MXPA97003741A - Specific regulator element for microespo - Google Patents

Abstract

A novel DNA regulatory element conferring gene expression specific to microspores has been discovered, isolated and characterized. The specific regulatory element for microspores can be used to control the expression of a foreign gene that dissociates the microspore function. Therefore, pulley production control can be achieved by using the specific microspore regulatory element to produce male sterile plants. Various methods can be used to restore male fertility in the F1 generation of said male sterile plants. In addition, specific regulatory element for microspores can be used to confer resistance to viral and insect pests

Description

SPECIFIC REGULATOR ELEMENT FOR MICROSPORES BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel regulatory element that confers microspore specificity for gene expression. In particular, this invention is directed to a specific regulatory element for microspore of the Brassica napus Bnm1 gene, and to the use of said regulatory element to produce transgenic male sterile plants. This invention is also directed to a method for restoring male fertility in the progeny of male sterile plants. In addition, the present invention relates to the use of a specific regulatory element for microspores to protect it against viral and insect pests. 2. Background Pollen fertility control is essential in the production of hybrid crops. Traditional methods for regulating pollen fertility include manual mutilation of plants that are to be used as the female mother and the application of chemical compositions. According to the latter method, hybrid seeds are produced by cross-fertilization of female plants treated chemically with pollen from untreated plants. However, both approaches are hard work. In addition, it is preferred to avoid the introduction of toxic chemicals into the environment.

Another approach to fertility control is based on the use of a cytoplasmic gene for male sterility. The problem with this approach is that the expression of certain cytoplasmic male sterility genes is accompanied by an increased susceptibility to fungal pathogens. For example, the extensive use of cmsT cytotype in corn leads to an epiphytic outbreak of southern corn leaf plague in the early 1970s. Although the additional cytoplasmic male sterility cytotypes have become available, their use did not spread due to concern on possible susceptibility to pathogens. The ability to produce hybrid lines has particular economic importance for oily seed crops. For example, Brassica napus F ~ ¡hybrid lines normally produce yields that are greater than 20 to 70%, compared to established lines. Thompson, Adv. Appl. Biol. 7: 1 (1983); Johnston, Euphytica 20: 81 j (1971). The most economical and flexible approach to controlling fertility in Brassica uses a system of self-incompatibility that occurs in nature in Brassica species. Gowers, Euphytica 24: 537 (1975). However, the self-incompatibility system is not reliably effective under certain environmental conditions, such as at elevated temperatures. Therefore, there is a need for a method to control pollen production without relying on male sterility that occurs in nature or self-incompatible genes, or traditional manual and chemical methods. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for producing sterile male plants using an expression vector that disrupts the function of microspores. It is a further objective of this invention to provide a DNA regulatory element that confers expression of specific genes for microspores. These and other objects are achieved, according to one embodiment of the present invention, by the provision of an isolated DNA molecule, wherein the DNA molecule comprises a nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 8, (b) a nucleotide sequence having substantial sequence similarity to SEQ ID NO: 8, and (c) a functional fragment of (a) or (b), wherein the DNA molecule is a specific regulator element for microspheres. The present invention also contemplates expression vectors comprising a regulatory element specific for microspores. said expression vectors may further comprise a promoter, wherein the function of the promoter is under the control of a regulatory element specific for microspores.Examples of suitable promoters include a Bnm1 promoter, an anther-specific promoter and the CaMV 35S core promoter.

The present invention is also directed to an expression vector that further comprises a foreign gene that is operably linked to a promoter, wherein the foreign gene product disrupts the microspore function. The present invention further contemplates a method for using said expression vectors to produce a male sterile plant, comprising the step of introducing an expression vector into cells of embryogenic plants, wherein the foreign gene is selected from the group consisting of a structural gene. , a contrasense gene, a ribozyme gene and an external guide sequence gene. For example, an expression vector can be introduced into embryogenic plant cells of Brassica nupus. Suitable structural genes encode a protein selected from the group consisting of diphtheria toxin, scab antiviral protein, Aspergillus oryzae ANase-T1, barnase, and the product of the roIB gene. A suitable product for a sense gene is selected from the group consisting of actin counter-sense RNA, tubulin counter-sense RNA, ubiquitin counter-sense RNA, ubiquitin-conjugated enzyme-contrasts, ubiquitin-bearing protein counter-sense RNA. , Elongation factor counter-sense RNA and chalcone synthase counter-sense RNA. In addition, the ribozyme genes or outer leader sequence genes may comprise nucleotide sequences selected from the group consisting of actin nucleotide sequences, tubulin nucleotide sequences, ubiquitin nucleotide sequences, nucleotide sequences of enzyme conjugated ubiquitin , ubiquitin vehicle protein nucleotide sequences, elongation factor nucleotide sequences and chalcone synthase nucleotide sequences. The present invention is also directed to a method for producing a male sterile plant, comprising: (a) constructing an expression vector comprising a microspore-specific regulatory element, a promoter, and a foreign gene, wherein the specific regulatory element for microspore comprises a nucleotide sequence selected from the group consisting of (i) SEQ ID NO: 8, (ii) a nucleotide sequence having substantial sequence similarity to SEQ ID NO: 8, and (iii) fragments of (i) or (ii), wherein the specific regulatory element for microspore together with the promoter controls the expression of the foreign gene and wherein the foreign gene product disrupts the microspore function, thereby producing a sterile male plant. Said method may further comprise the step of (b) introducing the expression vector into the cells of embryogenic plants.

The present invention further contemplates a method for using a microspore-specific regulatory element to produce a male fertile hybrid plant, comprising: (a) producing a first male sterile male plant, comprising an expression vector comprising a regulatory element specific for microspores , a promoter and a first foreign gene, wherein the specific regulatory element for microspores together with the promoter controls the expression of the first foreign gene and wherein the product of the first foreign gene interrupts the microspore function; (b) producing a second parent plant comprising an expression vector comprising the regulatory element specific for microspores, a promoter and a second foreign gene, wherein the specific regulatory element for microspores together with the promoter controls the expression of the second foreign gene; and (c) cross-fertilizing the first mother with the second mother to produce a hybrid plant, wherein the microspores of the hybrid plant express the second foreign gene, wherein the product of the second foreign gene prevents the interruption of the function of microspores by the product of the first foreign gene thus producing a male fertile hybrid plant.

For example, the first foreign gene can encode barnase and the second foreign gene can encode a barnase inhibitor.

