WO2000018926A1

WO2000018926A1 - USE OF BIFUNCTIONAL α-AMYLASE SUBTILISIN INHIBITOR PROMOTER SEQUENCE OF BARLEY TO CONFER EXPRESSION IN SEEDS

Info

Publication number: WO2000018926A1
Application number: PCT/AU1999/000823
Authority: WO
Inventors: Agnelo Furtado; Robert James Henry; Kenneth John Scott
Original assignee: The University Of Queensland; Southern Cross University; Grains Research And Development Corporation
Priority date: 1998-09-25
Filing date: 1999-09-24
Publication date: 2000-04-06
Also published as: AUPP617598A0; CA2343933A1

Abstract

The present invention provides a novel promoter that is functional in the seeds of plants, in particular the endosperm and/or aleurone and/or the scutellum. Preferably, the isolated promoter sequence of the invention further modulates the expression of a structural gene in response to the phytohormones ABA and/or GA. Alternatively or in addition, the promoter sequence of the present invention is capable of being repressed or otherwise down-regulated in response to GA. The present invention further encompasses genetic constructs capable of expressing a structural gene operably under the control of the inventive promoter sequence, and transgenic plants carrying the genetic constructs. The promoter sequence of the present invention is particularly useful for modifying the seed traits of plants.

Description

USE OF BIFUNCπONAL α-AMY ASE SUBTILISIN INHIBITOR PROMOTER SEQUENCE OF BARLEY TO CONFER EXPRESSION IN SEEDS

FIELD OF THE INVENTION

The present invention relates generally to genetic sequences which confer expression in plant seeds and/or tissues or organs of a plant. In particular, the present invention provides a promoter sequence that is capable of conferring expression on a genetic sequence to which it is operably connected in the starchy endosperm cells of an immature seed and/or the aleurone cells of a mature seed and/or in the scutellum and/or in response to the phytohormone abscisic acid. Alternatively or in addition, the promoter sequence of the present invention is capable of being repressed or otherwise down-regulated in response to the phytohormone gibberellic acid (GA). The invention further encompasses transgenic plants carrying genetic constructs expressing a structural gene, such as a structural gene which encodes a cytotoxin, antisense, ribozyme, abzyme, co-suppression, reporter molecule, polypeptide hormone or other polypeptide, placed operably under the control of the inventive promoter sequence. The present invention is particularly useful for expressing desirable structural genes in the seeds of plants and in particular, in the starchy endosperm and/or mature aleurone cells of plants (collectively referred to as "endosperm"). The present invention is further useful in a wide range of applications involving the expression of desirable genes in seeds, including the production of seeds having modified protein and amino acid composition, modified flour quality for breads, noodles and pasta, amongst others, modified malting and brewing characteristics of grains and reduced propensity of grains for pre-harvest sprouting.

GENERAL

Those skilled in the art will be aware that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such 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 said steps or features.

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

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

This specification contains nucleotide and amino acid sequence information (referenced herein by the prefix "SEQ ID NO:<400>"), prepared using the programme Patentln Version 2.0. The Sequence Listing is presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the Sequence Listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1 , <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1 , <400>2, etc).

The designation of nucleotide residues referred to herein are those recommended by the lUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

Amino acid designations referred to herein are listed in Table 1.

TABLE 1

Amino Acid Three-letter One-letter Abbreviation Symbol

Alanine Ala A Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamine Gin Q Glutamic acid Glu E

Glycihe Gly G

Histidine His H

Isoleucine He

Leucine Leu L Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S Threonine Thr T

Tryptophan Trp w

Tyrosine Tyr Y

Valine Val V

Any amino acid as above Xaa X

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

BACKGROUND TO THE INVENTION A major problem in the genetic improvement of agriculturally-important crops, in particular those crops producing metabolites (proteins, amino acids, starches and secondary metabolites, amongst others), is the manipulation of gene expression to produce plants which exhibit novel characteristics. More particularly, the expression of novel characteristics is often required to be effected in specific cell types, tissues or organs of the plant, or under specific environmental or developmental conditions.

Advances in biotechnological research have produced an explosion of information in relation to the number of genetic sequences identified which, if appropriately expressed, are useful to produce improved crop plants, for example plants in which reproductive development is controlled, plants having altered shape or size characteristics, plants capable of rapid regeneration following harvest, or plants having improved resistance to pathogens, amongst others.

However, the application of biotechnology to the production of plants expressing novel traits is limited by the availability of genetic sequences which are capable of conferring appropriate expression patterns upon structural genes. Clearly, in the absence of appropriate regulatory sequences to confer expression in a particular cell type at a particular stage of development and/or in response to specific environmental and hormonal stimuli, the potential of genetic sequences which encode novel proteins to express those proteins in planta cannot be realised.

SUMMARY OF THE INVENTION

In work leading up to the present invention the inventors sought to isolate useful regulatory sequences which were capable of conferring expression on structural gene sequences to which they are operably connected in the seeds of plants in a developmentally-regulated and/or hormonally-regulated manner. The inventors isolated the bifunctional α-amylase subtilisin inhibitor (BASI) gene promoter sequence from a monocotyledonous plant species and demonstrated that this promoter sequence is at least capable of conferring expression on a structural gene sequence in seeds, in particular in the endosperm cells of immature seeds and/or the aleurone cells of mature seeds and/or in the scutellum.

Accordingly, one aspect of the present invention provides an isolated promoter sequence derived from a plant cell which is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in the seeds of a plant, wherein said isolated promoter sequence comprises a sequence of nucleotides which is at least about 20% identical to the nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary nucleotide sequence thereto.

Preferably, the promoter sequence of the invention is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in the endosperm and/or aleurone and/or scutellum of a plant, or one or more cells thereof.

Those skilled in the art will be aware that expression of the BASI gene is also regulated by the phytohormones abscisic acid (ABA) and giberrellic acid (GA), Accordingly, an alternative embodiment of the present invention provides an isolated BASI gene promoter sequence which is capable of regulating or otherwise modulating the expression of a structural gene in the seeds of a plant and preferably, in the endosperm and/or aleurone and/or scutellum cells of said seeds, in response to ABA and/or GA.

The promoter sequence of the invention preferably includes one or more c/s-acting regulatory sequences selected from the list comprising gibberellic acid responsive element (GARE), abscisic acid responsive element (ABRE), Sph element, CA-rich element, sugar-responsive element (SRE), MYB-transcription factor binding site, endosperm box and TT-box, or alternatively or in addition, comprises a nucleotide sequence having at least about 20% identity to the BASI promoter sequence exemplified herein as SEQ ID NO: <400> 1. The invention clearly extends to isolated promoter sequences which comprise nucleotide sequences that are complementary to the nucleotide sequences of said regulatory sequences.

Preferably, the promoter sequence of the invention comprises at least two of the regulatory sequences selected from the list comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB-transcription factor binding site, endosperm box and TT- box. More preferably, the promoter sequence of the invention comprises at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six and even more preferably at least seven of said regulatory sequences.

In a particularly preferred embodiment of the invention, there is provided an isolated promoter sequence that comprises regulatory sequences selected from the list comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB-transcription factor binding site, endosperm box and TT-box.

In a further alternative embodiment of the invention, there is provided an isolated promoter sequence that is capable of regulating expression in the seeds of a plant, wherein said promoter sequence is up-regulated by abscisic acid and/or down- regulated by gibberellin and comprises a nucleotide sequence selected from the list comprising:

(i) a nucleotide sequence having at least about 20% identity to the nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary nucleotide sequence thereto;

(ii) a nucleotide sequence that is capable of hybridising under at least low stringency, preferably medium stringency, and more preferably under high stringency hybridisation conditions to the nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary nucleotide sequence thereto; and (ii) a nucleotide sequence that includes any one of the regulatory sequences selected from the list comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB-transcription factor binding site, endosperm box and TT-box.

A second aspect of the present invention is directed to a genetic construct comprising the isolated promoter sequence herein described.

A further aspect of the present invention provides a transfected or transformed cell, tissue, organ or whole organism which expresses a recombinant polypeptide or a ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co- suppression molecule under the control of the promoter sequence described herein. Preferably, the transfected or transformed cell or tissue is a plant seed cell or tissue, more preferably in an endosperm cell or tissue such as aleurone or starchy endosperm. The organ according to this embodiment is preferably a plant seed that comprises cells or tissues, more preferably endosperm cells or tissues such as aleurone or starchy endosperm, which express the recombinant polypeptide or a ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co- suppression molecule under the control of the promoter sequence of the invention. Similarly, the whole plant is preferably a plant that comprises such plant seed or seed cells or seed tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation showing the nucleotide sequence of the BASI promoter with the locations and nucleotide sequences of the c/^'s-acting elements designated GARE-like, GARE, CA-rich element, SRE, pyrimidine box, Sph element, ABA- responsive element (ABRE), MYB binding site (myb), Endosperm box and TT- box, in addition to the location and sequence of the TATA box, all indicated in boldface type. Nucleotide positions corresponding to the nucleotide positions of SEQ ID NO: <400>1 are indicated at the left-hand and right-hand sides of the nucleotide sequence. Numbers in brackets refer to nucleotide positions relative to the transcription start site (+1 ) of the BASI gene.

Figure 2 is a graphical representation showing the restriction enzyme map of the BASI promoter region (top) and the location of the amplified 1200 bp Seal fragment (below). The restriction enzyme sites PvuW, Seal, Dral and EcoRV, in addition to the location of the BASI protein-coding region basi gene) are indicated at the top of the Figure. The positions at which the amplification primers GSP1 and GSP2 hybridize to the BASI gene are also indicated.

Figure 3 is a schematic representation of plasmid pA17, comprising an approximately 1200 bp Seal fragment containing the BASI gene promoter sequence, including nucleotides -959 to +74 relative to the transcription start site, inserted into T-overhangs of the plasmid vector pGEM-Teasy (Promega). This promoter sequence thus comprises nucleotides 1 to 1033 of SEQ ID NO: <400>1. The positions of the ampicillin resistance gene (Amp-r), Xmnl and Seal restriction sites in plasmid pA17 are also indicated.

Figure 4 is a schematic representation of plasmid pAGN, containing the structural gfp gene encoding a red-shifted variant of wild-type green fluorescent protein (GFP; Prasher et al., 1992; Chalfie et al., 1994; Inouye and Tsuji, 1994; and Cormack et al. (1996), which has been optimised for brighter fluorescence, operably connected to the nos gene terminator sequence, in the plasmid vector pGEM3Zf (Promega). The positions of the ampicillin resistance gene (Amp-r), and the HindlU, Sph\, Pst\, EcoRI, Nco\, Sail, Aval, Xho\, Apa\, Kpn\, Sac\, Xmnl and Seal restriction sites in plasmid pAGN are also indicated.

