WO2010048666A1 - Transcriptional control elements and uses therefor - ii - Google Patents

Transcriptional control elements and uses therefor - ii Download PDF

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WO2010048666A1
WO2010048666A1 PCT/AU2009/001407 AU2009001407W WO2010048666A1 WO 2010048666 A1 WO2010048666 A1 WO 2010048666A1 AU 2009001407 W AU2009001407 W AU 2009001407W WO 2010048666 A1 WO2010048666 A1 WO 2010048666A1
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sequence
seq
nucleic acid
complement
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

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 ⁇ which comprises core promoter elements, 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.
  • 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] each of a, b, c, d, e, g, i, k and m is an integer from 0-1 ;
  • each of h,j and n is an integer from 0-2;
  • [0032] / is an integer from 0-3;
  • N ⁇ comprises 0 bases or is A; N& comprises 0 bases or is K; N c comprises 0 bases or is K; N ⁇ comprises 0 bases or is A; N e comprises 0 bases or is T; N/ comprises 0 bases or a sequence selected from ATTT or ACTC; Ng comprises 0 bases or is T; N / , comprises 0 bases or is K; N, comprises 0 bases or is A; Ny comprises 0 bases or is the sequence TC; N 7 comprises 0 bases or is W; N ⁇ comprises 0 bases or is a sequence selected from GA or CT; N / comprises 0 bases or is the sequence TGC; N w comprises 0 bases or is A; and N n comprises 0 bases or is the sequence AT.
  • sequences according to SEQ ID NO: 1 are suitably selected from the group consisting of: [0036] TGAGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTAATTGTTG
  • 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:
  • each of p, s, t, x, y and z is an integer from 0-1 ;
  • each of q and r is an integer from 0-2;
  • o is an integer from 0-4;
  • w is an integer from 0-5; [0052] each of v and aa is an integer from 0-6; and [0053] u is an integer from 0-23.
  • N V is the sequence ATCTC
  • N w is A or a sequence selected from TCAAA, GGCAC or TTTTT
  • N* is D
  • N 7 is A
  • N* is A
  • N ⁇ is the sequence AATCAA.
  • any one or more ofN o , N p , N ? , N r , N J5 N,, N M , N v , N w , N ⁇ , N y , N 2 or N ⁇ fl comprises 0 bases.
  • sequences according to SEQ ID NO: 8 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: [0063] (g) WYHAWCRKTAYWWRAN a ⁇ AGRAGYRWSN ⁇ c YATGASAGCATR
  • A, C 5 G, T, M, R, W, S, Y, K, V 5 H 5 D 5 B and N are as defined above;
  • each of ae, ⁇ /and ag is an integer from 0-1 ;
  • ad is an integer from 0-9.
  • any one or more of N 0 ⁇ 5 N ⁇ c , N 0 ⁇ , N ⁇ e , N ⁇ /or N ⁇ g comprises 0 nucleotides.
  • Representative examples of sequences according to SEQ ID NO: 20 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:
  • A, C, G, T, M, R, W, S 5 Y, K, V, H, D, B and N are as defined above; [0086] each of ah and al is an integer from 0-1 ;
  • aj is an integer from 0-2;
  • ai is an integer from 0-5;
  • ak is an integer from 0-8.
  • N 0/ is G; N ⁇ , is a sequence selected from AATAA, AATA or TAGGT; N 0/ is the sequence AA; N 0/t is a sequence selected from TATGTTTA or AATTAAAC; or N 0/ is C. In some embodiments, any one or more of N 0 /,, N ⁇ ;, N 0/ , N 0 /t or N 0 / comprises 0 bases.
  • Representative sequences according to SEQ ID NO: 27 may be selected from the group consisting of: [0092] AACTAATATCTCAGTAAATAGTATAGCAATAAATGAGTCTATAA
  • 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: [0098] (m) YWYTYWRWKTGKSYKWYWDMAAGWRWSYATWN em RMKWT RWRRTBTWGAAGMWTBKAATWRYSMCATRTATAN ⁇ TAAAGAAWARN ⁇ AN ⁇ TK ACAYTAYWWGWAYAWYAYATSTARRYAMWKGTCATWYATRRAMMMTWWYM YAWWKAKWN ⁇ ?
  • A, C, G 5 T, M, R, W, S, Y 5 K 5 V, H, D 5 B and N are as defined above;
  • each of am and ao is an integer from 0-1 ;
  • ap is an integer from 0-2;
  • N om is W; N ⁇ /7 is a sequence selected from AAACGCAAAAT [SEQ ID NO: 34], AAACGCAAAAC [SEQ ID NO: 35] or TAAAGAGTATA [SEQ ID NO: 36]; N 00 is T; N ⁇ is a sequence selected from TA or AA; or K q is a sequence selected from GCAATAAATGAGTCTATAATATGCAC [SEQ ID NO: 37] or GGGGGTGGAACCTTCCTTCAC [SEQ ID NO: 38] .
  • any one or more of N flW , N ⁇ , !5 N ao , N ⁇ p or N o? comprises 0 nucleotides.
  • sequences according to SEQ ID NO: 33 include:
  • 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 150 to about 250 nucleotides, typically from about 180 to about 220 nucleotides and suitably from about 190 to about 210 nucleotides. In illustrative examples of this type, the intervening sequence is selected from the group consisting of:
  • SEQ ID NO: 45 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: 45 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 45 or a complement thereof, under at least medium or high stringency conditions; and
  • SEQ ID NO: 46 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: 46 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 46 or a complement thereof, under at least medium or high stringency conditions.
  • the promoter sequence has a structure represented by formula (I):
  • ⁇ may be present or absent and is selected from any one of (m), (n) or (o);
  • ⁇ may be present or absent and is selected from any one of Q), (k) or (1);
  • Z is selected from any one of (g), (h) or (i);
  • ⁇ is an optional spacer or intervening sequence; [0125] ⁇ is selected from any one of (d), (e) or (f); and
  • ⁇ is selected from any one of (a), (b) or (c).
  • the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
  • the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
  • CTGCAGGCGGCCGCGAATTCACTAGTGATTACTATAGGGCACGC GTGGTCGACGGCCCGGGCTGGTATCGGGGCGTAGATTCCTCTCAACATATCAGCG TTGGAATATGTTTGCCTATTGAGTGGCCAGCACCAAGTACCATAGCCCCAGCTAA
  • Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 47, or that hybridize to the sequence set forth in SEQ ID NO: 47 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 48, 49, 52, 53, 54, 55 and 58.
  • Representative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 48, or that hybridize to the sequence set forth in SEQ ID NO: 48 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 49, 52, 53, 54, 55 and 58.
  • Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 49, or that hybridize to the sequence set forth in SEQ ID NO: 49 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 48, 52, 53, 54, 55 and 58.
  • Illustrative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 52, or that hybridize to the sequence set forth in SEQ ID NO: 52 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 48, 49, 53, 54, 55 and 58.
  • Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 50, or that hybridize to the sequence set forth in SEQ ID NO: 50 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 51, 56 and 57.
  • Representative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 51, or that hybridize to the sequence set forth in SEQ ID NO: 51 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 50, 56 and 57.
  • 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: [0144] (i) a nucleotide sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50,
  • (iii) a nucleotide sequence that hybridizes to the sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof, under at least medium or high stringency conditions.
  • promoter elements positioned upstream or 5' to the TATA box in segment ⁇ of the exemplified promoter sequences set forth in SEQ ID NO: 47-58 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 that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence of the at least one segment or a complement thereof, or
  • the nucleotide sequence represented by (1) comprises segment ⁇ . In other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ and ⁇ , and optionally segment ⁇ . In still other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , ⁇ and Z, and optionally segment ⁇ . In yet other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , ⁇ , Z and ⁇ , and optionally segment ⁇ . In still other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , ⁇ , Z, ⁇ and ⁇ , and optionally segment ⁇ . In other embodiments, the nucleotide sequence represented by (1) comprises segment ⁇ , and optionally segment ⁇ .
  • nucleotide sequence represented by (1) comprises segments ⁇ and Z, and optionally segment ⁇ . In yet other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , Z and ⁇ , and optionally segment ⁇ . In still other embodiments, the nucleotide sequence represented by (1) comprises segments ⁇ , Z, ⁇ and ⁇ , and optionally segments ⁇ .
  • 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 1248-nt sequence set forth in SEQ ID NO: 59 is a transcribable nucleic acid sequence comprising an ORF, which codes for a 415-amino acid sequence, as set forth in SEQ ID NO: 60.
  • 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: 59 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: 59.
  • 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 2500 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: 59 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 ScCIPKl 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, hybridized to a probe comprising nt 960-1248 of SEQ ID NO: 59.
  • Figure 2 is a diagrammatic representation of a sequence alignment of the promoter sequences corresponding to promoter alleles A, B, 5, 49, 51 and 77.
  • the nucleotide sequences delineating segments ⁇ , ⁇ , Z, ⁇ , ⁇ , ⁇ and ⁇ are shown.
  • “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, I 5 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 full-length promoter sequence.
  • chimeric construct chimeric nucleic acid or chimeric DNA
  • chimeric construct chimeric nucleic acid sequence, either single- or double-stranded, comprising at least two nucleic acid sequences from species which do not combine nucleic acids such as DNA under natural conditions, or which nucleic acid sequences are positioned or linked in a manner which does not normally occur in the native genome of the untransformed plant.
  • 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, In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G.
  • match and mismatch 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.
  • mature tissue refers to tissue with more than 80%, 85%, 90% or 95% of the maximum stored carbohydrate concentration achieved during plant development. For example, in sugarcane, maturation increases initially with internode age, so that if internodes are counted from the internode attached to the youngest emerged leaf as number 1 , maturation typically occurs up to about internode number 11 and older internodes are mature, as reviewed by Moore, 1995, Aust. J. Plant Physiol. 22, 661.
  • 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,
  • a naturally-occurring nucleic acid molecule can encode a protein that occurs in nature.
  • obtained from is meant that 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 valiant 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' 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 subsequence 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.
  • Constant 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.
  • recombinant 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.
  • Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. These relationships are commonly analysed by use of 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 1 ) 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 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because 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.uk/Tools/emboss/align/index.htmL and GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage sequence identity over the comparison window) generated by any of the various methods selected.
  • 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.
  • the percentage of 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, California, USA) using standard defaults as used in the reference manual accompanying the software, which is incorporated herein by reference in its entirety.
  • 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.
  • Stringency 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.
  • Those skilled in the art will recognise that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of similarity between target and probe sequences.
  • Other examples of stringency conditions are described in Section 4 below.
  • 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. Such modifications include, but are not limited to, insertions, deletions and substitutions of one or more nucleotides.
  • the 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.
  • 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.
  • the promoter sequences of the present invention were first isolated through their linkage to a transcribable sequence, which comprises an ORF whose sequence is set forth in SEQ ID NO: 59, and which was found to be transcribed at high levels in 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: 59 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.
  • a microarray screening procedure as described below, may be used to identify genes expressed differentially in various tissues.
  • the present invention is not restricted to use of any particular method for identifying such differentially expressed genes.
  • alternative procedures for identifying genes expressed differentially in various tissues include, but are not restricted to: hybridization screening as for example described in patent specification PCT/AU99/01033; cDNA and genomic subtractive hybridization as for example described by Bulman andNeill (1996, In "Plant Gene Isolation: Principles and Practice", G.D. Foster and D.
  • 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: 47 to 58. 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 2.
  • 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 ⁇ as well as segment ⁇ , which represents the portion of segment ⁇ , which is upstream of the TATA box comprise promoter elements, which preferentially direct transcription in plant sink tissue, including stem tissue.
  • transcription is directed in stem tissue, including mature 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 are particularly useful for preferentially expressing nucleic acids in carbohydrate storage tissue, and more particularly in mature plant carbohydrate storage tissue, illustrative examples of which include the sucrose-storage tissue of plants that store sugars, such as but not limited to sugarcane, sugarbeet and sweet sorghum.
  • 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: 47 to 58, 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.
  • 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: 47 to 58). 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 PlantJ. 3:619), Sambrook e/ a/. (1989, supra) and McPherson e/ 1 ⁇ /. (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, EMBO J. 15: 3732). The combination of segments most effective for particular developmental patterns of promoter activity is thereby identified through comparison of the selected constructs. These segments may then be used for the construction of vectors for the expression of heterologous genes.
  • 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.
  • the present invention also contemplates fusing any one or more of segments ⁇ , ⁇ , ⁇ , Z, ⁇ , ⁇ 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.
  • 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: 61- 63.
  • Representative biologically-active fragments of the promoter sequences set forth in SEQ ID NOS: 47 to 58 may comprise at least about 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500 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 7, which correspond to segment ⁇ , respectively, of alleles 77, 5, 51, A, B and 49, as described herein.
  • biologically active 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: 47-58.
  • Illustrative examples of biologically active fragments according to the present invention are listed in Table 1 below.
  • 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 0 C.
  • Low 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 room temperature.
  • 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
  • 0.5% BSA 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at room temperature.
  • 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 0 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 0 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 0 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 0 C
  • 2 x SSC 0.1% SDS
  • 0.5% BSA 1 mM EDTA
  • 40 mM NaHPO 4 pH 5% SDS for washing at 60-65 0 C.
  • the polynucleotide variants hybridize to a reference polynucleotide under high stringency conditions.
  • 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°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 (logio 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 0 C for high stringency, or T m - 30 0 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: 47-58 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 M 13 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 al., 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 al., supra, in particular Chapters 8.2A 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 1 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 at, 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).
  • 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)
  • 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.
  • 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 pB S -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. ScL 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. Examples include 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 5 -enolshikimate-3 -phosphate synthase
  • a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988, Science, 242:419); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al, 1988, J Biol Chem., 263:12500); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP-A-154 204); a mutated anthranilate synthase gene that confers resistance to 5 -methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.
  • a nitrilase gene such as bxn
  • 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 al, 1983, Proc. Natl. Acad. Sci. USA 80:1101), which encodes a catechol dioxygenase that can convert chromogenic catechols.
  • the isolated promoters sequences of the invention may be used, inter alia, 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. [0267] 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. ScL 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 Cre-lox 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 K-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-/ox-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-mediated 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 (iii) 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).
  • 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. In 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.
  • the sequence of the ScCIPKl coding region is set forth in SEQ ID NO: 59.
  • Transcripts hybridizing to a probe derived from SEQ ID NO: 59 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.
  • Sequencing of a BAC clone containing ScCIPKl allele A revealed that this allele is linked to the ScCIPKl A promoter allele (SEQ ID NO 56). Therefore this promoter is potentially useful for driving strong, mature-stem expression of transgenes.
  • ScCIPKl promoter alleles may be used to drive different transgene expression levels and developmental patterns within sink tissues.
  • RT-PCR analysis in transgenic sugarcane cell lines showed reporter gene expression driven from the ⁇ fragment of the ScCIPKl A promoter, providing functional confirmation that the ⁇ sequence comprises a core promoter region.
  • Multiple independent transgenic sugarcane lines containing the ScCIPKl A or B promoter alleles fused to the LUC reporter gene showed LUC activity in regenerating shoots in tissue culture, and in stem and root tissues of transgenic plants grown in a containment glasshouse for 4 months. This confirmed the ability of the isolated ScCIPKl promoters to drive heterologous gene expression in transgenic plant cells.
  • promoters described in the present invention in gene constructs designed to (i) preclude read-through into or out of the transgene ⁇ e.g. through the use of flanking transcriptional terminators); (ii) eliminate known silencing signals including sequence features (such as inverted repeats) or motifs (such as targets for recipient plant small regulatory RNAs); (iii) exclude unnecessary sequences that might be sources of unknown silencing signals; and (iv) ensure efficient transcriptional termination. It will also be advantageous to use gene transfer parameters designed to (i) maximise the proportion of simple, intact integration events of the transferred expression cassette; and (ii) preferably integrate at a known locus that has been shown experimentally to be neutral for transgene expression (e.g.
  • 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).
  • allelic Variation and Expression The degree of allelic variation for ScCIPKl was assessed by sequencing homologous clones from a genomic BAC library prepared from sugarcane cultivar Q200. To identify expressed alleles, RT-PCR products derived from mature stem were amplified using the primers 5'-GCCATAACACTAAGACGAGCGCCAACC-S ' and 5'- CGACACCCACTCCGGGAACTCGAACTCC-3' and sequenced. For RT-PCR, 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 ScCIPKl were amplified using a PCR- based Genome Walker strategy (Clontech), using primers GSPl (5'- CCTCCTCTGTCGGTTCCGCTGGTCGAAC AAGCCG-3') and GSP3B (5'- GTGCGTCGTCAGCGTCGCGAGGGCAAC -3') derived from the ScCIPKl coding region, or primers GWl (5'-CCTTGGCGAACGTGCCTCGTC-S') and GW2 (5'- CGTGCCTCGTCCC AGC AAGC-3') derived from the 5' end of the ScCIPKl transcript.
  • Genome Walker libraries prepared using restriction enzymes Oral, EcoRY, Pvull, Seal, Stul, BsrB ⁇ , EclU ⁇ ll, and Ssp ⁇ . These and all other PCR amplification reactions used Expand High Fidelity polymerase (Roche) according to the manufacturer's instructions. Amplified products were cloned into pGem-T Easy (Promega) prior to sequencing. Additionally, promoter alleles were obtained by subcloning from clones in a Q200 genomic BAC library. The transcriptional start site was determined by 5'RACE using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions.
  • Promoter sequences with native 5'UTRs were excised from Genome Walker and BAC clones using Pst ⁇ and iVc ⁇ l and cloned in front of the /wc + NF (Promega) reporter gene. Constructs containing the same reporter gene driven by the maize Ubi-1 promoter (Christensen et al, 1992) were included as positive controls. All constructs included the nos terminator.
  • BSES Meringa Sugar Experiment Station, Gordon vale, QId was done as previously described (Bower et al. 1996). Plants were grown in a containment glasshouse under natural light intensity, at 28°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
  • 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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Abstract

This invention discloses transcriptional control elements for use in plant genetic engineering. More particularly, the present invention relates discloses tissue-specific promoters for expression of heterologous nucleic acids in plants including monocotyledonous plants. The invention also discloses 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.

Description

"TRANSCRIPTIONAL CONTROL ELEMENTS AND USES THEREFOR - II"
FIELD OF THE INVENTION
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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. [0003] Recent advances in genetic engineering have enabled the incorporation of a selected gene (or genes) into plant cells to impart a desired quality (or qualities) to a plant of interest. 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.
[0004] In order for the newly incorporated gene to express the transcript and/or protein for which it codes in a plant cell, the proper regulatory signals must be present and in the proper location with respect to the gene. These regulatory signals include at least a promoter, which is a region involved in the regulation of transcription. Other regulatory signals that common in protein-coding genes for example include a 5' non-translated leader sequence and a 3' polyadenylation signal. [0005] Generally, the efficiency of gene expression is governed largely by the promoter used to express the gene. 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. By corollary, 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.
[0006] 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. For example, 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.). Isolated regions of about 40-90 nt upstream of the transcription start site and containing such conserved sequence motifs commonly have little or no promoter activity alone. However, when combined with upstream regulatory sequences from the same promoter or from other promoters, they can show promoter activity in a pattern determined substantially by the upstream regulatory sequences (for example, see Puente, Wei and Deng, 1996, EMBO J. 15: 3732).
[0007] 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).
[0008] It has been shown that certain 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. For example, in the case of an enzyme for conversion of an endogenous storage compound into an alternative industrial product, it may be useful to drive expression preferentially in mature storage tissues, to avoid interference with other plant functions in non-storage tissues. [0009] There is currently a dearth of promoters that can be used for effective expression of foreign or endogenous coding sequences in desired developmental patterns in plants in general, and monocotyledonous plants in particular.
