CN102888408B - Application of paddy rice RAD51C gene in fertility control - Google Patents

Abstract

The invention relates to the technical field of plant gene engineering, especially to isolation, cloning and functional verification of the paddy rice fertility control gene RAD51C. The RAD51Cgene belongs to a RecA/RAD51 protein family, and can control fertility of rice pollen and embryo sac, so knock-out of the gene enables abnormal meiosis of the paddy rice, thereby causing infertility of the pollen and the embryo sac. Male infertility or male and female infertility rice materials can be built through tissue-specifically inhibiting the expression of the RAD51Cgene.

Description

The application of paddy rice RAD51C gene in fertility control

Technical field

The present invention relates to plant genetic engineering field.Be specifically related to separating clone and the functional verification of a fertility controlling gene RAD51C.RAD51C, by participating in the maiotic regulation and control of paddy rice, controls paddy pollen fertility and blastular fertility.Biological function verification result to this gene shows, this gene can be used as sterile gene, produces and fertility control for paddy rice cross breeding seed.

Background technology

Rice Production has very important status in China's grain-production.Since nineteen seventies, the popularization of a series of hybrid rice, has had per unit area yield and increases substantially, and has alleviated to a certain extent China's population and increase rapidly the pressure of the demand sharp increase to grain.Hybrid rice is selected the good rice varieties that has different in heredity, is that method production has heterotic cross-fertilize seed with three series (sterile line, maintenance line and restorer), some bilinear methods that are derived and one.Hybrid vigour be two different parents of genetic background hybridize generation the first generation of hybrid growth potential, vitality, reproductivity, the aspects such as output are better than parents' phenomenon.In three series, sterile line is can not self-pollination solid, be mainly gynoecium grow normal, and growth degeneration or the abortion of stamen.The Pistil And Stamen of maintenance line is all grown normally.Authorize sterile line by the pollen of maintenance line and can successfully obtain seed, but the plant being produced by this seed is still male sterile.Authorize with the pollen of restorer the plant that seed that sterile line produces grows and recovered again fertility.

The utilization of male sterile line is the important step of preparation cross-fertilize seed, but finds sterile line few from nature, and seed selection male sterile line and restorer also exist certain difficulty, utilize engineered method to obtain sterile line and restorer prospect tempting.Pollen development is an extremely complicated process, needs multiple genes jointly to participate according to certain spatial and temporal expression pattern, could complete smoothly, and pollen sterility appears extremely all can causing in any one process.The approach of creating male sterile line by engineered method comprises: (1) utilizes the promoter expression cytotoxin gene of flower pesticide or pollen-specific, some tissue of selective destruction pollen development; (2) can block the expression of pollen development genes involved by gene silencing, cause pollen abortion; (3) in change pollen development process, the spatial and temporal expression of key enzyme or functional protein can obtain male sterile material; (4) create (Miao Ying etc. 2000 such as cytoplasmic male sterile line by transfered cell matter male sterility gene or upset mitochondrial function; Song Jianghua etc. 2008); The maintenance line of corresponding male sterile line can be the adjoining tree that there is no conversion, can be also the maintenance line obtaining by genetic engineering modified; In addition, can also keep sterile line by vegetative propagation.

In the Eukaryotic life history, experience diplontic sporophyte epoch and haploid gametophyte epoch.In the sporophyte epoch, eukaryote carries out reduction division and forms haploid gamete; Occurring to merge from the female and male gametophyte of Parent makes offspring return to diplontic level.Reduction division forms in haploid process and is playing the part of important role in Eukaryotic syngenesis, and joint conference's exchange that in this process, karyomit(e) occurs, has increased hereditary diversity greatly.Closely during the last ten years, by the maiotic research to model animals yeast, identified the maiotic gene of much control.In addition, people have cloned tens by method reverse or that forward genetics is learned and have controlled maiotic gene (Schwarzacher, 2003 in model plant Arabidopis thaliana; Ma, 2005; Mercier and Grelon, 2008).Relatively the reduction division of yeast and plant is found, maiotic mechanism is relatively conservative in eukaryote.

Eukaryotic RAD51 is a class and albumen very similar on intestinal bacteria (Escherichia coli) recombinant protein RecA structure and function, in mitotic division and reduction division process, play an important role (Shinohara etc., 1992).In to the research of recA/RAD51 gene family, find: RAD51 gene family is due to gene redundancy and gene level transfer, and formation RAD51, RAD51B, RAD51C, RAD51D, DMCl, XRCC2, XRCC3, multiple members such as RecA (Lin etc., 2006).In eukaryote Yeast genome, comprise multiple RecA and RAD51 homologous protein, and in Mammals, comprise five homologous proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), these albumen and RAD51 or have each other 20% to 30% sequence homology (Lovett, 1994; Sonoda etc., 2001).In paddy rice, have 13 homologous proteins at least, comprising: RAD51A1, RAD51A2, RAD51B, RAD51C, RAD51D, DMC1A, DMC1B, XRCC2, XRCC 3 and four RecA homologous proteins (http://rice.plantbiology.msu.edu/).

