MXPA04011042A - Nucleotide sequences and methods for the specific expression of genes in the female gametophyte, female reproductive cells, pollen grain and/or male reproductive cells of plants. - Google Patents

Abstract

The invention relates to the localisation and expression patterns of promoters (regulatory nucleotide sequences) which act in a specific manner in the female gametophyte, the female gametes, the male gametophyte and/or the male gametes of Arabidopsis thaliana. The invention also relates to the methods that were used to identify the aforementioned promoters as well as the methods of using said promoters in expression constructions and to manipulate the reproductive development of plants. According to the invention, the identified, isolated promoters can be used to regulate the genic expression of selected genes in plants, so that such genes can be efficiently transcribed in the female or male reproductive cells of same. Said promoters can be used to prevent the transfer of transgenic traits to plants through in-field pollen in order to provide the seed with additional nutrients and to confer resistance to pathogens and diseases to the seed.

Description

Nucleotide sequences and methods for the specific expression of genes in the female gametophyte, female reproductive cells, pollen grain and / or male reproductive cells of plants Field of the invention. This invention is obtained in the field of genetic engineering and the molecular biology of plants. More specifically, the invention relates to the characterization of novel nucleotide sequences specific to the female gametophyte, to the female gametes, to the male gametophyte, and / or to the male gametes of the plants. These sequences allow the specific temporal and spatial expression of gene products in the male or female gametophyte, or the male or female reproductive cells of transgenic plants.

Background. The expression of genes (gene expression) depends on a series of processes that originate in the DNA molecule. The control and regulation of gene expression can occur from different mechanisms. It is generally considered that the initiation of the transcription of a gene (or gene) constitutes the predominant control of gene expression. Transcriptional controllers (better known as promoters) are generally short nucleotide sequences that are present at the 5 'border or the upstream region of a transcribed gene. There are promoter sequences that affect gene expression in response to environmental stimuli, to the availability of nutrients, or to adverse conditions that include high temperatures, anaerobiosis or the presence of heavy metals in the growth substrate of plants. There are also DNA sequences that control gene expression during the development of an organism, and this specifically in certain cells or tissues.

The promoters contain the signals required by the RNA polymerase enzyme to initiate transcription so that protein synthesis is obtained. There are nuclear DNA binding proteins that interact specifically with the promoter sequence to promote the formation of a transcriptional complex and eventually initiate the gene expression process. The region containing all the elements necessary to ensure the production of sufficient levels of transcript can be between 100 base pairs and up to 1, 000 base pairs in length.

The existence of promoters that confer activity in plant cells has been reported in the literature, including the promoters of nopaline synthase (NOS) and octopine synthase (OCS, both present in the plasmids of Agrobacterium tumefaciens), the promoters 19S and 35S of cauliflower mosaic virus, the light-inducible promoter of the small subunit of ribulose carboxylase bisphosphate (ssRUBISCO), and the sucrose synthase promoter. All these promoters have been used to create different types of DNA constructs that have been expressed in plants 1.

However, the most valuable promoters are those that act specifically in certain tissues or cells. This type of promoters can activate gene sequences only in specific tissues in which the expression of a protein is desired. This type of promoters can also be expressed only during a certain period of plant development. For example, the tomato E8 promoter is only transcriptionally active during the stage of fruit ripening, and therefore can be used to activate gene expression during the maturation process of tomato 2. The activity of the E8 promoter is not limited to the fruit of the tomato, if not that is thought to be compatible with any biological process that can be activated by ethylene. Other promoters that act during the development of the fruit are fragment 1.45 3 and the promoter of the tomato polygalacturonase.

Perhaps the most important specific promoters are those that regulate gene expression in embryos and in seeds and in their cellular precursors, the male or female reproductive cells contained in the male or female gametophyte. This type of promoters can be used to modulate the size of the seed, to produce seeds without the need for fertilization (by parthenogenesis), to activate the autonomous development of the embryo or the endosperm, or simply to obtain information on when and where it occurs. gene regulation in the reproductive phase of the life cycle of plants. This type of promoters includes regulatory sequences of genes that are expressed in maternal tissue such as the seed coat, the pericarp, or the ovule. One of these promoters is the promoter of the gene that codes for the α subunit of soybean β-conglycinin5 that is expressed in the premature endosperm of the seed. Some additional promoters include the Napin6 gene promoter to direct the expression of a gene that increases oil production in the seed; the DC3 promoter that is expressed in the early and middle stages of embryo development7; the promoter of the gene that encodes a phaseolin8 and that is only expressed in Phaseolus vulgaris during the development of the seed.

As can be seen from the previous paragraphs, there is a need in the field of agricultural biotechnology and plant molecular biology for specific promoters to direct the expression of nucleic acids in the reproductive cells contained in the male gametophyte and the female gametophyte of the plants.

Description of the figures. Figure 1. The sequence of the pFM promoter is observed. Figure 2. The sequence of the pES1 promoter is observed. Figure 3. The sequence of the pPol promoter is observed. Figure 4. The sequence of the pPo2 promoter is observed. Figure 5. The isolated DNA fragment is observed for the cloning of the pFM1 promoter. Figure 6. The activity patterns of each of the promoters described in this invention are observed. Each of the promoter sequences of the invention was fused to the uidA reporter gene (GUS) and Arabidopsis thaliana plants were transformed with said construction. The tissue of the transformant plants was analyzed to detect the presence of the reporter protein (blue), (a) - (c), activity of the pFM1 promoter in the female gametophyte and the female gametes; the pFM1 promoter is activated at the beginning of the female gametogenesis and is maintained in the gametophyte and the female gametes (d), activity of the pES1 promoter in the female gametophyte and the female gametes, (e); (f), pPo2 promoter activity in the male gametophyte and male gametes. Figure 7. The isolated DNA fragment is observed for the cloning of the pES1 promoter. Figure 8. The isolated DNA fragment is observed for the cloning of the pPol promoter. Figure 9. The isolated DNA fragment is observed for the cloning of the pPo2 promoter. Figure 10. The effect of the pFM1 promoter on the development of the female gametophyte is observed. The female gametophyte stops its development when it is altered by the activation of an RNAi sequence under the control of the pFM1 promoter described in this invention.

Objectives of the invention. The main objective of the invention presented here is to provide regulatory elements that direct the expression of nucleotide sequences in female gametophyte cells, female reproductive cells (female gametes), male gametophyte and / or male reproductive cells ( male gametes) of plants.

Another objective of this invention is to provide transcriptional units that allow the expression of genes during the formation of the female gametophyte, of the female reproductive cells (the female gametes), of the male gametophyte and / or of the male reproductive cells (the male gametes) of the plants.

Another objective of this invention is to provide vehicles for the transformation of plant cells, including viral vectors or plasmids that include the new regulatory sequences that are described in this invention.

Another objective of this invention is to provide bacterial cells that contain the vectors described in the previous paragraph and that allow the maintenance, replication and transformation of plants.

Another objective of this invention is to provide expression constructs, sequences and transformed cells for the creation of transgenic plants. Another objective of this invention is to provide genetic materials that can be used in breeding programs that serve to produce commercial crops from desirable exogenous genes.

Another objective of this invention is to provide sequences that serve as labels for the fine mapping of genes of Arabidopsis plants as well as to establish assays of activation or inactivation of genes during the development of the male and female gametophyte.

These and other objectives will be amply described and justified in the next paragraphs.

Description of the invention The invention described here includes the isolation and characterization of new regulatory nucleotide sequences that allow the expression of genes during the development of male and female gametophytes (including male gametes and female gametes) of Arabidopsis and other species. The invention also includes controlling the spatial and temporal expression of nucleotide sequences using the regulatory elements described herein.

The invention also includes expression constructs that include one or more of the promoters described in this invention, a structural gene whose expression is desirable in plant cells, and a polyadenylation signal or transcription stop. This type of constructions can be included in plasmid or viral vectors used for the transformation of plant cells.

The invention also includes transformed bacterial cells for the maintenance and replication of the vector described above, as well as transformed plant cells and transgenic plants or all germplasm derived therefrom.

At another level, the invention also includes the identification and localization of new nucleotide sequences that express or regulate expression that allows the activation or inactivation of genes during the development of the male and female gametophyte. Said sequences can be used as mapping markers, for the manipulation of gene expression during male or female gametogenesis, and for assays and protocols that seek to silence genes in the male or female gametophyte, or during the development of the seed.

I. Definitions. For purposes of this application the following terms will have the definition that is detailed below. Units, prefixes and symbols may be mentioned using their abbreviation accepted by the International System (SI). Unless explicitly indicated, the sequence of the nucleic acids is written from left to right in the orientation 5 'to 3'; the amino acid sequence is written from left to right in the orientation of the amino edge to the carboxy-terminal edge. The numerical ranges are inclusive of the numbers that define the range and include each of the integral numbers that define the range. The amino acids can be designated either by their known three-letter symbols or by their one-letter symbols that have been recommended by the. Naming Commission of the IUPAC-IUB. In the same way, nucleotides can be designated by their commonly accepted unique letter code. Software, electrical and electronic terms are used as defined in the New IEEE Dictionary for Electrical and Electronic Terms9.

