WO2010019996A1

WO2010019996A1 - Seed active transcriptional control sequences

Info

Publication number: WO2010019996A1
Application number: PCT/AU2009/001059
Authority: WO
Inventors: Sergiy Lopato; Nataliya Kovalchuk; Ainur Ismagul
Original assignee: Australian Centre For Plant Functional Genomics Pty Ltd
Priority date: 2008-08-18
Filing date: 2009-08-18
Publication date: 2010-02-25
Also published as: AU2009284691B2; AU2009284691A1

Abstract

The present invention relates generally to transcriptional control sequences tor effecting expression of a nucleotide sequence of interest in a plant. More particularly, the present invention relates to nucleotide sequences 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 seed, wherein said transcriptional control sequence is derived from a GL7 gene.

Description

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SEED ACTIVE TRANSCRIPTIONAL CONTROL SEQUENCES

PRIORITY CLAIM

This patent application claims priority to Australian provisional patent application 2008904229, filed 18 August 2008,, the contents of which is hereby incorporated by- reference.

FIELD

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 one or more parts of a plant seed.

BACKGROUND

The primary emphasis in genetic modification has been directed to prokaryotes and mammalian cells. For a variety of reasons,, plants have proven more intransigent than other eukaryotic cells to genetically manipulate. However, in many instances, it is desirable to effect transcription of an introduced nucleotide sequence of interest in a plant.

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 [issues or organs is desired, a tissue -specific promoter may be used.

Aicordingly, there is a substantial interest in identifying transcriptional control sequences, such as promoters or enhancers, which are active in plants. Frequently, it is also desirable to specifically or preferentially direct transcription in particular plant organs, tissues or cell types, or at particular developmental stages of plant growth.

Thus, isolation and characterisation of transcriptional control sequences, which can serve as regulatory regions for the expression of nudeotide sequent es of interest in particular cells, tissues or organs of a plant, would be desirable for use in the genetic manipulation of plants.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the i ommon general knowledge in any country.

SUMMARY OF I¹HE IX VHM ION

The present invention is predicated, in part, on the identification and lunctional i

preferentially- direct expression of an operably connected nudeotide sequence in one or more parts of a plant seed.

Thus, in a first aspect, the present invention provides an isolated nucleic acid i om prising:

(i) a nudeotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nudeotide sequence in one or more parts of a plant seed, wherein said transcriptional control sequence is derived from a GL7 gene; and/or (ii) a nucleotide sequence defining a functionally active fragment or variant of the nucleotide sequence defined at (i).

In some embodiments, the GlJ gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: ^'! or a homolog thereof.

In some embodiments, the transcriptional control sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3 or a functionally active fragment or variant thereof,

In a second aspect, the present invention also provides a nucleic acid construct comprising an isolated nucleic acid according to the first aspect of the invention.

In a third aspect, the present invention provides a cell comprising a nucleic acid construct according to the second aspect of the invention.

In a fourth aspect, the present invention contemplates a multicellular structure comprising one or more cells according to the third aspect of the invention.

In some embodiments, the multicellular structure comprises a plant or a part, organ or tissue thereof. In some embodiments of the fourth aspect of the invention, the plant or part, organ or tissue thereof comprises a seed,

In some embodiments of the fourth aspect of the invention, a nucleotide sequence of interest may be o per ably connected to the transcriptional control sequence or the functionally active fragment or variant thereof, such that the nucleotide sequence of interest is specifically or preferentially expressed in a seed, or in a particular cell or tissue type thereof, and optionally at a particular developmental stage of the seed,

In a fifth aspect, the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant 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 first aspect of the invention.

Nucleotide and amino acid sequences are referred to herein by a 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.

Table 1 - Summary of Sequence Identifiers

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description. In some embodiments the present invention is predicated, in part, on the cloning of the gene and promoter of a GL7 gene from wheat. Wheat, barley and rice were stably transformed with promoter-GUS fusion constructs and spatial and temporal activity of the promoter was studied using whole-mount and histological assays. It was demonstrated that TaGLJ promoter is preferentially active in the seed.

As used herein, the term "transcriptional control sequence" should be understood as a nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence. As such, 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. As referred to herein, the term "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.

As used herein, the term "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. For example, promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to 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 from the transcription 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 which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Thus, in a first aspect, the present invention provides an isolated nucleic acid comprising:

(i) a 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 seed, wherein said transcriptional control sequence is derived from a GL7 gene; and/or

(ii) a nucleotide sequence defining a functionally active fragment or variant of the nucleotide sequence defined at (i).

In the present invention, "isolated" refers to material removed from its original environment (e.g. the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, 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 (e.g. polymerase chain reaction and the like).

The isolated nucleic acid of the present invention may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, 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. In addition, 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 acid" also embraces chemically, enzymatically, or mefaboMcally modified forms of DNA and RNA.

As set out above, 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 seed.

As referred to herein, a plant "seed" should be understood to refer to a mature or immature plant seed. As such, the term "seed" includes, for example,, immature seed carried by a maternal plant or seed released from the maternal plant. In some embodiments, the term "seed" may encompass any seed plant sporophyte between the developmental stages of fertilisation and germination.

As would be appreciated, the term "seed" may also encompass the various cells and tissues that make up the mature or immature seed. For example, mature seeds may include tissue types such as the embryo, embryo surrounding region, endosperm transfer layer, starchy endosperm, aleurone layer, pericarp and the like. Meanwhile, immature seeds may include, for example, fertilised egg cells, zygotes, fertilised central cells, embryos, the endosperm coenocyte, the endosperm syncytium and the like.