RAD51 gene family member great majority are played an important role in reduction division, and sudden change may occur in various degree abnormal with post-meiotic division.Yeast DMC1 gene is a reduction division specific gene of expressing at meiosis prophase I, and the albumen of coding is that the pairing of reduction division homologous chromosomes is necessary.In Chinese mouse cell lines CL-V4B, irs1, irs1SF, RAD51C, after XRCC2 and XRCC3 sudden change, to DNA linking agent sensitivity, the spontaneous abnormal frequency of karyomit(e) obviously increases (Cui etc., 1999; Godthelp etc., 2002).Mouse RAD51B, after RAD51D and XRCC2 sudden change in Embryonic Stages lethal (Shu etc., 1999; Deans etc., 2000; Pittman and Schimenti, 2000).Nematode RAD51C, the restructuring of XRCC3 sudden change post-meiotic division causes partial sterility extremely, and to DNA double chain damage agent sensitivity (Abdu etc., 2003).In Arabidopis thaliana, after AtRAD51 sudden change, nourish and grow significantly not abnormal with mitotic division, and reduction division is subject to serious impact (Mercier etc., 2003).In the mutant of AtRAD51, karyomit(e) zygotene stage joint conference normally, cannot form synaptonemal complex and meiosis prophase I and occur rhexis (Mercier etc., 2003).AtRAD51C and AtXRCC3 suddenly change later and the mutant of AtRAD51 has similar phenotype: nourish and grow normal; Abnormal Circumstances In Meiosis, causes abortion (Bleuyard etc., 2006) thereby can not form normal female and male gametophyte.AtRAD51B, AtRAD51D and AtXRCC2 sudden change have improved the susceptibility to silk Streptomycin sulphate C (mitomycin C) later, but reduction division significantly not abnormal (Bleuyard etc., 2005; Durrant etc., 2007).AtDMC1 suddenlys change can not form bivalent in post-meiotic division process, but can repair double-strand break (Couteau etc., 1999).Corn RAD51A1 and RAD51A2 participate in the pairing of homologous chromosomes joint conference and double-strand break repair process, can see the pairing of nonhomologous chromosome, joint conference and exchange in double-mutant.In paddy rice, there is the DMC1 of two copies, respectively DMC1A and DMC1B, the pairing of homologous chromosomes in DMC1 Gene Handling rice cell reduction division process, after this gene is suppressed, nourish and grow normal, but in reduction division process karyomit(e) carry out unequal separation and sporulation random, thereby make pollen sterility, setting percentage reduces (Ding etc., 2001; Deng and Wang, 2007).

Also there is no at present the report about the function of paddy rice RAD51C gene.The present invention utilizes the T-DNA insertion mutation body of RAD51C to study the effect of this gene in reduction division process in great detail, for the control of plant fertility provides new approach, also provides help for we understand maiotic mechanism in depth.

Summary of the invention

The object of the invention is one of separating clone and control the maiotic gene of paddy rice, relate to the DNA fragmentation that separates a kind of RAD51C of comprising gene, thereby identify by T-DNA insertion mutation body and the genetic complementation of this fragment the fertility that shows this fragment control paddy rice reduction division, affects the growth control paddy rice of female and male gametophyte.The clone of this gene is conducive to understand the maiotic process of paddy rice, and is that Artificial Control paddy pollen fertility or blastular fertility are provided fundamental basis to produce cenospecies.Wherein, described fragment, as shown in sequence table SEQ ID NO:1, or is equivalent to the DNA sequence dna shown in SEQ ID NO:1 substantially, or its function is equivalent to the subfragment of sequence shown in SEQ ID NO:1.

Another object of the present invention is that the expression by controlling paddy rice meiotic gene RAD51C obtains sterile transgenic paddy rice, and then utilize male sterile rice to produce cross-fertilize seed, for increasing rice yield, improve rice quality, improve resistance and adaptability valuable source is provided, therefore on rice breeding, have important value, especially on rice heterosis utilization, certificate has important practice significance.Be exactly the method for utilizing genetic modification specifically, utilize the promotor of organ or tissue's specifically expressing, this gene of specific inhibition at megagamete or/and thereby the expression of microgamete obtains all sterile plant of female sterile or male sterile or male and female, create new female or/and male sterile line, these sterile lines can be for the production of hybrid rice, as the cultivation of hybrid rice seed.Also can be for controlling the fertility of other plant.In addition, the pollen sterility of all right render transgenic plant of these sterile lines, eliminates the risk that in transgenic plant, foreign gene is escaped.