For purposes of this patent application, "female gametophyte" means the organized set of female reproductive cells and their precursor cells in plants. In most plants, including Arabidopsis thaliana, the cells of the embryo sac differentiate from four different development schemes to form either the synergies (2), the ovocell (1), the central cell (1), or the antipodes (variable number). The ovocell and the synergies are organized in a triangle located at a pole of the central cell. Synergies are highly specialized cells that participate in the attraction of the pollen tube and the transport of sperm cells to the egg cell and the central cell. Due to unknown causes, one of the two synergies begins to degenerate before fertilization. The ovocell is located next to the synergies, and its nucleus is located in the apical part, next to the plasma membrane that separates it from the central cell. Its cytoplasm is highly vacuolized and has a metabolic activity inferior to that of the synergies. Unlike the other embryonic sac cells, the central cell contains two nuclei that fuse before fertilization. The origin of these two nuclei is found in the two poles of the embryo sac (micropilar and apical). The differentiation of the three antipodes takes place in the apical region.

For purposes of this patent application, "female reproductive cell" or "female gamete" means any of these cells: the synergies, the ovocell, the central cell or the antipode cells.

For purposes of this patent application, "male gametophyte" means the organized set of male reproductive cells and their precursor cells characteristic of plants. In the anther, the mother microspore before the first meiotic division. In grasses, a cell wall occurs between the products of meiosis I; the second division is perpendicular to the first and the four microspores form a tetrad. In many other families, cell wall formation only begins after meiosis II. During maturation of the pollen grain, the haploid nucleus in each of the microspores is divided (by mitosis) asymmetrically to form a vegetative nucleus of predominant size, and a dense and often elongated nucleus (called the generative nucleus), but smaller. The differentiation of the generative nucleus gives rise to the formation of a small cell with double plasmic membrane. The generative cell contains very few organelles, including mitochondria and plastes. It is considered, without clear experimental evidences, that the absence of organelles is due to an asymmetric distribution of the cytoplasm during the mitotic division of the haploid nucleus. In most species, the generative cell and its nucleus are divided before the dissemination of pollen to form a trinucleate grain, in which there are two sperm cells and a vegetative nucleus whose function remains a great unknown but a reason for abundant speculation . It is believed to act as a control and guide to the growth of the pollen tube, and during its growth, it is often found in close association with the cytoplasmic membranes of one of the sperm cells. In most grasses (eg maize), the mitotic division of the generative cell occurs after germination of the pollen grain begins inside the pollen tube.

For purposes of this patent application, "male reproductive cell" or "male gamete" means a vegetative cell or a sperm cell contained within the pollen of plants.

By "amplification" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the sequence of a nucleic acid using at least one nucleic acid sequence as the initial reference frame. Amplification systems include the polymerase chain reaction (PCR), the ligase-dependent chain reaction (LCR), the nucleic acid sequence-dependent amplification (NASBA, Canteen, Mississauga , Ontario), the Q-Beta Replicase system, the Transcription-dependent Amplification System (TAS), and the Strand Displacement Amplification System -SDA, by its acronym in English) 0. The amplification product is called an amplicon.

The "antisense orientation" refers to a double-stranded polynucleotide sequence that is functionally linked to a promoter in an orientation in which the antisense orientation of said molecule is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product so that translation of the endogenous transcript product is inhibited.

A "chromosomal region" refers to the length of a chromosome that can be measured by reference to the linear segment of DNA that said region includes. The region can also be defined from two unique DNA sequences or molecular markers.

The term "conservatively modified variants" applies to both nucleotide sequences and amino acid sequences. Referring to specific nucleotide sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any type of protein. For example, the GCA, GCC, GCG and GCU codons all encode the amino acid alanine. These variations of nucleic acid are silent variations and represent conservatively modified variants. Therefore, any nucleic acid sequence encoding a polypeptide in this document describes all possible silent variations of the nucleic acid. In the same manner, any silent variation of a nucleic acid sequence encoding a polypeptide is implicitly included in each polypeptide sequence of this document.

As for the amino acid sequences, it is recognized that individual substitutions, deletions or additions to a nucleic acid, a peptide, a polypeptide or a protein sequence that alters, adds or eliminates a single amino acid or a small percentage of amino acids in the The encoded sequence is also a "conservatively modified variant" in which the alteration results in the substitution of an amino acid for a biochemically similar amino acid. Therefore, any number of amino acid residues selected from the number of integrants ranging from 1 to 15 can be altered in the manner described above. For example, 1, 2, 3, 4, 5, 7 or 10 alterations can be created. Conservatively modified variants generally have a biological activity similar to that of the unmodified polypeptide sequence from which they were derived. For example, the specificity of the substrate, the enzymatic activity, or the binding properties between a receptor and its elicitor are generally 30% to 90% of the activity of the native protein and its native substrate. The tables of conservative substitutions are widely known in the area.

The following 6 groups contain amino acids that represent conservative substitutions within each of the groups: 1) Alanine (A), Serine (S), Threonine (T). 2) Aspartic acid (D), glutamic acid (E) 3) Aspargin (N), Glutamine (Q) 4) Arginine (R), Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M) ), Valine (V) 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) The term "encoded" or "encoding" in reference to a specific nucleic acid refers to the translation information of the corresponding protein. A nucleic acid encoding a protein may include untranslated sequences (introns, for example) contained within translated regions of the nucleic acid, or may not include such sequences (as in a cDNA, for example). The information of the encoded protein is specified in the use of codons. In general, the universal genetic code is used to determine the translation code of a nucleic acid sequence in amino acid sequence. However, there are variants of the universal code in the genetic information contained within the mitochondria of some plants, animals or fungi, the bacterioid organism Micoplasma caprícolum, the ciliate organism Macronucleus can be included within the corresponding organism.

When the nucleic acid is synthetically generated or altered, the codon preferences of the host organism in which the nucleic acid is intended to be expressed can be exploited. For example, although the nucleotide sequences of the invention described herein can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified taking into account the codon preferences and the preference for GC content that has been shown to differ in monocotyledons. and dicotyledonous11. Therefore, the codon predominant in corn for a specific amino acid can be derived from the known sequence of a corn gene.

The term "full-length sequence" of a specific polynucleotide or its encoded protein refers to the complete sequence of the amino acid chain of a native (non-synthetic), endogenous protein and in its biologically active form. The methods used to determine if a sequence is full length are widely known and as an example we can mention hybridizations of the "Northern" or "Western" type, extension of primers, or ribonuclease protection12. The comparison with homologous full length sequences (orthoiogas or paralogs) can also be used to identify the full length sequence of sequences included in the invention described herein. The consensus sequences generally present at the 3 'or 5' border of the untranslated regions of a messenger RNA molecule (mRNA) aid in the identification of the full length sequence of a polynucleotide. For example, the ANNNNAUGG consensus sequence, in which the underlined codon represents the methionine present at the N-terminal border helps determine if the polynucleotide has a complete 5 'terminal terminus. Consensus sequences at the 3 'border, such as the polyadenylation sequences, help determine if the terminal 3' border is complete.

The term "heterologous" refers herein to a nucleic acid derived from a different species or, if derived from the same species, a nucleic acid which is substantially modified from its native form. For example, a promoter that is operatively linked to a heterologous structural gene belongs to a different species from which the structural gene was originally obtained as long as it originates from a deliberate human intervention. In case of belonging to the same species, one to several heterologous genes must be substantially modified from their original form. A heterologous protein can originate from a different species, or from the same species as long as it originates from a deliberate human intervention.

The term "host cell" refers to a cell that contains a vector and that ensures replication and / or expression of said vector. The host cells can be prokaryotic cells such as e. Coli, or eukaryotic such as yeast, insect, amphibian or mammalian cells. Preferably, the host cells are monocotyledonous or dicotyledonous plants. A preferred cell host in monocots is the corn cell.

The term "hybridization complex" refers to a double strand structure of nucleic acid formed by two single-stranded nucleic acid sequences hybridized together selectively.

The term "introduced" in reference to the act of inserting a nucleic acid into a cell means "transfect" or "transform" and includes the incorporation of nucleic acids into a eukaryotic or prokaryotic cell in which nucleic acid can be incorporated into the genome of the cell (in the DNA of a chromosome, a plasmid, a plastid or a mitochondrion), or it can be converted into an autonomous replicon, or expressed transiently.

The term "isolated" refers to a material (nucleic acid or protein) that is: (1) substantially or completely free of the components that normally accompany it or interact with it in its natural form. The isolated material may optionally comprise another material that is not associated with the isolate in its natural form; O well (2) in case the material is in its natural environment, if the material has been altered in a synthetic way by a deliberate human intervention that modifies its composition or allocates it to a specific place in the cell (for example, an organelle ) different from where it is in its natural environment. The alteration that gives rise to the synthetic form of the material can be directed to the material (nucleic acid and / or protein) or depend on a removal from the natural environment. For example, a natural nucleic acid can be isolated if it is altered or if it is transcribed from DNA that has been previously altered from a deliberate human intervention in the cell from which the nucleic acid originated13. In the same way, a natural nucleic acid (for example, a promoter) is isolated if it is introduced by unnatural means into a locus of the genome that is not native to it. Nucleic acids that are "isolated" following this definition can be referred to as "heterologous" nucleic acids.

The term "located in a chromosomal region defined by" in reference to specific molecular markers refers to a chromosomal segment delimited by the corresponding markers, and includes said markers.

The term "marker" refers to a locus on a chromosome that serves to identify a unique position on said chromosome. A "polymorphic marker" refers to a marker that can have various forms (alleles) so that the different forms of the marker, when present in a pair of homologous chromosomes, allow the transmission of each one to be tracked. of the chromosomes of the pair. A genotype can be defined by one or more polymorphic markers or not.