In some embodiments, the term "seed" may also extend to floral and/or maternal garnetophyte tissues. For example, the term "seed" may include floral and/or maternal gametophyte structures that are precursors to, and/or ultimately develop into, a seed or an associated structure. An example of such a structure may include an ovary or embryo sac in a plant flower. Thus, in some embodiments of the invention, the present invention relates to expression in such tissues. it should be understood that reference herein to expression in a plant seed refers to the transcription and/or translation of a nucleotide sequence in one or more cells or tissues of a plant seed and/or at one or more developmental stages of the plant seed. This definition in no way implies that expression of the nucleotide sequence must occur in all cells of the plant seed or at all developmental stages of the seed, As set out later, the nucleic acids of the present invention may direct expression in particular parts of a seed and/or at particular developmental stages of a seed.

As set out above, the transcriptional control sequences contemplated by the present invention "specifically or preferentially" direct expression of an operably connected nucleotide sequence in a plant seed. As used herein, ''specifically expressing" means that the nucleotide sequence of interest is expressed substantially only in a plant seed

(or a particular tissue or cell type therein). "Preferentially expressing" should be understood to mean that the nucleotide sequence of interest is expressed at a higher level in a plant seed (or tissue or cell type therein) than in one or more other tissues of the plant, e.g. leaf tissue or root tissue. In some embodiments "preferential" expression in a plant flower includes expression of a nucleotide sequence of interest in a plant 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-seed tissue of the plant.

The transcriptional control sequence or functionally active fragment or variant thereof may effect specific or preferential expression in a seed from at least one seed plant species, including monocotyledonous angiosperm plants ("monocots"), dicotyledonous angiosperm plants ("dicots") or gymnosperm plants. For clarity, this should be understood as the transcriptional control sequence or functionally active fragment or variant thereof being able to effect specific or preferential expression in a seed in at least one plant species. 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 seed. Tints, 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 seed in all plants in which they are active.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a seed of a monocotyledonous plant.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a seed of a plant in the family Poaceae.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a seed of a cereal crop plant.

As used herein, 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 quinoa.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a seed of a wheat plant.

As referred to herein, "wheat" should be understood as a plant of the genus Trlticurn. Thus, the term "wheat" encompasses diploid wheat, tetraploid wheat and hexaploid wheat. In some embodiments, the wheat plant may be a cultivated species of wheat including, for example, T, aestizmm, T. durum, 7. monococcum or T. spelta. In some embodiments, the term "wheat" refers to wheat of the species Trhϊcuni aeshvwn. In some embodiments, the transcriptional control sequence directs expression of an opcrably connected nucleotide sequence in a seed of a barley plant.

As referred to herein, "barley" includes several members of the genus Hordeum. The term "barley" encompasses cultivated barley including two-row barley (Hordeum disϊichum), four-row barley (Hordeum tetrastichum) and six-row barley (Hordeum vulgβrc). in some embodiments, barley may also refer to wild barley,, {Hordeum spontaneum). In some embodiments, the term "barley" refers to barley of the species Hordeum vulgare.

in some embodiments, the transcriptional control sequence directs expression of an o per ably connected nucleotide sequence in a seed of a rice plant.

As referred to herein, "rice" includes several members of the genus Oryza including the species Oryza sativa and Oryza giaberrhna. The term "rice" thus encompasses rice eultivars 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.

As set out above, the nucleic acid of the first aspect of the present invention may also specifically or preferentially direct expression in a particular cell or tissue of a plant seed and/or specifically or preferentially direct expression at a particular developmental stage of a plant seed.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the endosperm, or a part thereof, in the seed.

The tissues of a plant encompassed by the term "endosperm" would be readily understood by one of skill in the art. However, this term should be understood to encompass at least the nutritive tissue, characteristic of flowering plants, which nourishes the embryo. The endosperm is typically formed after the fertilisation of the polar nuclei of the central cell by a sperm nucleus. In most plants the endosperm is a transient tissue absorbed by the embryo before maturity, whereas in cereals and grasses it contains storage reserves in the mature grain and is not absorbed until after germination.

Typically, the "endosperm" includes at least five ceil types,, namely, the central starchy endosperm (CSE), the sub-aleurone layer (SAL), the alcurone layer (AL), the endosperm transfer layer (ETL) and the embryo-surrounding region (ESR). The characteristics of each of these cell types are described in detail in the review of Olsen el ah (Trends in Plant Science 4(7): 253-257, 1999).

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the aleurone layer, or a part thereof, in the seed.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the embyro, or a part thereof, in the seed.

As referred to herein, 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. For example, dicotyledonous angiosperm embryos comprise two cotyledons, monocotyledonous angiosperm embryos comprise a single cotyledon (also referred to as the scutellurn), while gyrrmosperm embryos may comprise a variable number of cotyledons, typically ranging from 2 to 24. In light of the above, reference herein to an "embryo", particularly in the context of specific or preferential expression within an embryo (see later), may include expression in all of the embryo or expression in one or more cells, tissues or parts of the embryo. In some embodiments, the transcriptional control sequence directs expression ot an operably connected nucleotide sequence in a maternal gametophyte tissue In some embodiments, the maternal gametophyte tissue comprises an ovary or embryo sac.

As set out above, the present invention contemplates a transcriptional control sequence which specifically or preferentially directs expression ol an operably connected nucleotide sequence in one or more parts ot a plant seed, wherein said transcriptional control sequence is derived from a GJ 7 gene.

The term "derived from", as used herein, refers to a source or origin for the transcriptional control sequence. For example, a transcriptional control sequence ''derived from a GL7 gene" refers to a transcriptional control sequence which, in its native state, exerts at least some transc riptional c ontrol over a GI 7 gene. The term '' derived from" should also be understood to reter to the sourc e of the sequent e information for a transcriptional control sequence and not be limited to the source of a nucleic acid itself. Ihus, a transcriptional control sequence derived from a GL7 gene need not necessarily be directly isolated from the gene. For example, 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 GIJ gene should be considered derived from a GIJ gene

A "GL7 gene" as referred to herein encompasses any nucleotide sequence which encodes a GL7 polypeptide. As described later, CAJ polypeptides may be diaracterised as members ot the class IV of hυmeυdomain leucine zipper family of transcription factors.

