WO2009094704A1 - Expression spécifique des semences dans des plantes - Google Patents

Expression spécifique des semences dans des plantes Download PDF

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Publication number
WO2009094704A1
WO2009094704A1 PCT/AU2009/000091 AU2009000091W WO2009094704A1 WO 2009094704 A1 WO2009094704 A1 WO 2009094704A1 AU 2009000091 W AU2009000091 W AU 2009000091W WO 2009094704 A1 WO2009094704 A1 WO 2009094704A1
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Prior art keywords
plant
nucleic acid
nucleotide sequence
seq
cell
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PCT/AU2009/000091
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English (en)
Inventor
Sergiy Lopato
Nataliya Kovalchuk
Jessica Anne Smith
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The University Of Adelaide
Grains Research & Development Corporation
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Priority claimed from AU2008900432A external-priority patent/AU2008900432A0/en
Application filed by The University Of Adelaide, Grains Research & Development Corporation filed Critical The University Of Adelaide
Priority to AU2009208377A priority Critical patent/AU2009208377B2/en
Publication of WO2009094704A1 publication Critical patent/WO2009094704A1/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

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 embryo and/or embryo surrounding region (ESR) in a plant seed.
  • ESR embryo surrounding region
  • Expression of a DNA sequence in a plant is dependent, in part, upon the presence of an operably linked transcriptional control sequence, such as a promoter or enhancer, which is functional within the plant.
  • the transcriptional control sequence determines when and where within the plant the DNA sequence is expressed. For example, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilised. In contrast, where gene expression in response to a stimulus is desired, an inducible promoter may be used. Where expression in specific tissues or organs is desired, a tissue-specific promoter may be used.
  • transcriptional control sequences such as promoters or enhancers
  • isolation and characterisation of transcriptional control sequences which can serve as regulatory regions for the expression of nucleotide sequences of interest in particular cell, tissues or organs of a plant, would be desirable for use in the genetic manipulation of plants.
  • 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 an embryo and/or embryo surrounding region in a plant seed.
  • the present invention provides an isolated nucleic acid molecule comprising: (i) a nucleotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in an embryo and/or Embryo Surrounding Region (ESR) of a plant seed; or
  • the transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in the embryo and/or ESR of a plant seed is derived from a gene which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a homolog thereof.
  • the transcriptional control sequence is derived from a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2.
  • the transcriptional control sequence of the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 3 or a functionally active fragment or variant thereof.
  • the transcriptional control sequence may also comprise the nucleotide sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
  • the transcriptional control sequence may also comprise a cz ' s-element that activates, enhances or otherwise modulates the activity and/or expression pattern of the transcriptional control sequence, the cz ' s-element comprising the nucleotide sequence set forth in one or more of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the present invention also provides a nucleic acid construct comprising the isolated nucleic acid molecule of the first aspect of the invention.
  • the present invention provides a genetically modified cell comprising a nucleic acid construct of the second aspect of the invention or a genomically integrated form thereof.
  • the present invention contemplates a multicellular structure comprising one or more cells of the third aspect of the invention.
  • the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in an embryo and/or ESR in a plant seed, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of the nucleic acid of the first aspect of the invention.
  • the present invention provides an isolated nucleic acid selected from the list consisting of:
  • nucleic acid comprising a nucleotide sequence which is at least 50% identical to the nucleotide sequence mentioned in (i); (iii) a nucleic acid which hybridizes to the nucleic acid mentioned in (i) under stringent conditions; (iv) a nucleic acid comprising a nucleotide sequence which is the complement or reverse complement of any one of (i), (ii) or (iii); and (v) a fragment of any of (i), (ii), (iii) or (iv).
  • the present invention also provides a nucleic acid construct comprising the nucleic acid of the sixth aspect of the invention.
  • the present invention provides a genetically modified cell comprising the construct of the seventh aspect of the invention or a genomically integrated form of the construct.
  • the present invention provides a multicellular structure comprising a cell of the eighth aspect of the invention.
  • sequence identifier number SEQ ID NO:
  • a summary of the 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 an embryo and/or ESR in a plant seed.
  • 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, encompasses any nucleic acid that confers, activates or enhances expression of 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 nucleotide sequence of interest under the transcriptional control of the transcriptional control sequence.
  • promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to the promoter.
  • some variation in this distance can be accommodated without loss of promoter function.
  • the present invention provides an isolated nucleic acid molecule comprising:
  • nucleotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in an embryo and/or ESR of a plant seed;
  • isolated refers to material removed from its original environment (eg. the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide 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 polynucleotide.
  • 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-vitro amplification (eg. polymerase chain reaction and the like).
  • the isolated nucleic acid molecule of the present invention may comprise polyribonucleotides or polydeoxyribonucleotides, 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.
  • the 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 add” also embraces chemically, enzymatically, or metabolically modified forms of DNA and RNA.
  • the present invention contemplates a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in the embryo and/or ESR of a plant seed.
  • a plant “seed” should be understood to refer to a mature or immature plant seed.
  • the term “seed” includes, for example, immature seed carried by a maternal plant or seed released from the maternal plant.
  • seed should also be understood to include any seed plant sporophyte, together with any associated nutritive or protective tissues (which may or may not be clonal with the sporophyte embryo), between the developmental stages of fertilisation and germination.
  • the "embryo" of a plant seed refers to the part of a seed that comprises the precursor tissues of the leaves, stem (ie. hypocotyl), and root (ie. radicle), as well as one or more cotyledons.
  • the number of cotyledons comprised within the embryo can vary according to the plant taxon.
  • dicotyledonous angiosperm embryos comprise two cotyledons
  • monocotyledonous angiosperm embryos comprise a single cotyledon (also referred to as the scutellum)
  • gymnosperm embryos may comprise a variable number of cotyledons, typically ranging from 2 to 24.
  • reference herein to an "embryo" particularly in the context of specific or preferential expression within an embryo may include expression in all of the embryo or expression in one or more cells, tissues or parts of the embryo.
  • the "Embryo Surrounding Region" or “ESR” of a plant seed refers to the part of a seed that surrounds and/or is proximate to the embryo in the seed.
  • the ESR is typically characterized by small and densely cytoplasmic cells with a high content of endoplasmic reticulum and Golgi vesicles.
  • the ESR is thought to play a role in different types of exchanges between endosperm and embryo.
  • reference herein to the ESR particularly in the context of specific or preferential expression within the ESR (see later), may include expression in all of the ESR or expression in one or more cells, tissues or parts of the ESR.
  • telomere sequence of interest is expressed substantially only in a plant embryo and/or ESR in a plant seed.