Alternatively, the product of the first foreign gene may be diphtheria toxin and the product of the second foreign gene may be diphtheria toxin ribozyme. The present invention is also directed to a method of restoring fertility of a male sterile hybrid plant, which comprises treating the male sterile hybrid plant with a flavonol aglycone, wherein the male sterile plant comprises an expression vector consisting of (i) a regulatory element specific for microspores, (ii) a promoter, and (iii) a foreign gene, wherein the regulatory element specific for micospores together with the promoter, controls the expression of the foreign gene and where the foreign gene expresses RNA of contradictory of chalcone synthase thus producing defective flavonol microspores. Kaempferol is an example of suitable flavonol aglycone. The present invention is further directed to a method for producing transgenic plants resistant to the disease caused by virus or insect, comprising constructing an expression vector comprising a regulatory element specific for microspores, a promoter, and a foreign gene, wherein the element Specific regulator for microspores comprises a sequence of nucleotides selected from the group consisting of (i) Sec ID NO: 8, (ii) a nucleotide sequence having substantial sequence similarity to SEQ ID NO: 8, and (iii) fragments of (i) or (II), wherein the specific regulatory element for microspores together with the promoter controls the expression of the foreign gene and wherein the foreign gene product disrupts the function of the virus or encodes an insecticidal toxin, thus conferring resistance to diseases . The product of the foreign virus breaking gene can be selected from the group consisting of viral coat protein, 2'-5 'oligoadenylate synthetase, viral genome antisense RNA, and antiviral protein of a grana, while a suitable insecticidal toxin it is an endotoxin of Bacillus thuringiensis. BRIEF DESCRIPTION OF THE INVENTION Figure 1 presents the nucleotide sequence [SEQ ID NO: 1] of Bnm1 cDNA with its corresponding amino acid sequence [SEQ ID NO: 2]. The ATG start codon, the TGA stop codon and a putative polyadenylation signal sequence are underlined with two lines. Figure 2 presents the nucleotide sequence [SEQ ID NO: 5] of the Bnm1 genomic clone including the promoter, coding region and a single intron (in italics). The underlined region is identical to the Bnm1 cDNA sequence. The ATG start codon, the TGA stop codon and a putative polyadenylation signal sequence are indicated by asterisks. Figure 3 presents the nucleotide sequence [SEQ ID NO: 8] of the amplified PCR promoter fragment used to clone the Bnm1 promoter. The ATG start codon is present within the Ncol restriction site. Figure 4 presents a map of the DP5476 vector containing the Brassica napus microspore promoter (Msp Prom) driving the GUS-PINII gene terminator cassette grafted to the sSafl-EcoRI sites of pBlueScript®ll SK +. the vector also contains the ampicillin resistance gene (AmpR) as a selectable marker for E. coli transformations. Figure 5 presents a map of DP5477 containing the Brassica napus microspora promoter (MSp Prom), granting the terminator cassette of the gene-GUS-PINII inserted in the Sa1l-EcoRI sites of the binary vector DP1741. DP5477 also contains the CaMV 35S promoter directing the neomycin phosphotransferase (MPTII) gene which is used as a selection marker. The left and right T-DNA borders are labeled LB and RB, respectively. DETAILED DESCRIPTION 1. Definitions. In the following description, a number of terms are extensively used. The following definitions are provided to facilitate the understanding of the invention. A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide. A promoter is a DNA sequence that directs the transcription of a structural gene. Usually, a promoter is located in the 5 'region of a gene, near the transcription start site of a structural gene. If a promoter is a promoter that can be induced, then the transcription regime is increased in response to an inducing agent. In contrast, the transcription regime is not regulated by an induction agent if the promoter is a constitutive promoter. A core promoter contains essential nucleotide sequences for the promoter function, including the TATA box and the start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may increase activity or confer tissue-specific activity. For example, the core promoter of Cauliflower Mosaic Virus (CaMV) 35S, consists of approximately 33 nucleotides 5'-posterior of the start site of transcription of the 35S genome. A specific regulatory element for microspores is a DNA sequence that directs a higher level of transcription of an associated gene in microspores than in any or all other tissues of a plant. For example, the Bnm \ gene, described herein, is expressed in microspores during the binucleate and trinucleate stages of development. The specific regulatory element for microspums of the Bnm? it can direct the expression of a foreign gene in microspores, but not in the wall, pistil, or anther suede tissues. An isolated DNA molecule is a fragment of DNA that does not integrate into the genomic DNA of an organism. For example, the microspore-specific regulatory element of the Bnnt \ gene is a fragment of DNA that has been separated from the genomic DNA of Brassica napus. Another example of the isolated DNA molecule is a chemically synthesized DNA molecule that is not integrated into the genomic DNA of an organism. An enhancer is a DNA regulatory element that can increase the efficiency of transcription, without taking into account the distance or orientation of the enhancer in relation to the transcription start site. Complementary DNA (cDNA) is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a complementary primer for the mRNA portions is used to initiate reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of said single-stranded? DN 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, the expression involves the transcription of the structural gene into mRNA and the 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. Collation vectors usually contain one or a small number of restriction endonuclease recognition sites in which the foreign DNA sequences can be inserted in a determinable manner without losing an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the collation vector. Marker genes usually include genes that provide teracycline resistance or resistance to ampicillin. An expression vector is a DNA molecule that comprises a gene that is expressed in a host cell. Normally, the expression of the gene is placed under the control of certain regulatory elements including constitutive or inducing promoters, regulatory elements specific for tissue and enhancers. Such a gene is such that it is operably linked to the regulatory elements.A foreign gene in the present disclosure refers to a DNA sequence that is operably linked to at least one regulatory heterologous element.For example, any gene other than the structural gene of Bnnv is considered a foreign gene if the expression of that gene is controlled by a regulatory element specific for microsomes of the Bnm gene A recombinant host can be any prokaryotic or eukaryotic cell that contains either a collation vector or an expression vector This term also includes prokaryotic or eukaryotic cells that have been genetically treated to contain the gene (s) cloned in the chromosome or genome of the host cell.A transgenic plant is a plant that has one or more plant cells containing an expression vector In eukaryotes, RNA polymerase II catalyses the transcription of a structural gene to produce mRNA A DNA molecule can be designed to contain a RNA polymerase template li in which the RNA transcript has a sequence that is complementary to that of a specific mRNA. RNA transcription is called a nonsense RNA and a DNA sequence that encodes a nonsense RNA is called a nonsense gene. The antisense RNA molecules are capable of binding to the mRNA molecules, resulting in an inhibition of mRNA translation. A ribozoma is an RNA molecule that contains a catalytic center. The term includes RNA enzymes, self-dividing RNA, and self-separating RNAs. A DNA sequence that encodes a ribozyme is called a ribozyme gene. An external leader sequence is an RNA molecule that targets the endogenous ribozin, RNase P, for a particular species in intracellular mRNA, resulting in separation of the mRNA by RNase P. An ADn sequence encoding a leader sequence External is called an external guide sequence gene. Two nucleic acid molecules are considered to have a substantial sequence similarity if their nucleotide sequences share a similarity of at least 65%. The sequence similarity determinations can be carried out, for example, using the FASTA program (Genetics Computer Group; Madison, Wl). Alternatively, sequence similarity determinations can be performed using the BLASTP (Basic Local Alignment Search Tool) of the Experimental GENIFO (R) BLAST Network Service. See Altschul et al., J. Mo !. Biol. 215: 403 (1990). Also, see Pasternak et al., "Sequence Similarity Searches, Multiple Sequence Alignments, and Molecular Tree Building," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al., (Eds.), Pages 251-267 (CRC Press 1993). 2. General Review Specific genes for microspores are known in the art. See, for example, Wing and others, Plant Molec. Biol. 14: 17 (1989), Albani et al., Plant Molec. Biol. 15: 605 (1990), Guerrero et al., Mol. Gen. Genet. 224: 161 (1990), Twell et al., Development 109: 705 (1990), van Tunen et al., The Plant Cell 2: 393 (1990J, Albani et al., Plant Molec. Biol. 16: 501 (1991), Twell. and others, Genes &; Development 5: 496 (1991), and Albani et al., The Plant Journal 2: 331 (1992). According to the scheme of Mascarenhas, Ann. Rev. Plant Physiol. Plant Mol. Biol. 41: 317 (190), genes expressed during micro-porpogenesis can be characterized as "early" or "late" genes. Transcripts of early genes are detected first after meiosis and are reduced or not detected in mature pollen. In contrast, the expression of late genes starts around the time of mitosis of the microspores and continues as the pollen matures. In addition, it is possible that particular genes expressed during microsporogenesis can not be classified as early genes or late genes. Under the Mascarenhas classification scheme, the Bnm1 gene described herein is an early gene. Therefore, the regulatory element of the Bnm1 gene is functionally distinct from the regular elements of late genes such as the corn Zm13 gene (Guerrero et al., Supra), the tobacco NTP303 gene (Weterings et al., Plant Mol. Biol. 18: 1101 (1992)), the LAT51 tomato gene (McCormick, Trends Genet 7: 298 (1991)), the flavonone isomerase gene from petunia chalcone (chiA PA2 promoter, van Tunen et al., The Plant Cell 2: 393 (1990)), and the Bcp1 Brassica campestris gene (Theerakulpisut et al., The Plant Cell 3: 1073 (1991)). Functional differences can also be found within the group of early genes by comparing patterns of mRNA synthesis. For example, the LAT52 gene is expressed in the endosperm, LAT59 is expressed in the endosperm and roots, while LAT56 is expressed in roots. Twell et al., Mol. Gen. Genet. 217: 240 (1989); Wing and others, supra; McCormick, The Plant Cell 5: 1265 (1993). In contrast, the Bnm1 gene is expressed neither in roots nor in seeds of development. See Example 2. In addition, Northern analyzes indicate that the expression level of Bnm genes is higher than the expression level of Bp4, Bp19 and Bp19 genes of Brassicca napus. Albani et al. (1990, 1991, 1992), supra. See Example 2. This observation suggests that the Bnm'l promoter is stronger than the promoters of the Bp4, Bp19 and Bp10 genes. In addition, variations in the function of early gene regulatory elements can be revealed by the transformation studies. For example, the pollen-specific regulatory element of the SLG13 gene from Brassica olerácea induces the expression of genes in the pollen of transgenic plants mainly in the binucleate stage of development. Thorsness and others Develop. Biol. 143: 173 (1991); Dzelzkalns et al., The Plant Cell 5: 855 (1993). In addition, the regulatory element of S G13 does not induce the expression of the gene in the cap of transgenic plants. Thorsness and others, supra. In contrast, the regulatory element of Bnm induces the expression of genes in the microspores of transgenic plants starting in the uninucleate stage of development, as well as in cells of the tapeto. See Example 5. Therefore, Bnm1 can be distinguished on a functional basis from other early genes. In addition to this functional distinction, a comparison of nucleotide sequences in several databases did not reveal a nucleotide sequence that was equivalent or similar to the nucleotide sequence of the Bnm1 microspore-specific regulatory element shown in Figure 3 [SEQ ID NO: 8]. The absence of a coupling agrees with the fact that it has not been possible to develop generally accepted principles or structural criteria to recognize DNA sequences that confer specificity for microspores. This is particularly true for regulatory elements of specific genes for Brassica microspores. consequently, a specific regulatory element for Brassica microspores can not be isolated from a genomic bank by screening a consensus sequence that confers the expression of specific genes for microspores. Therefore, the novel microspore-specific regulatory element of the present invention was obtained by isolating cDNA molecules that encode genes specific for microspores and then using the cDNAs as probes to identify corresponding genes in a suitable genomic library. 3. Isolation of a Regulatory Element of a Gene Specific for Microspores A. Isolation of cDNA Molecules that Encode Genes Specific Microspore The first step in the construction of a cDNA library containing specific genes for microspores is to isolate total RNA from microspores. Preferably, the RNA is isolated from late uninucleated / binucleated early microspheres that have been cultured for four days to induce embryogenesis. More preferably, said microspores are obtained from Brassica napus.

Total RNA can be prepared from microspores using techniques well known to those skilled in the art. In general, RNA isolation techniques should provide a method for disrupting plant cells, a means of inhibiting RNA-directed RNase degradation, and a method for separating RNA from DNA, protein, and polysaccharide contaminants. For example, total RNA can be isolated from microspores by freezing the tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the microspores, removing the ground tissue with a phenol / chloroform solution to remove proteins, and removing RNA. of the remaining impurities by selective precipitation with lithium chloride. See, for example, Ausubel et al., (Eds.), CURRENT PROTOCOLS IN MOLCULAR BIOLOGY, pages 4.3.1-4.3.4 (Wiley Interscience 990) ["Ausubel"]. Also, see Sharrock et al., Genes and Development 3: 1745 (1989). Alternatively, the total RNA can be isolated from the microspores by extracting ground tissue with guanidinium isothiocyanate, extracting it with organic solvents and separating RNA from contaminants using differential centrifugation. See, for example, Strommer et al., "Isolation and characterization of Plant mRNA", in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (Eds.) Pages 49-65 (CRC Press 1993). In order to construct a cDNA library, poly (A) + RNA must be isolated from a total RNA preparation. Poly (A) + RNA can be isolated from total RNA using the normal oligo (dT) -cellulose chromatography technique. See, for example, Strommer and others, supra. Two-stranded cDNA molecules are synthesized from poly (A) + RNA using bine techniques known to those skilled in the art. See, for example, Ausubel on pages 5.5.2-5.6.8. In addition, commercially available kits can be used to synthesize double-stranded cDNA molecules. For example, such equipment is available from GIBCO / BRL (Gaithersburg, MD), Clontech Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and Stratagene® Cloning Systems (La Jolla, CA).

