JPH11512922A - Induction of plant male sterility by high levels of avidin expression - Google Patents

Abstract

  (57) [Summary] By increasing the endogenous concentration of avidin in plant tissues, it is possible to produce male-sterile plants. This effect can be achieved by creating a transgenic plant carrying an expression vector in which a DNA sequence encoding avidin is connected under the control of a promoter. Methods for restoring male fertility are also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION Induction of Plant Male Sterility by High Level Avidin Expression Background of the Invention The present invention relates to a method of controlling plant fertility using a DNA molecule encoding avidin. In particular, the present invention is directed to a method of producing transgenic male sterile plants that express avidin. The present invention further relates to a method of restoring male fertility in progeny of a male sterile plant. Controlling pollen fertility is essential for producing hybrid crops. See, for example, "JM. Poehlman, Breeding Field Crops, 3rd Edition (Van Norstrand and Reinhold, New York, NY, 1987)," which is incorporated herein by reference. For example, in producing hybrid corn, pollen fertility can typically be controlled by physically removing the male inflorescence or tuft before pollen spills. Removing the tufts manually is very hard work. Removing the tufts using a machine is less labor intensive than manual, but less reliable, and requires a later inspection of the crop and possibly additional manual tuft removal. Both methods of hair removal remove the viability of the female parent. However, most plants, primarily as crops of interest, have both functional male and female organs in the same flower; therefore, it is simple to perform emasculation It is not a process. Although it is possible to manually remove the pollen-forming organs before the pollen spills, the hybrid production by this method is very labor intensive and expensive. Pollen fertility can also be controlled by spraying leaves with the characteristics of male gametes. Cross et al., Sex Plant Reprod. 4: 235 (1991). For example, applying 2-chloroethyl sulfonic acid (esrel) to anthers causes extra pollen mitosis in wheat, degeneration of tapetocytes in barley, and hypoplasia and stunting in Lentinus. . Bennet et al., Nature 240: 566 (1972); Colhoun et al., Plant Cell Environ. 6:21 (1983); Berthe et al., Crop Sci. 18:35 (1978). However, the chemical approach is also labor intensive and has the potential problems of introducing chemical toxicity to the environment. In addition, the commercial production of hybrid seeds using gameticides is limited in terms of the price and effectiveness of chemicals, as well as the reliability and shelf life of chemicals. is there. A significant limitation of gametoids is that they have toxic effects on plants that vary in strength by genotype. Another limitation is that these chemicals may not be effective in long-flowering crops, because newly-grown flowers may not be affected . As a result, it is necessary to provide the chemical substance repeatedly. Current commercial hybrid seed production systems for field crops are often based on genetic means of pollination control. Plants used as females are either unable to disperse pollen, produce pollen that cannot be self-pollinated biochemically, or cannot produce pollen. Plants that cannot self-pollinate are called "self-incompatible". When using a self-incompatibility system, the usefulness and reproductivity of a self-incompatible female strain, and the stability of self-incompatibility become problems. In some cases, self-incompatibility can be resolved by manually pollinating immature buds before the chemical or biochemical mechanisms that inhibit pollen are activated. Self-incompatibility mechanisms that can be deactivated are often very susceptible to the effects of repressive climatic conditions, such as losing or reducing the effectiveness of biochemical inhibition of self-pollination. Of greater interest in commercial seed production are systems based on genetic pollen control that cause male sterility. These systems can be broadly divided into two types: (1) nuclear male sterility, that is, one or more nuclear gene mutations that prevent pollen formation, or (2) cytoplasmic inheritance. Sexual male sterility, often called "cytoplasmic male sterility" (CMS), is a change in cytoplasmic organelles (often mitochondria) that inhibits or defies pollen formation thing. Nuclear sterility can be either dominant or recessive. Dominant sterility can only be used to form hybrid seeds where asexual or parthenogenetic propagation of the female strain is possible. Recessive sterility can be used if it is easy to distinguish between sterile and fertile plants. However, dominant and recessive sterility systems have limited commercial utility, respectively, due to the high cost of parthenogenesis and the culling of female strains of self-pollinating plants. There are plans for hybridization, including the use of CMS, but their commercial value is limited. One example of a CMS system is when the mitochondria in the cytoplasm have specific mutations that, with the appropriate nuclear background, make it impossible to form mature pollen. In some cases, nuclear background can compensate for cytoplasmic mutations and cause normal pollen formation. A specific "restoring gene" in the nucleus enables pollen formation in plants with CMS mitochondria. In general, the use of CMS for commercial seed production involves the use of three hybrid strains: a male dominant sterile strain (female parent strain), other than having a fully functional mitochondria. Is a maintenance strain having the same genotype as the male sterile strain, and a male parent strain. The male parent strain may or may not have a specific restoring gene in the cytoplasm. For crops such as vegetables where recovering seed from hybrids is not important, a non-recoverable CMS system can be used. For crops in which the hybrid fruit or seed is a commercial product, it is necessary to restore the fertility of the hybrid seed by the recovery gene of the male parent strain or to pollinate the male sterile hybrid. Pollination of unrecovered hybrids can be achieved by including a small number of male fertile plants for pollination. In most species, all cytoplasmic organelles are inherited exclusively from egg cells, so the CMS trait is maternally inherited. CMS systems have the limitation that male plants must be produced as the only solution for producing sterile plants. For example, one particular CMS type of maize (T-cytoplasm) confers susceptibility to toxins produced by infection by certain fungi. Although still used in many crops, CMS systems can be destroyed under certain environmental conditions. From the above, it is clear that there is a great need for means to control pollen production other than manual, mechanical, chemical and traditional genetic techniques. Summary of the Invention Therefore, an object of the present invention is to provide a method for producing a male sterile plant that becomes sterile by heterologous expression of avidin. It is a further object of the present invention to provide a chimeric gene consisting of a DNA sequence encoding avidin, connected under the control of a plant promoter sequence. It is a further object of the present invention to provide a method of restoring male sterility caused by avidin expression. These and other objects are achieved according to one aspect of the present invention by providing an isolated DNA molecule consisting of a nucleotide sequence