WO2000012713A1

WO2000012713A1 - Plant promoter sequence and uses therefor

Info

Publication number: WO2000012713A1
Application number: PCT/AU1999/000692
Authority: WO
Inventors: Stephen Rowland Mudge; Robert George Birch
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 1998-08-26
Filing date: 1999-08-26
Publication date: 2000-03-09
Anticipated expiration: 2001-02-26
Also published as: AUPP549898A0

Abstract

The present invention provides isolated regulatory sequences of plants that are operable in dividing cells, in particular the meristem cells of plants. The regulatory sequences of the invention are useful in the genetic engineering of plants and, in particular, to facilitate or control the expression of foreign genes therein.

Description

PLANT PROMOTER SEQUENCE AND USES THEREFOR

FIELD OF THE INVENTION

The present invention relates generally to genetic sequences which confer expression in a plant cell, tissue or organ and 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 said genetic sequences. The present invention is particularly useful for expressing desirable structural genes in the meristematic tissue or dividing cells of plants.

GENERAL

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

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.

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

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentln Version 2.0. 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 IUPAC-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.

The designation of amino acid residues referred to herein, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission, 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

Glycine Gly G Histidine His H

Isoieucine He 1

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

BACKGROUND TO THE INVENTION

A major problem in the area of plant improvement is the manipulation of gene expression to produce plants which exhibit novel characteristics. More particularly, the expression of such 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 such technology is limited by the availability of genetic sequences which are capable of conferring appropriate expression upon a structural gene in a plant cell.

Notwithstanding that this is the case, those skilled in the art are aware that such regulatory sequences do not confer high levels of expression in appropriate cells of both monocotyledonous and dicotyledonous plants at all times. For example, promoter sequences derived from monocotyledonous plants, such as rice, are not always effective in conferring high levels of expression on structural genes in dicotyledonous ceils, and wee versa.

Moreover, regulatory sequences generally activate or enhance expression by binding one or more trans-acting protein factors, or transcription factors, at particular recognition motifs therein (i.e. c/s-acting nucleotide sequences). The level of such transcription factors in a particular cell at a particular time may be limiting, thereby placing an upper limit on the level of structural gene expression, in particular the level of transcription, that is conferred by a particular regulatory sequence to which the structural gene is operably connected. Accordingly, it is preferred to express multiple introduced structural genes, in the same cell, tissue or organ, from different regulatory sequences. This is because there may be competition between frans-acting proteins where expression of several introduced genes is placed operably under the control of a single regulatory sequence. Thus, even where a regulatory sequence is known to confer expression on a structural gene in a particular cell, tissue, or organ of a plant, a range of different regulatory sequences having the same expression-conferring profile in the plant is desirable to facilitate the expression of multiple introduced genes therein.

Accordingly, there is a need to identify additional regulatory sequences, in particular promoter sequences, that are capable of conferring expression in plant cells, tissues, or organs, at particular stages of development and/or in response to a range of environmental stimuli.

Several different regulatory sequences are available that are capable of conferring expression of a structural gene in the meristem of particular plants, for example the promoter of the Arabidopsis thaliana LEAFY gene (Weigel et al. , 1992); the A. thaliana knatl gene promoter (GenBANK Accession number AJ131822); the Malus domestica kn1 gene promoter (GenBANK Accession number Z71981); the A.thaliana CLAVATA1 gene promoter (GenBANK Accession number AF049870); and the Oryza sativa Proliferating Cell Nuclear Antigen (PCNA) gene promoter (Kosugi et al ., 1991 ; Kosugi and Ohashi, 1997).

In work leading up to the present invention, the inventors sought to identify nucleotide sequences that are capable of conferring expression in rapidly-dividing plant cells, such as the cells of meristematic tissue.

SUMMARY OF THE INVENTION

Accordingly, one aspect of the present invention provides an isolated genetic sequence derived from a plant cell which is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in a plant meristem cell, wherein said isolated genetic sequence comprises a sequence of nucleotides which is at least 40% identical to SEQ ID NO:1 or an analogue or derivative thereof.

This aspect of the invention extends clearly to any isolated plant meristem-expressible regulatory sequence, in particular a meristem-expressible promoter sequence, that is included in the nucleotide sequence set forth in SEQ ID NO:1 or a complementary nucleotide sequence thereto or an analogue or derivative thereof.

A second aspect of the present invention provides an isolated meristem-expressible regulatory sequence, in particular an isolated meristem-expressible promoter sequence, obtainable by the method of: a) hybridizing under at least low stringency conditions plant genomic DNA, or mRNA, or cDNA obtained therefrom, with one or more nucleic acid probes or primers that comprise a nucleotide sequence obtainable from SEQ ID NO:1 or a nucleotide sequence that is complementary thereto for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule; b) detecting the hybridised nucleic acid molecule; and c) isolating said hybridised nucleic acid molecule comprising the isolated meristem-expressible regulatory sequence or promoter sequence.

In an alternative embodiment, as exemplified herein, this aspect of the invention involves the steps of :

(a) selecting transformed or transfected meristem cells that express a reporter gene that has been introduced thereto by means of a reporter gene construct that does not contain a c/s-acting meristem-expressible regulatory sequence linked thereto and does not express said reporter gene in a meristem cell in the absence of said c/s-acting meristem-expressible regulatory sequence;

(b) hybridising under at least low stringency conditions genomic DNA, or mRNA, or cDNA obtained from said transformed or transfected meristem cells with one or more nucleic acid probes or primers that comprise a nucleotide sequence obtainable from said reporter gene or said reporter gene construct for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule; c) detecting the hybridised nucleic acid molecule; and d) isolating said hybridised nucleic acid molecule comprising the isolated meristem-expressible regulatory sequence.

This embodiment of the invention may further involve the first step of transforming or transfecting the meristem cells or a progenitor cell thereof with the reporter gene construct.

In both embodiments of this aspect of the invention, the use of probes and/or primers that hybridise to the non-coding region of a promoter derived from a plant meristem- expressible gene, or any other gene that is expressible in rapidly-dividing or dividing cells of a plant is clearly encompassed. Preferably, such probes and/or primers are derived from the exemplified sequence contained herein and used in the isolation procedure.

This aspect of the invention further encompasses the use of any known technology to isolate such sequences, including standard nucleic acid hybridisation approaches, a polymerase chain reaction (PCR) format, a PCR reaction equivalent such as, for example, rolling circle amplification (RCA), or isothermal RCA, amongst others.

A third aspect of the present invention is directed to a genetic construct comprising a genetic 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 meristem cell, wherein said genetic sequence preferably comprises the nucleotide sequence set forth in SEQ ID NO: 1 , or a functional derivative, part, fragment, homologue, or analogue thereof which is at least 40% identical thereto or at least 40% identical to the complementary strand of SEQ ID NO:1.

A fourth aspect of the invention provides a method of expressing a structural gene in a plant cell, said method comprising introducing into said plant cell a genetic construct comprising a regulatory genetic 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 meristem cell, wherein said genetic sequence preferably comprises the nucleotide sequence set forth in SEQ ID NO: 1 , or a functional derivative, part, fragment, homologue, or analogue thereof which is at least 40% identical thereto or a complementary sequence thereto, and wherein said structural gene is operably linked to said regulatory genetic sequence on said genetic construct.

A further aspect of the present invention provides a transfected or transformed cell, tissue, organ or whole organism that contains the isolated meristem-expressible regulatory sequence of the invention. Preferably, said cell, tissue, organ or whole organism expresses a structural gene operably under the control of said regulatory sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A is a copy of a photographic representation of a Southern blot of Hindlll- restricted genomic DNA from primary transformants, probed with the luc coding sequence. Lane 1 , untransformed Ti68; lane 2, pLUC19 transformant 040; lane 3, p35SLUC19 transformant #2; lane 4, p35SLUC19 transformant #12 (kanamycin resistant, LUC); lane 5, ten-copy reconstruction.

Figure 1B is a copy of a photographic representation of a Southern blot of genomic DNA from F₁ progeny of pLUC19 transformants 060 and 019, probed with the HindlW/EcoRV fragment of the luc coding sequence. Lane 1 , transformant 060, HindlW; lane 2, transformant 060, EcoRV; lane 3, transformant 019, Hindl ; lane 4, transformant 019, EcoRV; lane 5, untransformed Ti68, HindlW; lane 6, untransformed Ti68, EcoRV; lane 7, one-copy reconstruction.

Figure 2 is a representation of a map of the plasmid pEmu-luc, used as a positive control for inverse PCR experiments. The locations of primers Sn and As1 are shown by arrows, and the cleavage sites for the restriction enzymes Aatll and Pvull are shown. Other symbols represent: luc, luciferase coding sequence; nos 3', nopaline synthase terminator sequence; amp, β-lactamase gene.

Figure 3 is a representation of a map of iPCR clone 1 , which consists of the iPCR product amplified from pLUCI 9 transformant 060 DNA, cloned in pBluescript II. The region of amplified plant DNA is shown (060 DNA), along with the 5' region of the luc coding sequence (Iuc5') and the β-lactamase gene (amp). The left T-DNA border is shown as LB, and the positions of primers T₃, T₇, Sn (truncated) and As1 (truncated) are shown by white arrows.

Figures 4A, B & C is a representation of the DNA sequence of the plant DNA flanking the left border in pLUC19 transformant 060. This sequence extends from the HindlW site present in the flanking plant DNA to the Pvull site adjacent to the left border in the pLUC19 T-DNA. Vector sequences are from nucleotide position 201 to nucleotide position 269. The repetitive element with homology to the TS family of repeats is from nucleotide position -1167 to nucleotide position -919. Direct repeats are underlined. Double-underlining indicates the position of an inverted repeat with 92% homology to the TS repeat sequence. The putative TATA box and transcriptional start site (nucleotide 1 ) are indicated by boxes, and the internal A/col (nucleotide positions -8 to 68) and EcoRV (position -107) restriction sites are shown in italics. Arrows indicate the uORFs present in the plant DNA. This nucleotide sequence is also set forth herein as SEQ ID NO:1 (i.e. <400>1 ). The amino acid sequences encoded by the two uORFs are set forth as SEQ ID NOs: 2 and 3 (i.e. <400>2 and <400>3).

