CN106834305A - A kind of rice male sterility changing gene OsSTRL2 and its application - Google Patents

Abstract

The invention discloses a rice male sterility changing gene OsSTRL2 and its application, belong to plant biotechnology field, the nucleotide sequence such as SEQ ID NO of rice male sterility changing gene OsSTRL2:Shown in 1, the amino acid sequence such as SEQIDNO of the albumen of its coding：Shown in 2；The present invention also protects a kind of method for cultivating male sterile plants, fertile gene that the present invention is provided and based on the male sterile line produced by the gene mutation, the stable fertility of the sterile line, not by environmental influence, can be recovered by wild-type transgenic.The sterile line that the gene and the gene mutation are produced provides necessary element to build new crossbreeding system, has great importance in production practices.

Description

A kind of rice male sterility changing gene OsSTRL2 and its application

Technical field

The invention belongs to plant genetic engineering and field of plant breeding, specifically, it is related to a kind of rice male fertility to adjust Control gene OsSTRL2 and its application.

Background technology

Paddy rice (Rice) as grass one kind, be the most important work on human nutrition and energy intake Thing, there is provided 1/5th of the calorie consumed more than the mankind whole world.Paddy rice is also most extensive in most of population in the world The staple food of consumption, particularly in Asia.According to food and agricultural organization's statistics in 2012, paddy rice was the agriculture of the third-largest yield in the world Product, is only second to sugarcane and corn.Crop breeding refers to the hereditary capacity of Crop Improvement, to cultivate the skill of high yield and high quality kind Art, also known as improvement of crop cultivar.It is with science of heredity as theoretical foundation, and integrated application plant ecological, plant physiology, bioid Various subject knowledges such as, plant pathology and biometrics, are the biotechnologys of a small investment and high efficiency, and development is planted Industry production tool is of great significance.

Crossbreeding is the Main Means for cultivating new rice variety, is also the main path for improving rice yield and quality. Because paddy rice has obvious hybrid vigour phenomenon, vigorous, the well developed root system of growth, big panicle many grains per panicle, strong stress resistance are mainly manifested in Etc. aspect, therefore, using the hybrid vigour of paddy rice increasing substantially the rice yield always hope dreamed of of breeding man. But, Genus Oryza self-pollinated plant, Pistil And Stamen life in same grain husk is spent, because grain husk flower very little, and every flower are only tied One seed, therefore be difficult the method for manually emasculation hybridization and produce substantial amounts of first generation hybrid seed, so for a long time The hybrid vigour of paddy rice fails to be applied.

Plants male sterility is defined as pollen grain missing or does not work, and can not also be produced or release function including plant Property pollen grain.In hybrid rice seeds production, the use of male sterile material has very important significance, because it is eliminated The process of mechanical emasculation.That is, being awarded by human assistance as genetic tool by this male sterile rice female parent The method of powder, just can largely produce hybrid seed.But for a long time, the development of hybrid rice, by Hybrid resource The influence of limitation, just progressively reaches bottleneck.Therefore, scientist is always in the sterile gene new by separating clone, and initiative is new Sterile material extend the cell and molecular background of breeding of hybridized rice.

The content of the invention

In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of DNA sequence dna of paddy gene OsSTRL2, its volume The code sequence of albumen, the sequence of promoter and its application.OsSTRL2 genes have the function of adjusting and controlling rice fertility, to the gene Being mutated or suppressed its expression causes its afunction or expression quantity to be lowered, and this can produce new rice male-sterile plants system, Following breeding of hybridized rice practice in very important application value.

The purposes of the albumen of gene DNA sequence or the associated dna sequence coding of offer of the invention, is used for：

The gene OsSTRL2 that the present invention is provided, is a male sterility of rice related gene, the base sequence of OsSTRL2 Such as SEQ ID NO：Shown in 1, amino acid sequence such as SEQ ID NO：Shown in 2, the gene is located at No. 3 chromosome in paddy rice On.

Present invention additionally comprises following a)-c) in any described DNA sequence dna：A) have with above-mentioned OsSTRL2 gene orders At least 90% (preferably at least 95%) sequence similarity, and the DNA sequence dna with identical function；B) under strict conditions can With the DNA sequence dna of the DNA hybridization of sequence (a) described；C) DNA sequence dna complementary with any of the above-described sequence.