encoding avidin connected under the control of a plant promoter sequence. . In a preferred embodiment, the isolated DNA sequence is contained on an expression vector. In another preferred embodiment, the plant promoter sequence is a constitutive promoter, and in another preferred embodiment, the constitutive promoter is a ubiquitin promoter. According to yet another preferred embodiment of the present invention, there is provided a transgenic plant comprising an avidin gene and a heterologous nucleotide sequence that increases male avidin concentration in plant tissue to cause male sterility. In a preferred embodiment, the transgenic plant is a cereal, legume, or sunflower. In another preferred embodiment, the heterologous DNA molecule further comprises a constitutive promoter sequence, which in one preferred embodiment is a ubiquitin promoter sequence. According to yet another aspect of the present invention, the method comprises introducing an expression vector containing a promoter and an avidin gene into a plant cell, wherein the expression of the avidin gene causes male sterility by controlling the expression of the avidin gene. And a method for producing a transgenic male sterile plant. In a preferred embodiment, the transgenic plants are regenerated from plant cells. In another preferred embodiment, the promoter comprises a ubiquitin promoter or an inducible promoter. According to a further different aspect of the present invention, the creation of a first parent strain, a male sterile plant containing the DNA molecule described above, wherein the expression of avidin causes male sterility, a second transgenic expressing a second foreign gene Of a parent plant, and the second parent plant in which a first parent strain and a second parent strain are cross-pollinated to suppress the expression of avidin in a hybrid plant, thereby producing a male fertile hybrid plant. Provided is a method of using an avidin gene to produce a male-sterile hybrid plant, including producing a hybrid plant that expresses a foreign sperm gene. In a preferred embodiment of this aspect of the invention, the second exogenous gene is selected from the group consisting of an antisense gene, a ribozyme gene and an external guide sequence gene. In yet another preferred embodiment, the antisense gene comprises an avidin mRNA sequence under the control of an inducible promoter in yet another preferred embodiment. In yet another preferred embodiment, the ribozyme gene comprises an avidin mRNA sequence. In yet another preferred embodiment, the external guide sequence gene comprises an avidin mRNA sequence. In still another preferred embodiment, the DNA molecule of the first parent plant further comprises a LexA operator connected under the control of the promoter when the second exogenous gene is a LexA repressor gene. In another preferred embodiment, the promoter sequence is an answer selected from the group comprising the nucleotide sequence of SEQ ID NO: 1; the nucleotide sequence of SEQ ID NO: 2; the nucleotide sequence of SEQ ID NO: 3; and a functional fragment derived therefrom. Anther-specific promoter sequence containing a box (anther box). In yet another preferred embodiment of this aspect of the invention, the answer box has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the anther-specific promoter sequence further comprises a CaMV 35S core promoter. A nucleotide sequence of SEQ ID NO: 4; a nucleotide sequence of SEQ ID NO: 5; a nucleotide sequence of SEQ ID NO: 6; and a core promoter selected from a group including a functional fragment derived therefrom. In accordance with yet another aspect of the present invention, there is provided a method of restoring fertility of a plant that has become male sterile due to avidin expression, comprising spraying a biotin solution onto the plant. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the plasmid PHI5168 encoding avidin. FIG. 2 shows the plasmid PHI610 encoding the bar gene. Detailed description of preferred embodiments 1. Definitions In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the present invention. A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) and then translated into an amino acid sequence characteristic of a specific polypeptide. Exogenous gene, as used herein, refers to a DNA sequence connected under the control of at least one heterologous regulatory element. For example, any gene other than the maize 5126 structural gene is considered an exogenous gene if its expression is controlled by an anther-specific regulatory element of the 5126 gene. A promoter is a DNA sequence that governs the transcription of a gene, such as a structural gene, an antisense gene, a ribozyme gene, or an external guide sequence gene. Typically, a promoter is located in the 5 'region near the transcription start site of a gene. When the promoter is an inducible promoter, the rate of transcription increases with the inducing agent. In contrast, when the promoter is a constitutive promoter, the rate of transcription is not controlled by the inducing agent. A plant promoter is a promoter sequence that controls the transcription of a gene in a plant tissue. The core promoter contains a nucleotide sequence essential for promoter function, including a TATA box and a transcription initiation site. By this definition, a core promoter may or may not have detectable activity in the absence of a specific sequence that promotes its activity or is involved in tissue-specific activity. . For example, the SGB6 core promoter consists of about 38 bases 5 ′ to the transcription start site of the SGB6 gene, while the cauliflower mosaic virus (CaMV) 35s core promoter has about 33 bases 5 ′ to the transcription start site of the 35S genome. Consists of a base. A tissue-specific promoter is a DNA sequence that, when linked to a gene under its control, causes expression of that gene at a higher level in certain tissues of an organism than in some or all other tissues. is there. For example, an anther-specific promoter is a DNA sequence that causes high levels of transcription of the relevant gene in plant anther tissue. For example, the anther-specific promoter of SGB6 can cause exogenous gene expression in anther tissue, but not in root or coleoptile tissue. As used herein, an "anther-specific promoter" contains two functional elements: the "answer box" and the core promoter. Certain answer boxes may have the functional characteristics of classical enhancers. The combination of one answer box and one core promoter can promote gene expression to a greater extent than the core promoter alone. This is true even in the case of chimeric anther-specific promoters, including answerbox and core promoters from different genes. Such chimeric anther-specific regulatory elements are described below. An operator is a DNA molecule located in the 5 'direction of a gene and contains a nucleotide sequence that is recognized and bound by a repressor protein. Binding of the repressor protein to its cognate operator causes repression of transcription of the gene. For example, the LexA gene encodes a suppressor protein that binds to the LexA operator. An isolated DNA molecule is a DNA fragment that has been separated from the DNA of an organism. For example, a cloned DNA molecule that encodes an avidin gene is an isolated DNA molecule. Other examples of isolated DNA molecules include chemically synthesized DNA molecules or cDNA produced by enzymes that have not been integrated into the genomic DNA of an organism. Enhancers are DNA regulatory elements that can increase the efficiency of transcription. Complementary DNA (cDNA) is a single-stranded