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

Abstract

Male-sterile plants can be produced by increasing the endogenous concentration of avidin in the plant tissues. This effect can be achieved by producing transgenic plants containing an expression vector in which a promoter is operably linked to a DNA sequence encoding avidin. Methods for restoring male fertility are disclosed.

Description

By expressing high-caliber avidin inducing plant male infertility

Background of invention

The present invention relates to use the method for the dna molecular controlling plant fertility of coding avidin.Particularly, the present invention relates to produce the method for the transgenosis male sterile plants of expressing avidin.In addition, the present invention relates in the male sterile plants filial generation, recover the method for male fertile.

Being controlled in the hybrid crop production of pollen fertility is important, referring to for example J.M.Poehlman, and BREEDING FIELD CROP, 3rd ed. (Van Nostrand andReinhold, New York, NY, 1987), this book is attached to herein by reference.For example in hybrid corn produced, before pollen distributed, physics was removed male inflorescence or tassel usually, reached the control of pollen fertility.The workload of artificial emasculation fringe is very big.Though machinery castration fringe is littler than the workload of artificial emasculation fringe, its reliability is less, need check crop subsequently, and may need to carry out remedial artificial emasculation fringe.Castrate the loss that the fringe method all causes maternal output for two kinds.

Yet most of important staple crops have male and female two function organs in same spending; Therefore, castrating is not a simple operation.Form organ although can remove pollen by hand before pollen distributes, the workload that this mode hybrid produces is very big, and extremely expensive.

Also can have the leaf sprays control pollen fertility of microgamete character extremely by application.People such as Cross, Sex Plant Reprod.4:235 (1991).For example ethrel is spread on the flower pesticide, cause the extra mitotic division of wheat pollen, cause the barley tapetal cell to degenerate, cause thrush to belong to pollen deformity or pollen abortion.People such as Bennet, Nature 240:566 (1972), people such as Colhoun, Plant Cell Environ.6:21 (1983); People such as Berthe, Crop Sci.18:35 (1978).Yet the workload of chemical process is big, and has the potential problems that pharmaceutical chemicals toxicity imported environment.

In addition, the industrial production of the hybrid seed of use gametocide is subjected to the restriction of expense and pharmaceutical chemicals operability, also is subjected to the restriction of dispenser reliability and action time.A serious restriction of gametocide is that they have phytotoxic effect, and its seriousness depends on genotype.Other restriction comprises that these pharmaceutical chemicalss may be invalid than long crop to the florescence, because newly spending of producing may be unaffected.Therefore, need to reuse pharmaceutical chemicals.

The many industrial hybrid seed production system of field crop depends on the genetic method of pollination control at present.As maternal plant or can not loose powder, produce and can not carry out self-fertile pollen on biochemical, perhaps can not make pollen.Can not be called " selfing is not affine " by self-fertile plant.The difficulty relevant with using the self incompatibility system comprises selfing not operability and the breeding thereof of affine maternal side, and the stability of self incompatibility.In some cases, available chemical process overcomes self incompatibility, or before the biochemical mechanism of blocking-up pollen is activated the prematurity bud is carried out artificial pollination.The self incompatibility system that can lose efficacy is subject to the injury of adverse weather condition usually very much, and the latter destroys or reduce the effectiveness of biochemical blocking-up self-pollination.

The production of industry seed is extensively interested to be to cause male sterile system based on hereditary pollen control.There are two general types in these systems: (1) kernel male sterile, this is because one or more nuclear genes are undergone mutation, can not form pollen, or the male infertility of (2) plasma inheritance, be commonly referred to " cytoplasmic male sterility " (CMS), wherein because of the change of tenuigenin organoid (being generally plastosome), pollen forms and is blocked or pollen abortion.

The nuclear sterility can or dominance, or recessive.If can carry out maternal nourishing and generating or vegetative propagation, the dominance sterility can only be used for the formation of hybrid seed so.If sterility plant or fertility plant are easy to difference, can use recessive sterility so.Yet, the industrial applicibility of dominance sterility system and recessive sterility system be subjected to respectively vegetative expense and certainly flower can harvest the restriction of the maternal roguing of plant.

Relate to the crossing scheme that uses CMS although have, its industrial value is restricted.The example of a CMS system is a specific mutant that is arranged in cytoplasmic plastosome, causes forming mature pollen under suitable nuclear background.In some cases, the nuclear background can compensate cytoplasmic mutation, forms normal pollen.Specificity nuclear " recovery gene " makes that having the mitochondrial plant of CMS forms pollen.In general, use CMS to carry out industrial seed production and relate to three breeding systems of use: a male sterile line (female parent); A maintenance line with the male sterile line homology, but contains the plastosome of complete function; With a male parent system.Male parent system can carry or can not carry the tenuigenin specificity and recover gene.

For the seed such as hybrid the vegetables reclaims unessential crop, can use the CMS system, and need not recover.For the crop that the fruits and seeds of hybrid is Industrial products, must recover the fertility that gene recovers hybrid seed with the specificity of male parent, maybe must pollinate to the male sterile hybrid.Can finish by pollinating with the male-fertile plants of small proportion.In most of species, because all tenuigenin organoid is all only by ovum heredity, so the CMS feature is a maternal inheritance.

The CMS system has some restrictions, can hinder them as the unique method of producing male sterile plants.For example, a kind of special CMS type (T tenuigenin) of corn is given the susceptibility of the toxin that specific fungi infestation is produced.Though many crops are still used the CMS system, it may break down under some envrionment conditions.

Therefore, obviously be starved of the method that the control pollen except that artificial, machinery, chemistry and conventional genetic method is produced.

Summary of the invention

Therefore, an object of the present invention is to provide the method that produces male sterile plants, promptly make plant sterile by the heterogenous expression avidin.

Another object of the present invention provides a mosaic gene, and this gene comprises the dna sequence dna of the avidin of encoding, and functionally is connected with the plant promoter sequence.

Another object of the present invention is the method that proposes to reverse the male infertility that causes because of the expression avidin.

According to one embodiment of the invention,, reach these purposes and other purpose by the DNA isolation molecule that comprises the coding avidin nucleotide sequence that functionally is connected with plant promoter is provided.In a recommended embodiment, this separated DNA sequence is contained in the expression vector.In another recommended embodiment, this plant promoter sequence is a constitutive promoter, and in another recommended embodiment, constitutive promoter is the ubiqutin promotor.

According to another embodiment of the present invention, one transgenic plant are provided, and wherein this plant comprises heterologous nucleotide sequence, and the latter comprises the avidin gene, and this heterologous nucleotide sequence improves the concentration of avidin in the plant tissue, causes male infertility.In a recommended embodiment, transgenic plant are milpa, soybean plant strain or Sunflower Receptacle plant.In another recommended embodiment, this allogeneic dna sequence DNA molecule also comprises a constitutive promoter sequence, and the latter is the ubiqutin promoter sequence in a recommended embodiment.