Transcription factors containing a homeodomain (HO) together with a leucine zipper

(ZIP) motit constitute a large family ol plant specific transcription factors ( Il's). These factors ma v be classified into tour classes. The class IV is also known as ! ΪD-GL2 after the first identified gene from Arabidopsis GLABRA2 (GL2).

In some embodiments, GL7 polypeptides may also be characterised by the presence of a STeroidogenk: Acute Regulatory (STAR) related lipid transfer domain. The most striking feature of the STAR domain structure is a predominantly hydrophobic tunnel extending nearly the entire protein and used to binding a single molecule of large lipophilic compounds,, like cholesterol.

In some embodiments,, the GL7 polypeptide encoded by the GlJ gene contemplated in accordance with the present invention comprises the amino acid sequence set forth in SEQ ID NO: 1 or a homolog thereof.

The term "homolog", as used herein with reference to homologs of polypeptides comprising the amino add 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. In some embodiments, 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 whidi comprises at least 35% sequence identity, at least 40% sequence identity, at least 45% sequence identity, at least 50% sequence identity, at least 55% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

When comparing amino acid sequences to calculate a percentage identity, the compared sequences should be compared over a comparison window of at least 100 amino acid residues, at least 200 amino acid residues, at least 400 amino acid residues, at least 800 amino acid residues, or over the full length of SEQ ID NO: 1. 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) lor optimal alignment of the two sequences. Optimal alignment of sequences lor aligning a comparison window may be conducted by computerised implementation^ of algorithms such the BLAST family of programs as, for example, disclosed by Altsehul el Λ. (Nu ύ. Adds Res. 2x 3389-3402, J 997). A detailed discussion ot sequence analysis can be found in Unit 19.3 of Ausubel el ol. (Current Piotocols in Molecular Biology, John Wiley & Sons Inc., 1994-199«. Chapter 15, 199S).

The transcriptional control sequent es 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 lhe transcriptional control sequences contemplated herein are derived from a plant. In some embodiments,, the transcriptional c ontrol sequent es of the present invention are derived from a monocυt 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.

In some embodiments,, the transcriptional c ontrol sequent e is derived from a Tήticum species (for example T. aesήvum, T. durum, T. monococcum, T. die ocean, T. spells or T. γolonicum). In some embodiments, the transcriptional control sequence is derived from a tetraploid wheat (for example T. durum, 1. άicoccon, or I. polonicum). In some embodiments, the transcriptional control sequence is derived from a durum wheat, and in some embodiments, the transcriptional t ontrol sequence is derived from Tnlicum durum.

In some embodiments, 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 or a homolog thereof. One example of a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2 is a gene comprising the nucleotide sequence set forth in SEQ ID NO: 4.

The term "homolog", as used herein with reference to homologs of genes comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 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 SEQ ID NO: 2. In some embodiments, the homolog, ortholog, paralog, mutant or variant of a polypeptide comprising an open reading frame which comprises the nucleotide sequence set forth in SEQ ID NO: 2 comprises a nucleotide 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 55% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence 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 SEQ ID NO: 2.

When comparing nucleotide sequences to calculate a percentage identity, the compared sequences should be compared over a comparison window of 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: 2. 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 aligning a comparison window may be conducted by computerised implementations of algorithms sudi 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 fnc, 1994-1998, Chapter 15, 1998).

In some embodiments, 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.

As set out above, 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 SEQ ID NO: 3.

"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 a plant seed (or a particular cell or tissue type thereof as hereinbefore described) in at least one plant type. In some embodiments of the invention the functionally active fragment is at least 200 nucleotides (nt), at least 500 nt, at least 1000 nt, at least 1500 nt, at least 2000 nt or at least 2500 nt in length. In further embodiments, the fragment comprises at least 200 nt, 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.

"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 in a plant seed (or a particular cell or tissue type thereof as hereinbefore described) in at least one plant type. The term "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.

in some embodiments, 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 SEQ ID NO: 3.

When comparing nucleic acid sequences to calculate a percentage identity, the compared nucleotide sequences should be compared over a comparison window of 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 (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 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 Ausubei et al. (1998, supra).

In some embodiments, 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. In some embodiments, 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 SEQ ID NO: 3 under stringent conditions. As used herein, "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 30⁰C. Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. ϊn some embodiments, stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions. Exemplary low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, 1 M NaCl,. 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in Ix to 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodiυm citrate) at 50 to 55°C. 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°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. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridisation is generally less than 24 hours, usually 4 to ^'! 2 hours.

Specificity of hybridisation is also a function of post-hybridisation washes, with the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkofh and WaW (Anal Biocheni. 138: 267-284, 1984), i.e. Tm =81.5°C +16.6 (log M)+0.41 (% GQ-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA,, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs. 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 10⁰C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, high stringency conditions can utilise a hybridisation and/or wash at, for example, 1, 2, 3, or 4°C lower than the thermal melting point (Tm); medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, 8, 9, or 10⁰C lower than the thermal melting point (Tm); low stringency conditions can utilise a hybridisation and/or wash at, for example, 11, 12, 13, 14, 15, or 20⁰C lower than the thermal melting point (Tm). Using the equation, hybridisation and wash compositions, and desired I^'™, those of ordinary skill will understand that variations in the stringency of hybridisation and/or wash solutions are inherently described. If the desired degree of mismatching results in a T- of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridisation ot nucleic acids is found in πjssen (Laboratoi y Techniques in Biochemist! y and Molecular Biology Hybridisation with Nucleic Acid Probes, Pt I, Chapter 2, Elsevier, New York, 1993), Ausubel et ah, eds. (Current Protocols in MoUx alar Biology, Chapter 2, Greene Publishing and Wiley-interscience, New York, 1995) and Sambrook el al. (Molecular Cloning: A Laboratory Manual, 2^nJ ed., Cold Spring Harbor Laboratory Press, Plainview, NY, 1<»S9).