  • Preferentially expressing should be understood to mean that the nucleotide sequence of interest is expressed at a higher level in a plant embryo and/or ESR in a plant seed than in one or more other tissues of the plant, eg. leaf tissue, root tissue or another tissue of the grain such as the starchy endosperm.
  • preferential expression in a plant embryo and/or ESR in a plant seed includes expression of a nucleotide sequence of interest in a plant embryo and/or ESR in a plant seed at a level of at least twice, more preferably at least 5 times and most preferably at least 10 times the level of expression seen in at least one other tissue of the plant.
  • expression of an operably connected nucleotide sequence in a plant embryo and/or ESR in a plant seed refers to the transcription and/or translation of a nucleotide sequence in one or more cells of a plant embryo and/or ESR in a plant seed. This definition in no way implies that expression of the nucleotide sequence must occur in all cells of a plant embryo and/or ESR in a plant seed.
  • the transcriptional control sequence or functionally active fragment or variant thereof may effect specific or preferential expression in an embryo and/or ESR in a seed from any seed plant species, including monocotyledonous angiosperm plants ('monocots'), dicotyledonous angiosperm plants ('dicots') and gymnosperm plants.
  • the plant is a monocot.
  • the plant is a cereal crop plant.
  • the term "cereal crop plant” may be a member of the Poaceae (grass family) that produces grain.
  • 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 quinoa.
  • the plant is a barley plant.
  • the transcriptional control sequence specifically or preferentially directs the expression of an operably connected nucleotide sequence in the embryo and/or ESR of a barley seed at least between 6 DAP and 48 DAP.
  • barley includes several members of the genus Hordeum.
  • the term “barley” encompasses cultivated barley including two-row barley (Hordeum distichum), four-row barley (Hordeum tetrastichum) and six-row barley (Hordeum vulgare).
  • barley may also refer to wild barley, (Hordeum s ⁇ ontaneum).
  • the term “barley” refers to barley of the species Hordeum vulgar e.
  • the plant is a rice plant.
  • the transcriptional control sequence specifically or preferentially directs the expression of an operably connected nucleotide sequence in the embryo of the rice seed at least between 7 DAP and 69 DAP.
  • rice includes several members of the genus Oryza including the species Oryza sativa and Oryza glaberrima.
  • the term “rice” thus encompasses rice cultivars such as japonica or sinica varieties, indica varieties and javonica varieties.
  • the term “rice” refers to rice of the species Oryza sativa.
  • the plant is a wheat plant.
  • wheat should be understood as a plant of the genus Triticum.
  • 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. s ⁇ elta.
  • the term “wheat” refers to wheat of the species Triticum aestivum.
  • the transcriptional control sequences of the present invention may also specifically or preferentially direct the expression of an operably connected nucleotide sequence in a dicot.
  • dicots include, for example, Arabid ⁇ psis spp., Nicotiana spp., Medicago spp., soybean, canola, oil seed rape, sugar beet, mustard, sunflower, potato, safflower, cassava, yams, sweet potato, other Brassicaceae such as Thellungiella halo ⁇ hila, among others.
  • the transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in the embryo and/or ESR of a plant seed is derived from a gene which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a homolog thereof.
  • a transcriptional control sequence “derived from”, as used herein, refers to a source or origin for the transcriptional control sequence.
  • a transcriptional control sequence “derived from a gene which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1” refers to a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a gene which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in an organism.
  • homolog as used herein with reference to homologs of polypeptides comprising the amino 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, ortholog, paralog, mutant or variant of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 comprises an amino acid sequence which comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino add 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 acid residues, at least 60 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues or over the full length of SEQ ID NO: 1.
  • the comparison window may comprise additions or deletions (ie. 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 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. Acids 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 Inc, 1994-1998, Chapter 15, 1998).
  • 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.
  • the transcriptional control sequences contemplated herein are derived from a plant.
  • the transcriptional control sequences of the present invention are derived from a monocot plant species and 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 Triticum species (for example T. aestivum, T. durum, T. monococcum, T. dicoccon, T. s ⁇ elta or T. ⁇ olonicum).
  • the transcriptional control sequence is derived from a tetraploid wheat (for example T. durum, T. dicoccon, or T. ⁇ olonicum). In some embodiments, the transcriptional control sequence is derived from a durum wheat, and in some embodiments, the transcriptional control sequence is derived from Triticum durum.
  • the transcriptional control sequence is derived from a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2.
  • the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 3 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.
  • “Functionally active fragments” of the transcriptional control sequence of the invention include fragments of a transcriptional control sequence which retain the capability to specifically or preferentially direct expression of an operably connected nucleotide sequence in an embryo and/or ESR of a plant seed.
  • the functionally active fragment is at least 500 nucleotides (nt), at least 1000 nt, at least 1500 nt, at least 2000 nt or at least 2500 nt in length.
  • the fragment comprises at least 500 nt, at least 1000 nt, at least 1500 nt, at least 2000 nt or at least 2500 nt contiguous bases from the nucleotide sequence set forth in SEQ ID NO: 3.
  • SEQ ID NO: 3 examples include truncated forms of SEQ ID NO: 3, such as transcriptional control sequences which comprise the nucleotide sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
  • “Functionally active variants” of the transcriptional control sequence of the invention include orthologs, mutants, synthetic variants, analogs and the like which are capable of effecting transcriptional control of an operably connected nucleotide sequence and/or are capable of specifically or preferentially directing the expression of an operably connected nucleotide sequence in a plant embryo and/or ESR in a plant seed.
  • 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 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3.
  • the compared nucleotide sequences should be compared over a comparison window of at least 100 nucleotide residues, at least 200 nucleotide residues, at least 500 nucleotide residues, at least 1000 nucleotide residues, at least 1500 nucleotide residues, at least 2000 nucleotide residues, at least 2500 nucleotide residues or over the full length of SEQ ID NO: 3.
  • the comparison window may comprise additions or deletions (ie. 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 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. (1997, supra). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (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 add molecule comprising the nucleotide sequence set forth in SEQ ID NO: 3 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.
  • 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 about 24 hours, usually about 4 to about 12 hours.
  • Tm thermal melting point
  • 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. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridisation, and/or wash conditions can be adjusted to hybridise to sequences of different degrees of complementarity. For example, sequences with >90% identity can be hybridised by decreasing the Tm by about 10 0 C. Generally, stringent conditions are selected to be about 5°C lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH.
  • high stringency conditions can utilise a hybridisation and/or wash at, for example, 1, 2, 3, or 4°C lower than the T m ;
  • medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, 8, 9, or 10 0 C lower than the T m ;
  • 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 Tm.
  • the transcriptional control sequence may also comprise one or more cz ' s-elements that activate, enhance or otherwise modulate the activity and/or expression pattern of the transcriptional control sequence.
