MXPA99012109A - Male tissue-preferred regulatory region and method of using same - Google Patents

Abstract

The present invention relates to an isolated nucleic sequence encoding the Ms45 male tissue-preferred regulatory region. In one aspect this invention relates to the use of this male tissue-preferred regulatory region in mediating fertility. An example of such use is the production of hybrid seed such as in a male sterility system. The Ms45 male tissue-preferred regulatory region can be operably linked with exogenous genes, such as those encoding cytotoxins, complementary nucleotidic units and inhibitory molecules. This invention also relates to plant cells, plant tissue and differentiated plants which contain the regulatory region in this invention.

Description

PREFERRED REGULATORY REGION OF MALE TISSUE AND METHOD FOR USING THE SAME DESCRIPTION OF THE INVENTION The present invention is related to isolated DNA sequences that act as regulatory regions in eukaryotic cells. More specifically, the present invention relates to DNA sequences isolated from maize that act as regulatory regions of male tissue and play a role in the expression of male tissue genes. The present invention is also directed to a method for conferring on a gene that may or may not be expressed normally in male tissues, the ability to be expressed in a preferred manner of male tissue. Tissue gene expression and regulation and temporal specific are found, among others, during sexual reproduction in eukaryotes. In plant gametogenesis, important cytological and biochemical changes occur during pollen development when the asymmetric mitotic division of the monosomatic microspore results in the formation of two cells; each with different development destinations. The plant cell supports the growth of pollen while the generative cell supports mitosis and develops sperm cells. The specific messenger RNAs for both trajectories within the pollen have been identified in plants such as corn, tomato, tobacco, rice and thought; and specific messages for pollen or for one or more other types of cells within the anther such as membrane, epidermis and stoma have also been identified. . Pollen gene expression during differentiation involves an estimated 24,000 genes (Willing, et al., "An Analysis of the Quantity and Diversity of mRNA's From Pollen and Shoots of Zea mays" Theor. Appl. Genet.; Vol . ~ 75; pp. 751-753; (1988)), although only 10% of the clones from a cDNA library are male-specific (Stinson, et al. "Genes Expressed in the Male Gametophyte and Ther Isolation"; Plant Physiol; Vol. 83; pp. 442-447; (1987)) and the percentage of genes expressed in pollen that are specific to pollen is between 5% and ~ 80% (Willing, et al., "An Analysis of the Quantity and Diversity of mRNA's From Pollen and Shoots of Zea mays "Theor, Appl. Gene., Vol. 75; pp." 751-753; (1988).) This complex process of microsporogenesis involves the sequential production of many gene products. To date, male-specific genes have been cloned from plants: two of them, the maize Ms45 gene (US Patent No. 5,478,369 and the Arabidopsis Ms2 gene (Mark, G.M., et al., Nature: Vol. 363; - pp. 715-717; (1993)), have been shown to be required for the development of pollen. Other examples of male-specific promoters in plants include ZM13, PG, and SGB6. • The Zml3 promoter is described in the Patent North American No. 5,086,169. It consists of 1315 base pairs and 5 is of a specific pollen gene described by Hanson, et al., Plant Cell; Vol. 1; pp. 173-179; (1989). This gene hybridizes to mRNA found only in pollen. Another specific pollen promoter has been isolated and characterized upstream of the pollen-specific polyalacturonase 10 (PG) gene US Patent No. 5,412,085. This promoter region encompasses 2687 base pairs and is expressed predominantly in pollen and emerging tassel, but not in emerging pre-tassel. The patent No. 5,545,546, also from Alien, 15 describes another specific pollen promoter from the corn polygalacturonase gene. It is only expressed in pollen and in an emerging tassel. • U.S. Patent No. 5,470,359 describes a regulatory region from the SGB6 gene of maize that confers cell-layer specificity. The expression tissue, the cell layer, is a layer of cells that surrounds the microsporogenous cells in the anther and provides nutrients to it. Nine specific cDNAs and genomic clones from tobacco are described in US Pat. No. 5, 477,002. The cDNA clones were anther-specific by Northern analysis, although differentiated in developmental profiles. The Ant32 clone specific for memebrane. European Patent No. 0 420 8.19 A1 describes the method for producing male sterile plants with the wun gene 1. PCT document WO 90/08825 describes specific anthers cDNA TA13, TA26 and TA29 and their use in the male sterility system . _ "The .PCT WO 90/08825 explains the male sterility-pMSIO, pMS14 and pMS18 and its use with the GUS reporter gene. US Patent No. 5, 589, 610" details a promoter corresponding to anther specific cDNA and preferred anther cADN AC444. The use of a plant promoter and an exogenous gene to effect a change in the genetic conformation of plants is known in the art (US Patent Nos. 5,432,068, 5,412,085, 5,545,546, 5,470,359 and 5,478,369). These patents describe the cartridges of plant expression with a tissue-specific promoter linked to a gene to effect male sterility, fertility or otherwise express a gene in a specific tissue. However, these patents do not teach the use of this preferred male tissue regulatory region or the use of this preferred region of male tissue with an exogenous gene as a method for controlling male sterility. The present invention is directed to a specific regulatory region of male tissue and methods for using same. The expression of an exogenous gene in a preferred manner of male tissue can mediate male fertility and is useful in many systems such as in the production of hybrid seed. It is an object of the present invention to provide a method for expressing exogenous genes in a preferred manner of male tissue using an expression vector that confers the preferred expression of male tissue to an exogenous gene. This process can be used to restore (as in a human sterility system) or otherwise impact fertility, as in the production of hybrid seed. It is a further object of this invention to provide a DNA regulatory region that confers the expression of the preferred male tissue gene. It is also an object of the invention to provide a preferred male tissue regulatory region or those with sequence identity thereof preferably of about 70%, 75%, or 80%, most preferably about 85% or 90% and so more preferable of about 95% or 99%.

It is an object of the invention to provide an isolated nucleic acid sequence that encodes the regulatory regions of male Ms45 tissue. It is an object of the invention to provide nucleic acid sequence coding isolated from an Ms45 male tissue regulatory region from Zea mays comprising the nucleic acid sequence shown in SEQ. FROM IDENT. N0: 1 or those with sequence identity thereof. It is also an object of this invention to provide an isolated nucleic acid sequence encoding the preferred regulatory region of male Ms45 tissue from Zea mays ~ comprising a nucleic acid sequence shown in SEQ. FROM IDENT. NO: 2 or those with identity of sequences thereof. It is an object of this invention to provide a recombinant expression vector comprising the isolated nucleic acid sequence shown in SEQ. FROM IDENT. NO: 1, or those with a sequence identity thereof, operably linked to a nucleotide sequence that encodes an exogenous gene such that the nucleotide sequence is expressed in a preferred manner of male tissue in such a way as to promote expression of the gene exogenous It is an object of the invention to provide an exogenous gene, wherein the exogenous gene is Ms45.

