WO2010012034A1 - Séquences de contrôle de la transcription actives dans les fleurs et/ou les graines - Google Patents

Séquences de contrôle de la transcription actives dans les fleurs et/ou les graines Download PDF

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Publication number
WO2010012034A1
WO2010012034A1 PCT/AU2009/000968 AU2009000968W WO2010012034A1 WO 2010012034 A1 WO2010012034 A1 WO 2010012034A1 AU 2009000968 W AU2009000968 W AU 2009000968W WO 2010012034 A1 WO2010012034 A1 WO 2010012034A1
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Prior art keywords
plant
nucleic acid
seq
nucleotide sequence
transcriptional control
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PCT/AU2009/000968
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English (en)
Inventor
Sergiy Lopato
Nataliya Kovalchuk
Ming Li
Ainur Ismagul
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Australian Centre For Plant Functional Genomics Pty Ltd
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Priority claimed from AU2008904485A external-priority patent/AU2008904485A0/en
Application filed by Australian Centre For Plant Functional Genomics Pty Ltd filed Critical Australian Centre For Plant Functional Genomics Pty Ltd
Priority to AU2009276290A priority Critical patent/AU2009276290B2/en
Publication of WO2010012034A1 publication Critical patent/WO2010012034A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters

Definitions

  • the present invention relates generally to transcriptional control sequences for effecting expression of a nucleotide sequence of interest in a plant. More particularly, the present invention relates to transcriptional control sequences that direct specific or preferential expression of an operably connected nucleotide sequence of interest in a plant flower and/or seed or one or more particular cell or tissue types therein.
  • Enhancement of resistance of developing and germinating grain to pathogens requires identification and characterisation of suitable pathogen responsive (PR) and resistance (R) genes and strategies for modulation of their expression in transgenic plants.
  • PR pathogen responsive
  • R resistance
  • tissue specific promoters might be the most useful as they limit the gene expression to infection sites.
  • Promoters active in the pericarp of cereals would be beneficial for the targeting of transgenes to these tissues.
  • Such promoters for example, may have application in the mediation of fungal diseases aitecting wheat and barley crops, such as infections of Fusa ⁇ nm gramineanim.
  • promoters which can specifically or preferentially direct expression of a gene of interest in at least the pericarp of a plant seed would be desirable.
  • the present invention is predicated, in part, on the identification and functional characterisation of transcriptional control sequences which specifically or preferentially direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed.
  • the present invention provides an isolated nucleic acid comprising:
  • nucleotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression ot an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control sequence is derived from a gene which em odes a v-thi ⁇ nin; or
  • nucleotide sequence defining a functionally active fragment or variant of the nucleotide sequence defined at (i).
  • the transcriptional control sequence directs expression ot an operably connected nucleotide sequence in at least an ovary or ovule of a flower. In some embodiments, the transcriptional control sequence directs expression of an o per ably connected nucleotide sequence in at least the pericarp ot an immature seed.
  • the present invention also provides a nudeic acid construct comprising an isolated nucleic acid according to the tirst aspect ot the invention.
  • the present invention provides a cell comprising a nucleic acid construct according to the second aspect of the invention.
  • the cells contemplated by the third aspect of the invention include any prokaryotic or eukaryotic cell.
  • the cell is a plant cell
  • ⁇ he present invention contemplates a multicellular structure comprising one or more cells ai cording to the third aspei t ot the invention.
  • the multicellular structure comprises a plant or a part, organ or tissue thereof.
  • a nucleotide sequence of interest may be operably connected to the transcriptional control sequence or the functionally active tragment or variant thereof, such that the nucleotide sequence of interest is specifically or preferentially expressed in a flower and/or seed of the plant.
  • the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant flower and/or seed, ⁇ he method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control or a nudeic acid according to the first aspect of the invention
  • the nucleotide sequence of interest is expressed in at least an ovary or ovule of a ⁇ lower In some embodiments, the nucleotide sequence of interest is expressed in at least the pericarp of an immature seed.
  • Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of fhe sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification.
  • the present invention is predicated, in part, on the identification and functional characterisation of transcriptional control sequences which specifically or preferentially direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed.
  • the present invention is predicated, in part, on the cloning of y- thionin like defensins and their associated promoters from wheat and rice.
  • transcriptional control sequence should be understood as a nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence.
  • the transcriptional control sequences of the present invention may comprise any one or more of, for example, a leader, promoter, enhancer or upstream activating sequence.
  • transcriptional control sequence preferably at least includes a promoter, A ''promoter” as referred to herein, em om parses any nucleic acid that coolers, activates or enhances expression ot an operably connected nucleotide sequence in a cell.
  • operably connected refers to the connection of a transcriptional control sequence, such as a promoter, and a nucleotide sequence of interest in such a way as to bring the nuc leotide sequence of interest under the transcriptional control of the transcriptional control sequence
  • promoters are generally positioned 5' (upstream) of a nucleotide sequence to be opera bly connected to the promoter.
  • the promoter In the construction of heterologous transcriptional control sequence/nudeotide sequence of interest combinations, it is generally preferred to position the promoter at a distance trom the fransi ri prion start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e the gene from W ' hich the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss ol promoter 1 unction
  • nuileic acid comprising:
  • nucleotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control sequence is derived trom a gene which em ode « a v-thi ⁇ nin; or
  • isolated nucleic acid refers to material removed trom its original environment (e.g. the natural environment if it is naturally oicurring) and thus is altered “by the hand of man” from its natural state.
  • an isolated nucleic acid could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the nucleic acid.
  • An “isolated” nucleic acid molecule should also be understood to include a synthetic nucleic acid molecule, including those produced by chemical synthesis using known methods in the art or by in-vi ⁇ ro amplification (e.g. polymerase chain reaction and the like).
  • the isolated nucleic acid of the present invention may comprise any polyribonucleotide or poJydeoxyribonudeotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • the isolated nucleic acid molecules of the invention may comprise single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • the isolated nucleic acid molecules may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA, Hie isolated nucleic acid molecules may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified” bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus the term “nucleic acid” also embraces chemically, enzymatically, or metabolically modified forms of DNA and RNA.
  • the method of the present invention contemplates a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed.
  • flowers would be readily understood by a person skilled in the art. However, for clarity, flowers generally include at least some of the main structures as follows (although not all flowers within the meaning of the term as used herein will include all of the structures noted below):
  • Calyx the outer whorl of sepals; typically these are green, but are petal -like in Cowl In: the whorl of petals, which are usually thin, soft and colored to attract insects that help the process of pollination.
  • Andi oecium one or two whorls of stamens, each a filament topped by an anther where pollen is produced. Pollen contains the male gametes.
  • Gvnoecium one or more pistils.
  • the female reproductive organ is the i arpel: this contains an ovary with ovules (which contain female gametes).
  • a pistil may comprise a number of carpels merged together, in which case there is only one pistil to each flower, or of a single individual carpel (the flower is then called apocarpous).
  • the sticky tip of the pistil, the stigma is the receptor of pollen.
  • the supportive stalk, the style becomes the pathway for pollen tubes to grow from pollen grains adhering to the stigma, to the ovules, i arrying the repr ⁇ diu tive material.
