WO2001021785A2 - Megagametophyte transcriptional control elements and uses thereof - Google Patents

Abstract

In one aspect, the present invention provides plant information vectors comprising an enhancer element operative in a plant megagametophyte. The enhancer element (a) comprises a nucleic acid sequence that is at least 80 % identical to the nucleic acid sequence set forth in SEQ ID NO:1 or to the nucleic acid sequence set forth in SEQ ID NO:2; and (b) is operably linked to a nucleic acid sequence of interest. In another aspect, the invention provides transgenic cells and transgenic plants comprising a vector of the invention, as well as seeds obtained from such transgenic plants. In another aspect, the invention provides methods of expressing a nucleic acid sequence of interest in a megagametophyte of a plant. The vectors and methods of the invention can be used, for example, to induce apomixis in a plant.

Description

MEGAGAMETOPHYTE TRANSCRIPTIONAL CONTROL ELEMENTS

AND USES THEREOF

Field ofthe Invention This invention relates gu lly to gene elements involved in transcriptional control of genes in specific cell types. More specifically, it relates to enhancer elements that control transcription in megagametophytes of plants, and to vectors, transgenic plants, and methods employing such enhancer elements.

Background ofthe Invention Apomixis is plant reproduction involving the specialized reproductive structures of a plant, but not dependent upon fertilization. Apomixis typically involves the formation of a plant from one or more cells of the megagametophyte (i.e., the female gametophyte), such as cells of the embryo sac, including the egg. For example, parthenogenesis is one type of apomixis in which a plant embryo develops from an unfertilized egg. An advantage of apomixis is that numerous, genetically identical, progeny can be derived from a single plant that exhibits one or more desirable phenotypic traits. Thus, apomixis will allow high yield gains from hybrids to perpetuate, cheaply and undiminished. It will also provide the farmer with greater autonomy and choice in planting future generations and to capture the advantages of superior cultivars adapted to local conditions.

In order to transform sexually reproductive plants into apomictic plants, genes and regulatory elements are required that can be used to control gene activity in megasporogenesis (i.e., formation ofthe megaspore cell from which the embryo sac, including the egg, is derived) and the development of the female gametophyte and associated structures. Most research on the molecular biology of plant reproduction, however, has focused on floral development in the sporophyte phase, microsporogenesis and male gametophyte development due to accessibility of these structures. The present invention provides regulatory elements specific for megagametophytes, and more specifically, an embryo sac-specific enhancer element that controls gene expression in megagametophytes. The function of these regulatory elements can be exploited to control female gametophyte specific gene expression in plants, especially in cereals such as rice. Summary ofthe Invention

In one aspect, the present invention provides plant transformation vectors comprising an enhancer element operative in a plant megagametophyte. The enhancer element (a) comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or to the nucleic acid sequence set forth in SEQ ID NO:2; and (b) is operably linked to a nucleic acid sequence of interest. In some embodiments, the vectors of the invention comprise a plurality of enhancer elements operative in a plant megagametophyte, wherein each of the plurality of enhancer elements are operably linked to a common nucleic acid sequence of interest. In one embodiment, the gene of interest encodes a toxin, such as a ribosome inhibiting protein, Diphtheria Toxin A chain, a ribozyme, or an antisense molecule that is complementary to all or part of an mRNA molecule that is normally expressed in a megagametophyte. Expression ofthe toxin may be cytotoxic to the cell (i.e., kill the cell) or cytostatic, such that the cell can not replicate or synthesize new proteins. In another embodiment, the gene of interest encodes a product that stimulates embryogenesis, for example the Leafy Codyledon I (LEC1) gene product and thereby promotes apomixis.

Another aspect of the invention provides transgenic cells and transgenic plants (and plant parts, such as tissue and seeds) comprising a vector ofthe invention, as well as seeds obtained from such transgenic plants. In one embodiment, a transgenic plant is provided that is female sterile as a result of expressing a toxin gene under control of a megagametophyte enhancer. In other embodiments, transgenic, apomictic plants are provided, such as transgenic plants that exhibit embryogenesis in the megagametophyte as a result of expressing a gene stimulating embryogenesis under the control of a megagametophyte-specific enhancer. Another aspect ofthe invention provides methods of expressing a nucleic acid sequence of interest in a megagametophyte of a plant comprising the steps of (a) introducing a vector of the invention into a plant cell; and (b) regenerating a plant from the plant cell, the plant comprising a megagametophyte that expresses a nucleic acid sequence of interest under the control of an enhancer element operative in a plant megagametophyte. It is contemplated that these methods are generally applicable to the expression of structural genes in both monocotyledonous and dicotyledonous plants, such as rice, corn, wheat, cowpea, soybean, canola, and

Arabidopsis. The vectors and methods of the invention can be used to manipulate gene expression in a plant megagametophyte, for example to induce apomixis or to render a plant female sterile, or to otherwise alter the expression of one or more genes in a plant megagametophyte. The methods and materials of the invention can be used to characterize the development, cell biology, physiology and/or biochemistry of a plant megagametophyte.

All literature and patent citations herein are expressly incorporated by reference in their entirety.

Brief Description ofthe Drawings The foregoing aspects and many ofthe attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 shows embryo sac-specific GUS expression of Arabidopsis plant line ET253 flowers at different developmental stages. Flower 1, at floral stage 10 (before megasporophyte); flower 2 and flower 3, at floral stage 11 (megasporophyte to 1-nucleate embryo sac); flower 4, at floral stage early 12; flower 5, at floral stage mid-late 12 (4-8 nucleate embryo sac); flower 6, at floral stage 13 and 14 (mature embryo sac).

FIGURE 2 A shows a map of vector pWY-K 105.1 which is adapted for genetic transformation of Arabidopsis.

FIGURE 2B shows a map of vector pWY-O98.2 which is adapted for genetic transformation of rice.

FIGURE 2C shows a map of plasmid pWY-F68. FIGURE 2D shows a map of plasmid pWY-O93.1. FIGURE 3 shows 5¹ and 3' deletions of the 318 bp upstream sequence of the Ds flanking region in plant line ET253. +, sequence with megagametophyte-specific enhancer (MGSE) activity; -, sequence without megagametophyte-specific enhancer (MGSE) activity. FIGURE 4 shows the specific GUS expression pattern controlled by the megagametophyte-specific enhancer (MGSE) element, having the nucleotide sequence set forth in SEQ ID NO:l, in transgenic Arabidopsis plants. The arrow indicates the GUS stained area where the egg apparatus is located.