Those skilled in the art should know that restoring gene of the present invention is included with OsSTRL2 genes highly It is homologous, and the very high homology with same sterility changing function function equivalence body sequence.The function of the very high homology Equivalents sequence can be with the nucleotide sequence hybridization of OsSTRL2 genes disclosed in this invention under being included in high stringency conditions DNA sequence dna." high stringency conditions " used in the present invention are known, including such as in NaCl containing 400mM, 40mM PIPES (pH6.4) and in the hybridization solution of 1mM EDTA hybridize in 60 DEG C 12-16 hours, then with containing 0.1SDS and 0.1% at 65 DEG C The cleaning solution of SSC is washed 15-60 minutes.

Function equivalence body sequence also includes having at least 90% with the sequence shown in OsSTRL2 genes disclosed in this invention, 95%th, 96%, 97%, 98% or 99% sequence similarity, and with the DNA sequence dna of sterility changing function, can be from any plant Separated in thing and obtained.Wherein, the percentage of sequence similarity can be obtained by known bioinformatics, including Myers and Miller algorithms (Bioinformatics, 4 (1)：11-17,1988), Needleman-Wunsch overall comparison methods (J.Mol.Biol., 48 (3)：443-53,1970), Smith-Waterman Local Alignments method (J.Mol.Biol., 147：195- 197,1981), Pearson and Lipman similarity-searching (PNAS, 85 (8)：2444-2448,1988), Karlin and Algorithm (Altschul etc., J.Mol.Biol., 215 (3) of Altschul：403-410,1990；PNAS, 90：5873-5877, 1993).This is to those skilled in the art familiar.

Gene order of the present invention can be separated from any plant and obtained, including but not limited to Btassica, corn, Wheat, sorghum, two section shepherd's purse category, sinapsis alba, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus, peanut, lucerne Mu, oat, rapeseed, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheats (Spelt), emmer, flax, gramagrass (Gramma grass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sweet Sugarcane, crowberry, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, the stem of noble dendrobium, gladiolus, chrysanthemum, Liliaceae, cotton, Eucalyptus, sunflower, rape, beet, coffee, ornamental plant and conifer etc..Preferably, plant include corn and soybean, safflower, leaf mustard, Wheat, barley, rye, rice, cotton and sorghum.

Application of the present invention is：The invention provides by influence OsSTRL2 nucleotide sequence or by adjust The transcriptional expression of OsSTRL2 genes is controlled so as to influence the method for plant fertility, so that obtain new male sterible series of rice can be with For producing hybrid seed.The influence plant fertility refers to the expression by regulating and controlling OsSTRL2 genes, so that the plant Fertility change, such as cause plant male sterility.Specifically, depending on concrete application demand, can be by various methods To influence expression of the OsSTRL2 genes in plant, so as to reach the effect of regulation and control plant male fertile.More specifically, adjusting The expression for controlling OsSTRL2 genes can be carried out using instrument obtained by many those of ordinary skill in the art, for example, by prominent Introducing of change, mutagenesis, being transferred to of antisense gene, co-suppression or hairpin structure etc., may be used to destruction OsSTRL2 genes just Often expression, so as to obtain male sterile plant.On the other hand, the invention further relates to a kind of recovery rice male-sterile plants system The method of male-sterile character, comprises the following steps：Using conventional genetic means by the OsSTRL2 genes, it is transferred to as foregoing The rice male-sterile plants system that the application is obtained, and then cause that mutant recovers wild type phenotype.

Present invention also offers the sterile mutant sequence and its malesterile mutants material of a kind of OsSTRL2 genes. More specifically, the malesterile mutants material is the OsSTRL2 gene endogenous by being mutated paddy rice, or mutation is with it highly The nucleotide sequence of homologous gene, makes the plant lose the process of male fertile." mutation " include but is not limited to Lower method, such as uses gene mutation caused by method physically or chemically, and chemical method with mutagens such as EMS including processing caused Mutagenesis, the mutation can also be point mutation, or DNA missing or insertion mutation, can also be by RNAi, fixed point The gene silencing means such as mutation are produced.