Brief description of the drawings

Sequence table SEQ ID NO:1 and SEQ ID NO:2 are the aminoacid sequences of the sequence of paddy rice RAD51C gene and the protein of its coding.

Fig. 1: the schema of qualification of the present invention, separating clone and functional analysis paddy rice RAD51C gene.

Fig. 2: the qualification of the T-DNA insertion mutation body 4D-50016 of paddy rice RAD51C gene.(A) RAD51C gene structure and T-DNA on position schematic diagram.Wherein the genome structure of the structure of the non-translational region of RAD51C gene and RAD51C gene both sides is that DNA and cDNA (AK060971) sequence of the corresponding section fine according to GenBank database (http://www.ncbi.nlm.nih.gov/) intermediate keng rice kind Japan determined.(B) RAD51C gene T-DNA insertion mutation body T ₁the genotype detection of plant.WT: wild-type Dongjin (contrast).(C) expression analysis of RAD51C gene in wild-type Dongjin (WT), T-DNA insertion mutation body (M) and negative plant (N).Reference using the expression amount of Actin gene as sample size.Transgenosis family is T ₂generation.

Fig. 3: RAD51C knocks out the Phenotypic Observation of strain.In figure, scale is 150 μ M.(A) Dongjin (wild-type) and RAD51C knock out plant does not have notable difference in vegetative growth phase.(B) little Hua that Dongjin (wild-type) and RAD51C knock out plant does not have significant difference in appearance.(C) flower pesticide of Dongjin (wild-type) plant is full, and that RAD51C knocks out the flower pesticide of plant is relatively thin and hollow.(D) in the flower pesticide of Dongjin (wild-type) plant, be full of circular pollen granule, and that RAD51C knocks out in the flower pesticide of plant pollen granule is shrivelled.(E) normally loose powder of Dongjin (wild-type) plant, and RAD51C knocks out normally loose powder of plant.(F) pollen granule of Dongjin (wild-type) plant is normal, and RAD51C knocks out the pollen sterility of plant.

Fig. 4: T-DNA inserts the coseparation analysis with sterile proterties.Setting percentage is the mean value ± standard error of 3 tassel setting percentages.(A) BC of a RAD51C T-DNA insertion mutation body (4D-50016, Dongjin background) and Dongjin hybridization ₁f ₂the genotype of colony and setting percentage analysis.Contrast is Dongjin.(B) another RAD51C T-DNA insertion mutation body (4D-50016, Dongjin background) and the middle BC that spends 11 hybridization ₁f ₂the genotype of colony and setting percentage analysis.Contrast spends 11 in being.

Fig. 5: the structure of genetic transformation carrier pCAMBIA2301.

Fig. 6: RAD51C gene and the fertility coseparation analysis of D203Or genetic transformation plant (Dongjin genetic background) T1 family.Detect in genetically modified acceptor material and inserted T-DNA in RAD51C gene with PCR primer 39630-m-F and VectorR.Detect the importing situation of complementary fragment by a part for PCR primer 2 301-F and a part of section of rad51-com-R amplification pCAMBIA2301 carrier and the promoter region of RAD51C.Setting percentage is the mean value ± standard error of 3 tassel setting percentages.Contrast is japonica rice Dongjin.(A) setting percentage of D203Or-52T1 family and gene type assay.(B) setting percentage of D203Or-55 T1 family and gene type assay.

Fig. 7: wild-type (Dongjin) and RAD51C knock out Embryo Sac Development and the forming process of plant.In figure, scale is 50 μ M.(A) Embryo Sac Development of wild-type plant and forming process.In wild-type plant, sporogonium continues to grow formation megasporocyte (shown in arrow); Megasporocyte carries out reduction division and forms tetrad (shown in arrow), and three megaspores (shown in arrow) of nearly micropylar end degenerate in succession, only leave a megaspore of chalazal end; Function megaspore (shown in arrow) in blastular continues to grow formation monokaryon blastular.Progressively form afterwards two cores (shown in arrow) the Embryo Sac Development phase, four cores (shown in arrow) the Embryo Sac Development phase, the eight core Embryo Sac Development phases [being respectively polar core (PN) shown in three arrows, antipodal cell (AN) and synergid (SY)].In the blastular ripening stage, visible polar core (PN), antipodal cell (AN) and synergid (SY) in ripe blastular.(B) RAD51C knocks out Embryo Sac Development and the forming process of plant.Knock out in plant energy at RAD51C, grow and form normal megasporocyte (shown in arrow); In megasporocyte meiophase, the quadrantal core that megasporocyte reduction division forms is unintelligible, and quadrantal core all starts degraded (can not see four cores completely); Form the phase function megaspore, four megaspores all degenerate, and can not form function megaspore; In mature embryo sac, do not form normal polar core, antipodal cell and synergid, only have the vestige of the core of some degradeds.