The term "nucleic acid" or "nucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in their single or double strand form and comprises those analogous molecules which have the essential nature of the natural nucleotides of being able to hybridize to strand nucleic acids simple in a way similar to that of natural nucleotides.

The term "nucleic acid library" refers to a collection of RNA or DNA molecules that comprise and substantially represent the integrity of the transcribed fraction of the genome of a specific organism. Examples of library constructions, whether genomic libraries or cDNA libraries, are known in the literature14.

The term "operably linked" refers to a functional linkage between a promoter and a second sequence, in which the promoter sequence initiates and allows transcription of the DNA sequence corresponding to the second sequence. Generally, operatively linked means that the ligated sequences are contiguous, and in the case that it is necessary to join two sequences coding for the corresponding proteins, they are contiguous sequences and in the same reading frame.

In this document, the term "plant" may refer to complete plant organisms, parts of plants or organs (e.g., leaves, stems, roots, ovules etc.), plant cells, or seeds and plant offspring. Plant cells in this case include without limitation cells obtained from or present in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, buttons, meristems, gametophytes, gametes, sporophytes, pollen and microspores . Plant cells can also include cells modified as protoplasts obtained from the above-mentioned tissues. The type of plants that can be used for the present invention is generally as broad as the group of Angiosperms that can be modified by transformation techniques in both dicotyledonous and monocotyledonous. Particularly important plants are corn, soybean, sunflower, sorghum, cañola, wheat, alfalfa, cotton, tomato, potato, rice, barley, millet and Arabidopsis.

The term "polynucleotide" refers to a deoxyribonucleotide, a ribopolynucleotide, or its analogues having the essential properties of a natural ribonucleotide, such as the fact that they hybridize, under conditions of astringent hybridization, to essentially the same nucleotide sequences as the nucleotides. that occur naturally, or that allow translation into the same amino acids as natural nucleotides. A polynucleotide may be full-length or a sequence segment of a structural gene or a native or heterologous regulatory gene. Unless explicitly mentioned, the term includes the specified sequence as well as its complementary sequence. Therefore, DNA or RNA molecules containing segments that have been modified to increase their stability or for other reasons are also "polynucleotides" for purposes of the present invention. Additionally, DNA or RNA molecules that include infrequent nucleotide bases, such as inosine, or modified nucleotide bases, such as tritiated bases, to name just two examples, will also be considered "polynucleotides" for purposes of the present invention. It follows from this paragraph that a variety of modifications have been made to DNA or RNA molecules, and that such modifications serve multiple purposes. The term "polynucleotide" is used herein to designate forms of chemically-modified polynucleotides, enzymatically or metabolically, as well as the chemical forms of DNA and RNA characteristic of viruses and simple or complex cells.

The terms "polypeptide", "peptide", and "protein" are used herein interchangeably to designate a polymer of amino acids. The terms refer to amino acid polymers in which one or more of the amino acids is an analogue of a corresponding natural amino acid. The essential nature of such analogs is that when they are incorporated into a protein, said protein can be specifically recognized by antibodies designed to recognize that same protein when it is composed exclusively of natural amino acids. The terms "polypeptide", "peptide", and "protein" also include, without limitation, glycolisation, lipid binding, sulfation, carboxylation range of glutamic acid-containing residues, hydroxylation and ADP-ribosylation. mention that the polypeptides are not completely linear, for example, the polypeptides may be branched as a result of ubiquitination, they may be circular, with or without branching as a result of post-translational events such as natural processes and events caused by human manipulation that do not occur naturally. Circular and / or branched polypeptides can be synthesized by natural processes that do not depend on translation and completely synthetic processes. Additionally, this invention also includes terminal variants of amino acids with or without methionine of each of the proteins relating to the present invention.

The term "promoter" refers to a region of DNA located upstream of the start codon of transcription and which is involved in the recognition and binding of an RNA polymerase and other proteins necessary for the initiation of transcription. A "plant promoter" is a sequence derived from plants or from other organisms but capable of initiating transcription in plant cells. Some examples of plant promoters include those obtained from plant DNA, plant viruses and bacteria that contain genes that are expressed in plants such as Agrobacterium or Rhizobium. Examples of promoters that are under control of developmental states include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots or seeds. This type of promoters is called "tissue-preferred". Those promoters that only initiate transcription in certain tissues are called "specific promoters". A specific promoter of "cell type" is a promoter that only directs the expression in certain types of cells located in one or several organs, such as cells of the vasculature in leaves and roots. An "inducible" or "repressible" promoter is a promoter that responds to control signals that it emits from the environment. Some examples of environmental conditions that may have an effect on transcription dependent on inducible promoters are the presence of anaerobic conditions, or the effect of light. The tissue-specific promoters, the tissue-preferred promoters, the cell-type specific promoters and the inducible promoters constitute the class of "non-constitutive" promoters. The "constitutive" promoters are those that direct expression systemically and under most the environmental conditions.

The term "recombinant" refers to a cell or a vector that has been modified by the introduction of nucleic acid or that a cell is derived from another cell that was modified by said introduction. For example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or that express native genes that would otherwise be sub-expressed, expressed abnormally, not expressed to nothing as a result of human intervention. The term "recombinant" does not include the alteration of the cell or the vector by events that occurred naturally (for example, spontaneous mutation, or transformation, transduction or transposition) as all those that occur without human intervention.

The term "expression cassette" or "expression construct" refers to a nucleic acid construct (generated in recombinant or synthetic form) that contains a series of nucleic acid elements that allow the transcription of a particular nucleic acid in a cell Guest. The recombinate expression cassette can be incorporated in a plasmid, in a chromosome, in mitochondrial DNA, in platidic DNA, in a virus, or in a nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid to be transcribed and a promoter.

The terms "residue" or "amino acid residue" are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide or a peptide. The amino acid may be natural or may include synthetic amino acids analogous to natural amino acids that can function in a manner similar to natural amino acids.

The term "hybrid selectively" refers to the hybridization (under stringent hybridization conditions) of a nucleic acid sequence to a specific nucleic acid target sequence that can be distinguished by detection of its hybridization to a non-white nucleic acid sequence , which serves to substantially exclude non-white nucleic acids. Sequences that selectively hybridize typically have 90% shared sequence identity, and preferably 100% shared sequence identity.

The term "astringent conditions" or "astringent hybridization conditions" refers to conditions in which a probe hybridizes to its target sequence at a level higher than that of other sequences (i.e., at least 2 times greater than the background). The astringent conditions are dependent on the nature of the sequence and may vary depending on the circumstances. By controlling the conditions of astringency and washing, white sequences with 100% homology to the probe can be identified. Alternatively, the stringency conditions can be adjusted to allow certain non-homologous pairings in the sequences so that lower homology levels can be detected. Generally, a probe is less than 1000 nucleotides in length and optionally less than 500 nucleotides.

The conditions of astringency will generally be those in which the salt concentration is less than.% M of Na ions, and typically between 0.01 and 1.0 M concentration of Na ions (or other salts) at a pH of 7.0 to 8.3 and a temperature of at least 30 ° C for short probes (10 to 50 nucleotides) and at least 60 ° C for long probes (greater than 550 nucleotides). The astringent conditions can also be obtained from destabilizing agents such as formamide. Examples of stringent hybridization conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at a temperature of 50 to 55 ° C. Examples of moderate stringency conditions include hybridizations in 40-45% formamide, 1 M NaCl, 1% SDS at 37 ° C, and a wash in 0.1 X SSC at a temperature of 60 to 65 ° C.

The specificity is usually determined by the washings after hybridization, and the critical factors are the ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tm value can be approximated from the equation of einkoth and Wahl15: Tm = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L; where M is the molarity of the monovalent cations,% GC is the percentage of the guanosine and cytosine nucleotides in the DNA,% formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs . The Tm is the temperature (under certain conditions of ionic strength and pH) at which 50% of the hybrid white sequence to a probe that is perfectly homologous. The Tm is reduced by 1 ° C for every 1% of mating disparity between the probe and its target; therefore, the hybridization or washing conditions can be adjusted to hybridize with desired sequences. For example, if one seeks to detect sequences that have only 90% homology with the probe, the Tm value can be decreased by 0 ° C. Generally the conditions of astringency can be selected to be 5 ° C lower than the value of Tm corresponding to the specific sequence and its complement under certain conditions of pH and ionic strength. However, severe stringency conditions may use hybridization or washings of 1 to 4 ° C lower than Tm .; moderate stringency conditions can use hybridizations or washes of 11 to 20 ° C lower than Tm.

Using the aforementioned equation the hybridization conditions, the composition of the washing solutions and the desired Tm, those that have been routinely trained in molecular biology techniques may understand that the variations in the conditions of stringency of a hybridization or of the solutions of washing are clearly described. If the acceptable degree of mismatch between the two sequences results in a Tm value of less than 45 ° C (aqueous solution) or 32 ° C (formamide solution), it is preferable to increase the concentration of SSC in such a way that a temperature value Top can be used during hybridization and / or washings. An extensive guide to nucleic acid hybridization techniques can be found in the literature16.

The term "transgenic plant" refers to a plant that contains in its genome a heterologous polynucleotide. Generally said heterologous polynucleotide is stably integrated and is transmitted to the offspring of said plant. The heterologous polynucleotide can be integrated into the genome in isolation or as part of a recombination vector. The term "transgenic" includes here any cell, cell line, callus, tissue, part of the plant or plant, for which the genotype has been altered by the presence of a heterologous nucleic acid, including those transgenic elements that were created by transformation like those created by sexual crossings or asexual propagation from the initial transgenic. The term "transgenic" does not refer here to the alteration of the genome (chromosomal or extra-chromosomal) by conventional methods of genetic improvement or by natural events such as random crosses, non-recombinant viral infection, non-recombinant bacterial transformation, transposition non-recombinant or spontaneous mutation.

