CN1192784A - Induction of male sterility in plants by expressing high levels of avidin - Google Patents

Abstract

By increasing the concentration of endogenous avidin in plant tissues, male sterile plants can be generated. This effect can be obtained by producing transgenic plants containing an expression vector in which a promoter is operably linked to a DNA sequence encoding avidin. A method of restoring male fertility is also disclosed.

Description

Induction of male sterility in plants by expressing high levels of avidin

Background of the invention

The present invention relates to methods of controlling plant fertility using DNA molecules encoding avidin. In particular, the present invention relates to methods of producing transgenic male sterile plants expressing avidin. Furthermore, the present invention relates to methods for restoring male fertility in the progeny of male sterile plants.

Control of pollen fertility is important in hybrid crop production, see for example J.M. Poehlman, BREEDING FIELD CROP, 3rd ed. (Van Nostrand and Reinhold, New York, NY, 1987), which is incorporated herein by reference. For example, in hybrid maize production, pollen fertility control is usually achieved by physically removing male inflorescences or tassels before pollen shedding. Manual detasseling is a lot of work. While mechanical detasseling is less work than manual detasseling, it is less reliable, requires subsequent inspection of the crop, and may require remedial manual detasseling. Both methods of detasseling resulted in a loss of maternal yield.

However, most important staple crops have both male and female functional organs within the same flower; thus, detasseling is not a simple operation. Although pollen-forming organs can be removed by hand before pollen is shed, hybrid production in this way is labor-intensive and prohibitively expensive.

Pollen fertility can also be controlled by applying foliar sprays with andrgametocidal properties. Cross et al., Sex Plant Reprod. 4:235 (1991). For example, sprinkling ethephon on the anthers caused additional mitosis in wheat pollen, degeneration of tapetum cells in barley, and pollen deformity or pollen abortion in thrush. Bennet et al., Nature 240:566 (1972), Colhoun et al., Plant Cell Environ. 6:21 (1983); Berthe et al., Crop Sci. 18:35 (1978). However, chemical methods are labor intensive and have potential problems of introducing chemical toxicity into the environment.

Furthermore, the industrial production of hybrid seeds using gametocides is limited by cost and availability of chemicals, as well as by application reliability and duration of action. A serious limitation of gametocides is their phytotoxic effect, the severity of which depends on genotype. Other limitations include that these chemicals may not be effective on longer-flowering crops, as new flowers produced may not be affected. Therefore, chemicals need to be reused.

Many current industrial hybrid seed production systems for field crops rely on genetic methods for pollination control. Plants used as female parents are either unable to shed pollen, produce pollen that is biochemically incapable of self-fertilization, or are unable to produce pollen. Plants that cannot self-fertilize are called "self-incompatible". Difficulties associated with the use of self-incompatibility systems include the availability of self-incompatibility female lines and their reproduction, and the stability of self-incompatibility. In some cases, self-incompatibility can be overcome chemically, or immature shoots can be artificially pollinated before biochemical mechanisms that block pollen are activated. Disableable self-incompatibility systems are often very vulnerable to harsh climatic conditions that destroy or reduce the efficacy of biochemical blockade of self-pollination.

Of wider interest in industrial seed production are systems based on genetic pollen control that cause male sterility. There are two general types of these systems: (1) nuclear male sterility, which results from mutations in one or more nuclear genes that prevent pollen formation, or (2) cytoplasmic male sterility, often called "cytoplasmic male sterility." Male sterility" (CMS), in which pollen formation is blocked or pollen is aborted due to alterations in cytoplasmic organelles, usually mitochondria.

Nuclear sterility can be either dominant or recessive. Dominant sterility can only be used for hybrid seed formation if vegetative or vegetative reproduction from the maternal line is possible. Recessive sterility can be used if sterile or fertile plants are readily distinguishable. However, the industrial applicability of dominant and recessive sterile systems is limited by the cost of cloning and maternal selection of autogenous plants, respectively.

Although there are hybridization protocols involving the use of CMS, their industrial value is limited. An example of a CMS system is a specific mutation in the cytoplasmic mitochondria that, in the appropriate nuclear context, results in failure to form mature pollen. In some cases, the nuclear background can compensate for the cytoplasmic mutation, resulting in normal pollen formation. Specific nuclear "restorer genes" allow plants with CMS mitochondria to form pollen. In general, industrial seed production using CMS involves the use of three breeding lines: a male sterile line (the female parent); a maintainer line, homologous to the male sterile line but containing fully functional mitochondria; and a male sterile line Tie. The paternal line may or may not carry a cytoplasmic specific restorer gene.

For crops, such as vegetables, where seed recovery of hybrids is not important, a CMS system can be used without recovery. For crops in which the fruit or seeds of the hybrid are industrial products, the fertility of the hybrid seed must be restored with the male parent's specific restorer gene, or the male sterile hybrid must be pollinated. It can be done by pollination with a small proportion of male fertile plants. In most species, CMS traits are maternally inherited since all cytoplasmic organelles are inherited only by the egg cell.

CMS systems have several limitations that can prevent them from being the only method of producing male sterile plants. For example, a specific CMS type (T cytoplasm) in maize confers susceptibility to toxins produced by specific fungal infections. While many crops still use a CMS system, it can malfunction under certain environmental conditions.

Therefore, there is clearly a great need for methods of controlling pollen production other than manual, mechanical, chemical and conventional genetic methods.

Summary of the invention

It is therefore an object of the present invention to provide a method for producing male sterile plants by rendering the plants sterile by heterologous expression of avidin.

Another object of the present invention is to provide a chimeric gene comprising a DNA sequence encoding avidin operably linked to a plant promoter sequence.

Another object of the present invention is to propose a method for reversing male sterility caused by the expression of avidin.

According to one embodiment of the present invention, these and other objects are achieved by providing an isolated DNA molecule comprising an avidin-encoding nucleotide sequence operably linked to a plant promoter. In a preferred embodiment, the isolated DNA sequence is contained in an expression vector. In another preferred embodiment, the plant promoter sequence is a constitutive promoter, and in another preferred embodiment, the constitutive promoter is a ubiquitin promoter.

According to another embodiment of the present invention, there is provided a transgenic plant, wherein the plant comprises a heterologous nucleotide sequence comprising an avidin gene, and the heterologous nucleotide sequence increases avidin in plant tissue concentration, causing male sterility. In a preferred embodiment, the transgenic plants are maize plants, soybean plants or sunflower plants. In another preferred embodiment, the heterologous DNA molecule further comprises a constitutive promoter sequence, which in a preferred embodiment is the ubiquitin promoter sequence.

According to still another aspect of the present invention, there is provided a method for producing transgenic male sterile plants, comprising introducing into plant cells an expression vector comprising a promoter and an avidin gene, wherein the promoter controls the expression of the avidin gene , while expression of the avidin gene causes male sterility. In a preferred embodiment, transgenic plants are regenerated from the plant cells. In another preferred embodiment, the promoter comprises a ubiquitin promoter or an inducible promoter.