SUMMARY OF THE INVENTION
[0010] In work leading up to the present invention, studies were carried out to understand the molecular basis for developmental control of plant gene expression, and in particular to understand the basis for preferential expression of certain plant genes in sink tissues, sucrose-storage tissues and mature stem tissues. During the course of these studies, the present inventors surprisingly discovered a class of plant genes with developmental patterns of interest, which enabled the isolation of a structurally related class of promoter sequences that can be used to express heterologous genes in plants, including preferential expression in plant sink (e.g., stem) tissue. Sequence analysis has revealed that these promoter sequences share a common segmental architecture, which can be represented by the following formula:
[0011] 5'-Θ-Δ-Z-Ψ-Φ-Ω-3' (I)
[0012] in which segment Ω, which comprises core promoter elements, is more conserved across the class of promoter sequences than segment Φ, segment Φ is more conserved than segment Z, segment Z is more conserved than segment Δ, segment Δ is more conserved than segment Θ, and Ψ is an optional spacer or intervening segment. Based on studies investigating another class of promoter sequences with a similar segmental architecture, as disclosed in co-pending United States Provisional Application No 61/106,922 filed October 20, 2008, which is hereby incorporated by reference herein in its entirety, the present inventors propose that segment Ω is transcriptionally active in plant cells.
[0013] Accordingly, in one aspect of the present invention, isolated nucleic acid molecules are provided, which 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 Ω sequence ) selected from the group consisting of:
[0014] (a) YGASMTTKAYWAKTSGCTARRRARH7KTYRRAKRMRWARWT GKYRSRRNfiHTTYNcDMYTYKTNrfTTCCAASRGYTWRKMAAAYY AHRMTMCNeRA RGTATAKRWMAHAN/WRMATKWGSRRTYYTRRRHKA WTTHYWRMTYAMYMYA WTCAKYTYW AYWCWWTTYYYN^MKKWYWTN/A WMYYTSYMTTWTRRGWACM YKATTWRWRWKCYAAYCN(YRRTTRRNyAARYSCYARTYNATYGRATTTASWAACA AAAAYGKCAAATTGCATACCCAACTCAN/CYCATATAAAAMGKYACCAACCCAA
CATNWTN«TTYC [SEQ ID NO: I]; [0015] (b) 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: 1 or a complement thereof; and
[0016] (c) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 1 or a complement thereof, under at least medium or high stringency conditions,
[0017] wherein:
[0018] A, C, G and T represent the nucleic acid bases adenine, cytosine, guanine and thymine, respectively;
[0019] M is A or C; [0020] R is A or G;
[0021] W is A or T;
[0022] S is C or G;
[0023] Y is C or T
[0024] K is G or T; [0025] V is A or C or G;
[0026] H is A or C or T;
[0027] D is A or G or T;
[0028] B is C or G or T;
[0029] N is G or A or C or T; and [0030] each of a, b, c, d, e, g, i, k and m is an integer from 0-1 ;
[0031] each of h,j and n is an integer from 0-2;
[0032] / is an integer from 0-3; and
[0033] /is an integer from 0-5.
[0034] In some embodiments, Nα comprises 0 bases or is A; N& comprises 0 bases or is K; Nc comprises 0 bases or is K; N^ comprises 0 bases or is A; Ne comprises 0 bases or is T; N/ comprises 0 bases or a sequence selected from ATTT or ACTC; Ng comprises 0 bases or is T; N/, comprises 0 bases or is K; N, comprises 0 bases or is A; Ny comprises 0 bases or is the sequence TC; N7 comprises 0 bases or is W; N^ comprises 0 bases or is a sequence selected from GA or CT; N/ comprises 0 bases or is the sequence TGC; Nw comprises 0 bases or is A; and Nn comprises 0 bases or is the sequence AT.
[0035] Illustrative examples of sequences according to SEQ ID NO: 1 are suitably selected from the group consisting of: [0036] TGAGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTAATTGTTG
GGGCTTTTGACTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATAGAAAA TAATTTTGCATTAGGAATCTTAAACTATTTTCAAATTACCCTAATCATTTTTATACT TTTTTTTCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGG TTAATCAAACCCCAGTTTTCGGATTTAGAAACAAAAACGGCAAATTGCATACCCA ACTCATCGCCCATATAAAACGTTACCAACCCAACATATATTTCC [SEQ ID NO : 2] ;
[0037] TGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCATAATTGTTGG GGCTTTGACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATAGAAAAC ATGCATTAGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTTTATACTTTTTT TCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAAT CAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCA TCGCCCATATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 3];
[0038] TGAGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTG GGGGTTTTTGACTTTTTTCCAACAGTTTATAAAATCATGCTCCTAAAGTATAGAAA ACAATTTTGCATTAGGAATCCTAAACTATTTCTAAATTACCCTAATCATTTTTATA CTTTTTTTTCTTTCTTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGG TTAATCAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCA ACTCATCGCCCATATAAAACGGCACCAACCCAACATATATTTCC [SEQ ID NO: 4];
[0039] CGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATGGCA CAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATATGT CAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCTCAA CTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCCAAC CCAATTAGAAGTGCTAATCTTGAATTTACTAACAAAAATGGCAAATTGCATACCC AACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 5];
[0040] CGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATGGCA CAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATATGT
CAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCTCAA
CTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCCAAC CCAATTGGAAGTGCTAATCTTGAATTTACTAACAAAAATGTCAAATTGCATACCC AACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 6]; and
[0041] TGAGATTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGG GGCTTTGACTTTTTTCCCCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAAT TTTGCATTAGGAATCTTAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTT TTCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAA TCAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTC ATCGCTCATATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 7];
[0042] In some embodiments, the promoter sequence further comprises upstream of (a), (b) or (c) a nucleotide sequence (also referred to herein as segment Φ sequence ) selected from the group consisting of:
[0043] (d) YRWYWRWTAYSYCTWDGAWMRTYKTYRAWARAYMAN0RVC ARAKMTSRARWTTGTTGASKYNPYRYWRYAASCRTKGN9CACTYRAAYDVRYRKR YHSCWTA YTRWWGAAWMRMGAASMWRTSWWKMATWWRYYYAADMAMANrW MYKAGCWAGMRMRYYWWTCS ARKACTTKMCMRA YKRRRTRN.RAY ATRYTCM AYN/CAY AAKWGKWGMDSKAYCMAMMYYYMCYKARTAWMTTATWMYTTWG AGCNVYAATWGTTTNWAW AMAANXWYAYMRCWMAMAARN^GYTWTCAAARMW NzYARTTGGYWNflα [SEQ ID NO: 8];
[0044] (e) 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: 8 or a complement thereof; and
[0045] (f) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 8 or a complement thereof, under at least medium or high stringency conditions,
[0046] wherein: [0047] A, C, G, T, M, R, W, S, Y, K5 V, H5 D, B and N are as defined above;
[0048] each of p, s, t, x, y and z is an integer from 0-1 ;
[0049] each of q and r is an integer from 0-2;
[0050] o is an integer from 0-4;
[0051] w is an integer from 0-5; [0052] each of v and aa is an integer from 0-6; and [0053] u is an integer from 0-23.
[0054] In some embodiments, N0 is the sequence AGAC; Np is G; N9 is the sequence GC; Nr is a sequence selected from AA or TG; N15 is T; N, is A; N« is a sequence selected from AATTTCTAATCATGGTCAATATA [SEQ ID NO: 9], AATTTATAATCATGGCTAATATA [SEQ ID NO: 10], TATATATATATTAATCTATC [SEQ ID NO: 11], TATATATATTAATCTATC [SEQ ID NO: 12] or
AATTTCTAATCATGGCTAATATA [SEQ ID NO: 13]; NV is the sequence ATCTC; Nw is A or a sequence selected from TCAAA, GGCAC or TTTTT; N* is D; N7 is A; N* is A; or N∞ is the sequence AATCAA. In other embodiments, any one or more ofNo, Np, N?, Nr, NJ5 N,, NM, Nv, Nw, Nχ, Ny, N2 or Nαfl comprises 0 bases.
[0055] Representative examples of sequences according to SEQ ID NO: 8 are suitably selected from the group consisting of:
[0056] CAACTAATATCTCTATGAGCATCTTCGAAAGACAAAGACGGCAG ATCTCAAAATTGTTGAGGTCATAATAAGCATTGCACTCAAACAGGCATGTTGCTT ATTAAAGAATAGCGAAGCAATGAAGAATAAATCCAAGAAAATATGAGCAAGCAC GTCAATCGAGCACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGTGCTCA ATTTCTAATCATGGTCAATATAATCAAACCCCACCGAGTATATTATTACTTAGAGC ATCTCCAATAGTTTTCAAAAAAAAAATCATCGCAAAAAAAGTTTTCAAAAAAATA ATTGGTAAATCAA [SEQ ID NO: 14]; [0057] CAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGACAG
ATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTT ATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAAAAAAAATATGAGCAAGC ACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGC TCAATTTATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACTTAG AGCATCTCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAAAAA AATAATTGGTAAATCAA [SEQ ID NO: 15];
[0058] CAACTAATATCTCTATGAGCATCTTTAAAAGACAAAGACAACAG ATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAGCATGTCGCTT ATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATATGAGCAAGCAC ATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTC ATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTAAAAAAA ATCATCGCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAA [SEQ ID NO: 16]; [0059] TGTTAGTTACCCCTTAGAAAGTTGTTAATAAATCAGCCAAAGATG GAATTTGTTGACTCGTGCTACAACCATGGGCCACTTGAATTCATGGACACCATAC TGTTGAAACAAGAACATGTGTTTCATTTGCTTAATCACATGACCTAGCTAGAGAG CTTTTCCAATACTTTCCAGATGAAATGTAATATACTCCACACATAATTGTTGAAGT ATATATATATTAATCTATCACCCACATTTCCTTAATAACTTATACTTTTGAGCTAA TTGTTTGGCACATACAAGATACAACTCACAATGCTATCAAAGCTCAGTTGGCT [SEQ ID NO: 17];
[0060] TGTTAGTTACCCCTTAGAAAGTTGTTAATAAATCAGCCAAAGATG GAATTTGTTGACTCGTGCTACAACCATGGGCCACTTGAATTCATGGACACCATAC TGTTGAAACAAGAACATGTGTTTCATTTGCTTAATCACATGACCTAGCTAGAGAG CTTTTCCAATACTTTCCAGATGAAATGTAATATACTCCACACATAATTGTTGAGGT ATATATATTAATCTATCACCCACATTTCCTTAATAACTTATACTTTTGAGCTAATT GTTTGGCACATACAAGATACAACTCACAATGCTATCAAAGCTCAGTTGGCT [SEQ ID NO: 18]; and [0061] CAACTAATATGTCTATGAGCATCTTCAAAAGACAAAGACGACAG
ATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTT ATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAAAAAATATGAGCAAGCAC ATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTC AATTTCTAATCATGGCTAATATAATCAAACCCCACCCAGTATATTATTACTTAGAG CATCTCCAATAGTTTTTTTTAAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAAT AATTGGTAAATCAA [SEQ ID NO: 19].
[0062] Suitably, 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: [0063] (g) WYHAWCRKTAYWWRANaδAGRAGYRWSNβcYATGASAGCATR
WGTRTMMCWMATGTRN^YWTTCWTWRAAWATARYWWWYYTWYRRAWARWT RKTATAKYAATAAATSARWCWAWRAKKRAWTWWRTWKWTGAMTMRHKHTWY AAA YATCKRYCAMTYRN^AKWMATRRASWMCMTAMWRKTWW AAAAYRRAMW MAWGTRTTTGA WWNα/RHKTTTTMYWTYMATRAATNagAYA WYWWRWWTAYRS WHAWTCAWRWKMRYAYWYWYMCMTYATGRW [SEQ ID NO: 20]; 01407
[0064] (h) 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: 20 or a complement thereof; and
[0065] (i) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 20 or a complement thereof, under at least medium or high stringency conditions,
[0066] wherein:
[0067] A, C5 G, T, M, R, W, S, Y, K, V5 H5 D5 B and N are as defined above;
[0068] each of ae, α/and ag is an integer from 0-1 ;
[0069] ah is an integer from 0-3; [0070] ac is an integer from 0-8; and
[0071] ad is an integer from 0-9.
[0072] In some embodiments, Na& is the sequence CAT; Nαc is the sequence AATAAAA; Nflrf is the sequence GATAAAGAT; Nαe is G; N0/ is C; or Nαg is C. In other embodiments any one or more of N0^5 Nαc, N0^, Nαe, Nα/or Nβg comprises 0 nucleotides. [0073] Representative examples of sequences according to SEQ ID NO: 20 are suitably selected from the group consisting of:
[0074] TCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGTCCCT AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA AACAAAGATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGGATTC ATGGACACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCTTTTC ATGAATCACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGAA [SEQ IDNO: 21];
[0075] TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCT AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA AACAAAGATGGAATTTGTTGATGACTCGTGCTACAAACATCGGCCACTTGGAGTC ATGGACACCCTACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTC ATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ IDNO: 22];
[0076] TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCT AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA U2009/001407
AACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTC ATGGACACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTC ATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ ID NO: 23]; [0077] ATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGC
ATAAGTATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAA TAAATAGTATAGC AATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATT TAAATATCTATCAATCAATAAATAAAGTACATAAAAGTT AAAAATGAACTAAAGT ATTTGAAAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAA TATATTTACATCATGGT [SEQ ID NO: 24];
[0078] ATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGC ATAAGTATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAA TAAATAGTATAGC AATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATT TAAATATCTATCAATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGT ATTTGAAAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAA TATATTTACATCATGGT [SEQ ID NO: 25]; and
[0079] TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCT AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA AACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTC ATGGACACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTC ATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ ID NO: 26].
[0080] In some embodiments, the promoter sequence further comprises upstream of (g), (h) or (i) a nucleotide sequence (also referred to herein as segment Δ sequence ) selected from the group consisting of:
[0081] G) RRSKWMWAKYTYWRKAMWTRGTAYAKSRRTRMWYRWRTYT WTMARTAWTTAWWTWWRTAARW AWCTAYN0/, YDATWYAWWTARWDRNα;RAG KWSMYWSRWGWYAMMRAKGWRTTWATAKGWMTTYGWMAAYSTYWASATYR RTMARYWTAMTMTYTYAN^GGYWRRRKCMAAAKMAN^SATYATGGTYWRMW WWGKWKY YWKATWSTAKWMWWWTMCRKATWWT AWTICMAW ATMCWWMW TWMRWTWMTYTCAKATRYRRATMYAAATYMAGN0ZTARTKWRAAWTSAAWTM WGMRWTAWSMATTTTAKWTWCATACYYGRWWTGGRTGKKAGMAAKWWMKG AWTMRYWTWWWTYRRWSRKYTTSAWRWMCRKW [SEQ ID NO: 27];
[0082] (k) 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: 27 or a complement thereof; and
[0083] (1) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 27 or a complement thereof, under at least medium or high stringency conditions,
[0084] wherein:
[0085] A, C, G, T, M, R, W, S5 Y, K, V, H, D, B and N are as defined above; [0086] each of ah and al is an integer from 0-1 ;
[0087] aj is an integer from 0-2;
[0088] ai is an integer from 0-5; and
[0089] ak is an integer from 0-8.
[0090] In some embodiments, N0/, is G; Nα, is a sequence selected from AATAA, AATA or TAGGT; N0/ is the sequence AA; N0/t is a sequence selected from TATGTTTA or AATTAAAC; or N0/ is C. In some embodiments, any one or more of N0/,, Nα;, N0/, N0/t or N0/ comprises 0 bases.
[0091] Representative sequences according to SEQ ID NO: 27 may be selected from the group consisting of: [0092] AACTAATATCTCAGTAAATAGTATAGCAATAAATGAGTCTATAA
GTATTTAAATATATAAATATCTACTGATATAAATAAATAAATAAAAGTACATACA AGTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACATCAATAAATATACTA TTTTAGGCTAAATCAAAATAATATGTTTACATCATGGTTTACATAGTTGCTTTATT CTATACAAATACGGATAATATTGAATATCCATCGTACGTTTATTTCATATACGGAT CCAAATTCAGCTAATGAAAAATGAATTCAGAAATATCTATTTTATTTTCATACCTG AAATGGATGTGAGAAATATATGAATAATATAATTCAGCCAGCTTCATATCCATT [SEQ ID NO: 28];
[0093] AACGAATATATCAATAAATAGTATAGCAATAAATGAGTCTATAA
GTAATTAAATATATAAATATCTATCAATATAAATAAATAAAGTACATACAAGTTA CAAATGAATTAATAAGGTATTTGAAAACGTTTAAATCAATAAATATACTATTTTA GGCTAAATCAAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAATAC GGATAATATTGAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCT AATGAAAAATGAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTG AGAAATATATGAATAATATAATTCAGACAGCTTCATATCCATT [SEQ ID NO: 29]; [0094] AACGAATATCTCAATAAATAGTATAGCAATAAATGAGTCTATAA
GTAATT AAATATATAAATATCTATCGATATAAATAAATAAATAAAGTACATACAA GTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACATTAATAAATATACTAT TTTAGGCTAAATCAAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAA TACGGATAATATTGAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCA GCTAATGAAAAATGAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGAT GTGAGAAATATATGAATAATATAATTCAGACAGCTTCATATCCATT [SEQ ID NO: 30];
[0095] GGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAA TATTTATTTTAGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGAGGTGCCTGGT GACAACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCT CTCAAAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAG ATAGTAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCA AATATAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATAC TCGGTTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACG GA [SEQ ID NO: 31]; and
[0096] GGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAA TATTTATTTTAGTAAGAAACTATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGT GACAACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCT CTCAAAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAG ATAGTAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCA AATATAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATAC TCGGTTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACG GA [SEQ ID NO: 32].
[0097] In some embodiments, the promoter sequence further comprises upstream of Q), (k) or (1) a nucleotide sequence (also referred to herein as segment Θ sequence ) selected from the group consisting of: [0098] (m) YWYTYWRWKTGKSYKWYWDMAAGWRWSYATWNemRMKWT RWRRTBTWGAAGMWTBKAATWRYSMCATRTATAN^TAAAGAAWARN^AN^TK ACAYTAYWWGWAYAWYAYATSTARRYAMWKGTCATWYATRRAMMMTWWYM YAWWKAKWNα? [SEQ ID NO: 33]; [0099] (n) 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: 33 or a complement thereof; and
[0100] (o) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 33 or a complement thereof, under at least medium or high stringency conditions, [0101] wherein:
[0102] A, C, G5 T, M, R, W, S, Y5 K5 V, H, D5 B and N are as defined above;
[0103] each of am and ao is an integer from 0-1 ;
[0104] ap is an integer from 0-2;
[0105] an is an integer from 0-11; and [0106] aq is an integer from 0-26.
[0107] In some embodiments, Nom is W; Nα/7 is a sequence selected from AAACGCAAAAT [SEQ ID NO: 34], AAACGCAAAAC [SEQ ID NO: 35] or TAAAGAGTATA [SEQ ID NO: 36]; N00 is T; Nφ is a sequence selected from TA or AA; or Kq is a sequence selected from GCAATAAATGAGTCTATAATATGCAC [SEQ ID NO: 37] or GGGGGTGGAACCTTCCTTCAC [SEQ ID NO: 38] . In other embodiments, any one or more of NflW, Nα,!5 Nao, Nαp or No? comprises 0 nucleotides.