It is an object of this invention to provide a method for producing a transformed plant expressing an exogenous nucleotide sequence in a preferred manner of male tissue comprising the steps of introducing into a plant the exogenous nucleotide sequence operably linked to a preferred regulatory region of male tissue comprising a nucleotide sequence shown in SEQ. FROM IDENT. N0: 1 or those with sequence identity for it. The method wherein the introduction step can be executed by microprojectile bombardment, can use Agrobacterium or a transfer vector comprising a Ti plasmid. Also, there may be more than one copy of the exogenous nucleotide sequence operably linked to a preferred male tissue regulatory region. It is an object of this invention to provide a method wherein the regulatory region expresses in a preferred manner male tissue in tissues selected from the group consisting of pollen, cell layer, anther, tassel, pollen stem cells and microspores. It is an object of this invention to provide a transformed plant expression sequence or exogenous nucleotide in a preferred form of male tissue having an exogenous nucleotide sequence operably linked to a preferred male tissue regulatory region shown in SEQ ID NO. NO: 1 or that with sequence identities thereof .. Such a plant is a monocotyledon or a dicotyledon Any plant capable of being transformed may be used, including corn, sunflower, soy, wheat, canola, rice and sorghum. also provides the transformed tissue of the transformed plant As an example, the tissue may be pollen, spikes, ovules, anthers, tassels, stamens, pistils and plant cells.The transformed plant may contain more than one copy of the nucleotide sequence exogenously operable linked to a preferred male tissue regulatory region It is an object of this invention to provide a method of and fertility mediation in a plant wherein the preferred regulatory region of male tissue expresses the exogenous nucleotide sequence so that fertility is impacted. This exogenous nucleotide sequence may be any sequence that impacts male fertility and may be, by way of example, a complementary nucleotide unit such as antisense molecules such as antisense, callase, RNA, antisense, bamase, RNA, and chalcone synthase antisense, antisense Ms45. RNA, or ribosomes and external guide sequences or aptamers or individual linked nucleotides. The exogenous nucleotide sequence can also be auxins, rol B, cytotoxins, diphtheria toxin, DAM methylase, avidin, or can be selected from the prokaryotic regulatory system. Also, this exogenous nucleotide sequence is a male sterility gene or the structural gene Ms45 and this plant is a monocotyledone or dicotyledonous. It is an object of this invention to provide a method for producing hybrid seed, which comprises planting in this cross-pollinated position, a male fertile plant and a male infertile plant produced with the above method, allowing cross-pollination to occur and the seed to be harvested. resulting. The plants can be corn plants. These and other objects are achieved according to one embodiment of the present invention by providing an isolated DNA molecule wherein the DNA molecule comprises a nucleotide sequence shown in SEQ. FROM IDENT. NO: 1. According to an additional embodiment of the present invention, an expression vector has been provided which comprises an exogenous gene, wherein the expression of the exogenous gene is under the control of a preferred regulatory region of male tissue and wherein the exogenous gene product impacts male fertility. According to a further embodiment of the present invention there has been provided a method of using such an expression vector to produce a male sterile plant, comprising the step of introducing an expression vector into plant cells, wherein the exogenous gene of the Expression vector can be a complementary nucleotide unit such as antisense molecules (antisense callase RNA, antisense bamasa RNA and antisense chalcone synthase RNA, Ms45 antisense RNA), ribosomes and external guide sequences, an aptamer or individual interlaced nucleotides. The exogenous nucleotide sequence may also encode auxins, rol B, "cytotoxins, diphtheria toxin, DAM methylase, avidin, or may be selected from a prokaryotic regulatory system." According to a further embodiment of the present invention, it has been provided a method of using a preferred male tissue regulatory region to produce a male fertile hybrid plant comprising the steps of: a) producing a first male parent sterile plant comprising an expression vector comprising a preferred male tissue regulatory region and a first exogenous gene wherein the preferred regulatory region of male tissue controls the "expression of the first exogenous gene, and wherein the product of the first exogenous gene alters male fertility. b) producing a second parent plant comprising an expression vector comprising a second exogenous gene, wherein the regulatory region controls the expression of the second exogenous gene so that it can be expressed in male-tissues; c) cross fertilization of the first parent plant with the second parent plant to produce a hybrid plant, where the male tissues of the hybrid plant express the second exogenous gene and where the product of the second exogenous gene prevents the interruption of the "tassel" function by the product of the first exogenous gene, thereby producing a male fertile hybrid plant In accordance with a further embodiment of the present invention, a method of using a preferred male tissue regulatory region to produce a fertile male hybrid plant comprising the steps of: a) producing a first male sterile parent plant wherein a first gene involved in the expression of male fertility is altered, b) producing a second parent plant comprising a second expression vector that it comprises a preferred regulatory region of male tissue and an exogenous gene wherein the preferred tissue regulatory region male controls the expression "of the exogenous gene so that it can be expressed in male tissue and can functionally complement the function of the altered gene in a); c) cross-fertilize the first parent with the second parent to produce a hybrid plant, where the male tissues of the hybrid plant express the exogenous gene and where the product of the exogenous gene prevents alteration of the tassel function, producing this way a fertile male hybrid plant. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of a genomic clone Ac 4.1 Ms45 and the restriction sites. Figure 2 is a plasmid map of PHP6045. Figure 3 is an autoradiogram of the primer extension products that indicate the initiation of Ms45 transcription. The lines labeled G, A, T, C correspond to the sequencing reactions with dideoxynucleotides ddGTP, ddATP, ddTTP and ddCTP, respectively. Lines 1-4 correspond to the primer extension reactions with mRNA from (.1) tassels, (2) leaves, (3) anthers, and (4) leaves. "Figure 4 is a bar graph illustrating the stage specificity of the Male Ms45 Preferred Regulatory Region." Figure 5 illustrates the tissue specificity by the lack of activity in the non-male tissue. anther analysis mARN Northern gene hybridized with the male fertilization gene Ms45. Figure 7 shows the results of a TATA box mutation analysis.

All references are incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to one of ordinary skill in the art to which this invention pertains. Unless otherwise mentioned, the techniques employed or contemplated herein are standard methodologies well known to one skilled in the art. The materials, methods and examples are only illustrative and not limiting. In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate the understanding of the invention. 1. Definitions Identity or sequence similarity, as known in the art, are relationships between two polypeptide sequences or two polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relationship between two polypeptide or two polynucleotide sequences as determined by matching between two strands of such sequences. Such identity and similarity can be easily calculated (Computational Molecular Biology; Lesk, A.M., ed .; Oxford University Press, New York; (1988); Biocomputing: Informatics and Genome Projets; Smith, D.W. ed .; Academic Press, New York; (1993); Computer Analysis of Sequence Data (Part I); Griffin, A.M. and H.G. Griffín eds.; Humana Press, New Yersey; (1994); von Heinje, G., Sequence Analysis in Molecular Biology; Academic Press; (1S87); and Sequence Analysis Primer; Gribskov, M. and J. Devereux eds .; M. Stockton Press, New York; (1991)). While there are a number of methods for measuring the identity and similarity between two polynucleotide or two polypeptide sequences, both terms are well known to those skilled in the art (von Heinje, G., Sequence Analysis in Molecular Biology; Press; (1987); and Sequence Analysis Primer; Gribskov, M. and J. Devereux eds; M Stockton Press, New York; (1991); and Carrillo, H., and D. Lipman, SIAM, J ^ Kapplied Math;;; Vol. 48; pp. 1073; (1988)). The methods most commonly employed to determine the identity or similarity between two sequences include, but are not limited to, those described in Carrillo, H., and D. Lipman, SIAM J. Applied Math; Vol. 48; pp. 1073; (1988). The preferred methods for determining identity are designed to give the greatest equalization between the two sequences tested. The methods for identity and similarity are codified in computer programs. Preferred computer program methods for determining the identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12 (1); pp. 387). (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF, et al., J. Molec. Biol., Vol. 215, r pp. 403; (1990)). Male tissue consists of tissues made from collection and cells that are directly involved or sustained from the reproduction of male gametes such as pollen, cell layer, anther, tassel, pollen stem cells and microspores. The cellular layer is the tissue in the anther in closest contact with the pollen stem cells and the microspores and is probably involved with the nutrition of the pollen granules in development. Pollen stem cells undergo two meiotic divisions that produce a birth of four hapolid microspores. The 'microspores undergo maturation in a pollen grain. Pollen or pollen grains are mature male gametophytes that may have the ability to fertilize plants that are compatible. The anther is that portion of the stamen in which the pollen is produced, the rest of the stamen consists of the filament, on which the anther depends. The stamen is the male organ of the flower. The "preferred regulatory region of male tissue is a nucleotide sequence that directs a higher level of transcription of an associated gene in male tissues than in any or all of the tissues of a plant, eg, the Ms45 gene, described herein. , is detected in anthers during the quartet, quartet release and the early uninuclear stages of development .. For details regarding anther development stages see Chang, MT "and MG Neuffer," Maize Microsporogenesis "; Genome; Vol. 32; pp. 232-244; (1989). The preferred male tissue regulatory region of the Ms45 gene directs the expression of a gene operably linked in male tissues. Preferred expression tissues are male, not, for example, coleoptile root or tissue. The predominant expression is in male tissues such as, but not limited to, pollen, cell layer, anther, tassel, stem cells and pollen and microspores. This preferred expression of male tissue refers to higher levels of expression in male tissue, but not necessarily to the exclusion of other tissues. Mediating is influencing a positive or negative way or influencing emergence, such as fertility or any other space. Male fertility is impacted when fertility is experienced that is not normal; This can be as reduced fertility or increased fertility or fertility that is different in terms of timing or other characteristics. Isolated means altered "by the hand of man" from its natural state, that is, what happens in nature, has been changed or removed from its original environment or both. For example, a naturally occurring polynucleotide or a polypeptide that is naturally present in a living organism in its natural state is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated". ", as the term is used in the present. For example, with respect to polynucleotides, the term "isolated" means that it is separated from the chromosome and the cell in which it occurs naturally. As part of or after isolation, such nucleotides can be linked to other polynucleotides, such as DNAs, as mutagenesis, to form fusion proteins, and for propagation or expression in a host, for example. Isolated polynucleotides, alone or linked to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms introduced into host cells in culture or in whole organisms, such as DNAs that would be isolated, as the term is used. in the present, because they would not be in their natural occurrence or environment. Similarly, polynucleotides and polypeptides can occur in a composition such as media formulations, solutions for introducing polynucleotides or polypeptides, for example, into cells, compositions or solutions - for chemical or enzymatic reactions, for example, which are not of natural occurrence, and, in the present remain isolated polynucleotides or polypeptides within the meaning of that term as used herein. An exogenous gene is referred to in the present disclosure to a DNA sequence that is introduced or reintroduced into an organism. For example, any gene, including the structural gene Ms45, is considered to be an exogenous gene, if the gene is introduced or reintroduced into the body. 2. Isolation of a Preferred Regulatory Region of Male Tissue Although anther-specific promoters and genes active in male tissues are known in the art, (McCormick, et al., "Anther-Specific Genes: Molecular Characterization and Promoter Analysis in Transgenic Plants, "in Plant Reproduction: From Floral Induction to Pollination; Lord, et al. Eds .; pp. 128-135; (1989); Scott, et al., International Application Publication No. WO 92/11379 (1992); van der Meer, et al., The Plant Cell; Vol. 4; pp. 253; (1992)), there are no generally accepted principles or structural criteria for recognizing DNA sequences that confer male tissue expression to gene expression in corn. Consequently, it is not possible to isolate a preferred regulatory region of the male tissue directly from a maize genomic library by screening a consensus sequence that confers the preferred expression of male tissue. For example, the hybridization of such sequences can be carried out under conditions of reduced shortage, medium shortage or even highly scarce conditions (for example, conditions represented by a shortage of 35-40% formamide wash with Denhardt's 5X solution, 0.5% SDS and IxSSPE at 37 ° C, conditions represented by a shortage of Formamide wash 40-45% with 5X Denhardt solution, 0.5% SDS and IX SSPE at 42 ° C, and conditions represented by a shortage of washing of 50% formamide with 5X Denhardt solution, 0.5% SDS and IX SSPE at 42 ° C, respectively). The shortage of medium in a standard nucleic acid hybridization could be useful to identify the preferred regulatory regions of male tissue described herein as well as other genes (see for example Sambrook, J., et al., Molecular Cloning: a Laboratory-Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y .; (1982)). In general, the sequences with code for a preferred regulatory region of male tissue will have sequence identity therein of preferably 70%, 75% or 80%, more preferably 85%, or 90%, and most preferably 95% or 99%. The methods are readily available in the art for hybridization of nucleic acid sequences.