Fig. 8: wild-type (Dongjin) and RAD51C knock out the reduction division process of pollen mother cell in plant.In figure, scale is 25 μ M.(A) the reduction division process of the pollen mother cell of wild-type plant.In wild-type plant, enter maiotic cell in leptotene stage, Chromatin condensation is the fine rule of carefully growing; In zygotene stage, homologous chromosomes near and start joint conference; To the pachytene stage, chromatin further shortens chap, and homologous chromosomes completes joint conference, is thicker linear structure, and chromatin further shortens formation bivalent; Enter diplotene stage, can between chromatid, see cross knot; To the diakinesis stage, chromosome coiling degree is higher, can see clearly 12 bivalents; Meiosis I enters I in mid-term subsequently, and each bivalent is arranged on equatorial plate; At later stage I, homologous chromosomes is separated from each other and shifts to the two poles of the earth; At I in latter stage, homologous chromosomes arrives the two poles of the earth separately, and cytokinesis forms dyad; Each cell in dyad once divides again, forms tetrad, and wherein at II in latter stage, sister chromatid is separately shifted to the two poles of the earth.(B) RAD51C knocks out the reduction division process of the pollen mother cell of plant.Knock out in plant at RAD51C, reduction division leptotene stage and zygotene stage are significantly not abnormal; In the pachytene stage, homologous chromosomes is not completely closely affixed; In diplotene stage, can see obvious chromosome segment; In the diakinesis stage, there is chromosome segment, do not form obvious 12 bivalents; In the later stage I transition period, in cell, there is chromosome segment, and have chromosome bridge to occur; At later stage II, there is equally chromosome segment, finally form tetrad.

Embodiment

Further definition the present invention in following examples, Fig. 1 has described the flow process of qualification and separating clone RAD51C gene and checking RAD51C gene function.According to above description and the following examples, those skilled in the art can determine essential characteristic of the present invention, and in the situation that not departing from spirit and scope of the invention, can make various changes and amendment to the present invention, so that its applicable various uses and condition.

Embodiment 1: separating clone RAD51C gene and gene structure analysis

1. the full length DNA fragment of separating clone RAD51C gene from rice varieties Dongjin

According to structure design PCR primer OsRAD51C-F (5 '-ATGGAGATCGCCGACCTC-3 ') and the OsRAD51C-R (5 '-CATTACCCGGACTCGCTTG-3 ') of the RAD51C gene (RGAP database site name: LOC_Os01g39630) providing in paddy rice whole genome sequence database [RGAP (Rice Genome Annotation Project http://rice.plantbiology.msu.edu/)], adopt reverse transcription-PCR (reverse transcription-PCR, RT-PCR) technology, the full-length cDNA of amplification RAD51C from Rice cultivar Dongjin (Oryza sativa ssp.japonica).Concrete operation method is extracted total RNA from Dongjin leaf texture (Zhou etc., 2002).Get 1-5 μ g total for RNA DNaseI (American I nvitrogen company) process and pollute to remove genomic dna for 15 minutes, then with reference to the method for (Zhou etc., 2002) such as Zhou, use oligo (dT) ₁₅oligomerization primer and M-MLV ThermoScript II (Promega company of the U.S.) are carried out reverse transcription, obtain total cDNA.Taking total cDNA as template, obtain the cDNA of RAD51C gene with PCR primer OsRAD51C-F and OsRAD51C-R amplification.Utilize OsRAD51C-F and OsRAD51C-R primer and above-mentioned sequence measurement to check order to the cDNA of RAD51C gene.The genome sequence of comparative analysis RAD51C gene and cDNA sequence, find that the respective segments of the Japanese fine cDNA sequence A K060971 of RAD51C gene cDNA sequence in Dongjin and GenBank database (http://www.ncbi.nlm.nih.gov/) intermediate keng rice kind is in full accord.