According to another aspect of the present invention, the method that produces the transgenosis male sterile plants is provided, comprise and to comprise a promotor and avidin expression carrier imports in the vegetable cell, wherein promotor is controlled the avidin expression of gene, and the avidin expression of gene causes male infertility.In a recommended embodiment, by this vegetable cell regeneration of transgenic plant.In another recommended embodiment, this promotor comprises ubiqutin promotor or inducible promoters.

According to another aspect of the present invention, the method for using the avidin gene to produce the male sterile hybrid plant is provided, comprise producing the first parent's male sterile plants that comprises above-mentioned dna molecular that wherein the expression of avidin causes male infertility; Produce the second transgenosis stock plant of expressing second foreign gene; And first parent and second parent's cross fertilization, produce the hybrid plant of expressing second foreign gene, wherein the product of second foreign gene reduces the expression of avidin in the hybrid plant, produces the male-fertile hybrid plant thus.

In the recommended embodiment of the present invention aspect this, second foreign gene is selected from inverted defined gene, ribozyme gene and leads sequence (extemal guide sequence) gene outward.In another recommended embodiment, inverted defined gene comprises avidin mRNA sequence, in a recommended embodiment again under this mRNA sequence control at inducible promoters.In another recommended embodiment, ribozyme gene comprises avidin mRNA sequence.In a recommended embodiment again, lead sequence gene outward and comprise avidin mRNA sequence.In a recommended embodiment again, the dna molecular of first stock plant also comprises the LexA operon that functionally is connected with described promotor, and wherein second foreign gene is a LexA repressor gene.In another recommended embodiment, promoter sequence is the anther specific promoter sequence that comprises a flower pesticide frame, and the flower pesticide frame 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 their functional fragment.

In the again recommended embodiment of the present invention aspect this, the flower pesticide frame has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and anther specific promoter also comprises a core promoter, and this core promoter is selected from the nucleotide sequence of CaMV 35S core promoter, SEQ ID NO:4, the nucleotide sequence of SEQ ID NO:5, the nucleotide sequence of SEQ ID NO:6 and their functional fragment.

According to another aspect of the present invention, provide to make because of expressing the method for the male sterile plant recovery fertility of avidin, comprise to plant and spray biotin solution.

The accompanying drawing summary

Fig. 1 represents avidin coding plasmid PHI5168.

The plasmid PHI610 of Fig. 2 presentation code bar gene.

Detailed description 1. definition of recommended embodiment

In the following description, use in a large number a plurality of terms. For the ease of understanding the present invention, provide To give a definition.

Structural gene is for being transcribed into mRNA (mRNA), being translated as the ammonia of specific polypeptide then The dna sequence dna of base acid sequence feature.

Foreign gene refers to functionally regulate and control composition with at least one allos in this manual The dna sequence dna that connects. For example, if regulate and control the composition control by the anther-specific of 5126 genes System, any gene except corn 5126 structural genes all is considered to foreign gene so.

Promoter is for instructing gene (such as structural gene, antisense gene, ribozyme gene or lead order outward The dna sequence dna of row gene etc.) transcribing. Promoter is usually located at the gene 5 ' district, approaches to have transcribed The beginning site. If promoter is inducible promoters, speed is transcribed in increase corresponding to inducer so Rate. On the contrary, if promoter is constitutive promoter, transcription rate is not subjected to inducer so Regulation and control. Plant promoter is the promoter sequence that instructs genetic transcription in the plant tissue.

Core promoter contains the basic nucleotide sequence of promoter function, comprise the TATA frame and Transcriptional start point. According to this definition, core promoter lack enhanced activity or give organize special In the situation of the particular sequence of property activity, may have or not have detectable activity. For example, The SGB6 core promoter by SGB6 genetic transcription initiation site 5 ' direction (5 '-ward) about 38 Individual nucleotides forms, and the 35S core promoter of cauliflower mosaic virus (CaMV) is by the 35S base Because about 33 nucleotides of the transcription initiation site 5 ' direction of group form.

Tissue-specific promoter is so a kind of dna sequence dna, when it functionally with one When gene connected, the transcriptional level of this gene that it instructs was given birth to than this in organism particular organization Some or all of other tissues in the object are high. For example, anther specific promoter is planted for instructing The dna sequence dna that the related gene higher level is transcribed in the thing anther tissue. For example, SGB6 flower pesticide Specificity promoter can instruct foreign gene to express in anther tissue, and not root tissue or Express in the plumule tissue.

Here used " anther specific promoter " comprises two functional components: " a flower The medicine frame " and a core promoter. Specific flower pesticide frame may have the function of standard enhancer Feature. The combination of flower pesticide frame and core promoter can be than independent core promoter more The ground stimulated gene is expressed. Even derive from heterogeneic flower pesticide frame and core promoter containing Also be like this in the situation of chimeric anther specific promoter. The chimeric flower pesticide spy of this class is described below Opposite sex regulation and control composition.

Operon is the dna molecular that is positioned at the gene 5 ' direction, and it contains by aporepressor and identifies And the nucleotide sequence of combination. The combination of the operon of aporepressor and its identification causes gene to turn to The inhibition of record. For example, the LexA gene code aporepressor of being combined with the LexA operon.

The dna molecular that separates is the dna segment that is separated by organism DNA. For example, compile Code avidin gene cloning dna molecular is the dna molecular of a separation. Separate Another example of dna molecular be the dna molecular of chemical synthesis or enzyme process preparation CDNA, its unconformity enters the genomic DNA of organism.

Enhancer is for increasing the DNA regulation and control composition of transcribing efficient.

The single strand dna of complementary DNA (cDNA) for forming by the mRNA template with enzyme (ThermoScript II).Usually, the part complementary primer with mRNA is used for the initial of reverse transcription.Those skilled in the art also use term " cDNA ", are meant the double chain DNA molecule of being made up of this single strand dna and its complementary dna chain.

The biosynthesizing that is meant gene product expressed in term.For example, under the situation of structure gene, express to relate to that structure gene is transcribed into mRNA, then mRNA is translated as one or more polypeptide.

Cloning vector is a kind of dna molecular such as plasmid, clay or phage, it can be in host cell self-replicating.Cloning vector contains one or minority restriction enzyme enzyme recognition site usually, can insert exogenous DNA array in the mode of determining on these sites, and not lose the basic biological function of carrier; Also contain marker gene, the latter is applicable to identification and the screening with the cloning vector cell transformed.Marker gene generally includes the gene that tetracyclin resistance or amicillin resistance are provided.