Figure 5A is a copy of a photographic representation of a Southern blot of genomic DNA from transformed and untransformed tobacco, using the amplified DNA from pLUC19 transformant 060 as a probe. Lane 1 , untransformed Ti68, EcoRV; lane 2, untransformed Ti68, HindlW; lane 3, pLUC19 transformant 019, EcoRV; lane 4, pLUCI 9 transformant 019, HindlW; lane 5, pLUC19 transformant 060, EcoRV; lane 6, pLUC19 transformant 060, HindlW; lane 7, λ HindlW molecular weight standards. Figure 5B is a copy of a photographic representation of a Southern blot of genomic DNA from transformed and untransformed tobacco, using the internal A/col/EcoRV fragment of the amplified DNA from pLUC19 transformant 060 as a probe. Lane 1 , untransformed Ti68, HindlW; lane 2, pLUC19 transformant 019, HindlW; lane 3, pLUC19 transformant 060, HindlW; lane 4, λ HindlW molecular weight standards.

Figure 6 is a representation of the transcriptional fusion vector p060-GUS, showing the position of the putative meristem-specific promoter (060 promoter), β- glucuronidase gene (uidA), nopaline synthase terminator sequence (NOS 3'), and β- lactamase gene (amp). The black arrow shows the region sequenced using the GUS sequencing primer.

Figure 7 is a representation of the T-DNA present in the binary vector p060-GUS19, showing the right border (RB), nopaline synthase promoter (NOP), neomycin phosphotransferase gene (aphA), nopaline synthase terminators (πos3'), putative meristem-specific promoter (060 promoter), β-glucuronidase gene (uidA) and left border (LB). Cleavage site for the restriction enzymes Λ/col, EcoRV, HindlW, Spel, and EcoRI are also shown.

Figure 8 is a copy of a photographic representation showing the histochemical localisation of GUS activity in p060-GUS19 transformed tobacco. Panels(A) and (B): Arrows indicate GUS-positive regions in the shoot tip of plants transformed with p060- GUS19. Panel (C), Constitutive pattern of GUS activity in the region surrounding the shoot tip in a plant transformed with pBI121.

Figure 9 is a copy of a photographic representation showing the histochemical localisation of GUS activity in axillary buds of tobacco transformed with p060-GUS19. To the right of each photograph is an explanatory diagram (from Esau, 1977).

Figure 10 is a copy of a photographic representation showing the histochemical localisation of GUS activity in floral tissues. Panel (A), Anther from p060-GUS19 transformed tobacco, showing GUS activity only in pollen grains. Panel (B), GUS activity in mature pollen grains from p060-GUS19 transformed tobacco. Panel (C), Mature pollen grains from pBin19 transformed tobacco, showing no detectable GUS activity.

Figure 11 is a representation of a nucleotide sequence alignment showing the repetitive element present in the amplified DNA from pLUC19 transformant 060 to the five closest matches in the Genbank and EMBL databases, produced using the program PileUp (Feng and Doolittle, 1987). Sequences are as follows: "tobntl" - "tobnt4" are members of the TS repeat family isolated by Yoshioka et al. (1993); "ntaux35" is a insertion element present in the promoter of auxin-induced gene GNT35 (Van der Zaal et al, 1991 ); "060" is the repetitive element present in the amplified DNA from pLUC19 transformant 060. Direct repeats flanking the latter element are underlined. The nucleotide sequences set forth herein are also presented in SEQ ID NO: 4 (tobntl ); SEQ ID NO:5 (tobnt2); SEQ ID NO:6 (tobnt4); SEQ ID NO:7 (ntaux35); SEQ ID NO:8 (tobnt3); and SEQ ID NO:9 (060).

Figure 12 is a copy of a photographic representation showing the histochemical localisation of GUS activity in p060-GUS19 transformed Arabidopsis. The arrow points to the area showing GUS activity, indicated by dark staining, which is restricted to the region of the shoot apical meristem.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides an isolated genetic sequence derived from a plant cell which is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in a plant meristem cell, wherein said isolated genetic sequence comprises a sequence of nucleotides which is at least 40% identical to SEQ ID NO:1 or an analogue or derivative thereof.

Preferably, the genetic sequence of the invention is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in a shoot meristem cell.

More preferably, the genetic sequence of the invention is capable of conferring meristem-specific expression or shoot meristem-specific expression on a structural gene in a plant cell.

For the purposes of nomenclature, the sequence shown in SEQ ID NO: 1 is a genetic sequence derived from a tobacco meristem-specific gene, wherein said genetic sequence is capable of conferring, increasing or otherwise facilitating the expression of a structural gene in a shoot meristem cell.

Preferably, the invention includes genetic sequences which are at least 60-65% identical to SEQ ID NO:1. More preferably, the percentage identity to SEQ ID NO:1 is at least 70-75%. Yet still more preferably, the percentage identity is at least 80-90%, including at least 91% or 93% or 95%.

As used herein, the term "genetic sequence" shall refer to any single-stranded or double-stranded nucleic acid molecule which at least comprises the deoxyribonucleotides and/or ribonucleotides, including DNA, RNA, mRNA, or tRNA, amongst others. The combination of such molecules with non-nucleotide substituents derived from synthetic means or naturally-occurring sources is also contemplated.

"Analogues" of the genetic sequence of the invention shall be taken to refer to any isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence as described herein according to any embodiment, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others. "Derivatives" of the genetic sequence of the invention shall be taken to refer to any isolated nucleic acid molecule which comprises at least 10 contiguous nucleotides, and preferably at least 20 contiguous nucleotides, and more preferably at least 30 contiguous nucleotides, and even more preferably at least 50 or 100 contiguous nucleotides, derived from the genetic sequence as described herein according to any embodiment, in particular SEQ ID NO:1.

Generally, analogues or 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.

Those skilled in the art will be aware that meristem tissue is undifferentiated plant tissue from which new cells arise and that plant meristem cells are those cells which comprise meristem tissue. In general, meristem cells are small in size and more rapidly dividing than those cells which form the differentiated tissues and organs of the plant. Additionally, a meristem cell has the ability to differentiate into a particular cell type, thereby giving rise to new tissues and organs in an intact plant, for example the formation of a shoot, root, leaf or floral primordium, amongst others. Furthermore, as meristem tissue is undifferentiated, the developmental fate of the meristem may be altered by the application of an appropriate hormone or other stimulus, for example leading to the transition of a vegetative meristem to a floral meristem.

In the context of the present invention, a meristem cell shall also be taken to include any undifferentiated cell, whether in isolated form or tissue culture or in planta, wherein said cell has similar cell-division properties as a meristem cell in planta and/or the ability to differentiate in response to an internal developmental or hormonal stimulus, and/or an externally applied stimulus such as a hormone or other chemical substance and/or environmental stimulus such as temperature, hypoxia, anoxia, drought, flooding, metal or chemical substance. The term "shoot meristem cell" refers to a meristem cell as hereinbefore defined which is located within the epicotyl or which gives rise to the epicotyl or is derived from the epicotyl. Accordingly, during normal plant development a shoot meristem cell may eventually give rise to shoots, foliage, stems and/or floral structures. However, those skilled in the art will be aware that it is possible to produce shoot structures from root cells in tissue culture and, in such circumstances, the root cell which gives rise to the shoot structure is clearly to be regarded as a shoot meristem cell as defined herein.

The term "root meristem cell" as used herein refers to a meristem cell as hereinbefore defined which is located within the hypocotyl or which gives rise to the hypocotyl or is derived from the hypocotyl. During normal plant development, a root meristem cell will give rise to root structures and, in the case of nodulating plants, root nodules. The term "root meristem cell" shall clearly be taken to encompass any cell capable of producing, by direct or indirect means, cell types which form part of a plant root or are capable of forming part of a plant root, or are at least associated physically with the plant hypocotyl.

As used herein, the term "meristem-expressible" in relation to the regulatory sequence, or promoter sequence, or c/s-acting sequence of the present invention, shall be taken to indicate that said sequence is capable of conferring, increasing, activating or otherwise facilitating expression of a structural gene at least in a dividing cell or plant meristem cell, including the ability to confer, increase, activate or otherwise facilitate, meristem-specific expression of said structural gene.

As used herein, the term "meristem-specific" shall be taken to indicate gene expression which is substantially localised to one or more meristem cells as defined herein. The term "shoot meristem-specific" shall be taken to indicate gene expression which is substantially localised to one or more shoot meristem cells.

The genetic 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: a promoter sequence, a 5' non-coding region, a c/s-regulatory region such as a functional binding site for a transcriptional regulatory protein or translational regulatory protein, an upstream activator sequence, an enhancer element, a silencer element, a TATA box motif, a CCAAT box motif, or an upstream open reading frame (uORF), transcriptional start site, translational start site, and/or 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 meristem-expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of said gene, wherein said 5' non- coding region confers or activates or otherwise facilitates, at least in part, the meristem expression of said gene.

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 an expressible genetic sequence wherein the expression of the first genetic sequence is regulated, 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 genetic sequence described herein to confer, activate or otherwise regulate expression of a structural gene sequence in a plant meristem cell.

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 TS repeat (Yoshioka et al, 1993), the hexamer sequence 5'-TGACGT-3' (Terada et al., 1993; Lloyd er a/., 1991 ; Benfey er a/., 1989; Fromm et al, 1989; Tabata et al., 1991 ), the cell cycle box motif (Nasmyth and Dirick, 1991 ; Ogas et al., 1991 ), the 5'-TGTGG- 3' motif, 5'-TAGTAGT-3' motif, the 5'-CAACTCC-3' motif, the 5'-TCTGTT-3' motif, the 5'-CCACG-3' motif and the 5'-TATCT-3' motif, amongst others.

Alternatively, or in addition, the c/s-regulatory region may comprise one or more of the nucleotide sequences selected from the list comprising: (i)5'-TGACGT-3' (SEQ ID NO:10); (ii)5'-CAACTCC-3'(SEQ ID NO: 11 ); (iii)5'-TCTGTT-3' (SEQ ID NO: 12);

(iv)5'-TAGTAGT-3' (SEQ ID NO: 13); (v)5'-GTAGATT-3'(SEQ ID NO: 14); and (vi)5'-CATGCAA-3' (SEQ ID NO: 15). or a complementary sequence thereto.

Even more preferably, the c/s-regulatory regions according to the present invention comprises at least two, even more preferably at least four and still even more preferably at least six of the sequences listed supra. In a particularly preferred embodiment, a c/s-regulatory region which is at least capable of conferring, activating or otherwise regulating expression of a structural gene in a plant meristem cell may comprise all of the nucleotide sequence motifs listed supra.