[0108] Representative examples of sequences according to SEQ ID NO: 33 include:
[0109] TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTG AAGAATGGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACAC TATATGTATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAG CAATAAATGAGTCTATAATATGCAC [SEQ ID NO: 39];
[0110] TATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTG AAGAATGGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTA TAAGTATAATACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGA [SEQ ID NO: 40];
[0111] TATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTT GAAGAATTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACT ATAAGTATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGA [SEQ ID NO: 41];
[0112] CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATGTA GAAGCTTCTAATTGCGCCATGTATATAAAGAGTATATAAAGAAAAGTAAATTACA TTACTTGAATATCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTG GGGGTGGAACCTTCCTTCAC [SEQ ID NO: 42]; and
[0113] CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTA GAAGCTTCTAATTGCGCCATGTATATAAAGAAAAGTAAATTACATTACTTGAACA TCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAAC CTTCCTTCAC [SEQ ID NO: 43]. [0114] In some embodiments, an intervening sequence (also referred to herein as
"segment Ψ 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). Generally, the intervening sequence comprises from about 150 to about 250 nucleotides, typically from about 180 to about 220 nucleotides and suitably from about 190 to about 210 nucleotides. In illustrative examples of this type, the intervening sequence is selected from the group consisting of:
[0115] TCTATACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTT TTTTGGGTCCGGCCATCCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGC CCTTCTCTTTATATAATTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGT GAATAGGAATATTAATCCAAATAGAAAAAATATAATAATATGCA [SEQ ID NO: 44], 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: 44 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 44 or a complement thereof, under at least medium or high stringency conditions;
[0116] TTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATA TCCATCCCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTC AGATATATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGG TGTTTATAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTT [SEQ ID NO: 45] , 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: 45 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 45 or a complement thereof, under at least medium or high stringency conditions; and
[0117] TTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATA TCCATCCCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTC AGATATATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGG TGTTTATAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTT [SEQ ID NO: 46] , 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: 46 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 46 or a complement thereof, under at least medium or high stringency conditions.
[0118] In specific embodiments, the promoter sequence has a structure represented by formula (I):
[0119] 5'-Θ-Δ-Z-Ψ-Φ-Ω-3' (I) [0120] wherein:
[0121] Θ may be present or absent and is selected from any one of (m), (n) or (o);
[0122] Δ may be present or absent and is selected from any one of Q), (k) or (1);
[0123] Z is selected from any one of (g), (h) or (i);
[0124] Ψ is an optional spacer or intervening sequence; [0125] Φ is selected from any one of (d), (e) or (f); and
[0126] Ω is selected from any one of (a), (b) or (c).
[0127] In illustrative examples of this type, the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
[0128] TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTG AAGAATGGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACAC TATATGTATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAG CAATAAATGAGTCTATAATATGCACAACTAATATCTCAGTAAATAGTATAGCAAT AAATGAGTCTATAAGTATTTAAATATATAAATATCTACTGATATAAATAAATAAA TAAAAGTACATACAAGTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACAT CAATAAATATACTATTTTAGGCTAAATCAAAATAATATGTTTACATCATGGTTTAC ATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATATCCATCGTACGTTTA TTTCATATACGGATCCAAATTCAGCTAATGAAAAATGAATTCAGAAATATCTATT TTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAATATAATTCAGCCA GCTTCATATCCATTTCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGT CCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAA ATCAAACAAAGATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGG ATTCATGGACACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCT TTTCATGAATCACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGA ATCTATACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTTTTTTGGGTCC GGCCATCCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGCCCTTCTCTTT ATATAATTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGTGAATAGGAA TATTAATCCAAATAGAAAAAATATAATAATATGCACAACTAATATCTCTATGAGC ATCTTCGAAAGACAAAGACGGCAGATCTCAAAATTGTTGAGGTCATAATAAGCAT TGCACTCAAACAGGCATGTTGCTTATTAAAGAATAGCGAAGCAATGAAGAATAA ATCCAAGAAAATATGAGCAAGCACGTCAATCGAGCACTTGACCAACTGGGTAGA CATGTTCAATCACAAGAGGTGCTCAATTTCTAATCATGGTCAATATAATCAAACC CCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTTCAAAAAAAAAATCA TCGCAAAAAAAGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTACAAGTCG CTAAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTTGACTTTTTTCCAACAGTTTA TAAAATCACGCTCCTAAAGTATAGAAAATAATTTTGCATTAGGAATCTTAAACTA TTTTCAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTGTATATTTGCATTT TGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTTTCGGATTT AGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAACGTTAC CAACCCAACATATATTTCC [SEQ ID NO: 47], 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: 47 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 47 or a complement thereof, under at least medium or high stringency conditions; [0129] TATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTG AAGAATGGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTA TAAGTATAATACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGAAAC GAATATATCAATAAATAGTATAGC AATAAATGAGTCTATAAGTAATTAAATATAT AAATATCTATCAATATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATA AGGTATTTGAAAACGTTTAAATCAATAAATATACTATTTTAGGCTAAATCAAAAT AACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATA TCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATT CAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATA ATATAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATG AGAGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTT GTTATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGTGCTACAAAC ATCGGCCACTTGGAGTCATGGACACCCTACTGTTATAAAACGGAAACATGTGTTT GATTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCAC TCACACCTTATGGACAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGA CAGATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCG CTTATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAAAAAAAATATGAGCA AGCACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGG AGCTCAATTTATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACT TAGAGCATCTCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAA AAAAATAATTGGTAAATCAATGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCA TAATTGTTGGGGCTTTGACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAG TATAGAAAACATGCATTAGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTT TATACTTTTTTTCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAAT CATGGTTAATCAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCAT ACCCAACTCATCGCCCATATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 48], 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: 48 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 48 or a complement thereof, under at least medium or high stringency conditions;
[0130] TATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTT GAAGAATTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACT
ATAAGTATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGAAA CGAATATCTCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATTAAATATA TAAATATCTATCGATATAAATAAATAAATAAAGTACATACAAGTTACAAATGAAT TAATAAGGTATTTGAAAACGTTTACATTAATAAATATACTATTTTAGGCTAAATC AAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATT GAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAAT GAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATAT GAATAATATAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTG TCCATGAGAGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGG AAAGTTGTTATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGCGCT ACAAACATCGGCCACTTGGATTCATGGACACCATACTGTTATAAAACGGAAACAT GTGTTTGATTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGT GCGCACTCACACCTTATGGACAACTAATATCTCTATGAGCATCTTTAAAAGACAA AGACAACAGATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAG CATGTCGCTTATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATAT GAGCAAGCACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACA AGAGGAGCTCATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAG TTTAAAAAAAATCATCGCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAATG AGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTGGGGGTTTTTGACT TTTTTCCAACAGTTTATAAAATCATGCTCCTAAAGTATAGAAAACAATTTTGCATT AGGAATCCTAAACTATTTCTAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTC TTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCC AGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCAT ATAAAACGGCACCAACCCAACATATATTTCC [SEQ ID NO: 49], 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: 49 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 49 or a complement thereof, under at least medium or high stringency conditions;
[0131] CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATGTA GAAGCTTCTAATTGCGCCATGTATATAAAGAGTATATAAAGAAAAGTAAATTACA TTACTTGAATATCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTG GGGGTGGAACCTTCCTTCACGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCA TATTTTTCAATATTTATTTTAGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGA GGTGCCTGGTGACAACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTC
AGCTTAATCTCTCAAAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATA TGGATTCAGATAGTAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTC TCAGATGCAAATATAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTA GATACATACTCGGTTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTT GAAGAACGGAATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGC ATAAGTATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAA TAAATAGTATAGC AATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATT TAAATATCTATCAATCAATAAATAAAGTACATAAAAGTT AAAAATGAACTAAAGT ATTTGAAAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAA TATATTTACATCATGGTTTCATTAGGTCCTTTATTCTATACAAATACGAATAATAT TGAATATCCATCCCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAAT GAATTCAGATATATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAAT AGGGGTGTTTATAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTA GTTACCCCTTAGAAAGTTGTTAATAAATCAGCCAAAGATGGAATTTGTTGACTCG TGCTACAACCATGGGCCACTTGAATTCATGGACACCATACTGTTGAAACAAGAAC ATGTGTTTCATTTGCTTAATCACATGACCTAGCTAGAGAGCTTTTCCAATACTTTC CAGATGAAATGTAATATACTCCACACATAATTGTTGAAGTATATATATATTAATC TATCACCCACATTTCCTTAATAACTTATACTTTTGAGCTAATTGTTTGGCACATAC AAGATACAACTCACAATGCTATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGC TAGGGAAATTCAAATAAAAAGATGGCACAATATTCTTACTCGTATTCCAAGGGCT AGGCAAACTAAAATACGAGGTATATGTCAAAACTCAAAATGTGCGGTTTTGGGTG AATTATTGCTCAACACATTCAGCTCAACTCAATTCCCTAGGATATTTAACCCTCTC TTATAAGTACATTATTTATGTGCCAACCCAATTAGAAGTGCTAATCTTGAATTTAC TAACAAAAATGGCAAATTGCATACCCAACTCACCCATATAAAAAGGCACCAACC CAACATTATTTTC [SEQ ID NO: 50], 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: 50 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 50 or a complement thereof, under at least medium or high stringency conditions;
[0132] CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTA GAAGCTTCTAATTGCGCCATGTATATAAAGAAAAGTAAATTACATTACTTGAACA TCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAAC CTTCCTTCACGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAAT ATTTATTTTAGTAAGAAACTATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGTG
ACAACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCT 01407
CAAAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGAT AGTAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAA TATAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTC GGTTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGA ATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGTATAA CACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATAGTAT AGC AATAAATGAGTCTATAAGTAATT AAATATTTGAATAAATATTTAAATATCTA TCAATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGAAAA AGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTTACA TCATGGTTTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCA TCCCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGAT ATATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTT ATAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTA GAAAGTTGTTAATAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCA TGGGCCACTTGAATTCATGGACACCATACTGTTGAAACAAGAACATGTGTTTCAT TTGCTTAATCACATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATG TAATATACTCCACACATAATTGTTGAGGTATATATATTAATCTATCACCCACATTT CCTTAATAACTTATACTTTTGAGCTAATTGTTTGGCACATACAAGATACAACTCAC AATGCTATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAAA TAAAAAGATGGCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAAT ACGAGGTATATGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAAC ACATTCAGCTCAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTA TTTATGTGCCAACCCAATTGGAAGTGCTAATCTTGAATTTACTAACAAAAATGTC AAATTGCATACCCAACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 51], 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: 51 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 51 or a complement thereof, under at least medium or high stringency conditions; and [0133] TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCT
AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA AACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTC ATGGACACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTC
ATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGACAA CTAATATGTCTATGAGCATCTTCAAAAGACAAAGACGACAGATCTCAAGATTGTT GAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAAGAATAGCG AAGCAATCAAGAATAAATTCAAAAAAATATGAGCAAGCACATCAATCGAGGACT TGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTTCTAATCATGG CTAATATAATCAAACCCCACCCAGTATATTATTACTTAGAGCATCTCCAATAGTTT TTTTTAAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAATAATTGGTAAATCAAT GAGATTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTGACTTT TTTCCCCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAATTTTGCATTAGG AATCTTAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTCTTTCTTGT ATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCA GTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCTCATA TAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 52], 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: 52 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 52 or a complement thereof, under at least medium or high stringency conditions.
[0134] In other illustrative examples, the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
[0135] TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTG AAGAATGGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACAC TATATGTATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAG CAATAAATGAGTCTATAATATGCACAACTAATATCTCAGTAAATAGTATAGCAAT AAATGAGTCTATAAGTATTTAAATATATAAATATCTACTGATATAAATAAATAAA TAAAAGTACATACAAGTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACAT CAATAAATATACTATTTTAGGCTAAATCAAAATAATATGTTTACATCATGGTTTAC ATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATATCCATCGTACGTTTA TTTCATATACGGATCCAAATTCAGCTAATGAAAAATGAATTCAGAAATATCTATT TTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAATATAATTCAGCCA GCTTCATATCCATTTCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGT CCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAA ATCAAACAAAGATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGG ATTCATGGACACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCT TTTCATGAATCACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGA ATCTATACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTTTTTTGGGTCC GGCCATCCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGCCCTTCTCTTT ATATAATTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGTGAATAGGAA TATTAATCCAAATAGAAAAAATATAATAATATGCACAACTAATATCTCTATGAGC ATCTTCGAAAGACAAAGACGGCAGATCTCAAAATTGTTGAGGTCATAATAAGCAT TGCACTCAAACAGGCATGTTGCTTATTAAAGAATAGCGAAGCAATGAAGAATAA ATCCAAGAAAATATGAGCAAGCACGTCAATCGAGCACTTGACCAACTGGGTAGA CATGTTCAATCACAAGAGGTGCTCAATTTCTAATCATGGTCAATATAATCAAACC CCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTTCAAAAAAAAAATCA TCGCAAAAAAAGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTACAAGTCG CTAAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTTGACTTTTTTCCAACAGTTTA TAAAATCACGCTCCTAAAGTATAGAAAATAATTTTGCATTAGGAATCTTAAACTA TTTTCAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTGTATATTTGCATTT TGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTTTCGGATTT AGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAACGTTAC CAACCCAACATATATTTCCACCTAATGCCGTCGCCCAACTAATTGCCATAACACT AAGACGAGCGCCAACCTTCGGTGCTCTGCTTGCCTCGCTGTTGCTGTTGCCCATGG [SEQ ID NO: 53], 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: 53 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 53 or a complement thereof, under at least medium or high stringency conditions;
[0136] TCAAATAAATATAAATCAGGTAGTTCAAATTCAAATATACATTTA GATATGTATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTGAAGA ATGGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAG TATAATACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGAAACGAAT ATATCAATAAATAGTATAGC AATAAATGAGTCTATAAGTAATTAAATATATAAAT ATCTATCAATATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATAAGGT ATTTGAAAACGTTTAAATCAATAAATATACTATTTTAGGCTAAATCAAAATAACA TCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATATCCT TCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATTCAG AGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAATA TAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATGAG
AGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGT TATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGTGCTACAAACAT CGGCCACTTGGAGTCATGGACACCCTACTGTTATAAAACGGAAACATGTGTTTGA TTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCACTC ACACCTTATGGACAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGACA GATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCT TATTAAAGAATAGCGAAGC AATCAAGAATAAATTCAAAAAAAAATATGAGC AAG CACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAG CTCAATTTATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACTTA GAGCATCTCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAAAA AAATAATTGGTAAATCAATGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCATA ATTGTTGGGGCTTTGACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTA TAGAAAACATGCATTAGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTTTA TACTTTTTTTCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCA TGGTTAATCAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATAC CCAACTCATCGCCCATATAAAACGGCACCAACCCAACATATTTCCACCTAATGCC GTCGCCCAACTAATTGCCATAACACTAAGACGAGCGCCAACCTTGCGGTGCTCTG CTTGCCTCGCTGTTGCTGTTGCCCATGG [SEQ ID NO: 54], 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: 54 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 54 or a complement thereof, under at least medium or high stringency conditions;
[0137] TTGGATCAAATAAATATAAATCAAGTAGTTCAAATTCAAATATAC ATTTAGATATGTATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTT GAAGAATTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACT ATAAGTATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGAAA CGAATATCTCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATTAAATATA TAAATATCTATCGATATAAATAAATAAATAAAGTACATACAAGTTACAAATGAAT TAATAAGGTATTTGAAAACGTTTACATTAATAAATATACTATTTTAGGCTAAATC AAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATT GAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAAT GAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATAT GAATAATATAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTG TCCATGAGAGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGG
AAAGTTGTTATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGCGCT ACAAACATCGGCCACTTGGATTCATGGACACCATACTGTTATAAAACGGAAACAT GTGTTTGATTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGT GCGCACTCACACCTTATGGACAACTAATATCTCTATGAGCATCTTTAAAAGACAA AGACAACAGATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAG CATGTCGCTTATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATAT GAGCAAGCACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACA AGAGGAGCTCATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAG TTTAAAAAAAATCATCGCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAATG AGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTGGGGGTTTTTGACT TTTTTCCAACAGTTTATAAAATCATGCTCCTAAAGTATAGAAAACAATTTTGCATT AGGAATCCTAAACTATTTCTAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTC TTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCC AGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCAT ATAAAACGGCACCAACCCAACATATATTTCCACCTAATGCCGTCGCCCGACTAAT TGCCATAACTCTTAAAGACGAGCGCCAACCTTGCGGTGCTCTGCTTGCCTCGCTGT TGCTGTTGCCCATGG [SEQ ID NO: 55], 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: 55 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 55 or a complement thereof, under at least medium or high stringency conditions;
[0138] CTGCAGGCGGCCGCGAATTCACTAGTGATTACTATAGGGCACGC GTGGTCGACGGCCCGGGCTGGTATCGGGGCGTAGATTCCTCTCAACATATCAGCG TTGGAATATGTTTGCCTTTTGAGTGGCCAGCACCAAGTACCATAGCCCCAGCTAA CTAGCTCGAGTGGCCCTCGAGAATATCAGGGGTAGTTGCTATATACCAACATCAT GGTGAATTAACCTCAATGTCTTTCCTCACCAAAATATTTAAAGGTGTTTACAGGTG TCCCTTTCCCTGCTTTCATGTTGTAATCTTCAAGCTCTAGCAGCCAGGCGAACAAC CGAAGGAATTAAGAAGACGACTAGAAAGGCCTTGGTTGTATTTTTATTTTTGGAT GTTATTTTGTGTCAAGTTTCTCTCTTTCGTACTGGCTATTTACATATGAGACACTCT TTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATGTAGAAGCTTCTAATTG CGCCATGTATATAAAGAGTATATAAAGAAAAGTAAATTACATTACTTGAATATCA TATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTT CCTTCACGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATT TATTTTAGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGAGGTGCCTGGTGAC
AACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCA AAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAG TAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATA TAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGG TTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGAAT AATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGTATAACA CATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATAGTATAG CAATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATATCTATC AATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGAAAAAG TTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTTACATC ATGGTTTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATC CCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATA TATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTA TAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTAG AAAGTTGTTAATAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCAT GGGCCACTTGAATTCATGGACACCATACTGTTGAAACAAGAACATGTGTTTCATT TGCTTAATCACATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATGT AATATACTCCACACATAATTGTTGAAGTATATATATATTAATCTATCACCCACATT TCCTTAATAACTTATACTTTTGAGCTAATTGTTTGGCACATACAAGATACAACTCA CAATGCTATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAA ATAAAAAGATGGCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAA TACGAGGTATATGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAA CACATTCAGCTCAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATT ATTTATGTGCCAACCCAATTAGAAGTGCTAATCTTGAATTTACTAACAAAAATGG CAAATTGCATACCCAACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTC ACCTAGTGTCGTCGCCGAACTCATTGCCATAACACTAAGACGAGCGCCAACCTTG TGGTGCTCTTGCCTCGCTGTTGCTGTCGCCCATGG [SEQ ID NO: 56], 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: 56 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 56 or a complement thereof, under at least medium or high stringency conditions;
[0139] CTGCAGGCGGCCGCGAATTCACTAGTGATTACTATAGGGCACGC GTGGTCGACGGCCCGGGCTGGTATCGGGGCGTAGATTCCTCTCAACATATCAGCG TTGGAATATGTTTGCCTATTGAGTGGCCAGCACCAAGTACCATAGCCCCAGCTAA
CTAGCTCGAGTGGCCCTCGAGAATATCAGAGGTAGTTGCTATATACCAACATCAT GGTGAATTAACCTCAATGTCTTTCCTCACCAAAATATTTAAAGGTGTTTACAGGTG TCCCTTTCCCTGCTTTCATGGTGTAATCTTCAAGCTCTAGCAGGCAGGCGAACAAC CGAAGGAATTAAGAAGACGACTAGAAAGGCCTTGGTTGTATTTTTATTTTTGGAT GTTATTTTGTGTCAAGTTTCTCTCTTTCGTACTGGCTATTTAGATATGAGACACTCT TTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTAGAAGCTTCTAATTG CGCCATGTATATAAAGAAAAGTAAATTACATTACTTGAACATCATATCTAAATAC ATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTTCCTTCACGGGT TCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTATTTTAGTAA GAAACTATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGTGACAACGAGGTGTT TATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAGGTAGGGG CCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAGTATTTTCC ATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAAATCAAGT AGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTGGGTGGT AGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGAATAATCATTATA TAACATAGAAGTAAGGAATAAAATATGACAGCATAAGTATAACACATGTAGATA AAGATCATTCATAGAAAATAACATATTTACAAATAAATAGTATAGCAATAAATGA GTCTATAAGTAATTAAATATTTGAATAAATATTTAAATATCTATCAATCAATAAAT AAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGAAAAAGTTTTACATCAAT AAATATATCTAGTTTACGCTAAATCAAAATAATATATTTACATCATGGTTTCATTA GGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATCCCATATTGATTT CATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATATATCCAGCTTCA GATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTATAGGGGTGAGT ATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTAGAAAGTTGTTAA TAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCATGGGCCACTTGA ATTCATGGACACCATACTGTTGAAACAAGAACATGTGTTTCATTTGCTTAATCAC ATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATGTAATATACTCC ACACATAATTGTTGAGGTATATATATTAATCTATCACCCACATTTCCTTAATAACT TATACTTTTGAGCTAATTGTTTGGCACATACAAGATACAACTCACAATGCTATCA AAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATG GCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATA TGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCT CAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCC AACCCAATTGGAAGTGCTAATCTTGAATTTACTAACAAAAATGTCAAATTGCATA CCCAACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTCGCCTAGTGTCG TCGCCGAACTCATTGCCATAACACTAAGACGAGCGCCAACCTTGTGGTGCTCTTG CCTCGCTGTTGCTGTCGCCCATGG [SEQ ID NO: 57], 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: 57 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 57 or a complement thereof, under at least medium or high stringency conditions; and
[0140] TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCT AATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCA AACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTC ATGGACACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTC ATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGACAA CTAATATGTCTATGAGCATCTTCAAAAGACAAAGACGACAGATCTCAAGATTGTT GAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAAGAATAGCG AAGCAATCAAGAATAAATTCAAAAAAATATGAGCAAGCACATCAATCGAGGACT TGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTTCTAATCATGG CTAATATAATCAAACCCCACCCAGTATATTATTACTTAGAGCATCTCCAATAGTTT TTTTTAAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAATAATTGGTAAATCAAT GAGATTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTGACTTT TTTCCCCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAATTTTGCATTAGG AATCTTAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTCTTTCTTGT ATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCA GTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCTCATA TAAAACGGCACCAACCCAACATATTTCCACCTAATGCCGTCGCCCAACTAATTGC CATAACACTAAGTCGAGCGCCAACCTTGCGGTGCTCTGCTTGCCTCGCTGTTGCTG TTGCCCATGG [SEQ ID NO: 58], 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: 58 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 58 or a complement thereof, under at least medium or high stringency conditions. [0141] Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 47, or that hybridize to the sequence set forth in SEQ ID NO: 47 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 48, 49, 52, 53, 54, 55 and 58. Representative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 48, or that hybridize to the sequence set forth in SEQ ID NO: 48 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 49, 52, 53, 54, 55 and 58. Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 49, or that hybridize to the sequence set forth in SEQ ID NO: 49 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 48, 52, 53, 54, 55 and 58. Illustrative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 52, or that hybridize to the sequence set forth in SEQ ID NO: 52 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 47, 48, 49, 53, 54, 55 and 58.