In a sci ond aspect, the present invention also provides a nucleic acid i on struct 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 polydeoxyribonucleotide, which may be unmodified RNΛ or DNA υr modified RNA or DNA. For example, 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. In addition, 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.

In some embodiments, 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.

In some embodiments, 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.

The term "heterologous with respect to the transcriptional control sequence" refers to the nucleotide sequence of interest being any nucleotide sequence other than that whidi the transcriptional control sequence (or functionally active fragment or variant thereof) is operably connected to in its natural state. For example, 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,

In accordance with the definition above, it would be recognised that a nucleotide sequence of interest which is heterologous to a 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.

in some embodiments, the nucleic acid construct may further comprise a nucleotide sequence defining a transcription terminator. The term "transcription terminator^"' or ''terminator" refers to a DXΛ sequence at the end ol a transcriptional unit which signals termination of transcription. Terminators are generally 3'-non-translated DNA sequences and may contain a polyadenyJation 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 sequent es whuh may be useful in plant cells include: the nopalino synthase (nos) terminator, the CaMV 35S terminator, the octυpine synthase (oa) terminator,, potato proteinase inhibitor gene (pin) terminators, such as the pinJJ and pinIII terminators and the like.

In some embodiments the nucleic acid construct comprises an expression cassette i om prising the structure:

( [Nμ - TCS - IN] - Sol - [N|v - Tr - [N I )

wherein: [N]w 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;

[N]v comprises one or more nucleotide residues, or is absent;

Sol comprises a nucleotide sequence of interest which is operably connected to TCS;

\N], comprises one or more nucleotide residues, or is absent; TT comprises a nucleotide sequence defining a transcription terminator; - 11 -

[Njz comprises one or more nucleotide residues,, or is absent.

The nucleic acid constructs of the present invention may further comprise other nucleotide sequences as desired. For example, 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.

As used herein, 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 cells which are 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, kanamycin-resistance genes, the AURl-C" gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (e.g. nptl and nptlf) and hygromydn phosphotransferase genes (e.g. Jψt); herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transf^'erase-encoding genes (e.g. bar), glyphosate resistance genes including 3- enoyl pyruvyl shikimate 5-phosphate synthase-encoding genes (e.g. aroA), bromyxnil resistance genes including bromyxnil nitrilase-encoding genes, sulfonamide resistance genes including dihydropterate synthase-encoding genes (e.g. siύ) and sulfonylurea resistance genes including acetolactate synthase-encoding genes; enzyme-encoding reporter genes such as GUS-encoding and chloramphenicolacetyltransferase (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.

The constructs described herein may further include nucleotide sequences intended for the maintenance and/or replication of the construct in prokaryotes or eukaryotes - 71 -

and/or the integration of the construct or a part thereof into the genome of a eukaryotic or pro kary otic cell.

In some embodiments, the construct of the invention is adapted to be at least partially transferred into a plant cell via Agrobacterium-mediated transformation. Accordingly, in some embodiments, the nucleic acid construct of the present invention 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. However, the term "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 Agrobαcterium sp. cell into a plant cell susceptible to Agrobαcteήum-mcdiaLed transformation. By way of example, reference is made to the paper of Peralta and Ream (Proc, Nαtl. Acαd. Sd, USA, 82(15): 5112-5116, 19S5) and the review of Gelvin (Microbiology and Molecular Biology Reviews, 67(1): 16-37, 2003).

In some embodiments, the present invention also contemplates any suitable modifications to the construct which facilitate bacterial mediated insertion into a plant cell via bacteria other than Agi obacierium sp., for example, as described in Broolhaerls et al (Natm e 433: 629-633, 2005).

In some embodiments, the constructs of the second aspect of the invention may also comprise nucleotide sequences that encode regulatory microRNAs ("miRNA") and/or a target sequence for an miRNA, which may further modulate the expression pattern determined by the nucleotide sequence of the first aspect of the invention. A discussion of the 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)

Those skilled in the art will be aware of how to produce the constructs described herein, and of the requirements for obtaining the expression thereof, when so desired, - 2A -

in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a construct described herein or a derivative thereof in a prokaryotic cell such as an E. coH cell or a plant cell or an animal cell. Exemplary methods for cloning nucleic acid molecules are described in Sambrook el a!, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2000).

In a third aspect,, 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 he integrated into the genomic DNA of the cell.

As used herein, the term "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. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous plasmid DNA and the like. As such, the term "genomieally integrated" contemplates any of chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, or the like. A ''genomieally integrated form" of the construct may be all or part of the construct. However, in some embodiments the genomieally 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. In some embodiments, the cell is a plant cell. In some embodiments the cell is a monocot plant cell. In some embodiments the cell is a cell from a plant in the family Poaceae. In some embodiments the cell is a cereal crop plant cell. In some - ?S -

embodiments the cell is a wheat cell, a barley cell or a rice ceil.

In some embodiments, the cell may also comprise a prokaryotic cell. For example, the prokaryotic cell may include an Agrόbacterium sp. cell (or other bacterial cell), which carries the nucleic acid construct and which may, for example, be used to transform a plant. In some embodiments, the prokaryotic cell may be a cell used in the construction or cloning of the nucleic acid construct (e.g. an E. coli ceil).

In some embodiments, the multicellular structure comprises a plant or a part, organ or tissue thereof. As referred to herein, "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.

In some embodiments of the fourth aspect of the invention, the plant or part, organ or tissue thereof comprises reproductive material for a plant including, for example, seeds, flowers, vegetative plant material, explants, plant tissue in culture including callus or suspension culture and the like.

As would be appreciated from the remainder of the specification the plant or a part, organ or tissue thereof contemplated in the fourth aspect of the invention may include, for example, any of a rnonocot, a plant in the family Poaceae, a cereal crop plant, a wheat plant, a barley plant, or a rice plant or a part, organ or tissue of any of the foregoing.