Specifically, present invention also offers a kind of rice male sterility mutant, it contains the male sterility base after mutation Cause, the nucleotide sequence such as SEQ ID NO of the male sterility gene after the mutation：Shown in 15, amino acid sequence such as SEQ ID NO：Shown in 16.Compared with wild type, in sterile mutant, first sequence of extron of the gene is by such as SEQ ID NO： Sported such as SEQ ID NO shown in 17：Shown in 18, cause the termination in advance of the gene.

Present invention also offers a kind of promoter of OsSTRL2 genes, the promoter has the specifically expressing in pollen Function, the sequence of corresponding promoter is OsSTRL2 genes from ATG to the nucleotide sequence of upstream 2000bp or so.More specifically Ground, in paddy rice, the nucleotide sequence such as SEQ ID NO of the OsSTRL2 gene promoters：Shown in 6.By SEQ ID NO：6 Be connected with Reporter gene GUS, convert plant, detection and analysis transfer-gen plant in GUS expression activities and expression pattern, by The root of transfer-gen plant, stem, leaf and GUS staining analysis is all carried out in spending, as a result find that OsSTRL2 promoters drive gus gene It is main to be expressed in Rice Anther, specifically in the P9 phases specific expression high of anther development.Illustrate provided by the present invention SEQ ID NO：6 promoters are a promoters for anther-specific expression.

Plant pollen specific expression promoter provided by the present invention, containing SEQ ID NO in ordered list：Core shown in 6 Nucleotide sequence, or comprising with SEQ ID NO：Listed nucleotide sequence has the nucleotide sequence of more than 90% similitude in 6, or Comprising from SEQ ID NO：100 in 6 sequences and more than 100 continuous nucleotide fragments, and can drive and this The nucleotides sequence that promoter is operatively connected is listed in the expression in plant pollen.Expression vector, transgenosis containing above-mentioned sequence Cell line and Host Strains etc. belong to protection scope of the present invention.Expand SEQ ID NO disclosed in this invention：6 promoters Any nucleotide fragments primer pair also within protection scope of the present invention.

Promoter nucleotide sequence provided by the present invention can also be used to be separated accordingly from other plants beyond paddy rice Sequence, especially carries out homologous clone from other monocotyledons.The promoter sequence according to listed by these corresponding sequences with this paper Sequence homology between row, or the homology with this promoter gene, are differentiated using technologies such as such as PCR, hybridization and separate these Corresponding sequence.Therefore, the SEQ ID NO according to listed by them with the present invention：Sequence phase between 6 promoter sequences (or its fragment) The respective segments for like property separate, are also included within embodiment.

" promoter " of the present invention refers to a kind of DNA regulatory regions, and it generally comprises energy guide RNA polymerase II and exists The TATA boxes of the suitable transcription initiation site starting RNA synthesis of specific coding sequence.Promoter can also include other recognition sequences, These recognition sequences are usually located at the upstream or 5 ' ends of TATA boxes, commonly known as upstream promoter element, a regulatory transcription effect The effect of rate.Those skilled in the art should know, although identify the core for promoter region disclosed by the invention Nucleotide sequence, but separate and identify other tune of the TATA box upstream regions in the specific promoter region identified of the invention Control element is also within the scope of the invention.Therefore, promoter region disclosed herein is generally further defined as comprising upstream Controlling element, such as regulating and controlling the tissue expression of coded sequence and those elements, the enhancer of temporal expressions function etc..With Identical mode, can identify, isolate the promoter unit for making it possible to be expressed in destination organization (such as male tissue) Part, it is used together with other core promoters, the expression preferential to verify male tissue.Core promoter refers to initial transcription Required minimal sequence, for example, be referred to as the sequence of TATA boxes, this be the gene of coded protein promoter it is usual All have.Therefore, alternatively, the upstream promoter of OsSTRL2 genes can be with core that is its own or originating from other Promoter association is used.

Core promoter can be core promoter known to any one, and such as cauliflower mosaic virus 35S or 19S are opened Mover (United States Patent (USP) No.5,352,605), ubiquitin promoter (United States Patent (USP) No.5,510,474), IN2 core promoters are (beautiful State patent No.5,364,780) or figwort mosaic virus promoter.