Those skilled in the art will be aware that 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.

Preferred uORFs contained in the genetic sequence of the invention comprise a nucleotide sequence which is at least 40% identical to or redundant to the nucleotide sequence:

(i)5'-ATGCCACGTCTGAGGGTAATTCTGTAA-3' (SEQ ID NO: 16); and/or (ii)5'-ATGGACTCTCGCACGTTGTGGCCTTATTTACCGCTGC TTCAATCAGAACCAAGTCAGGACAAAATAGGTCAGTAA-3' (SEQ ID NO: 17).

Alternatively, or in addition, a preferred uORF will contain a sequence of nucleotides which is capable of encoding an amino acid sequence as set forth in any one of SEQ ID Nos:2 and/or 3 or which is at least 40% identical to said amino acid sequence.

Preferably, the regulatory sequence of the present invention is a promoter sequence.

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 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 derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, in a plant cell, in particular a meristem cell. Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a meristem cell, and/or to alter the timing of expression of a structural gene to which it is operably connected.

The term "operably in connection" in the present context means placing a structural gene under the regulatory control of the genetic sequence of the invention by positioning the structural gene such that the expression of the gene is controlled by the genetic sequence. Promoters and the like are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting, i.e., the gene from which the genetic sequence or 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 protein, polypeptide or a portion thereof or alternatively, an isolated nucleic acid molecule which does not necessarily encode a polypeptide, such as an antisense, ribozyme, abzyme or co-suppression molecule.

The term "structural gene" also refers to copies of a structural gene naturally found within the cell, but artificially introduced, or the structural gene may encode a protein not normally found in the plant cell into which the gene is introduced, in which case it is termed a heterologous gene. A heterologous structural gene may be derived in whole or in part from a bacterial genome or episome, eukaryotic genomic or plastid DNA, cDNA, viral DNA, or chemically synthesized DNA. It is possible that a structural gene may contain one or more modifications in either the coding or the untranslated regions which affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides.

Where the structural gene encodes a polypeptide, it may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate plant-functional splice junctions. The structural gene may be a composite of segments derived from a plurality of sources, naturally occurring or synthetic. The structural gene may also encode a fusion protein, as long as the experimental manipulations maintain functionality in the joining of the coding sequences.

"Co-suppression" is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. The present invention extends to the use of the subject genetic sequence to regulate the expression of any co-suppression molecule in a meristem cell, more particularly a shoot meristem cell, wherein said co- suppression molecule reduces, diminishes, delays or otherwise alters expression of a meristem-expressed gene sequence. An "antisense molecule" is an RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a polypeptide. The antisense molecule is therefore complementary to the sense mRNA, or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a polypeptide gene product. The present invention extends to the use of the subject genetic sequence to regulate the expression of any antisense molecule in a meristem cell, more particularly a shoot meristem cell, wherein said antisense molecule targets a sense mRNA encoding a polypeptide which is expressed in said cell, such that expression of the polypeptide encoded therefor is reduced, diminished, delayed or otherwise altered.

"Ribozymes" are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by Haseloff and Geriach (1988) and contained in International Patent Application No. WO89/05852. The present invention extends to the use of the subject genetic sequence to regulate the expression of any ribozyme molecule in a meristem cell, more particularly a shoot meristem cell, wherein said ribozyme targets a sense mRNA encoding a polypeptide which is expressed in said cell, such that it is no longer capable of being translated to synthesise a functional polypeptide product.

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. promoter, c/s-regulatory region or 5'-non-coding region, etc) to which the structural gene is operably connected. In particular, to achieve such an objective, the genetic 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 genetic 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 genetic sequence.

Nucleotide insertional derivatives of the genetic 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.

A further embodiment of the present invention provides an isolated nucleic acid molecule which is at least capable of activating, enhancing or otherwise conferring expression of a structural gene in a meristem cell and which is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 1.

More preferably, the stringency of hybridisation is at least moderate stringency, even more preferably at least high stringency.

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. For example, a low stringency may comprise a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28 °C or equivalent condition sufficient for annealing of primers in a polymerase chain reaction or hybridisation of oligonucleotide to DNA or RNA. A moderate stringency may comprise a 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. A high stringency may comprise a hybridisation and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C.

Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS in the hybridisation buffer or wash buffer and/or increasing the temperature at which the hybridisation and/or wash are performed. 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 can conveniently be made to pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

The present invention is particularly directed to a tobacco shoot meristem promoter, which confers, activates or enhances gene expression in any plant shoot meristem cell, at least, in particular a monocotyledonous plant or dicotyledonous plant. However, the invention clearly contemplates other sources of shoot meristem promoter sequences, such as but not limited to agricultural or horticultural crop plants or other suitable plant, the only requirement being that such sequences are capable of at least hybridising to the genetic sequence of the invention under at least low stringency conditions or are at least 40% identical to SEQ ID NO:1 or an analogue or derivative thereof.

Furthermore, it is to be understood that modifications may be made to the structural arrangement of specific enhancer and promoter elements of the genetic 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 plant-expressible enhancer and promoter elements without significantly affecting the function of the recombinant genetic 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 meristem cells, in particular shoot meristem cells. It will also be understood that the function of the genetic 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.

Accordingly, a further embodiment of the invention provides an isolated nucleic acid molecule which at least comprises one or more copies of each of the nucleotide sequence motifs:

(i)5'-TGACGT-3' (SEQ ID NO:10); (ii)5'-CAACTCC-3'(SEQ ID NO: 11); (iii)5'-TCTGTT-3' (SEQ ID NO: 12); (iv)5'-TAGTAGT-3' (SEQ ID NO: 13);

(v)5'-GTAGATT-3'(SEQ ID NO: 14); and (vi)5'-CATGCAA-3' (SEQ ID NO: 15). or a complementary sequence thereto, wherein said nucleic acid molecule is at least capable of conferring, increasing or otherwise facilitating the expression of a structural gene in a plant meristem cell, such as a shoot meristem cell.

Preferably, the isolated genetic sequence according to this embodiment further comprises or is complementary to a sequence of nucleotides which hybridises under at least low stringency conditions to SEQ ID NO:1 or is at least 40% identical thereto.

According to this embodiment, it will be understood that the subject genetic sequence may further comprise elements which are required for efficient transcription of a structural gene sequence in a plant cell, for example a TATA box and/or CCAAT box motif, however such sequences are not essential for the meristem-specificity of gene expression. A still further embodiment of the present invention extends to meristem-specific promoter sequences and any functional promoters, derivatives, parts, fragments or analogues thereof, or non-functional molecules which are at least useful as, for example genetic probes in the isolation of similar sequences, or primer sequences in the enzymatic or chemical synthesis of said genetic sequence or a related genetic sequence.

Accordingly, a second aspect of the invention relates to the use of the genetic sequence of the present invention or a fragment or part thereof in the identification and/or isolation of similar meristem-expressible regulatory sequences from other genes.

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 a meristem cell, such as a shoot meristem cell, said method comprising contacting genomic DNA, or mRNA, or cDNA, or parts of fragments thereof, or a source thereof, with a hybridisation-effective amount of the nucleotide sequence set forth in SEQ ID NO: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, the related genetic sequence originates from an agriculturally-important or horticulturally-important plant such as potato, tomato, barley, rye, oats, or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

The present invention clearly extends to an isolated nucleic acid molecule which comprises a sequence of nucleotides which overlaps with the sequence set forth in SEQ ID NO:1 and which encodes a polypeptide which is expressed in meristem cells. Such sequences are recognised by those skilled in the art to include transit peptide and signal peptide sequences which may be important, for example, in targeting polypeptides encoded by structural genes to appropriate compartments in the meristem cell, wherein expression of the structural gene is placed under operable control of the genetic sequence disclosed herein.

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

Preferably, the genetic sequence set forth in SEQ ID NO:1 , or a derivative or analogue thereof, is labelled with a reporter molecule capable of producing an identifiable signal (eg. a radio isotope such as ³²P, or ³⁵S, or a biotinylated molecule) to facilitate its use as a hybridisation probe in the isolation of related genetic sequences which are at least capable of conferring, activating or otherwise regulating gene expression in a meristem cell.

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: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 be 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. Preferably, the related genetic sequence originates from an agricultural or horticultural plant or other suitable plant species.

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 β-glucuronidase gene, or the chloramphenicol acetyl transferase gene, or the firefly luciferase gene, amongst others.

In an alternative embodiment, the structural gene sequence encodes, or is complementary to a structural gene sequence encoding, a cytotoxin or other gene product that, when produced in a plant cell, kills or significantly alters host cell metabolism to limit cell division.

In a further alternative embodiment, the structural gene sequence encodes, or is complementary to a structural gene sequence encoding, a hormone polypeptide or polypeptide which is involved in the biosynthesis of a hormone or other molecule, such that expression of said polypeptide in the meristem cell under control of the genetic sequence of the invention, alters the developmental fate of the cell. The invention particularly contemplates the expression of a phytohormone molecule under control of the meristem promoter set forth in SEQ ID NO:1 or an analogue or derivative thereof, to produce a high local concentration of said phytohormone in the undifferentiated cells which is sufficient to result in the development of a floral meristem or vegetative meristem, depending upon the nature of the phytohormone.

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

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 genetic sequence of the invention may further result in said gene being expressed in a cell-type or tissue- type specific pattern, in all cells other than meristem cells of the plant or intact plant organ. Accordingly, the present invention extends to a method of expressing a structural gene cell-type or tissue-type specific manner, in cells other than merely meristem 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 genetic 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 genetic 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 terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Tea mays, the Rubisco small subunit (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any r70-independent E. colt terminator, amongst others.

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 r^"7-ori 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 ampiciliin resistance gene (Amp^r), tetracycline resistance gene (Tc ), bacterial kanamycin resistance gene (Kan^r), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptll), hygromycin resistance gene, 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 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 ^ orpromoters, 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 genetic 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.