According to yet another aspect of the present invention, there is provided a method of producing a male sterile hybrid plant using the avidin gene, comprising producing a first parental male sterile plant comprising the above DNA molecule, wherein expression of the avidin causes male sterility producing a second transgenic parent plant expressing the second exogenous gene; and cross-fertilizing the first parent with the second parent to produce a hybrid plant expressing the second exogenous gene, wherein the product of the second exogenous gene reduces the hybrid plant expression of avidin, thereby generating male-fertile hybrid plants.

In a preferred embodiment of this aspect of the invention, the second exogenous gene is selected from antisense genes, ribozyme genes and external guide sequence (extemal guide sequence) genes. In another preferred embodiment, the antisense gene comprises an avidin mRNA sequence, which in yet another preferred embodiment is under the control of an inducible promoter. In another preferred embodiment, the ribozyme gene comprises an avidin mRNA sequence. In yet another preferred embodiment, the exogenous sequence gene comprises an avidin mRNA sequence. In yet another preferred embodiment, the DNA molecule of the first parental plant further comprises a LexA operon operably linked to said promoter, wherein the second foreign gene is a LexA repressor gene. In another recommended embodiment, the promoter sequence is an anther-specific promoter sequence comprising an anther box, and the anther box is selected from the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2, The nucleotide sequence of SEQ ID NO: 3 and a functional fragment thereof.

In yet another preferred embodiment of this aspect of the invention, the anther cassette has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the anther-specific promoter further comprises a core promoter promoter, the core promoter is selected from the CaMV 35S core promoter, the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence of SEQ ID NO: 6 and one of them Functional snippets.

According to a further aspect of the present invention, there is provided a method of restoring fertility to plants rendered male sterile by expression of avidin comprising spraying the plants with a biotin solution.

Brief description of the drawings

Figure 1 shows the avidin-encoding plasmid PHI5168.

Figure 2 shows plasmid PHI610 encoding the bar gene.

Detailed Description of the Recommended Implementation 1. Definitions

In the following description, various terms are used extensively. In order to facilitate the understanding of the present invention, the following definitions are provided.

A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) and then translated into the amino acid sequence characteristic of a specific polypeptide.

In this specification, exogenous gene refers to a DNA sequence operatively linked to at least one heterologous regulatory component. For example, any gene other than the maize 5126 structural gene is considered exogenous if it is controlled by an anther-specific regulatory component of the 5126 gene.

A promoter is a DNA sequence that directs the transcription of a gene (such as a structural gene, an antisense gene, a ribozyme gene, or an exogenous sequence gene, etc.). The promoter is usually located in the 5' region of the gene, close to the transcription initiation site. If the promoter is an inducible promoter, the rate of transcription is increased in response to the inducer. In contrast, if the promoter is constitutive, the rate of transcription is not regulated by the inducer. A plant promoter is a promoter sequence in plant tissues that directs the transcription of a gene.

The core promoter contains the essential nucleotide sequence for promoter function, including the TATA box and the start of transcription. According to this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that enhance activity or confer tissue-specific activity. For example, the SGB6 core promoter consists of approximately 38 nucleotides in the 5'-ward of the SGB6 gene transcription start site, while the 35S core promoter of cauliflower mosaic virus (CaMV) is composed of 35S genome transcription Consists of approximately 33 nucleotides 5' to the start site.

A tissue-specific promoter is a DNA sequence that, when operably linked to a gene, directs the level of transcription of that gene to be higher in a particular tissue of an organism than in some or all other tissues of the organism. For example, an anther-specific promoter is a DNA sequence that directs higher levels of transcription of an associated gene in the anther tissue of a plant. For example, the SGB6 anther-specific promoter can direct the expression of foreign genes in anther tissues but not in root or coleoptile tissues.

An "anther-specific promoter" as used herein includes two functional components: an "anther box" and a core promoter. Specific anther boxes may have functional features of canonical enhancers. The combination of the anther cassette and the core promoter can stimulate gene expression to a greater extent than the core promoter alone. This is even the case with chimeric anther-specific promoters containing anther cassettes and core promoters derived from different genes. Such chimeric anther-specific regulatory elements are described below.

An operator is a DNA molecule located in the 5' direction of a gene, which contains a nucleotide sequence that is recognized and bound by a repressor protein. Binding of the repressor protein to the operator it recognizes results in repression of gene transcription. For example, the LexA gene encodes a repressor protein that binds to the LexA operator.

An isolated DNA molecule is a segment of DNA separated from the DNA of an organism. For example, a cloned DNA molecule encoding the gene for avidin is one isolated DNA molecule. Another example of an isolated DNA molecule is a chemically synthesized DNA molecule or an enzymatically prepared cDNA that does not integrate into the genomic DNA of an organism.

Enhancers are DNA regulatory elements that increase transcription efficiency.

Complementary DNA (cDNA) is a single-stranded DNA molecule formed from an mRNA template using an enzyme (reverse transcriptase). Typically, a primer complementary to a portion of the mRNA is used to initiate reverse transcription. The term "cDNA" is also used by those skilled in the art to refer to a double-stranded DNA molecule consisting of this single-stranded DNA molecule and its complementary DNA strand.

The term expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA, which is then translated into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, or phage, that is capable of autonomous replication in a host cell. Cloning vectors usually contain one or a few restriction endonuclease recognition sites on which foreign DNA sequences can be inserted in a defined manner without losing the basic biological functions of the vector; they also contain marker genes, which are suitable for Identification and selection of cells transformed with cloning vectors. Marker genes typically include genes that confer tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule that contains a gene for expression in a host cell. Gene expression is usually under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. We say that this gene is "operably linked" to a regulatory element.

The recombinant host can be any prokaryotic or eukaryotic cell that either contains a cloning vector or contains an expression vector. The term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain one or more cloned genes in the chromosome or genome of the host cell.

A transgenic plant is a plant that has one or more plant cells containing an exogenous gene.

In eukaryotes, RNA polymerase II catalyzes the transcription of structural genes, producing mRNA. A DNA molecule can be designed such that it contains a template for RNA polymerase II in which the sequence of the RNA transcript is complementary to a certain specific mRNA. This RNA transcript is called an antisense RNA, and the DNA sequence encoding the antisense RNA is called an antisense gene. Antisense RNA molecules are capable of binding to mRNA molecules, resulting in inhibition of mRNA translation.

Ribozymes are RNA molecules that contain a catalytic center. The term includes RNases, self-splicing RNA and self-cleaving RNA. The DNA sequence encoding a ribozyme is called a ribozyme gene.

An exogenous sequence is an RNA molecule that directs an endogenous ribozyme (RNase P) to a specific class of intracellular mRNA, causing the mRNA to be cleaved by RNase P. The DNA sequence encoding the exogenous sequence is called exogenous sequence gene. 2. Overview

The present invention provides a method for producing male sterile plants by preparing transgenic plants expressing avidin. Avidin can be expressed constitutively in a non-tissue-specific manner, or can be expressed in an anther-specific manner. Methods of restoring male fertility are also provided.