Various collation vectors are suitable for the construction of a microspore cDNA side. For example, a cDNA library can be prepared in a vector derived from bacteriophage, such as a vector gtlO. Huynh et al., "Constructing and Screening cDNA Libraries in? Gt10 and? Gt11", in DNA Cloning: A Practical Approach, vol. I, Glover (ed.), Pages 49-78 (IRL Press, 1985). Alternatively, the double-stranded cDNA molecules can be inserted into a plasmid vector, such as a pBluescript® vector (Stratagene® Cloning Systems; La Jolla, CA), LambdaGEM®-4 (Promega Corp.) or other vectors commercially. available. Suitable collation vectors can also be obtained from the American Type Culture Collection (Rockville, MD). In order to amplify the cloned cDNA molecules, the cDNA library is inserted into a prokaryotic host, using normal techniques. For example, the microspore cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained from GIBCO / BRL (Gaithersburg, MD). Differential screening can be used to isolate cDNA clones that encode specific microspore genes from the cDNA library. For example, single-stranded cDNA probes can be synthesized in the presence of radioactive nucleotides poly (A) + RNA isolated from microspores that have been treated to induce embryogenesis ("Mi-RNA") or from microspores that have been treated to eliminate the embryogenic response ("Mx-RNA"). The cDNA clones that hybridize with cDNA probes synthesized from Mi-RNA, but not with Mx-cDNA probes, are isolated for further analysis. The differential screening technique is well known to those skilled in the art. See, for example, Sargent, "Isolation of Differentially Expressed Genes," in GUIDE TO MOLECULAR CLONING TECHNIQUES, Berger et al., (Eds.), Pages 423-432 (Academic Press, 1987); Teder et al., Proc. Nati Acad. Sci. USA 85: 208 (1988). Example 1 illustrates the use of this technique. The basic approach for obtaining microspore-specific cDNA clones can be modified by constructing a subtracted cDNA library that is enriched in microspore-specific cDNA clones. Hedrick et al., Nature 308: 149 (1984). For example, the double-stranded cDNA with EcoRI can be prepared from Mi-RNA and mixed with a 50-fold excess of small blunt-ended cDNA fragments prepared from Mx-RNA. The cDNA mixture is heated to fuse double-stranded cDNA, single-stranded cDNA molecules are allowed to hybridize and double-stranded cDNA molecules are inserted into the EcoRI site of a collation vector. Under these conditions, the Mi-cDNA molecules alone that tend to regenerate double-stranded fragments with an EcoRI site at each end are the cDNAs lacking complementary fragments in the Mx-cDNA. Ausubel on pages 5.8.9.-5.8.15. The cDNA clones can be analyzed using a variety of techniques such as Northern analysis, Southern analysis, restriction mapping and sequence analysis, as illustrated in Examples 1-3. B. Isolation of Specific Genes for Microspores of a Genomic Bank. The methodology, described above, can be used to isolate cDNA clones that encode proteins that are expressed in microspore tissue. A microspore-specific cDNA clone, such as the Bnm1 cDNA clone, can be used to dissociate the microspore function in non-sense constructs, following the general approaches described below. In addition, said cDNA clones can be used as probes to isolate the corresponding genes from a genomic library. A genomic DNA strand of plant can be prepared by means well known in the art. See, for example, Slightom and others "Construction of? Clone Banks," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al., (Eds.), Pages 121-146 (CRC Press, 1993). A preferred source of plant genomic DNA is Brassica napus DNA. A genomic DNA can be isolated from Brassica napus tissue, for example, by using plant tissue with the Sarkosil detergent, digesting the lysate with proteinase K, rinsing insoluble wastes from the lysate by centrifugation, precipitating lysate nucleic acid using isopropanol, and purifying DNA. resuspended in a cesium chloride density gradient. Ausubel on pages 5.3.2-5.4.4, and Slightom et al., Supra. Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, according to conventional techniques, such as restriction enzyme digestion to provide appropriate terminations, the use of alkaline phosphatase treatment to avoid binding undesirable DNA molecules and ligation with appropriate ligases. Techniques for such manipulation are described by Slightom et al., Supra, and are well known in the art. Also see Ausubel on pages 3.0.5-3.17.5. Alternatively, a plant genomic bank can be obtained from a commercial source such as Clontech Laboratories, Inc. (Palo Alto, CA) or Stratagene® Cloning Systems (La Jolla, CA). A library containing genomic clones is screened with one or more cDNA clones specific for microspores using standard techniques. See, for example, Ausubel on pages 6.0.3-6.6.1 .; Slightom et al., Supra; Raleigh et al., Genetics 122: 279 (1989). Example 4 presents the method that was used to screen a genomic bank of Brassica napus. C. Identification of a Microspore-Specific Regulatory Element Genomic clones can be analyzed using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis and DNA sequence analysis. The Initiator extension analysis or nuclease protection analysis S1, for example, can be used to locate the putative transcription start site of the cloned gene. Ausubel on pages 4.8.1-4.8.5; Walmsley and another, "Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protectin Assay". in METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray (de.), Pages 271-281 (Humana Press Inc. 1991). Example 4 illustrates the use of Southern analysis, restriction mapping and sequence analysis to characterize the Borni gene. The structural analysis by itself can not lead to the identification of a specific regulatory element for microspores associated with the cloned Bnm1 gene because a model for the microsepore-specific regulatory sequences of Brassica napus has not yet been developed. Therefore, the regulatory element must be identified using functional analysis. The general approach of said functional analysis involves subcloning fragments of the genomic clone into an expression vector containing a reporter gene, introducing the expression vector into various plant tissues and analyzing the tissue to detect the temporal expression of the report gene. The presence of a specific regulatory element for microspores in the genomic subclone is verified by observing the expression of the reporter gene in microspore tissue and the absence of expression of the reporter gene in tissue without microspores, such as pistil tissues or sepal. Methods for generating fragments of a genomic clone are well known. Preferably, enzymatic digestion is used to form nested deletions of genomic DNA fragments. See, for example, Ausubel on pages 7.2.1-7.2.20; And others, "Techniques for Isolation and Characterization Transcription Promoters, Enhancers, and Terminator" in METHOS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (Eds.) Pages 155-166 (CRC Press 1993). As an example, the possibility that the regulatory element resides "upstream" or "downstream" of the transcriptional starting site can be tested by subcloning a DNA fragment containing the upstream region, digesting the DNA fragment in any direction. 'a 3' or in the 3 'to 5' direction to produce nested deletions and subcloning the small fragments into expression vectors for temporal expression.The selection of an appropriate expression vector will depend on the method for introducing the expression vector into cells Host: Normally an expression vector contains: (1) Prokaryotic DNA elements that code for a bacterial origin of replication and an antibiotic resistance marker to provide growth and selection of the expression vector in the bacterial host; (2) elements of eukaryotic DNAs that control the initiation of transcription, such as a promoter; (3) DNA elements that it controls n the processing of transcripts, such as a termination / polyadenylation sequence; and (4) a report gene that is operably linked to the DNA elements that control the initiation of transcription. Useful report genes include β-glucuronidase, β-galactosidase, chloramphenicol acetyl transferase, luciferase and the like. Preferably, the reporter gene is β-glucuronidase (GUS) gene or the luciferase gene. General descriptions of expression vectors and reporter plants and genes can be found in Gruber et al., "Vectors for Plant Transformation", in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds.) pages 89-19 (CRC Press 1993). In addition, GUS expression vectors and GUS gene cassettes are available from Clontech Laboratories, Inc. (Palo Alto, CA), while luciferase expression vectors and luciferase gene cassettes are available from Promega Corporation (Madison, Wl). Expression vectors containing genomic test fragments can be introduced into protoplasts or into intact tissues or isolated cells. Preferably, the expression vectors are introduced into intact tissues. General methods for growing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al., (Eds.), Pages 67-88. (CRC Press, 1993).

Methods for introducing expression vectors into plant tissue include direct infection or co-cultivation of tissue from plants with Agrobacterium tumefaciens. Horsch et al., Science 227/1229 (1985). Descriptions of the Agrobacterium vector systems and methods for gene transfer mediated by Agrobacterium are provided by Gruber et al., Supra, Miki et al., Supra, and Moloney et al., Plant Cell Reports 8: 238 (1989). Alternatively, expression vectors are introduced into plant tissues using a direct gene transfer method such as microporyectil-mediated delivery, DNA injection, electroporation and the like. See, for example, Gruber et al., Supra; Miki and others, supra; Klein et al., Biotechnology 10: 268 (1992). For example, expression vectors can be introduced into plant tissues using microprojectile-mediated delivery with a biolistic device. Transformation studies have been used to identify DNA sequences that regulate gene expression in a specific manner for microspores. In particular, it has been found that a specific regulatory element for microspores resides within a DNA fragment with 280 base pairs that is located immediately upstream of the translational Bnm1 starting site. The nucleotide sequence of a regulatory element specific for microspores is provided in Figure 3 [SEQ ID NO: 8]. Therefore, the present invention encompasses a DNA molecule having a nucleotide sequence of SEQ ID NO: 8 and having the function of a regulatory element specific for microspores. Variants of the specific regulatory element for microspores can be produced by deleting, adding and / or substituting nucleotides for the nucleotides mentioned in SEQ ID NO: 8. Said variants can be obtained, for scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. Ausubel on pages 8.0.3-8.5.9. Also see generally, McPherson (de.), DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991). Therefore, the present invention also encompasses DNA molecules comprising nucleotide sequences that have substantial sequence similarity to SEQ ID NO: 8 and function as a specific promoter for microspores. In addition, suppression analyzes can be carried out to further locate one or more specific microspore regulatory elements with the specific microspore regulatory element of 820 base pairs. Therefore, the present invention also encompasses "functional fragments" of SEQ ID NO: 8 that can be used as specific regulatory elements for microspores 4. Use of a Specific Regulatory Element for Microspores to Control Pollen Production A. Plant Production Male sterile One object of the present invention is to provide a means to control the production of pollen using a specific regulatory element for microspores In particular, the present invention encompasses the production of male sterile plants using Bnmγ regulatory sequences to stimulate the expression of A strange gene in microspores The specific regulator element for Bnm1 microspores to stimulate the expression of a foreign gene in microspores The specific regulatory element for Bnm1 microspores induces the expression of genes of the technique immediately before the binucleate step through the trinucleate stage of my development Since the expression is confined to a limited tissue and stage, the use of the regulatory element of Bnm1 to induce male sterility makes obvious the regulatory aspects about the accumulation of a product of foreign genes in other different tissues, including edible plants. A general approach to induce male sterility is to construct an expression vector in which the microspore-specific regulatory element is operably linked to a nucleotide sequence that encodes a protein capable of dissociating the microspore function. proteins capable of dissociating the function of microspores include proteins that inhibit the synthesis of macromolecules that are essential for cellular function, enzymes that degrade the macromolecules that are essential for cellular function, proteins that alter the biosynthesis or metabolism of plant hormones and proteins that inhibit a specific function of microspores. For example, an expression vector can be constructed in which the regulatory element specific for microspores is operably linked to a nucleotide sequence which codes for a protein synthesis inhibitor. For example, diphtheria toxin is a well-known inhibitor of protein synthesis in eukaryotes. DNA molecules encoding the diphtheria toxin gene can be obtained from American Type Culture Collection (Rockville, MD), ATCC No. 39359 or ATCC No. 67011. As discussed below, the antiviral protein of grana is another inhibitor. adequate protein synthesis. Alternatively, disruption of the microspore function can be achieved using DNA sequences encoding enzymes capable of degrading a biologically important macromolecule. For example, Mariani et al., Nature 347: 737 (1990), have shown that expression in the TNase-T1 mat of Aspergillus oryzae or an RNase of Bacillus amyloliquefaciens, designated "barnase", induced the destruction of tapeto cells, resulting in male infertility. Therefore, the microspore function can be dissociated using an expression vector that contains a microspore-specific regulatory element that is operably linked to a barnase gene. Other suitable enzymes include DNases, proteases and lipases.