  • the term cz ' s-element, as referred to herein, should be understood as a part, region or fraction of the transcriptional control sequence itself that activates, enhances or otherwise modulates the activity and/or expression pattern of the transcriptional control sequence as a whole.
  • the cz ' s-element may comprise the nucleotide sequence set forth in one or more of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the transcriptional control sequence may also comprise a cz ' s-element that activates, enhances or otherwise modulates the activity and/or expression pattern of the transcriptional control sequence, the cz ' s-element comprising the nucleotide sequence set forth in one or more of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the present invention also provides a nucleic acid construct comprising an isolated nucleic acid molecule of the first aspect of the invention.
  • the nucleic acid construct of the second aspect of the present invention may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • the nucleic acid construct 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.
  • 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 may comprise, for example, a linear DNA molecule, a plasmid, a transposon, a cosmid, an artificial chromosome and the like. Furthermore, the nucleic acid construct 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 to 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 operably connected to in its natural state.
  • SEQ ID NO: 3 is operably connected to the nucleotide sequence set forth in SEQ ID NO: 4. Accordingly, in this example, 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.
  • nucleotide sequence of interest which is heterologous to the transcriptional control sequence (or functionally active fragment 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 sequence of interest may be a heterologous sequence from an organism of the same taxon.
  • the nucleic acid construct may further comprise a nucleotide sequence defining a transcription terminator.
  • transcription terminator or
  • Terminator refers to a DNA sequence at the end of 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 polyadenylate 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 cells, tissues or organs in which it is intended to be used. Examples of suitable terminator sequences which may be useful in plant cells include: the nopaline synthase
  • the nucleic acid construct comprises an expression cassette comprising the structure:
  • [N]w comprises one or more nucleotide residues, or is absent
  • TCS comprises a nucleic acid of the first aspect of the invention
  • [N] ⁇ comprises one or more nucleotide residues, or is absent
  • SoI comprises a nucleotide sequence of interest which encodes an mRNA or non- translated RNA, wherein the nucleotide sequence, SoI, 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.
  • 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 cells 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, kanamydn-resistance genes, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (eg.
  • hygromycin phosphotransferase genes eg. hpt
  • herbicide resistance genes including glufosinate, phosphinothridn or bialaphos resistance genes such as phosphinothricin acetyl transferase-encoding genes (eg. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase-encoding genes (eg. aroA), bromyxnil resistance genes including bromyxnil nitrilase-encoding genes, sulfonamide resistance genes including dihydropterate synthase-encoding genes (eg.
  • sul) and sulfonylurea resistance genes including acetolactate synthase-encoding genes; enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransf erase (CAT) encoding genes; fluorescent reporter genes such as the green fluorescent protein-encoding gene; and luminescence-based reporter genes such as the luciferase gene, amongst others.
  • enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransf erase (CAT) encoding genes
  • fluorescent reporter genes such as the green fluorescent protein-encoding gene
  • luminescence-based reporter genes such as the luciferase gene, amongst others.
  • the genetic constructs described herein may further include nucleotide sequences intended for the maintenance and/or replication of the genetic construct in prokaryotes or eukaryotes and/or the integration of the genetic 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 Agrobacterium-mediated transformation.
  • the nucleic acid construct comprises left and/or right T-DNA border sequences. Suitable T-DNA border sequences would be readily ascertained by one of skill in the art.
  • the term "T-DNA border sequences" should be understood to include, for example, any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrob ⁇ cterium sp. cell into a plant cell susceptible to Agrob ⁇ cterium- mediated transformation.
  • Peralta and Ream Proc. N ⁇ tl. Ac ⁇ d. ScL USA, 82(15): 5112-5116, 1985
  • Gelvin Gelvin
  • the present invention also contemplates any suitable modifications to the genetic construct which facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobacterium sp., for example, as described in Broothaerts et al. (Nature 433: 629-633, 2005).
  • the present invention provides a genetically modified cell comprising a nucleic acid construct of the second aspect of the invention or a genomically integrated form thereof.
  • a "genetically modified cell” includes any cell comprising a non- naturally occurring and/or introduced nucleic add.
  • the introduced nucleic acid comprises a construct of 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 (eg. 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 eg. 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 and the like.
  • the term “genomically integrated” contemplates chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, and the like.
  • the “genomically integrated form" of the construct may be all or part of the construct. However, in some embodiments the genomically 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 eukaryotic cell.
  • the cell is a plant cell.
  • the cell is a monocot plant cell.
  • the cell is a cereal crop plant cell.
  • the cell is a barley, rice or wheat 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 (eg. an E. coli cell).
  • the present invention contemplates a multicellular structure comprising one or more cells of 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 multicellular structure comprises a plant seed. In some embodiments, the multicellular structure comprises a plant embryo and/or ESR in a plant seed.
  • a nucleotide sequence of interest is operably connected to the transcriptional control sequence or the functionally active fragment or variant thereof, and the nucleotide sequence of interest is specifically or preferentially expressed in the embryo and/or ESR of the plant seed. In some embodiments, the level, rate and/or pattern of expression of at least one nucleotide sequence is altered in the embryo and/or ESR in said seed relative to the wild type form of said plant seed.
  • the plant or a part, organ or tissue thereof comprises a monocot plant or a part, organ or tissue thereof. In some embodiments the plant or a part, organ or tissue thereof comprises a cereal crop plant or a part, organ or tissue thereof. In some embodiments, the plant or a part, organ or tissue thereof comprises a barley, rice or wheat plant or a part, organ or tissue thereof.
  • the multicellular structure may comprise a plant seed.
  • the plant seed comprises a monocot plant seed.
  • the plant seed comprises a cereal crop plant seed.
  • the plant seed is a barley seed and in further embodiments the nucleotide sequence of interest is expressed in the barley seed at least between 6 DAP and 48 DAP.
  • the plant seed is a rice seed and in further embodiments the nucleotide sequence of interest is expressed in the rice seed at least between 7 DAP and 69 DAP.
  • the plant seed is a wheat seed.
  • the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in an embryo and/or ESR in a plant seed, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of the nucleic acid of the first aspect of the invention.
  • the present invention is predicated, in part, on effecting transcription of the nucleotide sequence of interest under the transcriptional control of 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 acid molecule may be introduced into the plant via 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 seed, either stably or transiently; a nucleic acid may be introduced into a seed 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 the particular plant species. Common methods include Agrobacterium- mediated transformation, microprojectile bombardment based transformation methods and direct DNA uptake based methods.
  • Roa-Rodriguez et al. Agrobacterium-medz ⁇ ted transformation of plants, 3 rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003
  • Other bacterial-mediated plant transformation methods may also be utilised, for example, see Broothaerts et ⁇ l. (2005, supra).
  • Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, are reviewed by Casas et al. (Plant Breeding Rev. 13: 235-264, 1995). Examples of direct DNA 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). In addition to the methods mentioned above, 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 Biotechnol 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 biomass, 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.
  • the nucleotide sequence of interest may alternatively encode a non- translated RNA, for example an siRNA, miRNA, antisense RNA and the like.
  • the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, as defined supra.
  • the method of the fifth aspect of the invention contemplates the specific or preferential expression of a nucleotide sequence of interest in the embryo and/or ESR in any suitable seed plant species.
  • the method contemplates expression in a monocot.
  • the method contemplates expression in a cereal crop plant.
  • the method contemplates expression in a barley plant, and in further embodiments the method contemplates expression in the embryo and/or ESR of a barley seed at least between 6 DAP and 48 DAP.
  • the method contemplates expression in a rice plant, and in further embodiments the method contemplates expression in the embryo of a rice seed at least between 7 DAP and 69 DAP.
  • the method contemplates expression in the embryo and/or ESR of a wheat plant.
  • the present invention provides an isolated nucleic acid selected from the list consisting of:
  • nucleic acid comprising a nucleotide sequence which is at least 50% identical to the nucleotide sequence mentioned in (i);
  • nucleic acid which hybridizes to the nucleic acid mentioned in (i) under stringent conditions;
  • nucleic acid comprising a nucleotide sequence which is the complement or reverse complement of any one of (i), (ii) or (iii); and (v) a fragment of any of (i), (ii), (iii) or (iv).
  • the isolated nucleic acid defined at (ii) comprises at least 50% sequence identity, more preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3.
  • the isolated nucleic acid of the sixth aspect of the invention comprises a nucleotide sequence which defines a transcriptional control sequence or a complement, reverse complement or fragment thereof.
  • the sixth aspect of the invention provides isolated nucleic acids which hybridize to any of the nucleic acids mentioned at (i) under stringent conditions.
  • exemplary stringent conditions include low stringency conditions, medium stringency conditions and high stringency conditions (as previously described).
  • the sixth aspect of the present invention also contemplates nucleic acid fragments.
  • the fragment comprises at least 500 nucleotides
  • fragments of SEQ ID NO: 3 contemplated in this aspect of the invention include, for example, truncated forms of
  • SEQ ID NO: 3 such as those nucleotide sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
  • the fragment may also comprise a cz ' s-element derived from SEQ ID NO: 3, such as the cz ' s-elements comprising the nucleotide sequences set forth in one or more of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • a cz ' s-element derived from SEQ ID NO: 3, such as the cz ' s-elements comprising the nucleotide sequences set forth in one or more of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the present invention also provides a nucleic acid construct comprising the nucleic acid of the sixth aspect of the invention.
  • the present invention provides a genetically modified cell (as hereinbefore described) comprising the construct of the seventh aspect of the invention; and/or a cell comprising a genomically integrated form of said construct.
  • the present invention provides a multicellular structure (as hereinbefore described) comprising a cell of the eighth aspect of the invention.
  • Figure 1 shows a Southern blot confirming the successful integration of pMDC164- TdPR61 into transgenic plant lines.
  • the coding region of Hygromydn phosphotransferase was used as a probe.
  • the number of bands reflects the number of integrated copies of the vector.
  • Figure 2 shows the activity of the TdPR61 promoter in barley grain at different stages of grain development in the transgenic plant line G81-2. The figure shows promoter activity at different ages of the grain (measured in DAP) and shows the transgenic grain in different orientations (crease up or crease down) and the grain sectioned in different orientations (longitudinal or lateral).
  • Figure 3 shows the activity of the TdPR61 promoter in barley grain at different stages of grain development in transgenic plant line G81-11.
  • the figure shows the age of the grain in DAP, the orientation of the grain (eg. crease up or crease down) and the orientation of the section (longitudinal (L) or lateral).
  • FIG. 4 shows isolated embryos and embryo surrounding regions (ESR) from the grain (10 DAP) of control and transgenic barley plants transformed with TdPR61 Promoter:GUS construct. At this stage of grain development GUS activity was detected in the ESR.
  • B - Embryos and ESRs from several grains of transgenic plants.
  • C Closer look on isolated ESR of transgenic plants.
  • FIG. 5 shows isolated embryos from the grain (15 DAP) of control (A) and transgenic (B - F) barley plants transformed with TdPR61 Promoter:GUS construct.
  • GUS activity can be seen as two strong points between embryo axis and scutellum (B) and three weaker points in scutellum (C). The same can be seen in the grains from three independent transgenic lines (D - F). E - embryo axis; S - scutellum.
  • FIG. 6 shows histological analyses of grain from transgenic barley plants transformed with the TdPR61 Promo ter-GUS construct.
  • GUS expression was detected in the embryo axis, in the part of coleorhiza close to connection of embryo axis to the scutellum.
  • Figure 7 shows the results of the promoter mapping of the TdPR61 promoter.
  • A First round of mapping using transient expression assay. Two promoter segments, containing putative proximal and distal to TATA box cz ' s-elements are shown as blue and brown boxes.
  • B Second round of mapping of the promoter segment, containing potential distal cz ' s-element. Positions of predicted embryo (EMB) and endosperm (END) specific cz ' s-elements are indicated with arrows; sequences of predicted putative cz ' s-elements are shown.
  • C shows the activity of the TdPR61 promoter deletions in colour forming units (cfu). CFU(GUS) per embryo is shown on the Y-axis.
  • Figure 8 shows examples of transient GUS expression in isolated wheat embryos driven by TdPR61 promoter. Promoter-GUS fusion constructs were bombarded into isolated embryo (12-17 DAP) and stained for GUS activity in 24 hours.
  • Figure 9 shows the activity of the TdPR61 promoter in To transgenic rice lines.
  • A B - rice grain at 7 DAP collected from the line with strong transgene expression. No GUS activity has been detected before 8 DAP.
  • D E - rice grain at 9 DAP. The strongest GUS expression was observed in embryo and in the opposite pole of the grain.
  • C was detected in vascular tissue of lemma, however, only in one plant from several analyzed transgenic lines with strong GUS expression in embryo. Grain from control plants is on the left side of each picture.
  • Figure 10 shows the activity of the TdPR61 promoter in To transgenic rice lines.
  • B F - longitudinal sections through the main vascular bundle of grain at 11 and 14 DAP, respectively.
  • Strong GUS activity has been detected in the embryo and along the main vascular bundle of the grain (and/or in ETC).
  • GUS activity in vascular bundle (ETC) is indicated with red arrows. Grain from control plants is on the left side of each picture.
  • Figure 11 shows the activity of the TdPR61 promoter in To transgenic rice lines.