  • the floral structure described above is considered the "typical" structural plan
  • plant species show a wide variety of modifications from this plan.
  • 1- 1 the two subclasses of flowering plants may be distinguished by the number of floral organs in eaih whorl: dicotyledons typii ally having 4 or 5 organs (or a multiple of 4 or 5) in each whorl and monocotyledons having three or some multiple of three.
  • flowers should also be understood to include immature flowers prior to anthesis, mature flowers after anthesis as well as fertilised flowers. Furthermore, the term “flower” should also be understood to include flowers which may comprise a mature or immature seed and structures. Such flowers generally include fertilised flowers, but may also include unfertilised flowers in which a seed develops, such as by the process of apomixis.
  • seed encompasses whole seeds as well as the various cells and tissues that make up a mature or immature seed.
  • seeds may include tissue types such as the embryo, embryo surrounding region, endosperm transfer layer, endosperm, aleurone layer, pericarp and the like.
  • immature seeds may include, for example, fertilised egg cells, zygotes, fertilised central cells, embryos, the endosperm coenocyte, the endosperm syncytium and the like.
  • seed as used herein may also include one or more immature plant structures associated with a germinating seed, including, for example, the embryo, scuteflurn or cotyledon(s), radicle, coleorhiza, coleoptile, hypocotyl and epicotyl.
  • the present invention contemplates plants of the grass family (Poaceae) and cereal crop plants.
  • the term "flower” should be understood to include a floret of such plants, which may include support structures such as the lemma and palea.
  • the term flower or floret should also be understood to include florets in the grass family which may include a seed, grain or caryopsis.
  • reference herein to expression in a plant flower and/or seed refers to the transcription and/or translation of a nucleotide sequence in one or more cells or tissues of a plant flower and/or seed and/or at one or more developmental stages of the plant flower and/or seed. This in no way implies that expression of the nucleotide sequence must occur in all cells of the plant flower and/or seed or at all developmental stages of the flower and/or seed. As set out later, the nucleic acids of the present invention may direct expression in particular parts of a flower and/or seed and/or at particular developmental stages of a flower and/or seed.
  • the transcriptional control sequences contemplated by the present invention direct expression of an operably connected nucleotide sequence in a plant flower and/or seed.
  • specifically expressing means that the nucleotide sequence of interest is expressed substantially only in a plant flower and/or seed (or a particular tissue or ceil type therein).
  • Preferentially expressing should be understood to mean that the nucleotide sequence of interest is expressed at a higher level in a plant flower and/or seed (or tissue or cell type therein) than in one or more other tissues of the plant, e.g. mature leaf or stem tissue.
  • preferential expression in a plant flower and/or seed includes expression of a nucleotide sequence of interest in a plant flower and/or seed (or a tissue or cell type therein) at a level of, for example, at least twice, at least 5 times or at least 10 times the level of expression seen in at least one other non-flower tissue and/or non-seed tissue of the plant.
  • the transcriptional control sequence or functionally active fragment or variant thereof may effect specific or preferential expression in a flower and/or seed from at least one flowering plant species, including monocotyledonous angiosperm plants ("monocots") or dicotyledonous angiosperm plants ("dicots").
  • monocots monocotyledonous angiosperm plants
  • dicotyledonous angiosperm plants dicotyledonous angiosperm plants
  • the transcriptional control sequence may or may not effect expression in one or more other plant species, and this expression may or may not be specific or preferential to the flower and/or seed.
  • the transcriptional control sequences of the present invention need not be active in all plant species, and need not necessarily direct specific or preferential expression in the flower and/or seed in all plants in which they are active.
  • the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a flower and/or seed of a monocotyledonous plant.
  • the transcriptional control sequence specifically or preferentially directs expression of an opcrably connected nucleotide sequence in a flower and/or seed of a plant in the family Poaceae.
  • the transcriptional control sequence specifically or preferentially directs expression of an operabiy connected nucleotide sequence in a flower and/or seed of a cereal crop plant.
  • the term "cereal crop plant” may be a member of the Poaceae (grass family) that produces grain.
  • Examples of Poaceae cereal crop plants include wheat, rice, maize, millets, sorghum, rye, triticale, oats, barley, teff, wild rice, spelt and the like.
  • the term cereal crop plant should also be understood to include a number of non- Poaceae plant species that also produce edible grain, which are known as the pseudocereals and include, for example, amaranth, buckwheat and q ⁇ inoa.
  • the transcriptional control sequence specifically or preferentially directs expression of an operabiy connected nucleotide sequence in a flower and/or seed of a wheat plant.
  • wheat should be understood as a plant of the genus Triticuin.
  • the term “wheat” encompasses diploid wheat, tetraploid wheat and hexaploid wheat.
  • the wheat plant may be a cultivated species of wheat including, for example, T, aestivum, T, durum, T, monococcum or T. spclta.
  • the term “wheat” refers to wheat of the species TrW cum aesthrum.
  • the transcriptional control sequence specifically or preferentially directs expression of an operabiy connected nucleotide sequence in a flower and/or seed of a rice plant.
  • "rice” includes several members of the genus Oryza including the species Oryza sativa and Oryza giaberrima. Hie term “rice” thus encompasses rice cuitivars such as japonica or sinica varieties, indica varieties and javonica varieties. In some embodiments, the term “rice” refers to rice of the species Oryza sativa.
  • the nucleic add of the first aspect of the present invention may also specifically or preferentially direct expression in a particular cell or tissue of a plant flower and/or seed and/or specifically or preferentially direct expression at a particular developmental stage of a plant flower and/or seed.
  • the nucleic acid according to the first aspect of the invention comprises a transcriptional control sequence which directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower at one or more developmental stages selected from before anthesis, after anthesis or after fertilisation.
  • the transcriptional control sequence directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower selected from: an ovary or ovule, the lemma and/or palea (in flowers having such structures) or an anther, In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in at least an ovary or ovule of a flower.
  • the nucleic acid according to the first aspect of the invention comprises a transcriptional control sequence which directs expression of an operably connected nucleotide sequence in one or more parts of a plant seed at one or more developmental stages selected from: immature seed or germinating seed,
  • the transcriptional control sequence directs expression of an operably connected nucleotide sequence in one or more parts of a plant seed selected from: an immature seed in a flower, one or both poles of an immature seed, the pericarp of a seed, an embryo in a seed, vascular tissue in a seed, or one or more structures associated with a germinating seed (including, for example, the embryo, scutellum or cotyledon(s), radicle, coleorhiza, coleoptile, hypocotyl and cpicotyl).
  • the transcriptional control sequence directs expression of an operabiy connected nucleotide sequence in at least the pericarp of an immature seed.
  • the "pericarp" of a seed should be understood to encompass...
  • the pericarp is the tissue that develops from the ovary wall of the flower and surrounds the seeds of a plant.
  • the pericarp itself is typically made up of three distinct layers: the exocarp, which is the most-outside layer, the mesocarp, which is the middle layer, and the endocarp, which is the inner layer surrounding the ovary or the seeds.
  • the grains of grasses are single-seed simple fruits wherein the pericarp (ovary wall) and seed coat are fused into one layer. This type of fruit may be referred to as a caryopsis.