Expression vector is included in a kind of dna molecular of the gene of expressing in the host cell.Genetic expression is usually under the control of (comprising constitutive promoter or inducible promoters, tissue specificity regulation and control composition and enhanser) of some regulation and control composition.We say that this gene " functionally is connected " with the regulation and control composition.

Recombinant host can be for any or contain a cloning vector or contain the prokaryotic cell prokaryocyte or the eukaryotic cell of an expression vector.This term also comprises those prokaryotic cell prokaryocytes or the eukaryotic cell that carries out genetic engineering, contains one or more clone genes in the karyomit(e) of host cell or genome.

Transgenic plant are to have one or more plants that contain the vegetable cell of foreign gene.

In eukaryote, rna plymerase ii catalytic structure gene transcription produces mRNA.Can design a dna molecular, make its template that contains rna plymerase ii, wherein the sequence of rna transcription thing and certain specific mRNA complementation.This rna transcription thing is called sense-rna, and the dna sequence dna of encoding antisense RNA is called inverted defined gene.Antisense rna molecule can combine with the mRNA molecule, causes the inhibition of mRNA translation.

Ribozyme is a kind of RNA molecule that contains a catalytic center.This term comprises the RNA enzyme, self splices RNA and self cutting RNA.The dna sequence dna of encoding ribozyme is called ribozyme gene.

Leading sequence outward is a kind of RNA molecule, and it causes this mRNA to be cut by RNA enzyme P mRNA in the born of the same parents of an endogenous ribozyme (RNA enzyme P) guiding particular types.The outer dna sequence dna of leading sequence of coding is called the outer sequence gene of leading.2. general introduction

The present invention provides the method that produces male sterile plants by the transgenic plant of preparation expression avidin.Avidin can be in non-tissue specificity mode with constitutive expression, or can express in the anther-specific mode.The method of recovering male fertile also is provided.

Separate the avidin gene, be inserted into and be suitable for importing in the expression vector of plant tissue.Expression vector also comprises a promotor, and the latter can be constitutive promoter, non-tissue-specific promoter (such as ubiqutin promotor etc.), tissue-specific promoter's (such as anther specific promoter etc.) or inducible promoters.Expression vector is imported in the plant tissue screening and breeding transgene plant by standard method.The transgenic plant of expressing avidin are male sterile.Suppress the avidin genetic transcription or suppress second gene that avidin mRNA translates by coexpression in transgenic plant, can recover male fertile.Perhaps, can recover male fertile by spraying the plant that grows with biotin solution.3. the separation of the dna molecular of coding avidin

The nucleotide sequence that can separate the coding avidin with currently known methods.For example, can be with people such as Gope, the described method of Nucleic Acids Res.15:3595 (1987) (this paper is attached to herein by reference) is separated the cDNA of the clear avidin of coding ovum gallinaceum from chicken salpingo cDNA library.

Perhaps, can be by the genomic clone that obtains the avidin gene in the genomic dna of the organism of known generation avidin.For example, can be with people such as Keinanen, the described method of Eur.J.Biochem.220:615 (1994) is by clone chicken avidin gene in the chicken genomic dna.

Also can adopt polymerase chain reaction (PCR), separate the avidin gene with standard method.Referring to Erlich, PCR TECHNOLOGY:PRINCIPLES ANDAPPLICATIONS FOR DNA AMPLIFICATION (Stockton Press, NY, 1989) and people such as Innis, PCR PROTOCOLS:A GUIDE TO METHODS ANDAPPLICATIONS (Academic Press, San Diego, 1990).Such as ELONGASE ^TMSystem (Life Technologies, Inc., Gaithersburg, MD) the pcr amplification method of and so on longer nucleotide sequence also can be used for obtaining long nucleotide sequence, such as the genomic clone of coding avidin.With 5 ' terminal and 3 ' terminal complementary PCR primer of known avidin gene order can be with commercially available oligonucleotide synthesizer, (Foster City, CA) Gong Ying those synthesizers synthesize such as Applied Biosystems.In a recommended embodiment, this primer comprises other nucleotide sequence that contains the restriction endonuclease cleavage site.The existence in this class site makes after handling with suitable restriction enzyme, with PCR product directed cloning in suitable cloning vector.Referring at CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, people Eds such as Ausubel (JohnWiley ﹠amp; Sons, New York, 1987) " the MolecularClonig of PCR Products " of Finny in the 15.7.1 page or leaf.

The template DNA of PCR can or cDNA or genomic dna, can be with method well-known in the art by the preparation of suitable organism.People such as Sambrook, MOLECULAR CLONING:A LABORATORY MANUAL, SecondEdition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989).Can use such as Triazol ^TM(Life Technologies, Inc., Gaithersburg, MD) and so on commercial reagent is directly by birds, Reptilia or Amphibians tissue preparation genomic dna.Perhaps, can (Pharmacia, Piscataway NJ), use the cDNA by the mRNA of chicken salpingo tissue extraction preparation coding avidin with commercially available medicine box.Use standard technique, use poly (dT) or sexamer primer at random, with this mRNA preparation as cDNA synthetic template.Referring to people such as Sambrook, supra.Then, carry out cDNA with commercially available medicine box (Pharmacia) and synthesize, use people such as Saiki, the method for Science 239:487 (1988) is directly used in PCR with cDNA.

Can be (suitably terminal with isolating genomic dna of aforesaid method or cDNA segment such as using the restriction enzyme enzyme liberating to produce according to routine techniques, use alkaline phosphatase treatment to avoid the inappropriate connection of dna molecular, and with suitable ligase enzyme connection etc.), insert in the carrier such as phage or cosmid vector.People such as Ausubel (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, 3.0.5-3.17.5 page or leaf (1990) [" Ausubel "] discloses the operative technique of this class carrier.

Perhaps, adopt and to cause long oligonucleotide mutually, the dna molecular of composite coding avidin, the nucleotide sequence of the avidin that can obtain to encode.Referring to people 8.2.8-8.2.13 pages or leaves such as for example Ausubel.Also referring to people such as Wosnick, Gene 60:115 (1987).Adopt the technology of polymerase chain reaction that the synthetic ability of 1.8 kilobase to the gene of length that be to the maximum is provided at present.People such as Adang, Plant Molec.Biol.21:1131 (1993); People such as Bambot, PCR Methods and Applications 2:266 (1993).The nucleotide sequence of synthetic avidin gene can be based on the dna molecular of any known coding avidin.Referring to people such as for example Gope, supra.