Screening of hybridization of DNA libraries placed on plates (see for example Sambrook, J., et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; (1982)) or sequences of coding and amplification using the polymerase chain reaction (see for example Innis, et al., PCR Protocols, to Guide to Methods and Applications; Academic Press; (1990)) are well-known techniques for isolating genomic DNA. Regulatory regions can be identified in genomic subclones using functional analysis usually verified by observing a reporter gene expression in anther tissue and reducing or absent a reporter gene expression in non-anther tissue. This general approach is illustrated in Example 3, below. The possibility of regulatory regions residing "upstream" or 5 'ward of the transcription initiation site can be tested by subcloning a DNA fragment containing the upstream region and subcloning small fragment expression vectors for expression experiments transient The smallest fragments that may contain regions essential for the preferred expression of male tissue are expected. For example, the essential regions of the CaMV 19S and 35S promoters have been identified in relatively small fragments derived from large genomic pieces as described in US Pat. No. 5,352,605. In general, the sequences encoded for a preferred regulatory region of male tissue will have sequence identity for the same preferably of 7Q%, 75% or 80%, more preferably of 95% or 90%, and more preferably of 95% or 99%. The deletion analysis can be presented for the 5 'and 3' ends of the regulatory region: the fragments can be obtained by linker screening mutagenesis, mutagenesis using the polymer chain reaction and the like (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)). The 3 'deletions can delineate the preferred regulatory region of the male tissue and identify the 3' end so that this essential region can be operably linked to a core promoter of choice. Once the essential region is identified, the transcription of an exogenous gene can be controlled by the preferred region of male Ms45 tissue plus a core promoter. The core promoter can be any of the known core promoters such as Cauliflower Mosaic Virus 35S or the 19S promoter (U.S. Patent No. 5,352,605), Ubiquitin (U.S. Patent No. 5,510,474), the IN2 core promoter (U.S. Patent No. 5,364,780), or a Figwort Mosaic Virus promoter (Gruber, et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology; Glick, et al., Eds., CRC Press; pp. 89-119; (1993 )). Preferably, the promoter is the core promoter of a preferred male tissue gene or the CaMV 35S core promoter. more preferably, the promoter is a promoter of a preferred male tissue gene and in particular the Ms45 core promoter. Further mutational analysis can introduce functionality modifications such as expression levels, expression synchronization or expression tissue. Mutations can also be silent and have no observable effect. 3. Insertion of the region into an expression vector The selection of an appropriate expression vector with which to test functional expression will depend on the host and the method of introducing the expression vector into the host and such methods are well known to someone with experience in the technique. For eukaryotes, the regions in the vector "include regions that control the initiation of transcription and control processing.These regions are operably linked to a reporter gene such as the ß-glucuronidase (GUS) gene or luciferase. Genral examples of plant expression vectors and reporter genes can be found in Gruber, et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology; Glick, et al., Eds., CRC Press; pp: 89- 119; (1993). Gus expression vectors and Gus gene cartridges are commercially available with Clonetech, Palo Alto, CA, while luciferase expression vectors and luciferase gene cartridges are available from Promega Corporation, Madison, Wl, the Ti plasmids and other Agrobacterium vectors are described in Ishida, Y., et al., Nature Biotechnology, Vol. 14; pp. 745-750; (1996) "and in U.S. Patent No. 5,591 , 616, Method for Transforming Monocotyledons, filed May 3, 1994. "Expression vectors containing regulatory regions located in genomic fragments can be introduced into intact tissues such as that represented in anthers, embryos or within calli. "DNA delivery methods include microprojectile bombardment, DNA injection, electrodeposition and Agrobacterium-mediated gene transfer (see Gruber, et al.," Vectors for Plant Transormation "in Methods in Plant Molecular Biology and Biotechnology; Glick, et al. Eds., CRC Press; (1993), U.S. Patent No. 5,591,616 Method for Transforming Monocotyledons, filed May 3, 1994, and Ishida, Y., et al., Nature Biotechnology; Vol. 14; pp. 745 -750; (1996)) The general methods of plant tissue culture are found in Gruber, et.al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology; Glick, et al., Eds .; CRC Press (1993) For the transient test system, the isolated anthers, by stages, are immediately placed in the tassel culture medium (Pareddy, DR and "JF" Petelino, Crop Sci. J.; Vol 29; 1564-1566; (1989)) solidified with 0.5% Phytagel (Sigma, St. Louis) or other med. solidification ios. The DNA expression vector is introduced for 5 hours preferably by means of microprojectile-mediated delivery with 1.2μ particles at 1000-1100 Psi. After the DNA supply, the anthers are incubated at 26 ° C on the same tassel culture medium for 17 hours and analyzed by preparing a whole tissue homogenate and testing for GUS or for luciferase activity (see Gruber, et al., "Vectors for Plant Transformation "in Methods in Plant Molecular Biology and Biotechnology; Glick, et al. Eds .; CRC Press; (1993)). The methods described above have been used to identify a DNA sequence that regulates gene expression in a preferred manner of male tissue. Such a region has been identified as the preferred regulatory region of the full-length male Ms45 tissue (SEQ ID NO: 1). A TATA box mutation with the sequence identity with the full length of tissue of the preferred regulatory region of male tissue Ms45 is identified in SEQ. FROM IDENT. No: 2. Thus, the present invention encompasses a DNA molecule having a nucleotide sequence of SEQ. - "DE IDENT No: 1 (or one with sequence identity) and having the function of a preferred regulatory region of male tissue A TATA box can be identified by primer extension analysis as described in Example 2 to continued- or in Current Protocols in Molecular Biology, Ausubel, FM, et al., eds., John Wiley and Sons, New York, pp. 4.8.1-4.8.5; (1987) 4. Use of Preferred Regulatory Region Male Fertility Control Tissue An object of the present invention is to provide a means for controlling fertility using a preferred regulatory region of male tissue Importantly, this preferred regulatory region of male tissue can control the expression of an exogenous gene in anthers from the quartet, through initial non-nucleated stages of development The practical significance of such synchronization is that the expression of a sterility-inducing gene during this stage of development will alter the growth of the anther sufficiently early to allow visual verification of the function of the field sterility induction system. It can also reduce the possibility that "switches" (anthers that shed pollen) will occur. Therefore, the effects of the sterility induction gene would be evident in the field of production at a sufficiently early stage of development to allow manual or mechanical overflowing or of any "fertile fugitive" that results from the partial or total separation of the system of induction of sterility. One approach to controlling male fertility is to manipulate gene expression in the cell layer. The cell layer is a layer of cells that surround the microsporogenous cells in the anther and also provide nutrients, such as reducing sugars, amino acids and lipids to the developing microspores (Reznickova, CR, Bulk, Sci., Vol.; pp. 1067; (1978); Nave, et al., J. Plant Physiol; Vol. 125; pp. 451; (1986); Sawnney, et al., J. Plant Physiol., Vol. 125; 467; (1986)). Ms45 is found to be highly expressed in the membrane layer. The membrane cells also produce β (1, 3) -glucanase ("callasse") that promotes the release of microspore (Mepham, et al., Protoplasma; Vol. 70; pp. 1; (1970)). However, there is a delicate relationship between the cell layer and the microsporogenous cells and any disruption of membrane function is likely to result in "dysfunctional" pollen grains.Indeed, the lesions in membrane biogenesis are recognized to result in male sterility mutants (Kaul, "Male Sterility in Higher Plants" in Monographs on Applied Genetics and Frankel et al., Eds. Springer Verlag, Vol. 10, pp. 15-95; (1988)). A premature or late appearance of callase during the development of the cell layer is associated with certain types of male sterility (Warmke, et al., J. Hered., Vol._63; pp. 103; (1972)). The callasse gene can be used to alter the function of male tissue Scott, et al., PCT WO 93/02197 (1993), describes the nucleotide sequence of a specific cell layer "callasse. Therefore, a failure of the microspores to develop into mature pollen grains can be induced by using a recombinant DNA molecule comprising a gene capable of altering the function of the membrane under the control of specific regulatory cell-layer sequences. A general approach to "impact male fertility is to construct an expression vector in which the preferred regulatory region of the male tissue is operably linked to" a nucleotide sequence that encodes a protein capable of altering the function of male tissue resulting in in infertility. Proteins capable of altering the function of male tissue include proteins that inhibit the synthesis of macromolecules that are essential for cell function, enzymes that degrade macromolecules that are essential for cell function, proteins that alter biosynthesis or metabolism of plant hormones, structural proteins, proteins expressed inappropriately and proteins that inhibit a specific function of male tissues. By. For example, an expression vector can be constructed in that the preferred regulatory region of male tissue is operably linked to a nucleotide sequence that encodes a protein synthesis inhibitor, which may be not limited to a cytotoxin. Diphtheria toxin, for example, is a well-known inhibitor of protein synthesis in eukaryotes. DNA molecules encoding the diphtheria toxin gene can be obtained from American Type Culture Colletion (Rockville, MD), ATCC "NO." 3"9359 or ATCC No. 67011 and see Fabijanski, et al., EP Appl. No. 90902754.2, "Molecular Methods of Hybrid Seed Production" for examples and methods of use DAM methylase, for example, is a well-known enzyme from Escherichia coli that encodes the adenine residue in the sequence 5 'GATC 3' to N6-methyl-adenine, Cigan and Albertsen -describe how DAM methylase could be used to impact fertility in transgenic plants (PCT / US95 / 15229 Cigan, A.M. and Albertsen, M.C.