  • Examples include cereal grains, such as wheat, barley, and rice.
  • expression in one or more particular parts of the flower and/or seed may occur at one or more specific developmental stages of the flower and/or seed, including, for example, the developmental stages of the flower and/or seed during which the particular part exists.
  • expression in a seed or caryopsis of the flower would be restricted to a developmental stage of the flower when a seed or caryopsis is present in the flower.
  • the present invention contemplates transcriptional control sequences which specifically or preferentially direct expression of an operabiy connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control sequence is derived from a gene which encodes a y-thionin
  • a transcriptional control sequence derived from a gene which encodes a ⁇ -thionin refers fo a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a gene which encodes a ⁇ -thionin in a plant.
  • the term derived from should also be understood to refer to the source of the sequence information for a transcriptional control sequence and not be limited to the source of a nucleic acid itself.
  • a transcriptional control sequence derived from a gene which encodes a ⁇ -thionin need not necessarily be directly isolated from the gene.
  • a synthetic nucleic acid having a sequence that is determined with reference to a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a gene which encodes a y-thionin should be considered derived from a gene which encodes a ⁇ -thionin.
  • Beta-thionins are small evolutionarily related proteins of plants that function as defensive proteins from pathogens and/or parasites. In their mature form, ⁇ -thionins generally consist of about 45 to 85 amino-acid residues. However, ⁇ -thionins of larger or smaller sizes may also fail within the scope of the term ⁇ -thionins as referred to herein.
  • Plant ⁇ -thionins have three dimensional structures similar to defensins from insects. Thus, they may also be referred to as "plant defensins" due to structural and functional similarities to animal defensins. Defensins demonstrate anti-bacterial and or antifungal activity as a result of pathogen membrane permeabilization, in high concentrations plant defensins can be potentially toxic to insects because of their ability to inhibit animal ⁇ -amylases and proteinases.
  • the folded structure of gamma-p ⁇ rothionin is characterised by a well-defined 3- stranded anti-parallel beta-sheet and a short alpha-helix. Three disulfide bridges are located in the hydrophobic core between the helix and sheet, forming a cysteine- stabilised alpha -helical motif.
  • This structure differs from that of the plant alpha- and beta-thionins, but is analogous to scorpion toxins and insect defensins. Examples of known y-thionins include:
  • Gamma- thionins from l riticum aestivum endosperm (gamma -purothionins) and gamma-hordothionins from Hordeum vulgare which are toxic to animal cell" and inhibit protein synthesis in cell free systems;
  • Antifungal proteins from the seeds of Brassicaceae species such as radish, mustard, turnip and Awbidopsis th ⁇ l ⁇ on ⁇ ;
  • Anther-specific protein SFlS from sunflower, which contains a gamma- thiorun domain at ils X -terminus and a proline- rich C -terminal domain;
  • the ⁇ -thionin contemplated in accordance with the present invention comprises the amino acid sequence set forth in SEQ ID NO: 1 or a homolog thereof
  • homoiog as used herein with reference to homologs of polypeptides comprising the ammo acid sequence set forth in SEQ ID NO 1, should be understood to include, for example, homologs, orthologs, paralogs, mutants and variants of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 1.
  • the homolog, orth ⁇ log, paralog, mutant or variant of a polypeptide comprising the amino acid sequence set forth in SFQ ID NO: 1 comprises an amino acid sequence which comprises at least °>5% sequence identity, at least 40% sequence identity, at least 45% sequence identity, at least 50% sequence identity, at least 55% sequence identify, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identitv, at least 75% sequence identity, at least 80 0 O sequent e identity, at least S5% sequence identity, ai least 90°/ « sequence identity or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • the compared sequences should be compared over a comparison window of at least 20 amino acid residues, at least 40 amino add residues, at least 60 amino acid residues, at least 80 amino acid residues, or over the full length of SEQ ID NO: 1.
  • the comparison window may comprise additions or deletions (i.e. gaps) or 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 aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. ⁇ Nucl. Adds Res. 25: 3389-3402, 1997). 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 !nc, 1994-1998, Chapter 15, 1998).
  • Examples of homologs of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 include for example: a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, which is derived from Jtiticum aesti ⁇ um. a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, whidi is derived from Qryza saliva; and a polypeptide comprising an amino acid sequence selected from the list consisting of SEQ ID NO: 13, SEQ ID NO: IS, SEQ ID NO: 23, SEQ ID NO: 28 or SEQ ID NO: 33, each of which are derived from T ⁇ ticuni durum.
  • the transcriptional control sequences of the present invention may be derived from any source, including isolated from any suitable organism or they may be synthetic nucleic acid molecules. In some embodiments, however, the transcriptional control sequences contemplated herein are derived from a plant. In some embodiments, the transcriptional control sequences of the present invention are derived from a monocot plant species. In some embodiments the transcriptional control sequences of the present invention are derived from a plant in the family Poaceae, In some embodiments, the transcriptional control sequences of the present invention are derived from a cereal crop plant species.
  • the transcriptional control sequence is derived from a TrW cum species (for example T. aesti ⁇ um, T. diu urn, T. monococcum, T. dicocvn, T. spelta or T. polonicum).
  • the transcriptional control sequence is derived from a tetraploid wheat (for example T. dw um, T. dicoccon, or T. polonicum).
  • the transcriptional control sequence is derived from a durum wheat, and in some embodiments, the transcriptional control sequence is derived from TrWc um durum.
  • the transcriptional control sequence is derived from a gene whidi comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a homolog thereof.
  • One example of a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SFQ ID ⁇ O: 2 is a gene comprising the nucleotide sequence set forth in SEQ ID NO: 4.
  • ''homolog as used herein with reference to homologs of genes comprising an open reading frame comprising the nucleotide sequence sot forth in SEQ ID XO: 2, should be understood to include, for example, homologs, orthologs, paralogs, mutants and variants of genes comprising an open reading frame which comprises the nucleotide sequence set forth in SuQ ID NO: 2.
  • the homolog, orfholog, paralog, mutant or variant of a polypeptide comprising an open reading frame which comprises the nucleotide sequent e set forth in SEQ ID NO: 2 c omprises a nudeoiide sequence which comprises at least 35% sequence identity, at least 40% sequence identity, at least 45% sequence identity, at least 50% sequence identity, at least 53% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequent e identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity or at least 95% sequence identity to the nucleotide sequence set forth in SHQ ID NO: 2.
  • the compared sequences should be compared over a comparison window of at least 20 nucleotide residues, at least ⁇ O nucleotide residues, at least 100 nucleotide residues, at least 150 nucleotide residues, at least 200 nucleotide residues or over the full length of SHQ ID NO: 2, lhe comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the relerence sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such the BI AST family of programs as, for evample, disclosed by Allsdiul et al. (Nucl. Acids Res, 25: 3389-3402. 1997). ⁇ detailed discussion of sequence analysis can be found in Unit l q .3 of ⁇ usubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1WS, Chapter 15, 1998).
  • One example of a gene comprising a homolog of an open reading frame comprising the nucleotide sequence set forth in SEQ IL) NO: 2 includes a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 7.