FIGURE 5 shows the genomic region (on chromosome 4) covering the MGSE (SEQ ID NO: 1) in Arabidopsis plant line ET253.

FIGURE 6 shows a schematic drawing ofthe 77 bp enhancer element having the nucleic acid sequence set forth in SEQ ID NO:l. The sequence is represented by seven blocks of 10 nucleotides and one block of 7 nucleotides. FIGURE 6 also shows regions of the 77 bp enhancer element, having the nucleic acid sequence set forth in SEQ ID NO:l, that are incorporated into plant transformation vectors identified as pWY-P5.1 through pWY-P5.6. The inserts also contain restriction site recognition sequences to facilitate cloning.

Detailed Description ofthe Preferred Embodiment In one aspect, the present invention provides plant transformation vectors comprising an enhancer element operative in a plant megagametophyte. The enhancer element (a) comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or to the nucleic acid sequence set forth in SEQ ID NO:2; and (b) is operably linked to a nucleic acid sequence of interest. The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.

An "enhancer" is a DNA sequence which stimulates promoter activity.

"A nucleic acid sequence of interest" refers to nucleic acid molecules that are desirably or advantageously expressed in a plant megagametophyte. Examples of "a nucleic acid sequence of interest" include: nucleic acid sequences encoding proteins expressible in at least one type of plant cell; antisense RNAs complementary to at least a portion of one or more genes expressed in plant cells; nucleic acid sequences encoding transcription factors originating from plant cells, or decoys comprising sequences, or analogous sequences, of binding sites for the transcription factors; ribozymes that cleave one or more plant cell mRNAs; and nucleic acid sequences that encode a protein capable of stimulating apomixis (e.g., by stimulating the development of a plant embryo from an egg cell).

"Toxin" or "toxic product" refers to any product that is toxic to the cells in which it is expressed. A toxin can be either cytotoxic or cytostatic to the cell in which it is expressed, such that the cell cannot perform all of its normal functions, such as replicate, grow, differentiate, synthesize new proteins, or the like. The term "toxin" includes, but is not limited to, polypeptide toxins, such as the A chain of Diphtheria toxin, and functional RNAs. A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated and that can be used to alter gene expression. Examples of ribozymes mentioned herein include any ribozymes which can cleave mRNAs for defined proteins to inhibit the translation of these defined proteins, such as hammer-head-type ribozymes, hairpin-type ribozymes, and delta- type ribozymes. A "nucleic acid sequence of interest" that "encodes a protein that stimulates embryogenesis" refers to any nucleic acid sequence (such as a gene) that is capable of stimulating embryogenesis when ectopically expressed in cells other than a zygote (for review see de Vries S.C. Trends Plant Sci. 3:41-452 [1998]). An example of a nucleic acid sequence of interest that encodes a protein that stimulates embryogenesis is the Leafy Cotyledon 1 (LEC1) gene, ectopic expression of which results in spontaneous somatic embryogenesis in leaf cells (Lotan T. et al., Cell 93: 1195-1205 [1998]).

"Reporter" or "marker" genes are used as indicators of gene activity. A reporter gene will typically encode an enzyme activity that is lacking in the host cell or organism which is to be transformed. This allows the measurement or detection of the enzyme activity which may be used as an indicator or "reporter" of the presence of expression of the newly introduced gene. A reporter gene may be put under the influence of a regulatory sequence, such as a promoter or an enhancer element. Successful expression of reporter gene product serves as an indicator of regulatory element activity. An example of a reporter gene that is widely used in plant cells is the β-glucuronidase gene.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. When the sense strand of coding sequence is used, the term "operably linked" means that the promoter sequence is positioned relative to the coding sequence such that the RNA polymerase is capable of initiating transcription of coding sequence from the promoter sequence. In such embodiments it is also preferred to provide appropriate ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences and polyadenylation sequences to produce an RNA transcript which can be translated into protein. When an antisense orientation of the coding sequence is used, all that is required is that the promoter be operably linked to transcribe the antisense strand. Thus, in such embodiments, only transcription start and termination sequences are needed to provide an RNA transcript capable of hybridizing with the mRNA or other RNA transcript from an endogeneous nucleic acid contained within a transformed plant cell. In addition to promoters, other expression regulation sequences, such as enhancers, can be added to the vector to facilitate the expression of a gene of interest.

The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a nucleic acid molecule to hybridize to a target nucleic acid molecule (such as a target nucleic acid molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. Typically, stringent hybridization conditions are no more than 25°C to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex. By way of non-limiting example, representative salt and temperature conditions for achieving stringent hybridization are: 1 M Na⁺ at 65°C; 5X SSC, 0.5% SDS, 5X Denhardt's solution, at 65°C, or equivalent conditions; see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

Tm for nucleic acid molecules greater than about 100 bases can be calculated by the formula Tm = 81.5 + 0.41%(G+C) - log(Na+). For oligonucleotide molecules less than 100 bases in length, exemplary hybridization conditions are 5 to 10°C below Tm. On average, the Tm of a short oligonucleotide duplex is reduced by approximately (500/oligonucleotide length)°C. For example, a 14 base oligonucleotide is hybridized at room temperature, 17 bases at 37°C, 20 bases at 42°C, and 23 bases at 48°C. The abbreviation "SSC" refers to a buffer used in nucleic acid hybridization solutions. One liter ofthe 20X (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.

"Sequence identity" is defined as the percentage of nucleic acid residues in a candidate polynucleotide molecule sequence that are identical with a subject polynucleotide molecule sequence (such as either of the polynucleotide molecule sequences set forth in SEQ ID NO:l and SEQ ID NO: 2), after aligning the sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the sequence identity. The candidate polynucleotide sequence (which may be a portion of a larger polynucleotide sequence) is the same length as the subject polynucleotide sequence, and no gaps are introduced into the candidate polynucleotide sequence in order to achieve the best alignment.

For example, if the 77 bp nucleic acid sequence set forth in SEQ ID NO:l is aligned with a 77 bp portion of a larger DNA molecule (such as a genomic clone), and 80% of the nucleic acid residues in the 77 bp nucleic acid sequence (SEQ ID NO:l) align with the identical nucleic acid residues present in the 77 bp portion of the larger DNA molecule, then the 77 bp portion ofthe larger DNA molecule is 80% identical to the 77 bp nucleic acid sequence set forth in SEQ ID NO: 1.