  • A B - rice grain at 18 DAP; B - longitudinal sections through the main vascular bundle of grain at 18 DAP. Very strong expression was observed in grain.
  • Weak expression of GUS still can be detected in ETC (indicated with arrow). Grain from control plant is on the left side of the picture.
  • Figure 12 shows the activity of the TdPR61 promoter in To transgenic rice lines. Al dose look on embryo isolated from control (left) and transgenic (right) grain at 26 DAP. C - endosperm after embryo isolation: no GUS staining at 26 DAP. Two different longitudinal sections at 30 DAP show strong GUS expression exclusively in embryo.
  • FIG 13 shows the activity of the TdPR61 promoter in To transgenic rice lines. Intact grain (A, C) and different longitudinal sections of grain (B, D-F) at 35 (A, B), 45 (C, D), and 50 DAP (E, F). Strong GUS staining was detected in the embryo and/or ESR of the grain only. Grain from control plants is on the left side of each picture.
  • Figure 14 shows the activity of the TdPR61 promoter in To transgenic rice lines. Longitudinal sections of grain from transgenic rice lines with strong (A, C and D) and weak (B, E) transgene expression. GUS activity has been detected only in the embryo and/or ESR of the grain. Grain from control plants is on the left side of each picture.
  • Figure 15 shows the activity of the TdPR61 promoter in transgenic barley lines.
  • A longitudinal section through the T2 transgenic barley grain at 20 and 40 DAP.
  • GUS activity can be observed in the middle of embryo and in the embryo surrounding region (ESR).
  • B histological analysis of Ti grain from transgenic barley plants transformed with TdPR61 promo ter-GUS construct: different longitudinal sections through embryos at 21, 24, 27, and 34 DAP. GUS staining is observed in endosperm surrounding region and in the connection between embryo axis and scutellum.
  • Figure 16 shows the activity of the TdPR61 promoter in T2 transgenic barley lines. Al- A6 - temporal pattern of TdPR61 expression in barley grain at 5, 8, 14, 20, 40, and 50 DAP, respectively. GUS activity was detected as early as 5 DAP (Al) in the close to embryo part of endosperm and was still detectible after 50 DAP. Grain from control plants is in the upper part of each picture. A7 - endosperm of transgenic barley grain at 11 DAP after isolation of embryo. GUS staining is clearly seen in the embryo surrounding region of endosperm.
  • GUS staining was observed as two close spots. Grain from control plants is on the left side of each of A8-A10 picture.
  • GUS staining is observed in the tissue, which connect embryo axis to scutellum.
  • B4, B5, B8, and B9 - sections were done from the side of embryo axis.
  • GUS staining is observed in root radical
  • the cDNA of TaPR61 was isolated from the cDNA library prepared from the liquid part of the syncytial endosperm of Triticum aestivum at 3-6 DAP. A single cDNA of TaPR61 was identified among about 200 cDNAs randomly selected for sequencing.
  • the amino acid sequence corresponding to the TaPR61 cDNA showed some homology to barley ENDl (Doan et ah, 20 Plant MoI. Biol. 31: 877-886, 1996). Furthermore, all multiple ESTs from databases with 100% sequence match to TaPR61 originate only from cDNA libraries prepared from early grain and early endosperm.
  • the full length cDNA sequence of TaPR61 was used to probe BAC libraries prepared from genomic DNA of Triticum durum cv. Langdon (described in Cenci et ah, Theor A ⁇ l Genet 107: 931-939, 2003) using Southern hybridisation as described in Example 2. Seven BAC clones which strongly hybridised with the probe were selected for further analysis. The T. durum homolog of TaPR61 (putatively contained within the BACs) was designated TdPR61.
  • Membranes were soaked in 5x SSC making sure that any residual precipitated SDS on pre-used filters had re-dissolved. The membranes were then placed into a bottle and approximately 30 ml of pre-hybridisation solution (see below) was added before incubating overnight at 65°C.
  • 300 ml of pre-hybridisation solution was prepared by mixing: 150 ml 10x SSC, 105 ml nanopure water, 30 ml Denhardt's III and 15 ml salmon sperm DNA (5mg/ml, autoclaved) followed by incubation at 55 - 65°C for 5 minutes.
  • the pre-hybridisation solution in the bottle was replaced with hybridisation solution prior to adding the labelled probe.
  • Hybridisation Solution 100 ml was prepared by mixing: 5 ml nanopure water, 30 ml 5x HSB buffer, 30 ml Denhardt's III, 30 ml 25% Dextran sulphate and 5 ml salmon sperm DNA (5mg/ml, autoclaved) followed by incubation at 55 - 65°C for 5 minutes.
  • DNA was isolated from positive clones selected according to the Southern hybridisation (described above) using the method set out below:
  • 7.5 ml of Luria Broth (LB) supplemented with chloramphenicol (Cm) was inoculated with a single colony before incubation overnight at 37°C with shaking at 225 rpm.
  • the cells were then resuspended in 400 ⁇ l Pl (QIAGEN #19051 - Tris.Cl-EDTA resuspension buffer) buffer by vortexing.
  • the pellet was then air-dried before being resuspended in 500 ⁇ l TE+5 ⁇ l RNAase cocktail (Geneworks, cat # AM-2286) followed by incubation at 37°C for 15 min.
  • 500 ⁇ l of (25:24:1) phenol: chloroform: isoamyl alcohol was then added followed by centrifugation at 15000 rpm for 10 min at room temperature. After centrifugation, the aqueous phase was removed and transferred to a new tube, to which 50 ⁇ l 3M Sodium Acetate pH 5.2 and 300 ⁇ l 100% isopropanol were added before incubation at -20 0 C for 60 min.
  • the sample was centrifuged at 15000 rpm for 15 min at room temperature and the supernatant removed.
  • the resulting DNA pellet was then washed with 1 ml of 70% ethanol followed by centrifugation at 15000 rpm for 5 min and removal of the supernatant.
  • the final pellet was then air-dried before being resuspended in 30 ⁇ l TE pH 8.
  • the promoter sequence was first identified on the BAC clone by several consecutive sequencing reactions. In the first sequencing reaction, reverse primers derived from the 5' end of the gene sequence were used. In subsequent reactions, primers were used that were derived from segments of DNA obtained during sequencing. As a result of such 'walking' along the DNA, about 3000 bp of sequence upstream from the translation start codon was obtained. This sequence was subsequently used to design forward and reverse primers for the isolation of the promoter segment.
  • a promoter with a full-length 5'-untranslated region of TdPR61 was isolated by PCR using AccuPrimeTM Pfx DNA polymerase (Invitrogen) from DNA of BAC clone W61-1 as a template using the primers shown below in Table 2.
  • the length of promoter used in constructs was 2669 bp.