  • genes comprising an open reading frame homologous to the nucleotide sequence set forth in SEQ ID NO: 2 include: a gene comprising an open reading frame comprising the nucleotide sequence set forth in SHQ ID NO: q ; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 11; a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 14; and/or a gene which comprises the nucleotide sequence set tenth in SEQ ID NO: 16; a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 19; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 21; a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 24; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 26; a gene comprising an open
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set torth in SEQ ID NO: 3 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 10 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 15 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 20 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 25 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 30 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 35 or a functionally active fragment or variant thereof.
  • the present invention also contemplates ''functionally active fragments or variants" of the transcriptional control sequences of the present invention, including (but not limited to) functionally active fragments or variants of a transcriptional control sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35.
  • the functionally active fragment is at least 200 nucleotides (nt), at least 500 nt, at least 1000 nt, at least 1500 nt or at least 2000 nt in length.
  • the fragment comprises at least 200 nt, at least 500 nt, at least 1000 nt or at least 1500 nt contiguous bases from the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ⁇ D NO: 20, SEQ ID NO: 25, SEQ ⁇ D NO: 30 or SEQ ID NO: 3
  • “Functionally active variants” of the transcriptional control sequence of the invention include orthoiogs, mutants, synthetic variants, analogs and the like which are capable of effecting transcriptional control of an operably connected nucleotide sequence in a plant flower and/or seed (or a particular cell or tissue type thereof as hereinbefore described) in at least one plant type.
  • variant should be considered to specifically include, for example, orthologous transcriptional control sequences from other organisms; mutants of the transcriptional control sequence; variants of the transcriptional control sequence wherein one or more of the nucleotides within the sequence has been substituted, added or deleted; and analogs that contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified” bases include, for example, tritylated bases and unusual bases such as inosine.
  • the functionally active fragment or variant comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleotide sequence identity to the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ TD NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35.
  • the compared nucleotide sequences should be compared over a comparison window of at least 200 nucleotide residues, at least 400 nucleotide residues, at least 1000 nucleotide residues, at least 1500 nucleotide residues or over the full length of any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35.
  • 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 - 11 -
  • aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et a (1997, supra). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et a (1998, supra).
  • the functionally active fragment or variant comprises a nucleic acid molecule which hybridises to a nucleic acid molecule defining a transcriptional control sequence of the present invention under stringent conditions.
  • the functionally active fragment or variant comprises a nucleic acid molecule which hybridises to a nucleic acid molecule comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ TD NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35 under stringent conditions,
  • stringent hybridisation conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8,3 and the temperature is at least 30 0 C. Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. In some embodiments, stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions.
  • Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5x to IxSSC at 55 to 60 0 C.
  • Exemplary high stringency conditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.IxSSC at 60 to 65°C.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridisation is generally less than 24 hours, usually 4 to 12 hours.
  • hybridisation is also a function of post-hybridisation washes, with the - 71 -
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe.
  • T- is reduced by about i°C for each 1% of mismatching; thus, T n , hybridisation, and/or wash conditions can be adjusted to hybridise to sequences of different degrees of complementarity.
  • sequences with >90% identity can be hybridised by decreasing the Tm by about 10 0 C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T-) for the specific sequence and its complement at a defined ionic strength and pH.
  • high stringency conditions i an utilise a hybridisation and/or wash at, for example, 1, 2, 3, or 4°C lower than the thermal melting point (T- );
  • medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, S, ⁇ J, or 10 0 C lower than the thermal melting point ( Im);
  • low stringency conditions can utilise a hybridisation and/or wash at, for example, 11, 12, 13, 14, 15, or 20 0 C lower than the thermal melting point (T-), Lsing the equation, hybridisation and wash compositions, and desired T-, those of ordinary skill will understand that variations in the stringency of hybridisation and/or wash solutions are inherently described.
  • the present invention also provides a nucleic acid construct comprising an isolated nucleic acid according to the first aspect of the invention.
  • the nucleic acid construct of the second aspect of the present invention may comprise any polyribonucleotide or polydeoxyribonudeotide, which may be unmodified RNA or DNA or modified RNA or DNA,
  • the nucleic acid construct of the invention may comprise single- and/or double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • the nucleic acid construct may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the nucleic acid construct may also comprise one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus the term “nucleic acid construct” embraces chemically, enzymatically, or metabolically modified forms.
  • the nucleic acid construct comprises DNA. Accordingly, the nucleic acid construct of the present invention may comprise, for example, a linear DNA molecule, a plasmid, a transposon, a cosmid, an artificial chromosome or the like. Furthermore, the nucleic acid construct of the present invention may be a separate nucleic acid molecule or may be a part of a larger nucleic acid molecule.
  • the nucleic acid construct further comprises a nucleotide sequence of interest that is heterologous with respect to the transcriptional control sequence or the functionally active fragment or variant thereof; wherein the nucleotide sequence of interest is operably connected to the transcriptional control sequence or functionally active fragment or variant thereof.
  • heterologous with respect to the transcriptional control sequence refers Io the nucleotide sequence of interest being any nucleotide sequence other than that which the transcriptional control sequence (or functionally active fragment or variant thereof) is operabiy connected to in its natural state.
  • SEQ ID NO: 3 is operabiy connected io the nucleotide sequence sei forth in SEQ ID NO: 4.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 4 should be considered heterologous with respect to SEQ ID NO: 3.
  • SEQ TD NO: 10 is operabiy connected to SEQ ID NO: 11 in its natural state.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 11 should be considered heterologous with respect to SEQ ID NO: 10.
  • SEQ TD NO: 15 is operabiy connected to SEQ ID NO: 16 in its natural state.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 16 should be considered heterologous with respect to SEQ ID NO: 15.
  • SEQ TD NO: 20 is operabiy connected to SEQ ID NO: 21 in its natural state.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21 should be considered heterologous with respect to SEQ ID NO: 20.
  • SEQ TD NO: 25 is operabiy connected to SEQ ID NO: 26 in its natural state.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 26 should be considered heterologous with respect to SEQ ID NO: 25.
  • SHQ ID ⁇ O 30 is operably connected Io SEQ ID XO: 31 in its natural stale. ⁇ s such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SIiQ ID KO 31 should be considered heterologous with respect to SFQ ID MO: 30.
  • SHQ ID ⁇ O 35 is operably connected to SEQ ID XO: 36 in its natural state.
  • any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SIiQ ID KO 36 should be considered heterologous with respect to SFQ ID MO: 35.
  • nucleotide sequence of interest which is heterologous to the transcriptional control sequence (or lunctionally active lragment or variant thereof) may be derived from an organism of a different taxon to the transcriptional control sequence (or functionally active fragment or variant thereof) or the nucleotide sequent e of interest may be a heterologous sequence from an organism ot the same taxon.
  • the nucleic acid construct may further comprise a nucleotide sequence defining a transcription terminator.
  • transcription terminator * ' or ''terminator refers to a DXA sequence at the end ot a transcriptional unit which signals termination of transcription. Terminators are generally 3'-non-translated DNA sequences and may contain a polyadenylation signal, which facilitates the addition of poiyadenylate sequences to the 3'-end of a primary transcript As with promoter sequences, the terminator may be any terminator sequence which is operable in the i ells, tissues or organs in which if is intended to be used.