Figure 5 is a schematic representation of plasmid pA57 containing the full-length BASI promoter driving gfp gene expression. Plasmid pA57 contains nucleotides 1 to 1033 of SEQ ID NO: <400>1 ( -959basi), which was amplified from plasmid pA17 (Figure 3) using a first primer BF1 (SEQ ID NO: <400> 20) containing a HindlU site, and a second primer BR (SEQ ID NO: <400> 21) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA57 are also indicated.

Figure 6 is a schematic representation of plasmid pA58. Plasmid pA58 contains 666bp 5 of the BASI gene promoter sequence (-592 basi), from position -592 to +74 relative to the transcription start site (i.e. nucleotides 368 to 1033 of SEQ ID NO: <400>1 ), which was amplified from plasmid pA17 (Figure 3) using a first primer BF2 (SEQ ID NO: <400> 22) containing a HindlU site, and a second primer BR (SEQ ID NO: <400> 21) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU

10 and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. Accordingly, plasmid pA58 contains a truncated BASI promoter that comprises the putative SRE (i.e. the pyrimidine box motif), the putative ABA responsive element (ABA-RE) or Sph element motif, two putative GARE motifs, and

15 the putative MYB-binding site, Endosperm box, TT-box and TATA-box motifs, driving gfp gene expression. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA58 are also indicated.

20 Figure 7 is a schematic representation of plasmid pA61. Plasmid pA61 contains 497bp of the BASI gene promoter sequence (-423 basi) , from position -423 to +74 relative to the transcription start site (i.e. nucleotides 537 to 1033 of SEQ ID NO: <400>1 ), which was amplified from plasmid pA17 (Figure 3) using a first primer BF3 (SEQ ID NO: <400> 23) containing a HindlU site, and a second primer BR (SEQ ID NO: <400>

25 21 ) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. Accordingly, plasmid pA61 contains a truncated BASI promoter that comprises two putative GARE motifs, and the putative MYB-binding site,

30 Endosperm box, TT-box and TATA-box motifs, driving gfp gene expression. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA61 are also indicated.

Figure 8 is a schematic representation of plasmid pA64. Plasmid pA64 contains 273bp of the BASI gene promoter sequence (-199 basi), from position -199 to +74 relative to the transcription start site (i.e. nucleotides 761 to 1033 of SEQ ID NO: <400>1), which was amplified from plasmid pA17 (Figure 3) using a first primer BF4 (SEQ ID NO: <400> 24) containing a HindlU site, and a second primer BR (SEQ ID NO: <400> 21) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. Accordingly, plasmid pA64 contains a truncated BASI promoter that comprises one putative GARE, and the putative MYB-binding site, Endosperm box, TT-box and TATA-box motifs, driving gfp gene expression. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA64 are also indicated.

Figure 9 is a schematic representation of plasmid pA67. Plasmid pA67 contains 221 bp of the BASI gene promoter sequence, from position -147 to +74 relative to the transcription start site (i.e. nucleotides 813 to 1033 of SEQ ID NO: <400>1 ), which was amplified from plasmid pA17 (Figure 3) using a first primer BF5 (SEQ ID NO: <400> 24) containing a HindlU site, and a second primer BR (SEQ ID NO: <400> 21 ) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. Accordingly, plasmid pA67 contains a truncated BASI promoter, that comprises the putative MYB-binding site, Endosperm box, TT-box and TATA-box motifs, driving gfp gene expression. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA67 are also indicated. Figure 10 is a schematic representation of plasmid pA70. Plasmid pA70 contains 173bp of the BASI gene promoter sequence, from position -99 to +74 relative to the transcription start site (i.e. nucleotides 861 to 1033 of SEQ ID NO: <400>1), which was amplified from plasmid pA17 (Figure 3) using a first primer BF6 (SEQ ID NO: <400> 26) containing a HindlU site, and a second primer BR (SEQ ID NO: <400> 21 ) containing a SspHI site, to facilitate sub-cloning of the amplified DNA into the HindlU and Λ/col sites, respectively, of plasmid pAGN (Figure 4), such that the promoter sequence is operably connected to the gfp gene coding region and nos terminator sequence therein. Accordingly, plasmid pA70 contains a truncated BASI promoter, that comprises the TT-box and TATA-box motifs, driving gfp gene expression. The positions of the ampicillin resistance gene (Amp-r), and the HindlU, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Seal restriction sites in plasmid pA70 are also indicated.

Figure 11 is a schematic representation showing the aligned linear maps of the constructs cloned into plasmids pA57, pA58, pA61 , pA64, pA67 and pA70. The positions of the TATA box, transcription start site (+1 ), 3'-terminal nucleotide position of SEQ ID NO: <400>1 (+74), translation start site (ATG), gfp open reading frame (gfp) and NOS terminator sequence (nos) are indicated at the top of the Figure. Numbering at the left of the Figure refers to the 5'-terminal nucleotide derived from the BASI promoter, relative to the transcription start site, that is present in each clone.

Figure 12 is a black and white copy of a colour photographic representation showing aleurone layers (half grains) of barley observed under a fluorescence microscope to detect cells transiently expressing GFP under the control of the Ubiquitin promoter. Tissue was co-bombarded with plasmid pA53, containing the Ubiquitin promoter regulating the expression of green fluorescent protein (gfp) in the aleurone tissue, and a control plasmid pDP687, which facilitates the production of red anthocyanin pigment (black grey spots) in transfected/transformed tissues and was used as a reporter construct to normalise variation in results between microparticle bombardments. Approximately 24 hr after bombardment of tissue, tissue samples were observed under blue light (490nm) to detect cells expressing GFP (G; white spots in the Figure) as indicated by the arrows. Full colour original photographic representations of this Figure are available on request.

Figure 13 is a black and white copy of a colour photographic representation showing aleurone layers (half grains) of barley observed under a fluorescence microscope to detect cells transiently expressing GFP under the control of the Ubiquitin promoter in plasmid pA53. Tissue was co-bombarded and incubated as described in the legend to Figure 12, however tissue was visualised under white and blue light to detect cells expressing anthocyanin pigment (black/grey spots indicated by black-flagged arrows) and/or GFP ( white spots as indicated by the arrows marked "G"). Full colour original photographic representations of this Figure are available on request.

Figure 14 is a black and white copy of a colour photographic representation showing leaf tissue of barley observed under a fluorescence microscope to detect cells transiently expressing GFP and anthocyanin pigment under control of the Ubiquitin promoter. Observations were made under blue light (490nm), 24 hr after co- bombardment with plasmids pA53 and pDP687. The Ubiquitin promoter in plasmid pA53 is able to direct GFP expression in the leaf tissue, as indicated by the arrows marked "G". The plasmid pDP687, which led to the production of red anthocyanin pigment in both the tissues, was used as a reporter construct to normalise variation in results between bombardments. Full colour original photographic representations of this Figure are available on request.

Figure 15 is a black and white copy of a colour photographic representation showing pericarp tissue of wheat observed under a fluorescence microscope to detect cells transiently expressing GFP and anthocyanin pigment under control of the Ubiquitin promoter. Observations were made under blue light (490nm), 24 hr after co- bombardment with plasmids pA53 and pDP687. The Ubiquitin promoter in plasmid pA53 is able to direct GFP expression in the pericarp tissue, as indicated by the arrows marked "G". The plasmid pDP687, which led to the production of red anthocyanin pigment in both the tissues, was used as a reporter construct to normalise variation in results between bombardments. Full colour original photographic representations of this Figure are available on request.

Figure 16 is a black and white copy of a colour photographic representation showing aleurone layers (half grains) of barley observed under a fluorescence microscope to detect cells transiently expressing GFP and anthocyanin pigment. Observations were made under blue light at 490 nm, 24 hr after co-bombardment with plasmids pA57 and pDP687. The 1033bp BASI promoter present in plasmid pA57 is able to direct GFP expression in the aleurone tissue, as indicated by the arrows marked "G". The plasmid pDP687, which led to the production of red anthocyanin pigment as indicated by the flagged arrows marked "A", was used as a reporter construct to normalise variation in results between bombardments. Full colour original photographic representations of this Figure are available on request.

Figure 17 is a black and white copy of a colour photographic representation showing aleurone layers (half grains) of barley observed under a fluorescence microscope to detect cells transiently expressing GFP under the control of the BASI promoter in plasmid pA57. Tissue was co-bombarded and incubated as described in the legend to Figure 16, however tissue was visualised under white and blue light to detect cells expressing anthocyanin pigment (black/grey spots indicated by flagged arrows marked "A") and/or GFP ( white/grey spots as indicated by the arrows marked "G"). Full colour original photographic representations of this Figure are available on request.

Figure 18 is a black and white copy of a colour photographic representation showing leaf tissue of barley observed under a fluorescence microscope to detect cells transiently expressing GFP and anthocyanin pigment. Observations were made under blue light (490nm), 24 hr after co-bombardment with plasmids pA57 and pDP687. Data indicate that the 1033bp BASI promoter present in plasmid pA57 does not confer detectable expression levels of GFP in the leaf, however anthocyanin pigment was detected, as indicated by the arrows marked "A". The plasmid pDP687, which led to the production of red anthocyanin pigment (A), was used as a reporter construct to normalise variation in results between bombardments. Full colour original photographic representations of this Figure are available on request.

Figure 19 is a black and white copy of a colour photographic representation showing pericarp tissue of wheat observed under a fluorescence microscope to detect cells transiently expressing GFP and anthocyanin pigment. Observations were made under blue light (490nm), 24 hr after co-bombardment with plasmids pA57 and pDP687. Data indicate that the 1033bp BASI promoter present in plasmid pA57 does not confer detectable expression levels of GFP in the pericarp, however anthocyanin pigment was detected, as indicated by the arrow marked "A". The plasmid pDP687, which led to the production of red anthocyanin pigment (A), was used as a reporter construct to normalise variation in results between bombardments. Full colour original photographic representations of this Figure are available on request.

Figure 20 is a black and white copy of a colour photographic representation showing an immature transgenic rice plantlet in tissue culture, that has been produced by bombardment with plasmids pA57 and pGL2. The plantlet was observed under blue light at 490 nm using a fluorescence microscope to detect cells that stably express GFP. The arrows indicate GFP expression in both the immature roots (RG) and immature shoots (SG) of transformed rice plantlets grown in these conditions, which expression is observable in the Figure as brightly-flaring tissue. Full colour original photographic representations of this Figure are available on request.