DNA molecule formed using mRNA as a template by an enzyme called reverse transcriptase. Typically, a primer complementary to a portion of the mRNA is used to initiate reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression includes transcription of the structural gene into mRNA and translation of the mRNA into one or more polypeptides. A cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one or a few restriction enzyme recognition sites into which foreign DNA can be inserted in a determinable manner without loss of the essential biological function of the vector. Contains marker genes suitable for use in the identification and selection of cells transformed by the vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance. An expression vector is a DNA molecule that contains a gene that is expressed in a host cell. Typically, the expression of a gene is under the control of specific regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is termed "operably linked" to a regulatory element. The term recombinant host can apply to all prokaryotic and eukaryotic cells, including either cloning or expression vectors. The term also includes prokaryotic or eukaryotic cells that have undergone genetic manipulation and contain the cloned gene (s) in the chromosome or genome of the host cell. A transgenic plant is a plant that has one or more plant cells containing an exogenous gene. In eukaryotes, RNA polymerase II catalyzes the process of transcribing structural genes to produce mRNA. DNA molecules containing an RNA polymerase II template can be designed such that the RNA transcript has a sequence complementary to a particular mRNA. This RNA transcript is named antisense RNA, and the DNA sequence encoding the antisense RNA is named antisense gene. The antisense RNA molecule can bind to the mRNA, such that translation of the mRNA is inhibited. Ribozymes are RNA molecules that contain a catalytically active center. The term includes RNA enzymes, self-splicing RNAs, and self-cleaving RNAs. The DNA sequence encoding the ribozyme is termed the ribozyme gene. The external guide sequence directs the endogenous ribozyme, RNase P, to a specific intracellular mRNA molecular species, which results in RNase P cleaving the mRNA. The DNA sequence encoding the external guide sequence is termed the external guide sequence gene. 2. Overview The present invention provides a method for producing a male sterile plant by preparing a transgenic plant that expresses avidin. Avidin can be expressed constitutively without tissue specificity or in an anther-specific manner. Methods for restoring male fertility are also provided. The avidin gene is isolated and inserted into an appropriate expression vector for introduction into plant tissue. Expression vectors further include a constitutive promoter, such as a tissue non-specific promoter, such as the ubiquitin promoter, and a tissue specific promoter, such as an anther specific promoter, or an inducible promoter. The expression vector is introduced into plant tissues by standard methods, and transgenic plants are selected for propagation. Transgenic plants that express avidin are male sterile. Male fertility can be restored by co-expressing a second gene in the transgenic plant that inhibits transcription of the avidin gene or inhibits translation of avidin mRNA. Alternatively, male fertility can be restored by spraying the growing plant with a biotin solution. 3. Isolation of DNA molecule encoding avidin The nucleotide sequence encoding avidin can be isolated by known methods. For example, cDNA encoding avian egg white avidin can be isolated from avian oviduct cDNA library by the method described in Gope et al., Nucleic Acids Res., 15: 3595 (1987), which is incorporated herein by reference. Can be released. Alternatively, a genomic clone of the avidin gene can be obtained from genomic DNA of an organism known to produce avidin. For example, the avian avidin gene can be cloned from avian genomic DNA by the method described in Keinanen et al., Eur. J. Biochem., 220: 615 (1994). The avidin gene can also be isolated using standard techniques, the polymerase chain reaction (PCR). Erlich, PCR TECHNOLOGY: Principles and Applica tions for DNA Amplification (PCR technique: application to DNA amplification) (Stockton Press, NY, 1989), and Innis et al., PCR Protocols: A GUIDE TO METH ODS AND APPLICATIONS ( PCR Protocols: Guide to Methods and Applications) (Aca demic Press, San Diego, 1990). Methods for amplification of long base sequences by PCR, such as the ELONGASE ™ system (Life Technologies, Inc., Gaithersburg, MD), also provide longer base sequences, such as genomic clones encoding avidin, Can be used. PCR primers complementary to the 5 'and 3' ends of the known avidin gene base sequence can be synthesized using a commercially available oligonucleotide synthesizer, such as that provided by Applied Biosystems (Foster City, CA). . In a preferred embodiment, the primer also contains an extra nucleotide sequence containing a restriction enzyme cleavage site. The presence of such a site allows the PCR product to be treated with an appropriate restriction enzyme and then unidirectionally cloned into an appropriate cloning vector. Finney, “Molecular Cloning of PCR Products”, CURRENT PROTOCOLS IN MOLECUTAR BIOLOGY (Current Protocol Collection of Molecular Biology), edited by Ausubel et al., (John Wiley & Sons, New York, 1987), p.15.7. 1. The template DNA used for PCR can be either cDNA or genomic DNA, and can be prepared from a suitable organism using methods known in the art. See Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Molecular Cloning: Laboratory Guidelines), Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989). Genomic DNA can be recovered directly from bird, reptile, or amphibian tissues using a commercially available drug such as Triazol ™ (Life Technologies, Inc., Gaithersburg,). Alternatively, cDNA encoding avidin can be prepared using mRNA extracted from avian oviduct tissue using commercially available kits (Pharmacia, Piscataway, NJ). The mRNA preparation is used as a template for cDNA synthesis by standard techniques using poly dT or random hexamer (random 6 base sequence) primers. See Sambrook et al., Supra. Thereafter, cDNA synthesis is performed using a commercially available kit (Pharmacia), and the cDNA is directly used for PCR according to the method of "Saiki et al., Science 239: 487 (1988)". The genomic DNA or cDNA fragment isolated by the above method is cleaved with a restriction enzyme to form an appropriate stump, treated with alkaline phosphatase so that the DNA molecule does not bind undesirably, and ligated with an appropriate ligase. And into a vector such as a bacteriophage or cosmid, according to conventional techniques. For details on techniques for handling such vectors, see "Ausubel et al. (Ed.