The function of the gene promoter can be analyzed by the following method：Can by promoter sequence and reporter gene It is operatively connected, forms transformable construct, then the construct is transferred in plant, in transgenic progeny is obtained, passes through Expression of the visual report gene in plant each histoorgan confirms its expression characterization；Or above-mentioned construct is sub- Clone into the expression vector for transient expression experiment, promoter or the function of its control region are detected by transient expression experiment Selection for test starting or the appropriate expression vector of regulatory region function will draw depending on host and by the expression vector The method for entering host, this kind of method is well known to those of ordinary skill in the art.For eucaryote, region in the carrier Region including control transcription initiation and control processing.These regions are operably connected to reporter gene, the report base Because including YFP, UidA, gus gene or luciferase.Expression vector comprising the presumption control region in genomic fragment can To be introduced into complete tissue, such as interim pollen, or callus is introduced, to carry out functional verification.

Additionally, promoter of the invention can be connected with the nucleotide sequence of not OsSTRL2 genes, it is heterologous to express other Nucleotide sequence.Promoter nucleotide sequence of the invention and its fragment and variant can be assembled in together with heterologous nucleotide sequence In one expression cassette, for being expressed in purpose plant, more specifically, being expressed in the male organs of the plant.The expression Box has suitable restriction enzyme site, for inserting the promoter and heterologous nucleotide sequence.These expression cassettes can be used for Genetic manipulation is carried out to any plant, to obtain desired corresponding phenotype.

OsSTRL2 promoters disclosed in this invention, the OsSTRL2 promoters more specifically in paddy rice, can be used to drive The expression of following heterologous nucleotide sequence is moved, so that the plant of conversion obtains male sterile phenotype.The heterologous nucleotide sequence Row codified promotes enzyme or modification enzyme, amylase, debranching enzyme and the pectase of carbohydrate degradation, more specifically such as α starch Enzyme gene, auxin (auxin), rot B, cytotoxin gene, diphtheria toxin, DAM methylases, Avidin, or it is optional From protokaryon regulator control system, dominant male sterility gene is can also be.

In some embodiments, the core for being operatively coupled on promoter downstream of the present invention being previously mentioned in the present invention Acid, wherein described " nucleic acid " can be operatively connected to structural gene, the regulation base on promoter disclosed herein Cause, the antisense gene of structural gene, the antisense gene of regulatory gene can interfere with the tiny RNA that endogenous gene is expressed.

More specifically, can be by sterility changing genes of SEQ ID NO provided by the present invention：1 is building up to promoter SEQ ID NO：6 downstream, so that the specifically expressing of the sterility changing gene in pollen is driven, or it is former by the technology of RNAi Reason, builds by SEQ ID NO：6 start can be with silence SEQ ID NO：The RNAi carrier of 1 gene, so as to obtain SEQ ID NO：The malesterile mutants of 1 gene.

Promoter sequence provided by the present invention is isolated from any plant, including but not limited to Btassica, corn, small Wheat, sorghum, two section shepherd's purse category, sinapsis alba, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus, peanut, clover, Oat, rapeseed, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheats (Spelt), Emmer, flax, gramagrass (Gramma grass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sugarcane, the red certain kind of berries Tongue, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, the stem of noble dendrobium, gladiolus, chrysanthemum, Liliaceae, cotton, eucalyptus, Xiang Certain herbaceous plants with big flowers, rape, beet, coffee, ornamental plant and conifer etc..Preferably, plant includes corn and soybean, safflower, leaf mustard, wheat, big Wheat, rye, rice, cotton and sorghum.

Organizing specific expression promoter can be used to target enhancing transcription and/or expression in specific plant tissue.Promoter Can express in targeted tissue also in other plant tissues express, can in targeted tissue strong expression and than other organize The much lower expression of degree, or highly preferred can express in targeted tissue.In one embodiment, promoter is preference The type of specifically expressing in the male or female tissue of plant.The present invention necessarily uses any specific male in method Tissue type of priority promoter, any in many such promoters well known by persons skilled in the art can use.Retouch herein The natural OsSTRL2 promoters stated are an examples of usable promoter.Another such promoter is 5126 startups Son, MS45 promoters, MS26 promoters, BS92-7 promoters, SGB6 controlling elements and TA29 promoters etc., it prefers to instruct Expression of the gene of its connection in male plant tissue.Gamete tissue precedence table up to startup can also be included in some constructs Son.Male gamete precedence table includes PG47 promoters and ZM13 promoters up to promoter.