Figure 21 is a black and white copy of a colour photographic representation showing the transgenic rice plant depicted in Figure 20, observed under white light. The arrows indicate the location of the roots (R) and shoots (S) of transformed rice. Full colour original photographic representations of this Figure are available on request.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides an isolated promoter sequence derived from a plant cell which is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene to which it is operably connected in the seeds of a plant and preferably, in the endosperm cells or tissues of said seeds.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or hormonal and/or environmental stimuli, or in a tissue-specific or cell-type- specific manner. A promoter is usually, but not necessarily, positioned upstream, or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule or a derivative of the nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary nucleotide sequence thereto, which possesses the same function as the promoter sequence of the invention as described herein.

For the purposes of nomenclature, the nucleotide sequence shown in SEQ ID NO: <400>1 comprises a functional promoter sequence derived from the barley bifunctional α-amylase subtilisin inhibitor (hereinafter referred to as the "BASI" gene).

Promoters encompassed by the invention include those promoters that are derived from SEQ ID NO: <400>1 or a part thereof or a complementary nucleotide sequence thereto and which possess the same function as said promoter sequence or part or complement, however comprise additional copies of one or more specific regulatory elements, derived from either the exemplified promoter sequence or a heterologous promoter sequence, to further enhance expression of a genetic sequence to which it is operably connected and/or to alter the timing of expression of a genetic sequence to which it is operably connected. For example, chimeric promoter sequences that comprise the nucleotide sequence set forth in SEQ ID NO: <400>1 may be modified by the inclusion of nucleotide sequences derived from the wheat glutenin gene promoter region to further enhance expression of a genetic sequence to which the promoter of the invention is operably connected in the endosperm of a seed. The performance of such embodiments is readily achievable by those skilled in the art when provided with the teaching herein.

The terms "operably connected", "operably in connection" or similar in the present context means placing a first genetic sequence, such as a structural gene that encodes a polypeptide, under the regulatory control of the promoter sequence of the invention by positioning the first genetic sequence such that its expression is controlled by the promoter sequence.

Promoters are generally positioned 5' (upstream) to the structural genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene 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 function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art and demonstrated herein with multiple copies of regulatory elements, some variation in this distance can occur.

As used herein, a "structural gene" shall be taken to refer to that portion of a gene comprising a DNA segment encoding a peptide, oligopeptide, polypeptide, protein or enzyme or a portion thereof. Accordingly, structural genes may include the protein- encoding regions of genomic genes, cDNA molecules and other DNA molecules, including fusion molecules. It is to be understood that the term "structural gene" may additionally include nucleotide sequences that do not encode a peptide, oligopeptide, polypeptide, protein or enzyme or a portion thereof.

Alternatively or in addition, a structural gene may comprise an isolated nucleic acid molecule that does not encode a polypeptide, for example an antisense molecule, ribozyme, abzyme, co-suppression molecule, gene-silencing molecule or gene- targetting molecule, amongst others.

The word "expression" as used herein shall be taken to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA, as detectable by the appearance of said mRNA and/or by the appearance of a peptide, polypeptide, oligopeptide, protein or enzyme molecule encoded by a sense mRNA molecule.

By "conferring, increasing or otherwise facilitating expression" of a structural gene is meant that the rate or steady-state level of transcription of mRNA encoded by said structural gene placed operably under the control of the promoter sequence is increased and/or the biological activity or steady-state level of a peptide, polypeptide, oligopeptide, protein or enzyme molecule encoded by said structural gene is increased in comparison to the level of expression that is detectable in the absence of the promoter sequence.

Those skilled in the art will be aware of whether expression is conferred, increased or otherwise facilitated by the promoter sequence of the invention, without undue experimentation.

For example, the level of expression of a particular structural gene may be determined by polymerase chain reaction (PCR) following reverse transcription of an mRNA template molecule, essentially as described by McPherson et al. (1991).

Alternatively, the expression level of a structural gene may be determined by northern hybridisation analysis or dot-blot hybridisation analysis or in situ hybridisation analysis or similar technique, wherein mRNA is transferred to a membrane support and hybridised to a "probe" molecule which comprises a nucleotide sequence complementary to the nucleotide sequence of the mRNA transcript encoded by the structural gene, labelled with a suitable reporter molecule such as a radioactively- labelled dNTP (eg [ -³²P]dCTP or [α-^SjdCTP) or biotinylated dNTP, amongst others. Expression of the structural gene may then be determined by detecting the appearance of a signal produced by the reporter molecule bound to the hybridised probe molecule.

Alternatively, the rate of transcription of a particular structural gene may be determined by nuclear run-on and/or nuclear run-off experiments, wherein nuclei are isolated from a particular cell or tissue and the rate of incorporation of rNTPs into specific mRNA molecules is determined.

Alternatively, the expression of the structural gene may be determined by RNase protection assay, wherein a labelled RNA probe or "riboprobe" which is complementary to the nucleotide sequence of mRNA encoded by said structural gene is annealed to said mRNA for a time and under conditions sufficient for a double-stranded mRNA molecule to form, after which time the sample is subjected to digestion by RNase to remove single-stranded RNA molecules and in particular, to remove excess unhybridised riboprobe.

Those skilled in the art will also be aware of various immunological and enzymatic methods for detecting the level of expression of a particular gene at the protein level, for example using rocket immunoelectrophoresis, ELISA, radioimmunoassay and western blot immunoelectrophoresis techniques, amongst others.

Such approaches are described by Sambrook et al. (1989) and Ausubel (1987).

In the present context, the word "seed" shall be taken to refer to any plant structure which is formed by continued differentiation of the ovule of the plant and to include a storage tissue such as a haploid female gametophyte or a triploid meternally-derived endosperm, an aleurone layer and embryo. As exemplified herein, the promoter sequence of the invention does not confer expression in the pericarp tissue of seeds and, as a consequence, the word "seed" in the context of the invention does not include such tissue.

Preferably, the promoter sequence of the invention is a seed-specific promoter.

As used herein, the term "seed-specific" shall be taken to indicate that the promoter sequence is capable of conferring expression on a structural gene sequence that is substantially localised to one or more cells or tissues of plant seeds as defined herein (i.e. the starchy endosperm and/or aleurone and/or embryo and/or scutellum).

Reference herein to "endosperm" shall be taken as comprising starchy endosperm and the aleurone layer(s).

Preferably, the promoter sequence of the invention is at least capable of conferring expression on a structural gene sequence in the endosperm tissues of the seed and cells derived therefrom or comprising same. Such expression may be in the starchy endosperm cells and/or the mature aleurone cells of the endosperm of a plant.

Alternatively or in addition, expression may be conferred in the scutellum.

Even more preferably, the promoter sequence of the invention is capable of conferring expression on a structural gene sequence during the coenocytic stage of endosperm development of a monocotyledonous plant species.

The term "mature aleurone cell" means those cells of the aleurone layer of a seed that have completed the phase of starch accumulation and preferably, a fully-developed seed. As will be known to those skilled in the art, a mature aleurone layer may be derived from a mature seed of a monocotyledonous plant species, following imbibing of seeds by soaking in a liquid medium such as water.

More preferably, the promoter sequence of the invention is endosperm specific which means that the promoter is starchy endosperm-specific and/or aleurone-specific.

The terms "endosperm-specific" and "aleurone-specific" shall be taken to indicate that the promoter sequence is capable of conferring expression on a structural gene sequence that is substantially localised to the starchy endosperm or aleurone, respectively.

In a particularly preferred embodiment, the promoter sequence of the invention is at least capable of conferring, increasing or otherwise facilitating expression of a structural gene sequence in the aleurone cells of a plant seed.

The promoter sequence of the invention is capable of conferring, increasing or otherwise facilitating expression of a structural gene to which it is operably connected in the seeds of any monocotyledonous or dicotyledonous plant species. Because the BASI gene is expressed in plants that produce high pi α-amylases, it is likely that the BASI gene promoter exemplified herein may confer expression in any plant tissue that produces high pi α-amylases to degrade starch reserves. Accordingly, the promoter sequence of the invention may also be operable in any plant species that produces a seed containing a starchy endosperm or a plant that produces another starch storage organ, such as a tuber.

Preferably, the promoter sequence of the invention is capable of conferring, increasing or otherwise facilitating expression of a structural gene in a monocotyledonous plant that produces a seed having agronomic importance, for example grain crops such as wheat, oats, maize, barley, rice, sorghum, millet or rye, amongst others.

In a particularly preferred embodiment, the promoter sequence of the invention is at least capable of conferring expression in the cells, tissues or organs of a wheat or barley plant.

Those skilled in the art will be aware that it is also possible to modify the level of structural gene expression and/or the timing of structural gene expression and/or the regulation of structural gene expression, by mutation of a regulatory genetic sequence (i.e. c/^'s-regulatory region or 5'-non-coding region, etc) within the promoter sequence to which the structural gene is operably connected. In particular, to achieve such an objective, the promoter sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.

Alternatively, or in addition, the arrangement of specific regulatory sequences within the promoter sequence may be altered, including the deletion therefrom of certain regulatory sequences and/or the addition thereto of regulatory sequences derived from the same or a different promoter sequence.

Accordingly, the present invention clearly encompasses derivatives of the nucleotide sequence set forth in SEQ ID NO: <400>1.

As used herein "derivatives" of the promoter sequence of the invention shall be taken to refer to any isolated nucleic acid molecule which comprises at least 10 and preferably at least 20 contiguous nucleotides, and more preferably at least 30 contiguous nucleotides, derived from the promoter sequence as described herein according to any embodiment.

As exemplified herein, the present inventors have shown that the minimum nucleotides of SEQ ID NO: <400>1 required for efficient seed expression of a structural gene to which said SEQ ID NO is operably connected is in the region downstream of nucleotide position 813. Accordingly, preferred derivatives of SEQ ID NO: <400>1 comprise nucleotides downstream of position 813 of SEQ ID NO: <400> 1. ln an alternative embodiment, a preferred derivative of the present invention will include those nucleic acid molecules which comprise only nucleotides upstream of position about 976 of SEQ ID NO: <400>1 and more preferably, nucleotides upstream of position about 961 , but not including nucleotides downstream of position 977 of SEQ ID NO: <400>1.

Alternatively or in addition, a derivative of SEQ ID NO: <400> 1 at least comprises nucleotides in the region of positions 813 to 961 , more preferably, positions 813 to 976.