The avidin gene is isolated and inserted into an expression vector suitable for introduction into plant tissue. The expression vector also contains a promoter, which can be a constitutive promoter, a non-tissue-specific promoter (such as a ubiquitin promoter, etc.), a tissue-specific promoter (such as an anther-specific promoter, etc.), or an inducible Promoter. Expression vectors are introduced into plant tissues by standard methods, and transgenic plants are selected and propagated. Transgenic plants expressing avidin are male sterile. Male fertility can be restored by coexpressing in transgenic plants a second gene that represses transcription of the avidin gene or represses translation of avidin mRNA. Alternatively, male fertility can be restored by spraying developing plants with a biotin solution. 3. Isolation of DNA Molecules Encoding Avidin

The nucleotide sequence encoding avidin can be isolated by known methods. For example, cDNA encoding chicken egg white avidin can be isolated from a chicken oviduct cDNA library using the method described by Gope et al., Nucleic Acids Res. 15:3595 (1987), which is incorporated herein by reference.

Alternatively, a genomic clone of the avidin gene can be obtained from the genomic DNA of an organism known to produce avidin. For example, the chicken avidin gene can be cloned from chicken genomic DNA using the method described by Keinanen et al., Eur. J. Biochem. 220:615 (1994).

The avidin gene can also be isolated by standard methods using polymerase chain reaction (PCR). See Erlich, PCR TECHNOLOGY: PRINCIPLES ANDAPPLICATIONS FOR DNA AMPLIFICATION (Stockton Press, NY, 1989) and Innis et al., PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS (Academic Press, San Diego, 1990). PCR amplification methods for long nucleotide sequences, such as the ELONGASE ^™ system (Life Technologies, Inc., Gaithersburg, MD), can also be used to obtain longer nucleotide sequences, such as genomes encoding avidin clone. PCR primers complementary to the 5' and 3' ends of known avidin gene sequences can be synthesized using commercially available oligonucleotide synthesizers, such as those supplied by Applied Biosystems (Foster City, CA) . In a preferred embodiment, the primer includes an additional nucleotide sequence comprising a restriction endonuclease cleavage site. The presence of such sites allows for directional cloning of PCR products into appropriate cloning vectors following treatment with appropriate restriction enzymes. See "Molecular Cloning of PCR Products" by Finny in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds (John Wiley & Sons, New York, 1987), p. 15.7.1.

The template DNA for PCR can be either cDNA or genomic DNA, prepared from an appropriate organism by methods well known in the art. Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989). Genomic DNA can be prepared directly from avian, reptile or amphibian tissue using commercially available reagents such as Triazol ^™ (Life Technologies, Inc., Gaithersburg, MD). Alternatively, cDNA encoding avidin can be prepared from mRNA extracted from chicken oviduct tissue using a commercially available kit (Pharmacia, Piscataway, NJ). This mRNA preparation was used as a template for cDNA synthesis using poly(dT) or random hexamer primers using standard techniques. See Sambrook et al., supra. Then, cDNA synthesis was performed using a commercially available kit (Pharmacia), and the cDNA was directly used in PCR using the method of Saiki et al., Science 239:487 (1988).

Genomic DNA or cDNA fragments isolated by the above methods can be digested with restriction endonucleases to generate appropriate ends, treated with alkaline phosphatase to avoid inappropriate ligation of DNA molecules, and ligated with appropriate ligases, etc. according to conventional techniques. ), inserted into vectors such as phage or cosmid vectors. Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pp. 3.0.5-3.17.5 (1990) ["Ausubel"] disclose techniques for the manipulation of such vectors.

Alternatively, the nucleotide sequence encoding avidin can be obtained by synthesizing a DNA molecule encoding avidin using reciprocally primed long oligonucleotides. See, eg, pages 8.2.8-8.2.13 of Ausubel et al. See also Wosnick et al., Gene 60:115 (1987). Current techniques employing the polymerase chain reaction provide the ability to synthesize genes up to 1.8 kilobase pairs in length. Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR Methods and Applications 2:266 (1993). The nucleotide sequence of the synthetic avidin gene can be based on any known DNA molecule encoding avidin. See eg Gope et al., supra.

These clones can be analyzed by a variety of standard techniques, such as restriction enzyme analysis, Southem analysis, primer extension analysis, and DNA sequence analysis. For example, primer extension assays or S1 nuclease protection assays can be used to map putative initiation sites of transcription of cloned genes. Ausubel pp. 4.8.1-4.8.5; Walmsley et al., "Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protection Assay". 4. Cloning the avidin gene into the vector and preparing transgenic plants

Once the avidin gene is isolated, it is placed into an expression vector using standard methods. See Sambrook et al., supra. Selection of an appropriate expression vector depends on the method of introducing the expression vector into the host cell. Expression vectors typically contain: (1) prokaryotic DNA components encoding bacterial origins of replication and antibiotic resistance genes for growth and selection of expression vectors in bacterial hosts; (2) for insertion of exogenous DNA sequences such as avidin (3) eukaryotic DNA components that control the initiation of transcription of foreign genes, such as promoters, etc.; (4) DNA components that control transcript processing, such as a transcription termination/plus poly(A) sequence, etc.; and (5) a gene encoding a marker protein (such as a reporter gene), wherein the gene is operably linked to a DNA component that controls transcription initiation. A general description of plant expression vectors and reporter genes is found in Gruber et al., "Vector for Plant Transformation," in METHODS IN PLANTMILECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds) pp. 89-119 (CRC Press, 1993).

In a preferred embodiment of the invention, the plant ubiquitin promoter system is used. Plant ubiquitin promoters are well known in the art. See for example European Patent Application 0 342 926, which is incorporated herein by reference. The ubiquitin promoter is a constitutive promoter.

In another preferred embodiment of the invention, an inducible promoter is used for the inducible regulation of the expression of avidin. An example of such an inducible promoter is the corn glutathione S-transferase system. See Weigand et al., Plant Mol. Biol. 7:235 (1986). This method is also used to reversibly induce male sterility. Thus, avidin expression and concomitant male sterility can be controlled by activating the promoter as desired. When the promoter is not activated, avidin is not expressed and the plants are male fertile.

Expression vectors containing the avidin gene can be introduced into protoplasts, or into intact tissues such as immature embryos and meristems, or into callus cultures, or into isolated cells. Expression vectors are preferably introduced into intact tissues. For example, "Procedures for Introducing Foreign DNA into Plants," by Miki et al. in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds) pp. 67-88 (CRC Press, 1993) and in CORN AND CORN IMPROVEMENT, 3rd Edition A general method for culturing plant tissue is provided in "Cell/Tissue Culture and In Vitro Manipulation" by Phillips et al., Sprague et al. (eds), pp. 345-387 (American Society of Agronomy, Inc. et al. 1988) .

The method for introducing the expression vector into plant tissue includes directly infecting plant tissue with Agrobacterium tumefaciens or co-cultivating plant tissue with Agrobacterium tumefaciens. Horsch et al., Science 227:1229 (1985). Preferably, a disarmed Ti plasmid is used as a vector for foreign DNA sequences. Transformation can be carried out using, for example, the procedure described in European Patent Applications 116 718 (1984) and 270 822 (1988). Recommended Ti plasmids contain foreign DNA sequences between the border sequences or at least upstream of the right border sequence.