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Current Protocol Collection of Molecular Biology), .A. 3.0.5 to 3.17.5 (1990)"["Ausubel"] It is described in. Alternatively, the avidin-encoding base sequence can also be obtained by synthesizing avidin-encoding DNA molecules using long mutually priming oligonucleotides. See, for example, Ausubel pages 8.2.8-8.2.13. See also Wosnick et al., Gene 60: 115 (1987). Current technology using the polymerase chain reaction allows genes up to 1.8 kilobases in length to be synthesized. Adang et al., Plant Molec. Biol. 21: 1131 (1993); Bambot et al., PCR Methods and Applications 2 (PCR and its applications 2): 226 (1993). Based on the nucleotide sequence of any known DNA molecule encoding avidin, the nucleotide sequence of the avidin gene can be synthesized. See, for example, Gope et al., Supra. These clones can be analyzed using various standard techniques such as restriction enzyme analysis, Southern blot analysis, primer extension analysis, and DNA sequence analysis. For example, analysis by the primer extension method or analysis by the S1 nuclease protection method can be used to estimate the transcription start site of the cloned gene. Ausubel et al., Pages 4.8.1-4.8.5; Walmsley et al., "Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protection Assay" (Quantitative and qualitative analysis of exogenous gene expression by S1 nuclease protection assay). Analysis), METHODS IN MOLECULAR BIOLOGY (Methods of Molecular Biology), Volume 7: GENE TRANSFER AND EXPRESSION PROTOCO LS (Collection of Protocols for Gene Transfer and Expression), Murray (eds.), Pages 271-281 (Humana Press Inc. 1991) ). 4. Cloning of avidin gene into expression vector and production of transgenic plant Once the avidin gene is isolated, it is incorporated into an expression vector by standard methods. See Sambrook et al., Supra. In order to select an appropriate expression vector, it is necessary to introduce the expression vector into a host cell. Typically, expression vectors include: (1) an origin of replication in bacteria, and encoding antibiotic resistance genes necessary for the expression vector to grow and be selected in bacterial hosts. A prokaryotic DNA element; (2) a cloning site for inserting an exogenous DNA sequence such as the avidin gene; (3) a eukaryote that controls the initiation of expression of the exogenous gene such as a promoter. (4) a DNA element that controls the processing of the transcript, such as a transcription termination / polyadenylation sequence; and (5) a marker protein, which is connected under the control of a DNA element that controls transcription initiation. (Eg, a reporter gene). General descriptions of plant expression vectors and reporter genes can be found in Gruber et al., "Vectors for Plant Transformation", ME THODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY (Molecular Biology and Biology of Plants). Engineering method) See Glick et al. (Eds.), Pages 89-119 (CRC Press, 1993). In a preferred embodiment of the present invention, a plant ubiquitin promoter system is used. Plant ubiquitin promoters are well known in the art. See, for example, European Patent Application 0 342 926, which is incorporated herein by reference. The ubiquitin promoter is a constitutive promoter. In another preferred embodiment of the present invention, an inducible promoter is used for inducibly controlling avidin expression. An example of such an inducible promoter is the corn glutathione S-transferase system. Wiegand et al., Plant Mol. Biol. 7: 235 (1986). The method can also reverse induce male sterility. Thus, expression of avidin and the accompanying male sterility can be controlled at will by activation of the promoter. If the promoter is not activated, avidin is not expressed and the plant is male fertile. Expression vectors containing the avidin gene can be introduced into protoplasts (protoplasts), or complete tissues such as immature embryos and meristems, or callus cultures, or isolated cells. Preferably, the expression vector is introduced into a complete tissue. For general methods of culturing plant tissues, see, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants", "METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY" Glick et al. (Eds.), Pages 67-88 (CRC Press, 1993), and Phillips et al., "Cell / Tissue Culture and In Vitro Manipulation" (cell / tissue culture and in vitro manipulation). CORN AND DORN IMPROBEMENT, 3rd edition, Sprague et al. (Eds.), Pages 345-387 (American Society of Agronomy, Inc. et al., 1988). Methods for introducing an expression vector into plant tissue include direct infection or cultivation of plant tissue in the presence of Agrobacterium soil bacteria. Horxch et al., Science 227: 1229 (1985). Preferably, the disarmed Ti plasmid is used as a vector for exogenous DNA sequences. Transformation can be performed using procedures such as those described in European Patent Application Nos. 116 718 (1984) and 270 822 (1988). Preferred Ti plasmid vectors contain exogenous DNA sequences between the marginal sequences or at least upstream of the right marginal sequence. Other types of vectors are also available for direct gene transfer (see, eg, Patent Cooperation Treaty application WO B5 / 01856 and European application 275 069), in vitro protoplast transformation (eg, US Pat. No. 4,684,611), plant virus-mediated transformation (Eg, EP 067553 and US Pat. No. 4,407,956) and are used to transform plant cells using procedures such as liposome-mediated transformation (eg, US Pat. No. 4,536,475). be able to. Suitable methods for maize transformation are described by Fromm et al., Bio / Technology 8: 833 (1990), and Gordon-Kamm et al., The Plant Cell 2: 603 (1990). Standard methods for rice transformation are described in Christou et al., Trends in Biotechnolgy 10: 239 (1992) and Lee et al., Proc. Nat'l. Acad. Sci. USA 88: 6389 (1991). Wheat can also be transformed using techniques similar to those used to transform corn or rice. Further, Casas et al., Proc. Nat'l. Acad. Sci. USA 90: 11212 (1993) describes a method for transformation of sorghum, while Wan et al., Plant Physiol. 104: 37 (1984) describes a method for barley transformation. In general, the direct transfer method is preferred for the transformation of monocotyledonous plants, especially cereals such as rice, corn, sorghum, barley or wheat. Suitable direct transfer methods include transfer via microparticles, DNA injection, electroporation, and the like. See, for example, Gruber et al., Supra, Miki et al., Supra, and Klein et al., Bio / Technology 10: 268 (1992). More preferably, the expression vector is introduced into monocotyledonous plant tissue using a particle mediated transmission via microparticles. 5. Control of Male Fertility A. Production of Male Sterile Plants In order to induce male sterility according to the present invention, the DNA sequence encoding avidin is controlled by the DNA sequence that controls the transcription of genes in plant tissues. Build the connected expression vector. General requirements for expression vectors are described above. In order to render male sterile by the expression of avidin, avidin is not only transiently expressed but also expressed in the embryonic tissue of plants in such a way that avidin is expressed at a late stage of development in the adult stage of plants. An expression vector is introduced. For example, mitotic stability can be imparted by using a plant virus vector that can replicate extrachromosomally. A preferred way to achieve mitotic stability is to integrate the sequence of the expression vector into the host chromosome. Such mitotic stability can be achieved by transferring the expression vector to plant tissue in microparticles, or by using other standard methods described above. For example, Fromm et al., Bio / Technology 8: 833 (1990), Gordon-Kamm et al., The Plant Cell 2: 603 (1990), and Walters et al., Plant Molec. Biol. 18: 189 (1992). Avidin transcription after introduction into plant tissue is preferably controlled by a constitutive promoter that non-specifically stimulates gene expression in plant tissue. Examples of constitutive promoters suitable for this purpose are, as mentioned above, the ubiquitin promoter. Avidin gene transcription can also be controlled by a promoter that stimulates gene expression in a tissue-specific manner. A particularly preferred anther-specific promoter is the 5126 