The term "vector" refers to a nucleic acid used for the transfection of a host cell into which a polynucleotide can be inserted. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid that has been inserted into them.

The terms indicated below are used to describe the relationship between the sequences of two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison interval", (c) "identity of the sequence ", (d) percentage of identity of the sequence" and (e) "substantial identity". (a) The term "reference sequence" is a defined sequence that is used as a basis for the comparison of sequences. A reference sequence may be a portion or a complete sequence; for example, a segment of a full-length cDNA, or the full length of the cDNA or an entire gene. (b) The term "comparison interval" refers to a specific and contiguous segment of a polynucleotide sequence that can be compared to a reference sequence and for which the comparative portion of the polynucleotide sequence can include additional or absent elements (eg. example, "gaps") when compared to the reference sequence (which has no additional or absent elements) so that the alignment of the two sequences is optimal. In general, the comparison interval is at least 20 contiguous nucleotides and can often include more than 100 nucleotides. Those who know how to perform this type of analysis understand that to avoid a high similarity with the reference sequence because of "gaps" in the polynucleotide sequence, penalty values for the "gap" can be used. Several methods of sequence alignment are well known. The optimal alignment of sequences for comparison can be obtained from the local homology algorithm of Smith and Waterman17, by the Needlman and Wunsch algorithm18, by the similarity search method of Pearson and Lipman19, by the computerized implementation of these algorithms, including but not limited to: CLUSTAL in the PC / Gene program of Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA and TFASTA in the package called Wisconsin Genetics Software, Genetics Computer Group (GCG), 575 Sciende Dr., Madison Wisconsin, USA; the CLUSTAL program of Higgins and Sharp20. The family of BLAST programs that can be used for comparative sequence searches includes: BLASTN for comparative search of nucleotide sequences compared to nucleotide sequences contained in public domain databases; BLASTP for comparative search of protein sequences compared to protein sequences contained in public domain databases; TBLASTN for the search of homologies between a protein sequence and a nucleotide sequence; and TBLASTX for a comparison of a nucleotide sequence with a set of nucleotide sequence databases21.

Unless otherwise explicitly stated, the values of identity or similarity of a sequence that are indicated in this document refer to the values obtained with the BLAST 2.0 version using the "default" parameters 22. The software to perform these analyzes is in the public domain and can be accessed or obtained through the website of the National Biotechnology Information Center of the United States of America. The algorithm begins by identifying pairs of sequences with a high degree of similarity from the identification of short words with a length of W in the search sequence; these words must be identical or very similar to that of a threshold of value T when they are aligned with a word of the same length contained in a database of the public domain. This initial similarity between two sequences gives rise to the beginning of a series of searches to find sequences of greater length and high degree of similarity. A T is called the word neighborhood value threshold23. The words found extend in both directions for each of the sequences as long as the cumulative alignment value continues to grow. For nucleotide sequences the cumulative values are calculated using the parameters (value of reward for a pair of matching residues, always greater than 0) and N (value of punishment for non-coincident residues, always less than 0). For amino acid sequences, a matrix value is used to calculate the cumulative value.

The length of the words found stops in each of the two directions when: a) the cumulative alignment value falls by the quantity X less than the maximum value obtained; b) the cumulative value becomes less than or equal to zero due to the. accumulation of one or more alignment residues with a negative value; or c) the end of each sequence is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the sequence alignment. The BLASTN program (for nucleotide sequences) uses as a default value a word length W of 11, a value of expectation E of 10, a threshold of 100, = 5, N = -4, and performs the comparison of both strands of nucleotide sequences24.

In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences25. A measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability with which the agreement between two nucleotide or amino acid sequences may occur at random.

BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences that may be enriched with one or more amino acids. Those regions of low complexity can be aligned from unrelated proteins although other regions of said proteins are completely different and do not show similarity. Some programs that function as low complexity filters can be used to reduce this type of alignments. For example, the program SEG26 and XNU27. These types of filters can be used individually or in combinations. (c) The terms "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues in both sequences that are the same when aligned to find the greatest correspondence within a window specific comparison. When the percentage of sequence identity is used in reference to proteins it is recognized that the positions of the residues that are not identical often differ by substitutions of conservative amino acids, in which the amino acids are substituted by other amino acids that have similar biochemical properties (charge or hydrophobicity) and therefore does not change the functional properties of the molecule. If the sequences differ in by the conservative nature of the substitution, the percentage of sequence identity can be adjusted upwards to correct the effect of the conservative nature of the substitution. It is said that those sequences that differ in conservative substitutions have "sequence similarity" or "similarity". The task of making these kinds of adjustments is routine for those with skills in the art. It usually involves assessing a conservative substitution as a partial and non-complete sequence discrepancy, thus increasing the percentage of sequence identity. For example, when an identical amino acid is assigned a value of 1 and a non-conservative substitution is assigned a value of zero, a conservative substitution is assigned a value between zero and 1. The value of the substitutions is calculated using the algorithm of Meyers and Miller28, as implemented in the PC / GENE program (Intellegentics, Mountain View, California, USA). (d) The term "identity sequence percentage" refers to the value determined from the comparison of two sequences optimally aligned from a specific comparative window, and in which the polynucleotide sequence portion in the comparison window may include additions or deletions (ie, absences) when compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of two sequences. The percentage is calculated by determining the number of positions from which the nucleotide base or the residue of > amino acids appears in both sequences to obtain the number of matching positions, dividing said number by the total number of positions in the comparison window and multiplying by 100 to obtain the percentage of identity sequence. (e) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence having at least 70%, preferably at least 80%, even more preferably 90% and ideally 95% sequence identity when compared to a reference sequence using one of the aforementioned alignment programs. It is recognized that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by 2 nucleotide sequences and taking into account codon degeneracy or amino acid similarity. For this purpose the substantial identity of amino acid sequences usually means an identity of at least 60%, or preferably 70%, 80%, 90% and ideally 95%.

Another indication that nucleotide sequences are substantially identical is the fact that two molecules hybridize with each other under stringent hybridization conditions. However, nucieic acids that do not hybridize to one another under astringent conditions remain substantially identical if the polypeptides they encode are substantially identical. This can occur when, for example, a copy of the nucleic acid is created using the maximum codon of degeneracy allowed by the genetic code. An indication that two sequences of nucieic acids are substantially identical is that the polypeptide encoded in the first nucleic acid is immunologically identical to the polypeptide encoded in the second nucleic acid.

The term "substantial identity" in reference to peptides indicates that a peptide comprises a sequence with at least 70%, preferably 80% or 85%, even more preferably 90%, and ideally 95% sequence identity with a reference sequence in a specific comparative window. Optionally, the optimal alignment is effected using the alignment algorithm of Needleman and Wunsch29. An indication that two peptide sequences are substantially identical is that a peptide is immunologically identical to the second peptide. For example, two peptides will be substantially identical if they only differ by a conservative substitution.

Detailed description. In this invention it is shown that applicants have identified promoters isolated from Arabidopsis DNA with ability to regulate gene expression in plants so that a selected gene can be efficiently transcribed in the female or male reproductive cells of plants, and / or in the female or male gametophyte (the pollen grain) at a precise moment and place, and in the amount necessary to obtain the desired effect. Promoters that direct the temporal and spatial expression in the ovule, in the female gametophyte, in the pollen, or in the sperm cells are particularly important and can be used with a great variety of transgenic protocols, to alter the reproductive mechanisms and eventually induce apomixis , or formation of asexual seeds genetically identical to the mother plant. Another important application of these promoters is the possibility of using them to implement technologies that prevent the transfer of transgenic traits through pollen in the field. They can also be used to provide additional nutrients to the seed and to confer resistance to pathogens and diseases in the seed, or to provide other features of high added value, to name a few examples.

The invention was obtained by generating transgenic plants that contain elements "gene trap" (gene trap) or "enhancer detector" (enhancer trap) 30. From these technologies, 2 regions were identified in the genomic DNA of Arabidopsis thaliana that confer transcriptional activity in diploid or haploid reproductive tissue of this plant. In one case the activity is restricted to the female tissues (the female gametophyte and the female gametes) and in the other to the male tissue (the pollen grain and the male gametes). Using this information and routine molecular biology techniques widely known in the area, the specific activity of these regulatory regions was confirmed in fusions of said regulatory regions with the reporter gene uidA (GUS) that encodes a beta-glucuronidase in Escherichia coli. These sequences can also be used as molecular markers to identify other genes or other regulatory regions as well as markers for molecular mapping.

In general, specific promoters such as those described for the ovule, the female gametophyte, the pollen grain or the male gametes can be isolated using known methods. These transcriptional control sequences are generally associated with genes that are activated only in the organs, tissues and times desired. In a normal plant, each organ or tissue contains thousands of messenger RNAs (mRNAs) that are absent in other tissues or organs31. The mRNAs are isolated to obtain probes that are used to subsequently obtain the appropriate genomic sequence containing the corresponding regulatory sequences. In this sense, an example of the use of cDNA associated with specific mRNA has been reported in avocado fruit32. In short, in this study the mRNA was isolated from the avocado fruit during its maturation process and was used to construct a cDNA library. The desired clones of the library were identified from their hybridization with radioactively labeled RNA and isolated from mature fruits; said probes did not hybridize with radioactively labeled RNA and isolated from immature fruits. Many of the clones obtained represented mRNAs encoded by genes that are transcriptionally active only in mature fruits.