Nucleic acid sequence identity can be determined in the following manner. The subject polynucleotide molecule sequence is used to search a nucleic acid sequence database, such as the Genbank database (accessible at Website http://www.ncbi.nlm.nih.gov/T3last ), using the program BLASTN version 2.1 (based on Altschul et al., Nucleic Acids Research 25: 3389-3402 (1997)). The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity as defined in Wootton, J.C. and S. Federhen, Methods in Enzymology 266: 554-571 (1996). The default parameters of BLASTN are utilized.

The term "vector" refers to a nucleic acid molecule, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing and translating the insert nucleic acid molecule into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. Many molecules of the polypeptide encoded by the insert nucleic acid molecule can thus be rapidly synthesized. Vectors that are functional in plants are preferably binary plasmids derived from Agrobacterium plasmids. Such vectors are capable of transforming plant cells. Briefly, these vectors typically contain left and right border sequences that are required for integration into the host (plant) chromosome. Typically, between these border sequences is the nucleic acid molecule (such as a cDNA) to be expressed under control of a promoter. In some embodiments, a selectable marker and a reporter gene are also included. The vector also may contain a bacterial origin of replication.

Enhancer elements useful in the practice of the present invention are operative in a plant megagametophyte (preferred enhancers are operative exclusively in a plant megagametophyte) and comprise a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or to the nucleic acid sequence set forth in SEQ ID NO:2. In some embodiments, enhancer elements useful in the practice ofthe present invention are at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO:l or to the nucleic acid sequence set forth in SEQ ID NO:2. In other embodiments, enhancer elements useful in the practice ofthe present invention are at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or to the nucleic acid sequence set forth in SEQ ID NO:2.

Additional enhancer elements useful in the practice of the present invention are isolated by using a variety of cloning techniques known to those of ordinary skill in the art. For example, nucleic acid molecules having the sequences set forth in SEQ ID NO:l or SEQ ID NO:2 can be used as hybridization probes utilizing, for example, the technique of hybridizing radiolabeled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E.F. Fritsch and T. Maniatis eds, the cited pages of which are incorporated herein by reference. The hybridization probes may be labeled with appropriate reporter molecules. Exemplary means for producing specific hybridization probes include oligolabeling, nick translation, end-labelling or PCR amplification using a labeled nucleotide. Appropriate hybridization conditions can be readily calculated by one of ordinary skill in the art as described supra in the discussion of the term "hybridize under stringent conditions". Oligonucleotides for hybridization screening may be designed based on the DNA sequence ofthe enhancer elements of SEQ ID NO:l or SEQ ID NO:2 herein.. Oligonucleotides for screening are typically at least 11 bases long and more usually at least 20 or 25 bases long. In one embodiment, the oligonucleotide is 20-30 bases long. Such an oligonucleotide may be synthesized in an automated fashion. To facilitate detection, the oligonucleotide may be conveniently labeled, generally at the 5' end, with a reporter molecule, such as a radionuclide, (e.g., ^^P), enzymatic label, protein label, fluorescent label, or biotin. A library is generally plated as colonies or phage, depending upon the vector, and the recombinant DNA is transferred to nylon or nitrocellulose membranes.

Hybridization conditions are tailored to the length and GC content of the oligonucleotide. Oligonucleotides for hybridization are typically at least 11 bases long, generally less than 100 bases long, and preferably at least 15 bases long, such as at least 20 bases long, or at least 25 bases long, and preferably 20-70, 25-50, or 30- 40 bases long. Washing is initially performed at the same conditions as hybridization. If the background is unacceptably high, washing temperature is increased a few degrees until background is acceptable.

Following denaturation, neutralization, and fixation of the DNA to the membrane, membranes are hybridized with labeled probe. Suitable hybridization conditions may be found in Sambrook et al., supra, Ausubel et al., supra, and furthermore hybridization solutions may contain additives such as tetramethylammonium chloride or other chaotropic reagents or hybotropic reagents (e.g., ammonium trichloroacetate; see for example, WO 98/13527) to increase specificity of hybridization. Following hybridization, suitable detection methods reveal hybridizing colonies or phage that are then isolated and propagated. Candidate clones or amplified fragments may be verified as containing a desired enhancer element by any of various means. For example, the candidate clones may be hybridized with a second, non-overlapping probe or subjected to DNA sequence analysis. Again, by way of example, enhancer elements useful in the practice of the present invention can be isolated by the polymerase chain reaction (PCR) described in The Polymerase Chain Reaction (K. B. Mullis et al., eds., Birkhauser Boston [1994]), incorporated herein by reference. Template genomic DNA can be obtained from any plant species, such as from rice, corn, wheat, cowpea, soybean, canola, and Arabidopsis. An exemplary method for extracting plant geneomic DNA suitable for use as a PCR template is the CTAB protocol set forth in Kleinhofs et al. Theoretical and Applied Genetics (1993) 86: 705-712.

By way of non-limiting example, representative PCR reaction conditions for amplifying enhancer elements useful in the practice ofthe present invention (such as amplifying sequences from plant genomic DNA) are as follows. The following reagents are mixed in a tube (on ice) to form the PCR reaction mixture: DNA template (e.g., up to 1 μg genomic DNA, or up to 0.1 μg cDNA), 0.1-0.3 mM dNTPs, 10 μl 10 X PCR buffer (10 X PCR buffer contains 500 mM KC1, 15mM MgCL₂, 100 mM Tris-HCl, pH 8.3), 50 pmol of each PCR primer (at least one of the PCR primer pair should correspond to a portion of one or both of the nucleic acid sequences set forth in SEQ ID NO:l or SEQ ID NO: 2 and be greater than 20 bp in length), 2.5 units of Taq DNA polymerase (Perkin Elmer, Norwalk, CT) and deionized water to a final volume of 50 μl. The tube containing the reaction mixture is placed in a thermocycler and a thermocycler program is run as follows. Denaturation at 94°C for 2 minutes, then 30 cycles of: 94°C for 30 seconds, 47°C to 55°C for 30 seconds, and 72°C for 30 seconds to two and a half minutes. Additionally, enhancers useful in the practice of the present invention can be synthesized in an automated fashion.

Regulatory element function (such as function of an enhancer element operative in a plant megagametophyte) during expression of a reporter gene under its regulatory control can be tested at the transcriptional stage using DNA-RNA hybridization assays (for example, by "Northern" blots or in situ hybridization), or at the translational stage using specific functional assays for the protein synthesized (for example, by enzymatic activity or by immunoassay ofthe protein). Nucleotide sequence variants of enhancer elements operative in a plant megagametophyte are useful in the practice of the present invention provided that they retain megagametophyte enhancer activity. Nucleotide sequence variants of enhancer elements refer to nucleic acid molecules with some differences in their sequences as compared to the corresponding, native, i.e., naturally-occurring, nucleic acid molecules. Ordinarily, the variants will possess at least about 70% identity with the corresponding native sequences, and preferably, they will be at least about 80% identical to the corresponding, native sequences. The nucleic acid sequence variants falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants may be used to attain desired enhanced or reduced activity, or altered temporal and spatial patterns of activity. Such sequence variants can be generated by a variety of art-recognized techniques.