[0142] Non-limiting examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 50, or that hybridize to the sequence set forth in SEQ ID NO: 50 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 51, 56 and 57. Representative examples of nucleotide sequences that share at least 80% sequence identity with the sequence set forth in SEQ ID NO: 51, or that hybridize to the sequence set forth in SEQ ID NO: 51 or a complement thereof, under at least medium or high stringency conditions are suitably selected from any one of SEQ ID NO: 50, 56 and 57.
[0143] In a related aspect, 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: [0144] (i) a nucleotide sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof;
[0145] (ii) 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 any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof; and 9 001407
[0146] (iii) a nucleotide sequence that hybridizes to the sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof, under at least medium or high stringency conditions.
[0147] In accordance with the present invention, it is predicted that promoter elements positioned upstream or 5' to the TATA box in segment Ω of the exemplified promoter sequences set forth in SEQ ID NO: 47-58 (e.g., promoter elements in any one or more of segments Φ, Ψ, Z, Δ, Θ or segment Λ that corresponds to the portion of segment Ω, which is upstream of the TATA box) 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. Accordingly, in another aspect, 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:
[0148] (1) a nucleotide sequence corresponding to at least one segment selected from segments Λ, Φ, Ψ, Z, Δ and Θ of SEQ ID NO: 47-58, or
[0149] (2) 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 of the at least one segment or a complement thereof, or
[0150] (3) a nucleotide sequence that hybridizes to the sequence s of the at least one segment or a complement thereof, under at least medium or high stringency conditions .
[0151] In some embodiments, the nucleotide sequence represented by (1) comprises segment Λ. In other embodiments, the nucleotide sequence represented by (1) comprises segments Λ and Φ, and optionally segment Ψ. In still other embodiments, the nucleotide sequence represented by (1) comprises segments Λ, Φ and Z, and optionally segment Ψ. In yet other embodiments, the nucleotide sequence represented by (1) comprises segments Λ, Φ, Z and Δ, and optionally segment Ψ. In still other embodiments, the nucleotide sequence represented by (1) comprises segments Λ, Φ, Z, Δ and Θ, and optionally segment Ψ. In other embodiments, the nucleotide sequence represented by (1) comprises segment Φ, and optionally segment Ψ. In still other embodiments, the nucleotide sequence represented by (1) comprises segments Φ and Z, and optionally segment Ψ. In yet other embodiments, the nucleotide sequence represented by (1) comprises segments Φ, Z and Δ, and optionally segment Ψ. In still other embodiments, the nucleotide sequence represented by (1) comprises segments Φ, Z, Δ and Θ, and optionally segments Ψ.
[0152] Generally, 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. Accordingly, in another aspect of the present invention, there is provided 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.
[0153] In some embodiments, 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. [0154] The heterologous nucleic acid sequence is heterologous with respect to the plant cell in which it is or will be introduced. In some embodiments, the heterologous nucleic acid sequence encodes a structural or regulatory protein, or alternatively, a transcript capable of modulating expression of a corresponding target gene. In illustrative examples of this type, the transcript comprises an antisense RNA or a ribozyme or other transcribed region aimed at downregulation of expression of the corresponding target gene. For example, 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.
[0155] Depending upon the heterologous nucleic acid sequence selected, 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. In specific embodiments, the promoter sequence is capable of regulating transcription of the heterologous nucleic acid preferentially in stem tissue of a plant. In illustrative examples of this type, 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. [0156] Plants falling within the scope of the present invention encompass any taxonomic grouping, including angiosperms, gymnosperms, monocotyledons and dicotyledons. In some embodiments, the plant is selected from monocotyledonous plants such as cereals, sugarcane, bananas and pineapples. In specific embodiments, the plant is a sucrose- accumulating plant such as sugarcane, sugarbeet or sweet sorghum.
[0157] In yet another aspect, 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.
[0158] In still another aspect, 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.
[0159] In a further aspect, 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.
[0160] In still a further aspect, 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.
[0161] In some embodiments, 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. In some embodiments, 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.
[0162] According to another aspect of the present invention, transformed plant cells are provided, containing a chimeric construct as broadly described above.
[0163] In still another aspect, 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.
[0164] In some embodiments, the expression of the chimeric construct renders the differentiated transgenic plant identifiable over the corresponding non-transgenic plant. [0165] In still a further aspect, the invention provides differentiated transgenic plants comprising plant cells containing a chimeric construct as broadly described above. In some embodiments, 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. Accordingly, 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.
[0166] The 1248-nt sequence set forth in SEQ ID NO: 59 is a transcribable nucleic acid sequence comprising an ORF, which codes for a 415-amino acid sequence, as set forth in SEQ ID NO: 60. This ORF is transcribed at high levels in stem tissues, including mature stem tissues, of sugarcane (Saccharum sp.). Accordingly, nucleotide sequences that correspond or are complementary to at least a portion of the sequence set forth in SEQ ID NO: 59 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. Thus, in another aspect, 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: 59. Advantageously, 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 2500 nts in length and usually greater than 250 nts in length. Suitably, 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: 59 under at least medium stringency conditions, and especially under high stringency conditions. BRIEF DESCRIPTION OF THE DRAWINGS
[0167] Figure 1 is a photographic representation of various Northern analyses showing expression of ScCIPKl 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, hybridized to a probe comprising nt 960-1248 of SEQ ID NO: 59. (b) Northern analysis using total RNA from internode 20 (IN20), mature roots (MR) and root tips (RT) from glasshouse-grown Ql 17 plants, hybridized to a probe comprising nt 960-1248 of SEQ ID NO: 59. (c) Northern analysis using total RNA from dissected rind (R), storage parenchyma (P) and vascular strands (V) from internode 8 of field-grown Ql 17 plants, hybridized to a probe comprising nt 960-1248 of SEQ ID NO: 59.
[0168] Figure 2 is a diagrammatic representation of a sequence alignment of the promoter sequences corresponding to promoter alleles A, B, 5, 49, 51 and 77. The nucleotide sequences delineating segments Θ, Δ, Z, Ψ, Φ, Λ and Ω are shown.
TABLE A
BRIEF DESCRIPTION OF THE SEQUENCES
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0169] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0170] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
[0171] By "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.
[0172] The term "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. [0173] The term "biologically active fragment", as applied to promoter sequences, refers to a fragment that has at least about 0.1, 0.5, I5 2, 5, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30% of the activity of a reference promoter sequence. It will also be understood that the phrase "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. In specific embodiments, "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. Included within the scope of the present invention are 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 full-length promoter sequence.
[0174] The terms "chimeric construct," "chimeric nucleic acid" or "chimeric DNA" and the like are used herein to refer to a gene or nucleic acid sequence, either single- or double-stranded, comprising at least two nucleic acid sequences from species which do not combine nucleic acids such as DNA under natural conditions, or which nucleic acid sequences are positioned or linked in a manner which does not normally occur in the native genome of the untransformed plant.
[0175] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
[0176] By "corresponds to" or "corresponding to" is meant 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.
[0177] The terms "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).
[0178] As used herein, the term "heterologous" refers to a nucleic acid sequence linked to a nucleic acid sequence to which it is not naturally linked.
[0179] "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, In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this regard, 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. [0180] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, 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. Suitably, an
"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. For example, in various embodiments, 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.
[0181] As used herein, "maturation" refers to the process of accumulating stored carbohydrate in sink tissue. Accordingly, the term "mature tissue" refers to tissue with more than 80%, 85%, 90% or 95% of the maximum stored carbohydrate concentration achieved during plant development. For example, in sugarcane, maturation increases initially with internode age, so that if internodes are counted from the internode attached to the youngest emerged leaf as number 1 , maturation typically occurs up to about internode number 11 and older internodes are mature, as reviewed by Moore, 1995, Aust. J. Plant Physiol. 22, 661.
[0182] By "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 (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by 'screening' (e.g., β-glucuronidase, neomycin phosphotransferase II, luciferase, or other enzyme activity not present in untransformed cells).
[0183] As used herein, 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. [0184] By "obtained from" is meant that a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source. For instance, the nucleic acid extract may be obtained from tissue isolated directly from a host plant. [0185] The term "oligonucleotide" as used herein 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). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring, it will be understood that 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. The exact size of the molecule can vary depending on the particular application. 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 valiant nucleic acid sequence. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.
[0186] The term "operably connected" or "operably linked" as used herein 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. In the construction of heterologous promoter/ transcribable nucleic acid combinations, it is generally desirable to position the promoter sequence at a distance from the transcription start site that is approximately the same as the distance between the promoter sequence and the gene it controls in its natural setting; /. e. , the gene from which the promoter sequence was derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, 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.
[0187] As used herein, "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.
[0188] The term "plant cell" as used herein 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.
[0189] By "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. [0190] The term "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. [0191] The terms "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. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.
[0192] "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.
[0193] By "primer" is meant 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. For example, depending on the complexity of the target sequence, 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' end of the template sequence. In certain embodiments, 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. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. Desirably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, 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. Alternatively, 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.
[0194] "Probe" refers to a molecule that binds to a specific sequence or subsequence 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.
[0195] The term "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. As used herein, 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.
[0196] "Constitutive promoter" refers to a promoter that directs transcription in many or all tissues of a plant.
[0197] By "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.
[0198] 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. [0199] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". These relationships are commonly analysed by use of 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.
[0200] As used herein, the term "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. It is to be understood that 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. Examples of 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 (31) to the site of transcription initiation, or within transcribed regions, or both. In the context of this disclosure, 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.
[0201] A "reference sequence" is at least 6 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because 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.uk/Tools/emboss/align/index.htmL and GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage sequence identity over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15. [0202] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a comparison window. Thus, 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. The percentage of sequence identity can be determined using the EMBOSS programs NEEDLE or WATER or other algorithms such as those described above. In specific embodiments, "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, California, USA) using standard defaults as used in the reference manual accompanying the software, which is incorporated herein by reference in its entirety. In relation to polypeptide sequences, "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. [0203] "Stringency" as used herein, 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. [0204] "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. Generally, in order to maximize the hybridization rate, non- stringent hybridization conditions are selected; about 20 to 25° C lower than the thermal melting point (Tm). The Tm 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. Generally, in order to require at least about 85% nucleotide complementarity of hybridized sequences, highly stringent washing conditions are selected to be about 5 to 15° C lower than the Tm. In order to require at least about 70% nucleotide complementarity of hybridized sequences, moderately stringent washing conditions are selected to be about 15 to 30° C lower than the Tm. Highly permissive (low stringency) washing conditions may be as low as 50° C below the Tm, allowing a high level of mis-matching between hybridized sequences. Those skilled in the art will recognise that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of similarity between target and probe sequences. Other examples of stringency conditions are described in Section 4 below. [0205] The term "transcribable DNA sequence" or "transcribed DNA sequence", excludes the non-transcribed regulatory sequence that drives transcription. Depending on the aspect of the invention, 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. Such modifications include, but are not limited to, insertions, deletions and substitutions of one or more nucleotides. The 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.
[0206] The term "transformation" means alteration of genotype by introduction of genetic material (e.g. , the chimeric construct of the present invention) into an organism.
[0207] As used herein, the term "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.
[0208] By "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. Accordingly, 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. Alternatively, 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.
[0209] The terms "wild type," "native" or "non-transgenic" 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.
2. Abbreviations
[0210] The following abbreviations are used throughout the application: nt nucleotide nts nucleotides kb =kilobase(s) or kilobase pair(s) ORF =open reading frame SNPs =single nucleotide polymorphisms
3. Transcribable DNA sequences useful for isolating promoter sequences
[0211] The promoter sequences of the present invention were first isolated through their linkage to a transcribable sequence, which comprises an ORF whose sequence is set forth in SEQ ID NO: 59, and which was found to be transcribed at high levels in mature stem tissues of sugarcane (Saccharum sp.). In accordance with the present invention, nucleotide sequences that correspond or are complementary to at least a portion of the sequence set forth in SEQ ID NO: 59 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.
[0212] 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. However, it will be understood that the present invention is not restricted to use of any particular method for identifying such differentially expressed genes. For instance, alternative procedures for identifying genes expressed differentially in various tissues include, but are not restricted to: hybridization screening as for example described in patent specification PCT/AU99/01033; cDNA and genomic subtractive hybridization as for example described by Bulman andNeill (1996, In "Plant Gene Isolation: Principles and Practice", G.D. Foster and D. Twell, eds Chichester, UK, Wiley, pp 369-397); multi -probe fluorescent analysis of microscopic cDNA arrays as for example described by Schena (1996 BioEssays 18:427-431); mRNA differential display as for example described by Liang and Pardee (1992, Science 257:967-970) and by Callard et al. (1994, BioTechniques 16:1096-1103); computer analysis of mRNA abundance based on frequency of occurrence of identical sequences emerging from large-scale sequencing of cDNA ends (ESTs) as for example taught by Cooke et al (1996, EST and genomic sequencing projects. In Plant Gene Isolation: Principles and Practice, supra, pp. 410-419); or promoter tagging by insertional mutagenesis with promoterless reporter genes as for example disclosed by Lindsey and Topping (1996, T-DNA-mediated insertional mutagenesis. In Plant Gene Isolation: Principles and Practice, supra, pp. 275-300) and Mudge and Birch (1998, Austral. J. Plant Physiol. 25:637-643).
4. Promoter sequences of the present invention
[0213] The present invention provides promoter sequences useful for expression of transcribable sequences in plants. In specific embodiments, 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: 47 to 58. Sequence analysis has revealed that these promoter sequences share a common segmental architecture, which can be represented by the following formula:
5'_θ_Δ-Z-Ψ-Φ-Ω-3' (I) in which segment Ω is more conserved across the class of promoter sequences than segment Φ, segment Φ is more conserved than segment Z, segment Z is more conserved than segment Δ, segment Δ is more conserved than segment Θ, and Ψ is an optional spacer or intervening segment, as illustrated in Figure 2. Segment Ω 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. In accordance with the present invention, it is predicted that segments Φ, Z, Δ and Θ as well as segment Λ, which represents the portion of segment Ω, which is upstream of the TATA box, comprise promoter elements, which preferentially direct transcription in plant sink tissue, including stem tissue. In certain embodiments, transcription is directed in stem tissue, including mature 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. In some embodiments, the promoter sequences of the present invention are particularly useful for preferentially expressing nucleic acids in carbohydrate storage tissue, and more particularly in mature plant carbohydrate storage tissue, illustrative examples of which include the sucrose-storage tissue of plants that store sugars, such as but not limited to sugarcane, sugarbeet and sweet sorghum.
[0214] 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.
[0215] Accordingly, the present invention contemplates that biologically-active fragments of any one of SEQ ID NOS: 47 to 58, 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. [0216] 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: 47 to 58). 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 PlantJ. 3:619), Sambrook e/ a/. (1989, supra) and McPherson e/1 α/. (U.S. Patent No. 5,164,316). For instance, 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. In this way, 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. Alternatively, 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, EMBO J. 15: 3732). The combination of segments most effective for particular developmental patterns of promoter activity is thereby identified through comparison of the selected constructs. These segments may then be used for the construction of vectors for the expression of heterologous genes.