The genes encoding said microspore disruption enzymes can be obtained by chemical synthesis using published nucleotide sequences. The genes of RNase-T1 and barnase can be obtained, for example, by synthesizing the genes with mutually initiating long oligonucleotides. See, for example Wosnick et al., Gene 60; 115 (1987). In addition, current techniques using the polymerase chain reaction provide the ability to synthesize genes as long as 1.8 kilobases in length. Adang et al., Plant Molec. Biol. 21: 1131 (1993); Bambot and others; PCR Methods and Applications 2: 266 (1993). Quaas et al., Eur. J. Biochem. 773: 617 (1988), describes the chemical synthesis of RNase-T1, whereas the nucleotide sequence of the barnase gene is described in Hartley, J. Molec. Biol. 202: 913 (1988). In an alternative approach, the pollen function is interrupted by altering the metabolism of plant hormones, such as auxins and gibberellins. For example, the ro1B gene from Agrobacterium rhizogenes codes for an enzyme that interferes with auxin metabolism by catalyzing the release of indoxyl-β-glucoside-free. Estruch et al., EMBO J: 11: 3125 (1991). Spena and others, Theor. Appl. Genet 84: 520 (1992), have shown that the anther-specific expression of the ro1B gene in tobacco resulted in plants having increased levels of indole-3-acetic acid, diminished gibberellin activity and wilted anthers in which the pollen production. Since the expression of the ro1B gene in microspores is expected to increase the levels of indole-3-acetic acid in anther tissue, the ro1B gene is another example of a gene that is useful for the control of pollen production. Slightom et al. J. Biol. Chem. 261: 108 (1985), describes the nucleotide sequence of the ro1B gene. In order to express a protein that disrupts the microspore function, an expression vector is constructed in which the DNA sequence encoding the protein is operably linked to the DNA sequences that regulate the transcription of genes in a specific form for microspores The general requirements of an expression vector are described above in the context of a temporal expression system, however, in the present the objective is to introduce the expression vector into embryonic tissue of plants in such a way that a foreign protein is expressed in a final stage of development in microspores of adult silver. Mitotic stability can be achieved using vectors of viral plants that provide epichromosomal replication. An alternative and preferred method for obtaining mitotic stability is provided by the integration of expression vector sequences in the host chromosome. Said mitotic stability can be provided by the Agrobacterium-mediated transformation technique illustrated in the following Example 5. The transcription of the foreign gene can be controlled by a promoter of a specific gene for microspores or by a viral promoter such as a promoter of the Virus of Cauliflower mosaic (CaMV), a promoter of the Scrofularia Mosaic Virus, and the like. Gruber and others, supra. Preferably, the promoter is a promoter of a specific gene for microspores or the core promoter CaMV 35S. More preferably, the promoter is a promoter of a specific gene for microspores and in particular, the Bnm1 promoter. Alternatively, transcription of the foreign gene can be controlled by a promoter of an anther-specific gene. In this way, foreign genes capable of dissociating cellular function can be expressed in microspores and anther cells. Promoters and anther-specific genes are known in the art, see, for example, McCormick et al., "Antheer-Specific Genes: Molecular Characterization and Promoter Analysis in Transgenic Plants," in PLANT REPRODUCTION: FROM FLORAL INDUCTION TO POLLINATION, Lord and others, (eds.), pages 128-135 (1989); Scott et al., International Application Publication No. WO 92/11379 (1992); van der Meer et al., The Plant Cell 4: 253 (1992). The methods described above can be used to chemically synthesize an anther-specific promoter having a published nucleotide sequence. In order to select transformed cells, the expression vector contains a selectable marker gene, such as a herbicide resistance gene or an antibiotic resistance gene. For example, such genes may confer resistance to phosphinothricin, glyphosate, sulfonylureas, anthrazine, imidazolinone or aminoglycoside antibiotics such as neomycin, kanamycin and G418 (genticin). Preferably, the selectable marker gene is the neomycin phosphotransferase gene (nptll gene). The expression vector may contain cDNA sequences that encode a foreign protein under the control of a microspore-specific regulatory element, as well as the selectable marker gene under the control of a constitutive promoter. Alternatively, the selectable marker gene can be delivered to the host cells in a selection expression vector separated by cotransformation with both vectors. In an alternative approach, male sterility can be induced by the use of an expression vector in which the microspore-specific regulatory element is operably linked to a nucleotide sequence encoding a sense RNA. Binding of counter-sense RNA molecules to target mRNA molecules results in decreased translational hybridization. Paterson, and others, Proc. Nati Acad. Sci. USA, 74: 4370 (1987). Therefore, a suitable counter-sense RNA molecule could have a sequence that is complementary to that of a species of mRNA that codes for a protein that is necessary for cellular function. For example, antisense RNA molecules can be used to inhibit the translation of actin, tubulin, ubiquitin, ubiquitin-conjugated enzyme, ubiquitin-bearing protein or elongation factors that encode mRNA. The DNA molecules that code for these genes can be isolated using normal techniques.

For example, plant actin genes are described by Shah et al., Proc. Nat'l Acad. Sci. USA 79: 1022 (1982) and by Baird et al., EMBO J. 6: 3223 (1987). Plant tubilin genes are described by Saha et al., Proc. Nat'l. Acad. Sci. USA 79: 1022 (1982) and by Baird et al., EMBO J. 6: 3223 (1987). The tubulin genes of plants are described by Raha et al., Plant Mol. Biol. 9: 565 (1987), Ludwig et al., Proc. Nat'l Acad. Sci. USA 84: 5833 (1987), and Yamada et al., Plant Physiol. 103: 1467 (1993). The genes of ubiquitin plants have been isolated by Gausing et al., Eur. J. Biochem. 158: 57 (1986), and by Xia et al., Plant Physiol. 104: 805 (1994). Pramanik et al., Plant Physiol. 104: 805 (1994). Pramanik et al., Plant Physiol. 102: 1049 (1993), reports the collation of alfalfa ubiquitin carrier protein while Picton et al., Plant Physiol. 103: 1471 (1993), isolated a ubiquitin conjugation enzyme from tomato. The collation of a barley elongation factor gene has been described by Sutton et al., Plant Physiol. 104: 807 (1994). In addition, DNA molecules that encode useful genes can be synthesized using published nucleotide sequences as discussed above. In addition, the antisense RNA molecules can be directed to mRNAs that encode proteins essential for pollen development. For example, chalcone synthase (CHS) catalyzes the initial step in flavonoid biosynthesis and a lack of Chs activity has been correlated with abnormal pollen development and / or function. Coe et al., J. Hered. 72: 318 (1981); Taylor et al., J. Hered. 83: 11 (1992); van der Meer et al., The Plant Cell 4: 253 (1992). Significantly, deficient flavonoid pollen does not work in autocruces. Taylor and others, supra. Therefore, sterility in male organisms can be induced by the inhibition of flavonoid biosynthesis using an expression vector that produces sense RNA for the 3 'untranslated region of chalice gene A synthase. van der Meer and others, supra. The collation and characterization of the A gene of chalcone synthase is described by Koes et al., Gene 81: 245 (1989), and by Koes et al., Plant Molec. Biol. 12: 213 (1989). Alternatively, an expression vector can be constructed in which the regulatory element specific for microspores is operably linked to a nucleotide sequence encoding a ribozyme. Ribozymes can be designed to express endonuclease activity that targets a certain target sequence in an mRNA molecule. For example, Steinecke et al., EMBO J. 11: 1525 (1992), achieved an inhibition of up to 100% of the expression of the neomycin phosphotransferase gene by ribozymes in tobacco protoplasts. More recently, Perrman et al., Antisense Research and Development 3: 253 (1993), chloramphenicol acetyl transferase activity inhibited in tobacco protoplasts using a vector that expressed a modified hammerhead ribozyme. In the context of the present invention, target RNA molecules suitable for ribozymes include mRNA species that encode proteins essential for microspore function, as described above. In a further alternative approach, expression vectors can be constructed in which a specific regulatory element for microspores is directed to the production of RNA transcripts capable of promoting P RNase-mediated separation of white mRNA target molecules. According to this approach, an external guide sequence can be constructed to direct the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently separated by the cellular ribozyme. Altman et al., Patent of E.U.A. No. 5,168,053. Yuan et al., Science 263: 1269 (1994). Preferably, the external leader sequence comprises a ten to fifteen nucleotide sequence complementary to a mRNA species encoding an essential protein for microspore function and a 3'-NCCA nucleotide sequence., wherein N is preferably a purine. ID. The external guide sequence transcribes the binding to the mRNA species targeted by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting the separation of mRNA by RNase P at the nucleotide located on the 5 'side of the base pair region. Id. Alternatively, the antisense genes, ribozyme genes and outer guide sequence genes can be regulated by a combination of a microspore-specific regulatory element and a promoter of an anther-specific gene. All the microspores of the transgenic plant can not express the foreign gene that interrupts cell function. However, Example 5 presents evidence that the specific regulatory element for Bnm1 microspores also induces the expression of a foreign gene in tapeto cells that are essential for the development of microspores. Therefore, the regulatory sequences of Bnm1 are particularly suitable to ensure complete male sterility. However, additional mechanisms can be adapted to dissociate the function of microspores that do not express the foreign gene. For example, the foreign gene can encode a protein that stimulates the production of a diffusible molecule capable of dissociating the function of viable microspores. An increase in the levels of indole-3-acetic acid by the expression of the ro1B gene or the production of the diphtheria toxin could achieve this objective. In addition, the specific regulatory element for Bnm1 microspores can be operably linked to DNA sequences encoding other functionally stable macromolecules that can degrade microspores, pollen, or structural components thereof (eg, cell walls). In this way, the macromolecules produced in a microspore are introduced into the surrounding loculum following the degradation of the microspore, and act on other microspores or pollen.

