTCTCCTAGACGCCAGT-S').
  • RT- PCR products derived from mature stem (amplified using the above primer set or the MYB R primer in combination with MYBF220: 5'-GCCTACTAYGGAGCTGCTGC-S') were sequenced to determine which alleles are expressed.
  • cDNA was synthesized using Superscript III (Invitrogen) and oligo dT primer using RNA from internodes 11-12 or 30. In all cases, the PCR was done using Expand High Fidelity polymerase (Roche) according to the manufacturer's instructions, and all amplified products were cloned into pGEM-T Easy (Promega) prior to sequencing.
  • Promoter alleles corresponding to ScRlMYBl were amplified using a PCR- based Genome Walker strategy (Clontech), using primers GSPl (5'- ATTGTTTCCACAACTGGAGCATTTCCTAGC-3') and GSP2 (5'- AACAAGGGCACAAGAAGTTGCGGTGTAGTA -3') derived from the 5' end of the ScRlMYBl transcript.
  • Promoter sequences with native 5'UTRs were amplified from Genome Walker clones, by use of PCR primers designed to incorporate Pstl and Ncol restriction sites. These restriction sites enabled cloning of the promoters in front of the GUSPlus (CAMBIA) and ZwC + NF (Promega) reporter genes. Constructs containing the same reporter genes driven by the maize Ubi-1 promoter (Christensen et al. 1992) were included as positive controls. All constructs included the nos terminator.
  • Transformation and regeneration of sugarcane cultivar Q 117 was done as previously described (Bower et al. 1996). Plants were grown in a containment glasshouse under natural light intensity, at 28 0 C and watered twice a day. Each plant was grown as a single stalk in a pot of 20 cm diameter and a density of 18 pots/m 2 , fertilized with Osmocote ® at 5 g/month for the first and the second months, then 10 g/month. Leaves were numbered from one for the top visual dewlap (TVD) with higher numbers for older leaves. Internodes were numbered according to the leaf attached to the node immediately above.
  • TVD top visual dewlap
  • tissues were vacuum infiltrated in assay buffer (50 mM sodium phosphate, pH 7.0; 0.1% Triton X-100; 0.5 mM potassium ferrocyanide; 0.5 mM potassium ferricyanide; 10 mM EDTA; 0.05% 5-bromo-4-chloro-3- indolyl glucuronide) and incubated at 37 0 C for 18 h. Green tissues were destained with ethanol.
  • fluorimetric GUS assays (Jefferson 1987), fluorescence measurements were taken at 0, 60, 120 and 180 minute timepoints using a Labsystems Fluoroskan Ascent microplate reader (Pathtech Diagnostics, Melbourne, Victoria, Australia).
  • Soybean mosaic virus helper component-protease enhances somatic embryo production and stabilizes transgene expression in soybean. Plant Physiology and Biochemistry 43(10-11):1014-1021.

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  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des éléments de contrôle transcriptionnel destinés à une utilisation dans le génie phytogénétique. Plus particulièrement, la présente invention concerne des promoteurs spécifiques aux tissus destinés à l'expression d'acides nucléiques hétérologues dans les plantes, notamment dans les plantes monocotylédones. L'invention concerne également des produits de recombinaison d'acide nucléique chimérique comprenant un promoteur selon l'invention, lié de manière opérationnelle à un polynucléotide étranger ou endogène codant pour une protéine d'intérêt ou un produit de transcription capable de moduler l'expression d'un gène cible. L'invention concerne également des cellules végétales transformées, ainsi que des plantes et des parties de plantes différenciées contenant le produit de recombinaison selon l'invention.
PCT/AU2009/001383 2008-10-20 2009-10-20 Eléments de contrôle transcriptionnel et leurs utilisations Ceased WO2010045679A1 (fr)

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US61/106,922 2008-10-20

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WO2010045679A1 true WO2010045679A1 (fr) 2010-04-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112813078A (zh) * 2021-04-08 2021-05-18 昆明理工大学 一种转录因子LbNAP在延迟百合花期中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004062366A2 (fr) * 2003-01-03 2004-07-29 The Texas A & M University System Promoteur de defense vegetale regule par la tige et ses utilisations dans l'expression specifique des tissus dans les monocotyledones

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004062366A2 (fr) * 2003-01-03 2004-07-29 The Texas A & M University System Promoteur de defense vegetale regule par la tige et ses utilisations dans l'expression specifique des tissus dans les monocotyledones

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 11 February 2009 (2009-02-11), "Saccharum hybrid cultivar Q117 R1MYB1 protein (R1MYB1)gene,.RIMYB1-Z1 allele", Database accession no. EU719199 *
MUDGE S.R. ET AL.: "Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species", PLANTA, vol. 229, no. 3, 2009, pages 549 - 558 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112813078A (zh) * 2021-04-08 2021-05-18 昆明理工大学 一种转录因子LbNAP在延迟百合花期中的应用

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