  • the tetranucleotide sequence CACC was introduced into the 5' ends of the forward primer.
  • the PCR product including the TdPR61 promoter sequence was directionally cloned into the pENTR-D-TOPO vector using pENTR Directional TOPO Cloning Kits (Invitrogen).
  • the construct was linearised with Mlul and used for cloning of the promoter by recombination into the destination binary vector for plant transformation, pMDC164 (Curtis and Grossniklaus, Plant Physiol. 133: 462-469, 2003), upstream of a ⁇ -glucoronidase (GUS) cDNA.
  • GUS ⁇ -glucoronidase
  • Agrobacterium tumefaciens-mediated transformation of barley was performed with plasmid pMDC164-TdPR61 promoter using the procedure developed by Tingay et ⁇ l. (Plant /. 11: 1369-1376, 1997) and modified by Matthews et al. (MoI Breed. 7: 195-202, 2001).
  • Developing spikes were harvested from donor plants grown in the glasshouse when the immature embryos were approximately 1-2 mm in diameter. The immature embryos were aseptically excised from the surface-sterilised grain, and the scutella were isolated by removing the embryonic axes.
  • Agrobacterium suspension (50 ml) was aliquotted onto the scutella, and the Petri dish was held at a 45° angle to drain away excess bacterial suspension. The explants were then turned over and dragged across the surface of the medium to the edge of the Petri dish. The scutella were transferred to a fresh plate of callus induction medium and cultured cut side-up for three days in the dark at 22-24°C.
  • the scutella were removed to fresh callus induction medium containing 95 ⁇ M hygromycin B (Becton Dickinson Bio sciences, Palo Alto, CA, USA) and cultured in the dark. The entire callus of an individual scutellum was transferred to fresh selection medium every fortnight for a further six weeks. At the end of the callus selection period, the callus derived from each treated scutellum was transferred to shoot regeneration medium. This medium is based on the FHG recipe of Wan and Lemaux (1994, supra).
  • the regenerated shoots were excised from the callus and transferred to culture boxes (Magenta Corporation, Chicago, IL, USA) that contained hormone-free callus induction medium, supplemented with 95 ⁇ M hygromydn B to induce root formation.
  • the tissue culture-derived plants were finally established in soil and grown to maturity.
  • the above media contain 150 mg/L Timentin (SmithKline Beecham, Pty. Ltd., Melbourne, Australia) to inhibit the growth of Agrobacterium tumefaciens following co- cultivation.
  • Seed embryo-derived callus of cv. Nipponbare (Oryza sativa ssp. japonica) was co- cultured with the Agrobacterium strain EHA105 or LBA4404 carrying the pMDC164- TdPR61 promoter plasmid following the procedure detailed in Sallaud et al. (Theor. Appl. Genet. 106: 1396-1408, 2003).
  • Dehulled seeds were sterilised, inoculated on NB medium and incubated for 18-21 days in the dark as described in Chen et al. (Plant Cell Rep. 18: 25-31, 1998).
  • Embryogenic nodular units (0.5-1 mm long), released from the primary embryo scutellum-derived callus at the explant/medium interface, were transferred onto fresh NB medium and incubated for an additional 10-15 days depending on the variety.
  • the calli were transferred to NBS medium. After 1 week of incubation, the protuberances developed into brownish globular structures, which were gently teased apart with forceps on the medium around the original callus and incubated for 10-15 days in the resealed petri dish. After co-culture, the globular structures developed into calli.
  • the putatively transgenic, hygromycin-resistant calli were gently picked out, placed on the PRAG pre-regeneration medium and incubated for a further week.
  • AU of the resistant calli originating from a single co-cultured embryogenic nodular unit were grouped in a sector of the PRAG dish, which can accommodate 40-50 resistant calli.
  • Immature seeds of wheat cv. Bob white were surface-sterilized by immersing into 70% ethanol for 2 min, followed by incubation in 1% Sodium Hypochlorite solution with shaking at 125 rpm for 20 min and finally by three washes in sterile distilled water.
  • Immature embryos (1.0-1.5 mm in length, semitransparent) were isolated aseptically and were placed, with the scutellum side up, on solid culture medium. Embryos developing compact nodular calli were selected using a stereomicroscope and used for bombardment 7-21 days after isolation. The cultures were kept in dark at 25°C on solid MS (Duchefa, M0222; Murashige and Skoog 1962) with 30 g/1 sucrose, 2 mg/1 2,4-D (MS2).
  • Plasmid constructs were purified using Macherey-Nagel or Qiagen kits according to the manufacturer's protocols.
  • a DNA-gold coating according to the protocol of Sanford et al. was performed as follows: 50 ⁇ l of gold powder (1.0 ⁇ m) in 50% glycerol (60 mg/ml) was mixed with 10 ⁇ l DNA (1 mg/ml), 50 ⁇ l CaCb (2.5M) and 20 ⁇ l of 0.1 M spermidine. For co-transformation the plasmids were mixed at a ratio 1:1 (5 ⁇ g + 5 ⁇ g). The mixture was vortexed for 2 min, followed by incubation for 30 min at room temperature, brief centrifugation, and serial washing in 70% and 99.5% ethanol. Finally, the pellet was resuspended in 60 ⁇ l of 99.5% ethanol (6 ⁇ l/shot). All manipulations were done at room temperature.
  • Microprojectile bombardment was performed utilizing the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad). Before bombardment, immature embryos were pre-treated for 4 hours on MS2 medium supplemented with 100 g/1 sucrose. Embryos (50/plate) were then placed in the centre of a plate to form a circle with a diameter of 10 mm. Bombardment conditions were 900 or 1100 psi, with a 15 mm distance from the macrocarrier launch point to the stopping screen and a 60 mm distance from the stopping screen to a target tissue. The distance between the rupture disk and the launch point of the macrocarrier was 12 mm.
  • MS selection medium For regeneration, two days after bombardment treated calli may be transferred to MS selection medium supplemented with 2.0 mg/1 2,4-D and 150 mg/1 hygromydn B. After 3-6 selections (4-6 months) greening callus tissues may be subcultured on MS regeneration medium supplemented with lmg/1 kinetin and 5-10 mg/1 zeatin. Regenerating plantlets may then be transferred to jars with the half-strength hormone- free MS medium supplemented with 50 mg/1 hygromydn B. Fully developed plantlets may be acclimated for 7-10 days at room temperature in a liquid medium containing four-fold diluted MS salts. Plants with strong roots are then transplanted into soil and grown under greenhouse conditions to maturity.
  • the presence of the transformation vector in the putative transformed plants was investigated by using Southern blotting to detect the kpt selectable marker in DNA isolated from putatively transformed plant tissue.
  • Figure 1 shows a Southern Blot confirming the successful integration of pMDC164- TdPR61 into transgenic plant lines.