"Reversible Nuclear Genetic System for Male Sterility in Transgenic Plants"). Another example of a protein that alters fertility is avidin as illustrated in US Patent Application No. 08 / 475,582"Induction of Male Sterility in Plants by Expression of High Levéis of Avidin" by Howard, J. and Albertsen, MC Alternatively , disruption of membrane function can be achieved by using DNA sequences encoding enzymes capable of degrading a biologically important macromolecule. For example, Mariani, et al., Nature; Vol 347; pp. 737; (1990), has shown that expression in the cell layer space of either Aspergillus oryzae Rnase-Tl or a Bacillus amyloliquefaciens Rnase, desingando "bamasa", of induced destruction of the membrane cells, results in male fertility. Quaas, et al., Eur. J. Biochem .; Vol. 173; pp. 617; (1988), describes the chemical synthesis of Rnase-Tl, so much that the nucleotide sequence of the bamasa gene is described in Hartley, J. Molec. Biol.; Vol. 202; pp. 913; (1988). The Rnase-Tl and bamasa genes can be obtained, for example, by synthesizing the genes with long oligonucleotides of mutual initiation. See for example, Current Protocols in Molecular Biology; Ausubel, F.M., et al., Eds .; John Wiley and Sons, New York; pp. 8.2.8 to 8.2.13; (1987). Also, see Wosnick, et al., Gene; Vol. 60; pp. 115; (1987). In addition, current techniques using the polymerase chain reaction provide the ability to synthesize very large genes (Adang, et al., Plant Molec. Biol., Vol. 21; pp. 1131; (1993); Bambot, et al. -, PCR Methods and Applications; Vol. 2; pp. 266 (1993)). In an alternative approach, pollen production is inhibited by altering the metabolism of plant hormones such as auxins. For example, the rolB gene of Agroba cterium rhizogenes codes for an enzyme that interferes with auxin metabolism by catalyzing the release of free indoles from indoxyl-β-glucosides. Estuch, et al., EMBO J.; Vol. 11; pp. 3125; (1991) and Spena, et al., Theor. Appl. Genet .; Vol. 84; pp. 520; (1992) have shown that anther-specific expression of the rolB gene in tobacco results in plants having wilted anthers in which pollen production was severely diminished. Therefore, the rolB gene is an example of a gene that is useful for the control of pollen production. Slighton, et al., J. Biol. Chem.; Vol. 261; pp. 108; (1985) describes the nucleotide sequence of the rolB gene. "To express a protein that abrogates the function of male tissue, an expression vector is constructed in which the DNA sequence encoding the protein is operably linked to the DNA sequences that regulate gene transcription in a preferred manner of male tissue. The general requirements of an expression vector were described above in the context of a transient expression system, however, the preferred mode is to introduce the expression vector into embryonic tissue of the plant in such a way that an exogenous protein will be expressed in a later stage of development in the male tissues of the adult plant. Mitotic stability can be achieved by using plant viral vectors that provide epicochromosomal reproduction. An alternative and preferred method of obtaining mitotic stability is provided by the integration of the expression vector within the host chromosome. Such stability. mitotic can be provided by the provision of microprojectiles of a de-expression vector to embryonic tissue (Gruber, et al., "Vector for Plant Transformation", in Methods in Plant Molecular Biology and Biotechnology; Glick, et al., CRC Press;; (1993)). The transformation methodology can be found for many plants, including but not limited to sunflower, soybean, wheat, canola, rice and sorghum (Knittel, N., et al., J. Plant Cell Rep. Springer ~ International, Berlin, W Germany; Vol. 14 (2/3); pp. 81-86; (1994); Chee, PP, et al., Plant Physiol., American Society of Plant Physiologists, Rockville, MD; Vol. 91 (3); pp. 1212-1218; (1989); Hadi, MZ, et al., J. Plant Cell Rep .; Springer International, Berlin, W. Germany; Vol. 15 (7); pp. 500-505; 1996), Perl, A., et al., Molecular land General Genetics, Vol. 235 (2-3), pp. 279-284, Zaghmout, OMF and NL, Trolinder, Nucleic Acids Res., IRL Press, Oxford; Vol. 21 (4); pp. 1048; (1993); Chen, JL and WD Beversdorf, Theor Appl. Genet; Springer International, Berlin, W. Germany; "Vol. 88 (2); pp. 187- 192; (1994); Sivamani, E., et al., Plant Cell Rep .; Springer International, Berlin, W. Germany; Vol. 15 (5); pp. 322-327; (1996); Hagio, T. "," et al., Plant C ell Rep .; Vol. 10 (5); pp. 260-264; "(1991)) and are also known to those skilled in the art In order to select" transformed cells, the expression vector contains a selectable marker gene such as a herbicidal resistance gene. For example, such genes _ can confer resistance to phosphinothricin, "" glyphosate, sulfonylureas, atrasine, idazolinine or kanamycin. Although the expression vector can contain cDNA sequences that encode an exogenous protein for the control of a preferred regulatory region of male tissue, as well as the selectable marker gene under the control of the constitutive promoter, the selectable marker gene can also be delivered to cells host in a separate selection expression vector, such as a __ cotransformation of the embryonic tissue with a test expression vector containing a preferred regulatory region of male tissue and an expression vector is illustrated below. 5. Induction of Sterility In an alternative approach, male sterility can be induced by the use of an expression vector in which the preferred regulatory region of male tissue is operably linked to a nucleotide sequence. which encodes a complementary nucleotide unit. The "binding of nucleic acid molecules complementary to the target molecule" can be selected to be "inhibitors." For example, if the target is a mRNA molecule, then the binding of a nucleotide unit complementary in this case as RNA results in Hybridization and in the arrest of the translation (Paterson, et al., Proc. Nat'l. Acad. Sci.; Vol. 74; pp. 4370; (1987)). Therefore, an appropriate antisense RNA molecule, such as a complementary one for Ms45 (US Patent No. 5,478,369), would have a sequence that is complementary to that of the mRNA species encoding a protein that is necessary for male sterility (Fabijanski in "Antisense Gene Systems of Pollination Control for Hybrid Seed Production", North American Patent Application No. 08 / 288,734). For example, the production of antisense callasa RNA would inhibit the production of the callase enzyme that is essential for the release of microspore. In addition, male sterility can be induced by the inhibition of flavonoid biosynthesis using an expression vector that produces an antisense RNA for the 3 'untreated region of chalcone synthase A gene (Van der Meer, et al., The Plant Cell; Vol. 4; pp. 253- (1992)). The cloning and characterization of chalcone synthase A gene is described by Koes, et al., Gene; Vol. 81; pp. 245; (1989), and by Koes, et al., Plant Molec. Biol.; Vol. 12; pp. 213; (1989). Alternatively, an expression vector can be constructed in which the preferred regulatory region of the male tissue is operably linked to a nucleotide sequence encoding a ribosome. Ribosomes can be designed to express endonuclease activity that is targeted to a certain target sequence in a "mRNA" molecule, eg Steinecke, et al., EMBO J., Vol. 11; pp. 1525; (1992), achieved up to 100% inhibition of the expression of the neomycin phosphothrasferase gene by ribosomes in tobacco protoplasts.Most recently, Perriman, et al., Antisense Research and Development; Vol. 3; pp. 253; (1993), inhibited Chloramphenicol Acetyl Transferase Activity in Tobacco Protoplasts Using a Vector Expressing a Modified Ribosome Hammer Head In the context of the present invention, target RNA molecules appropriate for ribosomes include mRNA species that encode proteins essential for male fertility, such as callasa mRNA and Ms45 mRNA In a further alternative approach, expression vectors can be constructed in which the preferred regulatory region of male tissue directs the production of transfer RNA capable of promoting RNase P-mediated divisions of target mRNA molecules. According to this approach, an external guide sequence can be constructed to direct the endogenous ribosome, RNase P, to a particular intracellular mRNA species, which is subsequently cleaved by the cellular ribosome (US Patent No. 5,168,053; Yuan, et al., Science; Vol. 263; pp. 1269; (1994)). Preferably, the external leader sequence comprises ten to fifteen complementary nucleotide sequences for a mRNA species encoding the male fertility essential protein and a 3'-RCCA nucleotide sequence, wherein R is preferably a purine. External guide sequence transcriptases bind to the target mRNA species by forming base pairs between the mRNA and the complementary outer guide sequences, thus promoting the mRNA division by RNase P at the nucleotide located on the 5 'of the base pair region. Another alternative approach is to use aptamer technology, where the complementary nucleotide unit is a nucleotide that serves as a ligand for a specified target molecule (US Patent No. 5472841). This objective can be an essential product for male fertility or a product that alters male fertility. Using this method, an aptamer can be • selected for the target molecule, Ms45 or avidin, for example, which would bind and inhibit target expression. The nucleotide sequence encoding the aptamer could be part of the expression vectors constructed such that a preferred regulatory region of male tissue directs the production of the aptamer. Sterility can also be induced by the interruption of a gene important in male fertility such as the Ms45 gene or the Ms2 gene (Mark, GM, et al., Nature; Vol. 363; pp. 715-717; (1993) ). Methods of gene disruption are well known in the art and include, but are not limited to, insertion of transposable element and induction of mutation. 6. Restoration of Male Fertility in Hybrid Fl The methods described above can be used to produce transgenic male sterile maize plants for the production of F1 hybrids in large-scale crosses between inbred lines. transgenic male sterile plants do not contain all the ex "oge" gene not "" that alters "the membrane function, then a proportion of hybrids Fl will have a male fertile phenotype. On the other hand, Fl hybrids will have a male-sterile phenotype if the exogenous gene is present in all egg cells of transgenic male sterile plants because the "sterility induced by the exogenous gene would be dominant." Therefore, it is desirable to use a restoration system of "male fertility to provide the production of male fertile F hybrids. Such a male restoration system has particular value when the harvested product is sown or when the crops are self-pollinated. Also, such a fertility restoration system has particular value when the preferred regulatory region of male tissue is operably linked to an inducible promoter such as in WO 89/10396"(Marianai, et al., Plants with Modified Stamen Cell) and The inducible pomotor is responsible for external controls. This preferred regulatory region of male tissue consists of a preferred regulatory region of male tissue, an inducible promoter and an exogenous gene. One approach to the restoration of male fertility would be transgenic male sterile plants crossed with transgenic male fertile plants containing a fertility restoration gene under the control of a preferred male tissue regulatory region. For example, Fabijanski in "Antisense Gene Systems of Pollution Control for Hybrid Seed Production", North American Patent No. 08 / 288,734, male fertile plants that expressed a bamase inhibitor, designated - "barstar", with sterile male plants - that They expressed Bamasa. Hartley, J. Mol. Biol.; Vol. 202: pp. 913; (1988), describes the nucleotide sequence of barstar. Another approach would be to cross male sterile plants containing an alteration in an essential male fertility gene, to transgenic male fertile plants containing the preferred regulatory region of male tissue operably linked to an unaltered copy of the fertility gene such as the Ms45 gene or Ms2. The complete sequence of the Ms45 gene is contained in U.S. Patent No. 5,478369 and Ms2 in Mark, G. M., et al., Nature; Vol. 363; pp. 715-717; (1993). Alternatively, restoration of male fertility can be achieved by expressing complementary nucleotide units such as toxin ribosomes or aptamers in male fertile plants to neutralize the effects of the toxin in male sterile plants. Therefore, male fertility can be restored in the Fl hybrids by producing a male fertile transgenic plant that synthesizes a particular species of RNA molecule or polypeptide to counteract the effects of the particular exogenous gene expressed in male sterile transgenic plants.