  • nucleic acid construct comprises an expression cassette comprising the structure:
  • jN]-, comprises one or more nucleotide residues, or is absent;
  • TCS comprises a nucleic acid according to any one of the first aspect of the invention; comprises one or more nucleotide residues, or is absent; SoI comprises a nucleotide sequence of interest which is operably connected to TCS; [N]y comprises one or more nucleotide residues, or is absent; TT comprises a nucleotide sequence defining a transcription terminator; [N]z comprises one or more nucleotide residues, or is absent.
  • nucleic acid constructs of the present invention may further comprise other nucleotide sequences as desired.
  • the nucleic acid construct may include an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts or the like.
  • the term ''selectable marker gene includes any gene that confers a phenotype on a cell, in which it is expressed, to facilitate the identification and/or selection of ceils which are transfected or transformed with a nucleic acid construct of the invention.
  • a range of nucleotide sequences encoding suitable selectable markers are known in the art.
  • Exemplary nucleotide sequences that encode selectable markers include: antibiotic resistance genes such as ampicillin-resistance genes, tetracycline- resistance genes, kanamyciri -resistance genes, the AURI-C gene which confers resistance to the antibiotic a ⁇ reobasidin A, neomycin phosphotransferase genes (e.g.
  • r ⁇ tl and r ⁇ tlF r ⁇ tl and r ⁇ tlF
  • hygromycin phosphotransferase genes e.g. hpt
  • herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transferase-encoding genes (e.g. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate ⁇ -phosphate synthase-encoding genes (e.g. aro ⁇ ), bromyxnil resistance genes including bromyxnil nitrilase-encoding genes, sulfonamide resistance genes including dihydropteratc synthase-encoding genes (e.g.
  • sul and sulfonylurea resistance genes including aeetolaetate synthase- encoding genes; enzyme-encoding reporter genes such as GUS-encoding and cWoramphenicoiacetyl transferase (CAT)-encoding genes; fluorescent reporter genes such as the green fluorescent protein-encoding gene; and luminescence-based reporter genes such as the ludf erase gene, amongst others,
  • constructs described herein may further include nucleotide sequences intended for the maintenance and/or replication of the construct in prokaryotes or eukaryotes and/or the integration of the construct or a part thereof into the genome of a eukaryotic or prokaryotic cell.
  • the construct of the invention is adapted to be at least partially- transferred into a plant cell via Agrohicterium-raediated transformation.
  • the nucleic acid construct of the present invention comprises left and/or right T-DNA border sequences. Suitable 1'-DNA border sequences would be readily ascertained by one of skill in the art.
  • T-DNA border sequences should be understood to at least include, for example, any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrobaclerium sp. ceil into a plant cell susceptible to Agrobacterium-mediated transformation.
  • the present invention also contemplates any suitable modifications to the construct whidi facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobaciieriim sp., for example, as described in Broothaerts et . JQ _
  • the constructs of the second aspect of the invention may also comprise nucleotide sequences that encode regulatory microRNAs which may further modulate the expression pattern determined by the nucleotide sequence of the first aspect of the invention.
  • regulatory activity of microRNAs in plants may be found in the review of Jones-Rhoades et al. (Annual Review of Plant Biology 57: 19-53, 2006)
  • the present invention provides a cell comprising a nucleic acid construct according to the second aspect of the invention.
  • the nucleic acid construct may be maintained in the cell as a nucleic acid molecule, as an autonomously replicating genetic element (e.g. a plasmid, cosmid, artificial chromosome or the like) or it may be integrated into the genomic DNA of the cell.
  • an autonomously replicating genetic element e.g. a plasmid, cosmid, artificial chromosome or the like
  • genomic DNA should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell.
  • genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous plasmid DNA arid the like.
  • the term “genomically integrated” contemplates chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, and the like.
  • a “genomically integrated form" of the construct may be all or part of the construct. However, in one particular embodiment the genorrucalJy integrated form of the construct at least includes the nucleic acid molecule of the first aspect of the invention.
  • the cells contemplated by the third aspect of the invention include any prokaryotic or eukaryotk cell.
  • the cell is a plant cell.
  • the cell is a monocot plant cell.
  • the cell is a cell from a plant in the family Poaceae.
  • the cell is a cereal crop plant cell and in one particular embodiment the ceil is a wheat cell.
  • the cell is a rice cell.
  • the cell may also comprise a prokaryotic cell.
  • the prokaryotic cell may include an Agrobacterium sp. cell (or other bacterial cell), which carries the nucleic acid construct and which may, for example, be used to transform a plant.
  • the prokaryotic cell may be a cell used in the construction or cloning of the nucleic acid construct (e.g. an E. coll cell).
  • the present invention contemplates a multicellular structure comprising one or more cells according to the third aspect of the invention.
  • the multicellular structure comprises a plant or a part, organ or tissue thereof.
  • a plant or a part, organ or tissue thereof should be understood to specifically include a whole plant; a plant tissue; a plant organ; a plant part; a plant embryo; and cultured plant tissue such as a callus or suspension culture.
  • the plant or part, organ or tissue thereof comprises reproductive material for a plant including, for example, flowers or parts thereof (including anthers and ovaries), seeds, vegetative plant material, explants, plant tissue in culture including callus or suspension culture and the like.
  • the plant or a part, organ or tissue thereof contemplated in the fourth aspect of the invention may include, for example, any of a monocot, a plant in the family l'oaceae, a cereal crop plant, a wheat plant or a rice plant or a part, organ or tissue of any of the foregoing.
  • the plant or part, organ or tissue thereof comprises a flower and/or seed.
  • a nucleotide sequence of interest may be operably connected to the transcriptional i ontrol sequence or the functionally active fragment or variant thereof, such that the nucleotide sequence of interest is specifically or preferentially expressed in a flower and/or seed, or in a particular cell or tissue type thereof, and optionally at a particular developmental stage, as described above with respect to the lirst aspect ot the invention.
  • the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant flower and/or seed, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of a nucleic acid according to the fir ⁇ t aspec t of the in v ention.
  • the present invention is predicated, in part, on effecting transcription of the nucleotide sequence of interest under the transcriptional control ot a transcriptional control sequence of the first aspect of the invention.
  • this is effected by introducing a nucleic acid molecule comprising the transcriptional control sequence, or a functionally active fragment or variant thereof, into a cell of the plant, such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence.
  • the nucleic add molecule may be introduced into the plant o ⁇ a any method known in the art.
  • an explant or cultured plant tissue may be transformed with a nucleic acid molecule, wherein the explant or cultured plant tissue is subsequently regenerated into a mature plant including the nucleic acid molecule; a nucleic acid may be directly transformed into a plant, either stably or transiently; a nucleic acid may be introduced into a plant via plant breeding using a parent plant that carries the nucleic acid molecule; and the like.
  • the nucleic acid molecule is introduced into a plant cell via transformation.
  • Plants may be transformed using any method known in the art that is appropriate for ⁇ he particular plant species. Common methods include Agrobactrrium- mediafed transformation, micro projectile bombardment based transformation methods and direct DNA uptake based methods.