The method according to this aspect of the invention is particularly useful for the expression of a wide range of foreign structural genes in the dividing cells of plants, such as in the meristem tissue, including a cell cycle control protein; an antibody- expressing gene, such as a SCAB gene; a selectable marker gene that confers resistance against kanamycin, phosphinothricin, spectinomycin or hygromycin, amongst others; a reporter gene including GUS, CAT and pigment genes, amongst others; a gene encoding a regulatory protein which modulates expression of a gene in plant cells; and a gene that encodes a developmental regulatory protein, such as, for example, a homeobox protein, that is involved in regulating the developmental fate of a cell. As will be apparent from the disclosure herein, the present method is clearly applicable to the expression of antisense molecules, ribozyme molecules, co- suppression molecules, gene-targeting molecules, or other molecules that are intended to modulate the expression of one or more endogenous plant genes in the dividing cells or meristem tissue of plants.

A further aspect of the present invention provides a transfected or transformed cell, tissue, organ or whole organism that contains the isolated meristem-expressible regulatory sequence of the invention. Preferably, said cell, tissue, organ or whole organism expresses a structural gene operably under the control of said promoter sequence.

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 genetic sequence which is at least 40% identical to SEQ ID NO:1 or alternatively, a genetic construct comprising said genetic sequence as described herein

The genetic construct of the present invention 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) microparticle bombardment, electroporation (Fromm et al, 1985), microinjection of DNA (Crossway et al, 1986), microparticle bombardment of tissue explants or cells (Christou er a/, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et a/.(1985), Herrera-Estrella et al. (1983a, 1983b, 1985).

For microparticle bombardment of cells, a 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 era/. (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 genetic 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.

Where 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.

Those skilled in the art will be aware of the methods for transforming, regenerating and propagating other type of cells, such as those of fungi. ln the case of plants, 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, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

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) transformants, 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 is further described by reference to the following non-limiting Examples. EXAMPLE 1 General Materials and Methods

Bacterial strains and media

The E. coli strains DH5α (Hanahan 1983), DH5α MCR (phenotype mcrA^' mcrBC; New England Biolabs) and XL2-Blue MRF' (Stratagene) were maintained on LB medium (Sambrook et al., 1989). Agrobacterium tumefaciens strain AGL1 (Lazo et al. 1991 ) was maintained on YEP medium (Ebert et al. 1987).

DNA extraction DNA was extracted from glasshouse-grown F₀ or F, plants using the method of Dellaporta et al. (1983). In order to further purify the DNA, it was redissolved in 7 mis of a solution containing 0.95 g ml^"1 CsCI and 0.35 mg ml¹ ethidium bromide and ultracentrifuged at 55000 rpm for 20 hrs using a Sorvall T-875 fixed angle rotor in a Sorvall OTD75B ultracentrifuge. Following ultracentrifugation, the ultracentrifuge tubes were punctured at the top and the base with a sterile needle, allowing the contents to drip out, and the fraction containing the genomic DNA band was collected. This fraction was then extracted several times with isoamyl alcohol to remove all traces of ethidium bromide, diluted 4-fold with sterile deionised water, and precipitated with 2 volumes of ethanol. This procedure yielded up to 0.7 mg DNA g^"1 leaf tissue.

The vector pBluescript II (Stratagene) was isolated using a large scale alkaline lysis method, and further purified by centrifugation in a CsCI gradient (Ausubel et al., 1989). Small scale isolations of plasmid DNA were done using the rapid alkaline extraction procedure (Birnboim and Doly, 1979).

DNA quantification

DNA concentrations were measured fluorometrically using Hoechst 33258 dye and a Jasco 820-FP spectrofluorometer according to the method of Cesarone et al. (1979), and visually confirmed by electrophoresis through agarose gels followed by ethidium bromide staining and uv transillumination. Restriction enzymes, DNA modifying enzymes and solutions All restriction enzymes, β-agarase 1 , calf intestinal alkaline phosphatase, T₄ DNA polymerase, T4 polynucleotide kinase and T4 DNA ligase were obtained from New England Biolabs, and used according to the manufacturer's instructions except where otherwise stated.

Southern analysis

Ten μg of high molecular weight genomic DNA was digested to completion with HindlW or EcoRV restriction endonucleases, and the fragments were separated by electrophoresis through a 0.8% (w/v) agarose gel. Following electrophoresis, DNA from the gel was transferred to Hybond-N⁺ nylon membrane (Amersham) using a Vacugene XL Vacuum Blotting System (Pharmacia), and the DNA was UV-cross linked to the membrane using a Bio-Rad Gene-Linker.

Probe DNA used to determine copy number of the luciferase gene consisted of either the luc coding sequence, obtained from a Hindl l/Xhol digest of pGEM-luc (Promega) and purified from low melting temperature agarose using the Prep-A-Gene kit (Bio-Rad), or the 1377 bp EcoRV fragment of luc, obtained from a HindlW/ EcoRV digest of pGEM-luc and purified from agarose using the Geneclean II kit (BIO-101 ). Probes used to determine copy number of the putative meristem-specific promoter amplified from transformant 060 included the 1559 bp H/πc/lll/Pvull fragment and the internal 763 bp Ncol/EcoRV fragment, both obtained from digests of inverse PCR (iPCR) clone 1 (see Figure 3) and purified from agarose using the Geneclean II kit (BIO-101 ).

Membranes were prehybridised for 10-24 hrs at 65-68 °C in hybridisation buffer, consisting of 5 x SSPE (SSPE contains 0.15M NaCI, I mM NaH₂P0₄.H₂0, 1.25 mM EDTA, pH 7.4), 5 x Denhardt's solution (Denhardt's solution contains 0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrrolidone, 0.02% (w/v) gelatine), 8% (w/v) dextran sulphate, 0.2% (w/v) SDS and 100 μg ml^"1 denatured, sheared salmon sperm DNA. Probe DNA was labelled with [α-³²P]dCTP by random primer extension using either the Megaprime or Rediprime kits (Amersham), after which any unincorporated nucleotides were removed by passing the labelling reaction through a Biospin 30 column (Bio-Rad), and the specific activity was measured in a Packard 1600TR liquid scintillation analyser. Labelled probe was added to the hybridisation solution at approximately 3-5 x 106 cpm ml^'1 hybridisation buffer, and hybridisation was allowed to proceed for 16 hrs at 65-68°C. After hybridising, membranes were washed twice with 5 x SSPE at room temperature for 10 min, once with 1 x SSPE, 0.1 % SDS at 65-68°C for 15 min., and twice with 0.1 x SSPE, 0.1 % SDS at 65-68°C for 15 min., before being exposed to X-ray film (Kodak X-Omat X-K1 ) with intensifying screens at -70°C, or exposed using a Phosphorlmager SI (Molecular Dynamics, Inc.).

DNA sequence analysis

Computer-assisted DNA sequence analysis was done using programs accessed through the Australian National Genomic Information Service (ANGIS).

Sequencing reactions were done using a PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit, using PEG-purified plasmid DNA template according to the manufacturer's instructions (Applied Biosystems, Inc.), and were analysed using an Applied Biosystems 373A DNA Sequencer.

Histochemical GUS assays

Histochemical detection of GUS activity in transformed cells or tissue was performed done using the substrate 5-bromo-4- chloro-3-indolyl glucuronide (X-gluc; Jefferson, 1987). Tobacco seedlings and tissues were immersed in assay buffer (containing 0.05% X-gluc, 50mM sodium phosphate pH7.0, 0.1% Triton X-100, 0.5 mM potassium ferricyanide, 0.5mM potassium ferrocyanide, and 10 mM EDTA), vacuum infiltrated twice, and incubated for 16 hrs at 37°C. Filters containing suspension culture cells were transferred to new Petri dishes containing 600111 assay buffer, and incubated at 37°C for 16 hrs. Following this incubation, green tissues were destained with ethanol, and blue stained cells were identified using a binocular microscope. EXAMPLE 2 Test screening and recovery of luc- expressing colonies

Overnight cultures of E. coli strain DH5α containing the plasmid pD0432 (Ow et al, 5 1986) or pUC18 (Boehringer Mannheim) were grown in LB medium supplemented with 50 μg ml^'1 ampicillin. The cell concentration in these cultures was estimated on the basis that an OD₆₀₀ of 1.0 corresponds to 8 x 10⁸ cells, and the cultures were diluted with LB medium and mixed at ratios of 1 :10⁵ and 1 :10⁶ of the pD0432-containing strain to the pUC18-containing strain. Volumes containing 3 x 10³or 3 x 10⁵ cells were then 0 spread on 9 cm diameter MacConkey agar plates containing 100 μg ml^"1 ampicillin, and the plates were incubated at 28°C for 2 days. The colonies obtained on these plates were assayed for luc expression using a liquid nitrogen-cooled CCD camera. The area corresponding to a glowing colony was collected on a sterile loop and streaked onto a fresh MacConkey plate with 100 μg ml^"1 ampicillin in order to give single colonies, 5 and this plate was grown for a further 2 days at 28°C and then re-assayed under the camera.

The results obtained from a test screening to determine whether /uc-expressing colonies can be detected from a background of non-expressing colonies are given in 0 Table 2. When a loopful of bacterial culture including one of the luc⁺ colonies in a confluent lawn of luc cells was restreaked and reassayed for luc expression, approximately half of the resulting colonies were found to express the luc gene.

TABLE 2

Detection of colonies containing a cloned luc gene in a background of non-glowing colonies using an in vivo luciferase assay.

Colony brightness is given as counts about background for a 100 second exposure.

EXAMPLE 3 Attempted promoter recovery via construction of a genomic library

The construction of a tobacco genomic library was attempted using three different strategies, as described below.

Strategy . Tobacco DNA was digested to completion with the restriction enzyme HindlW, and was then ligated into the vector pBluescript II (Stratagene) which had been digested with HindlW and dephosphorylated with calf intestinal alkaline phosphatase (CIP).

Strategy 2: H/ndlll-digested tobacco DNA (Strategy 1 ) was passed through an ultrafiltration membrane (Millipore Ultrafree-MC, 300 000 MW cutoff) to remove fragments smaller than 450 bp. The size-fractionated tobacco DNA was then ligated into vector as in Strategy 1.

Strategy 3: H/ndlll-digested tobacco DNA (Strategy 1 ) was size-fractionated through an agarose gel to enrich for fragments of similar size to the luc-hybridising band (revealed by Southern analysis). Fragments of appropriate size were isolated from the gel using β-agarase 1 digestion, according to the supplier's instructions. Purified fragments were then ligated into the vector pBluescript II as described in Strategy 1.