In an alternative method to restore male fertility, transgenic male sterile plants contain an expression vector that has a preferred regulatory region of male tissue, a prokaryotic regulatory region (from a prokaryotic regulatory system), and an exogenous gene that is able to alter membrane function. Transgenic male fertile plants are produced to express a prokaryotic peptide under the control of a preferred male tissue regulatory region. In the resultant F1 hybrids from the sterile, male-fertile male cross, the prokaryotic peptide binds to the prokaryotic regulatory sequence and represses the expression of the exogenous gene that is capable of altering male fertility. An advantage of this method of restoring fertility is that a transgenic male fertile plant form can be used to provide fertility Fl regardless of the identity of the exogenous gene that was used to alter membrane function in the transgenic male sterile plant . "" For example, the LexA gene / LexA operator system can be used to regulate gene expression consistent with the present invention. See US Patent No. 4,833,080 and Wang, et al., Mol. Cell. Biol .; Vol 13_; pp. 1805; (1993). More specifically, the expression vector of the male sterile plant would contain the LexA operator sequence, while the expression vector of the male fertile plant would contain the coding sequences of the LexA repressor. In the Fl hybrid, the LexA repressor would bind to the LexA operator sequence and inhibit the transcription of the exogenous gene that encodes a product capable of altering male fertility. This could include, but is not limited to, avidin, DAM methylase, diphtheria toxin, RNase T, bamasa, rol B and chalcone synthase A. LexA DNA carrier molecules can be obtained by, for example, synthesizing DNA fragments containing the operator sequence LexA well known. See, for example, U.S. Patent No. 4,833,080 and Garriga, et al., Mol. Gen. Geñet.; Vol. 236; pp. 125; (1992). The LexA gene can be obtained by the synthesis of a DNA molecule encoding the LexA repressor. Gene synthesis techniques were described above and the LexA gene sequences are described for example by Garriga, et al., Mol. Gen. Genet .; Vol. 236; pp. 125; (1992). Alternatively, DNA molecules encoding the LexA repressor can be obtained from plasmid pRB500, American Type Culture Collection Accession No. 67758. Those skilled in the art can readily devise other male fertility strategies using prokaryotic regulatory systems, such as repressor system the c / lac operon or the repressive system trp / trp operon. 7. Identification of the Essential Parts of the Regulatory Region Identification of the "essential parts of a regulatory region can be executed by removing, adding and / or replacing nucleotides in a regulatory region by methods well known to one skilled in the art. variants can be obtained, for example, by oligonucleotide directed mutagenesis, linker scanning mutagenesis and mutagenesis using the polymerase chain reaction (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)). a regulatory region must be constructed using the existing restriction sites The resulting promoter fragments can be tested for activity using an expression vector as previously described.Additional refinement and declination can be obtained by making minor changes, preferably of about 50 or 30 nucleotides, more preferably about 20 or 10 nucleotides and more preferably about 5 or 1 nucleotide, until the smallest restriction fragment that still confers adequate expression covers the reporter construct (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)). These can be introduced into the expression vector using introduced or natural restriction sites. A series of 3 'deletions can also be generated as described above either by PCR or by methods well known to one skilled in the art (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)). Further refinement and delineation may be obtained by making minor changes, preferably of about 50 or 30 nucleotides, more preferably of about 20 or 10 nucleotides and more preferably still of about 5 or 1 nucleotide up to the smallest restriction fragment that still confer adequate expression on construction (Directed Mutagenesis: A Practical Approach, IRL Press, (1991)). These 5 'and 3' deletions will therefore delineate the minimum essential region to limit the proper tissue and temporal expression of the larger regulatory region. In general, the sequences coding for this minimum region of a preferred regulatory region of male tissue will have sequence identity with it preferably of about 70%, 75% or 80%, more preferably about 85% or 90%, and more preferably about 95% or 99%. The following is presented by way of illustration and is not intended to limit the scope of the invention.