[0217] Based on sequence analysis of the promoter sequences set forth in SEQ ID NO: 47-58, it is predicted that promoter elements positioned upstream or 5' to the TATA box in segment Ω (e.g., promoter elements in any one or more of segments Λ, Φ, Ψ, Z, Δ and Θ) influence the transcription initiation rate and/or tissue (e.g., stem-specific) expression of operably connected nucleic acid sequences. Accordingly, the present invention also contemplates fusing any one or more of segments Λ, Φ, Ψ, Z, Δ, Θ 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. 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: 61- 63.
[0218] Representative biologically-active fragments of the promoter sequences set forth in SEQ ID NOS: 47 to 58 may comprise at least about 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500 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 7, which correspond to segment Ω, respectively, of alleles 77, 5, 51, A, B and 49, as described herein. Longer fragments can be produced by adding corresponding upstream sequences such as but not limited to Φ, Ψ, Z, Δ and Θ. Alternatively, biologically active fragments may include any one or more of segments Λ, Φ, Ψ, Z, Δ and Θ, 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: 47-58. Illustrative examples of biologically active fragments according to the present invention are listed in Table 1 below.
TABLEl
Figure imgf000054_0001
[0219] 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. [0220] According to such methods, 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. In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.
[0221] 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.
[0222] Typically, the following general procedure can be used to determine hybridization conditions. 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.
[0223] A skilled person will recognize that a number of factors influence hybridization. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 108 dpm/mg to provide a detectable signal. A radiolabeled nucleotide sequence of specific activity 108 to 109 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 μg. 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).
[0224] To achieve meaningful results from hybridization between a polynucleotide immobilized on a membrane and a labeled polynucleotide, a sufficient amount of the labeled polynucleotide must be hybridized to the immobilized polynucleotide following washing. Washing ensures that the labeled polynucleotide is hybridized only to the immobilized polynucleotide with a desired degree of complementarity to the labeled polynucleotide.
[0225] It will be understood that 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 420C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (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 NaHPO4 (pH 7.2), 5% SDS for washing at room temperature.
[0226] Suitably, 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 550C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 650C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 60-650C. [0227] In specific embodiments, the polynucleotide variants hybridize to a reference polynucleotide under high stringency conditions. 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°C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (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 NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C. [0228] Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al, supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to 1.104, which are incorporated herein by reference.
[0229] While stringent washes are typically carried out at temperatures from about 42°C to 680C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization typically occurs at about 20°C to 25°C below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm based on the length and sequence composition of duplex DNA and the composition of the hybridization solution are well known in the art (see Ausubel et al., supra at page 2.10.8). [0230] In general, the Tm of a perfectly matched duplex of DNA may be predicted as an approximation by the formula:
[0231] Tm= 81.5 + 16.6 (logio M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length)
[0232] wherein: M is the concentration ofNa+, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex.
[0233] The Tm 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 Tm - 15 0C for high stringency, or Tm - 30 0C for moderate stringency.
[0234] In a preferred hybridization procedure, a membrane (e.g. , a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 420C 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. 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 5O0C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 550C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-680C).
[0235] 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.
[0236] In general, 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. For example, polynucleotides according to any one of SEQ ID NO: 47-58 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. Thus, in addition to the unmodified promoter sequences set forth in SEQ ID NO: 47-58, the current invention encompasses functional variants of these sequences. For example, based on the information provided herein with respect to functional elements, one of skill in the art could derive functional promoters by deleting or modifying non-essential portions of the sequence without disrupting the essential functional elements. Such mutants may potentially have enhanced or altered function relative to the native sequence or alternatively, may be silent with regard to function.
[0237] 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. After hybridization, 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. [0238] Generally, 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. [0239] The DNA template can be generated by those vectors that are either derived from bacteriophage M 13 vectors, or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. (1987, Methods Enzymol. 153:3). Thus, 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).
[0240] Alternatively, the single-stranded template may be generated by denaturing double-stranded plasmid (or other DNA) using standard techniques.
[0241] For alteration of the native DNA sequence, 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. After 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.
[0242] Alternatively, 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. For example, reference may be made to Ausubel et al. , supra, (in particular, Chapter 8.4, incorporated herein by reference) which describes a first protocol that uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides is synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. 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 al., 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.
[0243] Region-specific mutagenesis and directed mutagenesis using PCR may also be employed to construct promoter variants according to the invention. In this regard, reference may be made, for example, to Ausubel et al., supra, in particular Chapters 8.2A and 8.5.
[0244] The activity of promoter sequence variants thus obtained can be determined by conventional methods known to those of skill in the art, illustrative examples of which are discussed above.
5. Chimeric nucleic acid constructs and expression vectors
[0245] 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. For the purposes of transformation and expression of the heterologous nucleic acid in plants, it is generally desirable that the chimeric construct includes regulatory sequences which influence expression of the heterologous nucleic acid in plants. Suitably, the chimeric construct is present in an expression vector which includes regulatory sequences that enable selective propagation in bacteria.
5.1 3' Non-translated region
[0246] 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-31 although variations are not uncommon. [0247] 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. Examples of 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 at, 1983, Nucl. Acid Res., 11:369) and the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens. Alternatively, 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. Alternatively, 3' non-translated regulatory sequences can be obtained de novo as, for example, described by An (1987, Methods in Enzymology, 153:292).
5.2 Optional sequences
[0248] The chimeric construct of the present invention can further include enhancers, either translation or transcription enhancers, as may be required. These 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.
[0249] Examples of 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.
[0250] As the DNA sequence inserted between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one can also employ a particular leader sequence. Preferred leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence. For example, such 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. However, 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. Thus, leader sequences (i) 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.
[0251] Regulatory elements such as the 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) can also be included where desired. Other such regulatory elements useful in the practice of the invention are known to those of skill in the art.
[0252] Additionally, 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. For example, 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. For example, 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. Thus, 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. 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.
[0253] 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 pB S -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. [0254] In general, 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. Alternatively, 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. To increase the likelihood of integration at a precise location, 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. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences.
[0255] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial 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. ScL USA 75:1433).
5.3 Marker genes
[0256] To facilitate identification of transformants, 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. 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.
[0257] Included within the terms selectable or screenable marker genes are genes that encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include 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., α-amylase, β-lactamase, phosphinothricin acety transferase).
5.3.1 Selectable markers
[0258] Examples of 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 (neό) gene conferring resistance to kanamycin, paromomycin, G418 and the like as, for example, described by Potrykus et al. (1985, MoI. Gen. Genet. 199:183); a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP-A 256 223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described WO87/05327, an acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP- A 275 957, a gene encoding a 5 -enolshikimate-3 -phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al. (1988, Biotech., 6:915), a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988, Science, 242:419); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al, 1988, J Biol Chem., 263:12500); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP-A-154 204); a mutated anthranilate synthase gene that confers resistance to 5 -methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.
5.3.2 Screenable markers [0259] Representative screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known; a β-galactosidase gene encoding an enzyme for which chromogenic substrates are known; an aequorin gene (Prasher et al, 1985, Biochem. Biophys. Res. Comm., 126:1259), which may be employed in calcium-sensitive bioluminescence detection; a green fluorescent protein gene (Niedz et al, 1995 Plant Cell Reports, 14:403); a luciferase (luc) gene (Ow et al, 1986, Science, 234:856), which allows for bioluminescence detection; a β-lactamase gene (Sutcliffe, 1978, Proc. Natl. Acad. Sci. USA 75:3737), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); an R-locus gene, encoding a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al, 1988, in Chromosome Structure and Function, pp. 263-282); an α-amylase gene (Ikuta et al, 1990, Biotech., 8:241); a tyrosinase gene (Katz et al, 1983, J. Gen. 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 al, 1983, Proc. Natl. Acad. Sci. USA 80:1101), which encodes a catechol dioxygenase that can convert chromogenic catechols.
6. Uses of the promoter sequences of the invention
[0260] The isolated promoters sequences of the invention may be used, inter alia, 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).
[0261] 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. [0262] Anti-sense regulation, co-suppression and the use of ribozymes and hairpin
RNA in plants are well known in the art. However, 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.
[0263] Alternatively, the foreign or endogenous nucleic acid sequence may encode a molecule which is readily detectable or measurable, e.g. β -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. desaturase or hydroxylase; 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, β-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.
7. Introduction of chimeric genes into plant cells
[0264] There are many plant transformation techniques well known to workers in the art, and new techniques are continually becoming known. The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce a chimeric DNA construct into plant cells is not essential to or a limitation of the invention, provided it achieves an acceptable level of nucleic acid transfer. Guidance in the practical implementation of transformation systems for plant improvement is provided by Birch (1997, Annu. Rev. Plant Physiol. Plant Molec. Biol. 48: 297-326), which is incorporated herein by reference.
[0265] In principle 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.
[0266] Introduction and expression of foreign or chimeric nucleic acid sequences in dicotyledonous (broad-leafed) plants such as tobacco, potato and alfalfa has been shown to be possible using the T-DNA of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (See, for example, Umbeck, U.S. Patent No. 5,004,863, and International application PCT/US93/02480). A construct of the invention may be introduced into a plant cell utilizing A. tumefaciens containing the Ti plasmid. In using an A. tumefaciens culture as a transformation vehicle, it is most advantageous to use a non-onco genie strain of the Agrobacterium as the vector carrier so that normal non-oncogenic differentiation of the transformed tissues is possible. It is preferred that the Agrobacterium harbors a binary Ti plasmid system. Such 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. [0267] Methods involving the use of Agrobacterium include, but are not limited to:
(a) co-cultivation of Agrobacterium with cultured isolated protoplasts; (b) transformation of plant cells or tissues with Agrobacterium; or (c) transformation of seeds, apices or meristems with Agrobacterium. [0268] Recently, rice, corn, pineapple and sugarcane, which are monocots, have been shown to be susceptible to transformation by Agrobacterium, for example as described in United States Patent No. 6,037,522, International Publication WO99/36637 and Arencibia et al. (1998, Transgenic Res. 7:213). However, some monocot crop plants have not yet been successfully transformed using Agrobacterium-mediated transformation. The Ti plasmid, however, 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.
[0269] In addition, gene transfer can be accomplished by in situ transformation by Agrobacterium, as described by Bechtold et al (1993, CR. Acad. ScL Paris, 316:1194). This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells. [0270] Alternatively, nucleic acids may be introduced using root-inducing (Ri) plasmids of Agrobacterium as vectors.
[0271] Cauliflower mosaic virus (CaMV) 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. After cloning, 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.
[0272] 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). In this technique, 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.
[0273] Another method for introducing nucleic acids into a plant cell is high velocity ballistic penetration by small particles (also known as particle bombardment or microprojectile bombardment) with the nucleic acid to be introduced contained either within the matrix of small beads or particles, or on the surface thereof as, for example described by Klein et al. (1987, Nature 327:70). Although typically only a single introduction of a new nucleic acid sequence is required, this method particularly provides for multiple introductions. [0274] Alternatively, nucleic acids can be introduced into a plant cell by contacting the plant cell using mechanical or chemical means. For example, a nucleic acid can be mechanically transferred by microinjection directly into plant cells by use of micropipettes. Alternatively, 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. [0275] Also contemplated are silicon carbide or tungsten whiskers, for example as described in United States Patent No. 5,302,523.
[0276] There are a variety of methods known currently for transformation of monocotyledonous plants. Presently, preferred methods for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation as, for example, described by Shimamoto et al. (1989, supra). Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar gene into embryo genie cells of a maize suspension culture by microprojectile bombardment (Gordon- Kamm, 1990, Plant Cell, 2:603-618). The introduction of genetic material into aleurone protoplasts of other monocotyledonous crops such as wheat and barley has been reported (Lee, 1989, Plant MoI. Biol. 13:21-30). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil, 1990, Bio/Technol. 8:429-434). The combination with transformation systems for these crops enables the application of the present invention to monocots. These methods may also be applied for the transformation and regeneration of dicots. Transgenic sugarcane plants have been regenerated from embryogenic callus as, for example, described by Bower et al. (1996, Molecular Breeding 2:239-249).
[0277] Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g., bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233). 8. Strategies to avoid transgene silencing
[0278] Transgenes driven by various promoter sequences can be efficiently silenced during the regeneration and growth of mature plants. To avoid undesired transgene silencing, 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,
8.1 Simple integration events and targeted integration sites
[0279] 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.
8.2 Recombinase-mediated resolution of complex integration events
[0280] Site-specific recombinase systems, such as Cre-lox from bacteriophage Pl, can be employed for resolution of complex integration events into simple patterns (Srivastava 1999, 2001; De Buck 2001a, 2001b, 2007).
8.3 Recombinase-mediated site-specific integration
[0281] 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 (favorable lox landing site, Cre recombinase enzyme activity and /øx-flanked gene for integration) 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 K-RS have shown promise in plants, but Cre-lox is the best characterized (Ow 2002; Ow 2007).
[0282] Direct gene transfer and Agrobacterium-mQdiated gene transfer systems have been applied for Cre-/ox site-specific integration (Albert 1995, Vergunst 1998, Srivastava 2002). The excision by site-specific recombinases is intrinsically more favorable than integration. Therefore, the strategy to obtain stable integrants typically involves use of (i) mutant lox sites that are compatible for targeted integration but less effective for excision of the integrated molecule, (ii) transient recombinase activity and (iii) selection based on reconstitution of a promoter - selectable marker expression unit through targeted integration. Conditions (ii) and (iii) can be accomplished together if the landing pad is a promoter-/ox-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).
[0283] Agrobacterium-mediated 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 (iii) 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).
8.4 Transposon-mediated integration
[0284] In contrast with random integration following Agr-obαcterium-mediated or direct gene transfer, maize Ac/Ds transpositions show a bias to regions that might be more amenable to transgene expression (Zhao 2006). When barley transformed using T-DNA that contained a bar selectable marker within a non-autonomous Ds element was crossed with Ac transposase-expressing plants, the όαr-containing Ds element transposed to different sites in the genome and segregated away from the initial T-DNA insertions in subsequent generations. The resulting plants that harboured a single copy of the 6αr-containing Ds element showed greatly enhanced bar transgene expression stability over several generations relative to the source lines (Koprek 2001). The requirement for crossing has subsequently been avoided by including the transposase gene and a negative selectable marker (cytosine deaminase) in the same T-DNA as the Ds .7 positive selectable marker element, and selecting against cells that incorporate the full T-DNA (Yan and Rommens 2007). 8.5 Restriction-site-directed integration
[0285] Co-expression of a rare-cutting restriction enzyme or a zinc finger nuclease, which induces double-stranded breaks in DNA, has been developed for site-specific integration of transgenes into a corresponding recognition site following protoplast or microprojectile or Agrobacterium- mediated transfer. The recognition site can be native to the recipient genome, or in a previously engineered 'landing pad' ( Wright 2005, D'Halluin 2008).
8.6 Use of S/MAR elements
[0286] Nuclear scaffold or matrix attachment regions (S/MARs) 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).
8.7 Elimination of silencing; triggers in the transgene sequence [0287] 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).
8.8 Minimal expression cassettes
[0288] It is useful to transfer the minimum DNA sequence needed to confer the desired transgenic trait. This eliminates the possibility that vector sequences might be unintended sources of signals to trigger silencing responses that can spread into the transgene. In the case of Agrobacterium-mQdiεAed transfer this is accomplished by use of minimal distance between the T-DNA borders. In the case of DGT, the transgene expression cassette may be isolated without vector backbone by either restriction digestion or PCR amplification (Agrawal 2005; Kumar 2006).
8.9 Codon Optimisation
[0289] Although 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.
[0290] 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).
8.10 Inhibiting RNAi pathways
[0291] One role for RNAi in plants is in antiviral defense, and many viruses encode proteins that interfere with RNAi mechanisms. Diverse viral suppressors act at several different steps in the RNAi pathway (Roth 2004; Voinnet 2005; Scholthof 2007). These suppressors can inhibit silencing of transgenes in addition to viral RNAs, as first demonstrated for the potyvirus helper component-protease, HC-Pro. 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).
9, Production and characterization of differentiated transgenic plants 9.1 Regeneration
[0292] 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. [0293] Regeneration from protoplasts varies from species to species of plants, but generally a suspension of protoplasts is first made. In 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.
[0294] In vegetatively propagated crops, 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.
[0295] In seed propagated crops, 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. [0296] 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. [0297] It will be appreciated that 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.
9.2 Characterization [0298] To confirm the presence of the heterologous nucleic acid in the regenerating plants, a variety of assays may be performed. Such 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.
[0299] Representative examples of various methods applicable to characterization of transgenic plants are provided in Chapters 9 and 11 of PLANT MOLECULAR BIOLOGY A Laboratory Manual Ed. M.S. Clark (Springer-Verlag, Heidelberg, 1997.
[0300] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting example.
EXAMPLES
EXAMPLE 1
IDENTIFICATION OF MULTIPLE ScCIPKl PROMOTER ALLELES IN SUGARCANE
Results Identification of a gene that is up-regulated in mature stem
[0301] The sequence of the ScCIPKl coding region is set forth in SEQ ID NO: 59. Transcripts hybridizing to a probe derived from SEQ ID NO: 59 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. Northern analysis using RNA from vascular, parenchyma and rind tissue dissected from internode eight of field-grown
Ql 17 (Rae et al, 2005) revealed the presence of transcript in all three tissue types within the stem (Figure Ic).
Sugarcane is highly heterozygous at the ScCIPKl locus
[0302] Sequence analysis of 13 Q200 genomic BAC clones hybridizing to the ScCIPKl coding region revealed the presence of at least eight haplotypes of ScCIPKl in this cultivar.
Multiple ScCIPKl alleles are differentially expressed in sugarcane
[0303] Sequence analysis of RT-PCR products indicates that at least four haplotypes are expressed in internode 12 of Ql 17. One haplotype was shown to comprise approximately 20% of RT-PCR clones from internode 12, and 100% (30/30) of RT-PCR clones in internode 30, consistent with this allele (designated as allele A) being most strongly expressed in mature stem tissues.
ScCIPKl promoter alleles
[0304] Using the Genome Walker single-specific-primer PCR procedure, in combination with BAC clone sequencing, we identified six distinct promoter variants for ScCIPKl. These sequences are represented by SEQ ID NOS: 53, 54, 55, 56, 57 and 58.
[0305] These six promoter alleles show at least 85% identity for 168 bp upstream of the start codon. A putative TATA box predicted using the program HCtata (Milanesi et al. 1996) occurs approximately 133-135 bp upstream of the start codon, and 23 bp upstream of the transcription start site determined by 5'-RACE. Beyond 168 bp upstream, the sequences diverge into 2 sub-families with many conserved motifs.
[0306] Sequencing of a BAC clone containing ScCIPKl allele A revealed that this allele is linked to the ScCIPKl A promoter allele (SEQ ID NO 56). Therefore this promoter is potentially useful for driving strong, mature-stem expression of transgenes. Other ScCIPKl promoter alleles may be used to drive different transgene expression levels and developmental patterns within sink tissues.
[0307] RT-PCR analysis in transgenic sugarcane cell lines showed reporter gene expression driven from the Ω fragment of the ScCIPKl A promoter, providing functional confirmation that the Ω sequence comprises a core promoter region. Multiple independent transgenic sugarcane lines containing the ScCIPKl A or B promoter alleles fused to the LUC reporter gene showed LUC activity in regenerating shoots in tissue culture, and in stem and root tissues of transgenic plants grown in a containment glasshouse for 4 months. This confirmed the ability of the isolated ScCIPKl promoters to drive heterologous gene expression in transgenic plant cells.