Using methods such as direct gene transfer (see for example PCT application WO 85/01856 and European application 275 069), in vitro protoplast transformation (for example US patent 4,684,611), plant virus-mediated transformation (for example European application 067 553 and US patent 4,407,956) and Procedures such as liposome-mediated transformation (eg, US Pat. No. 4,536,475), and other types of vectors can be used to transform plant cells. Suitable maize transformation methods are provided by Fromm et al., Bio/Technology 8:833 (1990) and Gordon-Kamm et al., The Plant Cell 2:603 (1990). Standard transformation methods for rice are described by Christou et al., Trends in Biotechnology 10:239 (1992) and Lee et al., Proc. Nat'l Acad Sci. USA 88:6389 (1991). Wheat can be transformed using techniques similar to those used to transform maize or rice. Furthermore, Casas et al., Proc. Nat'l Acad. Sci. USA 90:11212 (1993) describe a method for the transformation of sorghum, while Wan et al., Plant Physiol. 104:37 (1994) describe a method for the transformation of barley.

In general, direct gene transfer methods are preferred for the transformation of monocotyledonous plants, especially cereals such as rice, maize, sorghum, barley or wheat. Suitable direct transfer methods include microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, eg, Gruber et al., supra, Miki et al., supra and Klein et al., Bio/Technology 10:268 (1992). More preferably, the expression vector is introduced into monocotyledonous plant tissue by microprojectile-mediated delivery using a biolistic device. 5. Regulation of male fertility

A. Production of male sterile plants

In order to induce male sterility according to the present invention, an expression vector is constructed in which the DNA sequence encoding avidin is operatively linked to a DNA sequence regulating gene transcription in plant tissues. The general requirements for expression vectors are described above. In order to obtain male sterility by expressing avidin, it is preferable not to express avidin only in a transient manner, but to introduce the expression vector into plant embryonic tissue in such a way that avidin develops in mature plants. late expression. Mitotic stability can be achieved, for example, with plant viral vectors that provide epichromosomal replication.

The recommended method to provide mitotic stability is to integrate the expression vector sequence into the host chromosome. Such mitotic stability can be provided by microprojectile delivery of the expression vector into plant tissue, or by other standard methods as described above. See, eg, Fromm et al., Bio/Technology 8:833 (1990), Gordon-Kamm et al., The Plant Cell 2:603 (1990) and Walters et al., Plant Molec. Biol. 18:189 (1992).

Transcription of the avidin gene after introduction into plant tissue is preferably controlled by a constitutive promoter that non-specifically stimulates expression of the gene in plant tissue. An example of a constitutive promoter suitable for this purpose is the ubiquitin promoter described by supra.

Transcription of the avidin gene can also be controlled by a promoter that stimulates gene expression in a tissue-specific manner. A particularly recommended anther-specific promoter is the 5126 promoter isolated from the maize B73 inbred line. The 5126 promoter stimulates the expression of foreign genes in anthers from the tetrad stage to the early mononucleate microspore stage.

Another suitable anther-specific promoter is the SGB6 promoter, also isolated from the B73 line. The SGB6 promoter can induce the expression of foreign genes in anther tapetum cells from the tetrad stage to the middle mononucleate stage of microspore development.

Alternatively, anther-specific gene expression can be provided using a combination of anther cassette and core promoters. A particularly recommended anther box is the 5126 anther box, which contains the following nucleotide sequence:

5′GCGGCCGCGG ATCCGCTCAT CTGCTCGGTA CCAACCAGCC

CGCCGGCGCC TAGGCGAGTA GACGAGCCAT GGTTGGTCGG

TTTCCTATTA CCATGCACAG ATCT 3' [SEQ ID NO: 1].

AAAGGATAAT GGTACGTGTC TAGA

Another suitable anther box is located in a 94 base pair DNA segment defined by nucleotides 583-490 upstream of the SGB6 transcription start site. The nucleotide sequence of the SGB6 anther box of 94 base pairs is:

(-5 83) ACAGTTCACT AGATATGCAT GATCTTTAAC AATTGCTGCT

TGTCAAGTGA TCTATACGTA CTAGAAATTG TTAACGACGA

GGATTGTGCG GTTTCTTTTG GCACAAATGG CATGAACAGA

CCTAACACGC CAAAGAAAAC CGTGTTTACC GTACTTGTCT

GTAATCCGGG ACGC(-490)[SEQ ID NO:2].

CATTAGGCCC TGCG

Alternatively, a suitable anther cassette is obtained from the maize G9 promoter, which stimulates gene expression during the meiotic to tetrad stages of development. The G9 anther box comprises the following nucleotide sequences:

5′GCGGCCGCGG ATCCTGGCTG GATGAAACCG ATGCGAGAGA

CGCCGGCGCC TAGGACCGAC CTACTTTGGC TACGCTCTCT

AGAAAAAAAA ATTGTTGCAT GTAGTTGGCG CCTGTCACCC

TCTTTTTTTT TAACAACGTA CATCAACCGC GGACAGTGGG

AACCAAACCA GTAGTTGAGG CACGCCCTGT TTGCTCACGA

TTGGTTTGGT CATCAACTCC GTGCGGGACA AACGAGTGCT

TCACGAACeT AGATCT 3' [SEQ ID NO: 3].

AGTGCTTGCA TCTAGA

The 5126, SGB6 and G9 anther cassettes can be obtained by synthesizing oligonucleotides as described above. Those skilled in the art will appreciate that deletion analysis can be performed to map one or more other anther-specific regulatory sequences within the disclosed anther cassette. "Functional fragments" of such anther cassettes can also be used to regulate gene expression of avidin in an anther-specific manner.

Suggested core promoters are obtained from the 5126 core promoter, the SGB6 core promoter, the G9 core promoter, and the cauliflower mosaic virus 35S core promoter.

A particularly recommended core promoter is the 5126 core promoter, which contains the following nucleotide sequence:

5′ AGATCTAAGT AAGGTATATA TGTGCATAGT CTCCTAATTC

TCTAGATTCA TTCCATATAT ACACGTATCA GAGGATTAAG

TTCATTCTTCA ACCTCTAGCT GATTGATCTC TGGTATTTAC

AAGTAGAAGT TGGAGATCGA CTAACTAGAG ACCATAAATG

CACTCTTTCC TTCCTTCCTT CCTTCAATTC TAAATACCAC

GTGAGAAAGG AAGGAAGGAA GGAAGTTAAG ATTTATGGTG

AAATCAAAGT TGCTTTGCCA TG 3' [SEQ ID NO: 4].

TTTAGTTTCA ACGAAACGGT AC

The SGB6 core promoter consists of approximately 38 nucleotides upstream of the transcription initiation site of the SGB6 gene. A suitable SGB6 core promoter has the following nucleotide sequence:

5′ATCTCACCCT ATTAATACCA TGCTGACGAG

TAGAGTGGGA TAATTATGGT ACGACTGCTC

CCAATAGC 3'[SEQ ID NO: 5].

GGTTATCG

A suitable core promoter derived from the G9 gene comprises the nucleotide sequence:

5′AGATCTCTAT AAAACACGCA GGGACTGGAA AGCGAGATTT

TCTAGAGATA TTTTGTGCGT CCCTGACCTT TCGCTCTAAA

CACAGCTGAA AGCAGCAAA ACGCAGAAGC TGCACTGCAT

GTGTCGACTT TCGTCGGTTT TGCGTCTTCG ACGTGACGTA

ACATCGAGCT AACTATCTGC AGCCATG 3' [SEQ ID NO: 6].