As will be apparent to those skilled in the art, such derivatives may comprise one or more of the functional c/s-acting elements present in this region of the BASI promoter, operably connected to heterologous nucleotide sequences, such as, for example, the MYB-binding site and/or endosperm box and/or TT-box depicted in Figure 1 , in combination with a functional TATA-box motif. Additional integers are not excluded.

Nucleotide insertional derivatives of the promoter sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place.

Generally, derivatives of the nucleic acid molecule of the invention are produced by synthetic means or alternatively, derived from naturally-occurring sources. For example, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Accordingly, the promoter sequence of the invention may comprise a sequence of nucleotides or be complementary to a sequence of nucleotides which comprises one or more of the following sequences without complete loss of function:

(i) a 5' non-coding region; and/or (ii) one or more c/s-regulatory regions, such as one or more functional binding sites for a transcriptional regulatory proteins or translational regulatory proteins, one or more upstream activator sequences, enhancer elements or silencer elements; and/or

(iii) a TATA box motif; and/or (iv) a CCAAT box motif; and/or

(v) an upstream open reading frame (uORF);and/ or

(vi) a transcriptional start site; and/or

(vii) a translational start site; and/or

(viii) a nucleotide sequence which encodes a leader sequence.

As used herein, the term "5' non-coding region" shall be taken in its broadest context to include all nucleotide sequences which are derived from the upstream region of a seed-expressible gene, preferably derived from the BASI gene exemplified herein, other than those sequences which encode amino acid residues comprising the polypeptide product of said gene.

As used herein, the term "uORF" refers to a nucleotide sequence localised upstream of a functional translation start site in a gene and generally within the 5'-transcribed region (i.e. leader sequence), which encodes an amino acid sequence. Whilst not being bound by any theory or mode of action, a uORF functions to prevent over- expression of a structural gene sequence to which it is operably connected or alternatively, to reduce or prevent such expression.

As used herein, the term "c/s-acting sequence" or "c/s-regulatory region" or similar term shall be taken to mean any sequence of nucleotides which is derived from a promoter sequence wherein the timing, level or regulation of expression conferred by said promoter in a particular cell, tissue or organ is conferred at least in part by said sequence of nucleotides. Those skilled in the art will be aware that a c/s-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence to which it is operably connected. In general, a single c/s-regulatory region may be responsible for conferring one mode of regulation on a structural gene sequence to which it is operably connected, however the occurrence of several c/s-regulatory regions in operable connection with a single structural gene sequence may confer multiple regulatory modes on said structural gene, which are not necessarily the mere summation of the individual regulatory modes (i.e. there may be interaction between individual c/s-regulatory regions). Furthermore, such c/s-acting regions generally, but not necessarily, comprise a linear array of groups of nucleotides which each comprise at least four and preferably at least six contiguous nucleotide residues.

Accordingly, the present invention extends to isolated nucleic acid molecules which comprise one or more c/s-regulatory regions which act to contribute to the ability of the promoter sequence described herein to confer, activate or otherwise regulate expression of a structural gene sequence in a plant seed or a cell or tissue thereof.

Preferred c/s-regulatory regions according to the invention comprise a linear array of one or more silencer, enhancer, or upstream activating sequences, not necessarily juxtaposed, however in sufficiently close association to be at least capable of conferring, either in concert or independently of each other, one or more regulated modes of expression on a structural gene sequence to which they are operably connected.

Preferred c/s-regulatory regions according to the present invention include, but are not limited to, one or more of the sequences selected from the list comprising the GARE, ABRE, Sph element, CA-rich element, SRE, pyrimidine box, MYB-transcription factor binding site, endosperm box and TT-box. The invention clearly extends to isolated promoter sequences which comprise nucleotide sequences that are complementary to the nucleotide sequences of said regulatory sequences.

The pyrimidine box, TAACAAA box (set forth in SEQ ID NO: <400>2) and TATCCAC box (set forth in SEQ ID NO: <400>3) were first described in the context of the promoter sequence of the high pi α-amylase gene and are believed to mediate the responsiveness of that promoter to GAs (Skriver et al., 1991 ; Gubler and Jacobsen, 1992; Gubler et al., 1995). There is also evidence that the action of abscisic acid on the α-amylase promoter sequence is mediated via the same complex. Analyses of a barley low-pl amylase promoter, have shown that GA probably also acts through similar c/s-acting elements, but additional c/s-acting elements upstream of the pyrimidine box are also important (Lanahan et al., 1992). A GA-dependent binding factor was shown to bind specifically to sequences which coincide with the TAACAGA (set forth in SEQ ID NO: <400>4) and TATCCAT (set forth in SEQ ID NO: <400>5) boxes and proximal sequences in the α-amylase promoter.

Notwithstanding the presence of these sequences in the α-amylase promoter, the presently-described full-length promoter sequence is inducible by ABA and/or capable of repression by GAs, in contrast to the α-amylase promoter which is induced by GAs and repressed by ABA. Those skilled in the art will be aware that it may be possible to alter the mode of structural gene regulation by the promoter sequence of the invention by deleting or otherwise mutating the GARE or ABRE sequences therein. For example, by deleting the ABRE sequences, induction of gene expression by ABA may be lost. Alternatively, by deleting the GARE sequences, repression of gene expression by GAs may be lost. The presently described promoter encompasses all such variants.

As used herein, the term " gibberellic acid responsive element" or its abbreviation

"GARE" shall be taken to refer to any sequence of nucleotides that is capable of reducing, decreasing or repressing the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence which is identical to one or more of the nucleotide sequences 5'-ATAACTAAGTGGG-3' (set forth in SEQ ID NO: <400>6) or 5'-ATAGAGTGTA-3' (set forth in SEQ ID NO: <400>7) or 5'- TATCCA-3'(set forth in SEQ ID NO: <400>8) or 5'-TATAACATTGCTCTG-3' (set forth in SEQ ID NO: <400>9) or 5'-TCACAAA-3' (set forth in SEQ ID NO: <400>10) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term " pyrimidine box" shall be taken to refer to any sequence of nucleotides that is capable of regulating, at least in part, the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence which is identical to one or more of the nucleotide sequences 5'-TATCCA-3' (SEQ ID NO: <400>8) or 5'-TCACAAA-3' (SEQ ID NO: <400>10) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term " abscisic acid responsive element" or its abbreviation "ABRE" or "ABA-RE" shall be taken to refer to any sequence of nucleotides that is capable of conferring, increasing, or enhancing or inducing the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-CATGCAT-3'(set forth in SEQ ID NO: <400>11 ) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term "Sph element" shall be taken to refer to any sequence of nucleotides that is capable of regulating, at least in part, the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-CATGCAT-3'(set forth in SEQ ID NO: <400>11) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term " CA-rich element" shall be taken to refer to any sequence of nucleotides that comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-CATGTCATCAAAATCATC-3' (set forth in SEQ ID NO:

<400>12) or a complementary nucleotide sequence thereto or a homologue thereof. As used herein, the term "sugar responsive element" or its abbreviation "SRE" shall be taken to refer to any sequence of nucleotides that is capable of conferring, increasing, or enhancing or inducing the expression of a structural gene to which it is operably connected under conditions of low intracellular or extracellular sucrose and which comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-TATCCA-3' (SEQ ID NO: <400>10) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term "MYB-transcription factor binding site" shall be taken to refer to any sequence of nucleotides that is capable of regulating, at least in part, the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence that is related to the DNA-binding site of a MYB transcription factor, wherein said nucleotide sequence which is identical to the nucleotide sequence 5'-TTACTG-3' (set forth in SEQ ID NO: <400>13) or a complementary nucleotide sequence thereto. Preferably, a "MYB-transcription factor binding site" as used herein is further capable of binding a transcription factor belonging to the MYB class of proteins or a homologue thereof.

As used herein, the term " endosperm box" shall be taken to refer to any sequence of nucleotides that is capable of conferring, increasing, or enhancing or inducing the expression of a structural gene to which it is operably connected in the endosperm and which comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-TGTAAAGG-3' (set forth in SEQ ID NO: <400>14) or a complementary nucleotide sequence thereto or a homologue thereof.

As used herein, the term " TT-box" shall be taken to refer to any sequence of nucleotides that is capable of regulating, at least in part, the expression of a structural gene to which it is operably connected and which comprises a nucleotide sequence which is identical to the nucleotide sequence 5'-TTCCAGATCA-3' (set forth in SEQ ID NO: <400>15) or a complementary nucleotide sequence thereto or a homologue thereof. Preferably, the promoter sequence of the invention comprises a sequence of nucleotides which is identical to at least two of the regulatory sequences selected from the list comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB- transcription factor binding site, endosperm box and TT-box or a homologue thereof. More preferably, the promoter sequence of the invention comprises a nucleotide sequence which comprises to three, even more preferably at least four, even more preferably at least five, even more preferably at least six and even more preferably at least seven of said regulatory sequences.

In an even more preferred embodiment of the invention, there is provided an isolated promoter sequence that comprises regulatory sequences selected from the list comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB-transcription factor binding site, endosperm box and TT-box or a homologue thereof.

In a particularly preferred embodiment, the isolated promoter sequence of the invention comprises a nucleotide sequence that at least includes one copy of one or more of each of the nucleotide sequences set forth in the Sequence Listing as SEQ ID NO: <400>6 to SEQ ID NO: <400>15, as follows:

(i) SEQ ID NO: <400>6 5'-ATAACTAAGTGGG-3';

(ii) SEQ ID NO: <400>7 5'-ATAGAGTGTA-3';

(iii) SEQ ID NO: <400>8 5'-TATCCA-3';

(iv) SEQ ID NO: <400>9 5 '-TATAACATTGCTCTG-3 ' ;

(v) SEQ ID NO: <400>10: 5'-TCACAAA-3';

(vi) SEQ ID NO: <400>11 : 5'-CATGCAT-3';

(vii) SEQ ID NO: <400>12: 5 '-CATGTCATCAAAATCATC-3 ' ;

(viii) SEQ ID NO: <400>13: 5'-TTACTG-3';

(ix) SEQ ID NO: <400>14: 5'-TGTAAAGG-3'; and

(x) SEQ ID NO: <400>15: 5'-TTCCAGATCA-3', or a complementary nucleotide sequence thereto.

Even more preferably, the promoter sequence of the invention includes at least two or three, even more preferably at least four or five, still even more preferably at least six or seven, and even still more preferably at least eight or nine or ten, of the c/s- regulatory regions listed supra.