The genomic fragment of the RAD51C gene coding region in japonica rice Dongjin is made up of 4600 Nucleotide, and 8 introns that are inserted into are divided into 9 sections (Fig. 2 A).First paragraph coding region forms (the 1-112bp place that is positioned at sequence table SEQ ID NO:1) by 112 Nucleotide; First Intron forms (the 113-360bp place that is positioned at sequence table SEQ ID NO:1) by 248 Nucleotide; Second segment coding region forms (the 361-465bp place that is positioned at sequence table SEQ ID NO:1) by 105 Nucleotide; Second intron forms (the 466-748bp place that is positioned at sequence table SEQ ID NO:1) by 283 Nucleotide; The 3rd section of coding region forms (the 749-871bp place that is positioned at sequence table SEQ ID NO:1) by 123 Nucleotide; The 3rd intron forms (the 872-1111bp place that is positioned at sequence table SEQ ID NO:1) by 240 Nucleotide; The 4th section of coding region forms (the 1112-1145bp place that is positioned at sequence table SEQ ID NO:1) by 34 Nucleotide; The 4th intron forms (the 1146-1215bp place that is positioned at sequence table SEQ ID NO:1) by 70 Nucleotide; The 5th section of coding region forms (the 1216-1286bp place that is positioned at sequence table SEQ ID NO:1) by 71 Nucleotide; The 5th intron forms (the 1287-1365bp place that is positioned at sequence table SEQ ID NO:1) by 79 Nucleotide; The 6th section of coding region forms (the 1366-1592bp place that is positioned at sequence table SEQ ID NO:1) by 227 Nucleotide, and the 6th intron forms (the 1593-1788bp place that is positioned at sequence table SEQ ID NO:1) by 196 Nucleotide; The 7th section of coding region forms (the 1789-1920bp place that is positioned at sequence table SEQ ID NO:1) by 132 Nucleotide; The 7th intron forms (the 1921-2629bp place that is positioned at sequence table SEQ ID NO:1) by 709 Nucleotide; The 8th section of coding region forms (the 2630-2696bp place that is positioned at sequence table SEQ ID NO:1) by 67 Nucleotide; The 8th intron forms (the 2697-4424bp place that is positioned at sequence table SEQ ID NO:1) by 1728 Nucleotide; The 9th section of coding region forms (the 4425-4600bp place that is positioned at sequence table SEQ ID NO:1) by 176 Nucleotide.The 4601-4603bp place termination codon of sequence table SEQ ID NO:1.

The analysis of 2.RAD51C gene encoding production

The coding region of RAD51C gene is made up of 1047 Nucleotide, and the length of encoding is 349 amino acid whose protein.According to the RecA/RAD51 protein families evolutionary analysis of (2006) such as Lin, RAD51C belongs to RAD β subclass, comprises the RecA/RAD51A structural domain being made up of 230 amino acid; The Walker B die body (Hanson etc., 2005) that RecA/RAD51 structural domain comprises the conservative Walker A die body being made up of GXXXXGK (T/S) (" X " represents arbitrary amino acid) and is made up of hhhhDE (" h " represents hydrophobic amino acid).

The checking of embodiment 2:RAD51C gene T-DNA insertion mutation body

Acquisition and the checking of 1.T-DNA insertion mutation body

Utilize the promotor of RAD51C gene and coding region sequence retrieval rice mutant database RiceGE ( http:// signal.salk.edu/cgi-bin/RiceGE), find the mutant that exists a T-DNA to insert in this mutant library, its insertion point is arranged in the 3rd section of coding region (Fig. 2 A) of RAD51C gene; This mutant is numbered 4D-50016, and genetic transformation carrier is pGA2772, and genetic transformation acceptor rice varieties is Dongjin (Jeong etc., 2006).By commercial sources, we buy 4D-50016 mutant T from this Rice mutant pool ₁for seed.Seed, after germinateing, obtains 5 mutant plant.

In order to verify the exactness of obtained mutant, researchist of the present invention according to the sequences Design of RAD51C gene the PCR primer (Fig. 2 A) of a pair of RAD51C gene that is positioned at T-DNA insertion point both sides: 39630-m-F (5 '-GCTCCCCAAGAAACTTTTCC-3 ') and 39630-m-R (5 '-CCACTGTTCTTGGCATGTTG-3 ').In addition, according to T-DNA sequences Design PCR primer vectorR (5 '-ACCTGTAAGATTTAGCACCC-3 ') (Jeong etc., 2002).Utilize the T-DNA of these 3 PCR primer mutant that analysis obtains whether to be inserted in RAD51C gene.The ultimate principle of this analysis is: in homozygous mutation body (having the insertion of T-DNA in the pair of homologous karyomit(e)) plant of 4D-50016, due to the about 14kb (Wu etc. of T-DNA fragment that insert, 2003), utilize the primer 39630-m-F of RAD51C gene and 39630-m-R, the conventional PCR method of employing so large DNA fragmentation that cannot increase, and can amplify the known rice genome of one section of size and the hybrid DNA fragment of T-DNA with primer vectorR and 39630-m-R; In heterozygous mutant body (having T-DNA to insert in only having item chromosome in the pair of homologous karyomit(e)) plant of 4D-50016, with can the increase DNA fragmentation of the known rice genome of one section of size of primer 39630-m-F and 39630-m-R, by primer vectorR and 39630-m-R also can the increase known rice genome of one section of size and the hybrid DNA fragment of T-DNA; In the feminine gender of 4D-50016 (isolated in mutant family do not have T-DNA to insert) plant, by primer 39630-m-F and the 39630-m-R one section of oryza sativa genomic dna fragment that size is known that can increase, but with primer vectorR and 39630-m-R can not amplification of DNA fragments.To the T of 5 strain 4D-50016 familys ₁plant has carried out above-mentioned pcr analysis.Detected result shows that in this 5 strain, having 4 strains is heterozygous plant (4D-50016-2,3,4,5), the negative plant of 1 strain (4D-50016-1); The offspring of these 4D-50016 heterozygous plants is for the analysis of other embodiment.