Other components are may also include in above-mentioned construct, this depends primarily on the purpose and purposes of vector construction, for example may be used Further include selectable marker gene, targeting or regulating and controlling sequence, critical sequences or homing sequence, introne etc..Expression cassette will also Has functional transcription and translation terminator in 3 ' ends of desired heterologous nucleotide sequence are included in plant.Terminator can be The terminator of gene provided by the present invention, or the terminator from external source.More specifically, above-mentioned terminator can be rouge Fat propylhomoserin synthase or octopine synthase termination area.

Wishing to guide the expression product of heterologous nucleotide sequence into specific cells device, such as plastid, amyloplast, Huo Zheyin To endoplasmic reticulum, or in the case of cell surface or cell exocrine, expression cassette can also include the nucleosides for encoding transit peptides Acid sequence.Such transit peptides be it is known in the art, its including but not limited to the small subunit of Rubisco, plant EPSP synthase, Corn Brittle-1 chloroplast transit peptides etc..

During expression cassette is prepared, various DNA fragmentations can be operated, proper orientation is in provide, or DNA sequence dna in correct reading frame.To reach this purpose, adapter or joint can be used, DNA fragmentation is linked up, or Person further includes that other are operated, with restriction enzyme site of provides convenient etc..

Further, selectable marker gene is may also include in construct provided by the present invention, it is inverted for selecting Cell or tissue.The selectable marker gene includes assigning antibiotic resistance or the gene to Herbicid resistant.Suitable selection Marker gene is included but is not limited to:Chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistance Gene, sulfamido resistant gene, glyphosate gene, the careless bony resistant gene of fourth.The selectable marker gene can also be red Color fluorogene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene, flower The genes such as blue or green glucoside p1.

Expression cassette provided by the present invention or carrier can be inserted into plasmid, clay, yeast artificial chromosome, bacteria artificial dye Colour solid or other be adapted in any carrier being transformed into host cell.Preferred host cell is bacterial cell, is especially used In cloning or storage polynucleotides or bacterial cell for converting plant cell, such as Escherichia coli, Agrobacterium tumdfaciens and Agrobacterium rhizogenes.When host cell is plant cell, expression cassette or carrier can be inserted into the base of the plant cell being converted Because in group.Insertion can be positioning or random insertion.Preferably, such as homologous recombination is inserted through to realize.In addition, table It is external dyeing to be positively retained at up to box or carrier.Expression cassette of the invention or carrier may be present in core, chloroplaset, the line of plant cell In plastochondria and/or plastid.Preferably, expression cassette of the invention or carrier are inserted into the chromosomal DNA of plant nucleolus.

The purposes in the paddy rice production of hybrid seeds is tied up to the invention further relates to a kind of paddy rice sterile plant, is obtained with being applied as described in foregoing Rice male-sterile plants system is cross-breeding as female parent.Specifically, in some implementation methods applied, can apply OsSTRL2 genes provided by the present invention or its promoter realize OsSTRL2 or other similar fertility-related genes mutation institutes The breeding and holding of the male sterile line of acquisition.

More specifically, the breeding and holding of above-mentioned male sterile line, refer to be with homozygous recessive kernel male sterile mutant Transformation receptor material, 3 target genes of close linkage are converted into the sterile mutant recipient plant.3 targets Gene is respectively restoring gene, pollen inactivated gene and color mark screening-gene.Wherein, restoring gene can make not The transformation receptor fertility restorer educated, pollen inactivated gene can inactivate the pollen of the foreign gene containing conversion, that is, lose insemination Ability, screening-gene can be used for the sorting of transgenic seed and non-transgenic seed, and the non-transgenic seed for sorting out is used as Sterile line produce cenospecies, transgenic seed as maintainer come continuously, stably produce sterile line.

It is specific expressed in pollen that provided pollen-specific expression promoter of the invention can be used for foreign gene, So as to avoid foreign gene continuous expression adverse effect in plant its hetero-organization, plant pollen is can be also used for Grow the functional analysis and identification of related gene；Can be used for the establishment of male sterile line and restorer；And can be applied to flower It is male to plant so as to avoid by plant transgene drift or the brought bio-safety problem of pollen escape in powder abortion experiment The creation of property sterile line and restorer is significant.