promoter isolated from maize sublines B73. The 5126 promoter stimulates the expression of foreign genes, from tetrad to early mononuclear microspore stage anthers. Another suitable anther-specific promoter is the SGB6 promoter, which was also isolated from strain B73. The SGB6 promoter is capable of inducing the expression of foreign genes in anther tapetate cells from the stage of microspore development from the tetrad stage to the mid-mononuclear stage. Alternatively, anther-specific gene expression can be induced using a combination of an answer box and a core promoter. A particularly preferred answer box is the 5126 answer box, consisting of the base sequence described below: Another suitable answer box is within a 94 base pair DNA fragment defined by bases 583 to 490 upstream of the transcription start site of SGB6. The SGB6 94 base pair answer box consists of the following nucleotide sequence: Alternatively, a suitable answer box can be obtained from the maize G9 promoter, which stimulates gene expression during meiotic to tetrad development. The G9 answer box consists of the following nucleotide sequence: Answer boxes for 5126, SGB6 and G9 can be obtained by synthesizing oligonucleotides as described above. Those skilled in the art may be able to further position one or more anther-specific regulatory sequences within the indicated Anservox using analysis by deletion mutations. "Functional fragments" of such answer boxes can also be used to control avidin gene expression in an anther-specific manner. Preferred core promoters are derived from the 5126 core promoter, the SGB6 core promoter, the G9 core promoter and the cauliflower mosaic virus 35S core promoter. A particularly preferred promoter is a 5126 core promoter consisting of the following nucleotide sequence: The SGB6 core promoter consists of about 38 bases upstream of the transcription start site of the SGB6 gene. A suitable SGB6 core promoter has the following nucleotide sequence: A suitable core promoter derived from the G9 gene has the following nucleotide sequence: Functional fragments of the 5126, SGB6 and G9 core promoters also make suitable core promoters. Methods for obtaining such functional fragments have been described above. The use of a chimeric regulatory element consisting of an answer box from one gene and an anther-specific promoter from a second gene can also extend the stage of development in which the avidin gene is expressed. For example, the regulatory sequence of SGB6 stimulates gene expression through the developmental stages from quaternary to mid-mononuclear phase, while the G9 regulatory sequence stimulates gene expression through the developmental stages from meiosis to tetrad. Stimulates. However, the combination of the SGB6 answer box and the G9 promoter stimulates the transcription of foreign genes through developmental stages from meiosis to mid-nuclear. Thus, various combinations of answer boxes and anther-specific promoters are particularly useful in the present invention. The viral core promoter can also be used. Examples of suitable viral core promoters include the cauliflower mosaic virus (CaMV) core promoter, the Figwort mosaic virus core promoter, and the like. Gruber et al., Supra. Preferably, the viral core promoter is the CaMV 35S core promoter, or a variant derived therefrom. To select for transformed cells, the expression vector contains a selectable marker gene, such as a herbicide resistance gene. For example, such a gene may be involved in resistance to phosphinothricin, glyphosate, urea sulfonate, atrazine, or imidazolinone. Preferably, the gene for the selectable marker is a bar gene or a pat gene encoding phosphinothricin acetyltransferase. The nucleotide sequence of the bar gene can be found in Leemans et al., European Patent Application No. 0-242-246 (1987), and in White et al., Nucleic Acids Res. 18: 1062 (1990). Wohlleben et al., Gene 70: 25 (1988), provide the nucleotide sequence of the pat gene. When the Bar or pat gene is expressed, it acquires resistance to herbicides such as glufosinate (otherwise sold as BastaR and IgniteR) and bialaphos (sold as Herbi-aceR and LibertyR). Expression vectors can include avidin-encoding DNA sequences controlled by a constitutive or anther-specific promoter, as well as a selectable marker gene controlled by a constitutive promoter. Alternatively, embryonic tissue can be "co-transformed" with an independent selectable expression vector to deliver the selectable marker gene to the host cell. B. Restoration of Male Fertility in First-Generation Hybrids The above-described method is used to produce a first-generation hybrid of transgenic male-sterile plants by performing large-scale directional crosses between neighboring varieties. Can be used. If none of the eggs of the transgenic male sterile plant contain the recombinant avidin gene, some of the first hybrids will have a male fertile phenotype. On the other hand, if the recombinant avidin gene is present in all the eggs of the transgenic male sterile plant, the sterility induced by ethylene will be dominant, and the first hybrid will have a male sterile phenotype. Have. From this, it is considered preferable to use a male fertility recovery system that can produce a male fertile first-generation hybrid. Such a fertility restoration system is particularly valuable for inbred species when the harvest product is seed. In a transgenic male sterile plant line, the methods commonly used to restore fertility require the creation of a second "restored" line of the transgenic plant. For example, a transgenic plant that has become male sterile due to the expression of barnase is crossed with a male fertile plant that expresses a barnase inhibitor, as discussed above. Mariani et al., Nature 357: 384 (1992). In this case, as a similar approach, it is necessary to create a recovery system for transgenic plants expressing ribozymes targeting avidin mRNA or expressing antisense avidin. For example, a ribozyme can be designed to express an endonuclease activity on a specific target sequence of an mRNA molecule. See, for example, Steinecke et al. 11: 1525 (1992), ribozymes inhibit neomycin phosphotransferase gene expression to 100% in tobacco protoplasts. For a more recent example, see Perriman et al., Antisense Res. & Devel. 3: 253 (1993) uses a vector that expresses a modified hammerhead ribozyme to inhibit chloramphenicol acetyltransferase activity in tobacco protoplasts. In the context of the present invention, avidin mRNA is suitable as a target RNA molecule for a ribozyme. In a similar approach, fertility can be restored using an expression vector containing a base sequence encoding the antisense RNA. When the antisense RNA molecule binds to the target mRNA molecule, hybridization inhibits translation. Paterson et al., Proc. Natl. Acad. Sci. USA, 74: 4370 (1987). In the context of the present invention, a suitable antisense RNA molecule has a sequence that is complementary to avidin mRNA. In a preferred embodiment of the invention, the antisense RNA is controlled by an inducible promoter. Activating this promoter can restore male fertility. As yet another approach, expression vectors encoding RNA transcripts can be constructed that can facilitate RNase P-catalyzed cleavage of avidin mRNA molecules. Using this approach, an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to avidin mRNA and subsequent cleavage by the cellular ribozyme. Altman et al., US Patent No. 5,168,053; Yuan et al., Science 263: 1269 (1994). Preferably, the external guide sequence comprises a 10-15 base sequence complementary to avidin mRNA and a 3'-NCCA base sequence where N