Another important method to identify specific promoters and which was used to obtain the invention presented here, and which allows the identification of active promoters in single cells is the method of gene traps (gene trap) and enhancer detector (enhancer traps) 33. This method was initially developed in Drosophila and quickly adapted for mice and plants34.

In this type of strategies, transposable elements (or transposons) are used that are modified to serve as gene traps or detector of enhancers that are randomly integrated into the genome of plants to generate transgenic individuals that are later analyzed to identify genes that are locate in the immediate vicinity of the insertion site. From the identification of the moment and the site (organ, tissue or cell) where the expression of the reporter gene present in the transposon is activated, the sequence surrounding the insertion is routinely identified by means of TAIL-PCR35. From this sequence, the tagged gene and its corresponding promoter are identified. In some lines the enhancer detector element may have been inserted into the coding region of the tagged gene. In others the element can be directly inserted in the promoter of the corresponding gene. The promoters that direct the activity of the ovule gene, the female gametophyte, the pollen grain or the sperm cells can be isolated and evaluated using routine molecular biology techniques such as those described here. An example of the method begins with the identification of transcriptional units that surround the insertion site of the transposon, the subsequent isolation of regulatory sequences located in cis, and the fusion of said sequences with a reporter gene such as beta-glucuronidase GUS36. From these fusions transgenic plants are generated and it is identified which of the fragments confers activity in the tissues and the desired moment.

The applicants of this patent provide in this document sufficient data to characterize the promoters that were identified: the demonstration that they act in a specific way in the ovules, the female gametes, the pollen grain or the male gametes, and that they were isolated from of their identification by means of gene traps and the strategies described below: After having identified lines of gene traps that had expression patterns in the female or male gametophyte of Arabidopsis thaliana, the insertion site of the active transposon was identified by means of TAIL-PCR. The transposon may have been inserted into coding regions in the promoter region of transcriptional units predicted from programs and algorithms established for the analysis of the Arabidopsis thaliana genome. In both cases, the identification of regulatory sequences (promoters) is focused on the gene identified by the site of the transposon insertion. In most cases, regulatory sequences have been identified in the first 2 or 3 Kb immediately adjacent to the 5 'end of the coding region of the corresponding gene37. Therefore, the region located at the 5 'end of the coding region of the gene is amplified by PCR and cloned against an appropriate reporter gene (GUS or GFP). In some exceptional cases, regulatory sequences have been identified in introns or in regions 3'38. If the 5 'region does not confer the expected expression pattern, possibly introns or 3' regions of the same gene can be cloned against a minimal promoter to again monitor the expression of a reporter gene (such as the minimal promoter of the 35 gene used in the power detector constructions39). If the insertion of the transposon is located in a region located between two genes, the same analysis can be performed to determine which of the two genes is activated in an expression pattern identical to that identified by means of gene traps.

From the information presented herein, the transgenic plants are not necessary to obtain the invention since the location of the promoters and their sequence are shown in Figures 1 to 4, and the description of the activity patterns of each one. of the promoters is illustrated in Figure 6.

Once the promoters have been identified and isolated, they can be used for a multitude of applications to transform plants by genetic engineering strategies. The selection of structural genes, the transfer vector and the transformation method can be made by any person skilled in the art and therefore all these aspects fall within the protection limits of this invention.

STRUCTURAL GENES. In the same way, through the invention presented here, genes of agronomic interest can be expressed in transformed plants. In particular, plants can be transformed by genetic engineering methods to express several phenotypes of agronomic interest whose characteristics must be manifested in the female gametophyte, the female gametes, the pollen grain or the male gametes. Some non-exclusive examples are included below. 1. Genes that confer resistance to pests or diseases such as: A) Residence genes to diseases. The plant defenses are activated in a specific way from the interaction of the product of a disease resistance gene present in the plant (called R) and the product of an avirulence gene (Avr) present in the pathogen. A plant variety can be transformed with a disease resistance gene to generate transgenic plants resistant to a specific pathogen40.

B) A protein from Bacillus thuringiensis41, which reported the isolation and sequencing of a gene that codes for a Bt d-endotoxin. In addition, DNA molecules that encode? -endotoxins can be purchased from the American Type Cell Culture Collection (Rockville MD, USA), under catalog numbers Nos. 40098, 67136, 31995 and 31998.

C) A lectin42, which reported the nucleotide sequence of several ligase-mediated lectin-binding genes in Olivia miniata.

D) A vitamin binding protein, such as avidin43. The use of avidin and its homologues as larvicides against insects is shown.

E) An enzyme inhibitor such as an inhibitor of proteases or amylases44.

F) A specific hormone of insect or ferhormona like an ecdisteroide or a juvenile hormone, or one of its variants. An example of this is the expression of a juvenile esterase in baculovirus45.

G) An insect-specific peptide or neuropeptide that when expressed causes a physiological interruption in a pest46, such as an altostatin identified in Diploptera puntata. "In the literature, genes encoding insect-specific paralytic neurotoxins are reported47.

H) A specific poison for insects produced in nature by a viper or a wasp etc. In the literature, the heterologous expression in plants of a gene that encodes a insect-toxic scorpion peptide is reported48.

I) An enzyme responsible for the hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a derivative of phenylpropanol or another non-protein molecule that has insecticidal activity.

J) An enzyme involved in the modification of a biologically active molecule (including post-translational modifications); for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase or a glucanase , whether natural or synthetic. Scott reports the nucleotide sequence of a gene that encodes a callassa49. Molecules containing chitinase coding sequences can be obtained, for example, by the methods described by Kramer50, which shows the nucleotide sequence of a cDNA sequence encoding a tobacco borer worm chitinase, and by Kawalleck51, which report the nucleotide sequence of the ubi4-2 gene that codes for a polyubiquitin.

K) A molecule that stimulates transduction signaling. For example, see the report of Botella52 that describes a cDNA corresponding to a gene that codes for a calmodulin of beans, or that of Griess53, which reports the nucleotide sequence of a cDNA clone of corn calmodulin.

L) A hydrophobic peptide. Such as, for example, derivatives of tachyplesin peptides that inhibit the growth of fungal pathogens of plants54 and synthetic antimicrobial peptides that confer resistance to diseases55.

M) A membranal permease that generates or blocks the formation of cellular channels. For example, see the report by Jaynes56 corresponding to the heterologous expression of a lytic cecropin that. confers resistance against Pseudomonas solanacearum to tobacco plants.

N) A viral invasive protein or a complex of toxins derived. For example, protein accumulation of viral coverage in transformed plant cells confers resistance against viral infections57. This type of resistance has been in alfalfa plants that were given resistance against the tobacco mosaic virus, the cucumber mosaic virus, the potato X virus, the potato Y virus, or the mosaic virus. of tobacco, as examples.

O) An insect-specific antibody or an immunotoxin derived therefrom. For example, an antibody whose target is a metabolic function in the intestine of the insect that is lethal for the latter58. Enzymatic inactivation of transgenic tobacco from the production of single chain antibody fragments.

P) A virus-specific antibody. See, for example, the report by Tavladoraki59 showing that transgenic plants that express genes encoding recombinant antibodies are protected against viral attacks.

Q) A protein that stops the development of a pathogen and that is produced by a pathogen or a parasite. For example, endo-1 -4-D-polygalacturonase of fungal origin facilitates the colonization of fungi and the loss of plant nutrients by solubilizing homo-4D-galacturonase in the cell wall60. The cloning and characterization of a gene that encodes an endopolygalacturonase inhibitor protein is described in Toubaart61.

R) A protein that stops the development of a pathogen and that is produced by the plant. For example, Logemann62 shows that transgenic plants that express the ribosome inactivating gene in barley have a high resistance to fungal diseases.

Genes that confer resistance to a herbicide, such as: A) A herbicide that inhibits the growth point of meristems, such as imidazalinone or sulfonylurea. Examples of genes that confer resistance to this type of herbicide code for mutant versions of the ALS or AHAS enzyme as described by Miki63.

B) Glysophosphate (resistance conferred by a mutant of 5-enolpyruvyl-3-phosphiquimate synthase (EPSP) and the aroA genes, and other phosphorus compounds such as glufosinate (genes that encode a phosphinothricin acetyl transferase (PAT) or a phosphinothricin acetyl transferase) of Streptomyces hygroscopicus known as the bar gene), and pyridinoxy- or phenoxy propionic acids and cyclohexones (genes encoding ACCase inhibitors) Shah et al64 released the nucleotide sequence from an EPSP form that confers resistance to glyphosates. A mutant of the aroA gene can be obtained from the information reported by Comai65.

C) A herbicide that inhibits photosynthesis such as triazine (genes psbA and gs +) and benzonitrile (gene of nitrilase). See for example Przibilla66 where the transformation of Chalmydomonas with plasmids encoding psbA gene mutants is described. The nucleotide sequence of the genes encoding nitrilase was reported by Stalker67 and the molecules containing these genes are found under accession number ATTCC Nos. 53435, 67441 and 67442. The cloning and expression of the gene encoding an S-transferase is described by Hayes68.