Ligations were performed in the ligation buffer described by King and Blakesley (1986), and were incubated at 16 °C overnight. The bacterial strain DH5α MCR was used for all cloning experiments involving plant genomic DNA, in order to stabilise the cloned inserts. This strain was transformed using either electroporation (Dower et al., 1988) or a PEG/DMSO treatment (Chung and Miller, 1988).

Results Strategy 1: Approximately 60,000 - 70,000 insert-containing colonies obtained from Strategy 1 ligations with transformant 060 genomic DNA and 15 000 white colonies from ligations with transformant 019 genomic DNA were screened for luc expression, with no positives being identified. However, in 20 randomly selected colonies, approximately 60% of inserts were smaller than 500 bp, while 88% were smaller than 4 kb. Strategy 2 was tested in an attempt to increase the proportion of inserts larger than 4 kb.

Strategy 2: In 18 randomly selected white colonies obtained from Strategy 2, most inserts were between 500 and 1000 bp, but there were no inserts larger than 3.5 kb. More precise size fractionation through agarose gels was therefore undertaken (Strategy 3).

Strategy 3: Several attempts were made to clone tobacco genomic DNA fragments ranging from approximately 3.5 - 4.8 kb in size, which had been isolated from agarose gels using β-agarase 1 digestion. Although ligation products were visible after gel electrophoresis, only 2 recombinant colonies were obtained from these ligations, and both had inserts of only a few hundred bp. A control ligation using 2.0 and 2.3 kb fragments of a λ DNA HindlW digest which had been gel purified using the same procedure yielded 2.3 x 103 colonies, of which 48% were recombinant, and a control ligation using a λ DNA Hindl digest without gel purification yielded several thousand colonies, of which 98% were recombinant.

EXAMPLE 4

Amplification and cloning of upstream flanking plant DNA

The inverse PCR strategy is described by Silver (1991 ) and modifications to this method are within the capability of those skilled in the art.

In particular, CsCI-purified tobacco genomic DNA was digested to completion with the restriction enzyme HindlW, and then heated to 65°C for 30 minutes to inactivate the enzyme. Digested DNA was ligated under conditions favouring recircularisation (Collins and Weissman, 1984), in 400 μ\ reactions containing 600 ng DNA and 9 Weiss units of T₄ DNA ligase in 1 x ligation buffer (New England Biolabs), which were incubated for 16 hrs at 15°C. Ligated DNA was purified by extraction with phenoLchloroform and chloroform, precipitated with 0.1 volumes of 3M sodium acetate and 2 volumes of ethanol, and finally redissolved at a concentration of approximately 30 ng μl^"1.

Inverse PCR was carried out using primers:

Sn (SEQ ID NO: 18): 5'-GCATAAAGTGTAAAGCCTGGGGTGC-3'; and

As1 (SEQ ID NO: 19): 5 '-CTGTGATTTGTATTCAGCCC-3', which were designed using the computer program Primer Detective (Clontech). Primers were synthesised using a Beckman Oligosaccharide 1000 DNA Synthesiser, and were purified using a Beckman Ultrafast Cleavage and Deprotection Kit, according to the manufacturer's instructions. Reactions were conducted in a total volume of 50 μl. and contained 200 ng ligated genomic DNA, 0.2 mM dNTPs, 40 ng each primer, 2.5 mM MgCI₂ and 3 units of Tth plus DNA polymerase (Biotech International), in the reaction buffer supplied by the manufacturer. PCR was done using a PTC-100 programmable thermal cycler equipped with a hot bonnet (MJ Research, Inc.), and consisted of an initial denaturation at 94°C for 3 min., followed by 35 cycles of: 1 min. denaturation at 94°C; 2 min. primer annealing at 58°C; 2.5 min. extension at 72°C. These 35 cycles were followed by a final extension at 72°C for 10 min. Typically 10 μl of each reaction was electrophoresed on a 0.8 % agarose gel and stained with ethidium bromide to visualise amplification products.

To isolate sufficient inverse PCR product, the products from 3 reactions were pooled, concentrated by ethanol precipitation, and electrophoresed through a 0.8% (w/v) agarose gel. The amplified band was then purified from the gel, treated with T₄ DNA polymerase and T₄ polynucleotide kinase, and then repurified, according to the "Double Geneclean" protocol (BIO-101 ). The resulting fragment was ligated into pBluescript II (Stratagene) which had been digested with EcoRV and dephosphorylated with calf intestinal alkaline phosphatase, and the ligation was transformed into Epicurian Coli Ultracompetent cells (Stratagene) and plated on LB X-gal plates.

To identify clones containing the correct insert, white colonies were patched onto LB plates, duplicate colony lifts were made onto Hybond-N filters (Amersham), and these were probed with the luc sequencing primer according to the method of Woods (1984). A colony containing the plasmid pD0432 (Ow et al, 1986) was also patched onto each plate as a positive control. Briefly, filters were washed for a total of 20 hrs in 3 changes of 3xSSC/0.1% (w/v) SDS (SSC is 0.15 M NaCI, 15 mM trisodium citrate, pH 7.2) at 65°C, rinsed in 3xSSC and then prehybridised for 6 hrs in a solution consisting of: 6xSSC; IxDenhardts; 0.5% (w/v) SDS; 100 μg ml^'1 denatured, sheared salmon sperm DNA; and 0.05% (w/v) sodium pyrophosphate. The luc sequencing primer (100 ng) was end-labelled for 60 min at 37°C, in a 50 μl reaction containing 50 μCi [γ-³²P]ATP (Amersham) and 10 units of T₄ polynucleotide kinase in the supplied buffer, and hybridisation was carried out overnight at 42 °C in oligosaccharide hybridisation buffer (6xSSC; IxDenhardts; 20 μg ml^'1 tRNA; 0.05% (w/v) sodium pyrophosphate) containing the labelled probe. After hybridisation, filters were rinsed 3 times in 3xSSC at room temperature, washed at 42°C for 1 hr in 6xSSC7 0.05% (w/v) sodium pyrophosphate, washed at 48 °C for 10 min in preheated 6xSSC/ 0.05% (w/v) sodium pyrophosphate, and then washed at 52°C for 10 min in preheated 6xSSC/ 0.05% (w/v) sodium pyrophosphate. After this final wash, the filters were blotted dry and exposed using a Phosphorlmager SI (Molecular Dynamics, Inc.).

5 The sequences of oligonucleotides used as sequencing primers are shown in Table 3. Sequencing primers were designed and synthesised as described for PCR primers.

Results

Inverse PCR conditions were optimised using the plasmid pEmu-luc (Mudge & Birch, 10 1998). The PCR primers Sn and As1 , which were designed against the pLUC19 T-DNA sequence, were able to amplify a 2609 bp fragment from the larger (4455bp) recircularised >4afll fragment from pEmu-luc. In a dilution experiment to determine the sensitivity of the PCR, a 2.6 kb band is amplified from as little as 0.04 pg of this template, equivalent to less than a single copy reconstruction (0.064 pg for a 200 ng 15 tobacco template). A 0.2 pg aliquot of the recircularised pEmu-luc Aatll template was used as a positive control in subsequent iPCR using tobacco DNA templates.

A single 2.5 kb band was reproducibly amplified by iPCR on aliquots of 4 different batches of pooled ligations from pLUC19 transformant 060, and a 3.1 kb band was 20 reproducibly amplified from 2 different batches of template ligations from transformant 019. The yield of amplified fragment varied between batches of template. No visible bands were ever amplified from untransformed Ti68 tobacco template.

EXAMPLE 5 25 Construction ofp060-GUS and p060-GUS19

To enable functional identification of putative meristem-specific promoters, a transcriptional fusion was made between the iPCR products obtained as described in the foregoing examples and the uidA (β-glucuronidase) reporter gene. This was achieved by cloning the 1558 bp PvuWIHindlll fragment of the iPCR product (Figure 3) 30 into the vector pGEM4-GUS.3 digested with Smal and HindlW, creating p060-GUS (Figure 6). The vector pGEM4-GUS.3 consists of the uidA coding sequence and nopaline synthase terminator from the vector pB1101.3 (Clontech Laboratories), cloned as an Xba1/EcoRI fragment into pGEM-4Z (Promega). The transcriptional fusion was confirmed by sequence analysis using the GUS sequencing primer (Figure 6).

TABLE 3 Oligonucieotides used as sequencing primers

To create the binary vector p060-GUS19, the 3714 bp promoter-u/αy\-nos3' cassette from p060-GUS was cloned as an EcoRI/H/ndlll fragment into the binary vector pBin19 (Bevan, 1984). The 060 promoter-u/ctø-nos 3' cassette flanked by the T-DNA left and right border sequences in the binary vector p060-GUS19 is shown in Figure 7. EXAMPLE 6 Microprojectile bombardment

Target material used for microprojectile bombardment experiments consisted of suspension culture cells of Nicotiana plumbaginifolia and intact Ti68 tobacco seedlings. The suspension culture was maintained by subculturing 5 mis into 45mls of CSV medium (Gibson et al., 1976) weekly, and was shaken at 120 rpm in darkness at 28°C. Suspension culture cells were bombarded 2 days after subculturing. For each bombardment, 5 mis of suspension was collected on a 4.25 cm diameter sterile filter paper (Whatman No. 1 ) using vacuum filtration. Following bombardment, each filter was transferred to a stack of 3 new filters, which had been moistened with 1 ml of CSV medium, and incubated in darkness at 28 °C.

Tobacco seedlings were sterilised using methods known to those skilled in the art, and plated on MSO medium at a density of 70-80 per plate. These were incubated in the light at 28°C, and bombarded 8 days after germination.

The plasmids p060-GUS and pGEM4-GUS.3 were prepared as described above for sequencing. A CsCI-purified preparation of pGN1 (Rathus and Birch, 1992) was used as a positive control. The concentration of these plasmids was adjusted to 1 μg μl^'1 for use in microprojectile bombardment experiments.

The device used for bombardment consisted of the apparatus described previously (Franks and Birch, 1991 ), modified such that the barrel and stop plate were replaced with a gas control solenoid and syringe filter holder (Finer et al., 1992). Precipitation of DNA onto microprojectiles was as described (Finer and McMullen, 1990), except that 2 μl of plasmid was used for each precipitation. N. plumbaginifolia suspension culture cells were bombarded using a helium pressure of 2100 kPa, and intact tobacco seedlings using 500 or 1000 kPa, each with a pulse length of 50msec.