In some embodiments of the fourth aspect of the invention, the plant or part, organ or tissue thereof comprises a seed as hereinbefore defined. In some embodiments of the fourth aspect of the invention, a nucleotide sequence of interest may be operably connected to the transcriptional control sequence or the functionally active fragment or variant thereof,, such that the nucleotide sequence of interest is specifically or preferentially expressed in a seed, or in a particular cell or tissue type thereof, and optionally at a particular developmental stage, as described above with respect to the first aspect of the invention,

As set out above, in its fifth aspect, 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. In some embodiments, 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. For example, 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.

In some embodiments, 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 Agrobacierhim- mediatcd transformation, micro projectile bombardment based transformation methods and direct DN Λ uptake based methods. Roa-Rodriguez et al. (Λgrobaclerium mediated transformation of plants, 3^rd Fd. GAMBIA Intellectual Property Resource, Canberra, Australia, 200°>) review a wide array of suitable Λςrohicteήnm-medidted plant transformation methods for a wide range of plant species. Other bacterial-mediated plant transformation methods may also be utilised, for example, see Brooihaerts el ol. (2005, supra). Microprojeciile bombardment may also be used to transform plant tissue and method" for the transformation of plants particularly cereal plants, reviewed by Casas el al. (Plan! Breeding Rtv. 13: 235-264, 1995). Direct DXA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Gaibraiih 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 indude infiltration, electroporation of cell" and tissues, electroporation of embryos, microinjection, pollen-tube pathway-, "ilicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-rrojanowska (Cell. MoI. Biol. Lett. 7: «49-858, 2002). Λ range of other plant transformation methods may also be evident to {hose of skill in the art and, accordingly, the present invention should not be considered in any way limited to the partu ular plant transformation methods exemplified above.

As set out above, the transcriptional control sequence of the present invention is introduced into a plant ceil such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence and the present invention i ontem plates any method to effei t thR For example, 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, in another example, the nucleic acid sequence of the present invention may be inserted into the genome of a target cell such that if is placed in operable c onnection with an endogenous nucleic acid sequence. As would be recognised by one of skill in the art, 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 ef al. [Nat Biotedmos 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, GiJS, 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. Furthermore, the nucleotide sequence of interest may alternatively encode a non- translated RNA, for example an siRNA, rniRNA, antisense RNA and the like.

In some embodiments, the nucleotide sequence of interest may comprise, for example, a pathogen responsive (PR) gene, a resistance (R) gene or a defensin gene, In some embodiments, the nucleotide sequence of interest may encode a protein such as PDR5 or TRiIOl, Such proteins may be expressed in a seed-specific manner in crop plants, such as wheat, in order to lower the incidence of diseases such as head blight (caused by Fiisarium graminearum or GlbbereUa 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 seeds. For example, in some embodiments, the plant may be a monocotyledonous plant. In some . JQ _

embodiments, the plant may be a plant in the family Poaceae. In some embodiments, the plant may be a cereal crop plant. In some embodiments the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a wheat seed,, a barley seed and/or a rice seed.

As set out above, the method of the fifth aspect of the present invention may also be used to specifically or preferentially direct expression of a nucleotide sequence of interest in a particular cell or tissue of a plant seed and/or specifically or preferentially direct expression at a particular developmental stage of a plant seed.

In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a maternal gametophyte.

In further embodiments of the method of the fifth aspect of the invention, the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, as defined siφra.

Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various constructs described herein. See, for example, Maniatis ct ah, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1982) and Sambrook et al. (2000, supra).

The present invention is further described by the following non-limiting examples:

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows (A) an alignment of protein sequences of TaGL7 and TdGL7 to protein sequences of closest homologies from other plants. Identical amino acids are in black boxes, similar amino acids are in grey boxes. (B) shows a broader alignment of TaGL7 and TdGL7 with closest homologues and proteins from the same subfamily of HD-Zip transcription factors presented in the form of a phylogenetic tree.

Figure 2 shows the results of Q-PCR analysis of TaGLJ expression in different wheat tissues, at different stages of grain development and in several grain fractions.

Figure 3 shows the spatial and temporal GUS expression in wheat directed by the TdGL? promoter. Strong (Al, A2) GUS expression in the Ti grain from the same transgenic line at 5 DAP: Al - crease up, A2 - crease down. TdGlJ promoter activity in the whole Ti grain of the same transgenic line at the 6 DAP (A3). Hand cut longitudinal section at 11 DAP (A4). Two cross sections, the whole grain and longitudinal section at 12 DAP (A5 •^■ A8). Longitudinal sections at 14 DAP.

Figure 4 shows spatial and temporal GUS expression in the υn-cut (All and A16), hand -cut (Al 2 and A22) and 10 μm thick sections (histochemicai GUS assay counterstained with Safranin Orange) (A13-A15, A17-A21, A23-A30) of wheat grain at 2 (A11-A15), 5 (A16-A20), 10 (A21-23, 25, and A26), 11 (A24 and A27), and 14 (A28- A30) DAP. At 2 and 5 DAP GUS activity was observed in pericarp (A13-A15, A19-A20) and liquid and partially cellularized endosperm (Al 2, A19). At 10-15 DAP CUS activity in seed coat became faint, but it was detected throughout the starchy endosperm, it vv as stronger in endosperm transfer cell layer (A22 and A25) and in aleurone (A21-A23, A28-A30). At 10 DAP GIJS activity was also observed in the main vascular bundle of the grain (A25 and Alb).