By way of non-limiting example, the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech (Palo Alto, CA), may be employed for introducing site-directed mutations into enhancer elements useful in the practice ofthe present invention. Following denaturation ofthe target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage ofthe two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis ofthe initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be fully sequenced or restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J.T. Baker, Sanford, ME) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).

Again, by way of non-limiting example, the two primer system utilized in the QuikChange™ Site-Directed Mutagenesis kit from Stratagene (LaJolla, California), may be employed for introducing site-directed mutations into enhancer elements useful in the practice of the present invention. Double-stranded plasmid DNA, containing the insert bearing the target mutation site, is denatured and mixed with two oligonucleotides complementary to each of the strands of the plasmid DNA at the target mutation site. The annealed oligonucleotide primers are extended using Pfu DNA polymerase, thereby generating a mutated plasmid containing staggered nicks. After temperature cycling, the unmutated, parental DNA template is digested with restriction enzyme Dpnl which cleaves methylated or hemimethylated DNA, but which does not cleave unmethylated DNA. The parental, template DNA is almost always methylated or hemimethylated since most strains of E. coli, from which the template DNA is obtained, contain the required methylase activity. The remaining, annealed vector DNA incorporating the desired mutation(s) is transformed into E. coli.

As described above, the vectors ofthe invention include an enhancer element operative in a plant megagametophyte operably linked to a nucleic acid sequence of interest. The vectors of the invention can include a multiplicity of enhancer elements, each of which may be utilized in either possible orientation. Further, in the embodiments of the invention utilizing a multiplicity of enhancer elements, the enhancer elements utilized do not all have to be identical, and, indeed, each can have a different nucleic acid sequence from the other enhancer elements incorporated within the vector.

In one embodiment, the nucleic acid sequence of interest encodes a toxin that is toxic to the cells in which it is expressed. Expression of this toxic product results in ablation ofthe megagametophyte cells and/or the prevention of megagametophyte development. The toxic product can be, for example, an RNA or a polypeptide. Toxic polypeptides include but are not limited to: Diphtheria Toxin A-chain (DT-A), which inhibits protein synthesis, (Greenfield et al., Proc. Natl. Acad., Sci.:USA 80:6853 [1983]; Palmiter et al., Cell 50:435 [1987]); Pectate lyase pelE from Erwinia chrysanthemi EC 16, which degrades pectin, causing cell lysis (Keen et al., J. Bacteriol. 168:595 [1986)]); T-urfl3 (TURF-13) from cms-T maize mitochondrial genomes, which encodes a polypeptide designated URF13 which disrupts mitochondrial or plasma membranes (Braun et al., Plant Cell 2: 153 [1990]; Dewey et al., Proc. Natl. Acad Sci.:USA 84:5374 [1987]; Dewey et al., Cell 44:439 [1986]); Gin recombinase from phage Mu, a site-specific DNA recombinase which will cause genome rearrangements and loss of cell viability when expressed in cells of plants (Maeser et al., Mol Gen. Genet. 230:170-176 [199]); Indole acetic acid-lysine synthetase (iaaL) from Pseudomonas syringae, an enzyme that conjugates lysine to indoleacetic acid (IAA) (when expressed in the cells of plants, it causes altered development due to the removal of IAA from the cell via conjugation). (Romano et al., Genes & Dev. 5:438-446 [1991]; Spena et al., Mol Gen. Genet. 227:205-212 [1991]; Roberto et al., Proc. Natl. Acad Sci.:USA 87:5795-5801 [1990]); and CytA toxin gene from Bacillus thuringiensis Israeliensis, which encodes a protein that is mosquitocidal and hemolytic (when expressed in plant cells, it causes death of the cell due to disruption ofthe cell membrane) (McLean et al., J. Bacteriol., 169:1017- 1023 [1987]; U.S. Pat. No. 4,918,006). Other useful peptide toxins include ricin, exotonin A, and Herpes viridae thymidine kinase (Evans, G. A., Genes & Dev. 3:259-263 (1989)). In addition, a fungal ribonuclease may be used to cause male sterility in plants (Mariani, C. et al., Nature 347:737-741 [1990]).

Alternatively, the nucleic acid sequence of interest can produce a functional RNA that may, in turn, mediate control of gene expression by antisense, co- suppression or other mechanisms. For example, it is a well known plant phenomenon that the addition of extra copies of wild-type genes may result in co- suppression of all copies of the gene, both endogeneous and exogeneous. Again, by way of example, antisense RNA can be utilized which will hybridize with mRNA from a gene which is critical to megagametophyte development or fertility. In this manner, the anti-sense RNA will prevent expression of the necessary gene(s) resulting in aberrant megagametophyte development or sterility.

An anti-sense nucleic acid molecule is a DNA sequence that is inverted relative to its normal orientation for transcription and so expresses an RNA transcript that is complementary to a target mRNA molecule expressed within the host cell (i.e., the RNA transcript of the anti-sense nucleic acid molecule can hybridize to the target mRNA molecule through Watson-Crick base pairing). An anti-sense nucleic acid molecule may be constructed in a number of different ways provided that it is capable of interfering with the expression of a target gene. The anti-sense nucleic acid molecule can be constructed by inverting the coding region (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement, hence the RNAs encoded by the anti-sense and sense gene are complementary.

The anti-sense nucleic acid molecule generally will be substantially identical to at least a portion ofthe target gene or genes. The sequence, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter anti-sense nucleic acid molecule. The anti-sense nucleic acid molecule generally will be substantially identical (although in antisense orientation) to the target gene. The minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression ofthe endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred.

Furthermore, the anti-sense nucleic acid molecule need not have the same intron or exon pattern as the target gene, and non-coding segments ofthe target gene may be equally effective in achieving anti-sense suppression of target gene expression as coding segments. Normally, a DNA sequence of at least about 30 or 40 nucleotides should be used as the anti-sense nucleic acid molecule, although a longer sequence is preferable. Alternately, ribozymes can be utilized, such as ribozymes which target mRNA from a gene which is involved in megagametophyte development. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the antisense constructs.