Can use these clones of multiple standards technical Analysis such as restriction endonuclease analysis, Southem analysis, primer extension analysis and dna sequence analysis.For example primer extension analysis or S1 RNase protection analysis can be used for the supposition initiation site that clone gene is transcribed is located.Ausubel 4.8.1-4.8.5 page or leaf; People such as Walmsley, at METHODS INMOLECULAR BIOLOGY, VOL.7:GENE TRANSFER ANDEXPRESSION PROTOCOLS, Murray (ed.), " the Quantitative and Qualitative Analysis of ExogenousGene Expression by the S1 Nuclease Protection Assay " in the 271-281 page or leaf (HumanaPress Inc.1991).4. arrive the avidin gene clone in the carrier and the preparation transgenic plant

In case isolate the avidin gene, then put it in the expression vector with standard method.Referring to Sambrook etc., supra.Suitably the method that expression vector is imported host cell is depended in the selection of expression vector.Expression vector contains usually: the procaryotic DNA composition of (1) coding bacterium replication origin and antibiotics resistance gene is used for the growth and the screening of host bacterium expression vector; (2) be used to insert the cloning site of exogenous DNA array (for example avidin gene); (3) control transcription of foreign genes initial eukaryotic DNA composition is such as promotor etc.; (4) the DNA composition of control transcript processing is such as a Transcription Termination/the add sequence of poly (A) etc.; And the gene of (5) coded markings albumen (for example reporter gene), wherein this genetic manipulation ground is connected with the DNA composition of control transcription initiation.At METHODS IN PLANTMILECULAR BIOLOGY AND BIOTECHNOLOGY, people such as Glick (eds) 89-119 page or leaf (CRC Press, the general description that plant expression vector and reporter gene are arranged in people such as Gruber " Vector for PlantTransformation, " 1993).

In a recommended embodiment of the present invention, use plant ubiqutin promoter systems.Plant ubiqutin promotor is well-known in the art.Referring to for example european patent application 0 342 926, this patent application is attached to herein by reference.The ubiqutin promotor is a constitutive promoter.

In another recommended embodiment of the present invention, use inducible promoters antibiont fibroin to express and carry out induction regulating controlling.The example of this inducible promoters is the glutathione s-transferase system of corn.Referring to people such as Weigand, Plant Mol.Biol.7:235 (1986).This method also is used for the reversible male infertility of inducing.Therefore, the expression of avidin and the male infertility of following can be controlled by activating promotor as required.When not activating promotor, avidin is not expressed, and plant is a male-fertile.

Can import in the protoplastis containing the avidin expression carrier, or import in the complete tissue (such as immature embryo and meristematic tissue), or import in the callus culture thing, or import in the isolated cells.Expression vector preferably imports in the complete tissue.For example at METHODS IN PLANT MOLECULAR BIOLOGY ANDBIOTECHNOLOGY, people such as Glick (eds) 67-88 page or leaf (CRC Press, 1993) in people such as Miki " Procedures for Introducing Foreign DNA into Plants; " with at CORN AND CORN IMPROVEMENT, 3rd Edition, people such as Sprague (eds), the general method of culturing plants tissue is provided in people such as the middle Phillips of 345-387 page or leaf (American Society of Agronomy, Inc.et al.1988) " Cell/Tissue Culture and In Vitro Manipulation ".

The method that expression vector is imported plant tissue comprises agrobacterium tumefaciens direct infection plant tissue or plant tissue and agrobacterium tumefaciens is cultivated altogether.People such as Horsch, Science 227:1229 (1985).The most handy Ti-plasmids of disarming is as the carrier of exogenous DNA array.For example can adopting, step described in the european patent application 116 718 (1984) and 270 822 (1988) transforms.The Ti-plasmids of recommending between border sequence or at least on the right upstream of boundary's sequence contains exogenous DNA array.

Employing such as direct gene shifts (referring to for example PCT application WO 85/01856 and European application 275 069), (for example United States Patent (USP) 4 for external protoplast transformation, 684,611), the conversion of plant virus mediation (for example European application 067 553 and United States Patent (USP) 4,407,956) and liposome-mediated conversion (for example United States Patent (USP) 4,536,475) and so on step can be with the carrier transformed plant cells of other type.People such as Fromm, people such as Bio/Technology 8:833 (1990) and Gordon-Kamm, The Plant Cell 2:603 (1990) provides suitable corn method for transformation.People such as Christou, people such as Trends in Biotechnology10:239 (1992) and Lee, Proc.Nat ' l Acad Sci.USA 88:6389 (1991) has described the standard conversion method of paddy rice.Can employing and the similar technique transformed wheat of maize transformation or paddy rice.People such as Casas in addition, Proc.Nat ' l Acad.Sci.USA 90:11212 (1993) has described the method that transforms Chinese sorghum, and people such as Wan, Plant Physiol.104:37 (1994) has described the method that transforms barley.

In general, the direct gene transfer method is preferred for monocotyledonous conversion, particularly the cereal such as paddy rice, corn, Chinese sorghum, barley or wheat.Suitable direct transfer method comprises the transmission, DNA injection, electroporation of little bullet (microprojectile) mediation etc.Referring to people such as for example Gruber, people such as supra, Miki, people such as supra and Klein, Bio/Technology10:268 (1992).More preferably use biological trajectory (biolistic) device, the transmission that mediates by little bullet imports expression vector in the monocotyledons tissue.5. the regulation and control of male fertile

A. the generation of male sterile plants

In order to induce according to male infertility of the present invention, make up an expression vector, the dna sequence dna of the avidin of wherein encoding functionally is connected with the dna sequence dna of genetic transcription in the regulation and control plant tissue.The general requirement of expression vector has more than been described.In order to obtain male infertility by expressing avidin, avidin preferably is not only to express in instantaneous mode, but expression vector is imported the plant embryos tissue by this way, make avidin express late period in the growth of ripe plant.For example adopt the plant viral vector that the table chromosome duplication is provided, can reach mitotic stability.

It is that the expression vector sequence is incorporated in the host chromosome that the recommend method that obtains mitotic division stability is provided.Expression vector is sent in the plant tissue by little bullet, or adopts above-mentioned other standard method, this mitotic division stability can be provided.Referring to people such as for example Fromm, people such as Bio/Technology 8:833 (1990), Gordon-Kamm, people such as The Plant Cell 2:603 (1990) and Walters, Plant Molec.Biol.18:189 (1992).

The best constitutive promoter of being expressed in plant tissue by the nonspecific stimulation gene of transcribing behind the avidin gene transfered plant tissue is controlled.An example that is applicable to the constitutive promoter of this purpose is the described ubiqutin promotor of supra.