Figure 5 shows the spatial and temporal GUS expression in barley directed by the TaG L7 promoter. GUS expression in barley flowers before anthesis (Bl) and at anthesis (B2-B3). GUS expression is observed in the ovary and in the stigma. Strong CJUS expression was observed in the embryo sac, but no expression in the pericarp and flower tissue was observed at 1 DAP (B4, B5). Strong expression was observed in partially cellularized endosperm at 5 DAP, demonstrated in the longitudinal section (Bό) and cross sections (B7, B8). Strong GUS expression was observed in the embryo and moderate expression was observed in the endosperm at 10 DAP, as shown in the longitudinal (B9) and cross (BlO) section of grain at 10 DAP. Longitudinal sections are also shown at 15 DAP (BI l, B12), 20 DAP (B13, B14) and 30 DAP (Bl 5, B16).

Figure 6 shows the spatial and temporal expression in rice directed by the TdGL7 promoter, GUS expression was detected in grain and vascular tissue of palea at 2 DAP (Cl). Strong GUS expression was detected in all grain tissues at 2 (C2), 10 (C3), 15 (C4), 25 (C5), 35 (C6), 45 (C7), and 56 (CS) DAP.

Figure 7 shows a close-up view of GUS expression directed by the TdGLJ promoter in un-cut rice grain at 2 DAP.

EXAMPLE 1 CkinlngiifJhe.7gGI_i-7.g£llg

The full length cDNA of TaGL-7 was isolated using a yeast one-hybrid (YlH) screen from a cDNA library prepared from the whole grain of Tήticum aestiviini at 0-6 Days After Pollination ("DAP"), A quadruple repeat of the ris-element 5'-TAAATGCA-3', which is specific for HDZip IV transcription factors, was used as a bait.

Three clones containing the same length of the insert were selected in the screen. The size of the cloned cDNA was 3281 bp. It contained the full length open reading frame for the 883 aa long protein. A search through the databases using the TaGL7 protein sequence identified this protein as a member of the class IV of homeodomain leucine zipper family of transcription factors. Besides the homeodomain and leucine zipper, responsible for homo- and hetero-dimerization and DNA binding, TaGL7 contains a STeroidogenic Acute Regulatory (STAR) related lipid transfer domain.

The most striking feature of the STAR domain structure is a predominantly hydrophobic tunnel extending nearly the entire protein and used to bind a single molecule of a large lipophilic compound,, for example, cholesterol.

The expression of the JaGL? in different plant tissues and grain at different stages of development was demonstrated by quantitative PCR (Figure 2). Weak expression was found in all tested tissues. It was slightly higher in shoots of seedlings and in flowers during meiosis. Strong expression was detected in the liquid fraction of the syncytial endosperm at 5 DAP, but gene expression in pericarp at the same time was very low.

The 3' untranslated region (3'-UTR) of TaGL? was used as a probe to screen a bacterial artificial chromosome (BAC) library prepared from genomic DNA of Trhϊcum durum cv. Langdon (Cenci et ah, TJieor A>ψl Genetics 107: 931-939, 2003) using Southern blot hybridisation. Three BAC! clones were selected for further analysis on the basis of the strength of the hybridisation signals. BAC DNA was isolated and used as a template for PCR with several primer pairs derived from the coding region of TaGlJ. Two BAC clones gave the same predicted PCR product and one of them was used in a further work. The whole selected BAC clone was sequenced using 454 sequencing technology (Life Sciences) and the full length genomic clone (5370 kb) plus more than 4 kb of - Η -

promoter sequence of the JaLxL? orlhologue from T. durum was obtained as a non- interrupted contig. The cloned gene was designated TdGLJ.

The coding region of TdGLJ contains 9 introns. Alignment of the protein sequences of TaGL.7 and TdGL.7, deduced from the genomic sequence, shows 96.7% identity of protein sequences (Figure IA). The alignment of the protein sequence of TaGL.7 to TdG L7 and closest the homoiogues derived from databases are shown in Figures IA and B. The protein domains of the TaGL7 and TdGLZ proteins are underlined in the protein alignment in Figure IA.

EXAMPLE 2 Spatial and temporal activity of the TdGLZ promoter in wheat, barley and rice

The 454 sequencing data for the selected BAC clone were used to design forward and reverse primers for the amplification from BAC DNA of a promoter fragment corresponding to 3046 bp upstream of the translational start of TdGL? (Table 2).

Subsequently, the promoter fragment was cloned into the plant transformation vector pMDC164 (Curtis and Grossniklaus, Plant Physiol. 133: 462-9, 2003), which harbours a hygrυmycin resistance marker gene for selection of transgenic plants, to provide the transcriptional GL^S fusion promoter construct designated pTdGLZ.

For stable transformation experiments, the pTdGLZconstruct was transformed into the Agrobactcriurn tumifaciens strain AGLl and the presence of plasmid in selected colonies was confirmed by PCR using specific primers. Transformed Λgrobacteήum was subsequently used to introduce constructs into barley and rice.

The same plasmid was linearized with Fmcl and co-transformed together with a plant selectable marker cassette (Ubi-ZipMNlos) into wheat using microprojeclilc bombardment. The integration of promoter:GUS fusions in transgenic plants was confirmed by PCR using primers derived from promoter and GUS sequences.

Forty five confirmed transgenic To wheat lines were analysed. Twenty five Ti wheat lines were selected using the GUS staining assay, from which fifteen demonstrated strong GUS expression, four had moderate expression and six showed weak expression of the reporter gene. Four lines were sterile and analysis of these lanes w^ras not performed. Three lines, two with strong transgene expression and one with moderate expression were selected for further analysis. All positive lines demonstrated the same pattern of GUS expression.

Sixteen To lines of transgenic barley were analysed for GUS activity. Eleven lines demonstrated strong promoter activity and one showed weak GUS activity. All lines had the same pattern of gene expression. No expression was found in one line and one line was sterile and has not been analysed. Wild type plants and/or plants transformed with a vector containing only the selectable marker cassette were used as negative controls. No differences were found between wild type plants and plants transformed with the control vector.