Ribozymes useful in the practice of the invention typically comprise a hybridizing region of at least about nine nucleotides which is complementary in nucleotide sequence to at least part ofthe target RNA and a catalytic region which is adapted to cleave the target RNA (see, e.g., EPA No. 0 321 201; WO88/04300; Haseloff & Gerlach, Nature 334:585-591 [1988]; Fedor & Uhlenbeck, Proc. Natl. Acad Sci: USA 87:1668-1672 [1990]; Cech & Bass, Ann. Rev. Biochem. 55:599-629 [1986]).

In some embodiments of the vectors of the invention, the gene of interest is one whose expression in the megagametophyte causes apomixis. The apomictic plants of this invention can be created, for example, by expressing an embryogenesis- inducing gene in megagametophytes, or, for example, by prolonging the embryonic state in megagametophytes.

An example of an embryogenesis-inducing gene is Leafy Cotyledonl (LEC1). Ecoptic expression of LEC1 results in spontaneous somatic embryogenesis in leaves (Lotan T. et al., Cell 93: 1195-1205 [1998]). In this embodiment of the invention, the expression of an embryogenesis-inducing gene under the control of a megagametophyte-specific regulatory element initiates an embryonic program of development in the megagametophyte leading to apomixis.

Apomictic plants of this invention can also be created by delaying the switch from an embryonic program into an adult program of development in megagametophytes. For example, inactivation of the pickle, clavatal or primordia timing genes produces an enhanced somatic embryo phenotype, probably as a consequence of retaining of embryo-characteristic traits during later development (for review see de Vries S.C. Trends Plant Sci. 3:41-452 [1998]). Therefore, in one embodiment, apomictic plants are created by inactivating the function of genes such as pickle in the megagametophyte, for example by antisense technology as described above.

The vectors of the present invention can also include other regulatory sequences, such as promoters, translation leader sequences, introns, and polyadenylation signal sequences. Indeed, the vectors of the invention include at least a minimal promoter operably linked to an enhancer. "Promoter" refers to a DNA sequence involved in controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The term "promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. The plant vectors of the invention can be constructed using conventional techniques well known to those skilled in the art. The choice of vector is dependent upon the method that will be used to transform host plants and the desired selection markers. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the vector (for details of exemplary expression vectors for transformation of Arabidopsis thaliana, see EXAMPLE 2 herein).

The construction of plant vectors is facilitated by the use of a shuttle vector which can be manipulated and selected in both plant and a convenient cloning host such as a prokaryote. Such shuttle vectors thus can include a gene for selection in plant cells (e.g. kanamycin resistance) and a gene for selection in a bacterial host (e.g. actinomycin resistance). Such shuttle vectors also contain an origin of replication appropriate for the prokaryotic host used and preferably at least one unique restriction site or polylinker containing unique restriction sites to facilitate vector construction. Examples of such shuttle vectors include pMON530 (Rogers et al., Methods in Enzymology 153: 253-277 [1988]) and pCGN1547 (McBride et al., Plant Molecular Biol. 14: 269-276 [1990]).

As will be apparent to those skilled in the art, any vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used in the practice of the invention. The vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Vectors typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). However, many other suitable vectors are available as well. These vectors may contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.

The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the DNA of interest utilize standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press [1989]). The recombinant DNA molecules of the invention may be prepared by manipulating the various elements to place them in proper orientation. Thus, adapters or linkers may be employed to join the DNA fragments. Other manipulations may be performed to provide for convenient restriction sites, removal of restriction sites or superfluous DNA. These manipulations can be performed by art-recognized methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press [1989].

Prokaryotes may also be used as host cells for routine genetic manipulation and/or construction of enhancer elements and vectors useful in the practice of the invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JMIOI, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are generally transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, W.J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp. (1990); and Hanahan et ., Meth. Enzymol, 204:63 (1991). In other aspects, the present invention provides cells (such as plant cells, including megagametophyte cells), plant tissue, fruit, seeds, plants (and selfed or hybrid progeny and any descendant of such a plant), that include one or more vectors of the invention. The invention also provides methods of expressing a nucleic acid sequence of interest in a megagametophyte of a plant, the methods comprising the steps of: (a) introducing a vector of the invention into a plant cell; and (b) regenerating a plant from the plant cell, the plant comprising a megagametophyte that expresses the nucleic acid sequence of interest under the control of the enhancer element.

Plant vectors of the invention can be introduced into plant cells using techniques well known to those skilled in the art. These methods include, but are not limited to, (1) direct DNA uptake, such as particle bombardment or electroporation (see, Klein et al., Nature 327:70-73 [1987]; U.S. Pat. No. 4,945,050), and (2) Agrobacterium-medi&ted transformation (see, e.g., U.S. Patent Serial Numbers: 6,051,757; 5,731,179; 4,693,976; 4,940,838; 5,464,763; and 5,149,645). Within the cell, the transgenic sequences may be incorporated within the chromosome. The skilled artisan will recognize that different independent insertion events may result in different levels and patterns of gene expression (Jones et al., EMBO J. 4:2411-2418 [1985]; De Almeida et al., MGG 218:78-86 [1989]), and thus that multiple events may have to be screened in order to obtain lines displaying the desired expression level and pattern. Transgenic plants can be obtained, for example, by transferring vectors that include a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature, 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices, or other tissues or cells, of the plant to be transformed as described by An et al., Plant Physiology, 81:301-305 (1986). Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens.

Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, for example, kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Florida [1993], incorporated by reference herein). Representative examples include electroporation-facilitated DNA uptake by protoplasts in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA (Rhodes et al., Science, 240:204-207 [1988]); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology, 13:151-161 [1989]); and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the cell wall (Klein et al., Plant Physiol. 91:440-444 [1989] and Boynton et al., Science, 240(4858): 1534-1538 [1988]). A method that has been applied to Rye plants (Secale cereale) is to directly inject plasmid DNA, including a selectable marker gene, into developing floral tillers (de la Pena et al., Nature 325:274-276 (1987)). Further, plant viruses can be used as vectors to transfer genes to plant cells. Examples of plant viruses that can be used as vectors to transform plants include the Cauliflower Mosaic Virus (Brisson et al., Nature 310: 511-514 (1984); Other useful techniques include: site-specific recombination using the Crellox system (see, U.S. Patent Serial No. 5,635,381); and insertion into a target sequence by homologous recombination (see, U.S. Patent Serial No. 5,501,967). Additionally, plant transformation strategies and techniques are reviewed in Birch, R.G., Ann Rev Plant Phys Plant Mol Biol, 48:297 (1997); Forester et al., Exp. Agric, 33:15-33 (1997). The aforementioned publications disclosing plant transformation techniques are incorporated herein by reference, and minor variations make these technologies applicable to a broad range of plant species. Positive selection markers may also be utilized to identify plant cells that include a vector of the invention. For example, U.S. Patent Serial Numbers 5,994,629, 5,767,378, and 5,599,670, describe the use of a beta- glucuronidase transgene and application of cytokinin-glucuronide for selection, and use of mannophosphatase or phosphmanno-isomerase transgene and application of mannose for selection.