In a particularly preferred embodiment, a promoter sequence which is capable of conferring, activating or otherwise regulating expression of a structural gene in a plant seed (including endosperm i.e. starchy endosperm cell and/or a mature aleurone cell), developmentally and/or which is regulatable by the phytohormones abscisic acid (ABA) and gibberellic acid (GA) will comprise at least nucleotide sequence motifs (viii) to (x) supra.

Those skilled in the art will recognise that the promoter according to this embodiment may require additional sequences for function, for example a TATA box motif, such as the TATA-box contained in the promoter sequence of the invention, which TATA box motif is set forth herein as SEQ ID NO: <400>16 (i.e. 5'-TATAAATA-3'), amongst others. Such nucleotide sequences will be readily supplied by persons skilled in the art without undue experimentation and the invention according to this embodiment clearly encompasses such alternatives.

It is to be understood that modifications may be made to the structural arrangement of specific enhancer and promoter elements of the promoter sequence described herein without destroying the improved enhancing activity of gene expression. For example, it is contemplated that a substitution may be made in the choices of enhancer and promoter elements without significantly affecting the function of the recombinant promoter sequence of this invention. Further, it is contemplated that nucleotide sequences homologous to the active enhancer elements utilized herein may be employed advantageously, either as a substitution or an addition to the recombinant promoter construct for improved gene expression in plant seeds, in particular in endosperm and/or aleurone and/or scutellum cells. It will be understood that the function of the promoter sequence of this invention also potentially results from the arrangement, orientation and spacing of the different enhancer elements with respect to one another, and with respect to the position of the TATA box.

The present invention clearly extends to functionally homologous promoters to the BASI promoter sequence exemplified herein. Preferred homologues of the BASI promoter include those promoters that share structural features with the BASI promoter that are useful in conferring the seed expression pattern on a structural gene in plants that is obtained using the BASI promoter.

In one embodiment, a homologue of the BASI promoter exemplified herein is capable of confeπing, increasing or otherwise facilitating the expression of a structural gene in the seeds of a plant, wherein said promoter sequence comprises a nucleotide sequence which is capable of hybridising under at least low stringency conditions, preferably medium stringency conditions, and more preferably under high stringency conditions, to a nucleotide sequence selected from the group consisting of: (i) the nucleotide sequence set forth in SEQ ID NO: <400>1 ;

(ii) a nucleotide sequence comprising at least 10 contiguous of SEQ ID NO: <400>1 ;

(iii) any one of the c/s-acting regulatory sequences selected from the group consisting of: gibberellic acid responsive element (GARE); abscisic acid responsive element (ABRE); Sph element; CA-rich element; sugar-responsive element (SRE); MYB-transcription factor binding site; endosperm box; and TT- box; and (iv) a complementary nucleotide sequence to any one or more of (i) to (iii).

For the purposes of defining the level of stringency, those skilled in the art will be aware that several different hybridisation conditions may be employed. As used herein, a low stringency hybridisation may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures in the range 25°C to 37°C or higher, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in a buffer comprising 6xSSC buffer, 0.1 % (w/v) SDS at ambient temperature, or equivalent annealing/hybridisation conditions. In the present context, references to "hybridisation" conditions clearly refer to both a standard "Southern" or "northern" type of hybridisation, and to the conditions required for annealing of a primer to template nucleic acid in a polymerase chain reaction.

As used herein, a medium stringency may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures in the range 37°C to 42°C or higher, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in 2xSSC buffer, 0.1% (w/v) SDS at a temperature in the range 45°C to 65°C or equivalent annealing/hybridisation conditions.

A high stringency may comprise the standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures higher than 42°C, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C, or equivalent annealing/hybridisation conditions.

As will be known to those skilled in the art, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS in a standard hybridisation, and/or increasing the temperature of the annealing/hybridisation of PCR or a standard hybridisation, and/or increasing the temperature of the wash in a standard hybridisation. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

As will be known to those skilled in the art, the specificity of PCR may also be increased by reducing the number of cycles, or the time per cycle, or by the use of specific PCR formats, such as, for example, a nested PCR, a format that is well-known to those skilled in the art. For the purposes of clarification of the parameters affecting the specificity of PCR, reference is made herein to McPherson et al. (1991 ) which is incorporated by way of reference.

Alternatively or in addition, a homologue of the BASI promoter comprises a nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence having at least about 20% identity to the nucleotide sequence set forth in SEQ ID NO: <400>1 ; (ii) a nucleotide sequence that includes nucleotide sequences having at least about 20% identity to any one of the regulatory sequences selected from the group consisting of: gibberellic acid responsive element (GARE); abscisic acid responsive element (ABRE); Sph element; CA-rich element; sugar- responsive element (SRE); MYB-transcription factor binding site; endosperm box; and TT-box; and

(iii) a complementary nucleotide sequence to (i) or (ii).

The words "identical " or "identity" as used herein includes exact identity between compared sequences at the nucleotide level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. For example, a c/s-acting regulatory sequence present in the inventive promoter sequence exemplified herein may be similar to a c/s-acting sequence in a functionally homologous promoter, albeit non precisely conserved so as to be considered identical when viewed in context of the consensus sequence for that c/s-acting sequence (i.e. there are variations from the accepted consensus), or even when viewed in the wider context of the minimum promoter required for function (i.e. there may be similar sequences and/or different arrangements of functionally identical c/s-acting elements present in distinct but functionally homologous promoters). Any number of programs are available to compare nucleotide sequences. Preferred programs have regard to an appropriate alignment. One such program is Gap which considers all possible alignment and gap positions and creates an alignment with the largest number of matched bases and the fewest gaps. Gap uses the alignment method of Skriver et al. (1991 ). Gap reads a scoring matrix that contains values for every possible GCG symbol match. GAP is available on ANGIS (Australian National Genomic Information Service) at website http://mel1.angis.org.au.

Alternative percentage identities contemplated by the present invention include at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% and at least about 90% or above compared to a reference sequence.

Preferably, the homologous sequence is regulatable by the phytohormones abscisic acid (ABA) and gibberellic acid (GA), such that expression of a structural gene to which said genetic sequence is operably connected is inducible by ABA and/or is capable of being repressed by GAs.

A still further embodiment of the present invention extends to derivatives, parts, fragments or analogues of the nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary nucleotide sequence thereto which are at least useful as genetic probes in the isolation of similar sequences, or primer sequences in the enzymatic or chemical synthesis of said promoter sequence or a related promoter sequence.

In a particularly preferred embodiment, the genetic sequence of the present invention is employed to identify and isolate similar genetic sequences from other plants, in particular grain crops such as wheat, oats, maize, barley, rice, sorghum, millet or rye, amongst others.

According to this embodiment, there is contemplated a method for identifying a related genetic sequence which is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in the cells of a plant seed, in particular an endosperm cell, such as a starchy endosperm or mature aleurone cell, said method comprising contacting genomic DNA or parts of fragments thereof, or a source thereof, with a hybridisation-effective amount of the nucleotide sequence set forth in SEQ ID NO: <400>1 , or a part, analogue or derivative thereof or a complementary sequence thereto, and then detecting said hybridisation.

The related genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.

Preferably, such isolated nucleic acid molecules comprise genomic DNA which is isolated using polymerase chain reaction or hybridisation approaches, based upon the nucleotide information disclosed in SEQ ID NO: <400>1.

In the performance of this embodiment of the invention, the genetic sequence set forth in SEQ ID NO: <400>1 , or a derivative or analogue thereof, is labelled with a reporter molecule capable of producing an identifiable signal (eg. a radioisotope such as ³²P, or ³⁵S, or a biotinylated molecule) to facilitate its use as a hybridisation probe in the isolation of related promoter sequences which are at least capable of conferring, activating or otherwise regulating gene expression in seeds.

An alternative method contemplated in the present invention involves hybridising a nucleic acid primer molecule of at least 10 nucleotides in length, derived from SEQ ID NO: <400>1 or a derivative or analogue thereof, to a nucleic acid "template molecule", said template molecule herein defined as genomic DNA, cDNA or RNA, or a functional part thereof. Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art and described in detail by McPherson et al (1991 ), which is incorporated herein by reference.

Preferably, the nucleic acid primer molecule or molecule effective in hybridisation is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form.

The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.

A second aspect of the present invention is directed to a genetic construct comprising a promoter sequence which is at least capable of conferring, increasing or otherwise regulating expression of a structural gene to which it is operably connected in a plant seed and preferably in the endosperm tissues of the seed, such as the starchy endosperm and/or the mature aleurone, wherein said promoter sequence preferably comprises the nucleotide sequence set forth in SEQ ID NO: <400>1 , or a functional derivative, part, fragment, or homologue thereof.

The present invention extends to genetic constructs in which the genetic sequence of the invention, or a functional derivative, part, fragment, homologue, or analogue thereof, is operably linked to a structural gene sequence. The invention is not to be limited by the nature of the structural gene sequence contained in such genetic constructs.

In one embodiment, the structural gene sequence is a reporter gene, such as but not limited to the green fluorescent protein (gfp) gene, the β-glucuronidase gene, the chloramphenicol acetyl transferase gene, or the firefly luciferase gene, amongst others.

In a further embodiment, the structural gene encodes a BASI peptide, polypeptide, oligopeptide or protein or enzyme. This embodiment of the invention is particularly useful for the purpose of over-expressing the BASI protein in the seeds of developing grains, in particular wheat grains or other grains which are subject to pre-harvest sprouting, with a view to inhibiting inappropriate α-amylase gene expression leading to such pre-harvest sprouting. In such applications, it may be necessary to boost the activity of the promoter sequence set forth herein by including additional regulatory sequences as described supra.

In a further embodiment, the structural gene encodes a peptide, polypeptide, oligopeptide or protein or enzyme, such as but not limited to enzymes involved in the malting process, for example high pi α-amylase, low pi α-amylase, EII-(1-3,1-4)-β- glucanase, Cathepsin β-like proteases, α-glucosidase, xylanase and arabinofuranosidases, amongst others or alternatively, a seed storage protein selected from the list comprising glutenins, glutelins, gliadins, hordeins and zeins, amongst others, or alternatively, proteins capable of influencing the nutritional and/or functional quality of grains, such as those that bind important minerals (eg. hemoglobins and ferritins) or are rich in essential amino acids such as lysine, amongst others.

Structural genes which may conceivably be placed in operable connection with the promoter sequence of the invention also include those structural genes that are involved in the metabolism of the seed and produce any product, bi-product or intermediate of the metabolism of the seed of a plant, including but not limited to amino acids, oils (i.e. fatty acids), starch and fibre amongst others.