Genetically modified plants of the invention use method for transformation known to plant biotechnology field technical staff to prepare.It is any Method can be used for being transformed into recombinant expression carrier in plant cell, to produce genetically modified plants of the invention.Method for transformation May include method for transformation directly or indirectly.Suitable direct method includes DNA intakes of polyethylene glycol induction, liposome-mediated Conversion, imported using particle gun, electroporation and microinjection, etc..In specific embodiment of the invention, the present invention The transformation technology based on agrobacterium has been used (reference can be made to Horsch RB etc. (1985) Science 225：1229；White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.15-38；The .Techniques such as Jenes B For Gene Transfer, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Agrobacterium bacterial strain (such as Agrobacterium tumdfaciens or hair root soil bar Bacterium) plasmid (Ti or Ri plasmids) and T-DNA elements are included, the plasmid and element are transferred to plant after with Agrobacterium transfection Thing, and T-DNA is integrated into the genome of plant cell.T-DNA can be located on Ri- plasmids or Ti- plasmids, or independently wrap It is contained in so-called binary vector.During agrobacterium-mediated method for transformation is described in for example.Agrobacterium-mediated conversion is most It is adapted to dicotyledon, but also is adapted for monocotyledon.During conversion of the agrobacterium to plant is described in for example.Conversion can be led Cause instantaneous or stabilization conversion and expression.Although nucleotide sequence of the invention can be inserted into fall into these broad varieties appoint In what plant and plant cell, but it is particularly suited for crop plants cell.

Compared with prior art, the present invention can be obtained including following technique effect：

1) present invention is sent out by controlling rice male fertility OsSTRL2 genes and its encoding proteins to obtain rice male reproduction The variant educated, realizes control paddy rice reproductive process.

2) rice mutant that the present invention is obtained is in trophophase and source parent's no significant difference, into generative growth phase Arrenotoky dysplasia, pollen abortion afterwards, obtains the sterile line of holandry infertility, the stable fertility of the sterile line, does not receive Environmental influence, can be recovered by wild-type transgenic.

3) sterile line that the gene and the gene mutation are produced provides necessity to build third generation crossbreeding system Element, the male sterile line that the gene mutation is produced, for producing hybrid seed, for breaking through and improves existing " three are " " two are " hybridization technique is significant, and is built in hybrid rice and have highly important application valency in agricultural production Value.

Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.

Brief description of the drawings

Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：

Fig. 1 is Subcellular Localizations of the OsSTRL2-YFP of the present invention in tobacco leaf epidermal cell, it is shown that infection 72 hours Confocal images of tobacco leaf epidermal cell afterwards.Wherein (A) 2x35S::The YFP signals of YFP are used as control；(B)2x35S:: The YFP signals of OsSTRL2-YFP；(C) the merging image of (B) and light field；The 2x35S of (D and E) coexpression::OsSTRL2-YFP YFP the and RFP signals marked with ER-；(F) the merging signal of (D and E) and light field.Scale=10 μm (A, D, E and F)；25μm(B And C)；

Fig. 2 is that the present invention turns OsSTRL2pro::GUS positive plants respectively organize GUS staining analysis；Wherein (A, B, C, D, E, F and H) be respectively root, stem, leaf, little Hua, little Hua (eliminate external application), small ear (eliminate external application and interior apply)) and GUS dyeing spend The cross section of medicine part；Scale=3mm (A-F)；30μm(H)；

Fig. 3 is the Phenotypic Observation of the malesterile mutants osstrl2 of CRISPR/Cas9 mediations of the present invention.Wherein, (A) The phenotype of wild type (left side) and osstrl2 (right side) in the maturity period compares；(B) wild type (left side) and osstrl2 (right side) are in heading rank The comparing of the little Hua of section；(C) in heading-stage wild type (left side) and the ratio of osstrl2 (right side) (being applied and external application in removal) little Hua Compared with；(D and E) is respectively the I2/KI pollen stainings of wild type and osstrl2；Scale=0.20mm (B and C)；30 μm (D and E)；

Fig. 4 is osstrl2 (complementary osstrl2 mutant) phenotype schematic diagram that fertility of the present invention is restored；Wherein, (A) it is wild type little Hua cut-away views；(B) it is wild-type mature pollen I₂/ KI coloration results；(C) for complementation osstrl2 is prominent The little Hua cut-away views of variant offspring；(D) be complementary osstrl2 Mutant progenies mature pollen I₂/ KI coloration results.