No GUS activity was observed above the threshold of detection for the histochemical assay following microprojectile-mediated delivery of the transcriptional fusion construct p060-GUS (Figure 6) into Nicotiana plumbaginifolia suspension culture cells or tobacco seedlings. Positive control bombardments using the plasmid pGN1 into suspension culture cells yielded large numbers of GUS-expressing cells (808, 178 and 751 blue foci from 3 bombardments). Bombardment of intact tobacco seedlings, however, proved to be inefficient, with less than 5 blue foci per bombardment with pGN1.

EXAMPLE 7 Stable transformation of plant material

The rapid-flowering mutant of Nicotiana tabacum cv. Wisconsin 38, Ti68 was transformed with A. tumefaciens strain AGL1 containing the binary vectors p060-GUS19, pBI121 (Clontech) or pBin19, using a leaf disk procedure.

Plants regenerated following transformation with the binary vector p060-GUS19 (Figure 7) were assayed for GUS activity using histochemical assays. Seven out of eight plants tested showed detectable GUS activity. A similar frequency of plants regenerated from the pBI121 transformation (5/6 tested) also showed GUS activity. In contrast to the pBI121 transformants, however, the GUS activity observed in the p060-GUS19 plants was confined to the shoot tip region, including the areas adjacent to the apical meristem, upper axillary meristems, and the youngest expanding leaves. GUS activity was also observed in mature pollen from 3 out of 4 transformants tested, but not in other floral tissues. None of the p060-GUS19 transformants tested showed detectable GUS activity in root tips. No GUS activity was observed in any vegetative of floral tissues from pBinlθ transformants. Examples of GUS activity in some of these lines are shown in Figures 8-10.

EXAMPLE 8

Southern analysis of selected transformants

H/ndlll-digested DNA from pLUC19 transformants 040, 024, 060, and 35SLUC19 transformants 2, 10, and 11 probed with the luc coding sequence showed single bands of approximately 7.5 kb, 5.0 kb, 4.2 kb, 5.9 kb, 11 kb, and 8 kb, respectively (e.g. Figure 1A). No hybridisation was observed to untransformed Ti68 DNA or DNA from 35SLUC19 transformant #12 H/ndlll-digested DNA from an F. progeny plant of pLUC19 line 060 (meristem- specific LUC activity) showed a single luc-hybridising band of 4.2 kb as in the primary transformant, and EcoRV digestion confirmed a single copy integration (Figure 1 B). In contrast, similarly digested DNA from F. progeny of 5 line 019 (expression during early stage of shoot regeneration) showed two bands with different intensity from each digestion, indicating multiple integrations and a possible tandem integration at one site (Figure 1B).

EXAMPLE 9 10 Discussion luc copy number in transgenic plants

The results of Southern analysis using the luc coding sequence as a probe indicate that the majority (six out of seven tested) of transgenic plants obtained in this study have single copy T-DNA insertions. Single copy transformants are preferred for the 15 isolation of tagged plant promoters, because only a single region of plant DNA, upstream of the single copy reporter gene, must be isolated to investigate the putative promoter. There may, however, be situations where remote enhancer sequences also play a role in the final expression pattern.

20 The high proportion of single copy transformants in the population of plants described herein is consistent with the relatively low promoter tagging frequency. High tagging frequencies are often accompanied by higher T-DNA copy numbers (e.g. Koncz et al, 1989, Lindsey er a/., 1993).

25 35SLUC19 transformant #12 showed no LUC activity in either in vivo or in vitro assays. Southern analysis indicates that this lack of activity is due to the absence of the luc coding sequence in this transformant (Figure 1A), presumably due to truncation of the T-DNA, rather than expression at a level undetectable by the assays. T-DNA truncation events such as this have been described before (e.g. Gheysen et al, 1990).

30

The hybridisation pattern obtained with pLUC19 transformant 019 suggests that this transformant may contain an inverted repeat plus an additional unιιnκed T-DNA copy. For an inverted repeat joined at the left border, the expected fragment sizes for a HindlW and EcoRV digest are 5694 bp and 4002 bp, respectively. In Figure 1B, the brighter band in each digest is approximately 1.1 kb smaller than this, suggesting that this band may be an inverted repeat with a truncation of 1.1 kb centred on the left border region. It is not uncommon for T-DNAs to be truncated at one of the borders, particularly the left one, and inverted repeats with truncations similar to this have been described previously (Jorgensen et al, 1987). Confirmation of this structure is obtained by Southern analysis of an EcoRM/HindlW double digest of 019 DNA, which should give the same banding pattern as the EcoRV digest alone. If such truncations are present, then the observed LUC phenotype will probably be due to a tagged promoter linked to the 5.2 kb HindlW fragment in Figure 1B.

Evaluation of a novel technique for the recovery of plant promoters tagged by promoterless luc genes

The in vivo LUC assay was sufficiently sensitive to facilitate the detection of any bacterial colony containing a cloned luc gene. The assay allows the relatively simple recovery of tagged plant promoters, via direct cloning of H/ndlll-digested DNA from the transformant of interest in plasmids, followed by high efficiency transformation into E. coli and screening of recombinant colonies for LUC activity.

The results given in Table 2 indicate that a single colony with LUC activity can readily be identified and recovered from a lawn containing hundreds of thousands of non-expressing colonies, and therefore, provided that sufficient colonies can be obtained, this approach should be feasible for promoter recovery. However, the very low efficiency of cloning of 4-4.5 kb fragments of plant DNA obtained in this study prevented the successful application of the technique. The preferential cloning of small fragments made direct cloning without size-fractionation very inefficient (Strategy 1 ), and this problem was not solved by removal of fragments smaller than 500 kb through an ultrafiltration membrane (Strategy 2). While size fractionation through agarose was much more precise, genomic DNA fragments obtained in this way were unable to be cloned (Strategy 3). Control cloning experiments done at the same time indicated that the cloning failure was not due to the vector used or the process of purification from the agarose. The cause of the problem, therefore, remains unclear.

Construction of genomic libraries in high-copy plasmids using size-fractionated, gel-purified DNA fragments has been previously described. Herman et al. (1986) and Claes et al. (1991 ) cloned relatively small DNA fragments (less than 1100 bp), obtaining 6000 and 1300 recombinant colonies, respectively. Gheysen et al. (1991 ) reported that size-fractionated fragments of up to 20 kb were successfully cloned, yielding 105 colonies. Using the formula of Clark and Carbon (1976), approximately 1.6 x 10⁶ colonies are required for a genomic library with an average insert size of 4.2 kb. in order to have a 99% probability of the luc-containing fragment being present. It is clear, therefore, that for this technique to be successful, a highly efficient system for generating large numbers of recombinant colonies is required.

The presence of ligation products including genomic DNA fragments in the attempts at direct cloning described above, indicates that the techniques of plasmid rescue and inverse PCR may allow recovery of the putative tagged promoters upstream of the luc gene. While plasmid rescue may be a useful method for recovering promoters tagged with the vector pRBLUCI9, it is not applicable for pLUCI9 transformants. Inverse PCR was therefore tested for the recovery of the putative tagged promoter from pLUC19 transformant 060.

Inverse PCR allows efficient amplification and cloning of plant DNA flanking the left border

The results obtained in the present study indicate that the inverse PCR technique can be used to readily isolate plant DNA sequences flanking T-DNA borders in plants, in agreement with several other recent reports (Jiang et al., 1992; Lindsey et al., 1993; Thomas et al. 1994; Ohba et al., 1995). The region flanking the left border in pLUC19 transformant 060 contains a short interspersed repetitive element

When the sequence of the amplified plant DNA from transformant 060 was compared to known nucleotide sequences in the Genbank and EMBL databases using the computer programs BLAST (Altschul et al., 1990) and FastA (Pearson and Lipman, 1988), a region with high homology to members of the TS family of short interspersed repetitive elements (SINEs) was identified. This region consists of 249 bp, and is flanked by direct 16 bp repeats, as shown in Figure 4. An alignment of this region to the five closest matches from the database searches above, produced using the program PileUp (Feng and Doolittle, 1987), demonstrates the high homology between these sequences (Figure 11 ).

Members of the TS (Tobacco SINE) family of short interspersed repetitive elements have previously been found within introns and flanking regions of many genes from plants belonging to the order Tubiflorae, and are present in at least 5.0 x 10⁴ copies per tobacco haploid genome (Yoshioka et al, 1993). The sequence "ntaux35" in Figure 11 , for example, is present in the promoter of an auxin-induced gene from tobacco which is expressed in root tips (Van der Zaal et al, 1991 ), as is the tagged promoter from transformant 060. Whether this element has any role in the regulation of expression is unknown. According to the diagnostic nucleotide positions described by Yoshioka et al. (1993), the repetitive element identified in the present study is a member of the TSa subfamily.

The presence of a highly repetitive element in the amplified plant DNA from transformant 060 is confirmed by the results of Southern analysis using the entire HindlW/ Pvull fragment as a probe, which hybridised to the whole smear of genomic DNA (Figure 5A).

Several other groups have also demonstrated the presence of highly repetitive DNA linked to T-DNA insertions using Southern analysis (Zambryski et al., 1982; Holsters et al., 1983; Gheysen et al., 1987; Matsumoto et al, 1990). This is not unexpected, due to the high copy number and random distribution of repetitive elements throughout the genome (Zimmerman and Goldberg, 1977).

The amplified plant DNA from pLUC19 transformant 060 contains a novel promoter element DNA sequencing of the cloned iPCR product indicates that a truncation of approximately 46 bases is present at the left border, similar to previously characterised left border-plant junctions (e.g. Gheysen et al., 1991 ; Topping et al., 1994). The amplified plant DNA is AT rich (61.5% A+T), which is a characteristic of 5' noncoding regions of plant genes (Fobert et al., 1994).

Other than the TS repeat, the amplified DNA from transformant 060 was not found to be homologous to any other nucleotide sequence in the databases. The possible translation products from all 6 reading frames showed no homology to sequences in the protein databases PIR, Swiss-Prot, and the Brookhaven Protein Databank, when compared using the BLAST program (Altschul et al., 1990).

Using the computer program Signal Scan (Prestridge, 1991 ), a putative TATA box was identified 288 bp upstream of the left border-plant DNA junction. This region, with the sequence TATATAA (SEQ ID NO: 35), is similar to the TATA boxes of several previously characterised plant genes (Joshi, 1987). A putative transcriptional start site, with the eukaryotic consensus sequence of CA followed by pyrimidines (Bucher and Trifonov, 1986), occurs 88 bp downstream of the TATA box, as shown in Figure 4.