EXAMPLE 1 Genomic Cloning and Ms45 Promoter Sequencing • The AC labeling of the Ms45 cDNA and the Northern analysis are described in US Pat. No. 5,478,369. An Ms45 cDNA was used to screen a B73 maize genomic library. This library was developed by cloning of partial SAU3A1 into a BAMHI digested genomic cloning vector (Lambda Dash II, Stratagene, La Jolla, CA). Approximately lxlO6 plates were screened using an E. coli strain suitable for genomic DNA (ER1647, New England Biolabs, MA) as the host. Clone AC4.1 was purified to homogeneity after three rounds of sieving. The restriction mapping of AC4.1 showed that the clone was approximately 13 kb in length and contained two internal BAMHI sites (Figure 1). One of those sites was also found in the partial csn Ms45. Two fragments BANHI were subcloned to a cloning vector (Bluescript SK +, Stratagene, La Jolla, CA). The clone of The 5 'end was approximately 3.5 kb in length and corresponded to the sequence upstream (5') of the internal BAMHI site. The 3 'end clone was 2.5kg and contained the sequence Ms45 downstream of the internal BAMHI site. Concurrently, a full length of Ms45 cDNA was isolated and secuced. By sequence comparison of the 5 'end clone and the Ms45 cDNA the translation start site was identified (Figure 1). Sequencing of the Ms45 promoter region was achieved using the dideoxy chain termination method of Sanger, F., et al., "DNA Sequencing with Chain terminating Inhibitors"; Proc. Nat'l. Acad. Sci .; Vol. 74; pp. 5463-5467; (1977). The genomic clone pac4.1-5 '(Figure 1) was sequenced using the universal oligo and others that were sequence specific using techniques well known in the art. The preferred regulatory region of male tissue had an NCOI site introduced into the initial bead and was cloned as an NCOI fragment into a Luci expression vector without promoter. This new reporter vector was designated as plasmid PHP6045 (Figure 2) ATCC No: 97828 (Deposited on December 12, 1996; American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852). EXAMPLE 2 Primer Extension Analysis Total RNA was isolated from the corn tassel containing the quartet through anthers of the initial non-nucleated stage. The total RNA was precipitated with ethanol and MgCl2. One milligram of total RNA was isolated and the poly A + mRNA was purified using oligo-dT cellulose. Poly A + RNA was also isolated directly from 6-day maize seed leaves and corn anthers used in conjunction with those known to those skilled in the art. A sequence run was prepared using a single chain of Ms45 oligonucleotide and the incorporation of 35S-dATP in a standard sequencing procedure, using protocols well known to one skilled in the art. The Primer Extension was made according to the following method: oligonucleotide primer labeled at the 1.5 'end. Combined: primer 5 pmol Ni1316 (PHL11916) in 1.0 μl. 5 μl (50 μCi) gamma 32 P-ATP (> 5000 Ci / mmol) 0.7 μl 10X kinase regulator 0.7 μl T4 polynucleotide kinase incubated 37 ° C, 45 min. Diluted with 20 μl TE and heated to 65 ° C to activate the enzyme. 10X Kinase Regulator To make 1 ml 0.5M Tris - HCl, pH 7.6-8.0"0.5 ml of 1M 5 'mM thickness 0.5 ml of 0.1 M 100 mM Mg" C12 0.1 ml of 1M 100 mM DTT 0.5 ml of 0.5M 0.1 mg / ml gelatin or BSA 50 μl 2 mg / ml 0.1 ml water II Tempered primer and RNA Kinase primers were hardened to mRNA from corn tassel, 6d corn seedling leaves, corn anthers and leaves of corn 6d, were mixed together in ice and there were 2μl mRNA, 1μl oligo kinase, 2 μl tempered 5X buffer (1.25 M KCl, 10 mM Tris, pH 7.9-8.15), and 1 μl 30 mM vanadyl. carried up to 10 μl with 10 mM Tris, pH 8.15 This mixture was heated to 65 ° C and cooled to 55 ° C for a period of 4 hours in a thermocycler heating block III Primer extension The extension mixture 23 μl primer (see recipe below) and 0.4 μl of reverse transcpptase (SuperScript, BRL, MD) were added to each tube. This was mixed by moderate pipetting and placed immediately at 48 ° C and incubated for 45 min. The Primer Extension Mixture consists of 10mM MgC12, 5mM DTT, 0.33mM each dATP, dCTP, dGTP, dTTP and DEPC water. 300 μl were added and precipitated in a freezer at -20 ° C overnight, then pellets were formed at 30 minutes in a microfuge. The pellets were dried in a Spedd Vac and dissolved in 6 μl of 0.1 NaOH / lmM EDTA. The contents of the tubes are mixed by pipetting and vortexing to ensure that the pellets dissolve. These were left at room temperature for 2.5 hours, and 6 μl sequence dye (stop the solution from the USB Sequencing kit) were added, and the solution was denatured at approximately 95 ° C. Half of the sample was loaded on the 6% denaturing polyacrylamide sequencing gene with stacking buffer and operated "a" 55 Watts for 2 hours. The gel was dried in a gel dryer and exposed to Kodak X-AR film. After a three-day exposure, a transcription product was observed in the corn tassel mRNA primer extension reaction corresponding to the deoxythymidine located upstream of the starter cord (Figure 3). This position was designated +1. A minor start-transcript was also identified in -3. EXAMPLE 3 Stage Determination and Tissue Specificity of the Preferred Regulatory Region of Male Tissue Ms45 The preferred regulatory region of total length male tissue (SEQ ID NO: 1) was fused to the reporter gene of luciferase from of luciferin, Pho tinus pyralis, (DeWit, TR, et al., Proc. Nat'l Acad. Sci. USA; Vol. 82; pp 7870-7873; "" ("1985)) with the region; Translated PinII-3 'from the potato (An, G., et al., "Functional Analysis of the 3' Control Region of the Patato Wound-Inducible Proteinase Inhibitor II Gene"; Plant Cell; Vol. 1; pp. 115-122; (1989)). "The anthers of corn in various stages of development were plated on the tassel culture medium (Pareddy, et al., Theoret, Appel. Genet, Vol.; pp. 521-526; (1989)), solidified with agar (Phytagar®, _Sigma, St. Louis). One of the three anthers from each floret was represented, and the anthers were joined by stage and placed on plates for microprojectile bombing; typically eight anthers per plate. The anthers were fired at 1100 p.s.i. with 1.8 μl tungsten particles on which the DNA of the preferred regulatory region of male tissue Ms45-luciferase reporter was precipitated. All the anthers on a given plate were in the same stage: premeiotic, meiosis I, meiosis II, "quartet, release of microspore, initial uninuclear microspore or uninuclear medium microspore." Three replications were fired on each stage.The anthers were incubated during the overnight at 26 ° C for 18 hours A crude extract with the anthers was prepared from each plate and tested for luciferase activity and protein content.The luciferase activity, normalized to the protein concentration is plotted in the Figure 4 as a function of the stage of development.The main stage was in the quartet and the stages of microespore release of the development, with activity lower% in meiosis I and II, and activity barely detectable in the initial uninuclear stage. significant antecedent activity in premeiotic or medium-uninuclear anthers.