Discussion
[0308] Sugarcane is highly heterozygous at the ScCIPKl locus, with eight coding region variants and six promoter variants identified in cultivar Q200. We have shown that the upstream genomic sequences isolated from ScCIPKl alleles are functional as promoters for heterologous reporter gene expression in transgenic sugarcane plants. It is reported that transgenes driven by various promoter sequences can be efficiently silenced during the regeneration and growth of mature plants in sugarcane (Hansom et al. 1999; Ingelbrecht et al, 1999; Wei et al, 2003). To avoid undesired transgene silencing, constructs should be designed and transferred so as to minimise insofar as possible any features likely to contribute to the production of aberrant transcripts and associated sequence-specific gene silencing responses in plants.
[0309] In particular, it will be advantageous to use the promoters described in the present invention in gene constructs designed to (i) preclude read-through into or out of the transgene {e.g. through the use of flanking transcriptional terminators); (ii) eliminate known silencing signals including sequence features (such as inverted repeats) or motifs (such as targets for recipient plant small regulatory RNAs); (iii) exclude unnecessary sequences that might be sources of unknown silencing signals; and (iv) ensure efficient transcriptional termination. It will also be advantageous to use gene transfer parameters designed to (i) maximise the proportion of simple, intact integration events of the transferred expression cassette; and (ii) preferably integrate at a known locus that has been shown experimentally to be neutral for transgene expression (e.g. away from heterochromatin, outside of any endogenous transcript and not unintentionally influenced by endogenous ds-acting enhancer or repressor elements). Following gene transfer into plants, it is generally necessary to screen transformants for those showing the desired expression pattern and lack of developmental silencing in primary transformants and in subsequent generations tested under typical commercial growing conditions. These considerations are well known in the art, as discussed in Section 8 above, and for example by Finnegan and McElroy 1994, Matzke 1994, Meyer 1995, Birch 1997, Matzke and Birchler 2005, Brodersen and Voinnet 2006, Vaucheret 2006, Chapman and Carrington 2007, Ossowski 2008.
Materials and Methods
Choice of Target Locus [0310] Sugarcane cDNA clones that are potentially differentially regulated in immature (sucrose catabolising) and maturing (sucrose loading) stems of sugarcane were identified initially by microarray analysis of EST libraries (Casu et αl, 2004). There are many uncertainties in the interpretation of microarray analyses, including differential expression of cross-hybridizing multi-gene family members, the presence of fusion events in EST clones and EST sequences too short to assign tentative gene functions. Further experimentation is required to determine which of many candidate EST clones indeed represent genes with particular expression patterns inferred from a microarray analysis. The present studies were conducted to search for genes expressed preferentially within the mature (sucrose storing) stems of sugarcane, a pattern not reported by Casu et αl. (2004). Analysis of many sugarcane EST clones in this context led the present inventors to a particular interest in a sugarcane gene locus designated below as ScCIPKL
RNA Extraction and Northern Analysis
[0311] RNA was extracted from field- or glasshouse-grown sugarcane cultivar Ql 17 tissues by CsCl ultracentrifugation (Chirgwin et αl, 1979). Following denaturation, approximately 10 μg RNA was fractionated on a 1.2% TBE agarose gel, and transferred onto Hybond N+ membrane (Amersham) using capillary transfer with 1 OxSSC. Hybridization was done for 16 h at 650C in 0.5 M NaH2PO4, 1 mM EDTA, 7% SDS; using 32P-dCTP-labelled probes prepared using a Megaprime kit (Amersham). Following hybridization, membranes were washed to high stringency (0.1 x SSC; 0.1% SDS at 650C), and analyzed using a Phosphorlmager SI (Molecular Dynamics).
DNA Extraction and Southern Analysis [0312] Sugarcane genomic DNA was isolated using a CTAB-based method
(Rogers and Bendich 1988). Ten μg of each sample was digested with restriction enzymes, fractionated on a 0.8% agarose TAE gel, and transferred onto Hybond N+ membrane (Amersham) using capillary transfer with either 0.4 M NaOH or 10 x SSC. Labeling of probes and hybridization conditions were as described for northern analysis.
DNA Sequencing
[0313] 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).
Analysis of Allelic Variation and Expression [0314] The degree of allelic variation for ScCIPKl was assessed by sequencing homologous clones from a genomic BAC library prepared from sugarcane cultivar Q200. To identify expressed alleles, RT-PCR products derived from mature stem were amplified using the primers 5'-GCCATAACACTAAGACGAGCGCCAACC-S ' and 5'- CGACACCCACTCCGGGAACTCGAACTCC-3' and sequenced. For RT-PCR, 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.
Isolation of Upstream Promoter Regions and PCR Amplification [0315] Promoter alleles corresponding to ScCIPKl were amplified using a PCR- based Genome Walker strategy (Clontech), using primers GSPl (5'- CCTCCTCTGTCGGTTCCGCTGGTCGAAC AAGCCG-3') and GSP3B (5'- GTGCGTCGTCAGCGTCGCGAGGGCAAC -3') derived from the ScCIPKl coding region, or primers GWl (5'-CCTTGGCGAACGTGCCTCGTC-S') and GW2 (5'- CGTGCCTCGTCCC AGC AAGC-3') derived from the 5' end of the ScCIPKl transcript. To maximize the chances of amplifying all promoter alleles in the polyploid genome of cultivar Ql 17, we used a total of eight Genome Walker libraries (prepared using restriction enzymes Oral, EcoRY, Pvull, Seal, Stul, BsrBΪ, EclUβll, and Sspϊ). These and all other PCR amplification reactions used Expand High Fidelity polymerase (Roche) according to the manufacturer's instructions. Amplified products were cloned into pGem-T Easy (Promega) prior to sequencing. Additionally, promoter alleles were obtained by subcloning from clones in a Q200 genomic BAC library. The transcriptional start site was determined by 5'RACE using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions.
Construction of Promoter-Reporter Gene Fusions
[0316] Promoter sequences with native 5'UTRs were excised from Genome Walker and BAC clones using Pstϊ and iVcøl and cloned in front of the /wc+NF (Promega) reporter gene. Constructs containing the same reporter gene driven by the maize Ubi-1 promoter (Christensen et al, 1992) were included as positive controls. All constructs included the nos terminator.
Sugarcane Transformation [0317] Transformation and regeneration of sugarcane cultivar Q 117 (obtained from
BSES Meringa Sugar Experiment Station, Gordon vale, QId) was done as previously described (Bower et al. 1996). Plants were grown in a containment glasshouse under natural light intensity, at 28°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/m2, 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.
Reporter Gene Assays
[0318] LUC assays were done as described (Luehrsen and Walbot 1993), using a BMG POLARstar OPTIMA luminometer. [0319] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[0320] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
[0321] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
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Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule comprising a promoter sequence that is operable in plant cells, wherein the promoter sequence comprises a nucleotide sequence selected from the group consisting of: (a) YGASMTTKAYWAKTSGCTARRIIARN0KTYRRAKRMRWARWTGKYR
SRRNfiHTTYNcDMYTYKTNrfTTCCAASRGYTWRKMAAAYYAHRMTMCNeRARGTAT AKRWMAHAN/WRMATKWGSRRTYYTRRRHKAWTTHYWRMTYAMYMYAWTCAK YTYWA YWCWWTTYYYNgMKKWYWTN/A WMYYTSYMTTWTRRGWACMYKATT WRWRWKCYAAYCHYRRTTRRN7AARYSCYARTYNiTYGRATTTASWAACAAAAA YGKCAAATTGCATACCCAACTCAN/C YCATATAAAAMGKYACCAACCCAACATN,,, TNnTTYC [SEQ ID NO: I];
(b) 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: 1 or a complement thereof; and (c) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO:
1 or a complement thereof, under at least medium or high stringency conditions, wherein:
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; each of a, b, c, d, e, g, i, k and m is an integer from 0-1; each of h,j and n is an integer from 0-2; / is an integer from 0-3; and /is an integer from 0-5.
2. A nucleic acid sequence according to claim 1, wherein Nα comprises 0 bases or is A.
3. A nucleic acid sequence according to claim 1, wherein N& comprises 0 bases or is K.
4. A nucleic acid sequence according to claim 1 , wherein Nc comprises 0 bases or is
K.
5. A nucleic acid sequence according to claim 1 , wherein N^ comprises 0 bases or is A.
6. A nucleic acid sequence according to claim 1 , wherein Ne comprises 0 bases or is T.
7. A nucleic acid sequence according to claim 1, wherein N/ comprises 0 bases or a sequence selected from ATTT or ACT.
8. A nucleic acid sequence according to claim 1 , wherein Ng- comprises 0 bases or is T.
9. A nucleic acid sequence according to claim 1 , wherein N/( comprises 0 bases or is
K.
10. A nucleic acid sequence according to claim 1, wherein N, comprises 0 bases or is A
11. A nucleic acid sequence according to claim 1 , wherein N7 comprises 0 bases or is the sequence TC.
12. A nucleic acid sequence according to claim 1, wherein N, comprises 0 bases or is W.
13. A nucleic acid sequence according to claim 1, wherein N^ comprises 0 bases or is a sequence selected from GA or CT.
14. A nucleic acid sequence according to claim 1, wherein N/ comprises 0 bases or is the sequence TGC.
15. A nucleic acid sequence according to claim I5 wherein Nn, comprises 0 bases or is A.
16. A nucleic acid sequence according to claim 1, wherein N,, comprises 0 bases or is the sequence AT.
17. A nucleic acid sequence according to claim 1, which is selected from the group consisting of:
TGAGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTT TGACTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATAGAAAATAATTTT GCATTAGGAATCTTAAACTATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTT CTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATC AAACCCCAGTTTTCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCAT CGCCCATATAAAACGTTACCAACCCAACATATATTTCC [SEQ ID NO: 2];
TGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCATAATTGTTGGGGCTTTG ACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATAGAAAACATGCATT AGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTTTATACTTTTTTTCTTTCT TGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCC CAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCA TATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 3];
TGAGATTTACAAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTGGGGGTTT TTGACTTTTTTCCAACAGTTTATAAAATCATGCTCCTAAAGTATAGAAAACAATTT TGCATTAGGAATCCTAAACTATTTCTAAATTACCCTAATCATTTTTATACTTTTTTT TCTTTCTTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCA AACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATC GCCCATATAAAACGGCACCAACCCAACATATATTTCC [SEQ ID NO: 4]; CGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATGGCACAATAT
TCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATATGTCAAAAC TCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCTCAACTCAATT CCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCCAACCCAATTA GAAGTGCTAATCTTGAATTTACTAACAAAAATGGCAAATTGCATACCCAACTCAC CCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 5];
CGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATGGCACAATAT TCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATATGTCAAAAC
TCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCTCAACTCAATT CCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCCAACCCAATTG GAAGTGCTAATCTTGAATTTACTAACAAAAATGTCAAATTGCATACCCAACTCAC CCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 6]; and
TGAGATTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTG ACTTTTTTCCCCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAATTTTGCAT TAGGAATCTTAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTCTTTC TTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACC CCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCTC ATATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: I].
18. A nucleic acid sequence according to claim 1 , wherein the promoter sequence further comprises upstream of (a), (b) or (c) a nucleotide sequence selected from the group consisting of:
(d) YRWYWRWTAYSYCTWDGAWMRTYKTYRAWARAYMAN0RVCARAKM TSRARWTTGTTGASKYNPYRYWRYAASCRTKGN9CACTYRAAYDVRYRKRYHSCW TA YTRWWGAA WMRMGAASMWRTSWWKMATWWRYYYAADMAMAN.WMYKA GCWAGMRMRYYWWTCSARKACTTKMCMRAYKRRRTRN.RAYATRYTCMAYNiCA YAAKWGKWGMDSNUA YCMAMMYYYMCYKARTA WMTTATWMYTTWGAGCNVY AATWGTTTNuAWAMAAN^WYAYMRCWMAMAARN^GYTWTCAAARMWN- YARTT GGYWNαα [SEQ ID NO: 8]; (e) 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: 8 or a complement thereof; and
(f) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 8 or a complement thereof, under at least medium or high stringency conditions, wherein:
A, C, G, T, M, R, W, S, Y, K, V, H, D, B and N are as defined above; each of p, s, t, x, y and z is an integer from 0-1; each of q and r is an integer from 0-2; o is an integer from 0-4; w is an integer from 0-5; each of v and aa is an integer from 0-6; and u is an integer from 0-23.
19. A nucleic acid sequence according to claim 18, wherein N0 is the sequence AGAC.
20. A nucleic acid sequence according to claim 18, wherein Np is G.
21. A nucleic acid sequence according to claim 18, wherein N9 is the sequence GC.
22. A nucleic acid sequence according to claim 18, wherein N,- is a sequence selected from AA or TG.
23. A nucleic acid sequence according to claim 18, wherein aN, is T.
24. A nucleic acid sequence according to claim 18, wherein N; is A.
25. A nucleic acid sequence according to claim 18, wherein N« is a sequence selected from AATTTCTAATCATGGTCAATATA [SEQ ID NO: 9],
AATTTATAATCATGGCTAATATA [SEQ IDNO: 10], TATATATATATTAATCTATC [SEQ ID NO: 11], TATATATATTAATCTATC [SEQ IDNO: 12] or AATTTCTAATCATGGCTAATATA [SEQ ID NO: 13].
26. A nucleic acid sequence according to claim 18, wherein Nv is the sequence ATCTC.
27. A nucleic acid sequence according to claim 18, wherein Nw is A or a sequence selected from TCAAA, GGCAC or TTTTT.
28. A nucleic acid sequence according to claim 18, wherein Nx is D.
29. A nucleic acid sequence according to claim 18, wherein Ny is A.
30. A nucleic acid sequence according to claim 18, wherein N= is A.
31. A nucleic acid sequence according to claim 18, wherein Nflα is the sequence AATCAA.
32. A nucleic acid sequence according to claim 18, wherein any one or more of N0, Np, N?, Nλ, N4, Nf, N«, Nv, Nw, Nx, Ny, Nz or N00 comprises 0 bases.
33. A nucleic acid sequence according to claim 18, which is selected from the group consisting of:
CAACTAATATCTCTATGAGCATCTTCGAAAGACAAAGACGGCAGATCTCA AAATTGTTGAGGTCATAATAAGCATTGCACTCAAACAGGCATGTTGCTTATTAAA GAATAGCGAAGCAATGAAGAATAAATCCAAGAAAATATGAGCAAGCACGTCAAT CGAGCACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGTGCTCAATTTCT AATCATGGTCAATATAATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTC CAATAGTTTTCAAAAAAAAAATCATCGCAAAAAAAGTTTTCAAAAAAATAATTG GTAAATCAA [SEQ ID NO: 14]; CAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGACAGATCTCA AGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAA GAATAGCGAAGCAATCAAGAATAAATTCAAAAAAAAATATGAGCAAGCACATCA ATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTT ATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACTTAGAGCATC TCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAAAAAAATAAT TGGTAAATCAA [SEQ ID NO: 15];
CAACTAATATCTCTATGAGCATCTTTAAAAGACAAAGACAACAGATCTCA AGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAGCATGTCGCTTATTAAA GAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATATGAGCAAGCACATCAAT CGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCATCAAA CCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTAAAAAAAATCATC GCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAA [SEQ ID NO: 16];
TGTTAGTTACCCCTTAGAAAGTTGTTAATAAATCAGCCAAAGATGGAATTT GTTGACTCGTGCTACAACCATGGGCCACTTGAATTCATGGACACCATACTGTTGA AACAAGAACATGTGTTTCATTTGCTTAATCACATGACCTAGCTAGAGAGCTTTTCC AATACTTTCCAGATGAAATGTAATATACTCCACACATAATTGTTGAAGTATATAT ATATTAATCTATCACCCACATTTCCTTAATAACTTATACTTTTGAGCTAATTGTTTG GCACATACAAGATACAACTCACAATGCTATCAAAGCTCAGTTGGCT [SEQ ID NO: 17];
TGTTAGTTACCCCTTAGAAAGTTGTTAATAAATCAGCCAAAGATGGAATTT GTTGACTCGTGCTACAACCATGGGCCACTTGAATTCATGGACACCATACTGTTGA AACAAGAACATGTGTTTCATTTGCTTAATCACATGACCTAGCTAGAGAGCTTTTCC AATACTTTCCAGATGAAATGTAATATACTCCACACATAATTGTTGAGGTATATAT ATTAATCTATCACCCACATTTCCTTAATAACTTATACTTTTGAGCTAATTGTTTGG CACATACAAGATACAACTCACAATGCTATCAAAGCTCAGTTGGCT [SEQ ID NO: 18]; and
CAACTAATATGTCTATGAGCATCTTCAAAAGACAAAGACGACAGATCTCA AGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAA GAATAGCGAAGCAATCAAGAATAAATTCAAAAAAATATGAGCAAGCACATCAAT CGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTTCT AATCATGGCTAATATAATCAAACCCCACCCAGTATATTATTACTTAGAGCATCTC CAATAGTTTTTTTTAAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAATAATTGG TAAATCAA [SEQ ID NO: 19].
34. A nucleic acid sequence according to claim 18, wherein the promoter sequence further comprises upstream of (d), (e) or (f) a nucleotide sequence selected from the group consisting of:
(g) WYHAWCRKTAYWWRANαδAGRAGYRWSNβcYATGASAGCATRWGTRTM MCWMATGTRN^YWTTCWTWRAAWATARYWWWYYTWYRRAWARWTRKTATA KYAATAAATSARWCWAWRAKKRAWTWWRTWKWTGAMTMRHKHTWYAAAYAT CKRYCAMTYRN^AKWMATRRASWMCMTAMWRKTWWAAAAYRRAMWMAWGT RTTTGA WWN«/RHKTTTTMYWTYMATRAATNagA YAWYWWRWWTAYRS WHAWT CAWRWKMRYAYWYWYMCMTYATGRW [SEQ ID NO: 20]; (h) 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: 20 or a complement thereof; and
(i) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 20 or a complement thereof, under at least medium or high stringency conditions, wherein:
A, C, G, T, M, R, W, S, Y, K, V5 H, D, B and N are as defined above; each of ae, α/and ag is an integer from 0-1; ab is an integer from 0-3; ac is an integer from 0-8; and ad is an integer from 0-9.
35. A nucleic acid sequence according to claim 34, wherein Na& is the sequence CAT.
36. A nucleic acid sequence according to claim 34, wherein Nαc is the sequence AATAAAA.
37. A nucleic acid sequence according to claim 34, wherein Nαc/ is the sequence GATAAAGAT.
38. A nucleic acid sequence according to claim 34, wherein Nfle is G.
39. A nucleic acid sequence according to claim 34, wherein Nα/is C.
40. A nucleic acid sequence according to claim 34, wherein Ηag is C.
41. A nucleic acid sequence according to claim 34, wherein any one or more of N0^5 Nαc, N0^, Nαg, Nα/ or Nαg- comprises 0 nucleotides
42. A nucleic acid sequence according to claim 34, which is selected from the group consisting of:
TCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGTCCCTAATGTG TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGGATTCATGGAC ACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCTTTTCATGAAT CACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGAA [SEQ ID NO:
21]; TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCTAATGTG
TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGTGCTACAAACATCGGCCACTTGGAGTCATGGAC ACCCTACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTCATGAAT CACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ ID NO: 22];
TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCTAATGTG TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTCATGGAC ACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTCATGAAT CACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ ID NO: 23];
ATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGT ATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATA GTATAGCAATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATA TCTATCAATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGA AAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTT ACATCATGGT [SEQ ID NO: 24];
ATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGT ATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATA GTATAGCAATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATA TCTATCAATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGA AAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTT ACATCATGGT [SEQ ID NO: 25]; and
TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCTAATGTG TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTCATGGAC ACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTCATGAAT CACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGA [SEQ ID NO: 26].