is preferably a purine. The same reference. The transcript of the external guide sequence binds to the target mRNA species by forming a base pair between the mRNA and the complementary external guide sequence, resulting in a 5 ′ position of the base-paired region. At these bases, the cleavage of mRNA by RNase P is promoted. The same reference. In another method of restoring male fertility, the transgenic male sterile plant has an expression vector that contains prokaryotic regulatory elements in addition to the promoter sequence connected to control the avidin gene. . A transgenic male fertile plant is produced that expresses the prokaryotic polypeptide under the control of its promoter. In the first hybrid, the prokaryotic polypeptide binds to prokaryotic regulatory sequences and suppresses avidin expression. For example, according to the present invention, a LexA gene / LexA operator system can be used to control gene expression. U.S. Pat. No. 4,833,080 ("'080 patent") and Wang et al., Mol. Cell. Biol. 13: 1805 (1993) More specifically, an expression vector for a male sterile plant comprises a LexA operator sequence. In contrast, the expression vector of the male fertile plant contains a sequence encoding the LexA repressor, In the first hybrid, the LexA repressor binds to the LexA operator sequence and inhibits transcription of the avidin gene. The LexA operator DNA molecule can be obtained, for example, by synthesizing a DNA fragment containing the known LexA operator sequence, eg, the '080 patent and Garriga et al., Mol. Gen. Genet. 236: 125 (1992). The LexA gene can be obtained by synthesizing a DNA molecule encoding the LexA repressor.Gene synthesis techniques are described above, and the base sequence of the LexA gene is, for example, Garri or a DNA molecule encoding the LexA repressor can be obtained from plasmid pRB500, American Type Culture Collection Accession number 67758. Yet Another Method to Restore Fertility Some use the high affinity of avidin for biotin by spraying a biotin solution onto growing plants.Biotin solutions also use a biotin solution, such as DMSO, to ensure complete dissolution of biotin. Contains a minimal amount of organic co-solvents, begins spraying during the meiotic phase of pollen development and repeats at regular intervals until pollen is scattered. In a preferred embodiment of the invention, the spraying is repeated every 3 to 5 days. Those skilled in the art will readily recognize that the lac repressor / lac operonsi Other methods of restoring male fertility can be set up using a prokaryotic regulatory system, such as a stem or trp repressor / trp operon system.The invention is described generally for this purpose. It is believed that the present invention may be more readily understood by reference to the following examples, which are provided by way of illustration, and not by way of limitation, of the invention: Example 1: By high level constitutive expression of avidin Production of Male Sterile Transgenic Maize A method for producing transgenic corn plants is described in European Patent Application No. 0 442 174A1, and is hereby incorporated by reference. The method is briefly described below. A transgenic corn plant was created using PHI5168, a vector containing the avidin gene under the control of the ubiquitin promoter and also containing the PNINII terminator sequence. The structure of PHI5168 is shown in FIG. By treating the bar gene under the control of the dual 35S promoter, and simultaneously using the expression plasmid PHI610, which also contains the PINII terminator sequence, together with the construct with avidin, with the transformation mixture containing bialophos. , Transgenic plants were selected. The structure of PHI610 is shown in FIG. Two expression vectors, type II, according to the method of Green et al., "Molecular Genetics of Plants and Animals", edited by Downey et al., Academic Press, NY, 20, 147 (1983). Embryogenic suspension cultures derived from embryogenic cultures were transformed. Cultures were prepared as described in Murashige et al., Physio. Plant; 15 volumes; pp. 453-497; Murashige and Skoog ("MS") medium described in (1962) were supplemented with 2 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g / L of saccharose. Maintained. Suspension cultures were sieved to 710 microns seven days before the experiment and the filtrate was maintained in MS medium. Cells were collected from the suspension culture by suction filtration (Whatman No. 614) through a Buchner funnel and 100 ml (as fresh weight) of the cells were transferred to a 3.3 cm Petri dish. The cells were diffused into 0.5 mL of fresh culture medium to form a thin layer of cells. The uncovered Petri dishes are then transferred to Biolistics Inc. (Geneva, NY) was placed in the sample chamber of a gene gun device. Using a suction pump, the pressure in the chamber was reduced to 0.1 atm, preventing the particles from slowing down due to the friction of air. The cells were bombarded with tungsten particles having an average diameter of about 1.2 microns (GTE Sylvania Precision Materials Group, Towanda, Pennsylvania). By adding 5 μl of a DNA solution (1 μg DNA / 100λ) prepared in TE buffer of pH 7.7 to 25 μl of a suspension of tungsten fine particles at a concentration of 50 mg per 1 ml of distilled water in a 1.5 ml Eppendorf tube. An equal mixture of PHI5168 and PHI610 was loaded on the microparticles. The particles aggregated and settled in the tube. Cultures of the transformed plant cells containing the foreign gene were cultured in 560R (medium) (N6 based medium supplemented with 1 mg / ml bialaphos) for 4-8 weeks. This medium selects cells that express the bar gene. Thereafter, embryo formation was induced by culture for embryo formation, and the cells were germinated into plants. To germinate somatic embryos found on callus maintenance media, two lines of media were used. Calli were first transferred to a medium containing 5.0 mg / L indoleacetic acid (IAA) (maturation medium) and cultured for 10-14 days while the callus continued to grow. Calli were seeded at 50 mg / plate to optimize the recovery per unit volume of material. The callus was then transferred from the "mature" medium to a second medium with a lower IAA content (1 mg / L) compared to the first medium. At this point, the culture was transferred to a lighted location. A characteristic of the germinating somatic embryo was that green shoots elongated with connected root access. Somatic embryos were then transferred to medium in culture tubes (150 x 25 mm) and cultured for an additional 10-14 days. At this point, the plants were approximately 7-10 cm long and large enough to actively cold acclimate under conditions in the greenhouse. To acclimate the regenerated plants to low temperatures, the plants were removed from the sterile container, the solidified agar medium was washed off the roots and transferred to a growth chamber. A commercially available potting mix was placed in a growth chamber equipped with a humidifier that maintained a relative humidity of about 100% so that the roots of the plant were not excessively wetted, and the seedlings were transferred thereto. After 3-4 weeks in a humidified chamber, the plants became robust enough to be transplanted to field conditions. The male sterility of the plant put out in the field was observed and analyzed. Selected plants were then further analyzed by PCR and Southern blotting for the presence of the avidin gene, and avidin expression was analyzed by ELISA using standard methods. Of 94 plants analyzed by PCR, 53 were fertile and 41 were sterile. When the fertility of each plant