TGTAGCTCGA TTGATAGACG TCGGTAC

Functional fragments of the core promoters of 5126, SGB6 and G9 also provide suitable core promoters. Methods for obtaining such functional fragments are described above.

The developmental window of avidin gene expression can be extended using chimeric regulatory elements (an anther cassette comprising one gene and an anther-specific promoter for a second gene). For example, the SGB6 regulatory sequence stimulates gene expression during the tetrad stage of development to the mesouninucleate stage, while the G9 regulatory sequence stimulates gene expression during meiosis and up to the tetrad stage of development. The combination of the SGB6 anther box and the G9 promoter stimulates the transcription of exogenous genes during meiosis and through the middle mononucleate stage of development. Accordingly, various combinations of anther cassettes and anther-specific promoters are particularly useful for the present invention.

A viral core promoter can also be used. Examples of suitable viral core promoters include the cauliflower mosaic virus (CaMV) core promoter, the Scrophulariaceae mosaic virus core promoter, and the like. Gruber et al., supra. The viral core promoter is preferably the CaMV 35S core promoter or a variant thereof.

For selection of transformed cells, the expression vector contains a selectable marker gene such as a herbicide resistance gene or the like. For example, such genes may confer resistance to phosphinothricine, glyphosate, sulfonylureas, atrazine, or imidazolinones. The selectable marker gene is preferably the bar gene or the pat gene encoding phosphinohomoalanine acetyltransferase. The nucleotide sequence of the bar gene can be found in Leemans et al., European Patent Application 0-242-246 (1987) and White et al., Nucleic Acids Res. 18:1062 (1990). The nucleotide sequence of the pat gene is disclosed by Wohlleben et al., Gene 70:25 (1988). Expression of the bar gene or the pat gene confers resistance to herbicides such as glufosinate (sold as ^Basta® and ^Ignite®, among others) and bialaphos ( ^sold as Herbi- ^ace® and Liberty®).

Expression vectors may contain a DNA sequence encoding avidin under the control of a constitutive or anther-specific promoter and a selectable marker gene under the control of a constitutive promoter. Alternatively, the selectable marker gene can be delivered into host cells by "co-transformation" of embryonic tissue in a separate selectable expression vector.

B. Restoration of male fertility in F1 hybrids

The methods described above can be used to generate transgenic male sterile plants for the production of -F1 hybrids in large scale directed crosses between inbred lines. If all egg cells of the transgenic male sterile plants do not contain the recombinant avidin gene, then a fraction of the F1 hybrids will have a male fertile phenotype. On the other hand, if the recombinant avidin gene is present in all egg cells of the transgenic male sterile plants, since ethylene-induced sterility is dominant, then the F1 hybrids will have a male sterile phenotype. Therefore, it may be necessary to employ a male fertility restorer system to produce male fertile F1 hybrids. This fertility restoration system is of particular value in self-fertilizing species when the harvested product is seeds.

Commonly proposed methods of restoring fertility in one transgenic plant line require the generation of a second "restorer" line of transgenic plants. For example, transgenic plants that are male sterile due to expression of the target enzyme are crossed with male fertile plants expressing an inhibitor of the target enzyme as described above. Mariani et al., Nature 357:384 (1992).

In the present case, a similar approach entails the production of transgenic restorer plants expressing a ribozyme targeting avidin mRNA, or expressing antisense avidin. For example, ribozymes can be designed to express endonuclease activity directed to a target sequence in an mRNA molecule. For example, Steinecke et al., EMBO J. 11:1525 (1992) achieved up to 100% inhibition of neomycin phosphotransferase gene expression by ribozymes in tobacco protoplasts. More recently, Perriman et al., Antisense Res. & Devel. 3:253 (1993) used a vector expressing a modified hammerhead ribozyme to inhibit chloramphenicol acetyltransferase activity in tobacco protoplasts. In the context of the present invention, avidin mRNA provides an appropriate target RNA molecule for the ribozyme.

Fertility can be restored in a similar manner using an expression vector containing a nucleotide sequence encoding an antisense RNA. Binding of the antisense RNA molecule to the target mRNA molecule results in a hybrid arrest of translation. Paterson et al., Pro Natl. Acad. Sci. USA 74:4370 (1987). In the context of the present invention, suitable antisense RNA molecules have a sequence complementary to avidin mRNA. In a preferred embodiment of the invention, the antisense RNA is under the control of an inducible promoter. Thus, activation of this promoter restores male fertility.

In another approach, expression vectors can be constructed wherein the expression vector encodes an RNA transcript capable of initiating RNase P-mediated cleavage of the avidin mRNA molecule. Following this approach, an exogenous sequence can be constructed to direct an endogenous ribozyme (RNase P) to the avidin mRNA, which is subsequently cleaved by the cellular ribozyme. US Patent 5,168,053 to Altman et al., Yuan et al., Science 263:1269 (1994). The exogenous sequence preferably comprises a sequence of 10-15 nucleotides complementary to avidin mRNA and a 3'-NCCA nucleotide sequence, wherein N is preferably purine. Id. The exogenous sequence transcript binds to the target mRNA by forming base pairs between the mRNA and the complementary exogenous sequence, thereby initiating RNase P to cleave the mRNA at nucleotides located 5' to the base pair region. Id.

In another method of restoring male fertility, transgenic male sterile plants contain an expression vector containing a prokaryotic regulatory element in addition to a promoter sequence operably linked to the avidin gene. Transgenic male sterile plants are produced which express a prokaryotic polypeptide under the control of the promoter. In the F1 hybrid, the prokaryotic polypeptide binds to the prokaryotic regulatory sequence, repressing the expression of avidin.

For example, the LexA gene/LexA operator system is used to regulate gene expression according to the present invention. See US Patent 4,833,080 (the '080 patent) and Wang et al., Mol. Cell Biol. 13:1805 (1993). More specifically, expression vectors for male sterile plants may contain a LexA operator sequence, while expression vectors for male sterile plants contain a coding sequence for a LexA repressor. In F1 hybrids, the LexA repressor binds to the LexA operator sequence, repressing the transcription of the avidin gene.

LexA operon DNA molecules can be obtained, for example, by synthesizing a DNA fragment containing the well-known LexA operon sequence. See, eg, the '080 patent and Garriga et al., Mol. Gen. Genet. 236:125 (1992). The LexA gene can be obtained by synthesizing a DNA molecule encoding the LexA repressor. Gene synthesis techniques are discussed above, eg Garriga et al., supra describe the LexA gene sequence. Alternatively a DNA molecule encoding the LexA repressor can be obtained from plasmid pBR500. American Type Culture Collection number 67758.

Yet another method of restoring fertility takes advantage of the high affinity of avidin for biotin by spraying developing plants with a biotin solution. The biotin solution also contained a minimal amount of an organic co-solvent (such as DMSO) to ensure complete dissolution of the biotin. Spraying was performed during the meiotic phase of pollen development and repeated at regular intervals until pollen shedding was observed. Intervals between sprayings varied from 1 to 7 days. In a preferred embodiment of the invention, spraying is repeated every 3-5 days.