Several previously characterised transcription factor binding sites were also identified in the amplified plant DNA from pLUCI9 transformant 060. The hexamer sequence TGACGT (SEQ ID NO: 10) occurs at position -337 relative to the transcriptional start site. This motif is believed to be responsible for the meristem-specific expression of the histone H3 promoter from wheat (Terada et al., 1993), the chloroplast FBPase promoter from wheat (Lloyd et al., 1991 ), and the 35S and OCS enhancer sequences (Benfey et al., 1989; Fromm et al., 1989), and has been shown to bind a family of related leucine zipper-type transcription factors including wheat HBP-lb, tobacco ASF-1 and maize OCSTF (Tabata et al, 1991 ). It is possible that this sequence is also involved in the regulation of the tagged promoter isolated in the present study. Sequences similar to the "cell cycle box" found in some yeast genes 5'-CACGAAAA- 3' (SEQ ID NO: 36) occur twice in the plant DNA (at -447 and -87). This sequence binds the transcription factors SWI4 and SWI6, which regulate cell-cycle specific transcription (Nasmyth and Dirick, 1991 ; Ogas et al., 1991 ). A promoter which is activated only at a specific stage of the cell cycle would be expected to direct highest reporter gene expression in tissues undergoing high rates of cell division, consistent with the phenotype of the 060 tagged promoter. The motif TGTGG, which occurs 3 times in the amplified plant DNA (at -919, +13 and +114), is conserved in a number of pollen-specific promoters (Ingersoll et al, 1994; Twell er a/., 1991 ).

The sequence 5'-CCACG-3' (SEQ ID NO: 37) occurs 3 times (at -1149, -536 and +45 relative to the putative transcriptional start site), and has been shown to bind a factor involved in activation of the maize alcohol dehydrogenase-l (Adh1) gene (Ferl and Nick, 1987). Other transcription factor binding sites that occur multiple times in the plant DNA include those for NIT2 (5'- TATCT-3'; SEQ ID NO: 38), at positions -1045, -396, -170, -108 and +92, which regulates genes involved in nitrate metabolism in the fungus Neurospora crassa (Fu and Marzluf, 1990); and GCN4 (5'-T(G/T) A(C/G)T-3'; SEQ ID NO: 39), at positions -1001 , -993, -727, -642, -616, +166), which activates yeast amino acid biosynthetic enzymes (Arndt and Fink, 1986). Whether any of these transcription factors plays a role in the regulation of the putative promoter isolated in the present study remains to be determined.

In addition to the motifs described above, several direct and inverted repeat structures were identified near the putative TATA box, some of which are shown in Figure 4. Computer searches indicated that some of these motifs are also present in other meristem-specific promoter sequences in the database (Table 4), and thus these may also be cis acting regulatory regions. Based on the putative transcriptional start site shown in Figure 4, the expected mRNA transcript size of the luc transcriptional fusion in this transformant would be approximately 2500 nucleotides. This is confirmed by Northern analysis, using the luc coding sequence as a probe. This transcript would have an untranslated leader sequence of 901 nucleotides, in which there are 6 upstream open reading frames (uORFs) of 8 to 24 amino acids. Two of these are present in the plant DNA (see Figure 4), and four in the vector pLUC19. In addition, there are 2 upstream out-of-frame ATG codons, positioned 35 and 31 bp upstream of the Luc ATG which do not have corresponding stop codons upstream of the Luc ATG.

The presence of uORFs in plant genes (as in other organisms) generally causes a decrease in expression levels of the gene. While most plant genes do not have uORFs, several transcription factor genes have been identified where uORFs are believed to play a role in translational regulation of the gene, preventing over expression of the transcription factor. These include the maize genes R-S (Perrot and Cone, 1989), Opaque-2 (Schmidt era/., 1990), OCSBF-I (Singh era/., 1990), Zmhoxla (Bellmann and Werr, 1992), and Lc (Damiani and Wessler, 1993), the parsley gene CPRFI (Feldbrugge et al., 1994), the Arabidopsis gene KNATI (Lincoln et a/., 1994), and the wheat gene HBP-la (Mikami et al., 1995). For two of these genes (Opaque-2 and Lc), as well as the yeast transcription factor GCN4 gene, the uORFs have been shown experimentally to mediate translational regulation (preventing over expression) of the gene (Lohmer ef a/., 1993; Damiani and Wessler, 1993; Mueller and Hinnebusch, 1986). Di-cistronic transcripts are able to be expressed in plant cells (Angenon et al., 1989; Koncz et al., 1989; lida et al, 1992). According to the scanning model for translation, the downstream ORF can be translated by the (relatively inefficient) process of ribosome reinitiation, provided that a stop codon occurs that is in-frame with the upstream ATG and upstream of the start codon for the downstream ORF (Kozak, 1987, 1989).

The sequence surrounding the upstream ATGs in the amplified plant DNA (and in the T-DNA) do not fit the consensus for plants at every nucleotide (Lutcke et al., 1987). However, there is considerable variation from this context among plant genes, and there is evidence suggesting that the effect of ATG context is less significant in plants than in animals (Luehrsen and Walbot, 1994; Lutcke et al., 1987). Furthermore, the upstream ATGs in the transcription factor genes described above also differ from the consensus.

The similarity between the uORFs in the putative leader sequence identified here and the previously characterised genes for the above transcription factors suggest that the gene that has been tagged in pLUCI9 transformant 060 may also be a transcription factor, whose expression is under translational control mediated by the uORFs. Isolation of the coding sequence of the gene (which flanks the right border of the T-DNA) would be required to determine if this is the case.

The presence of the uORFs within the vector pLUCI9 does not explain the relatively low promoter tagging frequency obtained with this vector, since the same uORFs are also present in the vector used by Topping et al. (1991 ), who reported a tagging frequency of 78%. However, the presence of upstream ATG codons with no in-frame stop codons before the luc start codon is likely to significantly reduce expression levels, and hence apparent tagging frequency (since reinitiation of translation does not occur under these conditions), and there are no such ATG codons present in the vector used by Topping et al. (see T-DNA sequence Wei et al., 1994). Removal of these upstream ATGs in pLUC19, therefore, is likely to increase the observable promoter tagging frequency.

Southern analysis using an internal fragment of the putative meristem-specific promoter from transformant 060 (which does not include the TS repeat) indicates that this is a unique sequence in the tobacco genome (Figure 5B). Kanamycin-resistant F, progeny derived from the transgenic plants are likely to be hemizygous, which explains the hybridisation pattern observed with DNA from the 060 F, seedling. In this plant, one locus is linked to the T-DNA (resulting in a band at 4.2 kb, to which the luc probe also hybridises), while the other locus is unlinked, resulting in the 4.6 kb band observed in untransformed tobacco.

The amplified DNA from transformant 060 contains a functional promoter The results of microprojectile-mediated transient expression analysis using the β-glucuronidase (GUS) transcriptional fusion construct p060-GUS indicate that the putative meristem-specific promoter from transformant 060 does not direct expression in suspension culture cells of N. plumbaginifolia at a level that is detectable by the histochemical GUS assay. The results obtained for bombardments into intact tobacco seedlings, however, are inconclusive, since the positive controls gave very low levels of expression. The problems associated with gene transfer into very localised tissue types (such as apical meristems and root tips) limit the applicability of transient expression analysis for the functional testing of promoters with spatially restricted expression patterns.

The results of histochemical GUS assays on tobacco plants transformed with the binary vector p060-GUS19, however, indicate that the amplified DNA from transformant 060 does contain a functional promoter which directs shoot tip-specific expression. The pattern of GUS activity revealed by these assays is not confined only to the meristematic regions, as generally observed in the LUC assays on pLUC 19 transformant 060, but tends to be in the stem adjacent to the meristem and in the youngest, expanding leaves (Figure 8). This difference may reflect the relative stability of the GUS and LUC enzymes. Previous studies suggest that the LUC protein is relatively unstable (Thompson et al., 1991 ), and that LUC activity closely follows mRNA induction (Millar er a/., 1992a). The GUS protein is relatively stable, and GUS activity has been shown to continue increasing as long as detectable mRNA is present, so that maximum activity can occur 2 days after maximum mRNA levels (Gatz et al., 1992; Hensgens et al., 1992).

GUS activity was also detected in mature pollen grains (Figure 10). Endogenous GUS-like activity has been reported in tobacco pollen (Plegt and Bino, 1989), although several other studies have reported the absence of such activity (e.g. Guerrero et al., TABLE 4

Sequence motifs present multiple times in the amplified DNA from pLUC19 transformant 060 that are also present in other meristem-specific promoters

^a Promoters shown are as follows: 060, the promoter sequence amplified from pLUC19 transformant 060; cyc07, the S-phase-specific cyc07 gene from Catharanthus roseus (Ito et al., 1994); ACTI, an actin gene isolated from Arabidopsis thaliana (An et al., 1996); FBPase, the chloroplast fructose-1 ,6-bisphosphatase gene from wheat (Lloyd et al., 1991); par, an auxin-regulated gene from tobacco (Takahashi er al., 1995); HMG2, the 3-hydroxy-3- methylglutaryl coenzyme A reductase gene from A. thaliana (Enjuto et al., 1995).

The position relative to the transcriptional start site is shown, except for the FBPase promoter, for which the position relative to the translational start site is shown. 1990). The absence of GUS activity in pollen from pBinl9-transformed plants observed here suggests that the amplified promoter fragment from pLUCI9 transformant 060 does direct expression in mature pollen.

Histochemical GUS assays on the p060-GUS19 transformed plants did not reveal any expression in root tips. In pLUCI9 transformant 060, however, LUC activity was observed in both the root and shoot tips. It is possible, therefore, that the sequences responsible for expression in the root tips are not present in the upstream region that was amplified by inverse PCR. These sequences may be present further upstream, or possibly downstream of the coding region.

Further characterisation of this promoter by deletion analysis and stable transformation studies can now be done. As well as deleting regions at the 5' end of the promoter, it would be interesting to test a promoter construct which lacks the uORFs present in p060-GUS19. A promoter fragment lacking these inhibitory ORFs is amplified by PCR using the primers SM4 (SEQ Dl NO: 26) and an oligonucleotide consisting of 5'-TGCTCTATTGACAGTGCC-3' (SEQ ID NO: 40) complementary to bases 16-33 in Figure 4, and this would be expected to direct stronger levels of expression.