The cells which have been transformed may be grown into plants by a variety of art-recognized means. See, for example, McConnick et al., Plant Cell Reports 5:81-84(1986) These plants may then be grown, and either selfed or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

The following are representative plant species that are suitable for genetic manipulation in accordance with the present invention. The citations are to representative publications disclosing genetic transformation protocols that can be used to genetically transform the listed plant species. Rice (Alam, M.F. et al., Plant Cell Rep. 18: 572-575 [1999]); maize (U.S. Patent Serial Nos. 5,177,010 and 5,981,840); wheat (Ortiz, J.P.A., et al., Plant Cell Rep. 15: 877-881 [1996]); tomato (U.S. Patent Serial No. 5,159,135); potato (Kumar, A., et al., Plant J. 9: 821-829 [1996]); cassava (Li, H-Q., et al., Nat. Biotechnology 14: 736-740 [1996]); lettuce (Michelmore, R., et al., Plant Cell Rep 6:439-442 [1987]); tobacco (Horsch, R.B., et al., Science 111: 1229-1231 [1985]); cotton (U.S. Patent Serial Nos. 5,846,797 and 5,004,863); grasses (U.S. Patent Serial Nos. 5,187,073 and 6.020,539); peppermint (X. Niu et al., Plant Cell Rep. 17:165-171 [1998]); citrus plants (Pena, L. et al., Plant Sci. 104: 183-191 [1995]); caraway (F.A. Krens, et al., Plant Cell Rep.,11: 39-43 [1997]); banana (U.S. Patent Serial No. 5,792,935; soybean (U.S. Patent Serial Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Patent Serial No. 5,952,543); poplar (U.S. Patent Serial No. 4,795,855); monocots in general (U.S. Patent Serial Nos. 5,591,616 and 6,037,522); brassica (U.S. Patent Serial Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Patent Serial No. 6,074,877).

The vectors used in this inventions are introduced into plant cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Progeny from the transformed plants may be tested to insure that the DNA package has been successfully integrated into the plant genome. The presence ofthe stably integrated elements into the transformed parent plants may be ascertained, for example, by southern hybridization techniques or PCR analysis, known in the art.

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

EXAMPLE 1 Characterization of Plant Line ET253 This example shows the characterization of Arabidopsis plant line ET253 which expresses a GUS reporter gene in the early stages of female gametophyte development.

Line ET253 is constructed as reported in Sundaresan et al. (Genes & Dev. 9: 1797-1810, 1995). Briefly, a two-element transposon system is used in which Arabidopsis plants containing an immobilized Ac element are crossed to plants carrying a Ds element. The Ac element comprises a CaMV 35S promoter regulating an Ac transposase. The transposase generates excision of Ds elements in trans, but cannot transpose itself. The Ds element contains a selectable marker and a GUS reporter gene whose expression is dependent on its insertion near a genomic enhancer sequence. Lines are screened by staining for GUS. Line ET253 is chosen because GUS staining is confined to embryonic cells and associated with ovule development.

GUS staining of ET253 flowers at different developmental stages reveals that GUS expression is not only specific to megagametophyte (embryo sac), but also associated with the stages of ovule development (FIGURE 1). The GUS activity is detectable as early as stage 11 (gynoecium length is about 0.5-1.0 mm and at around 1 -nucleate embryo sac stage). With the development of the ovule, the embryo sac elongates and the intensity of GUS staining increases but the GUS staining is mainly located at the egg apparatus. This covers stages 12 and 13. After fertilization (stage 14), the GUS activity decreases and no GUS expression is observed in mature seeds. This pattern indicates that the GUS expression in ET253 is restricted to the early stages of female gametophyte development.

EXAMPLE 2 Transformation Vectors for Testing Enhancer Activity This Example describes plant transformation vectors used to test the tissue specificity of plant enhancer elements.

Vectors pWY-K105.1 and pWY-O98.2 are shown in FIGURE 2A and 2B, respectively. Vector pWY-K 105.1 is specifically designed for testing tissue specific cis-regulatory elements in Arabidopsis, while vector pWY-O98.2 is specifically designed for testing tissue specific cis-regulatory elements in rice. pWY-K105.1 is constructed as follows: A 1.2 kb EcoRI fragment containing 1' promoter-iαr-35S terminator sequence is isolated from the plasmid pl'barbi (Mengiste et iλ.flant J., 12:945-948 [1997]) and inserted into the EcøRI site of the multi-cloning sites in pWY-K35.1 (a vector based on pCAMBIA1281Z with the CaMV 35S promoter driving the gusA gene replaced by a 65 bp minimal promoter, which is derived from the CaMV 35S promoter, between the HindJSL and Ncol sites) to generate pWY-K95.4, which has the bar gene in the same orientation as the gusA gene. pWY-K95.4 is then digested with Xhol and BsfXl to remove the CaMV 35S promoter-λyg sequence and treated to create blunt ends. The digested plasmid is then re-circularized to generate pWY-K105.1. pWY-O98.2 is constructed by replacing the bar gene in pWY-K105.1 with the hyg gene.

In both vectors, the gusA gene is driven by a minimal promoter but cannot be expressed without an enhancer element. Insertion of a DNA fragment with enhancer activity into the multi-cloning site leads to GUS expression in accordance with the expression pattern ofthe enhancer. The use ofthe 1' promoter in driving bar gene or hyg gene for selection eliminates the interference from non-specific GUS expression due to trans-activation by the strong CaMV 35S enhancer. All the deletion tests and enhancer function tests reported in the subsequent examples are carried out using one or other of these two vectors. Additionally, FIGURE 2C shows a map of plasmid pWY-F68 which is also useful (in addition to pWY-K105.1) for testing enhancer activity of plant genomic DNA fragments. FIGURE 2D shows a map of plasmid pWY-O93.1 which is useful for testing the ability ofthe MGSE having the nucleic acid sequence set forth in SEQ ID NO: 1 to drive embryo sac specific green fluorescent protein (GFP) expression, and therefore it has the gfp gene as a replacement for the gusA gene. EXAMPLE 3 A Megagametophyte-Specific Enhancer (MGSE is Located Directly Upstream ofthe Ds Insertion Site in Line ET253 This example describes the isolation and deletion analysis of a 77 bp genomic DNA fragment (SEQ ID NO: 1) that possesses megagametophyte-specific enhancer activity in Arabidopsis.

A large fragment (11.5 kb) of genomic DNA flanking the Ds insertion site in line ET253 is obtained by inverse amplification performed according to the following protocol. Genomic DNA is digested with HwdUI (or other appropriate restriction enzymes) overnight in 30 μl volume. The digested DNA is then purified by heating to 65 ° C for 20 minutes and extracting twice with phenol/chloroform (1 : 1) to remove Hrød ϋJ and other components. The supernatant is transferred to a new Eppendorf tube to which 1/10 (v/v) of 3M sodium acetate (pΗ 5.2) and 2 volume of 100% ethanol is added to precipitate the DNA. The DNA is recovered by centrifugation at 12,000 x g for 10 minutes and washed once with 70% ethanol. The pellet is briefly dried and then resuspended in Η₂O to a final concentration of approximately 2 μg/ml. To circularize the digested DNA, 20 μl of 10^χDNA ligase buffer and 2 μl of T DNA ligase (400units/μl, from NEB) is added and incubated overnight at 16 ° C. Following incubation, the reaction is heated to 75 ° C for 10 min to inactivate the ligase. 20 μl of sodium acetate (pH 5.2) and 400 μl of 100 % ethanol is added to the reaction, which is then chilled to -80° C for 15 minutes to precipitate DNA. DNA is collected by centrifugation at 12,000 x g for 10 minutes and washed once with 70% ethanol. The DNA pellet is briefly dried and then resuspended in 20 μl of TE (pH 8.0). Amplification is performed using a GeneAmp® XL PCR kit. The amplification reaction contains 6 μl of circularized; 15 μl of 3.3 XL buffer JJ; 4 μl of 10 mM dNTPs; 7 μl of 25 mM Mg(OAC)_2; 1 μl of 10 μM primer 1 (5'-CCA CGA TGC AAA TAT ATC GAT AAC G (SEQ ID NO:3)); 1 μl of 10 μM primer 2 (5'- ATT AAT CTT GGG GTA ACT TTA CTT C (SEQ ID NO:4)); 16 μl of H₂O to make a final volume of 50 μl. Amplification times are as follows: 1 cycle of 94 ° C for 3 min; 30-35 cycles of 94° C for 0.5 min, 58 ° C for 5 min, and 72 ° C for 10 min. An approximately 2.1 kb fragment that contains the enhancer is isolated.

Deletions of the 5' and 3' ends of the fragment are made and tested for enhancer activity in order to define a smaller region containing the enhancer. A 5' deletion test initially narrows down the region containing enhancer activity to a 318 bp sequence directly upstream of the Ds insertion site. A series of further 5' and 3' deletions are generated by amplification. The nucleic acid sequences of the primers used in amplification are shown in Table 1. Table 1

The combination of primers used to construct the deletion fragments shown in FIGURE 3 are set forth in Table 2.

Table 2

Each primer has a H dlJJ site at the 5' end to facilitate cloning. The amplified products are digested with H/^'wdlJI and cloned into the HwdHJ site in pWY-K105.1 (as shown in FIGURE 2A) to generate the 5' and 3' deletion constructs. The constructs are then tested for enhancer activity. The enhancer activity of each ofthe tested constructs is set forth in FIGURE 3.

The 77 bp fragment (5Δ77, FIGURE 3) (SEQ ID NO:l) is sufficient in conferring MGSE activity. This result is confirmed by deletion of 68 bp from the 3 'end of the 318 bp fragment, which abolishes MGSE activity completely (3Δ68, FIGURE 3). EXAMPLE 4

Basic Features ofthe MGSE A typical GUS expression pattern, in transgenic Arabidopsis plants, controlled by the MGSE having the nucleic acid sequence shown in SEQ ID NO:l is shown in FIGURE 4. The MGSE having the nucleic acid sequence shown in SEQ ID NO:l possesses the following features: 1) single copy in the genome according to Southern analysis; 2) enhancer activity is position- and orientation-independent, and 3) in tandem repeat (2x) it gives much stronger activity in driving embryo sac specific GUS expression.

This MGSE (SEQ ID NO: 1) is located at the top arm of chromosome 4, as shown in FIGURE 5. It is mapped by BLAST sequence similarity analysis of Arabidopsis sequences. The matching contig is F19F18. Genes located close to the MGSE (SEQ ID NO:l) are: upstream region— two peroxidase genes atpH21-l and atpH21-2, 2.1 kb and 5.2 kb away, respectively; downstream regio — a gene coding for a predicted protein, 1.4 kb away. Southern analysis shows that the gene encoding the predicted protein is a multi-copy gene (3 copies) but the region harboring the two peroxidase genes is unique in the genome. Southern analysis is performed using the 318 bp fragment (shown in FIGURE 3) and probing genomic DNA digested with either Ncol + Ndel, neither of which cut the 318 bp fragment, or E øRI, which cuts once inside the fragment. When digested with Ncol + Ndel, a single 535 bp fragment is observed; when digested with EcøRI, two fragments, approximately 5440 and 2080 bp, are observed.

Northern blotting of mRNA isolated from different tissues shows that the predicted protein-encoding mRNA is expressed mainly in floral tissues (gynoecium and other floral tissues) and trace amount can be seen in leaf tissue. However, the peroxidase gene atpH21-l related mRNA exists in all tissues examined (gynoecium, other floral tissues and leaf). These results suggest that the predicted protein- encoding gene is likely to be associated with the MGSE (SEQ ID NO:l). Interestingly, the 170 bp sequence directly downstream of the Ds insertion site is highly homologous to many sequences on all five chromosomes in different locations (more than 30 such sequences by BLAST search) and is quite likely to be a MITE- like transposable element. Therefore, the MGSE element (SEQ ID NO:l) is also possibly associated with this MITE-like transposable element.

EXAMPLE 5 Test of MGSE Function

This example describes the enhancer activity of fragments of the 77 bp enhancer element having the nucleic acid sequence set forth in SEQ ID NO: 1.

To further characterize the enhancer activity of the 77 bp enhancer element having the nucleic acid sequence set forth in SEQ ID NO:l, fragments of the 77 bp enhancer element (SEQ ID NO: 1) are cloned into plasmid pWY-K105.1.