  • Roa- Rodriguez et al. Agr ⁇ bac teriurn mediated transformation of plants, 3 rd Yd. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003
  • Other bacterial-mediated plant transformation methods may also be utilised, for example, see Broothaerfs et al. (2005, supra).
  • Microprojectilo bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, reviewed by Casas el al. ⁇ Plant Breeding Rev. 13: 235-264, 1995).
  • Direct D ⁇ A uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995).
  • a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway-, silicon carbide- and liposome mediated transformation.
  • the transcriptional control sequence of the present invention is introduced into a plant cell such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence and the present invention contemplates any method to effect this.
  • the subject transcriptional control sequence and a nucleotide sequence of interest may be incorporated into a nucleic acid molecule such that they are operably connected,, and this construct may be introduced into the target cell.
  • the nucleic acid sequence of the present invention may be inserted into the genome of a target cell such that it is placed in operable connection with an endogenous nucleic acid sequence.
  • the insertion of the transcriptional control sequence into the genome of a target cell may be either by non-site specific insertion using standard transformation vectors and protocols or by site-specific insertion, for example, as described in Terada et al. (Nat Bioteclmol 20: 1030-1034, 2002).
  • the nucleotide sequence of interest which is placed under the regulatory control of the transcriptional control sequence of the present invention, may be any nucleotide sequence of interest.
  • General categories of nucleotide sequences of interest include nucleotide sequences which encode, for example: reporter proteins, such as, GUS, GFP and the like; proteins involved in cellular metabolism such as Zinc finger proteins, kinases, heat shock proteins and the like; proteins involved in agronomic traits such as disease or pest resistance or herbicide resistance; proteins involved in grain characteristics such as grain biornass, nutritional value, post-harvest characteristics and the like; heterologous proteins, such as proteins encoding heterologous enzymes or structural proteins or proteins involved in biosynthetic pathways for heterologous products; "terminator" associated proteins such as barnase, barstar or diphtheria toxin.
  • reporter proteins such as, GUS, GFP and the like
  • proteins involved in cellular metabolism such as Zinc finger proteins, kinases, heat shock proteins and the like
  • nucleotide sequence of interest may alternatively encode a non- translated RNA, for example an si RNA, miRNA, antisense RNA and the like.
  • nucleotide sequence of interest may comprise, for example, a pathogen responsive (PR) gene, a resistance (R) gene or a defensin gene.
  • PR pathogen responsive
  • R resistance
  • the nucleotide sequence of interest may encode a protein such as PDR5 or TRIlOl.
  • proteins may be expressed in a flower or seed-specific manner in crop plants such as wheat in order to lower the incidence of diseases such as head blight (caused by Fusa ⁇ um grarmnearum or Gibberella zeae) and/or reduce mycotoxin levels within the seed.
  • the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a range of different plant flowers and/or seeds.
  • the plant may be a monocotyledonous plant.
  • the plant may be a plant in the family Poaceae.
  • the plant may be a cereal crop plant.
  • the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a wheat flower and/or seed.
  • the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a rice flower and/or seed.
  • the method of the present invention may be employed to specifically or preferentially effect expression of an opera bly connected nucleotide sequence in one or more parts of a plant flower and/or seed or at one or more developmental stages of a plant flower and/or seed as hereinbefore described.
  • the nucleotide sequence ol interest is heterologous with respect to the transcriptional control sequence, as defined bupta
  • Figure 1 shows (A) Northern blot hybridization of TuPRPI cDNA with total RMA isolated from different wheat tissues and gram at different stages of elopment.
  • B Expression ol OsPRPl in different rice tissues demonstrated by Quantitative PCR.
  • C Unrooted phylogenetic tree of wheal and rice PRPl piotein sequences and known or putative homologies from other plants (CADEM (Ace. ⁇ F442388), LePl (Ace AJ133601 1), I pSUN ( ⁇ cc ABO36641 ), 1 IaPI (Ace P82659), GmPI (Ace AAC97524), CaJl (Ace. X95363), CaPIl (Ace.
  • FIG. 1 shows Spatial and temporal GUS expression in wheat directed by the TdPRPI-
  • Control grain is on the right hand side of each picture, Al 7-A20 - transverse (A 17, A 19, and A20) and longitudinal (AlS) sections of T 1 grain at 3 DAP (Al 7), T 2 grain at 2 DAP (A19) and 5 DAP (A20) DAP, and Ti grain at 5 DAP (A18).
  • Figure 3 shows spatial and temporal GIJS expression in rice directed by the TdPRPl-IO promoter, GUS activity in rice detected in floral and grain tissues shortly before anthesis (Bl), at anthesis (B2), 5 DAP (B3), 8 DAP (B4), 15 DAP (Bo) and 18 DAP (B6).
  • B7-B11 - Histochemical GUS assay counters tained with safranin in: 10 ⁇ m thick longitudinal sections of anthers (B7) and ovule at anthesis (BS), grain at 5 DAP (B9, BIO), and in the vascular bundle of lemma (BIl). GUS expression in the vascular bundle of the anther and lemma and in the ovary is indicated with arrows.
  • FIG. 4 shows TdPRPl-I promoter activity in rice flower and grain.
  • FIG. 5 shows TdPRPl-Il promoter activity in rice flower and grain. GUS expression in flowers of two independent transgenic lines before anthesis (Dl and D2), in lemma at 1 DAP (D3), in flowers with removed lemma at 2 DAP (D4), 5 DAP (D5), and 9 DAP (D6).
  • FIG. 6 shows the activity of wheat promoters in germinating rice grain.
  • Figure 7 shows the activity of OsPRPl promoter in transgenic rice plants.
  • GUS activity detected 2-3 days before anthesis in vascular bundles of lemma and palea (Fl), the vascular bundles of filament, anthers, stamen and both poles of ovary of transgenic (F2) plants, but not in control plants.
  • Figure 8 shows the activity of the OsPRPI promoter in developing grain demonstrated by histochemicai GUS assay counterstained with safranin (G1-G4) in: 10 ⁇ m thick longitudinal section (Gl) and transverse sections (G2-G3) T2 grain of transgenic rice at 16 DAP.
  • GUS expression can be seen in the main vascular bundle and adjacent to vascular bundle cell layers. Magnification is shown in the corner of each picture.
  • the full length cDNA of TaPRPl was isolated from a cDNA library prepared from the whole grain of T ⁇ ti ⁇ um aestwuni at 0-6 DAP, A subsequent comparison of the nucleotide sequence of TaPKPl cDNA with wheat expressed sequence tags (ESTs) identified multiple ESTs with a high level of sequence identity (data not shown). Most of them originated from cDNA libraries prepared from ovary, pre-anthesis spikes, and 5-15 DAP spikes. The flower and grain specific expression of the gene was confirmed by northern blot hybridisation with RNA isolated from different wheat tissues ( Figure IA). A database search using TaPRPI protein sequence (SEQ ID NO: 1) identified this protein as a member of the v-thionin (clefensin) subfamily.