In a further alternative embodiment, the structural gene sequence may be a ribozyme, abzyme, antisense or co-suppression molecule which targets the expression of a seed- expressible gene. According to this embodiment, expression of such a structural gene under the control of the promoter sequence of the invention will partially or completely reduce, delay or inhibit the expression of said structural gene in the seed, in particular in the endosperm cells.

Wherein the structural gene being targeted is normally expressed in more than one cell type, the expression of said structural gene under control of the promoter sequence of the invention may further result in said gene being expressed in a cell-type ortissue- type specific pattern, in all cells other than seed cells of the plant. Accordingly, the present invention extends to a method of expressing a structural gene in a cell-type or tissue-type specific manner, in cells other than seed cells.

The genetic construct according to this aspect of the invention may further comprise a transcription termination sequence, placed operably in connection with the structural gene sequence.

In an alternative embodiment, the transcription termination sequence is placed downstream of the promoter sequence of the invention, optionally spaced therefrom by a nucleotide sequence which comprises one or more restriction endonuclease recognition sites, to facilitate the insertion of a structural gene sequence as hereinbefore defined between said promoter sequence and said transcription termination sequence.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the tumor morphology large (tml) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the ADP-glucose pyrophosphorylase gene terminator (t3'bt2) derived from Oryza sativa, the zein gene terminator from Zea mays, the HMW glutenin gene terminator derived from Triticum aestivum, the Rubisco small subunit (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any rho- independent E. coli terminator, amongst others.

Alternatively or in addition, the BASI 3'utr (i.e. 3' untranslated region of the BASI gene; SEQ ID NO: <400> 17) may be used, particularly to provide for optimum stability of the mRNA encoded by the structural gene placed under control of the promoter of the invention. For example, the modulation of expression by phytohormones under control of the BASI promoter may be enhanced by including the BASI 3' utr downstream of the translation stop codon of the structural gene, with or without additional transcription termination sequences placed downstream thereof.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

The genetic constructs of the invention may further include an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said genetic construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.

Preferred origins of replication include, but are not limited to, the f1-or and co/E1 origins of replication.

In a further alternative embodiment, the genetic construct of the invention further comprises one or more selectable marker gene or reporter gene sequences, placed operably in connection with a suitable promoter sequence which is operable in a plant cell and optionally further comprising a transcription termination sequence placed downstream of said selectable marker gene or reporter gene sequences.

As used herein, the term "selectable marker gene" includes any gene which 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 genetic construct of the invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp^r), tetracycline resistance gene (Tc^r), bacterial kanamycin resistance gene

(Kan^r), phosphinothricin resistance gene, neomycin phosphotransferase gene (npfll), hygromycin resistance gene (hph), β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

Those skilled in the art will be aware that the choice of promoter for expressing a selectable marker gene or reporter gene sequence may vary depending upon the level of expression required and/or the species from which the host cell is derived and/or the tissue-specificity or development-specificity of expression which is required.

Examples of further promoters suitable for use in genetic constructs of the present invention include promoters derived from the genes of viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in isolated plant cells or whole organisms regenerated therefrom, including whole plants.

The promoter may regulate the expression of the selectable marker gene or reporter gene constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others.

Examples of promoters include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed- specific promoter, P₃₂ promoter, BK5-T imm promoter, lac promoter, tac promoter, phage lambda λ_L or ή, promoters, CMV promoter (U.S. Patent No. 5,168,062), T7 promoter, lacUVδ promoter, SV40 early promoter (U.S. Patent No. 5,118,627), SV40 late promoter (U.S. Patent No. 5,118,627), adenovirus promoter, baculovirus P10 or polyhedrin promoter (U.S. Patent Nos. 5,243,041 ; 5,242,687; 5,266,317; 4,745,051 ; and 5,169,784), and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes are useful.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation. A still further embodiment contemplates a genetic construct which further comprises one or more integration sequences.

As used herein, the term "integration sequence" shall be taken to refer to a nucleotide sequence which facilitates the integration into plant genomic DNA of a promoter sequence of the invention with optional other integers referred to herein.

Particularly preferred integration sequences according to this embodiment include the left border (LB) and right border (RB) sequences of T-DNA derived from the Ti plasmid of Agrobacterium tumefaciens or a functional equivalent thereof.

A further aspect of the present invention provides a transfected or transformed cell, tissue, organ or whole organism which expresses a recombinant polypeptide or a ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co- suppression molecule under the control of the promoter sequence herein described.

The organ according to this embodiment is preferably a plant seed that comprises cells or tissues, more preferably endosperm cells or tissues such as aleurone starchy endosperm, which express the recombinant polypeptide or a ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co-suppression molecule under the control of the promoter sequence of the invention. Similarly, the whole plant is preferably a plant that comprises such plant seed or seed cells or seed tissues.

Preferably, the transfected or transformed cell or tissue is a plant seed cell and more preferably an endosperm.

This aspect of the invention clearly encompasses a transgenic plant such as a crop plant, transformed with a recombinant DNA molecule which comprises at least a promoter sequence which is at least 20% identical to SEQ ID NO: <400>1 or alternatively, a genetic construct comprising said promoter sequence as herein described. A chimeric gene comprising the isolated promoter of the present invention or a genetic construct comprising same may be introduced into a cell by various techniques known to those skilled in the art. The technique used may vary depending on the known successful techniques for that particular organism.

Means for introducing recombinant DNA into bacterial cells, yeast cells, or plant, insect, fungal (including mould), avian or mammalian tissue or cells include, but are not limited to, transformation using CaCI₂ and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990), electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford et al., 1987; Finer and McMullen, 1990; Finer et al., 1992; Sanford et al., 1993; Karunaratne et al., 1996; and Abedinia et al., 1997), vacuum-infiltration of tissue with nucleic acid, or T-DNA-mediated transfer from Agrobacterium to the plant tissue (An et al.1985; Herrera-Estrella et al., 1983a; 1983b; 1985).

For the transformation of monocotyledonous plants, microparticle bombardment of cells or tissues is preferred, particularly in cases where plant cells are not amenable to transformation mediated by A. tumefaciens. In such procedures, microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No.4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 0.5 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation. Once introduced into the plant tissue, the expression of a structural gene under control of the promoter sequence of the invention may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome.

Wherein the cell is derived from a multicellular organism and where relevant technology is available, a whole organism may be regenerated from the transformed cell, in accordance with procedures well known in the art.

Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, scutella, cotyledons, hypocotyls, megagametophytes, callus tissue including embryogenic callus, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon seed and hypocotyl seed).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

The regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformant, and the T2 plants further propagated through classical breeding techniques. The regenerated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed root stock grafted to an untransformed scion ).

The present invention clearly extends to the progeny plants and plant parts, in particular seed, derived from the transformed plants, the only requirement being that said progeny plants and plant parts also comprise the introduced promoter sequence of the invention.

The present invention is further described by reference to the accompanying non- limiting Figures and Examples.

EXAMPLE 1

Isolation of the BASI promoter

The BASI promoter sequence was isolated from barley leaf tissue using the Universal Genome Walker kit supplied by CLONTECH Laboratories, USA, essentially as described by the manufacturer. Briefly, the procedure involves the digestion of DNA with specific enzymes, the addition of adaptors and nested PCR using a first primer designed to anneal to the BASI gene downstream of the promoter sequence and a second primer designed to anneal to the adaptor sequence, to amplify the intervening promoter sequences.

In particular, genomic DNA was isolated from leaf tissue of Hordeum vulgare cv Grimmett and digested with restriction enzymes as indicated in Table 2 to produce gene fragments of pre-defined size classes following amplification. The following primers were used to amplify the BASI promoter sequence:

GSP1 :BASIPROMG: 5'-GCGGTTGGCCGAGAGGACGTAGTAGTTG-3' (set forth herein as SEQ ID NO: <400> 18); and GSP2:BASIPROMGN 5'-CGCGAGAGGGCGGTGCTGGCCAGAATAAGG-3'(set forth herein as SEQ ID NO: <400> 19).

TABLE 2

The Dral-fragment (Table 2) and the EcoRV-fragment (Table 2) possibly consisted of 450 bp and 300 bp respectively of the BASI promoter sequence and may not contain all the regulatory elements of the promoter. The Pvull-fragment (1600bp) was amplified in low amounts in the PCR reaction. The low yield of the Pwyll-fragment would have hindered the subsequent cloning procedures. The Seal-fragment (1200bp) was amplified in sufficient amounts and was thus selected for cloning (Figure 2).

The cloning of the Seal-fragment involved ligation of the Seal-fragment into a suitable vector and transformation of Escherichia coli DH5α-cells for plasmid preparation. In this regard, the Genome walker kit uses Tth as one of the DNA-polymerases in the PCR-amplification reaction, to preferentially add an extra deoxy-adenosine nucleotide at the 3'-end of the PCR-amplified product (i.e. to produce an "A-overhang"), thereby facilitating the cloning of fragments with A-overhang, in a suitable vector having T- overhangs.

The vector pGEM-Teasy from Promega (pGEM-Teasy kit) was used to ligate the Seal- fragment of the BASI promoter. The transformation of E-coli DH5α-cells was carried out and a number of white colonies were screened by PCR and by mini-preparation of the plasmid. In successful ligation and transformation reactions, it was expected that a 1500 bp fragment, including the Seal fragment, would be detected in the PCR reaction or alternatively, a recombinant plasmid having a size of 4200bp would be obtained from the cloning reaction. However, results of the screening experiments indicated that the Seal-fragment was not cloned. This experiment was repeated a number of times with appropriate modifications, and although the control DNA fragment (supplied in the pGEM-Teasy kit) was cloned the Seal-fragment was not cloned successfully using this approach.

Proceeding on the basis that the Seal-fragment may not have contained the requisite A-overhangs at one or both it's 3'-ends to facilitate ligation into the vector, an attempt was made to A-tail the Seal-fragment according to the manufactures protocol. The A- tailed Seal-fragment was then successfully cloned into the pGEM-Teasy vector. A number of white colonies were screened and three (ID No. P1C5, P1C17 and P1C4) were found to contain the expected recombinant plasmid containing the Seal-fragment.

EXAMPLE 2 Nucleotide sequence of the Seal-fragment containing the BASI promoter Plasmid DNA from the clone P1C5 described in the preceding Example was used as a template for sequencing the BASI promoter.

Sequencing reactions were carried out using the ABI-big dye kit according to manufacturers protocol.

The nucleotide sequence of the BASI promoter is set forth in SEQ ID NO: <400>1.