This approach is particularly attractive in the case of heterozygotes in which only 50% of the pollen grains in development express the dissociating gene. Again, the function of microspores or pollen grains that do not contain the foreign gene could be dissociated by the action of macromolecules released from microspores or pollen grains expressing the foreign gene. Since the regulatory element of Bnm1 stimulates the expression of the gene in a tissue-dependent manner, the remaining tissues of the sterile plant can develop normally. Finally, an expression vector of the foreign gene is controlled by a Bnm1 regulatory element and an anther-specific promoter, as discussed above. B. Restoration of Fertility of Male Organisms in the F1 Hybrid The methods described above can be used to produce sterile plants for transgenic male organisms for the production of F'l hybrids at large-scale crosses between lines of the same parents, if all the male gamete cells of the sterile plants for male organisms do not contain the foreign gene that dissociates the microspore function, so a proportion of F1 hybrids will have a fertile phenotype of male organisms. On the other hand, F1 hybrids will have a sterile phenotype for male organisms if the foreign gene is present in all male gamete cells of sterile plants for transgenic male organisms since the sterility induced by the foreign gene could be dominant. In addition, F1 hybrids will have a sterile phenotype for male organisms if the expression of the foreign gene results in the dissociation of neighboring cells. Therefore, it is convenient to use a male fertility restoration system to provide for the production of fertile F1 hybrids for male organisms. Said fertility restoration system has particular value when the cultivated product is sown. One approach to restoring fertility of male organisms could be to cross sterile plants into transgenic male organisms with fertile plants in transgenic male organisms that contain a fertility restorer gene controlling a specific regulatory element for microspores. For example, the restored fertility gene can encode a protein targeted specifically for the product of the cellular dissociating gene. As an illustration, Mariani et al., Nature 357: 384 (1992), fertile plants in crossed male organisms that expressed a barnase inhibitor in anther cells designated "barstar", with sterile plants in male organisms that expressed barnase in human cells. anther. Hartley, J. Mol. Biol. 202: 913 (1988), describes the nucleotide sequence of barstar. Alternatively, the fertility restoration gene can be used to produce RNA molecules that are designated for mRNAs that encode the cellular dissociative protein. For example, restoration of fertility can be achieved by expressing diphtheria toxin ribozymes or nonsense RNA in male fertile plants to neutralize the effects of diphtheria toxin in male sterile plants. Therefore, male fertility can be restored in the F1 hybrids by producing a male fertile transgenic plant that synthesizes a particular species of the RNA or polypeptide molecule to counteract the effects of the particular foreign gene expressed in the male sterile transgenic plants. In an alternative approach, male fertility can be restored by the application of a chemical compound. As discussed above, male sterility can be induced by blocking the expression of chalcone synthase to inhibit flavonoid biosynthesis. The application of flavonoid aglycones can be used to complement the biochemical defect and restore pollen function. Mo et al., Proc. Nat'l. Acad. Sci. USA 89: 1713 (1992); Vogt et al., The Plant Cell 6: 11 (1994). Preferred flavonol aglycones, such as kaempferol, have an unsaturated bond between the carbons in positions two and three in the C ring of the flavonoid backbone, as well as an unsubstituted hydroxyl group in the three position of the C. ring. Vogt et al. , supra. 5. Use of a Specific Regulatory Element for Microspores to Inhibit Plant Disease.

Another objective of the present invention is to provide means for controlling the disease caused by pests. In particular, the present invention encompasses the production of transgenic plants comprising expression vectors in which the regulatory sequences of Bnm1 stimulate the expression of macromolecules useful for defense against viruses and insects. Approximately 20% of plant viruses are transmitted from generation to generation in the seed. Matthews, PLANTA VIROLOGY, 3rd Edition, (Academic Press, Inc. 1991); Mink, Ann. Rev. Phytopathol. 31: 375 (1993). The transmission of seeds is achieved either by direct invasion of the embryo by an indirect invasion of the embryo via infected gametes. According to Mink, supra, there are at least nine viruses that seem to spread from plant to plant through pollen and at least four viroids that are transmitted through pollen to seeds and female plants. Therefore, the specific regulatory element for microsprads of Bnm1 is used to inhibit the transmission of these viruses and viroids. In an approach to provide protection against viral infections, a regulatory element of Bnm1 is used to stimulate the expression of a viral coat protein. The accumulation of viral coat proteins in cells of transformed plants provides resistance to viral infection and / or disease development by the virus from which the cover protein gene provides resistance to viral infection and / or development of disease by the virus of which the protein gene is derived, as well as by related viruses. See Beachy and others, Ann. Rev. Phytopathol. 28: 451 (1990); Beachy, "Virus Resistance Through Expression of Coat Protein Genes", in BIOTECHNOLOGY IN PLANT DISEASE CONTROL, 3rd Edition, Chet (De.), Pages 89-104 (Wiley-Liss, Inc. 1993). For example, resistance mediated by coating protein has been conferred on plants transformed against alfalfa mosaic virus, cucumber mosaic virus, tobacco vein virus, potato X virus, potato virus and tobacco etch virus, Tobacco Rattlesnake Virus, and Tobacco Mosaic Virus, Id. Alternatively, protection against viral disease can be achieved by using a vector comprising mammalian 2'-5'-oligoadenylate synthetase operably linked to a regulatory element specific for microspores. Truve et al., Bio / Technology 11: 1048 (1993), describes the collation and nucleotide sequence of a rat cDNA encoding 2'-5'-oligoadenylate synthetase, a component of the antiviral response induced by interferon in mammals . Truve et al. Also discloses that transgenic plants that express 2-5'-oligoadenylate synthetase are protected against viral infection under field conditions. In a third approach to provide protection against viral infection, a microspore-specific regulatory element of the present invention is used to direct the expression of viral genome counter-sense RNA. For example, the sense RNA may be used to confer resistance to the cucumber mosaic virus, as described by Rezaian et al., Plant Molec. Biol. 11: 463 (1988). Alternatively, the sense RNA may be used to confer resistance against tomato golden mosaic virus as described by Day et al., Proc. Nat'l. Acad. Sci. 88: 6721 (1991). In a fourth approach to provide protection against viral infection, a specific regulatory element for microspores is used to direct the expression of the antiviral protein of grana (PAP), a ribosome inhibitor protein found in the cell walls of Phytolacca americana. Lodge and others, Proc. Nat'l Acad. Sci USA 90: 7089 (1993) show that transgenic plants expressing PAP are resistant to a broad spectrum of plant viruses. Lodge and others also describe a method for isolating the PAP cDNA. The present invention also contemplates the use of a Bnm1 regulatory element to provide protection against insect pests., such as Thysanoptera, the arthropods that feed on pollen on the order of Thysanoptera. According to this approach, a regulatory element of Bnm1 is used to stimulate the expression of insecticidal toxin genes. For example, the gram-positive bacterium Bacillus thuringiensis produces polypeptides that are toxic to a variety of insect pests, but have no activity against vertebrates and beneficial insects. Thompson, "Biological Control of Plant Pests and Pathogens: Alternative Approaches", in BIOTECHNOLOGY IN PLANT DISEASE CONTROL, Chet (de.) Pages 275-290 (Wiley-Liss, Inc. 1993). Suitable Bacillus thuringiensis toxins include crylA d-endotoxins that are highly toxic to lepidopteran insects and crylllA d-endotoxins that are highly toxic to clophopper insects. Geiser et al., Gene 48: 109 (1986), describes the collation and nucleotide sequence of a crylA d-endotoxin gene. Transformation of plants with vectors comprising a crylA d-endotoxin gene has been described by Williams et al., Bio / Technology 10: 540 (1992), Koziel et al., Bio / Technology 11: 194 (1993), and Fujimoto et al. others, Bio / Technology 11; 194 (1993). Lereclus et al., Bio / Technology 10: 418 (1992), describes the construction of a plasmid which. it comprises structural genes that code for crylllA and crylAc. In addition, Adang and others, Plant Molec. Biol. 21: 1131 (1993), describes the nucleotide sequence of a synthetic crylllA gene that was designed for optimal expression in plant cells. In addition, DNA molecules that encode d-endotoxin genes can be purchased from the American Type Culture Collection (Rockville, MD), including the ATCC Access Nos. 40098, 67136, 31995 and 31998. As an illustration, the European corn weevil , Ostrinia nubialis, is a major maize pest in North America and Europe. After hatching, most of the maize weevil larvae of second hatchlings are fed with the accumulated pollen in the armpits of the leaf and in the tissues of the pod and neck. Once the larva begins to feed inside the neck, they are protected from the effects of chemical pesticides. Koziel et al., Supra, showed that transgenic maize plants expressing the crylA (b) gene were resistant to repeated severe infestations of the corn weevil. The following list provides other insecticidal toxins that are suitable for expression. (1) A vitamin binding protein such as avidin. See patent application of E.U.A. No. 07 / 911,864, which teaches the use of avidin and homologs of avidin and larvicides against insect pests, (2) An enzyme inhibitor, for example, a protease inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (cDNA nucleotide sequence encoding tobacco proteinase inhibitor I) and Sumitani et al., Biosci. Biotech Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor). (3) An insect-specific hormone or pheromone as an ecdysteroid and juvenile hormone, a variant thereof, an imitation based thereon or an antagonist or agonist thereof. See, for example, the description by Hammock et al., Nature 344: 458 (1990), of the expression of juvenile hormone esterase baculovirus, a juvenile hormone inactivator. (4) A peptide or neuropeptide specific for insects, which, garlic expression, dissociates the physiology of the affected pest. For example, see the descriptions of Regan, J. Biol. Chem. 269: 9 (1994) (the expression collation of DNA encoding insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an alostatin is identified in Diploptera puntata). See also patent of E.U.A. No. 5,266,317 to Tomalski et al., Who describes genes that encode insect-specific paralytic neurotoxins. (5) A specific poison for insects produced in the wild by a viper, a sighted one, etc. For example, see Pang et al., Gene 116: 165 (1992), for description of heterologous expression in plants of a gene encoding a toxic scorpion insect peptide. (6) An enzyme responsible for a hyperaccumulation of a monterpene, a sesuiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another molecule without protein with insecticidal activity. (7) An enzyme involved in the modification, including post-translational modification, of a biologically active molecule; for example, an enzymatic glycol enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See the PCT Application WO 93/02197 in the name of Scott et al., Which describes the nucleotide sequence of a calasus gene. DNA molecules containing sequences encoding chitinase, for example, from ATCC can be obtained under access Nos: 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23: 691 (1993), who teaches the nucleotide sequence of cDNA encoding tobacco worm chitinase and Kawalec et al., Plant Molec. Biol. 21: 673 (1993), who provides the nucleotide sequence of the ubiquitous polyubiquitin gene parsley. (8) A molecule that stimulates signal transduction. For example, see the description of Botella and others, Plant Molec. Biol. 24: 757 (1994), nucleotide sequences for cDNA clones of bean calmodulin and Griess et al., Plant Physiol. 104: 1467 (1994) who provides the nucleotide sequence of a corn calcomodulin cDNA clone. (9) An antibody specific for insects or an immunotoxin derivative thereof. Therefore, an antibody designated for a critical metabolic function in the insect's gut could inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract # 497, SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments). The present invention, generally so described, will be more readily understood by reference to the following examples which are provided by way of illustration and are not intended to limit the present invention. EXAMPLE 1 Construction and Screening of a Brassica napus MicroSpore cDNA Bank Brassica napus cv Topas uninucleate tardicles / early binucleated were cultured for four days at 32.5 ° C to induce embryogenesis. Poly (A) + RNA was isolated from the microspores using a guanidinium isothiocyanate procedure as described by Ouellet et al., Plant Journal 2: 321 (1992). Double-stranded cDNA of poly (A) + RNA was synthesized using the Riboclon® cDNA Synthesis System (Promega Corp., Madison, Wl) and cloned into the lambdaGEM®-4 collation vector (Promega Corp.). The cDNA library was screened differentially for microspore-specific clones by testing duplicate elevations of 32 P-labeled microspore arn used for bench construction (Mi-RNA) or with 32 P-labeled RNA from microspores subjected to 25 ° C for 24 hours and then at 32 ° C for three days to eliminate the embryogenic response (Mx-RNA). Three cDNA clones (Mi-cDNA 3, 4 and 5) that hybridized to Mi-RNA, but not to Mx-RNA, were purified and further characterized. A Southern plot of each isolated cDNA clone was hybridized with 32 P-labeled cDNA probes representing the total library of microspore cDNA. The Napin DNA and lambda vector arms were included on the graph as negative controls. Both My-cDNA 3 and My-cDNA 4 were hybridized with the total cDNA probe and found to contain inserts of approximately 800 base pairs. Nucleotide sequence analysis revealed that My-cDNA 3 and My-cDNA 4 were identical and probably contained full-length cDNA inserts since both cDNAs terminated in the same nucleotide. The cDNA clone specific for microspores was designated Bnm1. The nucleotide sequence of Bnm1 cDNA [SEQ ID NO: 1] with its corresponding amino sequence [SEQ ID NO: 2] is shown in Figure 1.