RAD51C genetic expression component analysis in 2.T-DNA insertion mutation body

At the offspring (T of 4D-50016 heterozygous plant ₂) middle screening homozygous mutation body strain.Expression amount with the methods analyst RAD51C gene of RT-PCR in mutant.Adopt the method in embodiment 1 to obtain total cDNA, the primer of pcr analysis is OsRAD51c-rt-f (5 '-TATGGTGGACTTGGTGGGAA-3 ') and OsRAD51C-rt-r (5 '-TCCGGCTGGAGCCTTGT-3 ').Reference with the expression amount of Actin muscle (actin) gene as sample size; The PCR primer of actin gene (GenBank number of registration: X15865) is Actin-F (5 '-TGTATGCCAGTGGTCGTACCA-3 ') and Actin-R (5 '-CCAGCAAGGTCGAGACGAA-3 ').In 4D-50016 homozygous lines (M), the expression of RAD51C gene do not detected, illustrate that the RAD51C genetic expression in these strains is knocked (Fig. 2 C), hereinafter claim that 4D-50016 homozygous lines is that RAD51C knocks out strain.

Embodiment 3:RAD51C knocks out the phenotype analytical of plant

RAD51C is knocked out to strain phenotype and observe discovery, in vegetative growth phase and not obviously difference (Fig. 3 A) of wild-type (Dongjin, contrast).At the heading stage of reproductive growth, RAD51C knocks out strain and also there is no significant difference (Fig. 3 B) to impinging upon fringe type and little floral shape aspect.But wild-type flower pesticide is full, and RAD51C knocks out the flower pesticide of plant relatively thin and hollow (Fig. 3 C).Get the flower pesticide at heading stage by bulk dyeing transparent laser scanning confocal microscopy method (Guo Haibin etc., 2006) observe, pollen granule in wild-type flower pesticide is normal circle, and RAD51C knocks out the pollen granule of plant shrivelled (Fig. 3 D).In addition, RAD51C knocks out normally loose powder of plant, shows as completely sterilely, and wild-type plant can normal loose powder (Fig. 3 E).Get flower pesticide iodine-potassiumiodide dye-binding assay of 1% (Lee from superfine, 1992), microscopic examination shows, contrast and compare with wild-type, it is the pollen (Fig. 3 F) of abortion that RAD51C knocks out in plant flower pesticide.

For confirming that RAD51C knocks out plant except male sterile, whether also there is female sterile.Researchist of the present invention knocks out (4D-50016 mutant isozygotys) plant with japonica rice Dongjin (wild-type contrast) and RAD51C and carries out reciprocal cross.Hybridize in contrast with isolated negative plant and Dongjin in 4D-50016 mutant family simultaneously.Result shows (table 1), and no matter RAD51C knocks out plant is as male parent or female parent, all shows as sterilely with the hybrid after Dongjin hybridization, and the setting percentage of hybrid in contrast is 42%-60%.It is all sterile that these presentation of results RAD51C knocks out the male and female of plant.

Table 1.Dongjin (wild-type) and RAD51C knock out the setting percentage of plant reciprocal hybrids

The common separate authentication of embodiment 4:T-DNA and mutant character

Due to T ₁few and there is no a homozygous plants for individual plant number, be further this mutant phenotype of checking and genotypic corresponding relation, use from 5 strain T ₁the T of individual plant (Fig. 2 B) results ₂carry out common separation detection for seed.Each T ₂for family plantation 20～40 strains.

Wherein 4D-50016-1 T ₁plant is negative, its T ₂family is all negative plant, and the fertility of these plant and Dongjin (wild-type) are without significant difference.4D-50016-2 ,-3 ,-4 ,-5T ₁plant is all heterozygous genes type, its T ₂in offspring, the fertility of negative plant and Dongjin be without significant difference, and the most of fertility of heterozygous plant and Dongjin do not have notable difference, and that homozygous plants shows as is completely sterile.