was compared with the presence or absence of the avidin gene analyzed by PCR, a 98% correlation was observed between the presence of the gene and the sterility of the plant. Southern blotting was performed on five plants to analyze the presence of the avidin gene in detail. Analysis by Southern blot showed that three plants had the avidin gene. All three plants were sterile. Two plants that did not have the avidin gene were completely fertile. From these results, there was a 100% correlation between the presence and sterility of the avidin gene. Example 2: Use of an Agrobacterium strain having a binary vector containing a DNA sequence encoding avidin for the production of transgenic soybean plants of male sterility. A method for producing transgenic soybean plants is described in US patent application Ser. No. 07 / 920,409, which is hereby incorporated herein by reference. Soybean (Glycine max) seeds of Pioneer variety 9341 are exposed to chlorine gas generated in a bell-shaped glass vessel, and the surface is sterilized. The gas is generated by adding 3.5 ml of hydrochloric acid (34-37% w / w) to 100 ml of sodium hypochlorite (5.25% w / w). Exposure is for 16-20 hours in a container approximately 1 cubic foot in volume. Seeds whose surface has been sterilized are stored in a Petri dish at room temperature. 1/10 strength agar solidification medium from Gaulborg (0.32 gm / L minimum organics, Sigma Chemical Cat. No. G5893, 0.2% w / v saccharose, (3.0 mM) 2- (N-morpholino) ethanesulfonic acid (MES) In the B5 basic medium added, seeds were seeded on a plant growth regulator-free one, and at 28 ° C, about 20 ^-2 S ^-1 The cells are cultured with a white fluorescent lamp for 16 hours as an irradiation time, and germinated. After 3-4 days, prepare the seeds for co-culture. The seed coat is removed and the growing roots are removed 3-4 mm below the cotyledon. Agrobacterium soil bacterial strain LBA4404 with a modified binary plasmid containing the avidin gene is grown overnight, grown in minimal A medium containing 1 μg / ml tetracycline to logarithmic growth phase, pooled and pooled at 550 nm. Measure the absorbance. 1-2x10 in each centrifuge tube ^Ten Enough 10 to harvest cells ⁹ Transfer the cells / mL culture into a 15 mL conical centrifuge tube. Centrifuge at 6,000 xg for 10 minutes to pellet the cells. After centrifugation, the supernatant is decanted and the centrifuge tube is stored at room temperature for up to one hour until needed. Inoculation is performed in pairs so that the seeds on each plate can be treated with freshly suspended Agrobacterium pellets. B5 salts (G5893) 3.2 g / L, 2.0% w / v saccharose, 45 μm 6-benzylaminopurine (BAP), 0.5 μM indole butyrate (IBA), 100 μM acetosyringone, pH 5.5 at 10 mM MES Resuspend each bacterial pellet in 20 ml of the inoculation medium adjusted to. Vortex to resuspend. The inoculum is then poured into a petri dish containing the ready seeds and the cotyledonous nodes are damaged with a surgical razor. This is done by protecting the two cotyledons intact and bisecting the seed in the longitudinal direction through the shoot apex. From the bud top that has been split in two, each cotyledon is then dug out and cut off with a surgical razor. The cotyledons are then damaged with a surgical razor by repeatedly scoring them symmetrically along the axis. Be careful not to cut the implant completely through the shaft. After preparing the explant in about 5 minutes, inoculation is carried out for 30 minutes without rocking at room temperature in the presence of bacteria. After 30 minutes, the explants are transferred to plates of the same medium solidified with 0.2% w / v Gelrite (Merck & Company Inc.). Implant the implant at the same height as the surface of the culture medium, with the axial side facing up, about 20 μEm ^-2 S ^-1 Cultured at 22 ° C for 3 days under white fluorescent light. Three days later, contain 3.2 g / l B5 salts (G5893), 2% w / v saccharose, 5 μM BAP, 0.5 μM IBA, 200 μg / ml vancomycin, 500 μg / ml cefotaxime, and reach pH 5.7 with 3 mM MES. Transfer the explant to conditioned, liquid reverse selection medium. Each petri dish containing the graft is swirled gently continuously and washed for 4 days at room temperature. Change the reverse selection medium four times. Then, 3.2 g / l B5 salts (G5893), 2% w / v saccharose, 5.0 μM BAP, 0.5 μM IBA, 50 μg / ml kanamycin sulfate, 100 μg / ml vancomycin, 30 μg / ml cefotaxime, 30 μg / ml The explant is transferred to a selective medium containing Timentin, adjusted to pH 5.7 with 3 mM MES and solidified on agar. The selection medium is solidified with 0.3% w / v Seakem Agarose. Implants are implanted in media with the axial side down and at 28 ° C, 60-80 μEM ^-2 S ^-1 Culture for 16 hours under a white fluorescent lamp. After two weeks, the explants are again placed on a swing rocker and washed with liquid medium. Washing is performed overnight in a reverse selection medium containing 50 μg / ml kanamycin sulfate. The next day, transfer the explants to agar-solidified selection medium. They are embedded in the medium with the axial side down and cultured for a further 2 weeks. After culturing for one month in the selection medium, the transformed tissue can be observed as a green fan-shaped regenerated tissue against a background of decolorized and unhealthy tissue. The graft without the green sector is discarded, and the implant with the green sector is replaced with 3.2 g / l B5 salt (G5893), 2% w / v saccharose, 3.3 μM IBA, 1.7 μM gibberellic acid, 100 μg Transfer to elongation medium containing / ml vancomycin, 30 μg / ml cefotaxime, and 30 μg / ml timentin, adjusted to pH 5.7 with 3 mM MES. The elongation medium is solidified with 0.2% w / v Gelrite. Embed the green sector with the axial side up and incubate as before. Continue culturing in this medium, transferring to new plates every two weeks. When the shoots are 0.5 cm long, they are cut from the base and placed in rooting medium in 13 x 100 ml test tubes. The rooting medium contains 3.2 g / l B5 salts (G5893), 15 g / l saccharose, 20 μM nicotinic acid, 900 mg / l pyroglutamic acid (PGA) and 10 μM IBA. The root medium is adjusted to pH 5.7 with 3 mM MES and solidified with 0.2% w / v Gelrite. After 10 days, transfer the shoots to the same medium without IBA or PGA. Shoots will emerge and be stored in these tubes under the same environmental conditions as before. Once the rooting system is well established, the seedlings are transferred to sterile soil. The temperature, irradiation time and luminosity are kept the same as before. Avidin expression in transgenic soybean plants is confirmed and quantified using ELISA, and the presence of the gene is confirmed by PCR and Southern blotting. The stability of expression is evaluated in successive generations in the same way. It can be seen that sterility correlates with avidin expression in soybean. Example 3 Generation of Male Sterile Sunflower Plants by Expression of Avidin An expression cassette encoding avidin is used to generate transgenic sunflower plants and seeds. The DNA sequence encoding avidin is inserted into an expression cassette under the control of a ubiquitin promoter. This expression cassette is then subcloned into a binary vector such as PHI 5765 using an EcoRI site. Next, the binary vector is introduced into a helper strain of Acrobacterium soil bacteria. Sunflower plants were purchased from Bidney et al., Plant Mol. Bio .; 18; p.301; 1992, followed by microparticle bombardment followed by transformation with Agrobacterium strain LBA4404. Briefly, the seed coat is removed from the seeds of Pioneer Sunflower Line SMF-3 