  • the number of bands hybridising with the probe reflects the number of integrated copies of the vector.
  • Leaf samples were homogenised in Eppendorf tubes with a sand powder in 0.3-5.0 ml of hot (55°C) 2x CTAB solution. Equal volumes of the CTAB solution and 0.6 - 10.0 ml of chloroform-isoamyl alcohol mixture (24:1 v/v) were added to the extracts. The tubes were incubated on a shaker (Mild Mixer PR-12, TAITEC) at a speed 5 at room temperature for 15-30 min. Phases were separated by centrifugation (3600-15000 rprn, 20 0 C, 5 min) and the supernatants were carefully transferred into new tubes with 0.6- 10.0 ml isopropanol.
  • DNA pellets (12 000-15 000 rpm, 20 0 C, 20 min) were washed by 70% ethanol, resuspended in 0.3-1.0 ml TE and RNAse treated for 30 min. After two sequential chloroform extractions DNA samples were pelleted by adding 0.1-0.33 ml of 10 M NH 4 Ac and 0.3-1.0 ml of isopropanol (15 000 rpm, 20 0 C, 20 min). Pellets were washed by 70% and 99.5% ethanol and redissolved in 20-500 ml of 0.1 TE. Southern Blotting
  • Isolated plant DNA was subjected to agarose gel electrophoresis and staining with ethidium bromide.
  • a Southern transfer assembly was constructed as follows: a sponge soaked in 0.4 M NaOH was placed in a Perspex tray. One sheet of filter paper soaked in 0.4 M NaOH was then placed over the sponge. The agarose gel was then overlaid on the soaked filter paper. A Hybond N+ membrane was then serially soaked in nanopure water and in 0.4 M NaOH for 30 seconds before being overlaid on the agarose gel. A further sheet of filter paper soaked in 0.4 M NaOH was then placed on top of the membrane, followed by a 10 cm stack of dry paper towels. A glass plate was then placed on top of the stack and the perspex tray was filled with 0.4 M NaOH.
  • the DNA was then allowed to transfer for at least 2 hours before disassembly of the transfer assembly.
  • the membrane was then rinsed for 1 minute in 100 ml 2x SSC and blotted dry on filter paper.
  • the membrane was then probed with a 1.1 kb fragment of the hygromycin phosphotransferase gene (kpt) amplified from the vector pCAMBIA1380 using standard techniques.
  • kpt hygromycin phosphotransferase gene
  • FIG. 2 The expression pattern of the TdPR61 promoter was observed in barley transformed with GUS under the control of the TdPR61 promoter.
  • Figures 2 and 3 show the expression of a GUS reporter under the control of the TdPR61 promoter in two transgenic barley lines, G81-2 and G81-11.
  • GUS expression in the transgenic barley lines was observed predominantly in the embryo surrounding region and later in the embryo. GUS expression was observed at 6 DAP and continued to be observed at 48 DAP, with the strongest expression being seen between 12 DAP and 15 DAP in the embryo surrounding region. Later expression of the GUS reporter is seen mainly in the embryo. No detectable expression was observed in other tissues.
  • ESRs embryos and embryo surrounding regions isolated from transgenic barley grain (8-15 DAP). The isolated ESRs and embryos were stained to detect GUS activity. Results are shown in Figures 4 and 5.
  • ⁇ -glucuronidase activity in transgenic plants was analysed by histochemical staining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl- ⁇ -glucuronic acid (X- Glue) (Bio Vectra) as described by Hull and Devic (Methods MoI Biol. 49: 125-141, 1995).
  • X- Glue 5-bromo-4-chloro-3-indolyl- ⁇ -glucuronic acid
  • Hull and Devic Methodhods MoI Biol. 49: 125-141, 1995.
  • Different plant organs, whole grain and grain sections of different ages were immersed in a lmg/ml X-Gluc solution in 5OmM sodium phosphate, pH 7.0, 1OmM Na EDTA, 2mM FeK 3 (CN)6, 2mM K 4 Fe(CN)e and 0.1% Triton X-100.
  • the sectioning and mounting was carried out on a Leica RM2265 Microtome. Each individual grain segment was cut into 12 ⁇ m-thick sections and the ribbons were mounted onto saline-coated slides. The slides were dried on a 42°C slide warmer overnight and deparaffinised.
  • the slides were soaked in xylene for 10 min and moved to fresh xylene for another 10 min or until the specimens were clear.
  • the specimen on each slide was mounted in DPX medium and covered with a cover slip.
  • the slides were air- dried in the fume hood overnight. The specimens were then observed using a compound microscope under bright-field illumination.
  • the histological assays showed GUS expression in the embryo surrounding region, particularly at the interface between the scutellum and the remainder of the embryo (24 DAP). Later, expression is also seen in particular regions of the scutellum.
  • FIGs 15 and 16 show the activity of the TdPR61 promoter in transgenic T2 generation barley lines. As shown in Figures 15 and 16, the TdPR61 promoter was active (observed as GUS staining) in the embryo and in the embryo surrounding region (ESR) in the T2 generation barley plants.
  • ESR embryo surrounding region
  • Mapping of the promoter regions was also performed using biolistic bombardment of promoter constructs into embryos and ESR tissues isolated from wheat (Triticum aestivum cv. Bobwhite) at 12-14 DAP.
  • TdPR61-l The full length promoter (TdPR61-l; SEQ ID NO: 3) and four truncated versions of the full length promoter, TdPR61-2 (SEQ ID NO: 13), TdPR61-3 (SEQ ID NO: 14), TdPR61-4 (SEQ ID NO: 15) and TdPR61-5 (SEQ ID NO: 16) were individually cloned in the pMDC164 binary vector upstream of a GUS gene. The resultant constructs were used for biolistic bombardment.
  • TdPR61-3 deletion (possibly close to or on the border with TdPR61-2 deletion) contains a cz ' s-element that is able to activate the promoter (cz ' s-element 1; SEQ ID NO: 17); while a fragment between the full length promoter and TdPR61-2 deletion contains a cz ' s-element defining a putative enhancer/modulator of activity (cz ' s-element 2; SEQ ID NO: 18), which increases promoter activity approximately 5 fold.
  • TdPR61-l.l SEQ ID NO: 19
  • TdPR61-1.2 SEQ ID NO: 20
  • TdPR61-1.3 SEQ ID NO: 21
  • TdPR61- 1.4 SEQ ID NO: 22
  • TdPR61-1.5 SEQ ID NO: 23
  • the pair of predicted embryo specific elements (AAACCCACACACG; SEQ ID NO: 25) were adjacent one to another and had single base pair overlap, which is shown in bold in the sequence above.