Thirty one To lines of transgenic rice were analysed for GUS activity. Twenty one lines exhibited strong promoter activity, four lines exhibited relatively strong promoter activity and three lines exhibited weak promoter activity. Three lines were sterile and were not analysed. All positive plants showed similar pattern of expression. Wild type plants and/or plants transformed with a vector containing only the selectable marker cassette were used as negative controls. No differences were found between wild type plants and plants transformed with the control vector.

In barley, wheat and rice the promoter starts to work before fertilisation in the embryo sac (Figure 5). In rice, promoter activity was detected in the same tissue at the second - ^S -

clay after fertilisation (Figure 7). Later in seed development, strong expression was detected in the syncytium and the cellularised endosperm of wheat and barley (Figures 3-5), These data correlate with Q-PCR data obtained for wheat grain fractions: the strongest expression of TaGlJ was detected in the liquid fraction of endosperm (Figure 2). On the fifth day strong expression was also observed in the aleurone and embryo (Figure 4). In rice, polarised expression in both ends of grain was observed at 4-5 DAP. However,, starting from 8 DAP, GUS activity was detected in the endosperm and embryo, At the end of ceUularization, GUS expression in the endosperm of both wheat and barley declines,, but appears again at approximately 10 DAP in wheat and 15 DAP in barley. Expression in both the endosperm and embryo was observed until at least 30 DAP and until 56 DAP for rice (Figure 6). However, at 30 DAP GUS expression in the wheat and barley embryo declined and could be seen only in some regions, while in rice it remained in the entire embryo,

In addition, as shown in Figure 5, expression was also observed in barley flowers prior to fertilisation. In particular, expression was observed in the ovary of the flower. GUS expression was also detected in the ovaries of transgenic rice plants before fertilization (data not shown). In rice, GUS expression was observed in vascular bundles of the lemma and palea before (and for a short time after) fertilisation (Figure 7), but was not detected in other flower tissues.

Although a low level of JaGL? expression was demonstrated by Q-PCR in other wheat tissues, GUS activity was not detectable in leaves and stems of wheat, barley and rice. Also, no GUS activity was detected in any other tissues. This discrepancy in results of Q-PCR and promoter-GUS activity assay can be explained by possible simultaneous detection of expression of two or three homeoiogues and/or close homologues, the promoter activities of which may be different. Another possible explanation for the difference in results could be the selected length of the promoter; that is the promoter may contain c/s-elements for grain-specific expression, but not contain elements for the weak constitutive expression in other tissues. EXAMPLE 3 Materials and Methods

(i) Gene cloning and piasmid construction

The full length cDNA of TdGLZ was isolated in the YlH screen of the cDNA library from wheat grain at 0-6 DAP (Lopato et ah, Plant MoI Biol 62: 637-53, 2006). The sequence derived from 3'UTR of JdGLJ cDNA was used to probe a BAC library prepared from the genomic DNA of Tiiticum durum cv. Langdon (Cenci et al., 2003, supra) using Southern blot hybridisation as described elsewhere (Sambrook and Russell Molecular Cloning: A Laboratory Manual 3rd Edition. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 2001). Piasmid DNA from three BAC clones, which strongly hybridised with the probe, was isolated using a Large Construct Kit (QlAGEN). The T. durum homolog of TaGL? was identified by PCR using BAC DNA as template and primers derived from the coding region of TaGlJ cDNA. The gene and TdGL? promoter sequence were obtained using 454 sequencing technology (454 Life Sciences, Branford, USA; Margulies ct al, Nature 437: 376-380, 2005). In this manner, the sequence of the full length genomic clone (5562 bp) and more than 4000 bp of sequence upstream from the TdGL-7 translation start codon were obtained. This sequence was subsequently used to design forward and reverse primers for the isolation of the promoter segment. A 3046 bp fragment of promoter with a full- length 5^"-untranslated region of TdGL? was amplified by PCR using AccuPrime^{3 M} Pfx DNA polymerase (Invilrogen) from DNA of BAC clone #1094 Mil as a template. The fragment was cloned into the pE NTR-D -TOPO vector (Invitrogen) and the cloned insert was verified by sequencing before being suhdoned into the pMDC164 vector (Curtis and Grossniklaus, 2003, supra) using recombination cloning. The resulting construct was designated pTdGL7. Selectable marker genes in the construct conferred hygromycin resistance in plants and kanamycin resistance in bacteria. The resulting binary vector was introduced into Agrobactεrium tumcfaciens AGLl strain by electroporation. (H) Plant transformation and analysis

The construct pTdGL7 was transformed into rice (Oryza sativa L. ssp, Japonica cv. Nipponbare) and barley (Hordeum vulgare cv. Golden Promise) using Agrobacterium- mediated transformation and the method developed by Tingay et al. (Phmt J 11 : 1369- 1376, 1997) and modified by Matthews et al. (MoI. Breed. 7: 195-202, 2001).

Wheat (Triticum aestivum L. cv. Bobwhite) was transformed using biolistic bombardment according to the following protocol. Immature seeds of wheat 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, semi trans parent) were isolated aseptically and were placed, with the scutellum side up, on solid culture medium. Embryos developing compact nodular calls were selected using a stereomkroscope and used for bombardment 7-21 days after isolation. The cultures were kept in the dark at 25°C on solid MS (Duchefa, M0222; Murashige and Skoog, Physloiogia Plan tar urn 15: 473-477, 1962) with 30 g/i sucrose and 2 mg/1 2,4-D (MS2).

The pTdGL7 construct was linearised with Pmel and co-transformed together with the Ubi-lrpt-Nos cassette into wheat using microprojecrile bombardment.