and the surface is sterilized. Seeds are placed on filter paper moistened with water and allowed to absorb moisture at 26 ° C. for 18 hours in the dark. Remove cotyledons and roots, and add 374 BGA medium (MS salts, Shephard vitamins, 40 mg / L adenine sulfate, 3% saccharose, 0.8% plant agar pH 5.6, 0.5 mg / L BAP, 0.25 mg / L IAA And 0.1 mg / L GA). Twenty-four hours later, the primary lobe was removed to expose the apical meristem, and the graft was placed 2 cm centered at 60 mm using a 20 mm Petri dish with aqueous agar, apical hemisphere up Put in a circle. The graft is bombed twice with tungsten particles suspended in TE buffer or particles to which an expression plasmid containing the avidin gene has been attached. The meristem explants are co-cultured on 374BGA medium under light irradiation at 26 ° C. for an additional 72 hours. Agrobacterium-treated meristems were co-cultured for 72 hours and then cultured in 374 medium containing 250 μg / ml cefotaxime (containing 1% sucrose, BAP, IAA or GA _Three (374BGA not containing). The seedlings are further cultured for 2 weeks at 26 ° C. under the conditions of 16 hours of sunshine, and are colored green or decolorized. The seedlings are transferred to a medium containing kanamycin and grown. The presence of avidin in the plant is confirmed and quantified as described in Example 2. It can be seen that the presence of male sterility correlates with the expression of avidin in the plant. While the preamble refers to particularly preferred embodiments, it should be understood that the invention is not limited thereto. It is believed that one of ordinary skill in the art can make various modifications to the embodiments disclosed herein, and such modifications are defined in the following claims. It is intended to be included within the scope of the present invention. All publications and patent applications mentioned herein are indicative of the level of skill of those in the art to which this invention belongs.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I L, IS, JP, KE, KG, KP, KR, KZ, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, TJ, TM, TR , TT, UA, UG, UZ, VN (72) Inventor Albertson Mark C.             United States Iowa West             Des Moines S. 60s Street 1930

Claims (1)

[Claims]   1. Avidin-encoding bases operably linked to plant promoter sequences An isolated DNA molecule comprising a sequence.   2. An expression vector comprising the isolated DNA molecule of claim 1.   3. 3. The method according to claim 2, wherein the plant promoter sequence is a constitutive promoter. Present.   4. 4. The constitutive promoter sequence is a ubiquitin promoter sequence. An isolated DNA molecule as described.   5. The heterologous nucleotide sequence increases the concentration of avidin in the tissue of the plant,       Increasing the avidin concentration causes male sterility of the plant,       A transgenic plant having a heterologous nucleotide sequence containing an avidin gene.   6. The transgenic plant according to claim 5, wherein the plant is a corn plant. .   7. 6. The transgenic plant according to claim 5, wherein the plant is a soybean plant.   8. 6. The transgenic plant according to claim 5, wherein the plant is a sunflower plant.   9. The method according to claim 5, wherein the heterologous nucleotide sequence further comprises a constitutive promoter sequence. Lancegenic plants.   Ten. 10.The constitutive promoter sequence is a ubiquitin promoter sequence. The transgenic plant as described.   11． The promoter controls the expression of the avidin gene, and thereby the avidin gene Promoter and avidin genes cause male sterility by expression of gene Transgenic male, comprising introducing an expression vector containing A method of producing a sterile plant.   12． 12. The method of claim 11, further comprising regenerating the transgenic plant from the plant cell. The described method.   13. 12. The method of claim 11, wherein said promoter comprises a ubiquitin promoter.   14. The hybrid plant expresses a second foreign gene, and wherein the second foreign gene product is Suppresses avidin expression in plants, thereby producing male fertile hybrid plants The   To produce a male fertile hybrid plant using the avidin gene, including the following steps Law:     (a) The DNA of claim 1, wherein the expression of avidin causes male sterility. Creating a first parental male sterile plant containing the molecule;     (b) creating a second transgenic parent plant that expresses a second foreign gene Doing; and     (c) cross-fertilizing the first parent with the second parent to produce a hybrid plant Stage.   15． The second foreign gene is an antisense gene, a ribozyme gene, and a foreign gene. 15. The method according to claim 14, which is selected from the group consisting of a partial guide sequence gene.   16． 16. The method of claim 15, wherein the antisense gene comprises a sequence of avidin mRNA.   17． 17. The method according to claim 16, wherein the ribozyme gene comprises a sequence of avidin mRNA.   18． 17. The method of claim 16, wherein the external guide sequence gene comprises the sequence of avidin mRNA. .   19. A LexA operet in which a DNA molecule of a first parent plant is operably linked to a promoter. The second foreign gene is a LexA repressor gene. Item 14. The method according to Item 14.   20. The promoter sequence is selected from the group consisting of the following items. 3. The expression vector according to claim 2, which is an anther-specific promoter sequence containing auxin.     (a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1;     (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2;     (c) a DNA molecule having the base sequence of SEQ ID NO: 3; and     (d) A functional fragment of (a), (b) or (c).   twenty one. The answer box has the base sequence of SEQ ID NO: 1, and contains an anther-specific promoter. Further comprises a core promoter selected from the group consisting of: The expression vector according to item 20,     (a) CaMV 35S core promoter;     (b) a DNA molecule having the base sequence of SEQ ID NO: 4;     (c) a DNA molecule having the base sequence of SEQ ID NO: 5;     (d) a DNA molecule having the base sequence of SEQ ID NO: 6;     (e) A functional fragment of (b), (c) or (d).   twenty two. The answer box has the base sequence of SEQ ID NO: 2 and is an anther-specific promoter. Further comprises a core promoter selected from the group consisting of: The expression vector according to item 20,     (a) CaMV 35S core promoter;     (b) a DNA molecule having the base sequence of SEQ ID NO: 4;     (c) a DNA molecule having the base sequence of SEQ ID NO: 5;     (d) a DNA molecule having the base sequence of SEQ ID NO: 6;     (e) A functional fragment of (b), (c) or (d).   twenty three. The answer box has the base sequence of SEQ ID NO: 6, and contains an anther-specific promoter. Further comprises a core promoter selected from the group consisting of: The expression vector according to item 20,     (a) CaMV 35S core promoter;     (b) a DNA molecule having the base sequence of SEQ ID NO: 4;     (c) a DNA molecule having the base sequence of SEQ ID NO: 5;     (d) a DNA molecule having the base sequence of SEQ ID NO: 6;     (e) A functional fragment of (b), (c) or (d).   twenty four. Male by expression of avidin, including spraying biotin solution on plants A method for restoring fertility in a plant that has become sterile.   twenty five. 12. The method of claim 11, wherein the promoter comprises an inducible promoter.   26. The antisense gene is operably linked to an inducible promoter. The method according to 15.