The avidin gene transcription also can be by controlling with tissue specificity mode stimulated gene expression promoter.The anther specific promoter of special recommendation is by isolating 5126 promotors of corn B73 self-mating system.5126 promotors stimulate the flower pesticide expression alien gene from the tetraspore phase to mononuclear microspore phase morning.

Another suitable anther specific promoter is the SGB6 promotor, and it is also separated by B73 system.The SGB6 promotor can be in the expression of tetraspore phase of microspore development induction exogenous gene to the flower pesticide tapetal cell of middle monokaryotic stage.

Perhaps, adopt the combination of flower pesticide frame and core promoter, the genetic expression of anther-specific can be provided.The flower pesticide frame of special recommendation is 5126 flower pesticide frames, comprises 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 flower pesticide frame is arranged in the dna segment of 94 base pairs, and this segment is by the 583-490 position Nucleotide definition of SGB6 transcription initiation site upstream.The nucleotides sequence of the SGB6 flower pesticide frame of these 94 base pairs is classified as:

(-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

Perhaps, obtain suitable flower pesticide frame by corn G9 promotor, it is expressed at meiophase to the tetraspore phase stimulated gene of growing.G9 flower pesticide frame comprises following nucleotide sequence:

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

Can be according to the method described above by synthetic oligonucleotide acquisition 5126, SGB6 and G9 flower pesticide frame.Skilled person in the art will appreciate that and to carry out deletion analysis, with one or more other anther-specific regulating and controlling sequence location in the disclosed flower pesticide frame." functional fragment " of this class flower pesticide frame also can be used for the genetic expression of avidin of anther-specific mode.

The core promoter of recommending derives from 5126 core promoters, SGB6 core promoter, G9 core promoter and cauliflower mosaic virus 35S core promoter.

The core promoter of special recommendation is 5126 core promoters, and it comprises following nucleotide sequence:

5′AGATCTAAGT?AAGGTATATA?TGTGCATAGT?CTCCTAATTC

TCTAGATTCA?TTCCATATAT?ACACGTATCA?GAGGATTAAG

TTCATCTTCA?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 is made up of about 38 Nucleotide of SGB6 genetic transcription initiation site upstream.Suitable SGB6 core promoter has following nucleotide sequence:

5′ATCTCACCCT?ATTAATACCA?TGCTGACGAG

TAGAGTGGGA?TAATTATGGT?ACGACTGCTC

CCAATAGC 3′[SEQ?ID?NO：5].

GGTTATCG

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

5′AGATCTCTAT?AAAACACGCA?GGGACTGGAA?AGCGAGATTT

TCTAGAGATA?TTTTGTGCGT?CCCTGACCTT?TCGCTCTAAA

CACAGCTGAA?AGCAGCCAAA?ACGCAGAAGC?TGCACTGCAT

GTGTCGACTT?TCGTCGGTTT?TGCGTCTTCG?ACGTGACGTA

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

TGTAGCTCGA?TTGATAGACG?TCGGTAC

5126, the functional fragment of the core promoter of SGB6 and G9 also provides suitable core promoter.The method that obtains this class functional fragment has been described above.

Can adopt chimeric regulation and control composition (comprising the flower pesticide frame of a gene and the anther specific promoter of second gene), extend the growth window of avidin genetic expression.For example, the SGB6 regulating and controlling sequence is expressed at tetraspore phase to the middle monokaryotic stage stimulated gene of growth, and the G9 regulating and controlling sequence is during reduction division and to the tetraspore phase stimulated gene expression of growing.The middle monokaryotic stage that is combined in during the reduction division and extremely grows of SGB6 flower pesticide frame and G9 promotor stimulates transcribing of foreign gene.Therefore, the various combinations of flower pesticide frame and anther specific promoter are particularly useful to the present invention.

Also can use virus core promoter.The example of suitable virus core promoter comprises cauliflower mosaic virus (CaMV) core promoter, figwort mosaic virus core promoter etc.People such as Gruber, supra.Virus core promoter is preferably CaMV 35S core promoter or its varient.

In order to screen transformant, expression vector contains a selected marker such as herbicide resistance gene etc.For example, this genoid can be given the resistance to methylphosphine base high lactamine (phosphinothricine), glyphosate, sulfonylurea, atrazine or imidazolone.The selected marker is the pat gene of bar gene or coding methylphosphine base high lactamine Transacetylase preferably.People such as Leemans, people such as european patent application 0-242-246 (1987) and White can find the nucleotide sequence of bar gene among the Nucleic Acids Res.18:1062 (1990).People such as Wohlleben, Gene 70:25 (1988) discloses the nucleotide sequence of pat gene.Bar gene or pat expression of gene give to such as glufosinate (in other weedicide with Basta ^And Ignite ^Sell) and bialaphos (with Herbi-ace ^And Liberty ^Sale) resistance of and so on weedicide.

Expression vector can contain the dna sequence dna and the selected marker under constitutive promoter control of coding avidin under constitutive promoter or anther specific promoter control.Perhaps, the selected marker can be in independent selective expression's carrier, and " cotransformation " by embryonic tissue is delivered in the host cell.

The recovery of male fertile in the B.F1 hybrid

Aforesaid method can be used for producing the transgenosis male sterile plants, with production-F1 hybrid in the extensive judicious mating between self-mating system.If all ovum of transgenosis male sterile plants do not contain reorganization avidin gene, so a part of F1 hybrid will have male fertile phenotype.On the other hand, if reorganization avidin gene is present in all ovum of transgenosis male sterile plants, because be dominance by ethene inductive sterility, the F1 hybrid will have male sterile phenotype so.Therefore, may need to adopt the male fertile recovery system, to produce male-fertile F1 hybrid.When the product of results is kind of a period of the day from 11 p.m. to 1 a.m, this fertility restorer system has special value in the autogamy species.

Usually it is transgenic plant that the method for recovering fertility in a transgenic plant system that proposes need produce second " recovery ".For example, will hybridize as stated above because of male sterile transgenic plant of the expression of target enzyme and the male-fertile plants of expressing the target enzyme inhibitors.People such as Mariani, Nature 357:384 (1992).

Under this situation, it is plant that similar method need be produced genetically modified recovery, and its expresses target is the ribozyme of avidin mRNA, or the antisence avidin.For example, ribozyme can be designed to express the endonuclease activity that points to a certain target sequence in the mRNA molecule.For example, people such as Steinecke, EMBO J.11:1525 (1992) reach maximum 100% genetic expression that suppresses neomycin phosphotransferases by the ribozyme in the tobacco protoplast.Recently, people such as Perriman, Antisense Res.﹠amp; Devel.3:253 (1993) adopts the carrier of expressing the hammerhead ribozyme of modifying, and suppresses chloramphenicol acetyl transferase activity in tobacco protoplast.In text of the present invention, avidin mRNA provides suitable target RNA molecule for ribozyme.