Genes that confer or contribute to obtain a feature of high added value as the following non-exclusive examples: A) Modifications to the metabolism of fatty acids, as for example, when transforming with an anti-sense gene that codes for a desaturase that increases the Stearic acid content in the plant69.

B) A reduction in the phytate content: a) Introducing a gene that codes for a phytase and that contributes to the degradation of the phytate, increasing the free phosphate in the transforming plant. See, for example, Van Hartingsveldt70 for the report of a nucleotide sequence of a gene encoding an Aspergillus niger phytase. b) A gene that reduces the phytate content. In corn, this can be achieved by cloning and re-introducing DNA associated with a single allele responsible for mutants that are characterized by a low level of phytate71. C) Modifications of the carbohydrate content, for example, when transforming plants with a gene that codes for an enzyme that alters the branching pattern of the starch. See for example the report of - Shiroza72 where the nucleotide sequence of a gene encoding a fructosyltransferase of Streptococcus mutans, of Steinmetz73 where the nucleotide sequence of a levansucrase of Bacillus subtilis is used), of Elliot74 where a nucleotide sequence of a gene encoding an invertase is used of tomato), or of Fischer75 that reports the nucleotide sequence of a gene that encodes a branching enzyme of endosperm starch. D) Induction of programmed cell death of transgenic pollen. A transferred transgenic trait behaves as a dominant trait whose inheritance may be undesirable under certain conditions. In the case of plants that allow cross-pollination, this type of involuntary transfer can have legal and ecological consequences that seriously compromise the implementation of transgenic crops in any country in the world. In many species, the horizontal transfer of a transgenic trait to non-transgenic plants of the same species can cause serious confusion between plants for industrial production and plants intended for human consumption. Additionally, relatives close to crop plants can also be affected by inter-specific crosses that transfer undesirable transgenic characteristics to F1 hybrids, which can represent serious risks for breeding programs that use introgression methods. This last problem is particularly important in countries such as Mexico that represent the center of origin of many species of agricultural interest. To eliminate the horizontal transfer of transgenic traits, lethal gametophytic genes can be used that, linked in a molecular construction to the transgenic character, eliminate the possibility that pollen grains containing the transgenic allele participate in seed formation. The gametophytic lethal gene would be present only in the genome pollen grains that contain the transgenic construction (50% of the pollen grains). The rest of the pollen (50%) will not carry the transgenic trait, and since it is completely viable, it will ensure cross-pollination (and self-pollination), leading to the formation of seeds free of transgenic traits.

Genes that control the growth and development of the seed, such as: A) Genes that control cell proliferation and the growth of the embryo and / or the endosperm as they can be regulators of the cell cycle76, genes that affect cell proliferation in the embryo and in the endosperm to increase the size of the seed, generate biomass change or minimize the content of certain compounds in the seed77. B) Genes that promote the autonomous proliferation of the endosperm in the absence of fertilization, an essential component of apomixis (or asexual reproduction from seeds), such as MEA, FIS2 or FIE (see references in the previous paragraph).

C) Genes that promote the formation of the embryo, another of the components of apomixis (asexual reproduction from seeds) such as SOMATIC EMBRYOGENESIS RELATED KINASE (SERK) 78 or LEAFY COTYLEDONS. 5. Genes that generate programmed cell death of transgenic pollen to avoid the transfer of transgenic traits in crop plants, such as: A) The gene that codes for, a T179 RNAse and the gene that codes for the A chain of diphtheria toxin. Pseudomonas aeruginosa80. It has been widely demonstrated that the expression of each of these two genes is capable of causing the death of plant cells with great specificity81.

PROMOTERS The promoters described herein and which are the subject of this patent application may be used in association with natural versions of transcribed sequences of a desired structural gene or with any other transcribed sequence that is critical for the formation or function of genes.

It may also be desirable to include introns sequences in the constructs containing the promoter since the inclusion of introns sequences may result in amplification of the expression or specificity of the promoter. It may be advantageous to bind DNA sequences to a promoter sequence containing the first intron and the first exon of a polypeptide that is unique to cells and tissues critical for seed formation.

Additionally, certain regions of a promoter can be linked to regions of a second promoter different from the first to obtain the desired activity, giving rise to a chimeric promoter. Synthetic promoters that regulate gene expression can also be used.

The expression system can also be further optimized by using supplementary elements such as transcriptional terminators or enhancer elements.

OTHER REGULATORY ELEMENTS. In addition to the sequence of a promoter, a construct must also contain a transcription terminator region adjacent to the desired structural gene, and this in order to provide an efficient transcriptional termination. The terminator region or the polyadenilization signal can be obtained from the same gene to which the promoter used belongs or from the terminator region of other genes. Polyadenylation sequences include but are not limited to the termination signal of the gene encoding the octopine synthase of Agrobacterium tumefaciens82, or the terminator signal of the gene encoding nopaline synthase83.

MARKERS GENES. Recombinant DNA molecules containing any of the sequences and promoters described in this patent application may contain additional marker genes that encode a selection gene product that confers on the cells of the plants resistance to a chemical agent or to a physiological stress, or that confer a distinctive phenotypic characteristic to plant cells transformed with recombinant DNA molecules that can be recognized by means of a selection agent. One of the selection markers routinely used is the gene that codes for neomycin phosphotransferase (NPTII), which confers resistance to kanamycin and the antibiotic G-418. Cells transformed with these selection markers can be selected by determining the in-vitro presence of kanamycin resistance in the seedlings, using techniques widely described in the literature or determining the presence of the mRNA corresponding to the NPTII gene using a Northern blot from tissue of the transformed plant. The polymerase chain reaction (PCR) can also be used to identify the presence of a transgene using routine RT-PCR strategies to monitor marker gene expression. Other selection markers that are routinely used are the ampicillin resistance gene, the tetracycline resistance gene and the hygromycin resistance gene. Cells from transformed plants can be induced to differentiate into plant structures that can eventually give rise to whole plants. It is understood that a marker gene can also be endogenous to a plant, as is the case of endosperm pigmentation genes that can be used as marker genes in corn.

TRANSFORMATION. A recombinant DNA protein that is designed to inhibit expression or to induce expression and that contains the DNA sequences and / or promoters described herein can be integrated into the genome of a plant by introducing it into the cells of said plants. by some of the widely known methods and their variants. Generally the recombinant DNA molecule is initially introduced into an appropriate vector and said vector is used to introduce the recombinant DNA molecule into a plant cell.

The use of cauliflower mosaic virus (CaMV) 84 and some geminiviruses as vectors85 have been suggested as vectors, but the most successful vector has been developed with Agrobacteria sp86.

Methods for the use of Agrobacterium-based transformation systems have already been described for many different plant species. Generally bacterial strains contain modified versions of the Ti plasmid so that the DNA is transferred to the host plant without causing the subsequent formation of tumors. These methods depend on the insertion within the edges of the Ti plasmid of the DNA that is desired to be inserted into the genome of the plant and which includes a selection marker, generally a marker gene as one of those described above. Numerous plant tissues can serve as targets for transformation with Agrobacterium, and several have been specifically described for use in Crucifera plants, family to which Arabidopsis belongs. These tissues include thin layers of cells87, hypocotyls88, discs in leaves89, cotyledons90 and embryos91, or even whole plants using infiltration methods available for Arabidopsis and Medicago sp. but that are continuously standardized for other plant species. It is understood, however, that there are other transformation methods that may be more convenient for other crops.

Other methods that have been used to introduce recombinant molecules into plants include direct inoculation with DNA, through liposomes, electroporation92 and micro-injection93. The possibility of using microprojectiles and a cannon or other type of instrument to force particles covered with DNA inside cells has received considerable attention94.

It is often convenient to contemplate within the framework of the invention that here is presented the possibility of generating homozygous parental plants that require more than one transformation event with the same product. It can also be contemplated that a plant cell is transformed with a recombinant DNA molecule containing at least two different sequences or transformed with more than one recombinant molecule at different transformation events. The DNA molecules can be physically bound in the same vector, or separated and in different vectors. A cell can be simultaneously transformed with more than one vector as long as each vector has a unique selection marker. Alternatively, a cell can be transformed with more than one vector allowing an intermediate regeneration step after transformation with the first vector. Additionally, it may be possible to perform a sexual cross from plants or plant lines containing different DNA sequences or different recombinant DNA molecules and select in the offspring of said crossbreeding plants containing both desirable sequences of endogenous or recombinant DNA.

The expression of recombinant DNA molecules in transforming plants from the promoters and sequences described herein can be monitored using Northern or Southern Blot techniques and PCR methods that are known to all those practicing molecular biology.

The regeneration of viable plants from individual cells that have been transformed has been successfully carried out for a large number of plant species. For example, regeneration has been demonstrated in dicotyledons for at least the following species: apple (Malus pumila) 95, raspberry and its derived hybrids (Rubus sp.) 96, carrot (Daucus carota) 97, cauliflower (Brassica oleracea) 98, cucumber (Cucumis sativus) 99, celery (Apium graveolens) l00, eggplant (Solanum melonoea) 101, lettuce (Lactuca sativa) 102, potato (Solanum tuberosum) 103, soybean (Glycine canescens) 104; strawberry (Fragaria x anannassa) 105, tomato (Lycopersicon esculentum) 106, walnut (Junglans regia) 107, melon (Cucumis melo) 108, grapevine (Vitis vinifera) 109, mango (Magnifera indica) 110; and for the following monocotyledonous species: rice (Oryza sativa) III, cornll2 and Sécale cerealell3.