Be further male parent and Dongjin hybridization with RAD51C heterozygous plant, the positive obtaining hybridization plant backcrosses with Dongjin again, the positive BC obtaining ₁f ₁plant selfing obtains BC ₁f ₂seed.Utilize 39630-m-F, the 39630-m-R and the vectorR primer that in embodiment 2, relate to, to BC ₁f ₂genotype and the fertility of plant are analyzed.Result in Fig. 4 A shows, is for 2,3,6,13,14,16 and No. 20 (being that the RAD51C knocks out) plant of isozygotying, and is for 7 and No. 8 negative plant, and all the other 12 strains are heterozygous plants.To these BC ₁f ₂the setting percentage of plant is investigated and is found, all RAD51C knock out plant and show as completely sterilely, and heterozygous plant and negative plant show as and can educate (Fig. 4 A).Adopting the same method that backcrosses, is in male parent and rice varieties, to spend 11 hybridization with RAD51C heterozygous plant, to BC ₁f ₂genotype and the fertility of plant are analyzed.The result of Fig. 4 B shows, is for 19,22 and No. 23 (being that the RAD51C knocks out) plant of isozygotying, and 2,3,5,7,8,11,13,16,18 and 20 strains are negative plant, and all the other 12 strains are heterozygous plants.It is all completely sterile that all RAD51C knock out plant, and heterozygous plant and negative plant can educate (Fig. 4 B).

Above result of study explanation, the sterile of mutant plant caused owing to having inserted T-DNA (gene function is knocked) in RAD51C gene.

Embodiment 5: adopt further checking RAD51C gene function of genetic complementation experiment

According to the sequence of RAD51C gene and website ( http:// www.genome.arizona.edu) on rice varieties Japan fine bacterial artificial chromosome (BAC) library information, confirm that BAC clone OSJNBa0004A24 comprises RAD51C gene.From the Japan fine BAC library (Chen etc. that buy, 2002) in, obtain OSJNBa0004A24 cloned plasmids, use after restriction enzyme PstI and SacI complete degestion plasmid, electrophoresis reclaims the 8.9kb fragment (Fig. 2 A) that comprises whole RAD51C gene, vector plasmid pCAMBIA2301 (Fig. 5) after simultaneously cutting with PstI and SacI enzyme, it is conventional rice transformation carrier (Chang etc., 2009).Enzyme cuts complete, with chloroform: primary isoamyl alcohol (volume ratio 24: 1) extracting, purifying enzyme are cut product.Do ligation with the endonuclease bamhi that comprises RAD51C gene and the good carrier of purifying, the method that connection product transforms by electricity imports in intestinal bacteria DH10B (purchased from Promega company), cut and sequence verification positive colony by enzyme, the recombinant plasmid of acquisition is named as D203O.

Adopt agriculture bacillus mediated genetic transforming method (Lin and Zhang, 2005) that D203O is imported to RAD51C and knock out rice material.The genetic transformation plant obtaining is named as D203Or.According to identical genetic transforming method by empty carrier plasmid pCAMBIA2301 import RAD51C knock out rice material, the genetic transformation plant of acquisition in contrast, called after D204Or.

The present invention obtains 60 strain independence D203Or genetic transformation plant and 12 strain independence D204Or genetic transformation plant (contrast) altogether.With above-mentioned PCR primer 39630-m-R and vectorR and 2301-f (5 '-CCAGGCTTTACACTTTATGCTTC-3 ') and the positive transformed plant of rad51-com-R (5 '-CGGGTCTCCGACTACTGTGC-3 ') augmentation detection.Result demonstration, in 60 strain D203Or positive plants, 48 strains show fertility restorer (table 2) in various degree; And still sterile (table 2) of the negative plant of 8 strain D204Or positive plants and 4 strain D204Or.The further T to two D203Or plant ₁genotype and the fertility of family (D203Or-52 and D203Or-55) are analyzed.At D203Or-52T ₁in family, be for No. 12 the negative plant of genetic transformation, show as completely sterile; Remaining 22 strain is genetic transformation positive plant, and its fertility is 29.0%～58.1% (Fig. 6 A).At D203Or-55T ₁in family, the the the the the the the the the the the 1st, 3,5,6,7,8,9,10,11,12,13,14,15,16,17,19,20 and 21 is genetic transformation positive plant, and its fertility is 35.9%～53.1% (Fig. 6 B); And the the the 2nd, 4,18,22,23 be the negative plant of genetic transformation, show as completely sterile (Fig. 6 B).These presentation of results, proceed to RAD51C gene, and the sterile proterties that can make RAD51C knock out plant is restored, and further prove that the mutant character of RAD51C mutant (4D-50016-1) is inserted in RAD51C gene and is caused by T-DNA.