Use contains the expression vector of the nucleotide sequence of encoding antisense RNA, can recover fertility with similar methods.Antisense rna molecule stops with the hybridization that combining of said target mrna molecule causes translating.People such as Paterson, Pro Natl.Acad.Sci.USA 74:4370 (1987).In text of the present invention, suitable antisense rna molecule has and avidin mRNA complementary sequence.In recommended embodiment of the present invention, sense-rna is under the control of inducible promoters.Therefore, activating this promotor makes male fertile recover.

In other method, can construction of expression vector, wherein the rna transcription thing of expression vector codes can start the cutting of the avidin mRNA molecule of RNA enzyme P mediation.According to this method, can make up the outer sequence of leading, with endogenous ribozyme (RNA enzyme P) guiding avidin mRNA, cut this mRNA by this cell ribozyme subsequently.People such as people's such as Altman United States Patent (USP) 5,168,053, Yuan, Science 263:1269 (1994).Lead sequence outward and preferably comprise sequence and 3 '-NCCA nucleotide sequence with 10-15 Nucleotide of avidin mRNA complementary, wherein N is preferably purine.Id。The formation base pair combines between the sequence by leading outside mRNA and complementation with target class mRNA to lead the sequence transcript outward, starts RNA enzyme P thus and is being positioned at the Nucleotide cutting mRNA of base pair zone 5 ' end.Id。

In recovering the other method of male fertile, the transgenosis male sterile plants contains an expression vector, the latter except that contain functionally with promoter sequence that the avidin gene is connected, also contain a protokaryon and regulate and control composition.Produce the transgenosis male sterile plants, it expresses a protokaryon polypeptide under this promotor control.In the F1 hybrid, this protokaryon polypeptide combines with the protokaryon regulating and controlling sequence, checks the expression of avidin.

For example, LexA gene/LexA operon system is used for regulation and control according to genetic expression of the present invention.Referring to United States Patent (USP) 4,833, people such as 080 ( 080 patent) and Wang, Mol.CellBiol.13:1805 (1993).More particularly, the expression vector of male sterile plants can contain the LexA operon sequence, and the expression vector of male-fertile plants contains the encoding sequence of LexA repressor.In the F1 hybrid, the LexA repressor combines with the LexA operon sequence, suppresses the avidin gene transcription.

For example, can obtain LexA operon dna molecular by the synthetic dna segment that contains well-known LexA operon sequence.Referring to for example people such as ' 080 patent and Garriga, Mol.Gen.Genet.236:125 (1992).The dna molecular of composite coding LexA repressor can obtain the LexA gene.Gene synthesis technology has been discussed above, people such as Garriga for example, supra has described the LexA gene order.The dna molecular of LexA repressor of perhaps encoding can be obtained by plasmid pBR500.U.S. typical case culture collection registration number is 67758.

Recover another method of fertility and utilize the high-affinity of avidin, spray the plant that grows with biotin solution vitamin H.Biotin solution also comprises the organic cosolvent (such as DMSO) of minimum to guarantee the dissolving fully of vitamin H.Spray the meiophase at pollen development, carries out repetition with regular interval, distributes until observing pollen.Being spaced apart 1-7 days and not waiting between the sprinkling.In a recommended embodiment of the present invention, every 3-5 days repeat to spray once.

Those skilled in the art can adopt protokaryon regulator control system (such as Lac repressor/lac operon system or trp repressor/trp operon system etc.), easily design other male fertile recovery policy.

Therefore, by reference following examples, can be more readily understood the present invention of general description, provide embodiment by explanation, these embodiment are not in order to limit the present invention.Embodiment 1: the height constitutive expression by avidin prepares the male sterile transgenic corns

The method that forms transgenic corn plant has been described among european patent application 0 442 174A1 (being attached to herein by reference).Its method is summarized as follows.

PHI5168, a carrier that carries the avidin gene under the control of ubiqutin promotor and contain a PINII terminator sequence forms transgenic corn plant with it.Fig. 1 has shown the structure of PHI5168.PHI610 is one and carries the bar gene under two 35S promoter controls and carry the expression vector of a PINII terminator sequence, it is constituted thing (construct) cotransformation with avidin, by handling transformation mixture, carry out the screening of transgenic plant with bialophos.Fig. 2 has shown the structure of PHI610.According to people such as Green, Molecular Genetics of Plants and Animals, people such as editors Downey, Academic Press, NY, 20,147 (1983) method is transformed into two expression vectors in embryo's generation suspension culture (deriving from II type embryo generation culture).People such as Murashige, Physio.Plant; Murashige described in Vol 15, the 453-497 pages or leaves (1962) and Skoog (" MS ") substratum (add in the 2,4 dichlorophenoxyacetic acid (2,4-D) and the sucrose of 30g/L) of 2mg/L and keep culture.In experiment preceding 7 days, suspension culture remained on filtrate in the MS substratum by 710 tm screen.

By on B (Whatman No.614), carrying out vacuum filtration,, place 3.3cm to accompany the Ti Shi flat board in 100ml (fresh weight) cell by the suspension culture harvested cell.Cell is scattered in the fresh substratum of 0.5ml, forms cell thin.The unlapped Ti Shi of accompanying flat board is placed in the sample chamber of particle gun device (by Biolistics Inc., Geneva NY makes).With vacuum pump the sample chamber internal pressure is decreased to 0.1 normal atmosphere, to reduce the atomic deceleration that causes owing to air friction.With tungsten particle (mean diameter is about 1.2 microns) (GTE SylvaniaPrecision Materials Group, Towanda, Pennsylvania) bombardment cell.By TE damping fluid (pH7.7) solution (1 μ g DNA/100 λ) of 5 μ lDNA being added in the suspension of 25 μ l 50mg tungsten particle/ml distilled water in the 1.5ml Eppendorf pipe, the equal amount of mixture of PHI5168 and PHI610 is loaded on the micropartical.Particle coacervation also precipitates in pipe.

The culture that will contain the transformed plant cells of foreign gene is cultivated 4-8 week in 560R (substratum) (the N6 base substratum with 1mg/ml bialaphos).The cell of Bar gene is expressed in this substratum screening.

Inducing embryo forms in embryo's generation culture then, cell is sprouted be plant.Sprout in proper order at callus with two substratum and to keep observed somatic embryo in the substratum.At first callus is transferred to the substratum (maturation medium) 10-14 days that contains 5.0mg/L indolylacetic acid (IAA), to continue the propagation callus.The callus that shifts is the 50mg/ flat board, to optimize the yield of per unit tissue block.