Additionally, the regeneration of whole plants from cells that have not necessarily been transformed has been successful in apricots (Prunus armeniaca) 114; Asparagus (Asparagus officinalis) 115; banana (Musa acuminata) 116, beans (Phaseolus vulgaris) ll7, cherry (Prunus sp.) 118, grapevine (Vitis vinifera) 119, mango (Mangifera indica) 120, melon (Cucumis melo) 121, okra (Abelmoschus esculentus) l22, onion (Allium sp.) i23, orange (Citrus senensis) l24, papaya (Car ate papaya) l25, peach (Prunus persica and Prunus domestica) 126, pear (Pyrus comunis) 127, pineapple (Ananas comosus) 128, watermelon (Citrullus vulgaris) 129 and wheat (Triticum aestivum) 130.

The regenerated plants are transferred to the soil and cultivated in a conventional manner. After the construction is stably incorporated into the genome of transgenic plants, it can be transferred to other plants of the same species or phylogenetically close species from a sexual cross. Several breeding techniques can be used, depending on the species you want to cross.

It may be useful to generate a number of transformed plants from any recombinant DNA construct to recover plants that do not exhibit undesirable effects due to the position of the transgenic insert. It may also be desirable to obtain plants containing more than one copy of the recombinant molecule.

According to the preferential incorporation, the transgenic plant that is used for the commercial production of a recombinant protein is corn. In another preferred embodiment of this invention, the biomass of interest are the seeds. For the relatively small number of plants showing high levels of expression, a genetic map can be generated mainly from the Polymorphic Length Restriction Fragment (RFLPs) strategy, from the polymerase reaction chain (PCR), and simple repeated sequences (SSRs) that identify the chromosomal location of the recombinant DNA molecule. For examples of these methodologies, see Glick and Thompson131. Map information regarding chromosomal location is useful for protection of the intellectual property of a transgenic individual in plants. If an unauthorized propagation of said transgenic plant is carried out by crossing it with another type of germplasm, the map of the integration region can be compared with other maps of suspicious plants, to determine if the plants suspected of having been propagated without authorization have relatives in common with protected transgenic plants. The comparison of maps is made from hybridizations, RFLPs, SSRs, PCRs and sequencing experiments, all techniques and technologies widely known in the environment.

As indicated above, it may be desirable to produce transgenic plant lines that are homozygous for a particular gene. In some species this can be done routinely from the m-vitro culture of anthers or microspores. This is particularly important in the case of the cañola, Brassica napus132. Using these techniques, it is possible to produce a haploid line that has the gene of interest integrated and duplicate the chromosome number spontaneously or from the use of colchicine. This results in a plant homozygous for the gene of interest, which can be easily tested if the inserted gene includes an appropriate selection marker for the detection of transgenic plants. Alternatively, the plants can be self-fertilized, resulting in a seed mixture containing 25% homozygous, 50% heterozygous and 25% null (non-transgenic) for the gene of interest. Although it is relatively easy to identify plants that contain and do not contain the gene of interest, in practice homozygous and heterozygous plants can also be identified from a Southern analysis in which special attention is paid to the amount of genomic DNA It is loaded in the gel before its hybridization. It is suggested to verify the results of the Southern Blot analysis by letting each independent transformant self-fertilize, since any additional evidence of homozygosis can be obtained from the simple fact that, if the plant is homozygous for the inserted gene, all the plants that result from the seed generated by self-fertilization will inherit and contain the gene, whereas if the plant is heterozygous, certain plants resulting from the seeds generated by self-fertilization will not inherit or contain the gene of interest. For this reason, transgenic homozygous plants can be selected from a simple self-fertilization.

The production of transgenic parental plants allows the generation of hybrids and seeds that contain a modified protein component. The homozygous parental transgenic plants are maintained so that each of the parents contains either the sequence of the first or the second recombinant molecule linked to an active promoter. This scheme also incorporates the advantages of cultivating a hybrid, including the advantage of having several agronomically interesting characteristics combined in the same individual.

The following examples serve to better illustrate the invention described herein and are not intended to limit the invention in any way.

EXAMPLES In our laboratory, a series of Arabidopsis promoters with activity in the embryo sac were identified, isolated and characterized. These promoters were identified through the use of gene-trap and enhancer-trap lines. In which transposing lines were searched with GUS expression schemes associated to the embryo sac in development and in lines that presented reproductive defects. The insertion site of the transposon was determined and the possible regulatory sequence responsible for the observed expression scheme was identified. Said sequence was fused to GUS and in the T1 transformant plants its expression scheme was determined.

The promoter pFM1 (FUNCTIONAL MEGASPORE-1) see SEQ ID1, was identified from a line "enhancer detector", or in English "enhancer-trap". This line presents a GUS expression scheme that starts from the functional megaspore and extends to the mature embryo sac. By means of TAIL-PCR it was determined that the Ds element was inserted in an intergenic region, at 788 bp of the At4g12250 (nucleotide sugar epimerase) gene and to approximately 2000 bp of the At4g12260 gene (similar to transposable element Ac-Iike). To identify the regulatory sequence responsible for the expression scheme observed in the transposing line, an 880 bp sequence was amplified by PCR, which comprises the nucleotide region -817 to +63, taking as reference the start codon (ATG) (see figure). 5).

The primers used in sense and in antisense (see sequences underlined in Figure 5) were added to the restriction sites Hindlll and BamHI respectively (in bold). This sequence was cloned in the PCRII-TOPO vector. This fragment was digested with the restriction enzymes mentioned, purified and cloned into the vector pB1101. The pBI101 vector has a multiple cloning site upstream of the GUS gene, which allowed to generate a transcriptional fusion; promoter-pFM1 :: GUS.

Plants of Arabidopsis thaliana ecotype Columbia were transformed using the floral dipping technique. GUS stains performed on transforming T1 plants presented an expression scheme identical to the transposing line (Figure 6). The expression scheme of the pFM1-GUS construct shows activity after meiosis and starts in the functional megaspore until the mature embryo sac and is restricted to the polar nucleus and the egg apparatus, no activity was detected in the antipodal cells (Figure 6a to 6c ). After fecundation, GUS expression is observed in the globular state, in the suspensor cell closest to the micropyle (data not shown).

The promoter pES1 (EMBRYO SAC 1), see SEQ ID2, was identified from another line "enhancer detector" or "enhancer-trap". This line presents an outline of GUS expression during megagametogenesis from the nuclear 4-8 state of the embryo sac to the mature embryo sac where it is active in antipodal cells, polar nucleus and egg apparatus (Figure 1d). By means of TAIL-PCR it was determined that the Ds element was inserted in the 5'-untranslated region (5'-UTR) of the At2g41050 gene coding for a hypothetical protein having homology with a transmembrane protein of Saccaromyces cerevisae (access no. : YDR352W). To identify the regulatory sequence responsible for the expression scheme observed in the transposing line, a 600 bp sequence comprising the nucleotide region -597 to -3, from the start codon (ATG) (see figure 7) was amplified by PCR. . The primers used in sense and in antisense are shown as sequences underlined in Figure 7.

The promoters pPol (see SEQ ID3) and pPo2 (see SEQ ID4) (POLLEN PROMOTER 1 and 2) were identified from 2 lines "additional enhancer detector" or "enhancer trap" showed expression patterns of the reporter gene in the grain of pollen (the male gametophyte) and the spermatic cells (the male gametes) (Figure 1e and 1f). In one of them the insertion was found in the 3'UTR region of a gene that codes for a mannose dehydrogenase (At4g37970), and in the other one it was found within the 5 'regulatory region of a gene that encodes a transcription factor of the type WUSCHEL (At3g11260). With the help of specific primers the sequences shown in figures 8 and 9 were amplified.

The sequences shown in Figures 8 and 9 were donated in the PCRII-TOPO vector. Using the restriction sites Xbal and BamHI present in this vector, this sequence was released and purified and cloned in the pBI101 vector. The pBH 01 vector has a multiple cloning site upstream of the GUS gene, which allows to generate a transcriptional fusion; promoter-pES1 :: GUS. Plants of Arabidopsis thaliana ecotype Columbia were transformed using the floral dipping technique. GUS stains performed on T1 transforming plants presented an expression scheme identical to the transposing line. The expression scheme of the pES1-GUS construct showed specific activity of GUS from the 4 and 8-nuclear states, to the mature embryo sac and an additional expression in small areas of the root.

Plants of Arabidopsis thaliana ecotype Columbia were transformed by the technique of "floral dipping" (floral transformation). GUS stains performed on T1 transformant plants showed that the activity of this sequence is of constitutive type, since GUS staining was observed in all tissues at seedling level and in inflorescences it shows activity in all cells of the ovule.

Additionally, the pFM1 promoter (see SEQ ID1) can be used successfully to alter the development of the female gametophyte and female reproductive cells in Arabidopsis thaliana. For this, a sequence of the CHR11 gene of Arabidopsis thaliana cloned against the pFM1 promoter in sense and anti-sense orientation was used, in order to post-transcriptionally silence the CHR11 gene by the strategy known as interferent RNA (RNAi). CHR11 encodes a chromatin remodeling protein essential for the nuclear divisions that result in the formation of female gametes in the female gametophyte. The transformant plants under the control of the pFM1 promoter showed altered female gametophytes (Figure 10) but no other defect in their vegetative or reproductive growth, demonstrating that the pFM1 promoter confers specific gene activation in the female gametophyte.

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Sequence Listing SEQ ID 1 Promoter Name: pFM1 Location: chromosome 4; coordinates 7290780 to 7291659 corresponding gene: locus At4g12250 Sequence: tgtatccaca catactagca tagtattgtt cacattataa gtagaaaatt cttattaaaa 60 aataaaaaaa tcactacatt ttttaatgca attttattgt ttacctccta aaaaacagat 120 aaataaaaaa caatttcata taactcgtaa taaaattcct cgattttgta caaaaataac 180 ttatttactt ttttgtcaat cgatataaat tagaactaaa aaaagttaat cccgaaagat 240 attatttata tatatgacat gatacgatcg attttatatc tatttttatc attattaccc 300 ccgttatttc acatttctta taaccttttg ttattgtata atttttcttt ttattatcaa 360 gaaatacaga tcggaacaaa aaaagtagta agttaaaagc aatattatta aacaattatt 420 atagtgtata gcgtgtaaaa tgtgatcgtg tacaaaagta catgcataac atgatcaata 480 tagaatgcaa tacaactata caaatgtcaa caatctacaa acacgtttta actttgaaaa 540 gcaatctaaa attcatgaat ctaaatatac tctctggcag ctttcttatt accatatctt 600 aaagattgaa ccaaataaat aaatttacat cattaaccaa attaaaccat ccacatttaa 660 tttaattaca acaagtaagt aaaagatcgt ctctttcttc aatcctctca tttcccggaa 720 tttttgactt gctcaatttt gacccaaaaa gcttccttaa tccaaaccta accctaatcg 780 acattttctc cgatttctaa atcctcctcc ggtgaaaatg tctcaccttg atgatcttcc 840 ttctactccc ggaaagtaca aaaccgataa agttccacc 879 SEQ ID 2 Promoter Name: pES1 Location: chromosome 2; coordinates 17132704-7133304 corresponding gene: locus At2g4 050 Sequence: cccggataat tcttccaagt ataaacccgt cgactcattc attcacgggt ttcgtttctt 60 tcttctggaa aaggtgacaa cttttttctg tttttttttt ataataaaag gtgacaactt 120 tctcttccat tgatgtgtaa acgaatagtc gaatacgaat tcgtttctct taatgtatta 180 gggaatcgat ttctcttgct ttttatgaaa ccggatattt caggctgcga atagattatt 240 ttaacaatgg tgttgatatg tgaatgtcga aaccagattt tgtgtaacgt tttcgatttt 300 gttgctttta atggaagaat ctgaatcctg agggtgtgat ttgggttttg ggggaatatt 360 cactttgtgg ggtctttttg tttgtgaatc tagtatattg aattattgtc ggaattagaa 420 atctttttta attctctagg attcgttttc tactctttca ttctagtgta aatgtggtca 480 aatgtttttc cttttcgtct tctaacactt ttttctagtt tgcacaatca aaaagacgtt 540 acctttgctc attaagggac ttgtttgatt tttgggttta acaggttttt ggctgtgaag 600 SEQ ID 3 Name promotor Ppol Location: Chromosome 3; Coordinates: 3526561-3527612 corresponding gene: Locus Sequence At3g1 260: aatggctttt tacaaatatt ttggtgtagg acttatatta tacatgtgtg tggcgaacct 60 tctggttcta tctattaaat gccttttctt tttgtagttt tttatgattc agaattcaga 120 tatttgattt tgtaatatta tattttctta taaaagagaa taaaatttca aatttcctgt 180 ctacacttgc cgaccaacgt tcctcaagtg tcacatcttc tgtgcaagag agacaaaact 240 acaatctgtt cacacaatgt catgatgttt ataattaatc ttatcatctt cattcatatc 300 aaactaatat cggccatcca tccgtctcaa taatgtaatt gcatacaatc tttgtttagg 360 acttgctagc tatttcaact tattattgat tacagattag atctacaatt tgtaaagaac 420 ttctaacttc aaaacaaata caaaaaaaag aaagtgaatg taaaatttct agtgtcataa 480 tttggaatag ttgaagcatt gaagttacaa tatcgaacgt taaaatattg acactagata 540 gtacattacc accaaggctt tctgattatt cttatcatgt tatatgaata gtaagaaatt 600 acgtagaacg caatttaagt gtgtgcgagt caccacaaat taaaggagaa caaattcaat 660 gtttcaatcg ctggttccga tatacaactt atgcatgcca gcgagatcca tggcatgaat 720 attatatact aatatataca tatgtaaata cacaaaaaca tagatggaac agaagcctag 780 ataggttagg ataaagaaaa cga tcaaatc tgcaaagatc agtctctccc aaatccccaa 840 aaaaaacaaa gcatgcattt cgtaataaac aactcatcat aaaacgacac ataactcgaa 900 aacctctcct cccgacattt catcaattca tttctctttt tattttcgaa aagatgaaaa 960 cttaataaat tattatgaac aatcttacta tatatacaca tatatatgga ggccctaaaa 1020 cgtaaaacag ttgaggactt tacatctgaa catg 1054 SEQ ID 4 Promoter Name: pPo2 Location: Chromosome 4; Coordinates: 17849276- 17849669 corresponding gene: At4g37970 Locus Sequence: tatttggcct attttagttt ttcttttgtt attaatgtaa aactaatcat tcgataagtt 60 catcaagttc ttcatacttt agagtatttt taattaaaaa caaaaaagtg gccaaaactg 120 atttataaag catatagtta tatcaaatag tacaataatc acgatgcatg atatatttgt 180 tagtatatga acattataag taatatgttt atgttaaata tgttaagaaa aaatacatat 240 atgtaagtca acttctgatt ggtatgagag acctaaagtc aaaacgatat ttctcaaacg 300 aaacgtcagc gtttagcccc atttatgttc tcactctttt ctatataaaa agaaaggtac 360 tctagctcgc ttaattgttc gaaacaaagg gagtgagaga tg 402

Claims (1)

1. Revindications. 1. A regulatory nucleotide sequence isolated and purified from Arabidopsis, and having the following characteristics: capable of directing the expression in a plant cell in the male gametophyte, the male gametophyte, the female gametes and / or the male gametes; and which is identifiable from one of the sequences identified with the identification number SEQ ID 1, 2, 3, 4. 2. An expression construct comprising a nucleotide sequence in accordance with claim 1, and which has been ligated operatively to a structural gene. 3. A vector capable of transforming or transfecting a host cell, which vector comprises an expression construct as described in claim 2. 4. The vector of claim 3 wherein said vector is a plasmid. 5. The vector of claim 3 wherein said vector is a viral component. 6. A eukaryotic or prokaryotic cell transformed or transfected with the vector described in claim 3. 7. The host cell of claim 6 wherein said cell is a plant cell. 8. A purified and isolated regulatory sequence of Arabidopsis capable of directing expression in a plant cell, and whose sequence is characterized by the following: a) Directs the expression of a transcriptional unit in the female gametophyte, the female gametes, the male gametophyte and / or male gametes. b) It is located in some of the following chromosomal ranges: On chromosome 2; between the nucleotide coordinates 17132704 and 17133304 (SEQ ID1) On chromosome 3, between the nucleotide coordinates 3526561 and 3527612 (SEQ ID2). On chromosome 4, between nucleotide coordinates 17849276 and 7849669 (SEQ ID3) On chromosome 4; between the nucleotide coordinates 7290780 and 7291659 (SEQ ID4) 9. A construction comprising a nucleotide sequence corresponding to any of those described in claim 8, and operatively linked to a structural gene. 10. A vector capable of transforming or transfecting a host cell, and comprising an expression construct as described in claim 9. 11. The vector of claim 10 wherein said vector is a plasmid. 12. The vector of claim 10 wherein said vector is a viral component. 13. A eukaryotic or prokaryotic cell transformed or transfected with the vector described in claim 10. 14. The host cell described in claim 13 wherein said cell is a plant cell. 15. A purified and isolated regulatory sequence of Arabidopsis capable of directing expression in a plant cell, and whose sequence is characterized by the following: a) It directs the expression of a transcriptional unit in the female gametophyte, the female gametes, the male gametophyte and / or male gametes. b) It is characterized by conferring specific expression to the transcriptional unit that contains the genomic sequences identified with the identification number SEQ ID 1, 2, 3, 4, or that contains closely linked sequences (sequences that include or are neighboring) to the sequences genomes identified with the identification number SEQ ID, 2, 3, 4. 16. An expression construct comprising a nucleotide sequence as described in claim 15, and which is operatively linked to a structural gene. 17. A vector capable of transforming or transfecting a host cell, and comprising an expression construct as described in the claim 16. 18. The vector of claim 17 wherein said vector is a plasmid. 19. The vector of claim 17 wherein said vector is a viral component. 20. A eukaryotic or prokaryotic cell transformed or transfected with the vector described in claim 17. 21. The host cell described in claim 13 wherein said cell is a plant cell. 22. A method to identify a regulatory sequence that is capable of directing expression to the female gametophyte, the female gametes, the male gametophyte, and / or the male gametes of the plants and comprising the identification of a regulatory sequence from a tagging sequence selected from the group of genomic sequences identified with the identification number SEQ ID 1, 2, 3, 4. 23. The method described in claim 22 and comprising the identification of neighboring transcriptional unit to any of the regions identified with the identification number SEQ ID 1, 2, 3, 4. 24. A novel nucleotide composition of Arabidopsis comprising a sequence selected from the group comprising the sequences identified with the identification number SEQ ID 1, 2, 3, 4.