Those skilled in the art can easily design other male fertility restoration strategies using prokaryotic regulatory systems (such as Lac repressor/lac operator system or trp repressor/trp operator system, etc.).

Accordingly, the invention generally described will be understood more readily by reference to the following examples, which are provided by way of illustration and not limitation of the invention. Example 1: Production of male sterile transgenic maize by highly constitutive expression of avidin

Methods of forming transgenic maize plants have been described in European Patent Application 0 442 174A1 (incorporated herein by reference). The method is briefly described as follows.

PHI5168, a vector carrying the avidin gene under the control of the ubiquitin promoter and containing a PINII terminator sequence, was used to form transgenic maize plants. Figure 1 shows the structure of PHI5168. PHI610, an expression vector carrying the bar gene under the control of a double 35S promoter and carrying a PINII terminator sequence, was co-transformed with an avidin construct, and transgenic plants were made by treating the transformation mixture with bialophos filter. Figure 2 shows the structure of PHI610. According to the method of Green et al., Molecular Genetics of Plants and Animals, editors Downey et al., Academic Press, NY, 20, 147 (1983), the two expression vectors were transformed into embryogenic suspension cultures (obtained from type II embryos in cultures). Murashige and Skoog ("MS") medium (supplemented with 2 mg/L of 2,4-dichlorophenoxyacetic acid ( 2,4-D) and 30 g/L sucrose). Seven days before the experiment, the suspension culture was passed through a 710 micron sieve and the filtrate was kept in MS medium.

Cells were harvested from suspension cultures by vacuum filtration on a Buchner funnel (Whatman No. 614) and 100 ml (fresh weight) of cells were plated in 3.3 cm Petri plates. Cells were dispersed in 0.5 ml of fresh medium to form a thin layer of cells. The uncovered Petri plate was placed in the sample chamber of a particle gun device (manufactured by Biolistics Inc., Geneva NY). Use a vacuum pump to reduce the pressure in the sample chamber to 0.1 atmospheres to reduce the deceleration of microparticles due to air friction. Cells were bombarded with tungsten particles (average diameter approximately 1.2 microns) (GTE Sylvania Precision Materials Group, Towanda, Pennsylvania). Load an equal mixture of PHI5168 and PHI610 onto microparticles by adding 5 μl of DNA in TE buffer (pH 7.7) (1 μg DNA/100λ) to a suspension of 25 μl of 50 mg tungsten particles/ml distilled water in a 1.5 ml Eppendorf tube . Particles agglomerate and settle in the tube.

Cultures of transformed plant cells containing the exogenous gene were grown in 560R (medium) (N6-based medium with 1 mg/ml bialaphos) for 4-8 weeks. This medium selects for cells expressing the Bar gene.

Embryo formation is then induced in embryogenic cultures and the cells are germinated into plants. The somatic embryos observed in the callus maintenance medium were sequentially germinated with both media. First, the callus was transferred to a medium (maturation medium) containing 5.0 mg/L indole acetic acid (IAA) for 10-14 days to continue to proliferate the callus. Transferred callus was 50mg/plate to optimize the yield per unit tissue piece.

Then, the calli were transferred from the "maturation" medium to a second medium (containing a lower concentration of IAA (1 mg/L) compared to the first medium). At this point, the culture was placed in the light. Germinated somatic embryos are characterized by elongated green shoots attached to the root entrance. Somatic embryos were then transferred to medium in culture tubes (150 x 25 mm) and cultured for an additional 10-14 days. At this point, the plants are approximately 7-10 cm tall and of sufficient size and vigor to thrive under greenhouse conditions.

To strengthen the regenerated plants, the plants to be transferred to the growth chamber were removed from the sterile containers and the solidified agar medium from the roots was washed away. The plantlets were placed in commercial potting mix in a growth chamber equipped with a misting device to maintain a relative humidity close to 100% without over-wetting the plant roots. After 3-4 weeks in the humid chamber, the plants were strong enough to be transplanted into field conditions.

Plants in the field were analyzed by observing male sterility. The plants were then further screened for the presence of the avidin gene by PCR and Southern blotting and for the expression of avidin by ELISA using standard methods.

94 plants were analyzed by PCR, of which 53 were found fertile and 41 sterile. When the fertility of each plant was compared to the presence of the avidin gene detected by PCR, a 98% correlation was found between the presence of the gene and plant sterility. Five plants were analyzed in detail for the presence of the avidin gene by Southern blotting. Three plants appeared to contain the avidin gene by Southern analysis. All 3 plants had sterility problems. The 2 plants without the avidin gene were fully fertile. Therefore, there is a 100% correlation between the presence of the avidin gene and infertility problems. Example 2: Generation of male sterile transgenic soybean plants using an Agrobacterium strain containing a binary vector including a DNA sequence encoding avidin

The method for forming transgenic soybean plants is that described in US Patent Application 07/920,409 (incorporated herein by reference). Seeds of soybean (Glycine max) Pioneer variety 9341 were surface disinfected by exposure to sodium hypochlorite emitted in a glass bell jar. 3.5ml of hydrochloric acid (34-37%w/v) was added to 100ml of sodium chloride (5.25%w/v) to generate gas. Expose for 16-20 hours in a container of approximately 1 cubic foot volume. Surface sterilized seeds were stored in Petri dishes at room temperature. Agar solidified medium (B5 basal medium containing a minimum amount of organic matter, Sigma Chemical Catalog No.G5893, 0.32gm/L sucrose, 0.2% w/v 2 -(N-Morpholino)ethanesulfonic acid (MES) (0.3mM)) was plated and cultured at 28°C, with a sunshine length of 16 hours and a cool-white daylight illumination of about 20μEm ^-2 S ^-1 to germinate the seeds . After 3 or 4 days, the seeds for co-cultivation were prepared. The seed coat was removed, and the elongated root 3-4mm below the cotyledon was removed.

Overnight cultures of Agrobacterium tumefaciens strain LBA4404 containing a modified double plasmid (containing the avidin gene), grown to log phase in minimal A medium (containing 1 μg/ml tetracycline), were pooled and incubated at 550 nm Measure the optical density. Place a sufficient volume of culture into 15 ml/conical centrifuge tubes so that upon sedimentation, 1-2 x ¹⁰¹⁰ cells at ¹⁰⁹ cells/ml are collected per tube. Centrifuge at 6,000 xg for 10 minutes to pellet. After centrifugation, discard the supernatant, and keep the centrifuge tube at room temperature until the inoculum is needed, but not more than 1 hour.

Inoculation was performed in batches such that each seed plate was treated with a freshly resuspended Agrobacterium pellet. The cell pellets were resuspended in 20 ml seeding medium containing 3.2 g/LB salt (G5893), 2.0% w/v sucrose, 45 μM 6-benzylaminopurine (BAP), 0.5 μM indolebutyric acid (IBA) , 100 μM acetosyringone (AS), and buffered to pH 5.5 with 10 mM MES. Resuspension is achieved by vortexing. The inoculum was then poured into Petri dishes containing the prepared seeds and the cotyledonary nodes were separated with a scalpel. Separation was accomplished by splitting the seed in half by cutting the shoot apex lengthwise, keeping the two cotyledons intact. Then use a scalpel to pry the two halves of the seedlings away from the corresponding cotyledons. Then, separate the cotyledonary nodes with a scalpel by repeatedly scoring along the axis of symmetry. Be careful not to cut completely through the explant to the axial end. Explants were prepared in approximately 5 minutes and then incubated with bacteria for 30 minutes at room temperature without agitation. After 30 minutes, the explants were transferred to the same media plate solidified with 0.2% w/v Gelrite (Merck & Company Inc.). Embed the explants with their adaxial ends up and flush with the surface of the medium, and culture them at 22°C for 3 days under cool white daylight at approximately 20 μEm ^-2 S ^-1 .