In addition, embryogenic calluses, cultured in the MS medium containing 2.0 ug / ml of 2,4-D were bombarded in one manner, except at 650 p. s. i. with particles coated with a luciferase reporter fused to either the preferred regulatory region of male Ms45 tissue or to a maize ubiquitin promoter (US Patent No. 5,510,474) and a uidA (GUS) reporter fused to a maize ubiquitin promoter. Luciferase was normalized to β-glucuronidase. As shown in Figure 5, the preferred regulatory region of tissue Ms45 was unable to promote transient expression in embryogenic calluses and shots, even through the ubiquitin promoter were expressed. Similarly, corn seeds, impregnated and germinated in distilled water for two days and placed on wet filters, were subjected to microprojectile bombardment and their hypocotyls tested for luciferase and β-glucuronidase. The ubiquitin regulatory region (promoter) was active, although the preferred regulatory region of male Ms45 tissue was not. These results were compared by the results of the RNA hybridization analysis. The anthers of corn at various stages of development were collected and treated as follows. One of the three anthers from each flower was fixed in (3: 1 ethanol: glacial acetic acid) in a well in a microtitre plate, and two were frozen in liquid nitrogen in a well in the corresponding position of another microtitre plate. The fixed anthers were staggered; then the corresponding frozen anthers were combined per stage and polyA + RNA were isolated from 20 anthers (RNA equipment Micro-Quick Prep, Pharmacia Uppsila Sweden). Identical RNA volumes from anthers in each combined step were electrophoresed on 1.2% aryl agarose in MOPS + formaldehyde. The RNA samples were transferred by spotting a nylon membrane, fixed by UV entanglement (Stratalinker, Stratagene Inc., La Jolla), and hybridized to a 32P labeled probe fragment consisting of the entire Ms45 cDNA coding region and the 3 'region. The results shown in Figure 4 confirm the stable state of the Ms45 transcriptor detectable in the quartet through the uninuclear stages, and possibly as soon as, but before, telephase II at meiosis. Any levels of transcription that result from the preferred regulatory region of the Ms45 male tissue and its activity during non-accumulated meiosis in a manner sufficient to be detected by RNA hybridization, or the preferred regulatory region activity of the male meiotic stage tissue observed in transient trials it does not occur in plants.

Therefore, the preferred regulatory region of male tissue Ms45 (SEQ ID NO: 1) was characterized by • have the preferred expression of male tissue from at least the developmental quartet stage, from anther through the release of quartet with the lowest possible expression level in the initial and meiotic uninuclear stages. EXAMPLE 4 Analysis of TATA Box 10 Within the '1388bp fragment of the DNA encoding • the preferred regulatory region of male Ms45 tissue the main start of transcription has been identified at +1, a minor start of transcription has been identified at -3 with reference to the main start of transcription and a TATA box has been identified in -33 (CATTAAA). It was observed that the TAAAGAT sequence at -30 could also be a candidate for the actual TATA box. This 1388bp fragment was operably linked to a reporter gene cartridge comprising the "luciferase" coding region from Luciferin (Pareddy, et al., Theoret, Appl. Genet, Vol. 77; pp. 521-526; "(1989)) followed by the region not translated 3 'from the gene" inhibitor II of potato proteinase. (An, G., et al., "Functional Analysis of the 3 'Control Region of the Potato Wound-Inducible Proteinase Inhibitor II" Gene "; Cell; Vol. "1; pp. 115-122; (1989)).

One way "that is well known in the art-to analyze TATA boxes is through mutation." In another derivative, from one to six nucleotides of the putative TATA box were changed into a given derivative. at -38 by altering the putative TATA box from CATTAAA to TATTAAA, which is a match closer to the canonical TATA box sequence TATATAA It will be appreciated by someone with "experience in the art that certain substitutions within the TATA box can affect -the expression of the promoter without influencing the specificity of the tissue. As shown in Figure 7, the change in the TATA box associated with the BglII site introduced at -38 dramatically increased transient expression levels in anthers and further suggests that the sequence at -33 is the authentic TATA box. The introduction of a BGLII site at -40, -43, -51 or -53 does not increase the activity of the promoter (data not shown), providing that the increase observed at the introduction of BGLII site 38 was not related to the BGLII site per se. . "7 Ot ás" modifications of the TATA box were introduced to further test its functionality. The alteration of the "TATA box sequence from CATTAAA to GATTAAA, CATGGAA or GGGCCCA was reduced to the level of transient expression in anthers, in addition to suggesting the importance of this sequence as a TATA box.

Surprisingly, none of the mutations eliminated the transient activity; however, there were reports of transient activity in other systems in the absence of a sequence similar to TATA and even promoters lower than TATA (Guan, 'L., and JG Scandalios, Plant J .; Vol. 3; pp. 527-536 (1993); Cióse PS, "Cloning and Molecular Characterization of Two Nuclear Genes for Zea Mays Mitochondrial Chaperonin 60" (Dissertation), Iowa State University, Ames, Iowa, pp. 92, 128; (1993)). While the foregoing describes the preferred embodiments of the invention, it will be understood by those skilled in the art that variations or modifications can be made and will remain within the scope of the invention.

SEQUENCE LIST (1) GENERAL INFORMATION (i) APPLICANT: Pioneer Hi-Bred International, Inc. (ii) TITLE OF THE INVENTION: PREFERRED REGULATORY REGION OF MALE TISSUE AND METHOD FOR USING THE SAME (iii) NUMBER OF SEQUENCES: 2 ( iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: PIONEER Hl BRED INTERNATIONAL, INC. (B) STREET: 800 Capital Square, 400 Locust Street (C) "CITY: Des Moines (D) STATE: Iowa (E) COUNTRY: UNITED STATES OF AMERICA (F) POSTAL CODE: 50309 (v) METHOD OF READING IN THE COMPUTER: '(A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: compatible with an IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Paten In Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: "(A) APPLICATION NUMBER: PCT NOT ASSIGNED" (B) DATE OF SUBMISSION: CONCURRENTLY WITH THIS (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME : "Sweeney, Patricia A.