A DNA-goid coating was prepared according to the protocol of Sanford et al. (Methods in Enzymology 217: 483-509, 1993), Microprojecrile 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 macrocamer was 12 mm, 16 hours after bombardment, the calli were transferred to MS2 medium and grown in the dark for one week I wo days alter bombardment the treated calli were transl erred to MS selection medium supplemented with 2.0 mg/1 2,4- D and 150 mg/1 hygrυmycin B. Aiter 3-6 selections [4-6 months) greening callus tissues were subcultured on MS regeneration medium supplemented with lmg/1 kinetin and 5-10 mg/1 zeatin. Regenerating piantlets were then transferred to jars with hail-strength hormone-tree MS medium supplemented with 50 mg/1 hygromycin B ^rl he fully developed piantlets were aci limated for 7-10 days at room temperature in a liquid medium containing tour-fold diluted MS salts Plants with strong roots were then transplanted into soil and grown under greenhouse conditions to maturity. Transgene integration was confirmed by FCR using GUS specitic primers

Histochomical and hi^tologu al GUS assays were performed as described by Ii et al. (Plant Biott eh J, 6: 465-476, 2008).

(lii⁾ Quantitative PCR

Q-PCR was carried out according to the method of Burton et al. (Plant Physiol 134: 224- 236, 2004) using the primer combinations shown in I able 2

[io) Primers

1 able 2 below shows a list of primer sequences used in Rf-PCR, Q-PCR and promoter cloning described herein

Table 2 - Primers

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes ail of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise. Thus, for example, reference to "a nucleotide sequence of interest" includes a single nucleotide sequence as well as two or more nucleotide sequences; "a plant cell" includes a single cell as well as two or more cells; and so forth.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An isolated nucleic acid comprising:

2. The nucleic acid of claim 1 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in one or more parts of a monocotyledonous plant seed.

3. The nucleic acid of claim 2 wherein the monocotyledonous plant is a plant in the family Poaceae,

4. The nucleic acid of claim 2 or 3 wherein the monocotyledonous plant is a cereal crop plant,

5. The nucleic acid of any one of claims 2 to 4 wherein the cereal crop plant is a wheat plant, a barley plant or a rice plant.

6. The nucleic acid of any one of claims 1 to 5 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the endosperm, or a part thereof, in the seed,

7. The nucleic add of any one of claims 1 to 6 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the embyro, or a part thereof, in the seed.

8. The nucleic acid of any one of claims 1 to 7 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the aleurone layer, or a part thereof, in the seed.

9. The nucleic add of any one of claims 1 to 8 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a ma ternal gametophy te.

10. The nucleic acid of any one of claims 1 to 9 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in an ovary.

11. The nucleic acid of any one of claims 1 to 10 wherein the transcriptional control sequence is derived from a monocotyledonous plant.

12. The nucleic acid of claim 11 wherein the transcriptional control sequence is derived from a plant in the family Poaceae.

13. The nucleic acid of claim 11 or 12 wherein the transcriptional control sequence is derived from a cereal crop plant.

14. The nucleic acid of any one of claims 11 to 13 wherein the transcriptional control sequence is derived from a Tήticum sγ. plant.

15. The nucleic acid of any one of claims 1 1 to 14 wherein the transcriptional control sequence is derived from a Triticum durum plant,

16. The nucleic acid of any one of claims 1 to 15 wherein the GL7 gene encodes a protein which comprises the amino acid sequence set forth in SEQ ID NO: 1 or a homolog thereof.

17. The nucleic add of any one of claims 1 to 16 wherein 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, or a hυmolυg thereof.

18. The nucleic acid of any one of claims 1 to 17 wherein the transcriptional control sequence is derived from a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 4, or a homolog thereof.

19. The nucleic acid of any one of claims 1 to 18 wherein the transcriptional control sequence comprises the nucleotide sequence set forth in SEQ ID NO: ? or a functionally active fragment or variant thereof.

20. A nucleic acid construct comprising the isolated nucleic acid of any one of claims ! to 19.

21. The construct of claim 20, wherein the nucleic acid construct further comprises a nucleotide sequence of interest operably connected to the nucleic acid of any one of claims 1 to 19.

22. The construct of claim 21, wherein the nucleotide sequence of interest is heterologous with respect to the nucleic acid of any one of claims 1 to 19.

23. The construct of any one of claims 20 to 22 wherein the nucleic acid construct further comprises a nucleotide sequence defining a transcription terminator.

24. A cell comprising a nucleic acid construct according to any one of claims 20 to

23.

25. The cell of claim 24 wherein the cell is a plant cell.

26. The cell of claim 25 wherein the plant is a monocotylcdonous plant.

27. The cell of claim 25 or 26 wherein the plant is a plant in the family Poaeeae.

28. The ceil of any one of claims 25 to 27 wherein the plant is a cereal crop plant.

29. The cell of any one of claims 25 to 28 wherein the plant is a wheat plant, a barley plant or a rice plant.

30. A multicellular structure comprising one or more ceils according to any one of claims 24 to 29.

31. The multicellular structure of claim 30 wherein the multicellular structure comprises a plant or a part, organ or tissue thereof.

32. The multicellular structure of claim 31 wherein the plant or a part, organ or tissue thereof comprises a seed or a part thereof.

33. A method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant 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 any one of claims 1 to 19.

34. The method of claim 33 wherein the plant is a monocotyledonous plant.

35. The method of claim 33 or 34 wherein the plant is a plant in the family Poaeeae.

36. The method of any one of claims 33 to 35 wherein the plant is a cereal crop plant.

37. The method of any one of claims 33 to 36 wherein the plant is a wheat plant, a barley plant or a rice plant.

38. The method of any one of claims 33 to 37 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the endosperm, or a part thereof, in the seed.

39. The method of any one of claims 33 to 38 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the ernbyro, or a part thereof, in the seed.

40. The method of any one of claims 33 to 39 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in the aleurone layer, or a part thereof, in the seed.

41. The method of any one of claims 33 to 40 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in a maternal gametophyte.

42. The method of any one of claims 33 to 41 wherein the transcriptional control sequence directs expression of an operably connected nucleotide sequence in an ovary.

43. The method of any one of claims 33 to 42 wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence.