CA2401858C - Embryo sac-specific genes - Google Patents

Abstract

The present invention relates to isolated nucleotide sequences useful for the production of plants with a modified embryo sac, embryo and/or endosperm development, e.g. female sterility, parthenogenetic embryo and/or autonomous endosperm development.

Description

Embryo Sac-Specific Genes Description The present invention relates to isolated nucleo-tide sequences useful for the production of plants with a modified embryo and/or endosperm develop-ment, to vectors containing the nucleotide se-quences, to proteins encoded by the nucleotide se-quences, to methods for obtaining the nucleotide sequences, to methods for isolating embryo sac-specific genes or proteins from a plant and to methods for producing agronomically interesting plants exhibiting female sterility or allowing apo-mictic propagation.
Diploid sporophytic and haploid gametophytic gen-erations alternate in the life cycle of higher and lower plant species. In contrast to lower plant species such as mosses or green algae in which the haploid gametophyte is the dominant generation, the gametophyte in higher plant species is dramatically reduced (Reiser and Fischer, 1993; Drews et al., 1998). Both male (pollen) and female (embryo sac) gametophytes have developed from spores, the hap-loid products of meiosis from spores (micro- and megaspores). In angiosperms, male gametophytes (pollen) are simple two to three-celled organisms consisting of one vegetative and one or two sperm cells, which are species-specific (Bedinger, 1992;
McCormick, 1993). Three of the four megaspores in most angiosperms degenerate and the surviving one

-2-forms the female gametophyte after three mitotic divisions (Reiser and Fischer, 1993; Russel, 1993).
The predominant female gametophyte, the Polygonium type, which occurs in about 70% of the angiosperm species (Webb and Gunning, 1990; Reiser and Fischer, 1993), is deeply embedded in sporophytic tissue and consists of only seven cells: the egg cell, two synergids, a central cell and three an-tipodals. In maize and several other species, the antipodal cells continue to proliferate until a group of about 20 to 40 cells is formed (Kiessel-bach, 1949).
The main function of the gametophytes is to supply the gametes: male and female gametes fuse during fertilisation, combine their different genomes, and thus form a new sporohytic generation. Thus, sex-ual reproduction in angiosperms is initiated when pollen grains start to germinate on the female flower organ, the stigma (Cheung, 1996). The fe-male gametophyte might then function in (i) direct-ing the pollen tube to the ovule (HUlskamp et al., 1995; Ray et al, 1997), (ii) directing one sperm cell to the egg cell and the other to the central cell (Russel, 1992), (iii) generating a barrier to polyspermy (Faure et al., 1994; Kranz et al., 1995), (iv) preventing autonomous embryo (partheno-genesis) and endosperm development (Grossniklaus et al., 1998; Luo et al., 1999; Chad et al., 1999) and finally (v) accumulating stores of maternal mRNAS
to facilitate the rapid initiation of embryo and endosperm development after fertilisation (Dressel-haus et al., 1999b).

-3-Morphological and structural studies of female ga-metophyte development as well as fertilisation and early embryo/endosperm development have been em-ployed with many plant species (e.g. with maize:
Kiesselbach, 1949; Diboll 1968; Huang and Sheridan, 1994 and Arabidopsis: Webb and Gunning, 1990, 1991;
Muriga et al., 1993). In contrast, "The identities and specific functions of the haploid-expressed genes required by the female gametophyte are almost completely unknown" (Drews et al., 1998). This re-flects the technical difficulty of identifying mu-tants and of gaining access to certain developmen-tal stages for molecular analyses.
Many mutants have been described that affect female gametophyte development and function, especially in maize and Arabidopsis, suggesting that a large num-ber of loci is essential for embryo sac development (Vollbrecht and Hake, 1995; Drews et al. 1998;
Grossniklaus and Schneitz, 1998). A few maternal genes functioning in the embryo sac as repressors of autonomous embryo (pathenogenesis) and/or en-dosperm development have been recently cloned in Arabidopsis. Mea/fisl (medea/fertilisation inde-pendent seed 1) is a gametophyte maternal effect gene probably involved in regulating cell prolif-eration in endosperm and partially in the embryo as well (Grossniklaus et al., 1998; Luo et al., 1999).
Fis2 shows a similar phenotype and encodes a puta-tive zinc-finger transcription factor (Luo et al., 1999): Autonomous endosperm development was ob-served in the fie (fertilisation independent en-dosperm/fis3 mutant. Mea/fisl and fie/fis3 display homology to polycomb proteins (Grossniklaus et al.

-4-1998; Ohad et al., 1999), proteins which are in-volved in long-term repression of homeotic genes in Drosophila and mammalian embryo development (Pir-rotta, 1998).
At a low frequency, auxin (2,4 D) treated sexual eggs from maize can be triggered to initiate embryo development (Kranz et al., 1995), and some egg cells initiate parthenogenetic development sponta-neously. In wheat, lines have been described pro-ducing up to 90% parthenogenetic haploids (Matzk et al., 1995). The molecular mechanisms underlying these processes are completely unknown. One pro-tein (a-tubulin) was identified whose expression is associated with the initiation of parthenogenesis in wheat (Matzk et al., 1997). De novo transcrip-tion from the zygotic genome occurs relatively soon after fertilisation in maize (Sauter et al., 1998;
Dresselhaus et al., 1999a), indicating that the store of maternal mRNA and the maternal control of embryo development is not as relevant as it is in animal species, for example Drosophila, Xenopus or Zebrafish (Orr-Weaver 1994; Newport and Kirschner 1982; Zamir et al. 1997).
An important biological process linked to flower and seed development is apomixis (asexual reproduc-tion through seeds: Koltunow et al., 1995; Vielle-Calzada et al., 1996). Due to the enormous economi-cal potential of apomixis once controllable in sex-ual crops, its application was named after the 'Green Revolution' as the 'Asexual Revolution' (Vielle-Calzada et al., 1996). Up to now all ap-proaches to isolate the 'apomixis genes' from apo-

-5-mictic species failed. Genes involved in autonomous endosperm development once inactivated were re-cently isolated from Arabidopsis (see Ohad et al., 1999; Luo et al., 1999). Autonomous embryo develop-ment (via parthenogenesis), a further component of apomixis will be necessary to engineer the apomixis trait in sexual crops. E.g. in wheat, lines have been described producing up to 90% parthenogenetic haploids (Matzk et al., 1995). Almost no molecular data concerning parthenogenesis is available for higher plants: one protein (a-tubulin) was identi-fied from the above described wheat lines whose ex-pression is associated with the initiation of par-thenogenesis (Matzk et al., 1997). Nevertheless, such a 'house keeping gene' will not be a valuable tool for genetic engineering of the induction of parthenogenesis. Regulatory genes are needed.
Thus, from an agronomical point of view it is highly desirable to provide plants, in particular agronomically important plants, which allow im-proved hybrid breeding, apomictic propagation and/or plants having seedless fruits, as well as providing female sterile plants.
Thus, it is considered particularly important to develop and provide means and methods that allow the production of plants exhibiting a modified em-bryo and endosperm development, in particular plants exhibiting a modified female gametophyte de-velopment. Such plants may prove particularly use-ful in commercial breeding programmes.

-6-The technical problem underlying the present inven-tion is to provide nucleotide sequences and pro-teins for use in cloning and expressing genes in-volved in embryo and endosperm development, in par-ticular for use in monocotyledonous plants which allow for the production of plants with a modified embryo and endosperm development, in particular which allow the production of female sterile plants or plants capable of apomictic propagation.
The present invention solves the technical problem underlying the present invention by providing iso-lated and purified nucleotide sequences for use in cloning or expressing an embryo sac-specific nu-cleotide sequence selected from the group consist-ing of a) the nucleotide sequence defined in any one of SEQ ID No. 1 to 8 and SEQ ID No. 13 to 31, a part or a complementary strand thereof, -b) a nucleotide sequence which hybridises to the nucleotide sequence defined in a), a part or a complementary strand thereof, c) a nucleotide sequence which is degenerated as a result of the genetic code to the nucleotide se-quence defined in a), b), a part or a complemen-tary strand thereof and d) alleles, functional equivalents or derivatives of the nucleotide sequence defined in a), b), c), a part or a complementary strand thereof.

-7-The nucleotide sequences set out in SEQ ID No. 1 to

8 and 13 to 31 represent nucleotide sequences which are essential for embryo and endosperm development, and which are active in the mature embryo sac of plants, such as maize. Thus, the present invention is inter alia based upon the finding, isolation and characterisation of genes, in the following also termed ZmES (Zea mays embryo sac) genes, which are specifically expressed in the cells of female ga-metophytes of a higher plant species, in particular in the ovary or mature egg apparatus, e.g. egg cell, central cell and synergides. Expression of the genes of the present invention in cells outside the female gametophyte was not detected. Further-more, the ZmES genes of the present invention are expressed in a temporarily specific manner, in par-ticular their expression is switched off after fer-tilisation and expression cannot be detected in the 2-cell or subsequent embryo stages.
The nucleotide sequence as set out in SEQ ID No. 1 to 8 and 13 to 31 represent nucleotide sequences, in particular DNA sequences for use in cloning or expressing an embryo sac-specific nucleotide se-quence which is essential for embryogenesis and en-dosperm development and is active in the embryo sac. Thus, these nucleotide sequences play a par-ticularly important role in embryogenesis and ga-metophyte development. Accordingly, the nucleotide sequences of the present invention are useful for cloning, in particular isolating, embryo sac-specific nucleotide sequences, in particular regu-latory elements, gene transcripts, coding sequences and/or full length genes in plants, in particular in monocotyledonous plants. Thus, the present in-vention provides a means for the isolation of em-bryo sac-specific coding sequences and/or tran-scription regulatory elements as well as gene tran-scripts that direct or contribute to embryo sac-specific preferred gene expression in plants, in particular in monocotyledonous plants, such as maize.
The nucleotide sequences of the present invention are both regulatory and protein coding nucleotide sequences.
The present invention thus relates to nucleotide sequences which are regulatory sequences, in par-ticular transcription regulatory elements capable of directing embryo sac-specific expression of a nucleotide sequence of interest, the regulatory se-quence being selected from the group consisting of a) the nucleotide sequence defined in any one of SEQ ID No. 13 to 31, a part or a complementary strand thereof, b) the nucleotide sequence which hybridise to the nucleotide sequence defined in a), a part or a complementary strand thereof and c) the alleles, functional equivalents or deriva-tives of the nucleotide sequence defined in a) or b), a part or a complementary strand thereof.
The regulatory sequences, in particular transcrip-tion regulatory sequences, are 5' or 3' regulatory sequences for instance promoters, transcribed, but . .

-9-untranslated regions (UTR) enhancers, or 3' tran-scription termination signals and may prove par-ticularly useful in directing embryo sac-specific expression of genes, in particular protein coding sequences, of interest in plants including the pro-tein coding sequences of the present invention.
They are in particular useful for directing embryo sac-specific transcription of heterologous struc-tural and/or regulatory genes in plants, for in-stance DNA sequences encoding proteins modulating, inducing, repressing or suppressing embryogenesis and/or endosperm development, e.g. Mea/Fisl, Fis2, Fie/Fis3, PICKLE, LEC1 or BBM1 (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al., 1999;
Ogas et al., 1999; Lotan et al., 1998.
Thus, the present invention provides regulatory elements such as promoters, enhancers, UTRs and 3' transcription termination signals providing for em-bryo sac-specific expression of a gene of interest including the ZmES coding sequences of the present invention. Further, regulatory elements of this specificity may be obtained by using the nucleotide sequences of the present invention to isolate in a genomic DNA library hybridising sequences encom-passing further regulatory elements.
In a particularly preferred embodiment of the pre-sent invention the above defined promoter of the present invention is expressed in a spatially and temporally specific manner, preferably in the em-bryo sac. Accordingly, the proteins encoded by a gene of interest cloned downstream from the pro-

-10-moter may be accumulated in embryo sacs or fruits.
In a further particularly preferred embodiment, the present invention relates to a DNA construct with a promoter, enhancer, UTR and/or a 3' regulatory ele-ment of the present invention operably linked to a coding sequence for a toxic protein such as Diphte-ria toxin A, Exotoxin A, Barnase or RNase Ti (Day et al., 1995; Koning et al., 1992; Mariani et al., 1990) specifically inhibiting the formation of em-bryo sac tissue. The genes of interest or coding sequences of interest and/or transcribed but un-translated regions (UTR) of interest may be cloned in sense or antisense orientation to the regulatory sequences of the present invention.
The transcription regulatory elements of the pre-sent invention exhibiting the above identified em-bryo sac-specificity, that is for instance embryo sac-specific promoters of the present invention, may be combined to nucleotide sequences encoding proteins capable of inducing or repressing embryo-genesis and/or endosperm development. Inducing em-bryogenesis and/or endosperm development may prove particularly useful for the production of plants, for example hybrid plants capable of apomictic propagation, that is propagation without fertilisa-tion. The production of plants exhibiting a re-pressed and/or abortive embryo and/or endosperm de-velopment allows the production of for instance fe-male sterile plants. Such plants may form sterile seed or seedless fruit. Thus, the present inven-tion may prove useful for all economically impor-tant plants which up until now have not been capa-ble of apomixis and/or plants which do not provide

-11-naturally occurring female sterility. The nucleo-tide sequences of the present invention are useful for expressing or suppressing an embryo sac-specific protein and/or its coding sequence of plants such as monocotyledonous, such as maize or dicotyledonous plants such as sugar beet, including but not limited to the proteins or coding sequences of the present invention. The nucleotide sequences of the present invention are accordingly in a par-ticularly preferred embodiment useful for express-ing or suppressing an embryo sac-specific protein, namely the ZmES protein or mutant variants thereof and its target genes in plants, in particular in the embryo sac of plants. Thus, the present inven-tion also provides a means to allow the expression or suppression of a particular embryo sac-specific or embryo sac-abundant gene in the embryo sac, thereby enabling the modification of the embryo sac and endosperm development, function and/or struc-ture. As explained above, the present invention thereby allows the production of plants, the em-bryos of which develop into plants without fertili-sation and allow apomixis, that is the asexual pro-duction of seeds.
The present invention also relates to isolated and purified nucleotide sequences which encode a pro-tein capable of modulating embryogenesis and en-dosperm development, function and/or structure in plants selected from the group consisting of a) the nucleotide sequence of any one of SEQ ID No.
to 8 and SEQ ID No. 13 and 14, a part or a complementary strand thereof,

-12-b) the nucleotide sequence encoding the amino acid sequence of any one of SEQ ID No. 9 to 12, a part or a complementary strand thereof, c) the nucleotide sequence which hybridise to the nucleotide sequence defined in a), b), a part or a complementary strand thereof, d) the nucleotide sequence which is degenerated as a result of the genetic code to the nucleotide sequence defined a), b), c), a part or a comple-mentary strand thereof, and e) the alleles, functional equivalents or deriva-tives of the nucleotide sequence defined in a), b), c), d), a part or a complementary strand thereof.
The nucleotide sequences specifically set out in SEQ ID No. 5 to 8 and SEQ ID No. 13 and 14 repre-sent nucleotide sequences encoding a protein, in the following termed the ZmES protein, which is es-sential for embryo and endosperm formation. ZmES
proteins are small, cysteine-rich proteins with an N-terminal signal peptide, most likely for translo-cation outside the cell. The ZmES proteins of the present invention, namely ZmES1, 2, 3 and 4 are highly homologous to each other.
The protein coding nucleotide sequences of the pre-sent invention may be useful in engineering geneti-cally manipulated plants exhibiting a modified em-bryogenesis and/or endosperm development, function and/or structure. In particular the proteins en-coded by the present nucleotide sequences may be

-13-considered to be defensins. Defensins appear to be involved in resistance systems against bacterial and fungal pathogens. Thus, the present invention may allow the specific modification of plants, the embryos of which exhibit a modified resistance, in particular improved resistance, against pathogens, for instance microbial pathogens. Of course, the present invention also relates to plants and meth-ods for their production which exhibit a modified resistance, in particular improved resistance against pathogens compared to a non-modified and non-transformed plant.
Plant defensins contain an N-terminal signal pep-tide and the mature peptides form four disulfide bridges. This protein family includes 7-thionins, proteinase inhibitors II and P322 and other (for review see Broekaert et al., 1995). The present invention provides a novel class of putative plant defensins, which is specifically expressed in the female gametophyte of maize. ZmES1-4 contains all structural components which classify them as plant defensins: they are small proteins, contain N-terminal signal peptides and eight Cys which proba-bly form four intramolecular disulfide bridges, the fourth one linking the N- and C-terminal regions of the mature proteins. The predicted secondary structure displays and a-helix and two 13-stands at the same position as in the antifungal protein RsAFP1 from radish seeds, whose three-dimensional structure has been determined by NMR spectrometry.
The same three-dimensional structure was also de-termined for charybdotoxin, a neurotoxin from scor-pion (Bontems et al., 1992), although this peptide

-14-is shorter at the N- and C-terminus and thus forms only three disulfide bonds. Predicted secondary and tertiary structures differ slightly, but the positions of a-helices, P-stands and eight Cys are conserved in all plant defensins. Mature ZmES pro-teins are longer than most other defensins, but all additional amino acids are located exclusively in coil-regions, neither in a-helix nor P-stands thus allowing the same three-dimensional structure than RsAFP1. Known plant defensins of diverse monocot and dicot species display higher homology among each other than with ZmES proteins.
The protein coding nucleotide sequences or the UTRs of the present invention may be cloned either in sense or antisense orientation to regulatory ele-ments, such as 5' or 3' regulatory nucleotide se-quences, including but not limited to the regula-tory nucleotide sequences of the present invention.
Thus, using for instance antisense or cosuppression technology the nucleotide sequences of the present invention, such as the protein coding sequences, transcribed, but not translated regions (UTRs) or parts thereof, it is possible to generate plants exhibiting a modified, in particular a distorted embryogenesis and/or endosperm development, func-tion or/and structure. Such a distorted embryo-genesis and/or endosperm development may cause fe-male infertility or contribute to generating plants capable of apomixis.
Thus, the present invention also allows the modifi-cation of structure or expression of the ZmES gene and/or protein which may lead for instance to

-15-parthenogenetic embryo development which is an im-portant component of engineering the apomixis trait. For instance, the coding sequence of the present invention may be overexpressed in trans-formed plants due to expression under control of a strong constitutive tissue or tissue-specific or regulated promoter. It is also possible to modify the coding sequence of the present invention so as to allow the production of a modified embryo sac-specific ZmES protein which in turn modifies in a desired manner embryo sac development and/or func-tion. Most importantly, the present invention pro-vides a means to specifically inhibit the formation of a protein essential for embryo sac and/or en-dosperm function or development namely the ZmES
protein by transforming plants with antisense con-structs comprising all or part of the coding se-quence or, transcribed but not translated regions of the ZmES gene or a part thereof in antisense orientation under the control of its wild-type or appropriate other regulatory elements so as to ef-fectively bind to wild-type ZmES mRNA and inhibits its translation. Such a construct may lead upon expression to the abolishment or elimination of the wild-type ZmES function thereby producing modified plants.
Of course, such an eliminating effect of natural gene function may also be obtained using cosuppres-sion technology. Accordingly, the nucleotide se-quences of the present invention, cloned in sense orientation to at least one regulatory element, such as a promoter into a suitable vector, are transformed into a plant, which in turn may exhibit

-16-a suppressed gene function of a wild-type ZmES
gene.
The present invention also relates to processes to restore the antisense effect obtained by using the antisense construct mentioned above. To be able to restore the antisense effect, a further DNA con-struct comprising an ZmES gene derived nucleic acid sequence in sense orientation under control of a switchable or inducible promoter could be used to transform the plant. After switching on the pro-moter, the antisense effect might be restored. An-other method for restoring the above described elimination effect is to utilise a DNA construct, in particular an antisense or co-suppression con-struct employing an inducible promoter to control the expression of the nucleic acid sequence derived from a ZmES gene, in particular in the antisense or co-suppression construct, via external factors.
In this context, it has to be understood that the antisense constructs of the present invention may not necessarily comprise all or an essential part of the coding sequence of the present invention in antisense orientation to regulatory elements, but in a particularly preferred embodiment it is suffi-cient to use parts of the coding sequences or of the UTRs which are considerably shorter than the full length coding sequence. The length of such a sequence must be sufficient to allow effective hy-bridisation to the target mRNA and may be a minimum length of 50 to 100 nucleotides.

-17-The present invention also relates to nucleotide sequences which hybridise, in particular under stringent conditions to the sequences set out in SEQ ID No. 1 to 8 and 13 to 31. In particular, these sequences have on the nucleotide level a de-gree of identity of 70% to the sequences of SEQ
ID No. 1 to 8 and 13 to 31.
In the context of the present invention, nucleotide sequences which hybridise to the specifically dis-closed sequences of SEQ ID No. 1 to 8 and 13 to 31 are sequences which have a degree of 60 to 70% se-quence identity to the specifically disclosed se-quence of the nucleotide level. In an even more preferred embodiment of the present invention, se-quences which are encompassed by the present inven-tion are sequences which have a degree to identity of more than 70%, and even more preferred, more than 80%, 90%, 95% and particularly 99% to the spe-cifically disclosed sequences of the present inven-tion on the nucleotide level.
Thus, the present invention relates to nucleotide sequences, in particular DNA sequences which hy-bridise under the hybridisation condition as de-scribed in Sambrook et al., (1989), in particular under the following conditions, to the sequences specifically disclosed:
Hybridisation buffer: 1 M NaCl; 1% SDS; 10%
dextran sulphate; 100 pg/ml sDNA
Hybridisation temperature: 65 C
First wash: 2 x SSC; 0.5% SDS at room tempera-

-18-ture Second wash: 0.2 x SSC; 0.5% SDS at 65 C.
More preferably, the hybridisation conditions are chosen as described above, except that a hybridisa-tion temperature and a second wash temperature of 68 C and, even more preferred, a hybridisation temperature and a second wash temperature of 70 C
is applied.
Thus, the present invention also comprises nucleo-tide sequences which are functionally equivalent to the sequences of SEQ ID No. 1 to 8 and 13 to 31, i.e. may have a different sequence but have the same or essentially the same function, in particu-lar sequences which are at least homologous to se-quences of SEQ ID No. 1 to 8 and 13 to 31. The in-vention also relates to alleles and derivatives =of the sequences mentioned above which are defined as sequences being essentially similar to the above sequences but comprising, for instance, nucleotide exchanges, substitutions - also by unusual nucleo-tides - rearrangements, mutations, deletions, in-sertions, additions or nucleotide modifications and are functionally equivalent to the sequences as set out in SEQ ID No. 1 to 8 and 13 to 31.
In the context of the present invention, a number of general terms shall be utilised as follows.
The term "promoter" refers to a sequence of DNA, usually upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for

-19-RNA polymerase and/or other factors required for transcription to start at the correct site. Pro-moter sequences are necessary, but not always suf-ficient to drive the expression of the gene.
"Nucleotide sequence" refers to a molecule which can be single or double stranded, composed of mono-mers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine. The nucleotide sequence may be cDNA, genomic DNA, or RNA, for in-stance mRNA.
Thus, the term "nucleotide sequence" refers to a natural or synthetic polymer of DNA or RNA which may be single or double stranded, alternatively containing synthetic, non-natural or altered nu-cleotide bases capable of incorporation into DNA or RNA polymers. In a particularly preferred embodi-ment, the nucleotide sequence of the present inven-tion is an isolated and purified nucleic acid mole-cule.
The term "gene" refers to a DNA sequence that codes for a specific protein and regulatory elements con-trolling the expression of this DNA sequence.
The term "coding sequence" refers to that portion of a gene encoding a protein, polypeptide, or a portion thereof, and excluding the regulatory se-quences which drive the initiation or termination of transcription. The coding sequence and/or the regulatory element may be one normally found in the cell, in which case it is termed "autologous", or it may be one not normally found in a cellular lo-cation, in which case it is termed "heterologous".

-20-A heterologous gene may also be composed of autolo-gous elements arranged in an order and/or orienta-tion not normally found in the cell into which it is transferred. A heterologous gene may be derived in whole or in part from any source known to the art, including a bacterial or viral genome or epi-some, eucaryotic nuclear or plasmid DNA, cDNA or chemically synthesised DNA. The structural gene may constitute an uninterrupted coding region or it may include one or more introns bounded by appro-priate splice junctions. The structural gene may be a composite of segments derived from different sources, naturally occurring or synthetic.
By "operably linked" it is meant that a gene and a regulatory sequence are connected in sense or an-tisense expression in such a way as to permit gene expression when the appropriate molecules (e.g.
transcriptional activator proteins) are bound to the regulatory sequence.
The term "vector" refers to a recombinant DNA con-struct which may be a plasmid, virus, or autono-mously replicating sequence, phage or nucleotide sequence, linear or circular, of a single or double stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product in sense or antisense orientation along with an appro-priate 3' untranslated sequence into a cell.

-21-"Plasmids" are genetic elements that are stably in-herited without being a part of the chromosome of their host cell. They may be comprised of DNA or RNA and may be linear or circular. Plasmids code for molecules that ensure their replication and stable inheritance during cell replication, and may encode products of considerable medical, agricul-tural and environmental importance. For example, they code for toxins that greatly increase the virulence of pathogenic bacteria. They can also encode genes that confer resistance to antibiotics.
Plasmids are widely used in molecular biology as vectors to clone and express recombinant genes.
Starting plasmids disclosed herein are either com-mercially available, publicly available, or can be constructed from available plasmids by routine ap-plication of well-known, published procedures.
Many plasmids and other cloning and expression vec-tors that can be used in accordance with the pre-sent invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
The term "expression" as used herein is intended to describe the transcription and/or coding of the se-quence for the gene product. In the expression, a DNA chain coding for the sequence of gene product is first transcribed to a complementary RNA, which is often an mRNA, and then the thus transcribed

-22-mRNA is translated into the above mentioned gene product if the gene product is a protein. However, expression also includes the transcription of DNA
inserted in antisense orientation to its regulatory elements. Expression, which is constitutive and possibly further enhanced by an externally con-trolled promoter fragment, thereby producing multi-ple copies of mRNA and large quantities of the se-lected gene product, may also include overproduc-tion of a gene product.
The term "suppression" refers to repression, inhi-bition or reduction of endogenous gene expression.
The term "directing expression" refers to inducing, controlling, regulating, modulating, contributing or enhancing expression of a nucleotide sequence.
In the context of the present invention, the term "protein" refers to any sequence length of amino acid, irrespective of its length. Thus, within the -present invention the term "protein" relates to peptides, polypeptides and proteins. The protein of the present invention may be modified by addi-tion of carbohydrates, fats or other proteins or peptides. The proteins of the present invention may also be modified by addition of isotopes, amino-, acyl-, allyl-, or other groups.
The proteins of the invention that do not occur in nature are isolated. The term "isolated" as used herein, in the context of proteins, refers to a polypeptide which is unaccompanied by at least some of the material with which it is associated in its

-23-natural state. The isolated protein constitutes at least 0.5%, preferably at least 5%, more preferably at least 25% and still more preferably at least 50%
by weight of the total protein in a given sample.
Most preferably the "isolated" protein is substan-tially free of other proteins, lipids, carbohy-drates or other materials with which it is natu-rally associated, and yields a single major band on a non-reducing polyacrylamide gel.
Substantially free means that the protein is at least 75%, pref-erably at least 85%, more preferably at least 95%
and most preferably at least 99% free of other pro-teins, lipids, carbohydrates or other materials with which it is naturally associated.
"Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognise an analyte (antigen). The rec-ognised immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu con-stant region genes, as well as the myriad immu-noglobulin variable region genes. Antibodies ex-ist, e.g. as intact immunoglobulins or as a number of well characterised fragments produced by diges-tion with various peptidases. The term "antibody", as used herein, also includes antibody fragments either produced by the modification of whole anti-bodies or those synthesised de novo using recombi-nant DNA methodologies. The term "antibody" in-cludes intact molecules as well as fragments thereof, such as Fab, F(aby)2, and Fv which are ca-pable of binding the epitopic determinant. These antibody fragments retain some ability to selec-

-24-tively bind with its antigen or receptor and are defined as follows:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule, can be obtained by treating a whole antibody with pep-sin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab'), the fragment of the antibody that can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Feb' fragments held together by two disulfide bonds;
.(4) Fv, defined as a genetically engineered frag-ment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), defined as a ge-netically engineered molecule containing the vari-able region of the light chain, the variable region of the heavy chain, linked by a suitable polypep-tide linker as a genetically fused single chain molecule.
Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies:

-25-A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)).
The term "host cell" refers to a cell which has been genetically modified by transfer of a chi-meric, heterologous or autologous nucleic acid se-quence or its descendants still containing this se-quence. These cells are also termed "transgenic cells". In the case of an autologous nucleic acid sequence being transferred, the sequence will be present in the host cell in a higher copy number than naturally occurring.
As used herein, "plant" refers to photosynthetic organisms, such as whole plants including algae, mosses, ferns and plant-derived tissues. "Plant derived tissues" refers to differentiated and un-differentiated tissues of a plant, including nodes, male and female flowers, fruits, pollen, pollen tubes, pollen grains, roots, shoots, shoot meris-tems, coleoptilar nodes, tassels, leaves, cotyle-dondous leaves, ovules, tubers, seeds, kernels and various forms of cells in culture, such as intact cells, protoplasts, embryos and callus tissue.
Plant-derived tissues may be in plants, or in or-gans, tissue or cell cultures. A "monocotyledonous plant" refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food. A "dicotyledonous plant" refers to a plant whose seeds have two cotyledons.
"Transformation" and "transferring" refers to meth-ods to transfer DNA into cells including, but not limited to, biolistic approaches such as particle bombardment, microinjection, whisker technology,

-26-permeabilising the cell membrane with various physical (e.g., electroporation) or chemical (e.g., polyethylene glycol, PEG) treatments; the fusion of protoplasts or Agrobacterium tumefaciens or rhizogenes mediated transformation. There are no specific requirements for the plasmids used for the injection and electroporation of DNA in plant cells. Plasmids such as pUC derivatives can be used. Selectable markers are not necessary. De-pending upon the method for the introduction of de-sired genes into the plant cell, further DNA se-quences may be necessary; if, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right border, often, how-ever, the right and left border of the Ti and Ri plasmid T-DNA must be linked as flanking region to the genes to be introduced.
If Agrobacteria are used for the transformation, the DNA to be introduced must be cloned into spe-cific plasmids, either into an intermediary vector or into a binary vector. The intermediary vectors can be integrated into the Ti or Ri plasmid of the Agrobacteria due to sequences that are homologous to sequences in the T-DNA by homologous recombina-tion. The Ti or Ri plasmid furthermore contains the vir region necessary for the transfer of the T-DNA into the plant cell. Intermediary vectors can-not replicate in Agrobacteria. By means of a helper plasmid, the intermediary vector can be transferred by means of a conjugation to Agrobacte-rium tumefaciens. Binary vectors can replicate both in E.coli and in Agrobacteria, and they con-tain a selection marker gene and a linker or poly-

-27-linker framed by the right and left T-DNA border region. They can be transformed directly into the Agrobacteria (Holsters et al., 1978). The Agrobac-terium serving as a host cell should contain a plasmid carrying a vir region. The Agrobacterium transformed is used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has been extensively examined and de-scribed in EP-A 120 516; Hoekema, (1985); An et al., (1985).
For the transfer of the DNA into the plant cell, plant explants can be co-cultivated with Agrobacte-rium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g., pieces of leaf, stem segments, roots, but also protoplasts or plant cells cultivated by suspension) whole plants can be regenerated in a suitable medium, which may contain antibiotics or biocides for the selection of trans-formed cells.
Alternative systems for the transformation of mono-cotyledonous plants are the transformation by means of electrically or chemically induced introduction of DNA into protoplasts, the electroporation of partially permeabilised cells, the microinjection of DNA into flowers, the microinjection of DNA into micro-spores and pro-embryos, DNA transfer by whisker technology, the introduction of DNA into germinating pollen and the introduction of DNA into embryos by swelling (Potrykus, (1990)).
While the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well-established, more

-28-recent research work indicates that monocotyledon-ous plants are also accessible for transformation by means of vectors based on Agrobacterium (Chan et al., (1993); Hiei et al., (1994); Bytebier et al., (1987); Raineri et al., (1990), Gould et al., (1991); Mooney et al., (1991); Lit et al., (1992)).
In fact, several of the above-mentioned transforma-tion systems could be established for various cere-als: the electroporation of tissues, the transfor-mation of protoplasts and the DNA transfer by par-ticle bombardment in regenerative tissue and cells (Jahne et al., (1995)). The transformation of wheat has been frequently described in the litera-ture (Maheshwari et al., (1995)) and of maize in Brettschneider et al. (1997) and Ishida et al.
(1996).
In a further preferred embodiment, the invention relates to nucleotide sequences specifically hy-bridising to transcripts of the nucleotide se-quences of the present invention. These nucleotide sequences are preferably oligonucleotides having a length of at least 10, particularly preferred of at least 15, most preferred of at least 50 nucleo-tides. The nucleotide sequences and oligonucleo-tides of the present invention may be used, for in-stance as primers for a PCR reaction or be used as components of antisense constructs or of DNA mole-cules encoding suitable ribozymes.
In a preferred embodiment of the present invention, the nucleotide sequence of the present invention is derived from dicotyledonous or monocotyledonous plants.

-29-In a particularly preferred embodiment of the pre-sent invention, the nucleotide sequence is derived from maize (Zea mays).
In a preferred embodiment of the present invention, the nucleotide sequence of the present invention is a DNA, cDNA or RNA molecule.
The present invention also relates to a vector com-prising the nucleotide sequences according to the above, in particular to a bacterial vector, such as a plasmid or a virus.
The present invention thus also relates to vectors comprising the above-identified nucleotide se-quences in particular comprising chimeric DNA con-structs or non-chimeric DNA constructs such as the wild-type ZmES gene, or derivatives thereof or parts thereof. The term DNA construct refers to a combination of at least one regulatory element and a coding sequence.
Thus, the present invention relates to recombinant nucleic acid molecules useful in the preparation of plant cells and plants as defined above by genetic engineering. In particular, the invention concerns chimeric DNA constructs comprising a coding DNA se-quence coding for a wild-type ZmES protein operably linked to a promoter wherein said promoter is dif-ferent to the promoter linked to said ZmES coding sequence in the wild-type gene i.e. either is a mu-tated wild-type promoter or a promoter from another gene and/or species. In a further preferred embodi-ment, the invention concerns chimeric DNA con-structs comprising a modified coding DNA sequence

-30-coding for a mutated ZmES protein, wherein the DNA-sequence is operably linked to a promoter which may be different from the promoter linked to said ZmES
coding seduence in the wild-type gene or the pro-moter is the wild-type ZmES promoter.
Of course, the present invention also relates to chimeric antisense constructs comprising a DNA se-quence encoding, at least partially, the natural, that is wild-type, or modified ZmES protein, or a part thereof, which is linked to a promoter wherein said promoter is different to the promoter linked to said ZmES coding sequences in the wild-type gene or is the wild-type promoter and wherein the orien-tation of the coding sequence to the promoter is vice versa to the wild-type orientation. In one embodiment of the present invention the DNA se-quence of the present invention used specifically to inhibit via antisense constructs the translation of ZmES expression from the wild-type gene is at least partially not derived from the ZmES coding sequence but rather contains sequences from un-translated regions of the ZmES transcribed region.
Both the ZmES coding sequence and the untranslated region of the ZmES gene are also termed ZmES de-rived sequences. Of course the invention also re-lates to DNA constructs comprising a DNA sequence coding for the non-chimeric wild-type ZmES protein operably linked to the wild-type promoter. These constructs may be used to transform plant cells and plants for which the DNA construct is autologous, i.e. is the source or natural environment for the DNA construct or for which the DNA construct is heterologous, i.e., is from another species. Plant

-31-cells and plants obtained by using the above listed DNA constructs may be characterised by ZmES an-tisense expression, multiple copies of the above DNA constructs in their genome, that means are characterised by an increased copy number of the ZmES gene in the genome and/or a different location in the genome with respect to the wild-type gene and/or the presence of a foreign gene in their ge-nome.
In the context of the present invention a chimeric DNA construct is thus a DNA sequence composed of different DNA fragments not naturally occurring in this combination. The DNA fragments combined in the chimeric DNA construct may originate from the same species or from different species. For example a DNA fragment coding for an ZmES protein may be op-erably linked to a DNA fragment representing a pro-moter from another gene of the same species that provides for an increased expression of the ZmES
coding sequence. Preferably however, a DNA fragment coding for an ZmES protein is operably linked to a DNA fragment containing a promoter from another species for instance from another plant species, from a fungus, yeast or from a plant virus or a synthetically produced promoter. A synthetically produced promoter is either a promoter synthesised chemically from nucleotides de novo or a hybrid-promoter spliced together by combining two or more nucleotide sequences from synthetic or natural pro-moters which are not present in the combined form in any organism. The promoter has to be functional in the plant cell to be transformed with the chi-meric DNA construct.

-32-The promoter used in the present invention may be derived from the same or from a different species and may provide for constitutive or regulated ex-pression, in particular positively regulated by in-ternal or external factors. External factors for the regulation of promoters are for example light, heat, chemicals such as inorganic salts, heavy met-als or organic compounds such as organic acids, de-rivatives of these acids, in particular its salts.
Examples of promoters to be used in the context of the present invention are the actin promoter from rice, the cauliflower mosaic virus (CaMV) 19S or 35S promoters, nopaline synthase promoters, patho-genesis-related (PR) protein promoters, the ubiq-uitin promoter from maize for a constitutive ex-pression, the HMG (High molecular weight glutemin) promoters from wheat, promoters from Zein genes from maize, small subunit of ribulose bisphospho-nate carboxylase (ssuRUBISC ) promoters, the 35S
transcript promoter from the figworm mosaic virus (FMV 35S), the octopine synthase promoter. etc. It is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of antisense mRNA or modified or wild-type ZmES
protein to produce flower and/or 'fruit modified plants. Of course for selective expression of the ZmES protein tissue specific promoters may be used.
However, in the most preferred embodiment of the present invention, i.e. the ZmES antisense con-structs, the promoter may be a constitutive strong promoter, since the embryo sac specificity of the antisense action is confined to the embryo sac due

-33-to embryo sac-specific expression of the target, i.e. the wild-type ZmES expression.
The DNA construct of the invention may contain mul-tiple copies of a promoter and/or multiple copies of the DNA coding sequences. In addition the con-struct may include coding sequences for markers and coding sequences for other peptides such as signal or transit peptides or resistance genes for in-stance against virus infections or antibiotics.
Useful markers are peptides providing antibiotic or drug resistance for example resistance to phosphin-strycine, hygromycin, kanamycin, G418, gentamycin, lincomycin, methotrexate or glyphosate. These mark-ers can be used to select cells transformed with the chimeric DNA constructs of the invention from untransformed cells. Thus, a useful marker gene is the herbicide resistance gene Pat (phosphinotrycine acetyl transferase). Of course other markers are markers coding peptidic enzymes which can be easily detected by a visible reaction for example a colour reaction for example luciferase, P-1,3-glucur-onidase or P-galactosidase.
Signal or transit peptides provide the ZmES protein formed on expression of the DNA constructs of the present invention with the ability to be trans-ported to the desired site of action. Examples for transit peptides of the present invention are chloroplast transit peptides or mitochondria tran-sit peptides, especially nuclear recognition/
localisation signal peptides and endoplasmatic re-ticulum signal peptides.

-34-In chimeric DNA constructs containing coding se-quences for signal or transit peptides these se-quences are usually derived from a plant, for in-stance from corn, potato, Arabidopsis or tobacco.
Preferably, transit peptides and ZmES coding se-quences are derived from the same plant, for in-stance corn. In particular such a chimeric DNA con-struct comprises a DNA sequence coding for a wild-type ZmES protein and a DNA sequence coding for a transit peptide operably linked to a promoter wherein said promoter is different to the promoter linked to said coding sequences in wild-type gene, but functional in plant cells. In particular, said promoter provides for higher transcription effi-ciency than the wild-type promoter.
The mRNA produced by a DNA construct of the present invention may advantageously also contain a 5' non-translated leader sequence. This sequence may be derived from the promoter selected to express the gene and can be specifically modified so as to in-crease translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs from suitable eucaryotic genes or a synthetic gene sequence.
Preferably, the coding sequence of the present in-vention is not only operably linked to 5' regula-tory elements, such as promoters, but is addition-ally linked to other regulatory elements such as enhancers and/or 3' regulatory elements. For in-stance, the vectors of the present invention may contain functional terminator sequences such as the terminator of the octopine synthase gene from Agro-

-35-bacterium tumefaciens. Further 3' non-translated regions to be used in a chimeric construct of the present invention to cause the addition of polyade-nylate nucleotides to the 3' end of the transcribed RNA are the polyadenylation signals of the Agrobac-terium tumefaciens nopaline synthase gene (NOS) or from plant genes like the soybean storage protein gene and the small subunit of the ribulose-1,5-bisphosphonate carboxylase (ssuRUBISCO) gene. Of course, also the regulating elements of the present invention deriving from the wild-type ZmES gene may be used.
The vectors of the present invention may also pos-sess functional units effecting the stabilisation of the vector in the host organism, such as bacte-rial replication origins. Furthermore, the chimeric DNA constructs of the present invention may also encompass introns or part of introns inserted within or outside the coding sequence for the ZmES
protein.
In a particularly preferred embodiment of the pre-sent invention, the nucleotide sequence e.g. the 5' and/or 3' regulatory elements of the present inven-tion contained in the vector, are operably linked to any desired gene or nucleotide sequence also termed a gene of interest, which in this context may also be a coding sequence which may be a het-erologous or autologous gene. Such a gene of in-terest may be a gene, in particular its coding se-quence, conferring for instance disease resistance, draught resistance, insecticide resistance, herbi-cide resistance, immunity and improved intake of

-36-nutrients minerals or water from the soil or a modified metabolism in the plant, particularly its embryo sac. In a particularly preferred embodi-ment, the vector defined above is comprised of fur-ther regulatory elements directing or enhancing ex-pression of the gene of interest, such as 5', 3' or 5' and 3' regulatory elements known in the art.
Regulatory elements concerned in the present inven-tion also encompass introns or parts of introns in-serted in or outside the gene of interest. In a particularly preferred embodiment of the present invention, the regulatory element is a promoter, in particular the cauliflower mosaic virus (CaMV) 35S
promoter or a promoter encoded by the nucleotide sequence selected from the group consisting of SEQ
ID No. 13 to 31.
Thus, the nucleotide sequences of the present in-vention are useful since they enable the embryo sac-specific expression of genes of interest of plants, in particular monocotyledonous plants. Ac-cordingly, plants are enabled to product useful products in their embryo or endosperm. The nucleo-tide sequence of the present invention may also be useful to regulate the expression of genes of in-terest depending upon the developmental stage of the transferred cell or tissue.
Furthermore, the present invention allows the specific modification of the metabolism in embryogenesis and endosperm development.
In a particularly preferred embodiment of the pre-sent invention, the vector furthermore contains T-DNA, in particular the left, the right or both T-

-37-DNA borders derived from Agrobacterium tumefaciens.
Of course, a sequence derived from Agrobacterium rhizogenes genes may also be used. The use of T-DNA sequences in the vector of the present inven-tion enables the Agrobacterium mediated transforma-tion of cells.
In a preferred embodiment of the present invention, the nucleotide sequence of the present invention, optionally operably linked to regulatory elements, is located within the T-DNA or adjacent to it.
The present invention also relates to a host cell transformed with the nucleotide sequence or the vector of the present invention in a particular plant, yeast or bacterial cells, in particular monocotyledonous or dicotyledonous plant cells.
The present invention also relates to cell cul-tures, tissue, calluses, etc. comprising a cell ac-cording to the above, for instance a transgenic cell and its descendants harbouring and preferably _expressing the nucleotide sequence or vector of the present invention.
Thus, the present invention relates to transgenic plant cells which were transformed with one or sev-eral nucleotide sequences of the present invention as well as to transgenic plant cells originating from such cells. Such plant cells can be distin-guished from naturally occurring plant cells by the observation that they contain at least one nucleo-tide sequence according to the present invention which does not naturally occur in these cells, or by the fact that such a sequence is integrated on the genome of the cell at a location where it does

-38-not naturally occur, that is in another genomic re-gion or by the observation that the copy number of the nucleotide sequence is different, in particular higher, than the copy number in naturally occurring plants.
Thus, the present invention also relates to trans-genic cells, also called host cells, transformed with the nucleotide sequence or vector of the pre-sent invention, in particular plant, yeast, or bac-terial cells, in particular monocotyledonous or di-cotyledonous plant cells. The present invention also relates to cell cultures, tissue, roots, flow-ers, calluses, propagation and harvest material, pollen seeds, stamen, cobs, nodes, seedlings, so-matic and zygotic embryos etc. comprising a cell according to the above, that is, a transgenic cell being stably or transiently transformed and being capable of expressing a nucleotide sequence of the present invention, for instance a regulatory ele-ment or a nucleotide sequence for encoding a pro-tein modifying the embryogenesis or endosperm de-velopment of the transformed plant. The transgenic plants of the present invention can be regenerated to whole plants according to methods known to the person skilled in the art. The regenerated plant may be chimeric with respect to the incorporated foreign DNA. If the cells containing the foreign DNA develop into either micro- or macrospores, the integrated foreign DNA will in one embodiment of the present invention be transmitted to a sexual progeny. If the cells containing the foreign DNA
are somatic cells of the plant, non-chimeric trans-genic cells are produced by conventional methods of

-39-vegetative propagation either in vivo, e.g. from buds or stem cutting or in vitro following estab-lished procedures known in the art.
The present invention also relates to a method of genetically modifying a cell by transforming it with a nucleotide sequence of the present invention or vector according to the above whereby the ZmES1, ZmES2, ZmES3 and/or ZmES4 coding sequence or fur-ther gene of interest operably linked to at least one regulatory element expressible in the cell, ei-ther according to the present invention or as con-ventionally used. In particular, the cell being transformed by the method of the present invention is a plant, bacterial or yeast cell. In a particu-larly preferred embodiment of the present inven-tion, the above method further comprises the regen-eration of the transformed cell to a differentiated and, in a preferred embodiment, fertile or non-fertile plant.
In a preferred embodiment of the present invention, the method to transform a cell involves direct up-take of the nucleotide sequence, in particular by microinjection of the nucleotide sequence, electro-poration, chemical treatment or particle bombard-ment.
The present invention also relates to a method of production of a protein having the activity of a protein modulating embryogenesis and/or endosperm development, wherein a host cell of the present in-vention is cultivated under conditions allowing the synthesis of the protein, and wherein the protein is isolated from the cultivated cell and/or the

-40-culture medium. Thus, the present invention also relates to a protein being preparable by a host cell of the invention or obtainable by a method for the production of a protein of the invention.
The present invention also relates to a protein ca-pable of modulating embyogenesis and/or endosperm development and being encoded by the nucleotide se-quences of the present invention.
The present invention also relates to derivatives of such a protein having essentially the same bio-logical activity. Such modifications may be modi-fications due to amino acid substitutions, inser-tions, deletions, inversions, etc. Such modifica-tions may also be constituted by glycosylation or other types of derivatisation.
The present invention also relates to an antibody or a fragment thereof which is reactive with a pro-tein of the invention. These antibodies may be used to screen expression libraries or to identify clones which produce the protein of the present in-vention. A used herein, the term "relates to an antibody" relates to detection, activation or inhi-bition of molecular and cellular pathways induced by the protein of the present invention, in par-ticular to modification of the embryogenesis and/or endosperm development. The term "antibody" relates to bivalent or monovalent molecular entities that have the property of interaction with the protein of the invention. As used herein, "antibody" re-fers to a protein consisting of one or more poly-peptides substantially encoded by immunoglobulin genes or fragments of immuno-globulin genes. Light

-41-chains are classified as either kappa or lambda.
Heavy chains are classified as gamma, mu alpha, delta or epsilon which in turn define the immu-noglobulin classes IgG, IgM, IgA, IgD and IgE, re-spectively (for details see definition of the terms). The phrase "specifically binds to", when referring to an antibody, refers to a binding reac-tion which is determinative of the presence of the domain and the presence of a heterogeneous popula-tion of proteins or other biologics. Thus, under designated immuno-assay conditions, the specified antibody binds to a. particular domain and does not bind to a significant degree to other proteins rep-resented in the sample. Specific binding to the domain under such conditions may require an anti-body that is selected for its specificity for the proteinof the invention. A variety of immuno-assay format may be used to select antibodies spe-cifically immuno-reactive with the ZmES1, ZmES2, ZmES3 and/or ZmES4 proteins. For example, solid ELISA immuno-assays are routinely used to select monoclonal antibodies specifically immuno-reactive with the domain. The immuno-assays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmuno-assays, immuno-precipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion as-says, agglutination assays, complement-fixation as-says, immunoradiometric-assays, fluorescent-immunoassays and protein A-immunoassays, to name but a few. Antibodies of the invention specifi-cally bind to one or more epitopes on the protein of the invention. Epitope refers to a region of

-42-the protein of the invention bound by an antibody, wherein the binding prevents association of a sec-ond antibody to the protein.
In an embodiment of the invention, the antibodies are polyclonal antibodies, monoclonal antibodies and fragments thereof. Antibody fragments encom-pass those fragments which interact with the pro-tein of the invention. Also encompassed are chi-meric antibodies typically produced by recombinant methods wherein a foreign sequence comprises part or all of an antibody which interacts with the pro-tein of the invention. Examples of chimeric anti-bodies include CDR-grafted antibodies. Also in-cluded are antibodies composed of an antibody of an animal and a lectin of an animal or plant, in par-ticular a lectin which recognises a modified carbo-hydrate of the membrane of cells of embryogenesis and/or endosperm development modified plants. An-tibodies of the invention may also have a detect-able label attached hereto. Such a label may be a fluorescent (e.g. fluorscein isothiocyanate, FITC) enzymatic (e.g. horse radish oxidates) affinity (e.g. biotin) or isotopic label (e.g. 1251). Also encompassed by the invention are hybridoma, cell lines producing a monoclonal antibody which inter-act with a protein of the invention. The antibod-ies of the present invention are useful in the de-tection of embryogenesis and/or endosperm modified development of plants. Antibodies may be used as a part of a kit to detect the presence of the protein of the invention in a biological sample. Biologi-cal samples include tissue, specimens and intact cells or extracts thereof. Such kits employ anti-

-43-bodies having an attached label to allow for detec-tion. The antibodies are useful for identifying non-modified embryogenesis and/or endosperm devel-opment of plants.
In an preferred embodiment of the present inven-tion, the antibody or the fragments thereof is modified, in particular used, oxidised and/or oli-gomerised.
The present invention also relates to a method for isolating embryo sac-specific genes from a plant, whereby a preferably labelled, for instance radio-actively or fluorescently labelled, nucleotide se-quence of the invention is used to screen gene li-braries containing nucleotide sequences derived from a plant, by hybridising the gene library with the labelled sequences of the present invention and detecting the hybridised probes.
The present invention also relates to a method for isolating embryo sac-specific proteins from a plant, whereby an antibody of the invention is used to screen and to isolate embryo sac-specific pro-teins derived from the plant.
Thus, the present invention also relates to trans-genic plants, parts of a plant, plant tissue, re-productive tissue, plant seeds, plant embryos, plant seedlings, plant propagation material plant harvest material, plant leaves and plant pollen, stamen, cobs, nodes, flowers, plant roots contain-ing the above identified plant cells of the present invention. These plants or plant parts are charac-terised by, as a minimum, the presence of the het-

-44-erologous transferred DNA construct of the present invention in the genome, or, in cases where the transferred nucleotide sequence is autologous to the transferred host cell, are characterised by ad-ditional copies of the nucleotide sequence of the present invention and/or a different location within the genome. Thus, the present invention also relates to plants, plant tissue, plant repro-ductive or vegetative tissue, plant seeds, plant seedlings, plant embryos, propagation, harvest ma-terial, leaves, nodes, cobs, stamen, fruits, flow-ers, pollen, roots, calluses, tassels, etc. non-biologically transformed which possess, stably or transiently integrated in the genome of the cells, for instance in the cell nucleus, plastides or mi-tochondria, heterologous and/or autologous nucleo-tide sequences containing a) a coding sequence of the present invention and/or b) a regulatory ele-ment of the present invention recognised by the po-lymerases of the cells of the said plant. In a preferred embodiment, the coding sequence of the present invention is operably linked in sense or antisense orientation to at least one regulatory element, for instance the regulatory sequence of the present invention. In a further preferred em-bodiment a regulatory element, in particular the regulatory sequence of the present invention is op-erably linked to a coding sequence of a gene of in-terest cloned in sense or antisense orientation to said regulatory sequence. The teaching of the pre-sent invention is therefore applicable to any plant, plant genus or plant species wherein the regulatory elements mentioned are recognised by the polymerases of the cell. Thus, the present inven-

-45-tion provides plants of many species, genera, fami-lies orders and classes, and is able to recognise the regulatory elements of the present invention or derivatives or parts thereof. Any plant is consid-ered, in particular plants of economic interest, for example plants grown for human or animal nutri-tion, plants grown for the contents of useful sec-ondary metabolites, plants grown for the content of fibres, and trees and plants of ornamental inter-est. Examples which do not imply any limitation as to the scope of the present invention are corn, wheat, barley, rice, sorghum, sugarcane, sugar beet, soybean, Brassica, sunflower, carrot, to-bacco, lettuce, cucumber, tomatoes, potato, cotton, Arabidopsis, Lolium, Festuca, Dactylis or poplar.
The present invention also relates to a process, in particular a microbiological process and/or techni-cal process, for producing a plant or reproduction material of said plant, including an heterologous or autologous DNA construct of the present inven-tion stably or transiently integrated therein, and capable of being expressed in said plants or repro-duction material, which process comprises trans-forming cells or tissue of said plants with a DNA
construct containing a nucleic acid molecule of the present invention, i.e. a regulatory element which is capable of causing the stable integration of the ZmES derived sequences in particular a coding se-quence in said cell or tissue and enabling the sense or antisense expression of a ZmES derived se-quence, in particular coding sequence or part thereof in said plant cell or tissue, regenerating plants or reproduction material of said plant or

-46-both from the plant cell or tissue transformed with said DNA construct and, optionally, biologically replicating said last mentioned plants or reproduc-tion material or both. The present invention also relates to the above process, wherein instead or in addition to the ZmES derived, in particular coding sequence, a regulatory element of the ZmES gene of the present invention is transformed into a plant, preferably operably linked to a coding sequence of interest.
The present invention also relates to a kit com-prising the nucleotide sequence of the present in-vention and /or the protein of the present inven-tion and/or the antibodies of the present inven-tion. The kit of the present invention is useful in detecting genes involved in embryogenesis and/or endosperm development. The present invention also relates to the use of the nucleotide sequence of the present invention and the protein and/or the antibody of the present invention for the produc-tion of embryo and endosperm development in modi-fied plants.
Further preferred embodiments of the present inven-tion are mentioned in the subclaims.
The invention may be more fully understood from the following figures and detailed sequence descrip-tions, which are part of the present teaching. The SEQ ID No. 1 to 45 are incorporated in the present invention. The numbering for each DNA sequence corresponds to the genomic clone of the gene in question.

-47-Figure 1 shows that mature ZmES1-4 peptides display structural homology to defensins.
(a): Homology between mature ZmES1/2 peptides, pro-teinase inhibitors (PI) and y-thionins (yThi). N-terminal signal peptides of all proteins show no or few homology among each other and were cleaved of.
The consensus sequence of the putative mature pep-tides is shown below the alignment. Accessions of proteins used to create the alignment are as fol-lows: putative proteinase inhibitors II and P322 from Arabidopsis thaliana, Oryza sativa, Brassica rapa, Solanum tuberosum, Glycine max (AtPI II:
AC005936; OsPI: AA317095; BrPI II: L31937; StPI
P322: P20346; GmPI P322: Q07502), y-thionins from Nicotiana tabacum, Picea abies (NtyThi: P32026;
PayThi: 0AA62761) and an a-amylase inhibitor from Sorghum bicolor (SbAAI: S13964).
(b): The predicted secondary and tertiary structure of mature ZmES peptides resemble the NMR structure of the plant defensin RsAFP1 from radish and charybdotoxin, a neurotoxin from scorpion. The pre-dicted (pred.) secondary structures are printed in grey (arrows indicate 3-strands, cylinder a-helices and lines coil regions). Tertiary structures of ma-ture RsAFP1 seeds (Terras et al., 1995; PDB acces-sion # lAYJ) and charybotoxin (Bontems et al., 1992) have been determined by NM?. crystallography (NM?.) and are printed in black. The lines below the sequences display the position of four (RsAFP1) or three (charybdotoxin) intramolecular disulfide bridges formed between cysteine residues. The posi-tions of all eight (six in the case of Charyb-

-48-dotoxin) Cys (C) are conserved in peptide sequences shown in (a) and (b) indicating that probably all plant defensins form four intramolecular disulfide bridges and thus probably function as monomers.
Figure 2 shows the expression of ZmES1-4 in differ-ent maize tissues, in embryo sac cells as well as in different stages of in vitro and in in vivo zy-gotes.
(a): Multiplex RT-PCR analysis using tissues and cells indicated. Gene specific primers were used to amplify cDNA of ZmES1 and Zmcdc2, respectively.
Zmcdc2 contains an intron between the primers used.
The corresponding genomic DNA was loaded onto the last lane. The ethidiumbromide gel was blotted and hybridized with the full length ZmES1 cDNA (below).
(b) and (c): RT-PCR analysis with cells of the fe-male gametophyte, of the ovule and leaf of maize, which were manually isolated. Different zygote and embryo stages were analysed after IVF. ZmES1 (b) or ZmES2/3/4 (c) transcripts were RT-PCR amplified with gene specific primers in the cells indicated, blotted after gel separation and hybridized with the full length ZmES4 cDNA. AP: antipodals, CC:
central cell, EC: egg cell, Emb: embryo (h/d after IVF), MC: leaf mesophyll cell, Nu: nucellus cells, SY: synergid, Z: zygote (h after IVF).
Figure 3 shows the expression of ZmES in the egg apparatus of maize.
(a) and (b): Median cut sections of ovules contain-ing the embryo sac were hybridized with a ZmES4 an-

-49-tisense probe. A purple signal shows the presence of ZmES4 clearly in the synergids and more faint in the egg and central cell. In nucellus and integu-ments no signal was detected.
(c): A similar section was hybridized with a ZmES4 sense probe, showing no hybridization signal.
(d): A median cut section of an ovule containing the embryo sac was stained with acridine orange to show nuclei and to monitor RNA content of sections used for in situ hybridization. CC: central cell, EC: egg cell, SY: synergid. Bars: 60 pm.
Figure 4 is a whole mount in situ hybridization showing that ZmES transcripts are uniformly dis-tributed in the cytoplasm of isolated female ga-metophyte cells.
(a): Egg and nucellus cells hybridized with a ZmES4 antisense probe.
(b): Egg cell hybridized with a ZmES4 sense probe.
(c): Central cell, synergid and nucellus cells hy-bridized with a ZmES4 antisense probe.
(d): Central cell and nucellus cells hybridized with a ZmES4 sense probe.
(e), (f) and (g): Acridine orange staining to dis-play total RNA distribution within synergid, cen-tral cell and nucellus cells, respectively. Bars:

50 pm.
Figure 5 shows green fluorescence protein (GFP) ex-pression in transgenic maize plants driven by 1594 bp promoter region upstream of the transcription start point of ZmES4.
(a): The expression pattern of ZmES4::GFP fusion protein in the ovary tissue around the embryo sac under light microscopy.
(b): The same preparation as in (a) under UV light microscopy.
(c): The same preparation as in (a) using CLSM.
SEQ ID No. 1 represents the full length cDNA se-quence of the ZmES1 (Zea mays embryo sac) gene, from and including position 619 towards the 5' end, up to and including position 1204.
SEQ ID No. 2 represents the full length cDNA se-quence of the ZmES2 gene, from and including posi-tion 1 towards the 5' end, up to and including po-sition 517.
SEQ ID No. 3 represents the full length cDNA-sequence of the ZmES3 gene, from and including po-sition 1 towards the 5' end, up to and including position 501.
SEQ ID No. 4 represents the full length cDNA se-quence of the ZmES4 gene, from and including posi-tion 1850 towards the 5' end, up to and including position 2430.
SEQ ID No. 5 represents the protein coding region of ZmES1, from and including position 702 towards the 5' end, up to and including position 977 (ex-cluding the stop-codon).

-51-SEQ ID No. 6 represents the protein coding cDNA re-gion of ZmES2, from and including position 77 to-wards the 5' end, up to and including position 349 (excluding the stop-codon).
SEQ ID No. 7 represents the protein coding cDNA re-gion of ZmES3, from and including position 78 to-wards the 5' end, up to and including position 350 (excluding the stop-codon).
SEQ ID No. 8 represents the protein coding region of ZmES4, from and including position 1927 towards the 5' end, up to and including position 2199 (ex-cluding the stop-codon).
SEQ ID No. 9 represents the amino acid sequence of the ZmES1 protein.
SEQ ID No. 10 represents the amino acid sequence of the ZmES2 protein.
SEQ ID No. 11 represents the amino acid sequence of the ZmES3 protein.
SEQ ID No. 12 represents the amino acid sequence of the ZmES4 protein.
SEQ ID No. 13 represents the full length genomic clone of the ZmES1 gene, from and including posi-tion 1 towards the 5' end, up to and including po-sition 1204; at the position 587 is a TATA se-quence, at the position 702 is a ATG sequence (start codon) at the position 978 is a TAA sequence (stop codon).

-52-SEQ ID No. 14 represents the full length genomic clone of the ZmES4 gene, from and including posi-tion 1 towards the 5' end, up to and including po-sition 2430; at the position 1817 is a TATA se-quence, at the position 1927 is a ATG sequence (start codon) and at the position 2200 is a TGA se-quence (stop codon).
SEQ ID No. 15 represents the full length promoter of the ZmES1 gene, from and including DNA sequence of the position 1 towards the 5' end, up to and in-cluding position 701.
SEQ ID No. 16 represents a partial DNA sequence of the promoter of the ZmES1 gene; the sequence spans the region from and including position 501 towards the 5' end, up to and including position 701.
SEQ ID No. 17 represents a partial DNA sequence of the promoter of the ZmES1 gene; the sequence spans the region from and including position 201 towards the 5' end, up to and including position 701.
SEQ ID No. 18 represents the transcribed 5'-untranslated region (UTR) of the ZmES1 gene, from and including position 619 towards the 5' end, up to and including position 701.
SEQ ID No. 19 represents the transcribed 5'-untranslated region of the ZmES2 gene, from and in-cluding position 1 towards the 5' end, up to and including position 76.
SEQ ID No. 20 represents the transcribed 5'-untranslated region of the ZmES3 gene, from and in-

-53-cluding position 1 towards the 5' end, up to and including position 77.
SEQ ID No. 21 represents the transcribed 5'-untranslated region of the ZmES4 gene, from and in-cluding position 1850 towards the 5' end, up to and including position 1926.
SEQ ID No. 22 represents the 3'- termination region including the Poly A addition sequence of the ZmES1 gene, from and including position 978 towards the 5' end, up to and including position 1223.
SEQ ID No. 23 represents the 3'-termination region including the Poly A addition sequence of the ZmES2 gene, from and including position 350 towards the 5' end, up to and including position 537.
SEQ ID No. 24 represents the 3'-termination region including the Poly A addition sequence of the ZmES3 gene, from and including position 351 towards the 5' end, up to and including position 519.
SEQ ID No. 25 represents the 3'-termination region including the Poly A addition sequence of the ZmES4 gene, from and including position 2200 towards the 5' end, up to and including position 2449.
SEQ ID No. 26 represents the full length DNA se-quence of the promoter of the ZmES4 gene, from and including position 1 towards the 5' end, up to and including position 1926.
SEQ ID No. 27 represents a partial DNA sequence of the promoter of the ZmES4 gene; the sequence spans

-54-the region from and including position 1699 toward the 5' end, up to and including position 1926.
SEQ ID No. 28 represents a partial DNA sequence of the ZmES4 gene; the sequence spans the region from and including position 1499 towards the 5' end, up to and including position 1926.
SEQ ID No. 29 represents the partial DNA sequence of the promoter of the ZmES4 gene; the sequence spans the region from and including position 999 towards the 5' end, up to and including position 1926.
SEQ ID No. 30 represents the partial DNA sequence of the promoter of the ZmES4 gene; the sequence spans the region from and including position 499 towards the 5' end, up to and including position 1926.
SEQ ID No. 31 represents the partial DNA sequence of the promoter of the ZmES4 gene; the sequence spans the region from and including position 199 towards the 5' end, up to and including position 1926.
SEQ ID No. 32 to 44 represent primers used in ob-taining the ZmES genes.
SEQ ID No. 45 represents a 1594 bp promotor region of ZmES4 that was used for monitoring expression of the promotor of ZmES4 after stable integration into the maize genome.

-55-The following examples are offered to more fully illustrate the invention, but are not construed as limiting the scope thereafter.
Examples:
Materials and Methods used throughout the examples Plant material, isolation of cells from the embryo sac, in vivo and in vitro fertilisation Maize (Zea mays) inbred line A188 (Green and Phil-lips, 1975) were grown under standard green house conditions.
Cells of the embryo sac were mechanically isolated from digested ovule tissues with glass needles and transferred using a hydraulic microcapillary system according to Kranz et al. (1991). In vitro zygotes were generated after fusing isolated gametes by a short electric pulse and cultivated as described (Kranz and Lorz, 1993). In vivo zygotes were gen-erated as described by Cordts et al. (2001). The cells were collected and fixed on glass slides or stored in 200 nl each at -800 until usage.
Light microscopy 2 mm thick slices of spikelets were fixed in 4%
paraformaldehyde in 0.005 M phosphate buffer, pH
7.2. The slices were washed in 0.1 M phosphate buffer, dehydrated in ethanol series and infil-trated in gradient steps of butyl-methyl methacry-late, followed by UV polymerisation (Wittich and Vreugdenhil, 1998). Sections of 3 pm were made

-56-with a Reichert Ultramicrotome, stretched on water, and dried on microscope slides at 60 C for 1 hour.
The resin was removed from the sections by washing the slides in pure acetone for 15 minutes. The slides were then washed in water and sections were stained with toluidine blue (O'Brien et al., 1965).
Differential plaque- and reverse Northern screening RT-PCR-based cDNA libraries generated from isolated egg cells and in vitro zygotes (Dresselhaus et al., 1994; 1996) were screened by differential plaque screening (Dresselhaus et al., 1996). Double plaque lifts were made from 15 cm plates of the egg cell library at a density of 500 p.f.u. (plaque-forming units). The filters were hybridised either with PCR amplified [32P]-cDNA from the egg cell or the zygote cDNA library. CDNA clones from the egg cell library selected by this screening were fur-ther analysed by a differential insert screening ("reverse Northern screening"; Dresselhaus et al., 1999 a/b). The cDNA clones were amplified by PCR, separated in agarose gels, blotted and hybridised either with the radiolabelled, PCR amplified cDNA
population of the egg cell library or the zygote library. The isolated cDNA clones were further hy-bridised, with uncloned cDNA populations of egg cells and zygotes as a control. The following gene specific primers were used to specifically amplify the different subgroups of the ZmES gene family:
ZmES1 (5'-CCOTTGGATTGGATTGGATCG-3' SEQ ID No. 32 and 5'-ACCACCGGTTTCCTGCTGTC-3' SEQ ID No. 33) and ZmES2/3/4 (5'-TCTTCACGAGGGAAGCTGTCT-3' SEQ ID No.
34 and 5'-GCACTGCA000ACCGCTCTT-3' SEQ ID No. 35).

. .

-57-RT-PCR
Total RNA from different maize tissues was isolated using TRIZOL (Gibco-BRL) after the manufacturers recommendations. For quantification, total RNA was separated in a formaldehyd-gel, transferred over-night with 10x SSC to Hybond N+ membrans (Amersham Pharmacia Biotech) and hybridized with a radio-labelled 18S raNA probe. RNA was quantified using a bioimager system (BAS-1000, Fuji). One pg quanti-fied total RNA of each sample was used for RT-PCR
analysis. To avoid ampification from remnant ge-nomic DNA in the sample, total RNA was treated prior RT reaction with DNaseI for 15 min at RT af-ter the manufacturers recommendations (Gibco-BRL).
The reaction was stopped by adding EDTA (25 mM) and by incubation for 10 min at 70 C. The RNA was primed with a T14- A/G/C-primer (Metabion) and re-verse transcribed in 20 pl final volume using 50 U
SuperscriptTM reverse transcriptase (Gibco-BRL) for 60 min. The reaction was stopped by incubating for .10 min at 70 C. Multiplex RT-PCR: 50 pl reactions containing 100 ng of total RNA and the primer pairs for ZmES1 (forward: 5'- CCCTTGGATTGGATTGGATCG-3' SEQ ID No. 32, reverse: 5'-GTCATTACCACCACAGACTTC-3' SEQ ID No. 42) and Zmcdc2 (forward: 5'-ACTCATGAGGTAGTGACATT-3' SEQ ID No. 43, reverse: 5'-CATTTAGCAGGTCACTGTAC-3' SEQ ID No. 44; Sauter et al., 1998) were run on a TGradient Cycler (Bio-metre). 30 cycles, with a first denaturation step at 96 C for 60 sec, were performed with the follow-ing parameters: 96 C for 30 sec, 58 C for 30 sec and 72 C for 60 sec, followed by a final extension at 72 C for 10 min before soaking at 4 C. Single-VR3010492.4 PCVEN1/02258

-58-cell RT-PCR analysis with one primer pair was car-ried out as described by Richert et al. (1996) us-ing primers SEQ ID No. 32-35 with a few modifica-tions as described by Cordts et. (2001).
DNA gel blot analyses Extraction of genomic DNA from 10-day old seedlings was performed according to Dellaporta et al.
(1983). 10 pg genomic DNA
was digested with the restriction enzymes indicated and resolved on 0.8%
agarose gels. DNA was transferred to Hybond membranes (Amersham Pharmacia Biotech) with 0.4 M
NaOH. Blots were hybridised with radioactive probes prepared by Prime-It Random Primer Labelling Kit (Stratagene, USA) in CHURCH buffer (7% SDS, 0.5 M NaH2PO4, pH 7.2, 1mM EDTA) containing 100 pg/ml salmon sperm DNA. Filters were washed with de-creasing concentrations of SSC, with a final wash at 65 C in 0.2 x SSC / 0.1% SDS. Filters were ex-posed at -70 C to Kodak X-Oma.rAR films using in-tensifier screens.
In situ hybridisation Ovule pieces containing embryo sacs were fixed in 4% formaldehyde, 0.25% glutaraldehyd and embedded in butyl-methyl-methacrylat (BMM) (Gubler 1989;
Baskin et al. 1992). The embedded tissues were sectioned on glass knives with an ultramicrotom at to 7 pm thickness.
A whole mount in situ hybridisation protocol was developed for isolated cells of the embryo sac.
Cells were temporarily collected after isolation in

-59-540-650 mosmol kg-1- mannitol and then placed in drops of fixation solution (540-650 mosmol kg-1 mannitol, 4% formaldehyde, 0.25 glutaraldehyd) on mounted glass coverslides (bindsilane; Wacker-Chemie). The cells were always submerged in liq-uids. After 30 min incubation, the samples were postfixed for 15 min by adding droplets of PBS-buffer containing 20% acetic acid. The samples were dehydrated by passage through a graded ethanol series (10% to 70%) and stored at 4 C or directly used for further steps. The solution was gradually substituted with hybridisation solution (10 mM
Tris-HC1 (pH 7,5), 300 mM NaCl, 50% formamid, 1mM
EDTA, 1 x Denharts and 10% dextransulphate) or in Dig-Easy-Hyb (Boehringer Mannheim) containing 250 ng/ml tRNA and 100 pg/ml poly(A) oligonucleotide.
The glass cover slides with sticking cells were placed in small (diameter of 35 mm) plastic petri dishes in a volume of 500 pl hybridisation solu-tion. 1 pg/m1 labelled probe was added to the hy-bridisation solution. Washing and detection steps were made by submerging the plastic dishes in lar-ger volumes of the appropriate solutions. Hybridi-sation, washing steps and detection were performed for sectioned material and whole mount cells in the same manner.
Antisense and sense RNA probes were labelled in vi-tro from cDNA inserts in pBluescript II SK- with digoxigenin-UTP by T7 or T3 RNA polymerase using a digoxygenin RNA Labelling kit (Boehringer Mann-heim).
Hybridisation was carried out at 43 C
overnight. Washing steps were performed as fol-lows: 10 min at 43 C, 30 min in 1 x SSC/0.01% SDS

= .

-60-and once 30 min in 0.5 x SSC/0.01% SDS followed by digestion with RNase A (Boehringer, Mannheim). Af-ter washing three times in 1% NaC1, detection was made using an anti-digoxigenin antibody conjugated with alkaline phosphatase and NBT/BCIP detection system (Boehringer Mannheim).
DNA and protein sequence analyses Selected cDNAs were excised from the XZAP XR vector according to the manufacturer's specifications (Stratagene). All clones were sequenced from both directions using Tag DNA polymerase FS Cycle Se-quencing Kit (PE APPLIED BIOSYSTEMS) and the 373A
and 377 automated DNA sequences (APPLIED
BIOSYSTEMS). DNA and amino acid sequence data were further processed using the PC DNASIS program soft-ware package (Hitachi Software Engineering).
Se-quence data were compiled and compared online with EMBL, GenBank, DDBJ, SwissProt, FIR and PRF data-bases with FASTA and BLAST algorithms (Pearson, 1990).
Protein alignment was performed with the CLUSTAL W program (Thompson et al., 1994). Predic-tion of protein localization sites was performed online using PSORT and the signal peptide cleavage site was identified after Nielson et al. (1997).
Secondary and tertiary structure prediction was performed and with PDB (protein data bank).
Isolation of genomic clones

-61-Genomic DNA was isolated from the maize inbred line A188 according to Dellaporta et al. (1983), par-tially digested with Sau3A and size fractionated using a saccharose gradient (Sambrook et al., 1989). DNA fragments between 13-23 kb were cloned into the BamHI site of the Lambda Dash II vector (Stratagene) according to the manufacturers speci-fications. Genomic clones containing ZmES1-4 se-quences were identified after using ZmES1-4 cDNA
clones as probes.
In order to obtain upstream sequences, the Univer-sal Genome Walker Kit (Clontech) was used. The protocol from the kit was modified as follows: to prepare adaptor ligated DNA, 2,5 pg of X-DNA was digested in 100 pl reaction volumes with 80 U of different restriction enzymes (Dral, EcoRV, Pvull, Scal and Stul) overnight at 37 C using buffers rec-ommended by Clontech. The DNA was extracted once with chloroform/isoamyl alcohol (24:1) vol./vol., once with chloroform, and then precipitated by ad-dition of 1/10 (vol/vol) 3 M Na0Ac (pH 4.5), 20 pg glycogen and 2 vol. of 95% Et0H. After vortexing, the tubes were immediately centrifuged at 15.000 rpm in a microcentrifuge for 5 min. The pellets were washed with 80% Et0H and immediately centri-fuged as above for 5 min, air dried and dissolved in 20 pl of 10 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA.
From each tube 4 pl of DNA was ligated to an excess of adaptor overnight at 16 C under the following conditions: 1.9 pl Genome Walker Adaptor (25 pM), 0.5 pl T4 DNA Ligase (1U/p1), 1.6 pl 5x ligation buffer in a total volume of 8 pl. The ligation re-action was terminated by incubation of the tubes at

-62-70 C for 5 min, then diluted 10-fold by addition of 72 pl of 10 mM Tris-HC1 (pH 7.4) and 1 mM EDTA (pH
7.4). The Biometra trioblock was used for all in-cubation reactions. FOR
amplifications were per-formed using TaqDNA Polymerase (Gibco Life tech-nologies). Primary PCR reactions were conducted in 50 pi volume containing 1 ul of ligated and diluted DNA, 5 pl 10X PCR buffer, lpl dNTP (10 mM each), 2.2 pl Mg(0Ac)2(25 mM), 1 ml adaptor primer (10 mM) API (5'-GTAATACGACTCACTATAGGGC-3', SEQ ID No. 36) and each 1 pl gene specific primer (10 mM) GSP1 (5'-CTTGACGCAGTAGCAGAGAATCCCGTC-3', SEQ ID No. 37) or GSP2 (5'-CAGTAGTCCGACCGCACGCACAG(A/g)TG-3', SEQ
ID No. 38), and 1.25 U Taq DNA Polymerase. The FOR
cycles were conducted as described by the manufac-turer. A
secondary FOR (nested PCR) reaction was performed with 1 pi of a 100 fold dilution of the primary FOR using adaptor primer AP2 (5'-ACTATAGGGCACGCGTGGT-3', SEQ ID No. 39) and nested GSP3 (5'-CAGACAGCTT000TCGTGAAGCT000ATTG-3', SEQ ID
No. 40) and GSP4 (5'-TCTG
(c/T)GTCAGGCAGTC(T/g)CGTGCCTCAAC-3', SEQ ID No.
41), respectively. The PCR
cycles of the second reaction were conducted as described by the manu-facturer. PCR products were cloned using TA clon-ing vectors (Invitrogen) and sequenced. Upstream sequences of ZmES1 and 4 could thus be cloned. The analysis of the genomic clones and genomic DNA fur-ther showed that ZmES1-4 gens contain no introns.

. V14301/602,4 . . -63-Biolistic Transformation and Analyses of Transgenic Maize Plants 1594 bp promoter region of ZmES4 (SEQ ID No. 45) was used for monitoring expression of the promoter of ZmES4 after stable integration into the maize genome. A construct consisting of SEQ ID No. 45, a part of the cDNA of ZmES4 (bp 2 to bp 351 of SEQ ID
No. 4), the coding region of GFP (pMon30049; Mon-santo) and the NOS-terminator (McElroy et al., 1995) was generated using the vector Litmus 29 (New England Biolabs). Immature embryos from maize in-bred line A188 were isolated 12 days after hand-pollination and co-bombarded with the construct de-scribed above and the p35S::pat vector containing phosphinotricin-acetyl-transferase as selection marker. Experimen-tal procedures followed the protocol of Brett-schneider et. al. (1997), except that embryos were bombarded with partial vacuum 28 inch Hg and gas pressure 1350 psi. Cultivation, regeneration and selection was carried out as described by Bret-tschneider et. al. (1997).
GFP analysis in transgenic maize plants Immature ears with silks of 15 cm length (counted from bottom part of the ear), were harvested from transgenic plants (lines containing full length in-tegrations of the pZMES::ZMES: :GFP: :NOS construct as analysed by gel blots; data not shown), kernel were excised and cut in the middle part with razor blades or scalpels. The part of kernels containing the embryo sac was transferred into a 650 mOsm man-nitol solution and the nucellar tissue dissected out of the submerged ovary tips. The embryo sac was preparated with fine-tipped glass needles using an inverted microscope. The preparations were analysed by light and UV microscopy, or a Confocal Laser Scanning Microscope (CLSM) for presence of fluores-cence in ovary tissues.
Example 1:
Isolation of the ZmES Gene Family from Maize Egg Cells The female gametophyte of maize is deeply embedded in the maternal tissues of the ovule. Gene expres-sion patterns in cDNA libraries of unfertilised egg cells and in vitro zygotes were compared as a starting point for molecular investigations. With the aim of identifying genes completely downregu-lated after IVF (in vitro fertilisation) and not expressed elsewhere in the plant, 29,000 pfu (plaque forming units) from the egg cell library were analysed. Double plaque lifts were hybridized with the egg cell cDNA population and either with cDNA from in vitro zygotes or cDNA from seedlings.
250 clones were selected and further analysed in reverse Northern blot analysis. 44 different cDNA
clones, which were highly represented in the egg cell library and not or weakly in the zygote li-brary, were fully sequenced. Ten cDNAs were highly homologous to each other and were further analysed.
These ten cDNAs represent four different genes (ZmES1-4) (see SEQ ID Nos. 1 to 4).

A reverse Northern blot indicates that the whole gene family is completely switched off after IVF
and minimal transcript amounts remain detectable 18 h after IVF.
DNA and protein sequence alignments display a high degree of sequence homology among the different ZmES gene family members; even 5' and 3' UTRs (un-translated regions) (SEQ ID No. 18 to 25) are highly conserved. ZmES1 is more distinct from the other ZmES members, both at the DNA and protein level, but all general features, such as transcrip-tion start point, two stop codons, putative CPE
element and poly(A) signal site at the DNA level, are identical. At the protein level, signal pep-tide cleavage site and cysteine residues are also identical. The longest cDNA clones of all ZmES
members start more or less at the same position with one or two Gs. These Gs are missing in ge-nomic clones of ZmES1 (SEQ ID No. 13) and ZmES4 (SEQ ID No. 14) and most likely result from the 7mG
cap at the 5' end of all messenger RNAs. This is a strong indication that the isolated cDNAs with SEQ
ID No. 1 to 4 are of full length.
ZmES1-4 encode small proteins of 92 and 91 aa (amino acids), shown in SEQ ID No. 9 to 12, respec-tively. A putative signal peptide is located at the N-terminus of all proteins. A
hydrophobic amino acid cluster at the N-terminus of the precur-sor protein is framed by two basic and one basic amino acid, respectively. The predicted cleavage site is after position 31 (ZmES1) (SEQ ID No. 9) or 30 (ZmES2/3/4) (SEQ ID Nos. 10-12). It is further predicted that the proteins are translocated out-side the cell, including the cell wall. The MW of ZmES1-4 precursor proteins is 9.7 kDa, and the pI
varies between 8.1 and 8.5, and is thus slightly basic. The mature proteins are cysteine-rich and extremely conserved, with little variation at the C-terminus and the predicted MW is 6.5 kDa, while the pI is between 7.9 and 8.3. The structural ho-mology to defensins is shown in figure 1.
To investigate the size of the ZmES gene family and their presence in other related genomes, genomic Southern blot analysis with two different maize in-bred lines and the diploid maize relative Trilosacum dactyloides was performed. All enzymes used do not cut within the cDNA, nor within the corresponding genomic sequences. Four bands were detected in A188, the maize line used to generate the cDNA li-braries. The same number of bands was detected in another inbred line (373), while two to three bands were detected in Trilosacum. According to the pre-sent invention, the whole gene family from the maize inbred line A188 was isolated.
SEQ ID Nos. 1 to 4 illustrate the full length cDNA
sequences, SEQ ID Nos. 5 to 8 the protein coding nucleotide sequences and SEQ ID Nos. 9 to 12 the amino acid sequence of ZmES1 to 4.
SEQ ID Nos. 13 and 14 represent the full length nu-cleotide sequences of the genomic clone of ZmES1 and 4, thus incorporating in 5' to 3' direction the promoter, the transcribed 5' untranslated region (UTR), the protein coding region and the 3' tran-scription termination region. SEQ ID Nos. 15 to 17 represent the full length promoter and promoters of reduced length of the ZmES1 gene.
SEQ ID Nos. 18 to 21 represent transcribed, but not translated regions of ZmES1 to 4 possibly function-ing as expression modulating elements. The UTR nu-cleotide sequence elements are included in most of the promoter fragments illustrated in SEQ ID Nos.
13 to 31. However, the present invention also en-compasses the promoters and fragments thereof indi-cated in SEQ ID Nos. 13 to 31 wherein the UTRs of SEQ ID Nos. 18 to 21 are missing.
SEQ ID Nos. 22 to 25 represent the 3' transcription termination sequences of ZmES1 to 4 containing pos-sibly important elements for the regulation of transcription.
SEQ ID Nos. 26 to 31 represent full length promo-ters and promoters of reduced length capable of promoting and/or enhancing transcription with em-bryo sac-specificity.
Example 2:
ZmES1-4 are specifically expressed in all cells of the embryo sac and switched off after IVF
In order to investigate whether ZmES1-4 are exclu-sively expressed in egg cells, total RNA, poly(A)+
RNA Northern blot and RT-PCR analysis was performed with many different tissues at distinct developmen-tal stages. No signal was obtained in any tissue tested (see figure 2 as an example).

Tissue in situ hybridisation was performed to in-vestigate the expression of ZmES1-4 in ovules at maturity; strong signals were detected in the cyto-plasm of two synergides already after short detec-tion time (see figure 3). Signals in nucellus cells, integuments or ovary tissues were never ob-served. A problem with in situ hybridisation of ovule tissue was that the structure after embedding in paraffin wax and sectioning was not conserved.
Tissues had to therefore be embedded in BMM (butyl-methyl-methacrylate), which however only allowed the generation of very thin sections. The struc-ture after BMM embedding is conserved, the sections still contain RNA, but cells contain only few cyto-plasm due to the slight thickness of each section, thus making in situ hybridisation less efficient.
In addition, all embryo sac cells are very large and highly vacuolated, thus making the detection of transcripts within these cells even more difficult.
The embryo sac in its different cell types was therefore dissected and single cell RT-PCR was ap-plied to investigate ZmES1-4 transcript contents.
As shown in figure 2 ZmES1-4 transcripts are ex-pressed at comparable levels in all egg cells tested and in most of the synergides and central cells. Some 15 antipodal cells were used under the same RT-PCR conditions for a single reaction, and a much smaller signal was detected, or no signal at all. After IVF, ZmES2/3/4 transcripts were detect-able at very low levels in few zygotes up to 42 h after IVF. ZmES1 transcript was detected until 18 h after IVF and 24 h after in vitro pollination in in vivo zygotes. After the first cell division, which generally occurs between 42 and 46h after IVF, transcripts were no longer detectable. No transcripts could be detected in different embryo stages, in nucellus or leaf mesophyll cells.
Example 3:
ZmES1-4 transcripts are uniformly distributed in cytoplasms of embryo sac cells An in situ hybridisation protocol with isolated em-bryo sac cells was developed to investigate whether ZmES1-4 transcripts are localized at different poles within the cells of the embryo sac: as shown in figure 4 transcripts were detected in egg cells, synergides and central cells. No detection was ob-served in nucellus cells adjacent to central cells.
It seems that ZmES1-4 transcripts are uniformly distributed in these cells, which is best seen in egg cells: in maize egg cells, numerous small vacu-oles are located in the periphery of the cells and give no signal. To monitor total RNA distribution, embryo sac and nucellus cells were stained with ac-ridine orange. Total RNA
displays a similar pat-tern than ZmES1-4 transcripts and is uniformly dis-tributed in the cytoplasm of the cells studied.
Example 4:
The ZmES4 promoter is exclusively active in embryo sac cells of transgenic maize lines As shown in Figure 5, some 1.6 kbp upstream of the transcription start point of ZmES4 is sufficient to drive a cell-specific expression of the ZmES4::GFP

fusion protein in the cells of the female gameto-phyte (embryo sac). Figure 5b shows a signal of the fusion protein in the two synergids and very strong signals around the filiform apparatus. CLSM analy-sis displays an expression also in the egg and cen-tal cell, but the strongest signals were observed in the region of the egg apparatus. All other cells/tissues of the ovary never showed any fluo-rescence of the GFP-fusion protein.

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SEQUENCE LISTING
<110> 1) Sudwestdeutsche Saatzucht.. 2)Advanta Seeds B.V
<120> Embryo Sac-Specific Genes <130> 23932 <140>
<141>
<150> 00104366.0 <151> 2000-03-02 <160> 45 <170> PatentIn Ver. 2.1 <210> 1 <211> 606 <212> DNA
<213> Zea mays <400> 1 gaatagttcc accacgttac ttccatatat atttcccttg gattggattg gatcgtcggc 60 gcccaaacga ataataatcc ggcaatggag ccttcacgag ggaagctgtc tgccgccgcc 120 gtcctcctgc tgatgacgac gctcctcgtg gtggccgcca tgcgggcggt tgaggcacgc 180 gactgcctga cacagagcac ccggttaccg gggcacctgt gcgtgcggtc ggactactgc 240 gcgatcgggt gcagggcgga gggcaagggc tacacgggcg gcaggtgcct catctctccc 300 atcccgctcg acgggattct ctgctactgc gtcaagccgt gcccatccaa cacgacgaca 360 taatgatgag acaaagagcg gtgggtgcag tgcacgctgg ccgggggtta tcagtccaca 420 catcctaccg tacgtgtctg tgttaataac ttttttttgt cttggaagtc tgtggtggta 480 atgactttta aatgtcttgg aataaaccgg gttctagtcc cttataagct agcagtactg 540 taacaattca gatcatcaaa gacagcagga aaccggtggt tgagttgaaa aaaaaaaaaa 600 aaaaaa 606 <210> 2 <211> 537 <212> DNA
<213> Zea mays <400> 2 gatagttcca ccacgttact tccatatatt tcccttggat tggatcgtcg gcgcccaaac 60 gaacaataat ccggcaatgg agtcttcacg agggaagctg tctgccgccg gcgtcctcct 120 gctaatgacg ctcctcatgg tggccgccat gcgggcggtt gaggcacgag actgcctgac 180 gcagagtacc cggttaccgg ggcatctgtg cgtgcggtcg gactactgcg caatcgggtg 240 cagggcggag ggcaagggct acacgggcgg caggtgcctc atctctccca tcccgctcga 300 cgggattctc tgctactgcg tcaagccttg cacatccacc acgacagaat gatgagacaa 360 gagcggtggg tgcagtgcag gctgatcggg gggttatcag ttatatatgg acatcctacc 420 gtgtctgtta ataacttgta aatatcttgg gaagtttgtg gtgataagtt ttaaatgtct 480 tggaataaaa tgggttctat atagacttct actcgttaaa aaaaaaaaaa aaaaaaa 537 <210> 3 <211> 519 <212> DNA
<213> Zea mays <400> 3 ggatagttcc accacgttac ttccatatat ttcccttgga ttggatcgtc ggcgcccaaa 60 cgaatactaa tccggcaatg aagtottcac aagggaagct gtctgccgcc gccgtcctcc 120 tgctaatgac gctccttatg gtggccacca tgcgggcggt tgaggcacac gactgcctga 180 06Z pelobloqoq ;2bbboebol ob000p000 qoqob.2c000 bqbbeobbo6 bboeopqobb 081 522obbbe5b obbbeobobb boq2bobobq opqopbboob bobgbobqbq oqeobbbboo OZT eqqbb000eo b26poeo2bq oobqo2bobo eobbebqlbb ob65obT2oo boobbqbbqp 09 413040boe5 q22qobooq ooqboobobb bobqoqbqob eebb8pboeo lqoq525bge L <006>
eiCew eaZ <E-TZ>
VNICI <ZTZ>
ELZ <TTZ>
L <OTZ>
ELZ 22b eoeboeoopo o;eoeobqqo obppoqbobq 06Z oeqobqoqcq qebbboebog ob0000p000 goqoqeoqoo bqbbpobbob bboebeqobb 081 bppobbbebb obbbeobqbb 5oq25obobq beb.o2bboqb bobgbobqbq oq2obbbboo OZT eqqbb000po 6b bob oobqo2bebo eobbpbqqbb obbbobgeob boobbqbbqe og oqoagobopb Tepqobqopq bogbobboob oobqoqbqob epbbbebopo qqoqbpbble 9 <OOP>
PaZ <ETZ>
VNCI <ZTZ>
ELZ. <TTZ>
9 <OTZ>
9LZ popboe bo2o22ooq2 000bgboobp pogbobqopq ofiz obqoqoqq25 563260.436o oogp000.40.4 oqpoqoobqb bpobbobbbo 2oeqobbb22 081 obbbebbobb b2o6q6b5oq pbobobqopq o2bboqb5o6 qbobqbqope obbbbooelq OZT 58=323525 2323p5go36 ;325060236 52_6.44550S5 Bobqeooboo bbqbbqbogo og oq35005325 Tebqobqopq 30;5306005 oo5qoqbqo5 22bbbeboeb qqoobebbqe g <OOP>
SiC2W 2aZ <gTZ>
VNU <ZTZ>
9LZ <TTZ>
S <OTZ>

qqqbqqqobq o2qq22552b qlge441-1.1 5b.o5qoego2 06S qpeq1eb54q bqqbeeqqbo qopqoqqopb 2o2120qqb bbgbeppqep bbqqoqbgpe 086 elqqq522q2 bqbbqbqqqb 22-2556qqoq bb,eppqbqqo 22qppqq5qo qbq5002.4o3 0Z6 42o266g2q2 geqq.buoqPq q5b565oo25 lo55po5b.22 obqbbbqbbo bebp2o2b22 ogE opbebqpbqe 22P0e5OPOO 2=q2b2obq goob22ogbo bqopqobqob. oqqpbbboeb 00E oqpbo2ogeo ooqoqoq2qq oobqbbeobb obbbopoe;o bbbpeobbbe bbobbbuobq 06Z 55boTebobo 5qo2goeb5o qbboblbobq bqoqpobbbb oopqqbb000 2q5pbeobop 081 Sloobqopbe boeobbeboq bbobbbobqp boboobbqbb qeoqboobbo ebqp2gob3o ozT oqooqbobbo oboobqoqbq ob2pbbbpbo eoqqoqbebb 122055=42 2422-42253e og pppoobobbo .450q2b5qq2 bbqqocoqqa 242.42ooqqo eq4P0eCOPO Oqqbeqegeb <00>
G/C2111 Paz <ETZ>
VNCI <ZTZ>
109 <TTZ>
6 <OTZ>

2222222pp 22222222p: 2-3,0q1bb55 222q2e55q;
08Voqb12222,qg q.52,22bgbb l6llq52256 5qb.0obb.222 ..5oo0e2;22 olblobbgbo opooqeo25 5.42opq2b.b eol2q;55b5 bbolPbb.obb eo5o5eobb.5 55o55ob2b2 ogE 2 2525l-ebb 2262026o-2o 0.200;ePO5 oo5000b obqo2oobbo b.00l2b155op 00E 5ow5obboe boboob.o4eo lob5o552ob bo66boeo2b bbbbepobbb e56obbb2ob oz qbbbooebob obqopqopbb oqbbobqbob q5qol2o566 boo2b.7455oo opob2bpoeo 8SZZO/I0d3113a ,Z61/9/10 OM

tgcgtcaaac cttacacatc caccacgaca gaa <210> 8 <211> 273 <212> DNA
<213> Zea mays <400> 8 atggagtctt cacgagggaa gctgtctgcc gccggcgtcc tcctgctaat gacgctcctc 60 atggtggccg ccatgcgggc ggttgaggca cgagactgcc tgacgcagag tacccggtta 120 ccggggcatc tgtgcgtgcg gtcggactac tgcgcgatcg ggtgcagggc ggagggcaag 180 ggctacacgg gcggcaggtg ccttatctct cccatcacgc tcgacgggat tctctgctac 240 tgcgtcaagc cttgcacatc caccacgaca aaa 273 <210> 9 <211> 92 <212> PRT
<213> Zea mays <400> 9 Met Glu Pro Ser Arg Gly Lys Leu Ser Ala Ala Ala Val Leu Leu Leu Met Thr Thr Leu Leu Val Val Ala Ala Met Arg Ala Val Glu Ala Arg Asp Cys Leu Thr Gin Ser Thr Arg Leu Pro Gly His Leu Cys Val Arg Ser Asp Tyr Cys Ala Ile Gly Cys Arg Ala Glu Gly Lys Gly Tyr Thr Gly Gly Arg Cys Leu Ile Ser Pro Ile Pro Leu Asp Gly Ile Leu Cys Tyr Cys Val Lys Pro Cys Pro Ser Asn Thr Thr Thr <210> 10 <211> 91 <212> PRT
<213> Zea mays <400> 10 Met Glu Ser Ser Arg Gly Lys Lou Ser Ala Ala Gly Val Leu Leu Leu Met Thr Lou Leu Met Val Ala Ala Met Arg Ala Val Glu Ala Arg Asp Cys Leu Thr Gin Ser Thr Arg Lou Pro Gly His Leu Cys Val Arg Ser Asp Tyr Cys Ala Ile Gly Cys Arg Ala Glu Gly Lys Gly Tyr Thr Gly Gly Arg Cys Leu Ile Ser Pro Ile Pro Lou Asp Gly Ile Leu Cys Tyr Cys Val Lys Pro Cys Thr Ser Thr Thr Thr Glu <210> 11 <211> 91 <212> PRT
<213> Zea mays <400> 11 Met Glu Ser Ser Arg Gly Lys Leu Ser Ala Ala Ala Val Leu Leu Leu Met Thr Leu Leu Met Val Ala Ala Met Arg Ala Val Glu Ala Arg Asp Cys Leu Thr Gln Ser Thr Arg Leu Pro Gly His Leu Cys Val Arg Ser Asp Tyr Cys Ala Ile Gly Cys Arg Ala Glu Gly Lys Gly Tyr Thr Gly Gly Arg Cys Leu Ile Ser Pro Ile Pro Leu Asp Gly Ile Leu Cys Tyr Cys Val Lys Pro Cys Thr Ser Thr Thr Thr Glu <210> 12 <211> 91 <212> PRT
<213> Zea mays <400> 12 Met Glu Ser Ser Arg Gly Lys Leu Ser Ala Ala Gly Val Leu Leu Leu Met Thr Leu Leu Met Val Ala Ala Met Arg Ala Val Glu Ala Arg Asp Cys Leu Thr Gin Ser Thr Arg Leu Pro Gly His Leu Cys Val Arg Ser Asp Tyr Cys Ala Ile Gly Cys Arg Ala Glu Gly Lys Gly Tyr Thr Gly Gly Arg Cys Leu Ile Ser Pro Ile Thr Leu Asp Gly Ile Leu Cys Tyr Cys Val Lys Pro Cys Thr Ser Thr Thr Thr Lys <210> 13 <211> 1204 <212> DNA
<213> Zea mays <400> 13 tggaagaatt cagatctact ggcggaaaca aaaaattrtt attcctcgaa cttcgatata 60 aaaagacata ggagaagcct cttatatttt aaaatagaat ttcacagaga tagacataat 120 gaagtattag aactctcaca gaagtcatac atagaaaagc actaaagaag tacagtatgc 180 atcagtgtaa gaccgcacct gcgccaaaag tcaagggtga taagtttggg aatcatcagt 240 gtccccagat tcagtgtcag aaaaatcaga taaagtcagt accatatgct tctactatca 300 aaagcattrt gtatgctcaa ataratattc accctgactt aaaatttact accgggatgc 360 ttgggagata tcgtgtatat ctggcaccta tgtttatgac agtgaagctc accatcttta 420 ttctattgac attcattrtc atgcaaatta cataacgtat ggtagaacga ccgaatgcaa 480 attactgcta ataaacatcc tgcgtgcgcg caatggtgca ccatctacca attaatagct 540 gtaacggtac tgcaaataat ggtcgaagcg ttataccgta caggattata tatatacaca 600 tgcctctcga acggcttcaa tagttccacc acgttacttc catatatatt tcccttggat 660 tggattggat cgtcggcgcc caaacgaata ataatccggc aatggagcct tcacgaggga 720 agctgtctgc cgccgccgtc ctccrgctaa tgacgacgct cctcgtggtg gccgccatgc 780 aggcggttga ggcacgcgac tgcctgacac agagcacccg gttaccgggg cacctgtgcg 840 tgcggtcgga ctactgcgcg atcgggtgca gggcggaggg caagggctac acgggcggca 900 ggtgcctcat ctctcccatc ccgctcgacg ggattctctg ctactgcgtc aagccgtgcc 960 catccaacac gacgacataa tgatgagaca aagggcggtg ggtacagtgc acgctggccg 1020 ggggttatca gtccacacat cctaccgtac gtgtctgtgt taataacttt tttttgtctt 1080 ggaagtctgt ggtagtaatg acttttaaat gtcttggaat aaaccgggtt ctagtccctt 1140 ataagctagc agtactgtaa caattcagat catcaaagac agcaggaaac cggtggttga 1200 gttg 1204 <210> 14 <211> 2430 <212> DNA
<213> Zea mays <400> 14 cgacggcccg ggctggtatc tgtgtgtatc tggcacctat gtttatgaca gagaaactca 60 ccatgtttat tctattgaca ttcattttca tgcaaattac ataacgtatg ctagaaccaa 120 atgcaaatta ctgctaataa acatgttgca taacgtactc tctgccgctg cggtcgccgt 180, gcctctcgcg tccattgatg cgatgtgaat catcgatgca gagactagac atcccttttc 240 tcgcgccgca acatgttatt ggttattgat ctactggtca tgtactcatg tcatttagat 300 gtaaggtcag atttgttcat ttgtacgtat tatttaggtg ccgtgaatgt gaagcgaatg 360 tctctgtatg cgtgtgagtt gggtgccgta gttctgatga gcgcatgtgg atatatacta 420 tgtatatatg acacgacaca aaacttcagt acagcgatgg attgtattta taccggtttg 480 gaatcagaat gcagcatcct gttttttttt ggattgattg atcctgcatg catggccgtg 540 tgttgggaga atctgagcgt caatcactgt gatgagtatc agtggtggtg ctttctgtga 600 aggcacggac ggtccgcaac gggaaactgg acggtccgtg acctggcgca gaggatacgg 660 tttccgtctg accagacgaa cggacggtcc gcacgtgcgc agggacaacg gagttcgccg 720 aacagtacct ggatcttgct ctccggagag acccgtcgag gaagagaaat cctaggattt 780 gtcttgaaat cagtaggcca cctaagacgc ctctaaacga gagaggtgaa gattagagag 840 agaaaactat gttactgtct actcctaggg aaaaaggtaa ataacagaag ataatttgat 900 tattgattcg attgttggtt cttcaatcgg tcgtacccct caaatatata aggggggtct 960 agatccattc caaaacgttc tccaacagct cccacgggat taaagggcta aacacacgag 1020 gagatagaaa ttttaaccgc ttgatttgat ctattcgtgg accgtctgca cctatgggcg 1080 gaccatccac gggccggacc atttagccgt gctcagtgtc acaaatggag ctcaacacat 1140 gccctcctgc ctttaggaga agctgagcga accaaaagca ctgacacagg ccggaaccga 1200 ctcgaaatgt tcacatcggt tctcttaagc atctgccaca tactagatga cgttaacaga 1260 aaatcacgtc aacgtctgca cagtcatggc tcgcccgagg tctccccagg atggcctcgc 1320 gcgagcgtga ctgtgtctcc cgtccgaggg tggcctcaag cgacaaacat agaaccatga 1380 tgtactatag atctatatct atgtttacag tacatcaaca gattatgaag tctatttcag 1440 gttgaatgag gtttatcctc ggacgagtga tatttgtcgt ttcatattta tgttttatat 1500 aaatttttac tctcgacaca atgcattgtc acataccgat tcaaattcaa atatatgtgc 1560 gattctgtgc tcatattgtg cgattctgcg ctcatatggc acctatgttt atgacagtga 1620 aactcacaat gtttattcta ttgacattca ttttcatgca aattacataa cgtatgctag 1680 aaccaaatgc aaattactgc taataaacat cctgcgtaag cgcaatggcg caccatttac 1740 caatagctgt aacggtgcaa gtacgtaata gttggagcgt tatgtttctc tcctctcttc 1800 ccaccgtaca ggatcatata tatacacatg cctctagaac ggcttcaata tatagttcca 1860 ccacattact tccatatatt tccottggat tggatcgtca gcgccaaaac gaataataat 1920 ccggcaatgg agtcttcacg agggaagctg tctgccgccg gcgtcctcct gctaatgacg 1980 ctcctcatgg tagccgccat gcgggcggtt gaggcacgag actgcctgac gcagagtacc 2040 cggttaccgg gacatctgtg cgtgaggtcg gactactgcg cgatcgggtg caaggcggag 2100 ggcaagggct acacgggcgg caggtgcctt atctctacca tcacactcga caggattctc 2160 tactactgag tcaagccttg cacatccacc acgacaaaat gatgagacaa gacaagagcg 2220 gtgggtgcaa tgcaggctga ccgggggtta tcagttatat atggacatcc taccgtgtct 2280 gttaataact tgtaaatgtc ttgggaaagt ttgtggtgat aagttttaaa tatcttggaa 2340 taaagtgggt tctatacaga cttctactcg ttaagttgtt ggattactac tactgctgtt 2400 ttttatttga ggaattactg ctttgttttt 2430 <210> 15 <211> 701 <212> DNA
<213> Zea mays <400> 15 tggaagaatt cggatctact ggcggaaaca gaaagttttt gttcctcgaa cttcgatata 60 aaaagacata ggagaagcct cttatgtttt aaaatagaat ttcacagaga tagacataat 120 gaagtattag aactctcaca gaagtcatac atagaaaagc actaaagaag tacagtatgc 180 atcagtgtaa ggccgcgcct gcgccaaaag tcaagggtga taagtttggg aatcatcagt 240 gtccccagat tcagtgtcag aaagatcaga taaagtcagt accatatgct tctactatca 300 gaagcatttt gtatgctcaa atatatattc accctgactt agaatttact accgggatgc 360 ttgggagata tcgtgtatat ctggcaccta tgtttatgac agtgaagctc accatcttta 420 ttctattgac attcattttc atgcaaatta cataacgtat ggtagaacga ccgaatgcaa 480 attactgcta ataaacatcc tgcgtgcgcg caatggtgca ccatctacca attaatagct 540 gtaacggtac tgcaagtaat ggtcgaagcg ttataccgta caggattata tatatacaca 600 tgcctctcga acggcttcaa tagttccacc acgttacttc catatatatt tcccttggat 660 tggattggat cgtcggcgcc caaacgaata ataatccggc a 701 <210> 16 <211> 201 <212> DNA
<213> Zea mays <400> 16 tgcgtgcgcg caatggtgca ccatctacca attaatagct gtaacggtac tgcaagtaat 60 ggtcgaagcg ttataccgta caggattata tatatacaca tgcctctcga acggcttcaa 120 tagttccacc acgttacttc catatatatt tcccttggat tggattggat cgtcggcgcc 180 caaacgaata ataatccggc a 201 <210> 17 <211> 501 <212> DNA
<213> Zea mays <400> 17 gcgccaaaag tcaagggtga taagtttggg aatcatcagt gtccccagat tcagtgtcag 60 aaagatcaga taaagtcagt accatatgct tctactatca gaagcatttt gtatgctcaa 120 atatatattc accctgactt agaatttact accgggatgc ttgggagata tcgtgtatat 180 ctggcaccta tgtttatgac agtgaagctc accatcttta ttctattgac attcattttc 240 atgcaaatta cataacgtat ggtagaacga ccgaatgcaa attactgcta ataaacatcc 300 tgcgtgcgcg caatggtgca ccatctacca attaatagct gtaacggtac tgcaagtaat 360 ggtcgaagcg ttataccgta caggattata tatatacaca tgcctctcga acggcttcaa 420 tagttccacc acgttacttc catatatatt tcccttggat tggattggat cgtcggcgcc 480 caaacgaata ataatccggc a 501 <210> 18 <211> 83 <212> DNA
<213> Zea mays <400> 18 aatagttcca ccacgttact tccatatata tttcccttgg attggattgg atcgtaggcg 60 cccaaacgaa taataatccg gca 83 <210> 19 <211> 76 <212> DNA
<213> Zea mays <400> 19 gatagttcca ccacgttact tccatatatt tcccttggat tggatcgtcg gcgcccaaac 60 gaacaataat ccggca 76 <210> 20 <211> 77 <212> DNA
<213> Zea mays <400> 20 ggatagttcc accacgttac ttccatatat ttcccttgga ttggatcgtc ggcgcccaaa 60 cgaatactaa tccggca 77 <210> 21 <211> 78 <212> DNA
<213> Zea mays <400> 21 gatatagttc caccacatta cttccatata tttcccttgg attggatcgt cggcgccaaa 60 acgaataata atccggca 78 <210> 22 <211> 246 <212> DNA
<213> Zea mays <400> 22 taatgatgag acaaagggcg gtgggtgcag tgcacgctgg ccgggggtta tcagtccaca 60 catcctaccg tacgtgtctg tgttaataac ttttttttgt cttggaagtc tgtggtggta 120 atgactttta aatgtcttgg aataaaccgg gttctagtcc cttataagct agcagtactg 180 taacaattca gatcatcaaa gacagcagga aaccggtggt tgagttgaaa aaaaaaaaaa 240 aaaaaa 246 <210> 23 <211> 188 <212> DNA
<213> Zea mays <400> 23 tgatgagaca agagcggtgg gtgcagtgca ggctgatcgg ggggttatca gttatatatg 60 gacatcctac cgtgtctgtt aataacttgt aaatgtcttg ggaagtttgt ggtgataagt 120 tttaaatgtc ttggaataaa gtgggttcta tatagacttc tactcgttaa aaaaaaaaaa 180 aaaaaaaa 188 <210> 24 <211> 169 <212> DNA

<213> Zea mays <400> 24 tgatgagaca agagcggtgg gtgcagtgca ggctgatcgg ggggttatca gttatatatg 60 gacatcctac cgtgtctgtt aataacttgt aaatgtcttg ggaagtttgt ggtgataagt 120 tttaaatgtc ttggaataaa gr_gggttcta taaaaaaaaa aaaaaaaaa 169 <210> 25 <211> 250 <212> DNA
<213> Zea mays <400> 25 tgatgagaca agacaagagc ggtgggtgca atgcaggctg accgggggtt atcagttata 60 tatggacatc ctaccgtgtc tgttaataac ttgtaaatgt cttgggaaag tttgtggtga 120 taagttttaa atgtcttgga ataaagtggg ttctatacag acttctactc gttaagttgt 180 tggattacta ctactgctgt tttttatttg aggaattact gctttgtttt taaaaaaaaa 240 aaaaaaaaaa 250 <210> 26 <211> 1926 <212> DNA
<213> Zea mays <400> 26 cgacggcccg ggctggtatc tgtgtgtatc tggcacctat gtttatgaca gagaaactca 60 ccatgtttat tctattgaca ttcattttca tgcaaattac ataacgtatg ctagaaccaa 120 atgcaaatta ctgctaataa acatgttgca taacgtactc tctgccgctg cggtcgccgt 180 gcctctcgcg tccattgatg cgatgtgaat catcgatgca gagactagac atcccttttc 240 tcgcgccgca acatgttatt ggttattgat ctactggtca tgtactcatg tcatttagat 300 gtaaggtcag atttgttcat ttgtacgtat tatttaggtg ccgtgaatgt gaagcgaatg 360 tctctgtatg cgtgtgagtt gggtgccgta gttctgatga gcgcatgtgg atatatacta 420 tgtatatatg acacgacaca aaacttcagt acagcgatgg attgtattta taccggtttg 480 gaatcagaat gcagcatcct gttttttttt ggattgattg atcctgcatg catggccgtg 540 tgttggggga atctgagcgt caatcactgt gatgagtatc agtggtggtg ctttctgtga 600 aggcacggac ggtccgcaac gggaaactgg acggtccgtg acctggcgca gaggatacgg 660 tttccgtctg accagacgaa cggacggtcc gcacgtgcgc agggacaacg gagttcgccg 720 aacagtacct ggatcttgct ctccggagag acccgtcgag gaagagaaat cctaggattt 780 gtcttgaaat cagtaggcca cctaagacgc ctctaaacga gagaggtgaa gattagagag 840 agaaaactat gttactgtct actcctaggg gaaaaggtaa ataacagaag ataatttgat 900 tattgattcg attgttggtt cttcaatcgg tcgtacccct caaatatata aggggggtct 960 agatccattc caaaacgttc tccaacagct cccacgggat taaagggcta aacacacgag 1020 gagatagaaa ttttaaccgc ttgatttgat ctattcgtgg accgtctgca cctatgggcg 1080 gaccatccac gggccggacc atttagccgt gctcagtgtc acaaatggag ctcaacacat 1140 gccctcctgc ctttaggaga agctgagcga accaaaagca ctgacacagg ccggaaccga 1200 ctcgaaatgt tcacatcggt tctcttaagc atctgccaca tactagatga cgttaacaga 1260 aaatcacgtc aacgtctgca cagtcatggc tcgcccgagg tctccccagg atggcctcgc 1320 gcgagcgtga ctgtgtctcc cgtccgaggg tggcctcaag cgacaaacat agaaccatga 1380 tgtactatag atctatatct atgtttacag tacatcaaca gattatgaag tctatttcag 1440 gttgaatgag gtttatcctc ggacgagtga tatttgtcgt ttcatattta tgttttatat 1500 aaatttttac tctcgacaca atgcattgtc acataccgat tcaaattcaa atatatgtgc 1560 gattctgtgc tcatattgtg cgattctgcg ctcatatggc acctatgttt atgacagtga 1620 aactcacaat gtttattcta ttgacattca ttttcatgca aattacataa cgtatgctag 1680 aaccaaatgc aaattactgc taataaacat cctgcgtaag cgcaatggcg caccatttac 1740 caatagctgt aacggtgcaa gtacgtaata gttggagcgt tatgtttctc tcctctcttc 1800 ccaccgtaca ggatcatata tatacacatg cctctagaac ggcttcaata tatagttcca 1860 ccacattact tccatatatt tcccttggat tggatcgtcg gcgccaaaac gaataataat 1920 ccggca 1926 <210> 27 <211> 227 <212> DNA
<213> Zea mays <400> 27 ctaataaaca tcctgcgtaa gcgcaatggc gcaccattta ccaatagctg taacggtgca 60 agtacgtaat agttggagcg ttatgtttct ctcctctctt cccaccgtac aggatcatat 120 atatacacat gcctctagaa cggcttcaat atatagttcc accacattac ttccatatat 180 ttcccttgga ttggatcgtc ggcgccaaaa cgaataataa tccggca 227 <210> 28 <211> 427 <212> DNA
<213> Zea mays <400> 28 taaattttta ctctcgacac aatgcattgt cacataccga ttcaaattca aatatatgtg 60 cgattctgtg ctcatattgt gcgattctgc gctcatatgg cacctatatt tatgacagtg 120 aaactcacaa tgtttattct attgacattc attttcatgc aaattacata acgtatgcta 180 gaaccaaatg caaattactg ctaataaaca tcctgcgtaa gcgcaatggc gcaccattta 240 ccaatagctg taacggtgca agtacgtaat agttggagcg ttatgtttct ctcctctctt 300 cccaccgtac aggatcatat atatacacat gcctctagaa cggcttcaat atatagttcc 360 accacattac ttccatatat ttcccttgga ttggatcgtc ggcgccaaaa cgaataataa 420 tccggca 427 <210> 29 <211> 927 <212> DNA
<213> Zea mays <400> 29 ttaaagggct aaacacacga ggagatagaa attttaaccg cttgatttga tctattcgtg 60 gaccgtctgc acctatgggc ggaccatcca cgggccggac catttagccg tgctcagtgt 120 cacaaatgga gctcaacaca tgccctcctg cctttaggag aagctgagcg aaccaaaagc 180 actgacacag gccggaaccg actcgaaatg ttcacatcgg ttctcttaag catctgccac 240 atactagatg acgttaacag aaaatcacgt caacgtctgc acagtcatgg ctcgcccgag 300 gtctccccag gatggcctcg cgcgagcgtg actgtgtctc ccgtccgagg gtggcctcaa 360 gcgacaaaca tagaaccatg atgtactata gatctatatc tatgtttaca gtacatcaac 420 agattatgaa gtctatttca ggttgaatga ggtttatcct cggacgagtg atatttgtcg 480 tttcatattt atgttttata taaattttta ctctcgacac aatgcattgt cacataccga 540 ttcaaattca aatatatgtg cgattctgtg ctcatattgt gcgattctgc gctcatatgg 600 cacctatgtt tatgacagtg aaactcacaa tgtttattct attgacattc attttcatgc 660 aaattacata acgtatgcta gaaccaaatg caaattactg ctaataaaca tcctgcgtaa 720 gcgcaatggc gcaccattta ccaatagctg taacggtgca agtacgtaat agttggagcg 780 ttatgtttct ctcctctctt cccaccgtac aggatcatat atatacacat gcctctagaa 840 cggcttcaat atatagttcc accacattac ttccatatat ttcccttgga ttggatcgtc 900 ggcgccaaaa cgaataataa tccggca 927 <210> 30 <211> 1427 <212> DNA
<213> Zea mays <400> 30 tgtttttttt tagattgatt gatcctgcat gcatggccgt gtgttggggg aatctgagcg 60 tcaatcactg tgataagtat cagtggtggt gctttctgtg aaggcacgga cggtccgcaa 120 cgggaaactg gacggtccgt gacctggcgc agaggatacg gtttccgtct gaccagacga 180 acggacggtc cgcacgtgcg cagggacaac ggagttcgcc gaacagtacc tggatcttgc 240 tctccggaga gacccgrcga ggaagagaaa tcctaagatt tgtcttgaaa tcagtaggcc 300 acctaagacg cctctaaacg agagaggtga agattagaga gagaaaacta rgttactgtc 360 tactcctagg ggaaaaggta aataacagaa gataatttaa ttattgattc gattgttgat 420 tcttcaatcg grcgtacccc tcaaatatat aaggggggtc tagatccatt ccaaaacgtt 480 ctccaacagc tcccacgaga ttaaagggct aaacacacga ggagatagaa attttaaccg 540 cttgatttga totattcgtg gaccgtctgc acctatgggc ggaccatcca cgggccggac 600 catttagccg tgctcagtgt cacaaatgga gctcaacaca tgccctoctg cctttaggag 660 aagctgagcg aaccaaaagc actgacacag accggaaccg actcgaaatg ttcacatcgg 720 ttctcttaag catctgccac atactagatg acattaacag aaaatcacgt caacgtctac 780 acagtcatgg ctcgcccgag gtctocccag gatggcctcg cgcgagcgtg actgtgtctc 840 ccgtccgagg gtgacctcaa gcgacaaaca tagaaccatg atgtactata aatctatatc 900 tatgtttaca gtacatcaac agattatgaa gtctatttca ggttgaatga ggtttatcct 960 cggacgagtg atatttgtcg tttcatattt atgttttata taaattttta ctctcgacac 1020 aatgcattgt cacataccga ttcaaattca aatatatgtg cgattctgtg ctcatattg: 1080 gcgattctgc gctcatatgg cacctatgtt tatgacagtg aaactcacaa tgtttattct 1140 attgacattc attttcatgc aaattacata acgtatgcta gaaccaaatg caaattactg 1200 ctaataaaca tcctgcgtaa gcgcaatggc gcaccattta ccaatagctg taacggtgca 1260 agtacgtaat agttggagcg ttatgtttct ctcctctott cccaccgtac aggatcatat 1320 atatacacat gcctctagaa cggcttcaat atatagttcc accacattac ttccatatat 1380 ttcccttgga ttggatcgtc ggcgccaaaa cgaataataa tccggca 1427 <210> 31 <211> 1727 <212> DNA
<213> Zea mays <400> 31 gcgatgtgaa tcatcgatgc agagactaga catccctttt ctcgcgccgc aacatgttat 60 tggttattga tctactggtc atgtactcat gtcatttaga tgtaaggtca gatttgttca 120 tttgtacgta ttatttaggt gccgtgaatg tgaagcgaat gtctctgtat gcgtgtgagt 180 tgggtgccgt agttctgatg agcgcatgtg gatatatact atgtatatat gacacgacac 240 aaaacttcag tacagcgatg gattgtattt ataccggttt ggaatcagaa tgcagcatcc 300 tgtttttttt tggattgatt gatcctgcat gcatggccgt gtgttggggg aatctgagcg 360 tcaatcactg tgatgagtat cagtggtggt gctttctgtg aaggcacgga cggtccgcaa 420 cgggaaactg gacggtccgt gacctggcgc agaggatacg gtttccgtct gaccagacga 480 acggacggtc cgcacgtgcg cagggacaac ggagttcgcc gaacagtacc tggatcttgc 540 tctccggaga gacccgtcga ggaagagaaa tcctaggatt tgtcttgaaa tcagtaggcc 600 acctaagacg cctctaaacg agagaggtga agattagaga gagaaaacta tgttactgtc 660 tactcctagg ggaaaaggta aataacagaa gataatttga ttattgattc gattgttggt 720 tcttcaatcg gtcgtacccc tcaaatatat aaggggggtc tagatccatt ccaaaacgtt 780 ctccaacagc tcccacggga ttaaagggct aaacacacga ggagatagaa attttaaccg 840 cttgatttga tctattcgtg gaccgtctgc acctatgggc ggaccatcca cgggccggac 900 catttagccg tgctcagtgt cacaaatgga gctcaacaca tgccctcctg cctttaggag 960 aagctgagcg aaccaaaagc actgacacag gccggaaccg actcgaaatg ttcacatcgg 1020 ttctcttaag catctgccac atactagatg acgttaacag aaaatcacgt caacgtctgc 1080 acagtcatgg ctcgcccgag gtctccccag gatggcctcg cgcgagcgtg actgtgtctc 1140 ccgtccgagg gtggcctcaa gcgacaaaca tagaaccatg atgtactata gatctatatc 1200 tatgtttaca gtacatcaac agattatgaa gtctatttca ggttgaatga ggtttatcct 1260 cggacgagtg atatttgtcg tttcatattt atgttttata taaattttta ctctcgacac 1320 aatgcattgt cacataccga ttcaaattca aatatatgtg cgattctgtg ctcatattgt 1380 gcgattctgc gctcatatgg cacctatgtt tatgacagtg aaactcacaa tgtttattct 1440 attgacattc attttcatgc aaattacata acgtatgcta gaaccaaatg caaattactg 1500 ctaataaaca tcctgcgtaa gcgcaatggc gcaccattta ccaatagctg taacggtgca 1560 agtacgtaat agttggagcg ttatgtttct ctcctctctt cccaccgtac aggatcatat 1620 atatacacat gcctctagaa cggcttcaat atatagttcc accacattac ttccatatat 1680 ttcccttgga ttggatcgtc ggcgccaaaa cgaataataa tccggca 1727 <210> 32 <211> 21 <212> DNA
<213> Zea mays <400> 32 cccttagatt ggattggatc g 21 <210> 33 <211> 20 <212> DNA
<213> Zea mays <400> 33 accaccggtt tcctgctgtc 20 <210> 34 <211> 21 <212> DNA
<213> Zea mays <400> 34 tcttcacgag ggaagctgtc t 21 <210> 35 <211> 20 <212> DNA
<213> Zea mays <400> 35 gcactgcacc caccgctctt 20 <210> 36 <211> 22 <212> DNA
<213> Zea mays <400> 36 gtaatacgac tcactatagg gc 22 <210> 37 <211> 27 <212> DNA
<213> Zea mays <400> 37 cttgacgcag tagcagagaa tcccgtc 27 <210> 38 <211> 26 <212> DNA
<213> Zea mays <400> 38 cagtagtccg accgcacgca cagrtg 26 <210> 39 <211> 19 <212> DNA
<213> Zea mays <400> 39 actatagggc acgcgtggt 19 <210> 40 <211> 30 <212> DNA
<213> Zea mays <400> 40 cagacagctt coctcgtgaa gctoccattg 30 <210> 41 <211> 28 <212> DNA
<213> Zea mays <400> 41 tctgygtcag gcagtckcgt gcctcaac 28 <210> 42 <211> 21 <212> DNA
<213> Zea mays <400> 42 gtcattacca ccacagactt c 21 <210> 43 <211> 20 <212> DNA
<213> Zea mays <400> 43 actcatgagg tagtgacatt 20 <210> 44 <211> 20 <212> DNA
<213> Zea mays <400> 44 catttagcag gtcactgtac 20 <210> 45 <211> 1594 <212> DNA
<213> Zea mays <400> 45 gttattggtt attgatctac tggtcatgta ctcatgtcat ttagatgtaa ggtcagattt 60 gttcatttgt acgtattatt taggtgccgt gaatgtgaag cgaatgtctc tgtatgcgtg 120 tgagttgggt gccgtagttc tgatgagcgc atgtggatat atactatgta tatatgacac 180 gacacaaaac ttcagtacag cgatggatrg tatttatacc ggtttggaat cagaatgcag 240 catcctgttt ttttttggat tgattgatcc tgcatgcatg gccgtgtgtt gggggaatct 300 gagcgtcaat cactgtgatg agtatcagtg gtggtgcttt ctgtgaaggc acggacggtc 360 cgcaacggga aactggacgg tccgtgacct ggcgcagagg atacggtttc cgtctgacca 420 gacgaacgga cggtccgcac gtgcgcaggg acaacggagt tcgccgaaca gtacctggat 480 cttgctctcc ggagagaccc gtcgaggaag agaaatccta ggatttgtct tgaaatcagt 540 aggccaccta aaacacctct aaacgagaga gatgaagatt agagagagaa aactatgtta 600 ctgtctactc ctagggaaaa aggtaaataa caaaagataa ttrgattatt gattcgattg 660 ttggttcttc aatcggtcgt acccctcaaa tatataaggg gggtcragat ccattccaaa 720 acgttctcca acagctccca cggaattaaa gggctaaaca cacgaggaga tagaaatttt 780 aaccgcttga trtgatctat tcgtggaccg tctgcaccta tgggcggacc atccacgggc 840 cggaccattt agccgtgctc agtgtcacaa atagagctca acacatgccc tcctgccttt 900 aggagaaact gagcgaacca aaagcactga cacaagccgg aaccgactcg aaatgttcac 960 atcggttctc rtaagcatct gccacatact agatgacgtt aacagaaaat cacgtcaacg 1020 tctgcacagt catggctcgc ccgaggtctc cccaggatgg cctcgcgcga gcgtgactgt 1080 gtctcccatc cgagggtggc ctcaagcgac aaacatagaa ccatgatgta ctatagatct 1140 atatctatgt ttacagtaca tcaacagatt argaagtcta tttcaggttg aatgaggttt 1200 atcctcggac gagtgatatt tgtcgtttca tatttatgtt ttatataaat ttttactctc 1260 gacacaatgc attgtcacat accgattcaa attcaaatat atgtgcgatt ctgtgctcat 1320 attgtgcgat tctgcgctca tatggcacct atgtttatga cagtgaaact cacaatgttt 1380 attctattga cattcatttt catgcaaatt acataacgta tgctagaacc aaatgcaaat 1440 tactgctaat aaacatcctg cgtaagcaca atggcgcacc atttaccaat agctgtaacg 1500 gtgcaagtac gtaatagttg gagcgttatg tttctctcct ctcttcccac cgtacaggat 1560 catatatata cacatgcctc tagaacggct tcaa 1594

Claims (6)

Claims

1. An isolated ZmES4 promoter comprising the DNA
sequence of SEQ ID No. 26.

2. An isolated ZmES4 promoter having a DNA
sequence identity of more than 95% to SEQ ID No. 26 and which has the same promoter activity as the ZmES4 promoter of SEQ ID NO:26.

3. An isolated ZmES4 promoter comprising the DNA
sequence of SEQ ID No. 45.

4. A vector comprising the isolated ZmES4 promoter of any one of claims 1 to 3.

5. An isolated host cell comprising the vector of claim 4.

6. The vector of claim 4, wherein the vector is bacterial or viral.