The sequencing data confirmed that the Seal-fragment contained 56bp of the non- translatable region of the BASI gene as published by Leah and Mundy (1989), suggesting that the Seal-fragment contained the linked promoter sequence of the BASI gene. EXAMPLE 3 Mapping of the BASI promoter sequence for putative DNA-elements

The sequence of the Seal-fragment (1033bp of nucleotide sequence upstream from the start codon of the BASI gene; SEQ ID NO: <400>1 ) was mapped to identify the putative TATA-box , using WEB-ANGIS to search the Transfec database . A mapping programme on WEB ANGIS was also used to identify the location of other putative c/s- acting DNA-elements. This resulted in the identification of the endosperm-box and the gibberellic acid and abscisic acid responsive elements (GARE and ABRE) [Figure 1].

EXAMPLE 4

Preparation of deletion constructs and plasmid maps

A total of six constructs containing the BASI promoter fused to the green fluorescent protein gene (gfp) in the plasmid pGEM3zf(+/-), were prepared using standard procedures. The construct comprising the full-length promoter sequence was designated pA57 and terminated at position -959 relative to the transcription start site of the BASI gene. Five of these constructs comprised 5 '-deletions of the promoter sequence set forth in SEQ ID NO: <400>1 , ending at positions -592 (pA58), -423 (pA61 ), -199 (pA64), -147 (pA67) and -99 (pA70), relative to the transcription start site of the BASI gene (Figures 5-11 ).

The sequences of each of the truncated BASI promoters in the deletions were compared to that of the BASI promoter sequence in pA57 and were found to be identical. All the six constructs were linearised to verify their size.

The above constructs were prepared such that the ATG of the gfp gene followed immediately after the nucleotide at position +74 of the BASI promoter. Thus the context sequence of the BASI promoter was immediately upstream of the translation start codon of the gfp gene. EXAMPLE 5 Endosperm-specific expression of the gfp gene under the control of the BASI promoter

Particle bombardment of aleurone layers derived from barley cv. Himalaya, was carried out to analyse transient expression of the gfp gene under control of the 1033 bp BASI promoter. Pericarp derived from immature wheat seeds and leaf tissue of barley (Hordeum vulgare cv. Grimmett) were also transfected as control tissues.

In control experiments, the plasmid pUbi.gfp (pA53) [Christensen and Quail, 1996] containing the Ubiquitin promoter driving gfp gene expression, was also used to transfect the same tissues (i.e. leaf, pericarp and aleurone tissue).

The genetic construct pDP687, containing genes of transcription factors required for the synthesis of the anthocyanin pigment, was used as reporter construct to standardise and check the efficiency of each transfection (Bowen, 1992). The plasmid pDP687 was precipitated onto tungsten particles (1.2 μm) in a ratio of 1 :2 with all other constructs.

For the transfection of leaf tissue, barley plants (2 weeks old) growing in soil, in 50ml falcon tubes, were placed in the particle bombardment chamber and a defined area of the leaf was bombarded. More precise details of the conditions used for biolistic bombardments of tissues in transfection assays are presented in Table 3.

Data presented in Figures 12 to 15 indicate that the ubiquitin promoter is capable of conferring expression on the gfp structural gene in the aleurone cells of barley, and in the leaf and pericarp tissue of wheat seeds. Data presented in Figures 16 to 19 indicate that the 1033 bp BASI promoter is capable of conferring expression on the gfp structural gene in the aleurone cells of barley, but not in the leaf, or in the pericarp tissue of wheat seeds.

TABLE 3

The results of transient expression assays were quantified and data are presented in Table 4. TABLE 4

The data presented in Table 4 confirm the conclusion from histochemical data present in Figures 16 to 19 that the 1033 bp BASI promoter sequence in the genetic construct pA57 directs gfp gene expression in the aleurone cells of barley seeds, nut not in the leaf tissue of barley or the pericarp tissue of wheat.

The five BASI promoter deletion constructs designated pA58, pA61 , pA64, pA67 and pA70 (Figures 6 to 11 ) were also tested for their ability to confer gfp gene expression in barley aleurone tissue, barley leaf tissue, and wheat pericarp tissue. Data presented in Table 4 indicate that the plasmids designated pA57, pA58, pA61 , pA64, pA67 and pA70 are able to direct expression of the gfp gene in mature aleurone tissue of barley, but not the leaf tissue of barley or the pericarp of immature wheat seeds. However, low levels of aleurone GFP expression were observed for plasmid pA70 (Table 4). Accordingly, the elements required for satisfactory aleurone-specific expression are present within the region from position -147 to position +74 of the BASI gene (i.e. nucleotides 813 to 1033 of SEQ ID NO: <400>1 ).

EXAMPLE 6 Stable expression of the gfp gene directed by the BASI-promoter sequence

I. Transformation of wheat Wheat is transformed by particle bombardment essentially according to Karunaratne etal (1996), with the following modifications:

Young caryopses are dissected from spikes of Triticum aestivum L. cv. Hartog, approximately 12 to 14 days post anthesis and surface sterilised with 10% Dairy-Chlor (100g/L available chlorine). Immature embryos are isolated and cultured in dark on MS medium (Murashige and Skoog, 1962) supplemented with 10 μM 2,4- dichlorophenoxyacetic acid. After 7 days of culture, the immature embryos are subjected to particle bombardment.

A plasmid containing a first ubiquitin promoter driving expression of the bar gene and the BASI promoter driving expression of the gfp gene is precipitated onto tungsten particles (1.2 μm) as described by Finer and McMullen (1990) with the following modifications. A 500mg/ml suspension (25 μL) of tungsten particles (1.2 μm) in distilled water is made in an Eppendorf tube, followed by the stepwise addition of the following: 5 μL of plasmid DNA (5 μg); 25 μL of calcium chloride (2.5M); and 10 μL of spermidine (0.1 M). The contents in the tube is mixed by finger vortexing and kept on ice. After 5 min, 30 μL of the supernatant is discarded and 300 μL ethanol (90%) is added, and the suspension is kept on ice after mixing the contents. After 1 min, the tube is centrifuged and all the supernatant discarded. This ethanol wash is repeated once, and the DNA-coated tungsten is finally suspended in 30 μL of ethanol (90%). The DNA-coated tungsten particles (2 μL) are delivered to target tissue using a particle inflow gun (Finer et al., 1992). The target tissue is placed on a shelf 14 cm from the screen of the filter holder, which carries a suspension of plasmid-DNA coated tungsten particles. After bombardment, the tissue is transferred to the original medium and cultured in the dark for 2 months with fortnightly subculture.

Embryogenesis, leading to plant regeneration, is stimulated by transferring clumps of embryogenic callus to MS medium devoid of hormones and containing Phosphinothricin (PPT) at a concentration of 5mg/L. After two weeks, PPT-resistant plants and callus are transferred to fresh medium and subcultured weekly. PPT- resistant plants, 4-5 cm in length, are transferred to soil and kept under water mist for two weeks. Plants are then transferred to larger pots and kept in the glasshouse under day and night temperature of 22° C and 19° C, respectively.

Transformed plants are analysed to determine the level of expression of the gfp gene in the seeds, and the specificity of expression in the aleurone and endosperm cells.

II. Transformation of barley

Hordeum vulgare cv Golden Promise is transformed by Agrobacterium tumefaciens- mediated transformation, using a binary genetic construct comprising the BASI promoter driving gfp gene expression, essentially according to Tingay et al (1997). Expression of GFP under the control of the BASI promoter is analysed in the leaf, and aleurone and endosperm cells, to confirm the expression of the gfp gene under control of the BASI promoter in the seed, including the immature endosperm and mature aleurone tissues.

III. Transformation of rice.

Rice plants were transformed particle bombardment essentially according to Abedinia et al. (1997).

Briefly, mature caryopses of rice were surface-sterilised using 1 % (v/v) sodium hypochlorite solution for 20 min, followed by washing five times in sterile distilled water.

The sterile caryopses were then incubated on callus induction medium (MSC) for 10 days after which the radicle and plumule (formed as a part of the germination process) and the swollen embryo axis was discarded. The scutellum with the callus was separated and cultured for a further twenty days on fresh MSC medium.

The embryogenic calli were bombarded according to Klein et al (1987) using a particle inflow gun (Finer et al., 1992). The embryogenic calli were transferred to medium containing osmoticum (MSCO). After four hours on MSCO medium, the embryogenic calli were subjected to particle bombardment and then left on the same medium for a further 16 to 20 hr.

After culture on MSCO medium, callus was transferred to selection medium (MSCS). After 15 days on MSCS medium, the surviving callus was transferred to the proliferation medium (MSP) and cultured for one month with subculture every 10 days.

The surviving calli and somatic embryos from the MSP medium were transferred to regeneration medium (MSR). Regenerated plantlets were transferred to 1/2 MS medium without hormones for additional growth of shoot and roots. Plants, 10 to 15 cms in length, were transferred to soil and kept for a week in the glasshouse under water mist. The plants were later transferred to potting mix and the pots kept half submerged in water.

Putative transformed rice plants that were resistant to hygromycin were tested for the presence of the BASI promoter and the gfp gene by PCR essentially according to Thompson and Henry (1995), using the following primers:

BASIPROMSC: SEQ ID NO: <400> 27:

5'- ATCGGAAGCTTACTGGGCTCGAAACTAAAATAAGAACATG-3'; and SGFPR1 : SEQ ID NO: <400> 28:

5'-GAAGTCGTGCTGCTTCATGTGG-3'.

The priming sites of the primers BASIPROMSC5' and SGFPR1 are in the 1033 bp BASI promoter (SEQ ID NO: <400> 1 ) and the gfp gene respectively, both of which are present in plasmid pA57. Use of 0.5 μM each of the above primers amplifies a 1300bp DNA fragment comprising 1033bp of BASI promoter plus 176bp of the gfp gene, thereby ensuring the detection of both the BASI promoter and the gfp gene.

Amplification reactions were performed using a Perkin Elmer Cetus 9600 under the following cycling conditions:

Cycle 1 : 94°C for 1min;

Cycles 2-11 : 94 °C for 30s, followed by 53 °C for 30s, followed by 72 °C for 45s per cycle; and

Cycles12-41 : 94°C for 30s, followed by 50°C for 30s, followed by 72°C for 45s per cycle.

Amplification products were electrophoresed on 1.0% (w/v) agarose gels containing ethidium bromide. Data obtained confirm the presence of the BASI promoter/gp gene construct.