Six pairs of either 30 base or 27 base oligomers that overlap by 20 bases are designed and synthesized with restriction sites at either end to facilitate cloning. The nucleic acid sequences of these oligomers are set forth in Table 3.

Table 3

When annealed these oligomers have a Sαcl-compatible overhang at the 5' end and a H/HdIU-compatible overhang at the 3 -end. The annealed oligomers are inserted into a Sacl - HϊwdJuT digested pWY-K105.1 vector to generate constructs pWY-P5.1 through pWY-P5.6. These constructs are used to transform Arabidopsis by a floral dip method (see, Clough and Bent The Plant J. 16, 735-743, [1998]; except that Basta (100 mg L) is used for the selection of transformants carrying the bar gene.) The Agrobacterium strain used for all transformation is EHA105. Reporter activity is assayed in floral parts. Of the six fragments shown in FIGURE 6, only the fragment cloned into pWY-P5.3 shows megagametophyte- specific enhancer activity. The sequence of this fragment is set forth in SEQ ID NO:2.

Tobacco is transformed essentially according to the protocol in: Svab Z, Hajdukiewicz P, Maliga P (1995). Generation of transgenic tobacco plants by cocultivation of leaf disks with Agrobacterium pPZP binary vectors. In "Methods in Plant Molecular Biology-A Laboratory Manual", P. Maliga, D. Klessig, A. Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press, except that lmg L bialaphos is used to select for transformants.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope ofthe invention.

Claims

The embodiments ofthe invention in which an exclusive property or privilege is claimed are defined as follows:

1. A plant transformation vector comprising an enhancer element operative in a plant megagametophyte, wherein said enhancer element

(a) comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:l or to the nucleic acid sequence set forth in SEQ ID NO:2; and

(b) is operably linked to a nucleic acid sequence of interest.

2. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.

3. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO:l.

4. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.

5. A plant transformation vector of Claim 1, wherein said enhancer element comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

6. A plant transformation vector of Claim 1, wherein said enhancer element consists ofthe nucleic acid sequence set forth in SEQ ID NO: 1.

7. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:2.

8. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO:2.

9. A plant transformation vector of Claim 1, wherein said enhancer element comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 2.

10. A plant transformation vector of Claim 1, wherein said enhancer element comprises the nucleic acid sequence set forth in SEQ ID NO:2.

11. A plant transformation vector of Claim 1, wherein said enhancer element consists ofthe nucleic acid sequence set forth in SEQ ID NO:2.

12. A plant transformation vector comprising an enhancer element operative in a plant megagametophyte, wherein said enhancer element:

(a) comprises a nucleic acid sequence that hybridizes under stringent conditions to the nucleic acid sequence set forth in SEQ ID NO:l, or to the complement ofthe nucleic acid sequence set forth in SEQ ID NO: 1; and

(b) is operably linked to a nucleic acid sequence of interest.

13. A plant transformation vector comprising an enhancer element operative in a plant megagametophyte, wherein said enhancer element:

(a) comprises a nucleic acid sequence that hybridizes under stringent conditions to the nucleic acid sequence set forth in SEQ ID NO:2, or to the complement ofthe nucleic acid sequence set forth in SEQ ID NO:2; and

(b) is operably linked to a nucleic acid sequence of interest.

14. A plant transformation vector of Claim 1, 2, 7, 12 or 13, said enhancer element being operative in the megagametophyte of a plant species selected from the group consisting of rice, corn, wheat, cowpea, soybean, canola, and Arabidopsis.

15. A plant transformation vector of Claim 1 comprising a plurality of enhancer elements operative in a plant megagametophyte, wherein each of said plurality of enhancer elements:

(a) comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or to the nucleic acid sequence set forth in SEQ ID NO:2; and

(b) is operably linked to a common nucleic acid sequence of interest.

16. A plant transformation vector of Claim 1, 2, 7, 12, 13 or 15, wherein said nucleic acid sequence of interest encodes a toxin.

17. A vector of Claim 16 wherein said toxin is a ribosome inhibiting protein.

18. A vector of Claim 16 wherein said toxin is Diphtheria Toxin A chain.

19. A plant transformation vector of Claim 1, 2, 7, 12, 13 or 15, wherein said nucleic acid sequence of interest codes for an RNA molecule that is at least 80% identical to the complement of a messenger RNA molecule that is normally expressed in a plant megagametophyte.

20. A plant transformation vector of Claim 1, 2, 7, 12, 13 or 15, wherein said nucleic acid sequence of interest codes for a ribozyme.

21. A plant transformation vector of Claim 1, 2, 7, 12, 13 or 15, wherein said nucleic acid sequence of interest encodes a protein that stimulates embryogenesis.

22. A plant transformation vector of Claim 21, wherein said nucleic acid sequence of interest is Leafy Cotyledon 1.

23. A cell comprising a vector of any one of Claims 1, 2, 7, 12, 13 or 15.

24. A plant cell of Claim 23.

25. A plant cell of Claim 24, wherein said plant cell is a megagametophyte.

26. A transgenic plant comprising a vector of any of Claims 1, 2, 7, 12, or 15.

27. A seed comprising a vector of any of Claims 1, 2, 7, 12, or 15.

28. A method of expressing a nucleic acid sequence of interest in a megagametophyte of a plant, said method comprising:

(a) introducing a vector of any one of Claims 1, 2, 7, 12, 13 or 15 into a plant cell; and (b) regenerating a plant from said plant cell, said plant comprising a megagametophyte that expresses the nucleic acid sequence of interest under the control of said enhancer element.

29. A method of Claim 28, wherein said nucleic acid sequence of interest encodes a toxin.

30. A method of Claim 29, wherein said toxin is a ribosome inhibiting protein.

31. A method of Claim 29, wherein said toxin is Diphtheria Toxin A chain.

32. A method of Claim 28, wherein said nucleic acid sequence of interest codes for an RNA molecule that is at least 80% identical to the complement of a messenger RNA molecule normally expressed in a plant megagametophyte.

33. A method of Claim 28, wherein said nucleic acid sequence of interest codes for a ribozyme.

34. A method of Claim 28, wherein said nucleic acid sequence of interest encodes a protein that stimulates embryogenesis.

35. A method of Claim 28, wherein said nucleic acid sequence of interest is Leafy Cotyledon 1.

36. A method of Claim 28, wherein said plant cell is from a plant species selected from the group consisting of rice, corn, wheat, cowpea, soybean, canola, and Arabidopsis.