Example 2 Northern analysis with the Bnm1 cDNA Probe Northern analyzes were carried out to characterize the expression pattern of the Bnm gene. These studies revealed that the Bnm1 cDNA hybridized with RNA from full buds, flowers and anthers, but it was not hybridized with RNA isolated from roots, stems, leaves, pistils and seeds in development. The expression of Bnm1 RNA was very strong in trinucleated and binucleated microspheres but essentially absent in uninucleate and tetrad stages of the development of microspores. The results of Northern analysis also indicate that the Bnm1 RNA was expressed to a greater degree through the development of microspores, compared to the microspore-specific clones of Brassica Bp4, Bp10 and Bp19. Albani et al., Plant Molecular Biology 15: 605 (1990); Albani et al., Plant Molecular Biology 16: 501 (1991); Albani et al., The Plant Journal 2: 331 (1992). To further characterize the Bnm1 clone, uninucleated microspores were cultured at 32.5 ° C for four days to induce embryogenesis or cultured at 24 ° C for four days to allow pollen development. The microspore embryos were isolated in globular, heart, torpedo and cotyledonary stages of development. Northern analysis of RNA samples revealed low levels of Bnm1 expression in microspores induced by embryogenesis with markedly higher levels of expression in pollen-induced microspores. RNA samples from the globular, heart, torpedo and cotyledonary stages of embryonic development do not hybridize with the Bnm1 probe. EXAMPLE 3 Southern Analysis with the Bnm1 cDNA Probe Hybridizations were carried out for Southern analysis and genomic screening, treated below, at 42 ° C using the following hybridization mixture: 50% formamide, 10x Denhadt's solution ( 100x: 10 grams of polyvinylpyrrolidone, 10 grams of bovine serum albumin and 10 grams of Ficoll 400 in 500 milliliters of sterile water), 5x of pHSSC buffer (20X: 3M of sodium chloride and 0.3M of Na32H2O citrate) , pH 7.0), 50 mM sodium phosphate (pH 7.0), 1% sodium dodecyl sulfate, and 5 mg / ml heat-denatured salmon sperm DNA. The spots were washed with great force (O.lxSSC, 65 ° C) and then incubated with X-ray film for a long time to obtain optimal exposure. In a series of experiments, Bnm1 was tested against a Southern blot of DNA samples obtained from B. campestris cv Candle, B. napus cv Topas, B. olerácea alboglabra, black spruce, Arabidopsis thaliana, sunflower, tobacco, parsley and corn . The results indicate that the Bnm1 gene is present in black parsley and crucifers, but not in sunflower, tobacco or corn. Example 4 Isolation of the Genomic Clone Bnm1 A genomic bank of Brassica napus was constructed partially by digesting fl. napus cv Westar genomic DNA with Sau3A and inserting DNA fragments (average insert size of about 8 kilobases) into the Sal site partially filling the space of plasmid pTZ18R. Mead et al., Protein Eng. 1: 67 (1986); Nantel et al., Plant Molecular Biology 16: 955 (1991). Following the method of Nantel and gold, above, the bank was collected in 42 fractions, an aliquot of each fraction was digested with EcoRI, and the EcoRi digestions were examined by Southern analysis to identify which fraction contained genomic clones corresponding to Bnm1 cDNA. . Approximately 10,000 colonies from each positive hybridization fraction were screened on Hybond-N filters (Amersham Corp., Arlington Heits, IL) using a 32P-dCTP randomized primer labeled cDNA probe. The hybridized colonies were harvested on duplicate colony grid analysis plates for secondary screening. Potential positives were taken through a final tertiary screening to isolate the unique positive colonies. After the third round of sieving, five clones were identified that showed strong positive signals. Restriction maps were generated for the five positive genomic clones using the entire Bnm1 cDNA with a probe. The results of these studies suggested that three of the five clones represented the same genomic fragment. One of these three clones plus the two remaining clones (Bnm1-5, 8 and 31) were further characterized. Restriction mapping and Southern analysis eliminated Bnm1-5 as a useful genomic clone. The lengths of the upstream region in Bnm1-8 and Bnm1-31 were determined by synthesizing the following oligonucleotides to amplify the 5 'end of the Bnm1 gene when used in combination with universal or reverse sequence primers: MSP-1 [SEQ ID NO. : 3]: 5 'GGCCGAATTCGCCGCCGCGGCGAAGGTAGA 3' EcoRI MSP-2 [SEQ ID NO: 4]: 5 'GGCCGAATTCCTGCCTTACCCGTCTCAGCCACG 3' EcoRI MSP-1 and MSP-2 were designed to generate fragments that differ in size by approximately 230 base pairs. The primers were annealed at 65 ° C in buffer solution of polymerase chain reaction (PCR) (Perkin Elmer Corp.; Norwalk, CT) containing 2.5 mM MgCl2. Two bands, 2.5 kilobases and 3.2 kilobases, were amplified from Bnm1-8 using primers Msp-1 and Msp-2, respectively, in combination with the reverse sequencing primer. The difference of 500 base pairs in size was thought to be due to the presence of an intron. The coding region of the genomic clone was sequenced and the presence of a single intron of 361 base pairs was confirmed. Figure 2 shows the nucleotide sequence [SEQ ID NO: 5] of the Bnm1 genomic clone with upstream and coding regions. To determine the nucleotide sequences of the RPC fragments, each fragment was digested with EcoRI and a 1.3 kilobase fragment was subcloned into the EcoR -Hinc site of pTZ18R. The ligated DNA molecules were introduced into the E. coli strain DHdaMCR-. The subcloned PCR fragments were sequenced using a Sequenase kit (United States Biochemical Corp., Cleveland, OH) using the dideoxy-mediated chain termination protocol of Sanger et al., J. Mol. Biol. 94: ??? (1975). It was found that the PCR fragments have only 535 base pairs of the sequence not translated upstream of 5 '. A fragment of Pvu \\ - Bam \ of 2.95 kilobases of the genomic clone Bnm? original was then subcloned to obtain additional upstream sequences. The walking initiator was used to sequence a total of 820 bases of the putative promoter region. Example 5 Transformation and Analysis of Cañóla Transgenic To examine the function of the Bnm1 promoter, the following groups of PCR primers were designed to add a Sa / I site at the 5 'end of the promoter and to introduce a? / Cabbage site into the promoter. codon ATG: MSP-8 [SEQ ID NO: 6]: -820 0 5 'GGCCGTCGACGCATCAAAGTGATGCGGAAGGAG 3' • Sa / I MSP-7 [SEQ ID NO: 7]: 'CCCGGGTCTAGAACGTTGCCATGGTCTTTGCGTCG 3' Xbal? / Col The MSP-8 primer consists of 23 nucleotides from the 5 'upstream region starting at nucleotide -820 and also includes an upstream Sa / I site to facilitate collation. The MSP-7 primer consists of 23 nucleotides facing the ATG start codon and was designed to work the? / Cab site at the start codon of the Bnm gene. "MSP-7 also includes an Xbal site to facilitate collation. Using these primers, a fragment of 844 base pairs was amplified, cloned and secreted.The DNA fragment included 820 base pairs of the promoter region directly upstream of the ATG starting site of the Bnm? Gene. The nucleotide sequence [SEQ ID NO: 8] of the 844 base pair fragment is shown in Figure 3. The promoter fragment was digested with? / Col-Sa / l ligated with a? / Col-EcoRI fragment containing a GUS reporter gene and the 3 'untranslated region (terminator) of a potato proteinase inhibitor gene (PINII), and cloned into the Sa / l-EcoRI sites of pBluescript®ll SK + (Stratagene® Cloning Systems, La Jolla, CA) and DP1741 to produce the vectors DP5476 (figure 4) and DP5477 (figure 5), respectively DP1741 differs from pBM 01.1 (Jefferson, Plant Molecular Biology Reporter 5: 387 (1987)) containing 35S CaMV regulatory sequences, in place of 5 'and 3' regulatory sequences of nopaline synthase, to regulate gene expression selectable marker NPTII. The binary construction DP5477 was transformed into B. napus cv Westar via Agrobacterium-mediated cocultivation of cotyledonary petioles using the general methodology of Moloney et al., Plant Cell Reports 8: 238 (1989). It was found that the isolated microspheres of three of four transgenic cañola analyzed for GUS activity stained intensely, while the fourth exhibited light blue staining. GUS expression seems to be localized mainly to microspores since other tissues including another wall, pistil and sepal were not stained. However, the matrix inside the locules that were stained blue indicated that they may have some expression of tapeto. Most of the expression of GUS seems to be associated with uninucleate and binucleated microspores, and the expression is probably post-meiotic, since they were not stained from 25 to 505 of the microspores.