  • ESTs wheat expressed sequence tags
  • the full length cDNA of TaPRPI (SEQ ID NO: 7) was used as a probe to screen a bacterial artificial chromosome (BAC) library prepared from genomic DNA of Triiicum durum cv. Langdon using Southern blot hybridisation. Eleven BAC clones were selected for further analysis on the basis of the strength of the hybridisation signals.
  • BAC bacterial artificial chromosome
  • BAC DNA was isolated and used as a template for PCR with primers derived from the beginning and the end of the coding region of TnPRPL Five BAC clones gave PCR products. Sequencing of the PCR products revealed that the cloned inserts are genomic clones of close homologues from T.
  • TdPRPl 1 durum of TaPRPl 1 which were designated TdPRPl- I 1 TdPRP '1-5, TdPRPI- 7, TdPRPTS, TdPRPI-IO, and TdPRPl- VL
  • TdPRPT? and TdPRPIS positioned one after another in tandem.
  • the coding regions of all cloned genes are interrupted with single introns of slightly different length.
  • the nucleotide sequences of eadi of the TdPRPI genes, together with the deduced protein and cDNA sequences, as well as the upstream promoter regions were identified and designated sequence identifiers as shown in Table 2, below.
  • TaPRPl and TdPRPlS are located on chromosomes IA and 2B of hexaploid wheat, respectively. The location of other cloned genes could not be identified precisely because several close homologues cross-hybridized with the probe.
  • the amino acid sequence of IaPRPI also allowed ⁇ he identification of a rice horn ⁇ logue, designated as OsPRPi, in FST databases.
  • RSTs for OsPRPl originated mainly from cDNA. libraries prepared trom the panicle, tlower, endosperm or pistil 1-4 weeks time after anthesis (Accession Numbers of ESI s: C1342CB8, C14952b4, CI719169, CI493678, CI454367. C1454009, CI673662S, CI453631, C14R1346, C14693R2, respectively) Quantitative PCR analysis confirmed preferential expression of OsPRPl in early panicles and demonstrated temporal similarity of expression to TaPRPI (Figure IB).
  • the nucleotide sequence of the FST con tig was used to identify the translational start site of the respective gene in the rice genomic database.
  • a F)KA, fragment of 1640 bp, upstream of the translational start site of OsPRPL was isolated by PCR using rice ⁇ Oiyza saliva ssp japonica cv. Nipponbare) genomic D ⁇ A as template. This sequence was designated the OsPRPJ promoter (SEQ ID ⁇ O: 10)
  • TaPRPl expression of TaPRPl in different wheat (T. aestivum cv. Chinese Spring) tissues was analysed using northern blot hybridisation.
  • the expression of the TaPRPl gene was detected in spikes starting from the very early stage of development, progressively increasing until it reached maximum at 2-5 DAP, and quickly diminishing after 11 DAP (Figure 1A).
  • Very low level of TaPRPJ expression was also observed in shoots of young seedlings ( Figure IA).
  • the TdPRPl-7 promoter contained the BSl element, - AGCGGG-, which is required for specific expression in vascular tissue (Lacombe el d. r Plant J, 23: 663-676, 2000).
  • the TdPRPl-5 promoter has the ds-elix identified among the promoters of the "core xylem gene set" in Arabidopsis (Ko el al. r Molecular Genetics and Genomics 276: 517-531, 2006).
  • the promoter sequences of TdPRPl-I, TdPRPl-IO, JdPRPl-Il, and OsPRPI were cloned into the plant transformation vector p MDC 164 to provide four transcriptional GUS fusion promoter constructs (pTdPRPI- 1,-10,-1 1, and pOsPRPl), which were used to transform rice.
  • pTdPRPI- ' iO was also linearised by restriction of the unique Pmel site and transformed into wheat using microprojectile bombardment.
  • promoter -GUS fusions in transgenic plants was confirmed by PCR using primers derived from promoter and CJIJS sequences.
  • Fourteen transgenic Tu wheat lines as confirmed by PCR were analysed.
  • Six To wheat lines were selected using the GUS staining assay, from which three demonstrated strong GUS expression and three showed weak expression of the reporter gene.
  • Three lines, two with strong transgene expression and one with weak expression, were selected for further analysis.
  • AU positive lines demonstrated the same pattern of CJUS expression.
  • TdPRPI- ' iO is one of the closest homologues of TnPRPI isolated so far from T. durum ( Figure 1 C and D), Protein sequence alignment shows differences in ten amino acid residues in unprocessed proteins and only three amino acid residues are different in mature proteins ( Figure ID). Analysis of the activity of the TdPRPl-IO promoter in transgenic wheat plants using a pr ⁇ moter-GUS fusion construct demonstrated good spatial and temporal correlation between the activity of the JdPRPl-IO promoter and expression of TaPRPl.
  • TdPRPl-IO promoter The activity of the TdPRPl-IO promoter was observed in ovules prior to fertilisation ( Figure 2 A1- A8) and later in grain with maximum activity between 5 and 8 DAP
  • TdPRPl-10 promoter activity was found in transgenic rice plants, except that the activity of the promoter was less specific than in wheat.
  • strong GUS expression was detected in ovaries and the vascular tissue of anthers, glume, lemma and palea, and in iodicuies (Fig 3 Bl and B2).
  • the GUS activity was found mainly in the pericarp ( Figure 3 B3-B6)
  • During grain elongation (2-7 DAP) strong activity was detected close to the grain poles and was not found in the middle part of the grain ( Figure 3 B3).
  • TdPRPTH promoter was weaker than TdPRPTlO promoter.
  • GUS expression in anthers increased a short time before pollination and remained on the same level until anthers became dry ( Figure 4 D4).
  • GUS expression in anthers increased a short time before pollination and remained on the same level until anthers became dry ( Figure 4 D4).
  • GUS was strongly expressed in the vascular tissue of the lemma and palea, everywhere in the pericarp and in anthers ( Figure 4 D3).
  • a polarised pattern of expression similar to that observed for other PRPl promoters was detected between 3 and 7 DAP ( Figure 4 D5).
  • the polarised pattern of expression of the TdPRPTH promoter disappeared from 7 DAP first from the tip ( Figure 4 D6) and a few days later from the rest of the grain,
  • the strength of GIJS expression in roots correlated with the strength of the respective promoters in grain.
  • the T ⁇ PRPI-1Q and TdPRPI-Il promoters were active in root vascular tissue and the TdPRPI-I promoter active in all root tissues.
  • no GUS expression was detected in the elongation zone of the root, but was observed in the root tip where it gradually diminished and vanished after 3-4 days.
  • No GUS activity was detected in root tips of seedlings where GUS was driven by the TaPRPIA promoter ( Figure 5 E7).
  • GUS activity eventually became restricted to the vascular bundle of the mature part of the root and finally disappeared in both roots and coleoptiles.
  • No activity of wheat promoters has been detected in the leaves of three week old seedlings of transgenic rice.
  • the full length cDNA of wheat defensin TaPRPl was identified as a gene whose product frequently generated false positives with a number of different unrelated bait proteins in the yeast 2-hybrid screen (data not shown). It was isolated from a c ⁇ NA library prepared from whole developing grain. Northern blot hybridization revealed specific expression ol this gene in flowers and grain until 12 D ⁇ P
  • the full length cDNA ot TaPRPJ was used as a probe to screen a BAC library prepared from tetraploid wheat and the screen resulted in the isolation ol six dose homoiogues to IaPRPl.