After 3 days, the explants were transferred to liquid counter-selection medium containing 3.2 g/l B5 salt (G5893), 2% w/v sucrose, 5 μM BAP, 0.5 μM IBA, 200 μg/ml vancomycin, 500 μg/ml cefotaxime, buffered to pH 5.7 with 3 mM MES. The explants were washed in each Petri dish for 4 days at room temperature with constant slow rotational agitation. The counter-selection medium was replaced 4 times.

The explants were then picked onto agar-solidified selection medium containing 3.2 g/l B5 salts (G5893), 2% w/v sucrose, 5.0 μM BAP, 0.5 μM IBA, 50 μg/ml Kanamycin sulfate 100 μg/ml vancomycin, 30 μg/ml cefotaxime, 30 μg/ml carboxyphene penicillin-clavulanic acid compound (timentin), buffered to pH 5.7 with 3 mM MES. Selection medium was solidified with 0.3% w/v Seakem agarose. The explants were embedded in the culture medium with the adaxial end down, and cultured at 28°C, under a cool-white daylight illumination of 60-80 μEm ^-2 S ^-1 with a daylight length of 16 hours.

After 2 weeks, the explants were washed again with liquid medium on a rotary shaker. Washing was performed overnight in counter-selection medium containing 50 μg/ml kanamycin sulfate. The next day, explants were picked onto agarose-solidified selection medium. They were embedded in medium adaxially down and cultured for another 2 weeks.

After 1 month on selection medium, regenerated tissue was visible as transformed tissue as green quadrants compared to yellowed non-healthy tissue. Discard explants without green quadrants and transfer explants with green quadrants to elongation medium containing 3.2 g/l B5 salts (G5893), 2% w/v sucrose, 3.3 μM IBA , 1.7 μM gibberellic acid, 100 μg/ml vancomycin, 30 μg/ml cefotaxime, 30 μg/ml carboxyphene penicillin-clavulanic acid complex, buffered to pH 5.7 with 3mM MES. Elongation medium was solidified with 0.2% w/v Gelrite. Embed the green fan-shaped body with its proximal end facing up, and culture it according to the aforementioned method. Cultures were continued on this medium, transferring to fresh plates every 2 weeks. When shoots were 0.5 cm in length, they were cut from the base and placed in rooting medium in 13 x 100 ml test tubes. Rooting medium contained 3.2 g/l B5 salt (G5893), 15 g/l sucrose, 20 μM niacin, 900 mg/L pyroglutamic acid (PGA) and 10 μM IBA. Rooting medium was buffered to pH 5.7 with 3 mM MES and solidified with 0.2% w/v Gelrite. After 10 days, shoots were transferred to the same medium without IBA or PGA. These shoots were rooted and kept in test tubes under the same environmental conditions as above.

When the root system is well established, the plantlets are transferred to sterile soil. The temperature, photoperiod and light intensity were kept the same as above.

Expression of avidin in transgenic soybean plants was confirmed and quantified by ELISA, and the presence of the gene was confirmed by PCR and Southern blotting. Expression stability can be assessed in successive passages using these same methods. Sterility problems were found to be associated with the expression of avidin in soybean. Example 3: Preparation of male sterile sunflower plants by expressing avidin

An expression cassette encoding avidin was used to generate transgenic sunflower plants and seeds. The DNA sequence encoding avidin is inserted into the expression cassette under the control of the ubiquitin promoter.

This expression cassette is then subcloned in a binary vector such as PHI 5765 using EcoRI sites. Then, the binary vector was transferred into the Agrobacterium tumefaciens helper strain.

Sunflower plants were transformed with Agrobacterium strain LBA4404 after microparticle bombardment as described by Bidney et al., Plant Mol. Bio. Vol. 18: p. 301, 1992. Briefly, seeds of Pioneer sunflower line SMF-3 were dehulled and surface sterilized. The seeds were imbibed on water-moistened filter paper for 18 hours at 26°C in the dark. Remove cotyledon and root, meristem explant is cultured in 374BGA medium (MS salt, Shephard vitamin, 40.mg/L adenine sulfate, 3% sucrose, 0.8% plant agar (phytagar) pH 5.6 add 0.5mg/L L BAP, 0.25mg/L IAA and 0.1mg/LGA). After 24 hours, the first leaf was removed, the apical meristem was exposed, and the explants were placed dome side up in a 2 cm circle in the center of a 60 mm x 20 mm Petri plate containing water agar. The explants were bombarded twice with tungsten particles suspended in TE buffer, or with particles attached to the expression plasmid containing the avidin gene. The meristem explants were co-cultured in 374BGA medium for an additional 72 hours at 26°C in the light.

After 72 hours of co-cultivation, Agrobacterium-treated meristems were transferred to 374 medium (374BGA with 1% sucrose, without BAP, IAA or GA ₃ ), supplemented with 250 μg/ml cefotaxime. The plantlets were allowed to develop green or yellow for 2 weeks under culture conditions of 16 hours day length and 26°C. The plantlets were transferred to medium containing kanamycin and allowed to grow. The presence of avidin in the plants was confirmed and quantified as described in Example 2. The presence of male sterility was found to correlate with plant expression of avidin.

While the foregoing description has referred to specific preferred embodiments, it should be understood that the invention is not so limited. Various modifications to the disclosed embodiments will occur to those skilled in the art and such modifications are intended to be included within the scope of the invention as defined in the following claims. All publications and patent applications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains.

Sequence Listing (1) General information:

(i) Applicant:

(A) Name: PIONEER HI-BRED INTERNATIONAL, INC.

(B) Street: 700 CAPITAL SQUARE, 400 LOCUST STREET

(C) City: DESMOINES

(D) State: IOWA

(E) Country: United States

(F) Zip code: 50309

(ii) Title of the invention: Inducing male sterility in plants by expressing high levels of avidin

(iii) Number of sequences: 6

(iv) Corresponding address:

(A) Recipient: Foley&Lardner

(B) Street: 3000 K Street, N.W., Suite 500

(C) City: Washington

(D) State: D.C.

(E) Country: United States

(F) Zip code: 20007-5109

(v) in computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: PatentIn Release#1.0, Version#1.30

(vi) The application data:

(A) Application number: still issued

(B) Application date:

(vii) Prior application data:

(A) Application number: US 08/475,582

(B) Application date: June 7, 1995

(viii) Information of attorney/agent:

(A) Name: Bent, Stephen A.