Although the foregoing relates to particularly preferred embodiments, it will be understood that the present invention is not limited in this way. It will occur to those skilled in the art that various modifications may be made to the embodiments described and that said modifications are intended to be within the scope of the present invention, which is defined by the following claims. All publications and patent applications mentioned in this specification indicate the level of experience of those skilled in the art to which the invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or parent application was specifically and individually indicated to be incorporated by reference in its entirety.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Pioner Hi-Bred International, Inc. (B) STREET: 700 Capital Square, 400 Locust Street (C) CITY: Des Moines (D) STATE OR PROVINCE: lowa (E) COUNTRY: United States of America (F) POSTAL CODE: 50390 (ii) TITLE OF THE INVENTION: REGULATOR ELEMENT SPECIFIC FOR MICROSPORES (iii) NUMBER OF SEQUENCES. 8 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT Foley & Lardner (B) STREET: 3000 K Street, N.W., suite 500 (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: USA (F) ZP: 20007-5109 (V) READABLE FORM OF THE COMPUTER: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reeléase # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA (A) APPLICATION NUMBER: US 08 / 345,756 (B) DATE OF SUBMISSION: 22-NOV-94 (viii) INFORMATION APPORTER / AGENT: (A) NAME: BENT, STEPHEN A. (B) REGISTRATION NUMBER: 29,768 (C) REFERENCE NUMBER / CASE: 233229/372 / PIHI (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (202) 672-5300 (B) TELEFAX: (202)672-5399 (C) TELEX: 904136 (2) INFORMATION FOR SEC ID NO: 1: (i) CHARACTERISTICS SEQUENCE: (A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 24.569 (ix) DESC RI PCI ÓN DE S EC U N Y: S EC ID NO: 1: ACAAAACTTC CGACGCAAAG AAA ATG GCA ACG TTC TCA GTT CTG TCT ACC 50 Met Aléi Thr Phe Ser Val Leu Ser Thr l 5 TTC GCC GCG GCG GCA ATT ACG TTG CAA CTA CTC CTA GTT CCA GCT TCA 98 Phe Ala Ala Ala Ala lie Thr Leu Ciln Leu Leu Leu Val Pro gAla Ser 10 15 20 25 GCC TCT CCT CAC ATG AAA TAC ATT GAC GCT ATC TGC GAT CGC TCC CAC 146 Wing Ser Pro His Met Lys Tyr lie Jisp? La lie Cys Asp Arg Ser His 30 35 40 GAC CAÁ GAT TAC TGC GTT gAAA ACA TTG ACC ACC AAC CCC CCT ACA GCT 194 Asp Gln Asp Tyr Cys Val Lys Thr Leu Thr Thr Asn Pro Pro Thr Wing 45 50 55 GCT CCC ATT GGC CTG AAT CCA CTG CiCC GAG GTG ATG GCG CTC ACC ATA 242 Wing Pro lie Gly Leu Asn Pro Leu Wing Glu Val Met the Leu Thr lie 60 S5 70 GCC CAC GCC GAG AAG ACA GCG GCT TTC GTG GCT GAG ACG GGT AAG GCT 290 Ala His Ala Glu Lys Thr Ala Ala Phe Val Ala Ala Chr Gly Lys Ala 75 80 85 GAT CA ?. ACG TTT ACT GAG TAC CAC? GCC TAC TTA GCC GTG GTG GCT 338 Asp Gln Thr Phe Thr Glu Tyr His Lys ida Tyr Leu Ma Val Val Ua 90 95 100 105 GAT CTC AAG AGC GCA AAC CTG AAG CTC AAG CAÁ TCC CCT GAC ACT GCT 386 Asp Leu Lys Ser Wing A = n Leu Lys Leu Lys Gln Ser Pro Asp Thr Wing 110 115 120 CAC TAC GAC GTT AGG TCT TCG ACC GAC CAG ATG AAG CGC GTG GAG GGA 434 His Tyr Asp Val Arg Ser Ser Thr Asp Gln Met Lys gArg Val Glu Gly 125 130 135 TTA GTT GCC AGC AAA AAT GAC CAG GCT TCA ACT ACT CTT GAA ATG 482 Leu Val Wing Ser Lys Asn Asp Gln Wing Ser Thr Thr Leu Lys Glu Met 140 145 150 ACG GTG CAG ATG GAG AAA CTT GAT CTT GCT GCT AGT GCC GCC GAT 530 Thr Val Gln Met Glu Lys Leu Leu Asp Leu Wing Wing Wing Wing Asp 155 160 165 GCT GTG GAC GAC GAT GAT GAC AAC ATC CAC CGT CGC GTC TGATTTTAAA 579 Wing Val Asp Asp Asp Asp Glu Asn lie His Arg Arg Val 170 175 180 CCGGTCCGGT TTCGTTTTTT TGTGTTCACA ATACAAAATA TAATAAATAA ATGAATATAC 639 ATATACACAC ACACAAATGT GTTGTGATAA ACTAGTAATT AAGTTTTTGA AATATTTGCA 699 GAACTAATGT TGTCAATATT TTTGGCATAT ATAAAGAGTC TGCTGTATTA TCTTTTTATA 759 AAACTAAATA TAAATCTGAT TTGTATC 786 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 182 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Wing Thr Phe Ser Val Leu Ser Thr Phe Wing Wing Wing Wing Thr 1 5 10 15 Leu Gln Leu Leu Leu Val Pro Wing Ser Wing Pro Pro His Met Lys Tyr 20 25 30 lie Asp Wing He Cys Asp Arg Ser Kis Asp Gln Asp Tyr Cys Val Lys 35 45 Thr Leu Thr Thr Asn Pro Pro Thr Wing Ala Pro He Gly Leu Asn Pro SO 55 60 Leu Ala Glu Val Met Ala Leu Thr He Ala Ala His Ala Glu Lys Thr Ala 65 70 75 80 Wing Phe Val Wing Glu Thr Gly Lys Wing Asp Gln Thr Phe Thr Glu Tyr 85 90 95 His Lys Ala Tyr Leu Ala Val Val Ala Asp Leu Lys Ser Ala Asn Leu 100 105 110 Lys Leu Lys Gln Ser Pro Asp Thr Ala His Tyr Asp Val Arg Ser Ser 115 120 125 Thr Asp Gln Met Lys Arg Val Glu Gly Leu Val Wing Ser Lys Asn Asp 130 135 14C Gln Wing Ser Thr Thr Leu Lys Glu Met Thr Val Gln Met Glu Lys Leu 145 15C 155 160 Leu Asp Leu Wing Wing Wing Wing Asp Wing Val Asp Asp Asp Asp Glu 165 170 175 Asn He Hrs Arg Arg Val 180 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGCCGAATTC GCCGCCGCGG CGAAGGTAGA 30 (2) INFORMATION FOR SEQ ID NO: 4: (i) ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGCCGAATTC CTGCCTTACC CGTCTCAGCC ACG 33TION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2039 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRITION: SEC I D NO: 5: GCCAGGGTTC CGCACCGCAT CAAAGTGATG CGGAAGGAGT AATTATTCTG TAAATTTAAA 60 TATTTAGTCT TACATTGTTC AAA r "l AT GTTTTATATT ATTTTATTTT TTGATTTTGA 120 CGATTTAAGT ATATTTGAAT TTTTTTgAAGA g * > AAATACATA ATTAAAATGG GTACCCGAAC 180 TCGAATCTGA ATCGAACCCA CAATGATTCG AACAAAATTC AAACCAAAAT TTATAAATAT 240 TCCAATACGA TTGAATTTTC TAATATAAGA ATCAGAAATC TGAATAGATT AACTGAATTC 300 AAACAGGTAT AAGAGTGTCC ACCCCAACAA ATCCCTTAGT ACAATATATA GTTATAAATA 360 ATTCAATAAA CTATTTCATT ATGCACAGCC! CGGACTACTA CTAGTATAGT ATAATGTATG 420 TCAAATAAAA CTTCAGTGAA ATGTGTTCAT ATTAGATTAG ACCACTTCTT TTCTATGATC 480 ACCAAGGACC TCAACACTTG TCACGACATA GCTCAATTTT ftAAGAAGATC 540 ACAGACGATT TTTTCGTTGA CTAAATCTAT ACAAACCACA TACTATTTAG ATAGGTTCTC 600 CgAAATTTAGC AAACTTTTAG TAAAAACCTT ACGCATTTTA CATCAATTCT 6r D TAATATAGTA GTTTCCAAGA ATATCAAACG TCCCTGACCA AGCCCTAGGT GTACTTGTAT 720 ATATACCCAC CCACAAACTA AAAGCAAATC AACATACAGA AAACTGAATA ACAACCGGAA 780 GAAAAAAGAG AAAAAAATAA ATAAAACAAA ACTTCCGACG CAAAGAAAAT GGCAACGTTC 840 TCAGTTCTGT CTACCTTCGC CGCGGCGGC *. ATTACGTTGC gAACTACTCCT AGTTCCAGCT 900 TCAGCCTCTC CTCACATGAA ATACATTGAC GCTATCTGCG ATCGCTCCCA CGACCAAGAT 960 TACTGCGTTA AAACATTGAC CACCAACCCC CCTACAGCTG CTCCCATTGG CCTGGTACTC 1020 ATCTTTAAAC CACTGTCTCT TTGTTTGCGT TgAgAATCACAG AAGAAATTTA CGTTTGAATT 1080 ATGGTTTATT CAGTTTATTT GGCAGTCCG3 TAATATGTAA TCCGAAAATC TTCTAACATT 1140 AGTCGAAAAA CATTTTAAAC AGACAATCCG ACAATGTGAT ACTTTTTTCC ACACTGTAGC 1200 ATCTAGTGTG TTTATACCGC AGCTGGCCGG ATTAGCTAGC TGCATATATA 1260 ATCATGTTTA CTTAATATGT TTCAAAAATA CAACTGCATA TGCTTTACGT GTGgAAAGAGC 1320 TTAAACGAGA ATGATCATTA GTATTAATAC TAATAAAATC TCTTTATTAT CTCTAGAATC 1380 CACTGGCCGA GGTGATGGCG CTCACCATAG CCCACGCCGA GAAGACAGCG GCTTTCGTGG 1440 CTGAGACGGG TAAGGCTGAT CAAACGTTTA CTGAGTACCA CAAGGCCTAC TTAGCCGTGG 1500 TGGCTGATCT CAAGAGCGCA AACCTGAAGC TCAAGCAATC CCCTGACACT GCTCACTACG 1560 ACGTTAGGTC TTCGACCGAC CAGATGAAGC GCGTGGAGGG ATT GTTGCC AGCAAAAATG 1620 ACCAGGCTTC AACTACTCTT AAGGAAATGA CGGTGCAGAT GGAGAAACTT CTTGATCTTG 1680 CAGCTAGTGC CGCCGATGCT GTGGACGACG ATGATGAGAA CATCCACCGT CGCGTCTGAT 1740 TTTAAACCGG TCCGGTTTCG TTTTTTTGTG TTCACAATAC AAAATATAAT AAATAATATGA 1800 ATATACATAT ACACACACAC AAATGTGTTG TGATAAACTA GTAATTAAGT TTTTGAAATA 1860 TTTGCAGAAC TAATGTTGTC AATATTTTTG GCATATATAA AGAGTCTGCT GTATTATCTT 1920 TTTATAAAAC TAAATATAAA TCTGATTTGT ATCAATTGTT GGACAACCCA AAAGCGCCAA 1980 GACATCACCT GGTACAAACA TATTGACTTT TGTAAGCTTA TCGATACCGT CGACCTCGA 2039 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGCCGTCGAC GCATCAAAGT GATGCGAAG GAG 33 (2) INFORMATION FOR SEQ ID NO: 7: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCCGGGTCTA GAACGTTGCC ATGGTCTTTG CGTCG 35 (2) INFORMATION FOR SEQ ID NO: 8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 859 base pairs (B) TYPE: nucleic acid (C) NO. ROWS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Genomic) (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CATAGTCGAT CTTAGGGCCG TCGACGCATC AAAGTGATGC GGAAGGAGTA ATTATTCTGT 60 AAATTTAAAT ATTTAGTCTT ACATTGTTCA TLATTTTTATG TTTTATATTA TTTTATTTTT 120 TGATTTTGAC GATTTAAGTA TATTTGAATT TTTTTgAAGAA gAAATACATAA TTAAAATGGG 180 TACCCGAACT CGAATCTGAA TCGAACCCAC AATGATTCGA ACAAAATTCA AACCAAAATT 240 TATAAATATT CCAATACGAT TGAATTTTCT AATATAAGAA TCAGAAATCT GAATAGATTA 300 ACTGAATTCA AACAGGTATA AGAGTGTCCA CCCCAACAAA TCCCTTAGTA CAATATATAG_360_ TTATAAATAA TTCAATAAAC TATTTCATTA TGCACAGCGC GGACTACTAC TAGTATAGTA 420 TAATGTATGT CAAATAAAAC TTCAGTGAAA TGTGTTCATA TTAGATTAGA CCACTTCTTT 480 TCTATGATCA CCAAGGACCT CAACACTTGT CACGACATAG CTCAATTTTC TAAACAAAGA 540 AAGAAGATCA CAGACGATTT TTTCGTTGAC TAAATCTATA CAAACCACAT ACTATTTAGA 600 TAGGTTCTCC íiAATTTAGCA AATATAACiCA AACTTTTAGT CGCATTTTAC 660 ATCAATTCTT AATATAGTAG TTTCCAACÍAA TATCAAACGT CCCTGACCAA GCCCTAGGTG 720 TACTTGTATA TATACCCACC CACAAACTAA AAGCAAATCA ACATACAGAA AACTGAATAA 780 CAACCGGAAG AAAAAAGAGA AAAAAATJAA TAAAACAAAA CTTCCGACGC AAAGACCATG 840 GCAACGTTCT AGACCCGGG 859