Then, with callus by on " maturation " media transfer to the second substratum (compare with first substratum, contain the IAA (1mg/L) of low concentration).At this moment, culture is placed under the light.The green seedling of elongation that is characterized as the connection root entry of the somatic embryo of sprouting.Then somatic embryo being transferred to culture tube (in the substratum in 150 * 25mm), cultivated 10-14 days again.At this moment, the about high 7-10cm of plant has enough sizes and vigor strongization under greenhouse experiment.

For strongization regeneration plant, the plant of intending being transferred in the growth room is taken out from sterile chamber, wash away the curing nutrient agar of root.Place the commercially available potting earth of growth room, this production chamber to be furnished with atomisation unit plantlet and keep relative humidity and don't excessive wetting plant root near 100%.3-4 is after week in damp camera, and plant is healthy and strong as to be enough to be transplanted under the field condition.

By observing male infertility, analyze big Tanaka's plant.Then, use standard method, by PCR and Southern blotting, the further existence of Analysis and Screening plant avidin gene, by ELISA, the expression of Analysis and Screening plant avidin.

By pcr analysis 94 plant, wherein 53 are found to be and can educate, 41 be sterile.When the fertility of each plant was compared with the existence of PCR detection avidin gene, finding had 98% dependency between gene existence and the plant sterility.By the Southern blotting, detailed analysis the existence of 5 plant avidin genes.Analyze by Southern, 3 plant show and contain the avidin gene.All 3 plant all have the sterility problem.There are not 2 plant of avidin gene can educate fully.Therefore, 100% relevant between the existence of avidin gene and the sterility problem.Embodiment 2: use the agrobacterium strains that contains two carriers (dna sequence dna that comprises the avidin of encoding), produce male sterile genetically engineered soybean plant

The method that forms the genetically engineered soybean plant is the method for describing in the U.S. Patent application 07/920,409 (being attached to herein by reference).The seed of soybean (Glycine max) Pioneer mutation 9341 carries out surface sterilization by being exposed to the clorox of emitting in the bell glass.3.5ml hydrochloric acid (34-37%w/v) is added generation gas in the 100ml sodium-chlor (5.25%w/v).In the container of about 1 cubic feet of volume, expose 16-20 hour.The seed of surface sterilization is housed in the Petri dish under room temperature.To solidify substratum according to the agar of 1/10 intensity of the Gambourg that does not contain plant-growth regulator and (contain the organic B5 basic medium of minimum, Sigma Chemical Catalog No.G5893,0.32gm/L sucrose, 0.2%w/v 2-(N-morpholino) ethyl sulfonic acid (MES) are (0.3mM)) pave plate, and be that 16 hours, cold daytime light modulation illumination are approximately 20 μ Em in 28 ℃, the duration of day ^-2S ^-1Following cultivation, germinating seed.After 3 or 4 days, preparation is used for the seed of common cultivation.Remove kind of a skin, remove the elongation root of 3-4mm under the cotyledon.

The overnight culture of agrobacterium tumefaciens bacterial strain LBA4404 that contains two plasmids (containing the avidin gene) of modification grows to logarithmic phase in basic A substratum (containing 1 μ g/ml tsiklomitsin), with its merging, measure optical density(OD) under 550nm.The culture of enough volumes is placed 15ml/ taper centrifuge tube, and when making precipitation, every pipe collects 10 ⁹The 1-2 of cell/ml * 10 ¹⁰Cell.Precipitated in centrifugal 10 minutes with 6,000 * g.After centrifugal, abandon supernatant liquor, centrifuge tube in be retained under the room temperature need inoculum till, but be no more than 1 hour.

Inoculate in batches, make each seed dull and stereotyped with resuspending Agrobacterium precipitation process just.Cell precipitation difference resuspending is in the 20ml inoculation medium, the latter is contained 3.2g/LB5 salt (G5893), 2.0%w/v sucrose, 45 μ M 6-benzyl aminopurines (BAP), 0.5 μ M indolebutyric acid (IBA), 100 μ M Syringylethanones (AS), and is buffered to pH5.5 with 10mM MES.Reach resuspending by vortex.Inoculum is poured in the Petri dish of the seed that contains preparation then, separated cotyledonary node with scalpel.Vertically cut in two by shoot apex, keep two complete cotyledons, finish separately seed.Then with scalpel with the two halves sledge of shoot apex from corresponding cotyledon.Then, by repeat delineation along symmetry axis, separate cotyledonary node with scalpel.Carefully do not cut to axle head by explant fully.Roughly preparing explant in 5 minutes, with bacterium incubation 30 minutes under room temperature, do not stirring then.After 30 minutes, explant is transferred to (the Merck ﹠amp with 0.2%w/v Gelrite; Company Inc.) in the solidified same medium flat board.The embedding explant upwards and with media surface flushes its proximal ends, at about 20 μ Em ^-2S ^-1Cold daytime light modulation light under cultivated 3 days in 22 ℃.

After 3 days, explant is transferred to liquid is counter to be selected in the substratum, the latter is contained 3.2g/l B5 salt (G5893), 2%w/v sucrose, 5 μ M BAP, 0.5 μ M IBA, 200 μ g/ml vancomycins, 500 μ g/ml cefotaximes, is buffered to pH5.7 with 3mM MES.Explant is in slowly rotating agitator treating 4 days with constant under the room temperature in each Petri dish.Change the anti-substratum of selecting 4 times.

Then, explant is chosen the agar solidified to be selected in the substratum, the latter is contained 3.2g/lB5 salt (G5893), 2%w/v sucrose, 5.0 μ M BAP, 0.5 μ M IBA, 50 μ g/ml sulphuric acid kanamycins, 100 μ g/ml vancomycins, 30 μ g/ml cefotaximes, 30 μ g/ml timentins (timentin), is buffered to pH5.7 with 3mM MES.Select substratum to solidify with 0.3%w/v Seakem agarose.The explant proximal ends being embedded in downwards in the substratum, cultivating down in 28 ℃, is 60-80 μ Em in cold daytime light modulation illumination ^-2S ^-1Under the duration of day be 16 hours.

After 2 weeks, explant washs with liquid nutrient medium on rotary shaker again.Washing is spent the night in the anti-selection substratum that contains 50 μ g/ml sulphuric acid kanamycins and is carried out.Second day, explant is chosen agarose solidify on the selection substratum.Their proximal ends are embedded in downwards in the substratum, cultivated for 2 weeks again.