43. A nucleic acid sequence according to claim 34, wherein the promoter sequence farther comprises upstream of (g), (h) or (i) a nucleotide sequence selected from the group consisting of:
G) RRSKWMWAKYTYWRKAMWTRGTAYAKSRRTRMWYRWRTYTWTMAR TA WTTAWWTWWRTAARWAWCTAYN0/, YDATWYAWWTARWDRNOjRAGKWSMY WSRWGWYAMMRAKGWRTTWATAKGWMTTYGWMAAYSTYWASATYRRTMARY WTAMTMTYTYAN07GGYWRRRKCMAAAKMAN^SATYATGGTYWRMWWWGKWK YYWKATWSTAKWMWWWTMCRKATWWTAWTKMAWATMCWWMWTWMRWT WMTYTCAKATRYRRATMYAAATYMAGN0ZTARTKWRAAWTSAAWTMWGMRWT AWSMATTTTAKWTWCATACYYGRWWTGGRTGKKAGMAAKWWMKGAWTMRY WTWWWTYRRWSRKYTTSAWRWMCRKW [SEQ ID NO: 27];
(k) 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: 27 or a complement thereof; and (1) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 27 or a complement thereof, under at least medium or high stringency conditions, wherein:
A, C, G, T, M, R, W, S, Y, K, V, H, D, B and N are as defined above; each of ah and al is an integer from 0-1 ; aj is an integer from 0-2; ai is an integer from 0-5; and ah is an integer from 0-8.
44. A nucleic acid sequence according to claim 43, wherein N0/, is G.
45. A nucleic acid sequence according to claim 43, wherein N0,- is a sequence selected from AATAA, AATA or TAGGT.
46. A nucleic acid sequence according to claim 43, wherein N0, is the sequence AA.
47. A nucleic acid sequence according to claim 43, wherein N0* is a sequence selected from TATGTTTA or AATTAAAC.
48. A nucleic acid sequence according to claim 43, wherein N0/ is C.
49. A nucleic acid sequence according to claim 43, wherein any one or more of N0/,,
N0,-, N0/, Nα/t or N0/ comprises 0 bases.
50. A nucleic acid sequence according to claim 43, which is selected from the group consisting of: AACTAATATCTCAGTAAATAGTATAGCAATAAATGAGTCTATAAGTATTTA AATATATAAATATCTACTGATATAAATAAATAAATAAAAGTACATACAAGTTACA AATGAATTAATAAGGTATTTGAAAACGTTTACATCAATAAATATACTATTTTAGG CTAAATCAAAATAATATGTTTACATCATGGTTTACATAGTTGCTTTATTCTATACA AATACGGATAATATTGAATATCCATCGTACGTTTATTTCATATACGGATCCAAATT CAGCTAATGAAAAATGAATTCAGAAATATCTATTTTATTTTCATACCTGAAATGG ATGTGAGAAATATATGAATAATATAATTCAGCCAGCTTCATATCCATT [SEQ ID NO: 28];
AACGAATATATCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATT AAATATATAAATATCTATCAATATAAATAAATAAAGTACATACAAGTTACAAATG AATTAATAAGGTATTTGAAAACGTTTAAATCAATAAATATACTATTTTAGGCTAA ATCAAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAAT ATTGAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAA AATGAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATA TATGAATAATATAATTCAGACAGCTTCATATCCATT [SEQ ID NO: 29];
AACGAATATCTCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATT AAATATATAAATATCTATCGATATAAATAAATAAATAAAGTACATACAAGTTACA AATGAATT AATAAGGTATTTGAAAACGTTTACATT AATAAATATACTATTTTAGG CTAAATCAAAATAACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGG ATAATATTGAATATCCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAA TGAAAAATGAATTCAGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAG AAATATATGAATAATATAATTCAGACAGCTTCATATCCATT [SEQ ID NO: 30];
GGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTAT TTTAGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGAGGTGCCTGGTGACAAC GAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAG GTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAG TATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAA ATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTG GGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGA [SEQ ID NO: 31]; and
GGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTAT TTTAGTAAGAAACTATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGTGACAAC GAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAG GTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAG TATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAA ATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTG GGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGA [SEQ ID NO: 32].
51. A nucleic acid sequence according to claim 18, wherein the promoter sequence further comprises upstream of (j), (k) or (1) a nucleotide sequence selected from the group consisting of:
(m) YWYTYWRWKTGKSYKWYWDMAAGWRWSYATWN^RMKWTRWRRT BTWGAAGMWTBKAATWRYSMCATRTATAN0nTAAAGAAWARNa0AN0PTKACAYTA YWWGWAYAWYAYATSTARRYAMWKGTCATWYATRRAMMMTWWYMYAWWK AKWN0? [SEQ ID NO: 33];
(n) 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: 33 or a complement thereof; and (o) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 33 or a complement thereof, under at least medium or high stringency conditions, wherein:
A, C, G, T, M, R5 W5 S5 Y5 K5 V5 H5 D, B and N are as defined above; each of am and ao is an integer from 0-1 ; ap is an integer from 0-2; an is an integer from 0-11; and aq is an integer from 0-26.
52. A nucleic acid sequence according to claim 51, wherein Now is W.
53. A nucleic acid sequence according to claim 51, wherein N0n is a sequence selected from AAACGCAAAAT [SEQ ID NO: 34], AAACGCAAAAC [SEQ ID NO: 35] or
TAAAGAGTATA [SEQ ID NO: 36].
54. A nucleic acid sequence according to claim 51, wherein N00 is T.
55. A nucleic acid sequence according to claim 51, wherein Nop is a sequence selected from TA or AA.
56. A nucleic acid sequence according to claim 51, wherein Nα? is a sequence selected from GCAATAAATGAGTCTATAATATGCAC [SEQ ID NO: 37] or GGGGGTGGAACCTTCCTTCAC [SEQ ID NO: 38].
57. A nucleic acid sequence according to claim 51, wherein any one or more of Naw, N0n, Nαo, 'Ngp or Naa comprises 0 nucleotides.
58. A nucleic acid sequence according to claim 51, which is selected from the group consisting of:
TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTGAAGAAT GGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACACTATATG TATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAGCAATAA ATGAGTCTATAATATGCAC [SEQ ID NO: 39];
TATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTGAAGAA TGGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGT ATAATACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGA [SEQ ID NO: 40];
TATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTTGAAGAA TTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGT ATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGA [SEQ ID NO: 41]; CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATGTAGAAGCT
TCTAATTGCGCCATGTATATAAAGAGTATATAAAGAAAAGTAAATTACATTACTT GAATATCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGT GGAACCTTCCTTCAC [SEQ ID NO: 42]; and
CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTAGAAGCT TCTAATTGCGCCATGTATATAAAGAAAAGTAAATTACATTACTTGAACATCATAT CTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTTCCT TCAC [SEQ ID NO: 43].
59. A nucleic acid sequence according to claim 51, wherein an intervening 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).
60. A nucleic acid sequence according to claim 59, wherein the intervening sequence is selected from the group consisting of: TCTATACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTTTTTTGGG TCCGGCCATCCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGCCCTTCTC TTTATATAATTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGTGAATAGG AATATTAATCCAAATAGAAAAAATATAATAATATGCA [SEQ ID NO: 44], 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: 44 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 44 or a complement thereof, under at least medium or high stringency conditions;
TTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATCC CATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATAT ATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTAT AGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTT [SEQ ID NO: 45] , 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: 45 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ
ID NO: 45 or a complement thereof, under at least medium or high stringency conditions; and
TTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATCC
CATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATAT
ATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTAT AGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTT [SEQ ID NO: 46] , 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: 46 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 46 or a complement thereof, under at least medium or high stringency conditions.
61. A nucleic acid sequence according to any one of claim 1 to 60, wherein the promoter sequence has a structure represented by formula (I):
Θ-Δ-Z-Ψ-Φ-Ω (I) wherein:
Θ is any one of (m), (n) or (o); Δ is any one of Q), (k) or (1);
Z is any one of (g), (h) or (i);
Ψ is an optional spacer or intervening sequence; Φ is any one of (d), (e) or (f); and Ω is any one of (a), (b) or (c).
62. A nucleic acid sequence according to claim 61, wherein the promoter sequence comprises a nucleotide sequence selected from the group consisting of: TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTGAAGAAT
GGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACACTATATG TATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAGCAATAA ATGAGTCTATAATATGCACAACTAATATCTCAGTAAATAGTATAGCAATAAATGA GTCTATAAGTATTTAAATATATAAATATCTACTGATATAAATAAATAAATAAAAG TACATACAAGTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACATCAATAA ATATACTATTTTAGGCTAAATCAAAATAATATGTTTACATCATGGTTTACATAGTT GCTTTATTCTATACAAATACGGATAATATTGAATATCCATCGTACGTTTATTTCAT ATACGGATCCAAATTCAGCTAATGAAAAATGAATTCAGAAATATCTATTTTATTT TCATACCTGAAATGGATGTGAGAAATATATGAATAATATAATTCAGCCAGCTTCA TATCCATTTCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGTCCCTAA TGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAA CAAAGATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGGATTCAT GGACACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCTTTTCAT GAATCACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGAATCTAT ACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTTTTTTGGGTCCGGCCAT CCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGCCCTTCTCTTTATATAA TTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGTGAATAGGAATATTAAT CCAAATAGAAAAAATATAATAATATGCACAACTAATATCTCTATGAGCATCTTCG AAAGACAAAGACGGCAGATCTCAAAATTGTTGAGGTCATAATAAGCATTGCACT CAAACAGGCATGTTGCTTATTAAAGAATAGCGAAGCAATGAAGAATAAATCCAA GAAAATATGAGCAAGCACGTCAATCGAGCACTTGACCAACTGGGTAGACATGTT CAATCACAAGAGGTGCTCAATTTCTAATCATGGTCAATATAATCAAACCCCACCG AGTATATTATTACTTAGAGCATCTCCAATAGTTTTCAAAAAAAAAATCATCGCAA AAAAAGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTACAAGTCGCTAAAA AAAGTTGGAGGCGTAATTGTTGGGGCTTTTGACTTTTTTCCAACAGTTTATAAAAT CACGCTCCTAAAGTATAGAAAATAATTTTGCATTAGGAATCTTAAACTATTTTCA AATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTGTATATTTGCATTTTGGGA ACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTTTCGGATTTAGAAA CAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAACGTTACCAACCC AACATATATTTCC [SEQ ID NO: 47], 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: 47 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 47 or a complement thereof, under at least medium or high stringency conditions;
TATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTGAAGAA TGGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGT ATAATACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGAAACGAATA TATCAATAAAT AGTATAGC AATAAATGAGTCTATAAGTAATTAAATATATAAATA TCTATCAATATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATAAGGTA TTTGAAAACGTTTAAATCAATAAATATACTATTTTAGGCTAAATCAAAATAACAT CATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATATCCTT CATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATTCAGA GATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAATAT AATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATGAGA GCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTT ATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGTGCTACAAACATC GGCCACTTGGAGTCATGGACACCCTACTGTTATAAAACGGAAACATGTGTTTGAT TCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCACTCA CACCTTATGGACAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGACAG ATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTT ATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAAAAAAAATATGAGCAAGC ACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGC TCAATTTATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACTTAG AGCATCTCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAAAAA AATAATTGGTAAATCAATGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCATAAT TGTTGGGGCTTTGACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATA GAAAACATGCATTAGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTTTATA CTTTTTTTCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATG GTTAATCAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCC AACTCATCGCCCATATAAAACGGCACCAACCCAACATATTTCC [SEQ ID NO: 48], 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: 48 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 48 or a complement thereof, under at least medium or high stringency conditions;
TATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTTGAAGAA TTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGT ATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGAAACGAATA TCTCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATTAAATATATAAATA TCTATCGATATAAATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATAA GGTATTTGAAAACGTTTACATTAATAAATATACTATTTTAGGCTAAATCAAAATA ACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATAT CCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATTC AGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAA TATAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATGA GAGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTG TTATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACA TCGGCCACTTGGATTCATGGACACCATACTGTTATAAAACGGAAACATGTGTTTG ATTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCACT CACACCTTATGGACAACTAATATCTCTATGAGCATCTTTAAAAGACAAAGACAAC AGATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAGCATGTCGC TTATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATATGAGCAAGC ACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGC TCATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTAAAAA AAATCATCGCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTAC AAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTGGGGGTTTTTGACTTTTTTCCAA CAGTTTATAAAATCATGCTCCTAAAGTATAGAAAACAATTTTGCATTAGGAATCC TAAACTATTTCTAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTATATTTG CATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTATCG GATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAAC GGCACCAACCCAACATATATTTCC [SEQ ID NO: 49], 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: 49 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 49 or a complement thereof, under at least medium or high stringency conditions; CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATGTAGAAGCT TCTAATTGCGCCATGTATATAAAGAGTATATAAAGAAAAGTAAATTACATTACTT GAATATCATATCTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGT GGAACCTTCCTTCACGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTT TCAATATTTATTTTAGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGAGGTGCC TGGTGACAACGAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTA ATCTCTCAAAGGTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATT CAGATAGTAGTATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGAT GCAAATATAAATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACA TACTCGGTTTGGGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAA CGGAATAATCATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGT ATAACACATGTAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATA GTATAGCAATAAATGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATA TCTATCAATCAATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGA AAAAGTTTTACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTT ACATCATGGTTTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATAT CCATCCCATATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCA GATATATCCAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGT GTTTATAGGGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCC CTTAGAAAGTTGTTAATAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACA ACCATGGGCCACTTGAATTCATGGACACCATACTGTTGAAACAAGAACATGTGTT TCATTTGCTTAATCACATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGA AATGTAATATACTCCACACATAATTGTTGAAGTATATATATATTAATCTATCACCC ACATTTCCTTAATAACTTATACTTTTGAGCTAATTGTTTGGCACATACAAGATACA ACTCACAATGCTATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAA TTCAAATAAAAAGATGGCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAAC TAAAATACGAGGTATATGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTG CTCAACACATTCAGCTCAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGT ACATTATTTATGTGCCAACCCAATTAGAAGTGCTAATCTTGAATTTACTAACAAA AATGGCAAATTGCATACCCAACTCACCCATATAAAAAGGCACCAACCCAACATTA TTTTC [SEQ ID NO: 50], 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; 50 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 50 or a complement thereof, under at least medium or high stringency conditions;
CTCTTTGAGTGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTAGAAGCT TCTAATTGCGCCATGTATATAAAGAAAAGTAAATTACATTACTTGAACATCATAT CTAAATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTTCCT TCACGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTAT TTTAGTAAGAAACTATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGTGACAAC GAGGTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAG GTAGGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAG TATTTTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAA ATCAAGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTG GGTGGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGAATAATC ATTATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGTATAACACATG TAGATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATAGTATAGCAAT AAATGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATATCTATCAATC AATAAATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGAAAAAGTTTT ACATCAATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTTACATCATG GTTTCATTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATCCCA TATTGATTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATATATC CAGCTTCAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTATAG GGGTGAGTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTAGAA AGTTGTTAATAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCATGG GCCACTTGAATTCATGGACACCATACTGTTGAAACAAGAACATGTGTTTCATTTG CTTAATCACATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATGTA ATATACTCCACACATAATTGTTGAGGTATATATATTAATCTATCACCCACATTTCC TTAATAACTTATACTTTTGAGCTAATTGTTTGGCACATACAAGATACAACTCACAA TGCTATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAAATA AAAAGATGGCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATAC GAGGTATATGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACAC ATTCAGCTCAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATT TATGTGCCAACCCAATTGGAAGTGCTAATCTTGAATTTACTAACAAAAATGTCAA ATTGCATACCCAACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTC [SEQ ID NO: 51], 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: 51 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 51 or a complement thereof, under at least medium or high stringency conditions; and
TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCTAATGTG TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTCATGGAC ACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTCATGAAT CACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGACAACTAATA TGTCTATGAGCATCTTCAAAAGACAAAGACGACAGATCTCAAGATTGTTGAGGTC ATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAAGAATAGCGAAGCA ATCAAGAATAAATTCAAAAAAATATGAGCAAGCACATCAATCGAGGACTTGACC AACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTTCTAATCATGGCTAAT ATAATCAAACCCCACCCAGTATATTATTACTTAGAGCATCTCCAATAGTTTTTTTT AAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAATAATTGGTAAATCAATGAGA TTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTGACTTTTTTCC CCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAATTTTGCATTAGGAATCT TAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTCTTTCTTGTATATTT GCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTATC GGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCTCATATAAAAC GGCACCAACCCAACATATTTCC [SEQ ID NO: 52], 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: 52 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 52 or a complement thereof, under at least medium or high stringency conditions.