Claims (26)

1. CLAIMS 1. An isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 8; (b) a nucleotide sequence with at least similarity of 65% with SEQ ID NO: 8; and (c) a functional fragment of (a) or (b); wherein said DNA molecule is a specific regulatory element for microspores.
2. 2. The DNA molecule of claim 1, wherein said DNA molecule consists of the nucleotide sequence of SEQ ID NO: 8.
3. 3. An expression vector comprising a microspore-specific regulatory element of claim 1.
4. The expression vector of claim 3, further comprising a promoter, wherein the function of said promoter is under the control of said regulatory element specific for microspores.
5. 5. The expression vector of claim 4, wherein said promoter is selected from the group consisting of the promoter. Bnm ?, a specific promoter for anther and the core promoter of CaMV 35S.
6. 6. The expression vector of claim 5, wherein said promoter is the Bn? Promoter.
7. 7. The expression vector of claim 4, further comprising a foreign gene, wherein said foreign gene is operably linked to said promoter.
8. 8. The expression vector of claim 7, wherein the product of said foreign gene dissociates the microspore function.
9. 9. A method for using the expression vector of claim 8, to produce a male sterile plant, comprising the step of introducing said expression vector into embryogenic plant cells, wherein said foreign gene is selected from the group consisting of a structural gene, a contrasense gene, a ribozyme gene and an external guide sequence gene.
10. The method of claim 9, wherein said structural gene encodes a protein selected from the group consisting of diphtheria toxin, antiviral protein of grana, RNase-T1 of Aspergillus oryzae, barnase, and the gene product of roIB.
11. The method of claim 9, wherein the product of said counter-sense gene is selected from the group consisting of actin counter-sense RNA, globulin counter-sense RNA, ubiquitin-sense RNA, conjugation enzyme counter-sense RNA of ubiquitin, ubiquitin vehicle protein antisense RNA, elongation factor counter-sense RNA and chalcone synthase counter-sense RNA.
12. The method of claim 9, wherein said ribozyme gene comprises nucleotide sequences selected from the group consisting of actin nucleotide sequences, tubulin nucleotide sequences, ubiquitin nucleotide sequences, nucleotide sequences of conjugation enzyme of ubiquitin, ubiquitin vehicle protein nucleotide sequences, elongation factor nucleotide sequences and chalcone synthase nucleotide sequences.
13. The method of claim 9, wherein said external guide sequence gene comprises nucleotide sequences selected from the group consisting of actin nucleotide sequences, tubulin nucleotide sequences, ubiquitin nucleotide sequences, nucleotide sequences of Ubiquitin conjugation enzyme, ubiquitin vehicle protein nucleotide sequences, elongation factor nucleotide sequences and chalcone synthase nucleotide sequences.
14. The method of claim 9, wherein said embryogenic plant cells are Brassica napus cells.
15. 15. A transgenic plant comprising the expression vector of claim 8.
16. 16. A method for producing a male sterile pineapple, comprising: (a) constructing an expression vector comprising a microspore-specific regulatory element, a promoter, and a foreign gene, wherein the specific regulatory element for microspore comprises a nucleotide sequence selected from the group consisting of (i) SEQ ID NO: 8, (ii) a nucleotide sequence having substantial sequence similarity to SEQ ID NO: 8, and (ii) fragments of (i) or (ii), wherein the microspore-specific regulatory element together with the promoter controls the expression of the foreign gene and where the product of the foreign gene disrupts the microspore function, thus producing a sterile male plant.
17. The method of claim 16, further comprising the step of: (b) introducing said expression vector into cells of embryogenic plants.
18. 18. A method for using a microspore-specific regulatory element to produce a male fertile hybrid plant, comprising: (a) producing a first male sterile, sterile plant, comprising an expression vector comprising a specific regulatory element for microspores, a promoter and a first foreign gene, wherein the specific regulatory element for microspores together with the promoter controls the expression of the first foreign gene and where the product of the first foreign gene disrupts the microspore function; (b) producing a second parent plant comprising an expression vector comprising the regulatory element specific for microspores, a promoter and a second foreign gene, wherein the specific regulatory element for microspores together with the promoter controls the expression of the second foreign gene; and (c) cross-fertilizing the first mother with the second mother to produce a hybrid plant, wherein the microspores of the hybrid plant express the second foreign gene, wherein the product of the second foreign gene prevents the interruption of the function of microspores by the product of the first foreign gene thus producing a male fertile hybrid plant.
19. The method of claim 18, wherein said foreign gene encodes barnase and said second foreign gene encodes a barnase inhibitor.
20. The method of claim 18, wherein said product of said first foreign gene is a diphtheria toxin ribozyme.
21. 21. A method for restoring fertility of a male sterile hybrid plant, which comprises treating the male sterile hybrid plant with a flavonol aglycone, wherein the male sterile plant comprises an expression vector consisting of (i) a specific regulatory element for microspores, (i) a promoter, and (iii) a foreign gene, wherein the regulatory element specific for microspores together with the promoter, controls the expression of the foreign gene and wherein the foreign gene expresses calcona synthase counter-sense RNA thus producing defective flavonol microspores.
22. 22. The method of claim 21, wherein said flavonol aglycone is Kaempferol.
23. 23. A method for producing transgenic plants resistant to disease caused by virus or insect, comprising: (a) constructing an expression vector comprising a specific regulatory element for microspore, a promoter, and a foreign gene, wherein the specific regulatory element for microspore comprises a nucleotide sequence selected from the group consisting of (i) SEQ ID NO: 8, (ii) a nucleotide sequence having substantial sequence similarity to SEQ ID NO: 8, and (iii) fragments of (i) ) or (ii), wherein the specific regulatory element for microspore together with the promoter controls the expression of the foreign gene and wherein the product of said foreign gene dissociates the function of said virus or encodes an insecticidal toxin, thus conferring resistance to the disease.
24. The method of claim 23, further comprising the step of: (b) introducing said expression vector into cells of embryogenic plants.
25. 25. The method of claim 24, wherein said foreign virus dissociation gene product is selected from the group consisting of viral coat protein, 2'-5'-oligoadenylate synthetase, viral genome antisense RNA and antiviral protein of cochineal.
26. 26. The method of claim 24, wherein said insecticidal toxin is an endotoxin of Bacillus thuringiensis.