After on the selection substratum 1 month, contrast with the non-health tissues of yellow, can see the regenerating tissues that transforming tissue is green segment.Abandon the explant that does not have green segment, the explant that will have green segment is transferred in the elongation medium, the latter is contained 3.2g/l B5 salt (G5893), 2%w/v sucrose, 3.3 μ M IBA, 1.7 μ M gibberic acid, 100 μ g/ml vancomycins, 30 μ g/ml cefotaximes, 30 μ g/ml timentins, is buffered to pH5.7 with 3mM MES.Elongation medium is solidified with 0.2%w/v Gelrite.The green segment of embedding makes its proximal ends upwards, cultivates according to preceding method.Continue to cultivate on this substratum, per 2 weeks are transferred on the fresh flat board.When seedling length is 0.5cm, they are downcut from base portion, place the root media of 13 * 100ml test tube.Root media contains 3.2g/l B5 salt (G5893), 15g/l sucrose, 20 μ M nicotinic acid, 900mg/L Pyrrolidonecarboxylic acid (PGA) and 10 μ M IBA.Root media is buffered to pH 5.7 with 3mM MES, and solidifies with 0.2%w/v Gelrite.After 10 days, seedling is transferred on the same medium that does not contain IBA or PGA.These seedling rootings remain on it in test tube under above-mentioned identical envrionment conditions.

When root system is set up well, plantlet is transferred in the aseptic soil.It is identical with above-mentioned condition that temperature, photoperiod and light intensity keep.

Expression with the avidin in ELISA confirmation and the quantitative assay genetically engineered soybean plant confirms the existence of this gene by PCR and Southern blotting.Can be with these identical methods, evaluation expression stability in the successive generation.The expression of finding the avidin in sterility problem and the soybean is relevant.Embodiment 3: prepare male sterile Sunflower Receptacle plant by expressing avidin

The expression cassette of coding avidin is used for producing transgenosis Sunflower Receptacle plant and seed.In the expression cassette under the dna sequence dna insertion ubiqutin promotor control of coding avidin.

Then, use the EcoRI site, this expression cassette of subclone in the two carriers such as PHI 5765.Then, this pair carrier is transferred in the agrobacterium tumefaciens supplementary strain.

The Sunflower Receptacle plant is according to people such as Bidney, and Plant Mol.Bio.Vol.18: the 301st page, 1992 described methods transform with agrobacterium strains LBA4404 after carrying out the micropartical bombardment.Say that simply the seed that is with pioneer Sunflower Receptacle SMF-3 removes shell and carries out surface sterilization.Seed in the dark, in 26 ℃ with imbibition on the filter paper of water-wet 18 hours.Remove cotyledon and foundation, the meristematic tissue explant is cultivated in 374BGA substratum (MS salt, Shephard VITAMIN, 40.mg/L adenine sulfate, 3% sucrose, 0.8% plant agar (phytagar) pH 5.6 add 0.5mg/L BAP, 0.25mg/L IAA and 0.1mg/LGA).After 24 hours, remove first leaf, the exposed top ends meristematic tissue, with this explant dome towards on place the 60mm * 20mm that contains water agar to accompany the 2cm circle at the dull and stereotyped center of Ti Shi.Explant be suspended in tungsten particle in the TE damping fluid or use with the particle bombardment 2 times that contains avidin expression of gene plasmid.The meristematic tissue explant was cultivated 72 hours in the 374BGA substratum more altogether in 26 ℃ under light.

After cultivating altogether in 72 hours, the meristematic tissue that Agrobacterium is handled is transferred to 374 substratum, and (374BGA with 1% sucrose does not contain BAP, IAA or GA ₃), add 250 μ g/ml cefotaximes.Allow plantlet under the culture condition of 16 hours durations of day and 26 ℃, grew for 2 weeks, grow and be green or yellow.Plantlet is transferred in the substratum that contains kantlex, allows its growth.Press the existence of avidin in embodiment 2 described methods confirmations and the quantitative assay plant.The existence of finding male infertility is relevant with plant expression avidin.

Though above-mentioned explanation relates to concrete recommended embodiment, it is really not so limited to understand the present invention.Those skilled in the art can expect and can carry out various modifications to disclosed embodiment that this class is revised in the scope of the invention that will be included in following claims definition.All publish an article and patent application shows one of ordinary skill in the art's of the present invention level of mentioning in this specification sheets.

Sequence table (1) physical data:

(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: the U.S.

(F) postcode: 50309

(ii) invention exercise question: by expressing high-caliber avidin inducing plant male infertility

(iii) sequence number: 6

(iv) corresponding address:

(A) addressee: Foley﹠amp; Lardner

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

(C) city: Washington

(D) state: D.C.

(E) country: the U.S.

(F) postcode: 20007-5109

(v) computer-reader form:

(A) media types: diskette

(B) computer: IBM PC compatible

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

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

(vi) the application's data:

(A) application number: still issue

(B) date of application:

(vii) previous request for data:

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

(B) date of application: June 7 nineteen ninety-five

(viii) attorney/proxy's data:

(A) name: Bent, Stephen A.

(B) number of registration: 29,768

(C) reference/agency's numbering: 33229/403/PIHI

(ix) telecommunication data:

(A) phone: (202) 672-5300

(B) facsimile recorder: (202) 672-5399

(C) fax: the data of 904136 (2) SEQ ID NO:1:

(i) sequence characteristic:

(A) length: 64 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: the data of SEQ ID NO:1:GCGGCCGCGG ATCCGCTCAT CTGCTCGGTA CCAACCAGCC TTTCCTATTA CCATGCACAG 60ATCT 64 (2) SEQ ID NO:2:

(i) sequence characteristic:

(A) length: 94 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: the data of SEQ ID NO:2:ACAGTTCACT AGATATGCAT GATCTTTAAC AATTGCTGCT GGATTGTGCG GTTTCTTTTG 60GCACAAATGG CATGAACAGA GTAATCCGGG ACGC 94 (2) SEQ ID NO:3:

(i) sequence characteristic:

(A) length: 136 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: the data of SEQ ID NO:3:GCGGCCGCGG ATCCTGGCTG GATGAAACCG ATGCGAGAGA AGAAAAAAAA ATTGTTGCAT 60GTAGTTGGCG CCTGTCACCC AACCAAACCA GTAGTTGAGG CACGCCCTGT TTGCTCACGA 120TCACGAACGT AGATCT 136 (2) SEQ ID NO:4:

(i) sequence characteristic:

(A) length: 142 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: the data of 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:

(i) sequence characteristic:

(A) length: 38 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: the data of SEQ ID NO:5:ATCTCACCCT ATTAATACCA TGCTGCGAG CCAATAGC 38 (2) SEQ ID NO:6:

(i) sequence characteristic:

(A) length: 107 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(xi) sequence description: SEQ ID NO:6:AGATCTCTAT AAAACACGCA GGGACTGGAA AGCGAGATTT CACAGCTGAA AGCAGCCAAA 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.

Images