63. A nucleic acid sequence according to claim 61, wherein the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
TATTCAATTTGGGTGATAGCAAGTATGCATTGCTTTATGGTTTTGAAGAAT GGAATAATCACATATATAAAACGCAAAATTAAAGAATAAATATGACACTATATG TATAATACATGTAGACAATGGTCATTCATAGAAAATAACATATTTATAGCAATAA ATGAGTCTATAATATGCACAACTAATATCTCAGTAAATAGTATAGCAATAAATGA GTCTATAAGTATTTAAATATATAAATATCTACTGATATAAATAAATAAATAAAAG TACATACAAGTTACAAATGAATTAATAAGGTATTTGAAAACGTTTACATCAATAA ATATACTATTTTAGGCTAAATCAAAATAATATGTTTACATCATGGTTTACATAGTT GCTTTATTCTATACAAATACGGATAATATTGAATATCCATCGTACGTTTATTTCAT ATACGGATCCAAATTCAGCTAATGAAAAATGAATTCAGAAATATCTATTTTATTT TCATACCTGAAATGGATGTGAGAAATATATGAATAATATAATTCAGCCAGCTTCA TATCCATTTCTACCGGTACTCGAAGGAGCGTCCATGAGAGCATGTGTGTCCCTAA TGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAA CAAAGATGGAATTTGTTGATGACTCGCTTTACAAACATCGGCCACTTGGATTCAT GGACACCATACTGTTATAAAACAGAAACATGTGTTTGATTCGTGTTTTCTTTTCAT GAATCACAATATAAATATAGATATTCATGTGCGCACTCACCCATTATGAATCTAT ACCTAATAATAAAGAGGCAAAATTTCTCTTCACCTGTTTTTTTTGGGTCCGGCCAT CCCTTAACTAACTTTGCGAATGTGAAAAACTGCTTATAGCCCTTCTCTTTATATAA TTAGGAATCATAATCCAATTAGATCTTTCTGATTTCGGGTGAATAGGAATATTAAT CCAAATAGAAAAAATATAATAATATGCACAACTAATATCTCTATGAGCATCTTCG AAAGACAAAGACGGCAGATCTCAAAATTGTTGAGGTCATAATAAGCATTGCACT CAAACAGGCATGTTGCTTATTAAAGAATAGCGAAGCAATGAAGAATAAATCCAA GAAAATATGAGCAAGCACGTCAATCGAGCACTTGACCAACTGGGTAGACATGTT CAATCACAAGAGGTGCTCAATTTCTAATCATGGTCAATATAATCAAACCCCACCG AGTATATTATTACTTAGAGCATCTCCAATAGTTTTCAAAAAAAAAATCATCGCAA AAAAAGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTACAAGTCGCTAAAA AAAGTTGGAGGCGTAATTGTTGGGGCTTTTGACTTTTTTCCAACAGTTTATAAAAT CACGCTCCTAAAGTATAGAAAATAATTTTGCATTAGGAATCTTAAACTATTTTCA AATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTGTATATTTGCATTTTGGGA ACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTTTCGGATTTAGAAA CAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAACGTTACCAACCC AACATATATTTCCACCTAATGCCGTCGCCCAACTAATTGCCATAACACTAAGACG AGCGCCAACCTTCGGTGCTCTGCTTGCCTCGCTGTTGCTGTTGCCCATGG [SEQ ID NO: 53], 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: 53 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 53 or a complement thereof, under at least medium or high stringency conditions; TCAAATAAATATAAATCAGGTAGTTCAAATTCAAATATACATTTAGATATG
TATTCAATTTGGGCGATATCAAGTATGCATTTGCTTTATGGTTTTGAAGAATGGAA TAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGTATAAT ACATGTAGACAATGGTCATTCATAGAACATAACATATTTAGAAACGAATATATCA ATAAATAGTATAGCAATAAATGAGTCTATAAGTAATTAAATATATAAATATCTAT CAATATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATAAGGTATTTGA AAACGTTTAAATCAATAAATATACTATTTTAGGCTAAATCAAAATAACATCATGG TTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATATCCTTCATAC GTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATTCAGAGATAT CCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAATATAATTCA GACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGT GTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTA ATAAATCAAACAAAGATGGAATTTGTTGATGACTCGTGCTACAAACATCGGCCAC TTGGAGTCATGGACACCCTACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTT TTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCACTCACACCTTA TGGACAACTAATATCTCTAGGAGCATCTTCAAAAGACAAAGACGACAGATCTCA AGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAA GAATAGCGAAGCAATCAAGAATAAATTCAAAAAAAAATATGAGCAAGCACATCA ATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTT ATAATCATGGCTAATATAATCAAACCCCACCGAGTATATTATTACTTAGAGCATC TCCAATAGTTTTCAAAAAAAAATTCATCGCAAAAAAAAGTTTTCAAAAAAATAAT TGGTAAATCAATGAGATTTACAAGTCGCTAAAAAGGTTGGAGGCATAATTGTTGG GGCTTTGACTTTTTTTCCAACAGTTTATAAAATCACGCTCCTAAAGTATAGAAAAC ATGCATTAGGAATCTTAAACTATTTTCAAATTACTCTAATCATTTTTATACTTTTTT TCTTTCTTGTATATTTGCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAAT CAAACCCCAGTTATCGGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCA TCGCCCATATAAAACGGCACCAACCCAACATATTTCCACCTAATGCCGTCGCCCA ACTAATTGCCATAACACTAAGACGAGCGCCAACCTTGCGGTGCTCTGCTTGCCTC GCTGTTGCTGTTGCCCATGG [SEQ ID NO: 54], 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: 54 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 54 or a complement thereof, under at least medium or high stringency conditions;
■ TTGGATCAAATAAATATAAATCAAGTAGTTCAAATTCAAATATACATTTAG ATATGTATTCAATTTGGGCGATAGCAAGTATGCATTTGCTTTATGGTTTTGAAGAA TTGAATAATCACATATATAAAACGCAAAACTAAAGAATAAATGACACTATAAGT ATAATACATGTAGGCAATGGTCATTCATAGAACATAACATATTTAGAAACGAATA TCTCAATAAATAGTATAGCAATAAATGAGTCTATAAGTAATTAAATATATAAATA TCTATCGATATAAATAAATAAATAAAGTACATACAAGTTACAAATGAATTAATAA GGTATTTGAAAACGTTTACATT AATAAATATACTATTTTAGGCTAAATCAAAATA ACATCATGGTTTACATAGTTGCTTTATTCTATACAAATACGGATAATATTGAATAT CCTTCATACGTTTATTTCATATATGAATCCAAATTCAGCTAATGAAAAATGAATTC AGAGATATCCATTTTATTTTCATACCTGAAATGGATGTGAGAAATATATGAATAA TATAATTCAGACAGCTTCATATCCATTTCCACCGTTACTTGAAGGAGTGTCCATGA GAGCATGTGTGTCCCTAATGTGTTTTCTTTAAATATAGTTATCCTTTGGAAAGTTG TTATATTAATAAATCAAACAAAGATGGAATTTGTTGATGACTCGCGCTACAAACA TCGGCCACTTGGATTCATGGACACCATACTGTTATAAAACGGAAACATGTGTTTG ATTCGCTTTTTCTTTTCATGAATCACAATATAAATATAGACATTCATGTGCGCACT CACACCTTATGGACAACTAATATCTCTATGAGCATCTTTAAAAGACAAAGACAAC AGATCTCAAGATTGTTGAGGTCATAGCAAGCGTTGCACTCAAACGAGCATGTCGC TTATTAAAGAATAGCGAAGCAATCAAGAATAAATTCAAGAAAATATGAGCAAGC ACATCAATCGAGGACTTGACCAACTGGGTAGACATGTTCAATCACAAGAGGAGC TCATCAAACCCCACCGAGTATATTATTACTTAGAGCATCTCCAATAGTTTAAAAA AAATCATCGCAAAAAAGGTTTTCAAAAAAATAATTGGTAAATCAATGAGATTTAC AAGTCGCTAAAAAAAGTTGGAGGCGTATTTGTTGGGGGTTTTTGACTTTTTTCCAA CAGTTTATAAAATCATGCTCCTAAAGTATAGAAAACAATTTTGCATTAGGAATCC TAAACTATTTCTAAATTACCCTAATCATTTTTATACTTTTTTTTCTTTCTTATATTTG CATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTATCG GATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCCCATATAAAAC GGCACCAACCCAACATATATTTCCACCTAATGCCGTCGCCCGACTAATTGCCATA ACTCTTAAAGACGAGCGCCAACCTTGCGGTGCTCTGCTTGCCTCGCTGTTGCTGTT GCCCATGG [SEQ ID NO: 55], 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: 55 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 55 or a complement thereof, under at least medium or high stringency conditions;
CTGCAGGCGGCCGCGAATTCACTAGTGATTACTATAGGGCACGCGTGGTC GACGGCCCGGGCTGGTATCGGGGCGTAGATTCCTCTCAACATATCAGCGTTGGAA TATGTTTGCCTTTTGAGTGGCCAGCACCAAGTACCATAGCCCCAGCTAACTAGCT CGAGTGGCCCTCGAGAATATCAGGGGTAGTTGCTATATACCAACATCATGGTGAA TTAACCTCAATGTCTTTCCTCACCAAAATATTTAAAGGTGTTTACAGGTGTCCCTT TCCCTGCTTTCATGTTGTAATCTTCAAGCTCTAGCAGCCAGGCGAACAACCGAAG GAATTAAGAAGACGACTAGAAAGGCCTTGGTTGTATTTTTATTTTTGGATGTTATT TTGTGTCAAGTTTCTCTCTTTCGTACTGGCTATTTACATATGAGACACTCTTTGAGT GTCTTTCTAAAAGAGACTATAAAAGATGAAATGTAGAAGCTTCTAATTGCGCCAT GTATATAAAGAGTATATAAAGAAAAGTAAATTACATTACTTGAATATCATATCTA AATACATGTCATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTTCCTTCA CGGGTTCAAGTTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTATTTT AGTAAGAAACTATGTTATTCATTTAGTAGTAGGTGAGGTGCCTGGTGACAACGAG GTGTTTATAGTGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAGGTA GGGGCCAAAGCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAGTATT TTCCATATTTTAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAAATCA AGTAGTTTGAATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTGGGT GGTAGCAAGTACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGAATAATCATT ATATAACATAGAAGTAAGGAATAAAATATGACAGCATAAGTATAACACATGTAG ATAAAGATCATTCATAGAAAATAACATATTTACAAATAAATAGTATAGCAATAAA TGAGTCTATAAGTAATTAAATATTTGAATAAATATTTAAATATCTATCAATCAATA AATAAAGTACATAAAAGTTAAAAATGAACTAAAGTATTTGAAAAAGTTTTACATC AATAAATATATCTAGTTTACGCTAAATCAAAATAATATATTTACATCATGGTTTCA TTAGGTCCTTTATTCTATACAAATACGAATAATATTGAATATCCATCCCATATTGA TTTCATATATAGATCCAAATTTAGCTAATAAAAATGAATTCAGATATATCCAGCTT CAGATCCATTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTATAGGGGTGA GTATGCGTGAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTAGAAAGTTGTT AATAAATCAGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCATGGGCCACTT GAATTCATGGACACCATACTGTTGAAACAAGAACATGTGTTTCATTTGCTTAATC ACATGACCTAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATGTAATATACT CCACACATAATTGTTGAAGTATATATATATTAATCTATCACCCACATTTCCTTAAT AACTTATACTTTTGAGCTAATTGTTTGGCACATACAAGATACAACTCACAATGCT ATCAAAGCTCAGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAA GATGGCACAATATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGG TATATGTCAAAACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTC AGCTCAACTCAATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATG TGCCAACCCAATTAGAAGTGCTAATCTTGAATTTACTAACAAAAATGGCAAATTG CATACCCAACTCACCCATATAAAAAGGCACCAACCCAACATTATTTTCACCTAGT GTCGTCGCCGAACTCATTGCCATAACACTAAGACGAGCGCCAACCTTGTGGTGCT CTTGCCTCGCTGTTGCTGTCGCCCATGG [SEQ ID NO: 56], 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: 56 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 56 or a complement thereof, under at least medium or high stringency conditions;
CTGCAGGCGGCCGCGAATTCACTAGTGATTACTATAGGGCACGCGTGGTC GACGGCCCGGGCTGGTATCGGGGCGTAGATTCCTCTCAACATATCAGCGTTGGAA TATGTTTGCCTATTGAGTGGCCAGCACCAAGTACCATAGCCCCAGCTAACTAGCT CGAGTGGCCCTCGAGAATATCAGAGGTAGTTGCTATATACCAACATCATGGTGAA TTAACCTCAATGTCTTTCCTCACCAAAATATTTAAAGGTGTTTACAGGTGTCCCTT TCCCTGCTTTCATGGTGTAATCTTCAAGCTCTAGCAGGCAGGCGAACAACCGAAG GAATTAAGAAGACGACTAGAAAGGCCTTGGTTGTATTTTTATTTTTGGATGTTATT TTGTGTCAAGTTTCTCTCTTTCGTACTGGCTATTTAGATATGAGACACTCTTTGAG TGTCTTTCTAAAAGAGACTATAAAAGATGAAATCTAGAAGCTTCTAATTGCGCCA TGTATATAAAGAAAAGTAAATTACATTACTTGAACATCATATCTAAATACATGTC ATATATGAACCCTTTTCCAAAGAGTGGGGGTGGAACCTTCCTTCACGGGTTCAAG TTTTAGACTTGGTACATGGGTGCTCATATTTTTCAATATTTATTTTAGTAAGAAAC TATGTTATTCATTTAGTGGTAGGTGAGGTGCCTGGTGACAACGAGGTGTTTATAG TGACTTCGTCAATCTCAAGATTGGTCAGCTTAATCTCTCAAAGGTAGGGGCCAAA GCAAATTAAACGATTATGGTCAGATATGGATTCAGATAGTAGTATTTTCCATATTT TAATTCAAATACTAATTTAAATACTCTCAGATGCAAATATAAATCAAGTAGTTTG AATTCAAATATGCATTAAGAATTTTAGATACATACTCGGTTTGGGTGGTAGCAAG TACGGATTCGCTTTTATTGATGGTTTTGAAGAACGGAATAATCATTATATAACATA GAAGTAAGGAATAAAATATGACAGCATAAGTATAACACATGTAGATAAAGATCA TTCATAGAAAATAACATATTTACAAATAAATAGTATAGCAATAAATGAGTCTATA AGTAATTAAATATTTGAATAAATATTTAAATATCTATCAATCAATAAATAAAGTA CATAAAAGTTAAAAATGAACTAAAGTATTTGAAAAAGTTTTACATCAATAAATAT ATCTAGTTTACGCTAAATCAAAATAATATATTTACATCATGGTTTCATTAGGTCCT TTATTCTATACAAATACGAATAATATTGAATATCCATCCCATATTGATTTCATATA TAGATCCAAATTTAGCTAATAAAAATGAATTCAGATATATCCAGCTTCAGATCCA TTTCCACCGTTACTTGAAGATGCTAATAGGGGTGTTTATAGGGGTGAGTATGCGT GAGAGCGTGTATGTCTGTACTTTGTTAGTTACCCCTTAGAAAGTTGTTAATAAATC AGCCAAAGATGGAATTTGTTGACTCGTGCTACAACCATGGGCCACTTGAATTCAT GGACACCATACTGTTGAAACAAGAACATGTGTTTCATTTGCTTAATCACATGACC TAGCTAGAGAGCTTTTCCAATACTTTCCAGATGAAATGTAATATACTCCACACAT AATTGTTGAGGTATATATATTAATCTATCACCCACATTTCCTTAATAACTTATACT TTTGAGCTAATTGTTTGGCACATACAAGATACAACTCACAATGCTATCAAAGCTC AGTTGGCTCGACCTTGATTATTGGCTAGGGAAATTCAAATAAAAAGATGGCACAA TATTCTTACTCGTATTCCAAGGGCTAGGCAAACTAAAATACGAGGTATATGTCAA AACTCAAAATGTGCGGTTTTGGGTGAATTATTGCTCAACACATTCAGCTCAACTC AATTCCCTAGGATATTTAACCCTCTCTTATAAGTACATTATTTATGTGCCAACCCA ATTGGAAGTGCTAATCTTGAATTTACTAACAAAAATGTCAAATTGCATACCCAAC TCACCCATATAAAAAGGCACCAACCCAACATTATTTTCGCCTAGTGTCGTCGCCG AACTCATTGCCATAACACTAAGACGAGCGCCAACCTTGTGGTGCTCTTGCCTCGC TGTTGCTGTCGCCCATGG [SEQ ID NO: 57], 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: 57 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 57 or a complement thereof, under at least medium or high stringency conditions; and
TCCACCGTTACTTGAAGGAGTGTCCATGAGAGCATGTGTGTCCCTAATGTG TTTTCTTTAAATATAGTTATCCTTTGGAAAGTTGTTATATTAATAAATCAAACAAA GATGGAATTTGTTGATGACTCGCGCTACAAACATCGGCCACTTGGATTCATGGAC ACCATACTGTTATAAAACGGAAACATGTGTTTGATTCGCTTTTTCTTTTCATGAAT CACAATATAAATATAGACATTCATGTGCGCACTCACACCTTATGGACAACTAATA TGTCTATGAGCATCTTCAAAAGACAAAGACGACAGATCTCAAGATTGTTGAGGTC ATAGCAAGCGTTGCACTCAAACGGGCATGTCGCTTATTAAAGAATAGCGAAGCA ATCAAGAATAAATTCAAAAAAATATGAGCAAGCACATCAATCGAGGACTTGACC AACTGGGTAGACATGTTCAATCACAAGAGGAGCTCAATTTCTAATCATGGCTAAT ATAATCAAACCCCACCCAGTATATTATTACTTAGAGCATCTCCAATAGTTTTTTTT AAAAAATCATCGCAAAAAAAAGCTTTCAAAAAAATAATTGGTAAATCAATGAGA TTTACAAGTCGCTAAAAAAGTTGGAGGCGTAATTGTTGGGGCTTTGACTTTTTTCC CCGTTTATAAAATCACGCTCCTAAAGTATAGAAAACAATTTTGCATTAGGAATCT TAAAATATTTTCAAATTACCCTAATCATTTTTATACTTTTTTTCTTTCTTGTATATTT GCATTTTGGGAACCCGATTAGAAATCTAATCATGGTTAATCAAACCCCAGTTATC GGATTTAGAAACAAAAACGGCAAATTGCATACCCAACTCATCGCTCATATAAAAC GGCACCAACCCAACATATTTCCACCTAATGCCGTCGCCCAACTAATTGCCATAAC ACTAAGTCGAGCGCCAACCTTGCGGTGCTCTGCTTGCCTCGCTGTTGCTGTTGCCC ATGG [SEQ ID NO: 58], 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: 58 or a complement thereof, or a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 58 or a complement thereof, under at least medium or high stringency conditions.
64. An isolated nucleic acid molecule, which comprises a promoter sequence that is operable in plant cells, wherein the promoter sequence comprises a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof;
(ii) 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 any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof; and
(iii) a nucleotide sequence that hybridizes to the sequence set forth in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58 or a complement thereof, under at least medium or high stringency conditions,
65. An isolated nucleic acid molecule comprising a chimeric promoter sequence that comprises an heterologous core promoter sequence in operable connection with a nucleotide sequence selected from the group consisting of :
(1) a nucleotide sequence corresponding to at least one segment selected from segments Λ, Φ, Ψ, Z, Δ and Θ of SEQ ID NO: 47-58, as defined herein, or (2) 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 of the at least one segment or a complement thereof, or
(3) a nucleotide sequence that hybridizes to the sequence s of the at least one segment or a complement thereof, under at least medium or high stringency conditions.
66. An isolated nucleic acid molecule according to any one of claims 1 to 65, comprising plant transcriptional regulatory elements able to confer expression preferentially in a plant sink tissue.
67. An isolated nucleic acid molecule according to claim 66, wherein the plant sink tissue is a mature plant carbohydrate storage tissue.
68. An isolated nucleic acid molecule according to claim 67, wherein the mature plant carbohydrate storage tissue is a sugarcane mature stem tissue.
69. A chimeric nucleic acid construct comprising an isolated nucleic acid molecule according to any one of claims 1 to 68 in operable connection with a heterologous nucleic acid sequence to be transcribed.
70. A chimeric construct according to claim 69, further comprising a 3' non-translated sequence that is operably connected 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.
71. A method for expression of a heterologous nucleic acid sequence, the method comprising introducing into a plant cell a chimeric construct according to claim 69 or claim 70.
72. A method for producing a transformed plant cell, the method comprising introducing into a plant cell a chimeric construct according to claim 69 or claim 70.
73. A method for producing transformed plant cells, the method comprising introducing into regenerable plant cells a chimeric construct according to claim 69 or claim 70 so as to yield transformed plant cells and identifying or selecting the transformed plant cells.
74. A method for selecting stable genetic transformants from transformed plant cells, the method comprising introducing into regenerable plant cells a chimeric construct according to claim 69 or claim 70 so as to yield transformed plant cells and identifying or selecting a transformed plant cell line from the transformed plant cells.
75. A method for producing a differentiated transgenic plant, the method comprising introducing a chimeric construct according to claim 69 or claim 70 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.
76. A method according to claim 75, wherein expression of the chimeric construct renders the differentiated transgenic plant identifiable over the corresponding non-transgenic plant.
77. A differentiated transgenic plant comprising plant cells containing a chimeric construct according to claim 69 or claim 70.
78. Cells, tissues, leaves, fruit, flowers, seeds or other plant reproductive material, material used for vegetative propagation, progeny plants including Fl hybrids, male-sterile plants and all other plants and plant products derived from the differentiated transgenic plant of claim 77.
79. An isolated nucleic acid molecule 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: 59.
PCT/AU2009/001407 2008-10-27 2009-10-27 Transcriptional control elements and uses therefor - ii Ceased WO2010048666A1 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 19 August 2003 (2003-08-19), Database accession no. CG066298. *
XIANG, Y. ET AL.: "Characterization of Stress-Responsive CIPK Genes in Rice for Stress Tolerance Improvement.", PLANT PHYSIOLOGY, vol. 144, no. 3, 2007, pages 1416 - 1428 *

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