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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:1 or a meristem- expressible homologue, analogue or derivative thereof; (ii) a meristem-expressible nucleotide sequence comprising at least 30 contiguous nucleotides of SEQ ID NO:1 ;

(iii) a meristem-expressible nucleotide sequence that contains at least four c/s-acting regulatory sequences selected from the group consisting of:

(a) 5'-TGACGT-3' (SEQ ID NO:10);

(b) 5'-CAACTCC-3'(SEQ ID NO:11 );

(c) 5'-TCTGTT-3' (SEQ ID NO:12);

(d) 5'-TAGTAGT-3' (SEQ ID NO:13);

(e) 5'-GTAGATT-3'(SEQ ID NO:14); and

(f) 5'-CATGCAA-3' (SEQ ID NO:15);

(iv) a meristem-expressible nucleotide sequence that contains a uORF sequence selected from the group consisting of:

(a) 5'-ATGCCACGTCTGAGGGTAATTCTGTAA-3' (SEQ ID NO:16);

(b) 5'-ATGGACTCTCGCACGTTGTGGCCTTATTTACCGCTGC TTCAATCAGAACCAAGTCAGGACAAAATAGGTCAGTAA-3' (SEQ ID NO:17); and

(c) a degenerate nucleotide sequence to (a) and/or (b); and

(v) a nucleotide sequence that is complementary to any one of (i) to (iv).

2. The isolated genetic sequence of claim 1 , comprising a meristem-expressible nucleotide sequence that comprises at least 30 contiguous nucleotides of SEQ ID NO:1.

3. The isolated genetic sequence of claim 2, comprising at least 50 contiguous nucleotides of SEQ ID NO:1.

4. The isolated genetic sequence of claim 2, comprising at least 100 contiguous nucleotides of SEQ ID NO:1.

5. The isolated genetic sequence of claim 1 , comprising a meristem-expressible nucleotide sequence that contains at least four c/s-acting regulatory sequences selected from the group consisting of:

(a) 5'-TGACGT-3' (SEQ ID NO:10);

(b) 5'-CAACTCC-3'(SEQ ID NO:11 );

(c) 5'-TCTGTT-3' (SEQ ID NO:12);

(d) 5'-TAGTAGT-3' (SEQ ID NO:13);

(e) 5'-GTAGATT-3'(SEQ ID NO: 14); and

(f) 5'-CATGCAA-3' (SEQ ID NO:15).

6. The isolated genetic sequence of claim 5, comprising at least five of said c/s- acting regulatory sequences.

7. The isolated genetic sequence of claim 5, comprising all of said c/s-acting regulatory sequences.

8. The isolated genetic sequence of claim 1 , comprising a meristem-expressible nucleotide sequence that contains a uORF sequence selected from the group consisting of:

(a) 5'-ATGCCACGTCTGAGGGTAATTCTGTAA-3' (SEQ ID NO:16);

(b) 5'-ATGGACTCTCGCACGTTGTGGCCTTATTTACCGCTGC TTCAATCAGAACCAAGTCAGGACAAAATAGGTCAGTAA-3' (SEQIDNO:17);and

(c) a degenerate nucleotide sequence to (a) and/or (b).

9. The isolated genetic sequence of claim 8, comprising a meristem-expressible nucleotide sequence that contains the two uORF sequences:

(a) 5'-ATGCCACGTCTGAGGGTAATTCTGTAA-3' (SEQ ID NO:16); and (b) 5'-ATGGACTCTCGCACGTTGTGGCCTTATΪTACCGCTGC

TTCAATCAGAACCAAGTCAGGACAAAATAGGTCAGTAA-3' (SEQ ID NO:17); or a degenerate nucleotide sequence to (a) and/or (b).

10. The isolated genetic sequence according to any one of claims 5 to 9, having at least 40% identity to SEQ ID NO:1.

11. The isolated genetic sequence according to claim 10, wherein the percentage identity to SEQ ID NO:1 is at least 60%.

12. The isolated genetic sequence of claim 1 , comprising the nucleotide sequence set forth in SEQ ID NO:1.

13. The isolated genetic sequence according to any one of claims 1 to 12, wherein said sequence confers expression on a structural gene in a shoot meristem cell.

14. The isolated genetic sequence according to any one of claims 1 to 12, wherein said sequence confers expression on a structural gene in a root meristem cell.

15. The isolated genetic sequence according to claims 13 or 14, wherein the expression conferred on the structural gene is meristem-specific.

16. An isolated meristem-expressible regulatory sequence obtainable by the method of: a) hybridising under at least low stringency conditions plant genomic DNA, or mRNA, or cDNA obtained therefrom, with one or more nucleic acid probes or primers that comprise a nucleotide sequence obtainable from SEQ ID NO:1 or a nucleotide sequence that is complementary thereto for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule; b) detecting the hybridised nucleic acid molecule; and c) isolating said hybridised nucleic acid molecule comprising the isolated meristem-expressible regulatory sequence or promoter sequence.

17. The isolated meristem-expressible regulatory sequence of claim 16 wherein said one or more nucleic acid probes or primers comprises a nucleotide sequence that hybridises to the non-coding region of a plant meristem-expressible promoter.

18. The isolated meristem-expressible regulatory sequence of claim 16 wherein detection and/or isolation of said promoter includes amplifying nucleic acid using said probes or primers in a PCR reaction or PCR reaction equivalent.

19. An isolated meristem-expressible regulatory sequence obtainable by the method of:

20. The isolated meristem-expressible regulatory sequence of claim 19 further comprising the first step of transforming or transfecting the meristem cells or a progenitor cell thereof with the reporter gene construct.

21. The isolated meristem-expressible regulatory sequence of claim 19 wherein detection and/or isolation of said promoter includes amplifying nucleic acid using said probes or primers in a PCR reaction or PCR reaction equivalent.

22. The isolated meristem-expressible regulatory sequence of claim 19 comprising a nucleotide sequence selected from the group consisting of:

(i) the nucleotide sequence set forth in SEQ ID NO:1 or a meristem- expressible homologue, analogue or derivative thereof; (ii) a meristem-expressible nucleotide sequence comprising at least 10 contiguous nucleotides of SEQ ID NO:1 ;

(a) 5'-TGACGT-3' (SEQ ID NO:10);

(b) 5'-CAACTCC-3'(SEQ ID NO:11 );

(c) 5'-TCTGTT-3' (SEQ ID NO: 12);

(d) 5'-TAGTAGT-3' (SEQ ID NO:13);

(e) 5'-GTAGATT-3'(SEQ ID NO:14); and

(f) 5'-CATGCAA-3' (SEQ ID NO:15);

(a) 5'-ATGCCACGTCTGAGGGTAATTCTGTAA-3' (SEQ ID NO:16);

(c) a degenerate nucleotide sequence to (a) and/or (b); and

(v) a nucleotide sequence that is complementary to any one of (i) to (iv).

23. An isolated meristem-expressible promoter sequence of plants that comprises the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary nucleotide sequence thereto or a meristem-expressible fragment thereof comprising at least one c/s-acting regulatory sequence obtainable from said nucleotide sequence.

24. A genetic construct comprising the isolated genetic sequence according to any one of claims 1 to 15 operably linked to a structural gene sequence.

25. The genetic construct of claim 24 suitable for expression in a plant cell.

26. A genetic construct comprising the isolated meristem-expressible regulatory sequence according to any one of claims 16 to 22 operably linked to a structural gene sequence.

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

28. A genetic construct comprising the isolated meristem-expressible promoter sequence of claim 23 operably linked to a structural gene sequence.

29. The genetic construct according to any one of claims 24 or 26 or 28 wherein the structural gene sequence is a protein-encoding structural gene sequence.

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

31. The genetic construct of claim 30 wherein the reporter gene is a luciferase gene, β-glucuronidase gene, or chloramphenicol acetyltransferase gene.

32. The genetic construct of claim 29 wherein the protein-encoding structural gene sequence encodes a cytotoxin or other protein that is capable of inhibiting cellular metabolism.

33. The genetic construct of claim 29 wherein the protein-encoding structural gene sequence encodes a hormone polypeptide or polypeptide involved in the synthesis of a hormone.

34. The genetic construct according to any one of claims 24 or 26 or 28 wherein the structural gene sequence is a molecule capable of targeting the expression of an endogenous meristem-expressible or meristem-specific gene in a cell.

35. The genetic construct of claim 34 wherein the structural gene sequence is an antisense molecule, co-suppression molecule, ribozyme, or abzyme molecule.

36. The genetic construct according to any one of claims 25 or 27 or 29 wherein the plant cell is a tobacco plant cell.

37. The genetic construct according to any one of claims 24 to 36 further comprising a transcription termination sequence downstream of the structural gene sequence.

38. The genetic construct according to any one of claims 24 to 37 further comprising an origin of replication.

39. The genetic construct according to any one of claims 24 to 38 further comprising a selectable marker gene sequence.

40. The genetic construct according to any one of claims 24 to 39 further comprising one or more integration sequences suitable for insertion into plant genomic DNA.

41. 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 24 to 40 for a time and under conditions sufficient for expression of the structural gene to occur.

42. A transfected or transformed cell, tissue, organ or whole organism that contains the isolated genetic sequence according to any one of claims 1 to 15 or a genetic construct comprising same introduced thereto.

43. A transfected or transformed cell, tissue, organ or whole organism that contains the isolated meristem-expressible regulatory sequence according to any one of claims 16 to 22 or a genetic construct comprising same introduced thereto.

44. A transfected or transformed cell, tissue, organ or whole organism that contains the isolated meristem-expressible promoter sequence according to claim 23 or a genetic construct comprising same introduced thereto.

45. The transfected or transformed cell, tissue, organ or whole organism according to claims 42 or 43 derived from a plant or comprising a plant or plant propagule.

46. The transfected or transformed cell, tissue, organ or whole organism according to claim 44 derived from a plant or comprising a plant or plant propagule.

47. Use of the isolated genetic sequence according to any one of claims 1 to 15 to produce a transgenic plant.

48. Use of the isolated meristem-expressible regulatory sequence according to any one of claims 16 to 22 to produce a transgenic plant.

49. Use of the isolated meristem-expressible promoter sequence according to claim 23 to produce a transgenic plant.