Table 2. have complementary functions genetic transformation plant (D203Or) and empty carrier genetic transformation plant (D204Or) T ₀for fertility analysis

The blastular that embodiment 6:RAD51C knocks out plant forms and growth course observation

The bulk dyeing transparent laser scanning confocal microscopy method of describing with reference to Guo Haibin etc. (2006), researchist's controlled observation of the present invention the RAD51C blastular that knocks out plant and contrast Dongjin (wild-type) plant form and growth course.In wild-type plant (Fig. 7 A), sporogonium forms under the epidermis near one end, the hole of bead; Then, form the phase at megasporocyte, the sporogonium formation megasporocyte of growing up; In megasporocyte meiophase, megasporocyte carries out reduction division and forms the megaspore of four linear arrangement; Form the phase function megaspore, three megaspores of nearly micropylar end in succession degenerate and finally leave the megaspore of chalazal end, i.e. function megaspore; Form the phase at monokaryon blastular, function megaspore grows up, and then at blastular m period, function megaspore carries out three mitotic division, forms successively two core blastulars, four core blastulars and eight core blastulars; In eight core Embryo Sac Development phases, both sides respectively have a core to shift to blastular intermediate formation polar core; Finally, in the blastular ripening stage, blastular increases to maximum, wherein comprises antipodal cell, polar core and synergid.

Knock out (Fig. 7 B) in plant at RAD51C, form the phase at megasporocyte, can form normal megasporocyte; But four megaspores that megasporocyte reduction division forms finally all can be degraded and can not form normal function megaspore.The Embryo Sac Development that RAD51C knocks out plant is obstructed, and does not observe the later developmental stage of normal function megaspore, has arrived mature embryo sac period, can only see the vestige of the core of empty blastocyst cavity and some degradeds in blastular.

Embodiment 7:RAD51C knocks out the observation of the pollen mother cells process of plant

It is undesired that RAD51C knocks out blastular formation and their the reduction division process of growth course hint of plant.For occur abnormal period in deep announcement reduction division, utilize pressed disc method (Lulong bucket etc., 2007) controlled observation RAD51C knock out the reduction division process of plant and contrast Dongjin (wild-type) plant pollen parent cell.In wild-type plant (Fig. 8 A), in leptotene stage, Chromatin condensation is the fine rule of carefully growing; In zygotene stage, homologous chromosomes near and start joint conference; To the pachytene stage, chromatin further shortens chap, and homologous chromosomes completes joint conference, is thicker linear structure under opticmicroscope; Chromatin further shortens formation bivalent, and each karyomit(e) of bivalent contains two chromatids; Enter diplotene stage, can between chromatid, see cross knot, the appearance of cross knot is the tangible result that exchange occurs; To the diakinesis stage, chromosome coiling degree is higher, and under opticmicroscope, diakinesis, chromosome dyeing was very dark; To observe chromosome number object best period; Enter I in mid-term from meiosis I subsequently, each bivalent is arranged on equatorial plate; At later stage I, the homologous chromosomes in bivalent is separated from each other and shifts to the two poles of the earth; At I in latter stage, homologous chromosomes arrives the two poles of the earth separately, and cytokinesis forms dyad; Each cell in dyad once divides again, forms tetrad.So far, reduction division process completes.Tetrad sporule continues to grow formation pollen granule.

Knock out (Fig. 8 B) in plant at RAD51C, when leptotene stage of meiosis prophase I, condense into filament shape, and in the time of zygotene stage, start joint conference.In the time of the pachytene stage, RAD51C knocks out plant appearance significantly extremely, and karyomit(e) now also presents thick line shape, but the pairing of condensing homologous chromosomes is incomplete, does not form the typical pachytene stage.Condensing in karyomit(e) continuation diplotene stage, there is obvious chromosome segment, and do not observe bivalent period at whole variant, the substitute is non-denumerable chromosome segment; To I in mid-term, Chromosomal arrangement is under the line on plate, and chromosome dyad fragment is not arranged on equatorial plate; At later stage I, homologous chromosomes separates, and can see the chromosome segment of many fractures in cell; At I in latter stage, in tenuigenin, be distributed with chromosome segment; At II in latter stage, can see the chromosome segment of many fractures in cell.

These presentation of results, RAD51C knocks out the sterile basic reason of plant and is that its karyomit(e) starts to occur the fragment of rhexis in the time of meiosis prophase I.Knock out after RAD51C, reduction division process occur extremely cause pollen sterility and blastular sterile.Therefore, the way that can adopt the special specificity promoter of organ or tissue to suppress RAD51C genetic expression creates male sterile or male and female sterile material, for the production of hybrid rice, or for cultivating the pollen sterility material of transgenic plant, eliminate the risk that in transgenic plant, foreign gene is escaped.

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Claims (1)

1. the RAD51C gene of separation, in an application of controlling in rice fertility, is characterized in that the nucleotide sequence of this gene is as shown in sequence table SEQ ID NO:1.

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