(B) Registration number: 29,768

(C) Reference/Agency Number: 33229/403/PIHI

(ix) Remote communication information:

(A) Tel: (202)672-5300

(B) Fax: (202)672-5399

(C) Telex: 904136 (2) Information of SEQ ID NO: 1:

(i) Sequence properties:

(A) Length: 64 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi) Sequence description: SEQ ID NO: 1: GCGGCCGCGG ATCCGCTCAT CTGCTCGGTA CCAACCAGCC TTTCCTATTA CCATGCACAG 60ATCT SEQ ID NO: 2 ( 2 )

(i) Sequence properties:

(A) Length: 94 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi) Sequence description: SEQ ID NO: 2: ACAGTTCACT AGATATGCAT GATCTTTAAC AATTGCTGCT GGATTGTGCG GTTTCTTTTG 60GCACAAATGG CATGAACAGA GTAATCCGGG ACGC    

(i) Sequence properties:

(A) Length: 136 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi)序列描述：SEQ ID NO：3：GCGGCCGCGG ATCCTGGCTG GATGAAACCG ATGCGAGAGA AGAAAAAAAA ATTGTTGCAT    60GTAGTTGGCG CCTGTCACCC AACCAAACCA GTAGTTGAGG CACGCCCTGT TTGCTCACGA    120TCACGAACGT AGATCT                                                    136(2)SEQ ID NO：4的资料：

(i) Sequence properties:

(A) Length: 142 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi)序列描述：SEQ ID NO：4：AGATCTAAGT AAGGTATATA TGTCATAGT CTCCTAATTC TTCATCTTCA ACCTCTAGCT    60GATTGATCTC TGGTATTTAC CACTCTTCC TTCCTTCCTT CCTTCAATTC TAAATACCAC    120AAATCAAAGT TGCTTTGCCA TG                                            142(2)SEQ ID NO：5的资料：

(i) Sequence properties:

(A) Length: 38 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi) Sequence description: SEQ ID NO: 5: ATCTCACCCT ATTAATACCA TGCTGCGAG CCAATAGC 38 (2) Information of SEQ ID NO: 6:

(i) Sequence properties:

(A) Length: 107 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(xi) Sequence description: SEQ ID NO: 6: AGATCTCTAT AAAACACGCA GGGACTGGAA AGCGAGATTT CACAGCTGAA AGCAGCAAA 60ACGCAGAAGC TGCACTGCAT ACATCGAGCT AACTATCTGC AGCCATG 107

Claims (26)

1. isolated DNA molecule comprises a nucleotide sequence of the coding avidin that operationally is connected with the plant promoter sequence.

2. expression vector comprises the isolated DNA molecule of claim 1.

3. according to the expression of claim 2, wherein said plant promoter sequence is a constitutive promoter.

4. according to the isolated DNA molecule of claim 3, wherein said constitutive promoter sequence is the ubiqutin promoter sequence.

5. transgenic plant, wherein said plant comprises a heterologous nucleotide sequence, and the latter comprises an avidin gene,

Wherein said heterologous nucleotide sequence increases the concentration of avidin in this plant tissue, and

Described thus higher avidin concentration makes described plants male sterility.

6. according to the transgenic plant of claim 5, wherein said plant is a milpa.

7. according to the transgenic plant of claim 5, wherein said plant is a soybean plant strain.

8. according to the transgenic plant of claim 5, wherein said plant is the Sunflower Receptacle plant.

9. according to the transgenic plant of claim 5, wherein said heterologous nucleotide sequence also comprises a constitutive promoter sequence.

10 transgenic plant according to claim 9, wherein said constitutive promoter sequence is the ubiqutin promoter sequence.

11. produce the method for transgenosis male sterile plants, comprise that an expression vector that will comprise a promotor and an avidin gene imports in the vegetable cell, wherein said promotor is controlled described avidin expression of gene, and described thus avidin expression of gene causes male infertility.

12., also comprise by described vegetable cell regeneration of transgenic plant according to the method for claim 11.

13. according to the method for claim 11, wherein said promotor comprises the ubiqutin promotor.

14. the method with avidin gene generation male-fertile hybrid plant may further comprise the steps:

(a) produce the first parent's male sterile plants comprise according to according to the dna molecular of claim 1, wherein the expression of avidin causes male infertility;

(b) produce the second transgenosis stock plant of expressing second foreign gene;

(c) described first parent and described second parent's cross fertilization, plant hybridizes.

Wherein said hybrid plant is expressed described second foreign gene, and the product of described second foreign gene reduces the expression of avidin in the described hybrid plant, produces the male-fertile hybrid plant thus.

15. according to the method for claim 14, wherein said second foreign gene is selected from inverted defined gene, ribozyme gene and leads sequence gene outward.

16. the method for claim 15, wherein said inverted defined gene comprise the mRNA sequence of avidin.

17. the method for claim 16, wherein said ribozyme gene comprise the mRNA sequence of avidin.

18. the method for claim 16, the wherein said outer mRNA sequence that sequence gene comprises avidin of leading.

19. the method for claim 14, the described dna molecular of wherein said first mother plant also comprises the LexA operon that operationally is connected with described promotor, and described second foreign gene is a LexA repressor gene.

20. the expression vector of claim 2, wherein said promoter sequence are to comprise the anther specific promoter sequence that is selected from following flower pesticide frame:

(a) has the dna molecular of SEQ ID NO:1 nucleotide sequence;

(b) has the dna molecular of SEQ ID NO:2 nucleotide sequence;

(c) has the dna molecular of SEQ ID NO:3 nucleotide sequence;

(d) (a) and (b) or a functional fragment (c).

21. the expression vector of claim 20, wherein said flower pesticide frame has the nucleotide sequence of SEQ ID NO:1, and described anther specific promoter also comprises and is selected from a following core promoter:

(a) CaMV 35S core promoter;

(b) has the dna molecular of SEQ ID NO:4 nucleotide sequence;

(c) has the dna molecular of SEQ ID NO:5 nucleotide sequence;

(d) has the dna molecular of SEQ ID NO:6 nucleotide sequence;

(e) (b), (c) or a functional fragment (d).

22. the expression vector of claim 20, wherein said flower pesticide frame has the nucleotide sequence of SEQ ID NO:2, and described anther specific promoter also comprises and is selected from a following core promoter:

(a) CaMV 35S core promoter;

(b) has the dna molecular of SEQ ID NO:4 nucleotide sequence;

(c) has the dna molecular of SEQ ID NO:5 nucleotide sequence;

(d) has the dna molecular of SEQ ID NO:6 nucleotide sequence;

(e) (b), (c) or a functional fragment (d).

23. the expression vector of claim 20, wherein said flower pesticide frame has the nucleotide sequence of SEQ ID NO:6, and described anther specific promoter also comprises and is selected from a following core promoter:

(a) CaMV 35S core promoter;

(b) has the dna molecular of SEQ ID NO:4 nucleotide sequence;

(c) has the dna molecular of SEQ ID NO:5 nucleotide sequence;

(d) has the dna molecular of SEQ ID NO:6 nucleotide sequence;

(e) (b), (c) or a functional fragment (d).

24. give the method for recovering fertility in the male sterile plant by the expression of avidin, comprise with biotin solution and spray described plant.

25. the method for claim 11, wherein said promotor comprises evoked promoter.

26. the method for claim 15, wherein said inverted defined gene operationally is connected with evoked promoter.

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