  • the first predicted embryo specific element (AAACCCA; SEQ ID NO: 26) shows homology to the SEF3 binding site from a soybean (Glycine max) consensus sequence from the 5' upstream region of beta-conglydnin (7S globulin) gene (AACCCA(-27bp- )AACCCA). This is the binding site of soybean embryo factors, SEF2 and SEF3.
  • the second predicted embryo specific element (ACACACG; SEQ ID NO: 27) shows homology to the binding site of bZIP transcription factors DPBF-I and 2 (Dc3 promoter-binding factor-1 and 2) from carrot. Dc3 expression is normally embryo- specific, and also can be induced by ABA.
  • CACGTAC The predicted endosperm specific element (CACGTAC; SEQ ID NO: 28) is situated relatively close to the embryo-specific elements. This element shows homology to an element found originally in GIuB-I gene in rice which is required for endosperm- specific expression and conserved in the 5'-flanking region of glutelin genes.
  • Plasmid constructs were purified using commercially available kits. DNA-gold compositions for Biolistic transformation were then produced as follows: 50 ⁇ l of gold powder (1.0 ⁇ m) in 50% glycerol (60 mg/ml) was mixed with 10 ⁇ l DNA (1 mg/ml), 50 ⁇ l CaCb (2.5M) and 20 ⁇ l of 0.1 M spermidine. For co-transformation plasmids were mixed at a ratio 1:1 (5 ⁇ g + 5 ⁇ g). The mixture was vortexed for 2 min, followed by incubation for 30 min at room temperature, brief centrifugation, washing by 70% and 99.5% ethanol. Finally, the pellet was resuspended in 60 ⁇ l of 99.5% ethanol (6 ⁇ l/shot). In some experiments spermidine and CaCb were replaced with 10 ⁇ l of PEG/Mg. All manipulations were done at room temperature.
  • Microprojectile bombardment was performed utilizing the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad). Immature embryos were pre-treated for 4 hours on MS2 medium supplemented with 100 g/1 sucrose. Embryos (50/plate) were placed in the centre of the plate to form a circle with a diameter of 10 mm. These embryos were then bombarded under the following conditions: 900 or 1100 psi, 15 mm distance from a macrocarrier launch point to the stopping screen and 60 mm distance from the stopping screen to a target tissue. The distance between the rupture disk and the launch point of the macrocarrier was 12 mm.
  • the embryos were acclimated in the same media and stained for GUS activity after 24hr of acclimation. Three repeats (independent bombardment events, three plates) with ten embryos on each plate were used in the experiment. After GUS staining, the tissues were examined for GUS colour forming units (CFU) and images were captured using a LEICA DC 300F camera attached to a LEICA MZ FLIII stereomicroscope. The CFU were counted by eye using the images viewed with Microsoft Office Document Imaging.
  • CFU GUS colour forming units
  • GUS activity was not detected before 7 DAP and during first 2-3 days could be seen mainly in the embryo and in the opposite pole of the grain. In one of transgenic plants (from 3 tested) it was also detected in vascular tissue of lemma at the 8 DAP (see Figure 9).

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Abstract

La présente invention concerne de manière générale des séquences de régulation transcriptionnelle pour réaliser l’expression d’une séquence de nucléotides d’intérêt dans une plante.  Plus particulièrement, la présente invention concerne des séquences de régulation transcriptionnelle qui dirigent l'expression spécifique ou préférentielle d’une séquence de nucléotides d’intérêt liée de manière fonctionnelle dans un embryon végétal et/ou dans une région environnante de l’embryon dans une semence de plante.
PCT/AU2009/000091 2008-01-31 2009-01-30 Expression spécifique des semences dans des plantes WO2009094704A1 (fr)

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WO2010019996A1 (fr) * 2008-08-18 2010-02-25 Australian Centre For Plant Functional Genomics Pty Ltd Séquences de commande transcriptionnelle active de graine
EP2507375A1 (fr) * 2009-12-03 2012-10-10 BASF Plant Science Company GmbH Cassette d'expression pour expression spécifique de l'embryon dans des plantes
WO2017141173A2 (fr) 2016-02-15 2017-08-24 Benson Hill Biosystems, Inc. Compositions et procédés de modification de génomes
WO2019030695A1 (fr) 2017-08-09 2019-02-14 Benson Hill Biosystems, Inc. Compositions et procédés de modification de génomes
WO2019049111A1 (fr) 2017-09-11 2019-03-14 R. J. Reynolds Tobacco Company Méthodes et compositions permettant d'augmenter l'expression de gènes d'intérêt dans une plante par co-expression avec p21
WO2021046526A1 (fr) 2019-09-05 2021-03-11 Benson Hill, Inc. Compositions et procédés de modification de génomes
WO2023119135A1 (fr) 2021-12-21 2023-06-29 Benson Hill, Inc. Compositions et procédés de modification de génomes

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010019996A1 (fr) * 2008-08-18 2010-02-25 Australian Centre For Plant Functional Genomics Pty Ltd Séquences de commande transcriptionnelle active de graine
EP2507375A1 (fr) * 2009-12-03 2012-10-10 BASF Plant Science Company GmbH Cassette d'expression pour expression spécifique de l'embryon dans des plantes
EP2507375A4 (fr) * 2009-12-03 2013-04-24 Basf Plant Science Co Gmbh Cassette d'expression pour expression spécifique de l'embryon dans des plantes
EP3002332A3 (fr) * 2009-12-03 2016-06-29 BASF Plant Science Company GmbH Cassettes d'expression pour expression spécifique à des embryons dans des plantes
WO2017141173A2 (fr) 2016-02-15 2017-08-24 Benson Hill Biosystems, Inc. Compositions et procédés de modification de génomes
EP4063501A1 (fr) 2016-02-15 2022-09-28 Benson Hill, Inc. Compositions et procédés de modification de génomes
EP4306642A2 (fr) 2016-02-15 2024-01-17 Benson Hill Holdings, Inc. Compositions et procédés de modification de génomes
WO2019030695A1 (fr) 2017-08-09 2019-02-14 Benson Hill Biosystems, Inc. Compositions et procédés de modification de génomes
EP4317443A2 (fr) 2017-08-09 2024-02-07 RiceTec, Inc. Compositions et procédés de modification de génomes
WO2019049111A1 (fr) 2017-09-11 2019-03-14 R. J. Reynolds Tobacco Company Méthodes et compositions permettant d'augmenter l'expression de gènes d'intérêt dans une plante par co-expression avec p21
WO2021046526A1 (fr) 2019-09-05 2021-03-11 Benson Hill, Inc. Compositions et procédés de modification de génomes
WO2023119135A1 (fr) 2021-12-21 2023-06-29 Benson Hill, Inc. Compositions et procédés de modification de génomes

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