The integration of the chimeric the BASI promoter/g p gene into the genome of rice is also confirmed using Southern blot analysis (Sambrook et al., 1989). High molecular weight DNA (at least 90% of total DNA) is digested using four or six base-cutting restriction enzymes, preferably a restriction enzyme having no restriction site substrates in the introduced plasmid DNA, to provide for the detection of integration of the the BASI promoter/g/p gene, as well as for a determination of the number of integration events in the rice genome. Preferably, the restriction enzymes EcoRV or Nrul or Spel, are used to digest genomic DNA containing the introduced plasmid pA57. Digested DNA, transferred onto a nylon Hybond membrane, is hybridised to radiolabelled probe and the signal detected using standard procedures. Data confirm the presence of the introduced transgene.

The transcription of the introduced the BASI promoter/g/p gene, integrated into the genome of rice, is detected using northern blot hybridisation according to standard procedures (Sambrook et al., 1989; Ausubel et al., 1987). Preferably, good quality (non-sheared) RNA is produced using the TRI-reagent method (GIBCO-BRL, USA). RNA (10 to 20 μg) is resolved on a 1% (w/v) agarose/formaldehyde gel and transferred onto nylon membrane, and hybridised using radio-labelled DNA or riboprobe containing nucleotide sequences complementary to the sense strand of the gfp structural gene. Data obtained confirm the expression of the gfp gene under control of the BASI promoter in transgenic rice seed.

EXAMPLE 7 Hormone-regulated expression of the gfp gene under the control of the

BASI promoter The regulation of expression of the BASI gene in immature endosperm and mature aleurone tissue of barley is mediated by the two phytohormones ABA and GA. It is known that ABA up-regulates the synthesis of BASI protein and GA down regulates it in both the endosperm and aleurone tissues.

To demonstrate efficacy of the BASI promoter in modulating the expression of a structural gene in planta, a genetic construct comprising the full-length BASI promoter operably connected to the gfp structural gene and placed upstream of the BASI 3'utr is introduced into wheat, barley and rice plants as described in the preceding examples. The expression of GFP under control of the BASI promoter is determined following the exogenous application of GA alone, ABA alone, or ABA plus GA, in accordance with standard procedures, such as those described in International Patent Application No. PCT/AU96/00383. Results of these experiments confirm the hormone- responsiveness of the full-length BASI promoter sequence.

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Claims

CLAIMS:

1. An isolated genetic sequence of plants comprising a nucleotide sequence selected from the group consisting of:

(i) the nucleotide sequence set forth in SEQ ID NO:<400>1 ;

(ii) a derivative of SEQ ID NO: <400>1 capable of conferring expression on a structural gene in a seed;

(iii) a nucleotide sequence that is at least 20% identical to the nucleotide sequence set forth in SEQ ID NO:<400>1 and capable of conferring expression on a structural gene in a seed;

(iv) a nucleotide sequence that contains at least one c/s-acting regulatory sequence selected from the group consisting of SEQ ID NO: <400>6 to SEQ

ID NO: <400>15 and capable of conferring expression on a structural gene in a seed;

(v) a nucleotide sequence that is capable of hybridising under at least low stringency conditions to at least 10 contiguous nucleotides of SEQ ID NO

<400>1 or capable of hybridising to the complement of SEQ ID NO: <400> 1 under said conditions and capable of conferring expression on a structural gene in a seed; and

(vi) a nucleotide sequence that is complementary to any one of (i) to (iii).

2. The isolated genetic sequence of claim 1 further capable of modulating the expression of a heterologous structural gene in response to ABA.

3. The isolated genetic sequence of claim 1 or 2 further capable of modulating the expression of a heterologous structural gene in response to GA.

4. The isolated genetic sequence according to any one of claims 1 to 3 comprising the nucleotide sequence set forth in SEQ ID NO:<400>1.

5. The isolated nucleotide sequence of claim 1 wherein the derivative of SEQ ID NO: <400>1 comprises at least nucleotides from about position 813 to about position 961 of SEQ ID NO: <400> 1.

6. The isolated nucleotide sequence of claim 5 wherein the derivative of SEQ ID NO: <400>1 comprises at least nucleotides from about position 813 to position 1033 of SEQ ID NO: <400> 1.

7. The isolated genetic sequence of claim 6, comprising at least nucleotides from about position 761 to position 1033 of SEQ ID NO: <400> 1.

8. The isolated genetic sequence of claim 6, comprising at least nucleotides from about position 537 to position 1033 of SEQ ID NO: <400> 1.

9. The isolated genetic sequence of claim 6, comprising at least nucleotides from about position 368 to position 1033 of SEQ ID NO: <400> 1.

10. The isolated nucleotide sequence of claim 1 wherein the derivative of SEQ ID NO: <400>1 comprises at least one copy of at least three c/s-acting regulatory sequences selected from the group consisting of SEQ ID NO: <400>6 to SEQ ID NO: <400> 15.

11. The isolated nucleotide sequence of claim 10 wherein the three c/s-acting regulatory sequences comprise the c/s-acting regulatory sequences set forth in SEQ ID NOs: <400> 13 to <400>15.

12. The isolated genetic sequence of claim 10, wherein at least two of the c/s- acting regulatory sequences comprise SEQ ID NO: <400>14 and SEQ ID NO: <400>15.

13. The isolated genetic sequence of claim 10, wherein the derivative of SEQ ID NO: <400>1 comprises at least one copy of the six c/s-acting regulatory sequences set forth in SEQ ID NO: <400>10 to SEQ ID NO: <400>15.

14. The isolated genetic sequence of claim 10, wherein the derivative of SEQ ID NO: <400>1 comprises at least one copy of the seven c/s-acting regulatory sequences set forth in SEQ ID NO: <400>9 to SEQ ID NO: <400>15.

15. The isolated genetic sequence of claim 10, comprising a nucleotide sequence that contains at least one copy of each of the ten c/s-acting regulatory sequences set forth in SEQ ID NOs: <400>6 to <400>15.

16. The isolated genetic sequence according to any one of claims 1 to 15, wherein said nucleotide sequence is capable of conferring expression on a heterologous structural gene in endosperm.

17. The isolated genetic sequence of claim 16 wherein expression is endosperm-specific.

18. The isolated genetic sequence according to any one of claims 1 to 15, wherein said nucleotide sequence is capable of conferring expression on a heterologous structural gene in aleurone.

19. The isolated genetic sequence of claim 16 wherein expression is aleurone- specific.

20. The isolated genetic sequence according to any one of claims 1 to 15, wherein said nucleotide sequence is capable of conferring expression on a heterologous structural gene in the scutellum.

21. An isolated genetic sequence of plants that is capable of conferring expression in seed or seed tissue of a plant and/or which is capable of modulating expression in response to GA and/or ABA, wherein said genetic sequence is obtainable by the method of: a) hybridising under at least low stringency conditions plant genomic DNA with one or more nucleic acid probes or primers of at least 10 nucleotides in length for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule, wherein said probes or primers comprise a nucleotide sequence obtainable from SEQ ID NO: <400>1 or a nucleotide sequence that is complementary thereto; b) detecting the hybridised nucleic acid molecule; and c) isolating said hybridised nucleic acid molecule comprising said genetic sequence.

22. The isolated genetic sequence of claim 21 wherein detection and/or isolation of said hybridised nucleic acid molecule includes amplifying nucleic acid using said probes or primers in a PCR reaction or PCR reaction equivalent.

23. The isolated genetic sequence of claim 21 or 22 wherein one or more of the probes or primers includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: <400>6 to <400>15.

24. The isolated genetic sequence of claim 21 or 22 wherein one or more of the probes or primers comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: <400>17 and <400>18.

25. A genetic construct comprising the isolated genetic sequence according to any one of claims 1 to 24 operably linked to a structural gene sequence.

26. The genetic construct of claim 25 suitable for expression in a plant cell.

27. The genetic construct of claim 25 or 26 wherein the structural gene sequence is a protein-encoding structural gene sequence.

28. The genetic construct of claim 27 wherein the protein-encoding structural gene sequence is the coding region of the BASI gene.

29. The genetic construct of claim 27 wherein the protein-encoding structural gene sequence is a reporter gene.

30. The genetic construct of claim 25 or 26 wherein the structural gene sequence is an antisense molecule, co-suppression molecule, ribozyme, or abzyme molecule.

31. The genetic construct according to any one of claims 25 to 30 further comprising a transcription termination sequence.

32. The genetic construct of claim 31 wherein the transcription termination sequence is the NOS terminator.

33. The genetic construct of claim 31 wherein the transcription termination sequence is the BASI 3' utr.

34. The genetic construct according to any one of claims 25 to 33 further comprising an origin of replication.

35. The genetic construct according to any one of claims 25 to 34 further comprising a selectable marker gene sequence.

36. The genetic construct according to any one of claims 25 to 35 further comprising one or more integration sequences suitable for insertion into plant genomic DNA.

37. A method of expressing a structural gene in a plant cell, said method comprising introducing into said plant cell the genetic construct according to any one of claims 25 to 36 for a time and under conditions sufficient for expression of the structural gene to occur.

38. A transfected or transformed cell, tissue, organ or whole organism that contains the isolated genetic sequence according to any one of claims 1 to 24 or a genetic construct comprising said genetic sequence introduced thereto.

39. The transfected or transformed cell, tissue, organ or whole organism according to claim 38 derived from a plant or comprising a plant or plant propagule.

40. The transfected or transformed cell, tissue, organ or whole organism of claim 39 wherein the plant is barley.

41. The transfected or transformed cell, tissue, organ or whole organism of claim 39 wherein the plant is rice.

42. The transfected or transformed cell, tissue, organ or whole organism of claim 39 wherein the plant is wheat.

43. Use of the isolated genetic sequence according to any one of claims 1 to 15 in the preparation of a gene construct for the genetic transformation of a plant.

44. A method of modifying α-amylase expression in the seeds of a plant comprising

(a) introducing a gene construct to a plant cell or tissue which construct comprises:

(i) the isolated genetic sequence according to any one of claims 1 to 24 operably connected in the sense orientation to the coding region of the BASI gene and placed upstream of a transcription termination sequence; and

(ii) a selectable marker gene operably connected to a second promoter sequence;

(b) expressing the selectable marker gene to facilitate the selection of transformed cells and tissues; (c) regenerating a whole plant from the cell or tissue and growing said plant to the stage of seed formation; and

(d) expressing said coding region of the BASI gene under control of said isolated genetic sequence for a time and under conditions sufficient for BASI protein to be produced in the seed formed.

45. A plant produced according to the method of claim 44.

46. The plant of claim 45 or seeds thereof having altered seed traits selected from the group consisting of: modified protein and amino acid composition; modified flour quality for breads, noodles and pasta; modified malting and brewing characteristics of grains; and reduced propensity of grains for pre-harvest sprouting.