(B) REGISTRATION NUMBER: 32,733 (C) REFERENCE NUMBER / RECORD: 0578-PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (515) 248-4800 (B) TELEFAX; (515) 334-6883 (2) INFORMATION FOR THE IDENTITY SEQUENCE NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1394 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM : simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1: CCATGGTGTC TCTATGAAAA AGATGAGTAC AATGTGTCTA TATCCGTTTT CTTAGGGTCC 60 CTTCTTCTGC CTTATTACTG ACTGAATCGG GGTTACAAAA AACTTCCACG GGTGCATGAT 120 CTCCATGTTC CACTTCTCCC ACCTCGCGTT GCACATTTCT TGGATGTCGG TGGTTCCCAT 180 CTGACCGAGG CCCATCAGAC ACCTTTCGGG ACACCCATCA AGGGCCTTTC GGATGGCCCA 240 CGAGACGTAT CGGGTCGTGG TGATCCAGGG GATATATGTC CCCCACAATC GTCACCTATA 300 TTATTATTCT TTAGATATTA TTTAATTTTT GGAAAAATAA CAAACTTATA CTTTTGTGTA 360 GGGCCTCAGC ATAGATTTTC GCTTAGGGCC CAGAAATGCG AGGACCAGCC ATGTCTAGTG 420 TCCACTATTG GCACTACCCA GAACAAGATT TAAAAAAATA ACCAAAGTAA CTAATCCACT 480 CGAAAGCTAT CATGTAATGT TTAAAGAAAC ATCTATTAAA ACCACGATCC TCTTAAAAAA 540 CAAGCATATT TCGAAAGAGA CAAATTATGT TACAGTTTAC AAACATCTAA GAGCGACAAA 600 TTATATCGAA AGGTAAGCTA TGACGTTCAG ATTTTTCTTT TTCATTCTTG TTATTTTGTT 660 ATTGTTTTTA TATACATTTT CTTCTCTTAC AATAGAGTGA TTTTCTTCCG ATTTTATAAA 720 • ATGACTATAA AGTCATTTTT ATATAAGAGC ACGCATGTCG TAGATTCTCG TTCAAAAATC 780 TTTCTGATTT TTTTAAGAGC TAGTTTGGCA ACCCTGTTTC TTTCAAAGAA TTTTGATTTT 840 TTCAAAAAAA ATTAGTTTAT TTTCTCTTTA TAAAATAGAA AACACTTAGA AAAATAGAGT 900 TGCCAGACTA GCCCTAGAAT GTTTTCCCAA TAAATTACAA TCACTGTGTA TAATTATTTG 960 GCCAGCCCCA TAAATTATTT AAACCGAAAC TGAAATCGAG CGAAACCAAA TCTGAGCTAT 1020 ^ faith TTCTCTAGAT TAGTAAAAAG GGAGAGAGAG AGGAAGAAAT CAGTTTTAAG TCATTGTCCC 1080 TGAGATGTGC GGTTTGGCAA CGATAGCCAC CGTAATCATA GCTCATAGGT GCCTACGTCA 1140 GGTTCGGCAG CTCTCGTGTC ATCTCACATG GCATACTACA TGCTTGTTCA ACCGTTCGTC 1200 TTGTTCCATC GTCCAAGCCT TGCCTATTCT GAACCAAGAG GATACCTACT CCCAAACAAT 1260 CCATCTTACT CATGCAACTT CCATGCAAAC ACGCACATAT GTTTCCTGAA CCAATCCATT 1320 AAAGATCACA ACAGCTAGCG TTCTCCCGCT AGCTTCCCTC TCTCCTCTGC CGATCTTTTT 1380 CGTCCACCAC CATG 1394 (2) INFORMATION FOR THE SEQUENCE OF I DENT I DAD NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1394 base pairs (B) TYPE: nucleic acid (C) FORM OF THE CHAIN : simple 25 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE DNA (genomic) (xi) DESCRITION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2: CCATGGTGTC TCTATGAAAA AGATGAGTAC AATGTGTCTA TÁTCCGTTTT CTTAGGGTCC 60 CTTCTTCTGC CTTATTACTG ACTGAATCGG GGTTACAAAA AACTTCCACG GGTGCATGAT 120 CTCCATGTTC CACTTCTCCC ACCTCGCGTT GCACATTTCT TGGATGTCGG TGGTTCCCAT 180 CTGACCGAGG CCCATCAGAC ACCTTTCGGG ACACCCATCA AGGGCCTTTC GGATGGCCCA 240 CGAGACGTAT CGGGTCGTGG TGATCCAGGG GATATATGTC CCCCACAATC GTCACCTATA 300 TTATTATTCT TTAGATATTA TTTAATTTTT GGAAAAATAA CAAACTTATA CTTTTGTGTA 360 GGGCCTCAGC ATAGATTTTC GCTTAGGGCC CAGAAATGCG AGGACCAGCC ATGTCTAGTG 420 TACACTATTG GCACTACCCA GAACAAGATT TAAAAAAATA ACCAAAGTAA CTAATCCACT 480 CGAAAGCTAT CATGTAATGT TTAAAGAAAC ATCTATTAAA ACCACGATCC TCTTAAAAAA 540 CAAGCATÁTT TCGAAAGAGA C.AAATTATTGT TACAGTTTAC AAACATCTAA GAGCGACAAA 600 TTATATCGAA ÁGGTAAGCTA TGACGTTCAG ATTTTTCTTT TTCATTCTTG TTATTTTGTT 660 ATTGTTTTTA TATACATTTT CTTCTCTTAC AATAGAGTGA TTTTCTTCCG ATTTTATAAA 720 ATGACTATAA AGTCATTTTT ATATAAGAGC ACGCATGTCG TAGATTCTCG TTCAAAAATC 780 TTTCTGATTT TTTTAAGAGC TAGTTTGGCA ACCCTGTTTC TTTCAAAGAA TTTTGATTTT 840 TTCAAAAAAA ATTAGTTTAT TTTCTCTTTA TAAAATAGAA AACACTTAGA AAAATAGAGT 900 TGCCAGACTA GCCCTAGAAT GTTTTCCCAA TAAATTACAA TCACTGTGTA TAATTATTTG 960 GCCAGCCCCA TAAATTATTT AAACCGAAAC TGAAATCGAG CGAAACCAAA TCTGAGCTAT 1020 TTCTCTAGAT TAGTAAAAAG GGAGAGAGAG AGGAAGAAAT CAGTTTTAAG TCATTGTCCC 1080 GGTTCGGCAG CTCTCGTGTC ATCTCACATG GCATACTACA TGCTTGTTCA ACCGTTCGTC 1200 TTGTTCCATC GTCCAAGCCT TGCCTATTCT GAACCAAGAG GATACCTACT CCCAAACAAT 1260 CCATCTTACT CATGCAACTT CCATGCAAAC ACGCACATAT GTTTCCTGAA CAGATCTATT 1320 AAAGATCACA ACAGCTAGCG TTCTCCCGCT AGCTTCCCTC TCTCCTCTGC CGATCTTTTT 1380 CGTCCACCAC CATG 1394

Claims (34)

1. CLAIMS 1. An isolated nucleic acid encoding a preferred regulatory region of male Ms45 tissue.
2. 2. An isolated nucleic acid encoding a preferred regulatory region of male Ms45 tissue characterized in that it comprises a nucleotide sequence of SEQ. FROM IDENT. NO: 1 or those with at least an identity of at least 70% with it.
3. 3. An isolated nucleic acid encoding a preferred regulatory region of male Ms45 tissue characterized in that it comprises a nucleotide sequence of SEQ. FROM IDENT. NO: 2 or those with at least 70% identity with it.
4. 4. The recombinant expression vector comprising the nucleic acid according to claim 2, characterized in that it is operably linked to a nucleotide sequence encoding an exogenous gene such as the exogenous gene that is expressed in a preferred manner of male tissue. "5. The exogenous gene according to claim 4, characterized in that the exogenous gene is Ms45. 6. A method of producing a transformed plant expressing "an exogenous nucleotide sequence in a preferred manner of male tissue characterized in that it comprises introducing into a plant the exogenous nucleotide sequence operably linked in a preferred regulatory region of male tissue comprising the nucleotide sequence of SEQ ID NO: 1 or that with at least 70% identity therewith 7. The method according to claim 6, characterized in that the introduction step is executed by bombardment of microprojectile 8. The method according to claim 6, characterized in that the introduction step uses Agrobacterium 9. The method of compliance with the claim 8, characterized in that the Agrobacterium comprises a Ti plasmid. 10. The method of compliance with the claim 6, characterized in that the regulatory region expresses in a preferred manner male tissue in tissues selected from the group consisting of pollen, cell layer, anther, tassel, pollen stem cells and microspores. 11. The method according to the claim 6, characterized in that a preferred expression portion of male tissue of the preferred regulatory region of male tissue is present in more than one copy. 12. The method of mediating fertility in a plant comprising the production of a transformed plant wherein the preferred regulatory region of the male tissue according to claim 2, operably linked to an exogenous gene, expresses the exogenous nucleotide sequence in a manner that fertility is impacted. 13. The method according to the claim 6, characterized in that the exogenous nucleotide sequence comprises a DNA sequence encoding a prokaryotic regulatory system. The method according to claim 6, characterized in that the exogenous nucleotide sequence encodes a cytotoxic gene. 15. The method according to claim 6, characterized in that the exogenous nucleotide sequence encodes avidin. "16. The method of compliance with the claim 6, characterized in that the exogenous nucleotide sequence encodes DAM methylase. 17. The method of compliance with the claim 6, characterized in that the exogenous nucleotide sequence encodes the preferred regulatory region of male tissue operably linked to a complementary nucleotide unit. 18. The method of compliance with the claim 17, characterized in that the complementary nucleotide unit is selected from the group consisting of antisense callasa RNA, antisense bamasa RNA, antisense chalcone synthase RNA and Ms45 antisense RNA. 19. The method according to claim 17, characterized in that the complementary nucleotide unit is selected from the group consisting of ribosomes and external guide sequences. 20. The method according to claim 12, characterized in that the exogenous nucleotide sequence encodes a product selected from the group consisting of aids, rolB and diphtheria toxin. 21. The method according to claim 12, characterized in that the exogenous nucleotide sequence is a male sterility gene. 22. The method according to claim 21, characterized in that the male sterility gene is Ms45, 23. " The method according to claim 12, characterized in that the plant is a monocotyledonous. 24. The method according to claim 12, characterized in that the plant is a dicot. 25. The hybrid seed production method, characterized in that it comprises the cross-pollinated juxtaposition plantation of a first male fertile plant and a second male infertile plant, the second male infertile plant produced according to the method of claim 6, allowing cross-pollination to occur and harvesting the resulting seed. 26. The method according to claim 25, characterized in that the plant is corn. 27. A transformed plant expressing an exogenous nucleotide sequence in a preferred manner of male tissue characterized in that it comprises an exogenous nucleotide sequence operably linked to a preferred regulatory region of male tissue comprising a nucleotide sequence of SEQ. FROM IDENT. I do not know: c. FROM IDENT. NO: 2. 28. The transformed plant according to claim 27, characterized in that the exogenous nucleotide sequence is operably linked to a preferred regulatory region of male tissue, a preferred expression portion of male tissue from which it can be present in more than one copy. 29. The transformed plant according to claim 27, characterized in that the plant is a monocot or a dicot. 30. The transformed plant according to claim 27, characterized in that the plant is selected from the group consisting of corn, sunflower, soybean, wheat, canola, rice and sorghum. 31. The transformed plant according to claim 28, characterized in that the plant is selected from the group consisting of corn, sunflower, soybeans, wheat, canola, rice and sorghum. 32. The tissue of the transformed plant according to claim 27 or claim 28. 33. The transformed fabric according to claim 32, characterized in that the fabric is selected from the group consisting of pollen, spikes, ovules, anthers, tassels, pistil stamens and plant cells. 34. The transformed plant cells of the transformed plant according to claim 27.