  • TdPRPl Because of the flower and early grain spedfii expression of TdPRPk (except TdPRPl 5, FSTs for which originated mainly from cDNA libraries prepared from root tissue) the promoters of TaPRPl homoiogues were characterised.
  • OsPRPL TnPRPI homologue
  • the promoters of three representative wheat genes TdPRPJ I 1 TdPRPl 10, and TdPRPJ 11 were selected for characterisation in transgenic rice.
  • the promoter of TdPRPl-IO was also characterised in transgenic wheat.
  • rice plants were transformed with an OsPRPl promo ter-G US construct, providing the opportunity to compare in one species, rice, the activity ot the promoters of similar wheat and rice genes.
  • JaPRPI I and TaPRPl 10 promoter activities were detec ted everywhere in pericarp about 2-2.5 weeks longer and were not observed in other grain tissues.
  • the JaPRPI I promoter was slightly activated in starchy endosperm and strongly activated in the vascular tissue of the embryo alter 16 DAF ( Figure 4), The JaPRPI I l promoter was, at the end of grain elongation, the weakest promoter.
  • TaPRPI-I 1 promoter activity during grain elongation was not sustained in the middle part of the pericarp and quickly vanished at the end of the second week after pollination, first at the top end of grain and then everywhere (Figure 4).
  • the pattern of GUS expression driven by the TaPRPJ-IO promoter in transgenic wheat flowers was more specific than the pattern driven by the same promoter in rice flowers, in wheat, the activity of the promoter was localised only to ovaries and later mainly to the upper epidermis layer ol the pericarp ( Figure 2). No GUS activity was detected in other flower tissues.
  • activity of the TaPRPI H) promoter was observed in the vascular tissue of anthers, lemma and palea ( Figure 3 and 4). At least at some stages of rice grain development the activity of the promoter was also detected in several layers of seed coat and even in aleurone ceils.
  • PRPI genes were not restricted to flower and pericarp tissues, but was also observed in the embryo and, in the case of TaPR.PI 1 1, in the scutellurn of germinating grain. In one week-old seedlings germinated on filter paper it was detected in coieoptiles and the vascular tissue of roots. An interesting observation was the absence of promoter activity not only in the elongating region of the pericarp, but also in the elongation zone ol root, coleoptile, and developing first leaf ( Figure 6 E7- F 12).
  • PRPI gene promoters in developing and germinating grain makes them valuable tools for the targeted expression of, for example, defensin, PR and R genes for engineered plant protection.
  • EXAMPLE 5 Materials and methods
  • the full length eDNA of TaVRPl was isolated from a yeast 2-hybrid cDNA library prepared from wheat grain at 0-6 DAP (Lopato et til, Plant Mol.Biol. 62: 637-653, 2006) as an occasional false positive gene.
  • the full length cDNA sequence of TaPRPl was used to probe a BAC library prepared from the genomic DNA of Triticum durum cv.
  • BAC DNAs Five BAC DNAs were combined and sequenced using the 454 sequencing method. The obtained gene sequences were subsequently used to design forward and reverse primers for the isolation of the promoter segments. Fragments of promoters of the three genes TdPRPl-I, TdPRPbIO, TdPRPl-VI with full-length 5'- untranslated regions were amplified by PCR using Accu PrimeTM P ⁇ DNA polymerase (Invitrogen) from DNA of BAC clones as a template. They were cloned into the pENTR-D-TOPO vector (Invitrogen); the cloned inserts were verified by sequencing and subdoned into the pMDC164 vector (Curtis and Grossniklaus, Plant Physiol.
  • the nucleotide sequence of the IiSl contig was used to identify the lranslational start site of the respective gene ( ⁇ cc. AP00S209, region 1694297 .1694622) in rice genomic databases.
  • a DN A fragment ot 1640 bp upstream of the translafional start site of OsPRPI was isolated by PCR using rice (Oryza saliva ssp. japonica cv Nipponbare) genomic DN ⁇ as template
  • the promoter sequence of OsPRPl was cloned into the plant transformation vector pMDCl ⁇ 4 to provide a transcriptional GUS fusion promoter construct, designated pOsPRPl, which was transformed into rice.
  • the constructs pTdPRP ⁇ -l, pTdPRPl-10, pTdPRPt-11 and pOsPRPl were transformed into rice (Oiyza saliva L. ssp. japonica cv. Nipponbare) using Agrobaaefium- ⁇ xcdiaied transformation and the method developed by Tingay et at. (Plant j, 14: 285-295, 1997) and modified by Matthews et al. (MoI. Breed. 7: 195-202, 2001). Wheat (Tiiticum aesti ⁇ um L. cv. Bob whitt') was transformed using biolistie bombardment as described in Kovalch ⁇ k et al.
  • RNA isolation and northern blot hybridisation was performed as described elsewhere (Sambrooi et ah, Molecular Cloning: 4 Laboratory Manual, 2 rd Fd , Cold spring Harbor Press, Cold Spring Harbor, New York, US ⁇ , 1989), The filter membrane was hybridised with a full length cDN ⁇ of JaPRPl Q-PCR analysis ot expression of the OsPKPi gene in different tissues of wild type rice was performed as described in Li et al. ⁇ Plan! Biotechnology journal 6: 465-476, 2008).

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Abstract

La présente invention porte d'une façon générale sur des séquences de contrôle de la transcription destinées à effectuer l'expression d'une séquence nucléotidique d'intérêt dans une plante. Plus particulièrement, la présente invention porte sur des séquences de contrôle de la transcription qui dirigent de façon spécifique ou préférentielle l'expression d'une séquence nucléotidique liée de façon fonctionnelle dans une ou plusieurs parties d'une fleur et/ou graine de plante.
PCT/AU2009/000968 2008-07-30 2009-07-30 Séquences de contrôle de la transcription actives dans les fleurs et/ou les graines WO2010012034A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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US20050177901A1 (en) * 2001-06-22 2005-08-11 Syngenta Participations Ag Identification and characterization of plant genes
US20070199106A1 (en) * 2003-09-11 2007-08-23 Rainer Stahl Novel endosperm-specific plant promoter for cultivated plants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050177901A1 (en) * 2001-06-22 2005-08-11 Syngenta Participations Ag Identification and characterization of plant genes
US20070199106A1 (en) * 2003-09-11 2007-08-23 Rainer Stahl Novel endosperm-specific plant promoter for cultivated plants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KARUNANANDAA, B. ET AL.: "Characterization of a predominantly pistil-expressed gene encoding a gamma-thionin-like protein of Petunia inflata", PLANT MOLECULAR BIOLOGY., vol. 26, 1994, pages 459 - 464 *
TREGEAR J.W. ET AL.: "Characterization of a defensin gene expressed in oil palm inflorescences: induction during tissue culture and possible association with epigenetic somaclonal variation events", JOURNAL OF EXPERIMENTAL BOTANY., vol. 53, no. 373, 2002, pages 1387 - 1396 *

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