Specific embodiment

Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.

The clone of embodiment 1, rice fertility control protein coding gene OsSTRL2 and functional analysis

1st, OsSTRL2 full length genes cDNA clone, expression vector establishment and Subcellular Localization

Inventor utilizes bioinformatics technique well-known to those skilled in the art, and one is determined from rice genome Containing complete rice male reproductive development control albumen OsSTRL2.Through software analysis and cDNA clone, its CDS is 1284bp, such as SEQ ID NO：Shown in 1, the rice male reproductive development control albumen of 427 amino acid of encoding full leng, its sequence such as SEQ ID NO：Shown in 2.Show through full nucleotide sequence analysis result:OsSTRL2 full length genes 2083bp (SEQ ID NO：3, comprising tune Control area and introne).

No. 9 RNA of kind are spent with normal wild type paddy rice as template, synthesizes the first chain cDNA, by the total length of OsSTRL2 CDNA is through primer pair OsSTRL2-YFP-F (SEQ ID NO：4) with OsSTRL2-YFP-R (SEQ ID NO：5) rear clone is expanded To in pA7-YFP carriers producing 2x35S::OsSTRL2-YFP expression cassettes, will OsSTRL2 C-terminal merged with YFP and Under the control of the double 35S promoters of mosaic virus；Then by whole 2x35S::OsSTRL2-YFP expression cassettes are imported pCAMBIA1300.Equally, by KDEL KDEL fusions and the C-terminal of RFP, the double 35S promoters of mosaic virus are placed in Control under as endoplasmic reticulum (ER) telltale mark.These plasmids are by agriculture bacillus mediated infiltration in tobacco leaf epidermal cell Middle single expression or coexpression.YFP and RFP signals infiltration 72 hours after with confocal scanning microscope (Nikon A1, Kanagawa, Japan) imaging.In Subcellular Localization result (see Fig. 1) display OsSRTL2 albumen is primarily targeted in cell Matter is online.

2nd, the promoter clone of OsSTRL2 genes and its expression pattern analysis

No. 9 DNA of kind are spent with normal wild type paddy rice as template, before the OsSTRL2 gene start codons Face about 1.7K fragments (SEQ ID NO：6) through primer pair OsSTRL2-promoter-F (SEQ ID NO：And OsSTRL2- 7) promoter-R(SEQ ID NO：8) amplification is cloned on carrier pCMBIYA1300 and produces OsSTRL2pro::GUS expression cassettes, Wherein gus gene is driven by natural OsSTRL2 promoters.OsSTRL2pro will be contained::The construct of GUS imports crown gall agriculture In bacillus strain EHA105 and it is transferred in wild rice by transgenosis and is spent in No. 9.Specifically, carrier is passed through into freeze-thaw method Import Agrobacterium EHA105 (preservation of Sichuan Agricultural University's paddy rice institute's Biotechnology Experiment room).Agrobacterium EHA105 flat boards (4 DEG C of guarantors Deposit) during picking single bacterium falls within YEP fluid nutrient mediums (each 50mg/L of Km and Rif), 28 DEG C of 12~18h of shaken cultivation, then take 1~ 5mL bacterium solutions are connected in 100mL YEP fluid nutrient mediums (100 μm of ol/L containing acetosyringone), OD values are surveyed after shaken cultivation 4h dilute To respective concentration (OD=0.5).By fresh bacterium solution in 8000rpm, 4 DEG C, 5min collects thallines, and it is resuspended in 1/3rd and refers to AAM culture mediums in.Addition soaks 25min in being placed with the sterilizing triangular flask of eugonic embryo callus, then dries up table Face bacterium solution, by callus transfer in co-culture base on, 28 DEG C of 2~3d of light culture.Callus is through sterilized water and contains for co-cultivation After the rinsed with sterile water of Cef 500mg/L, 4h or so is blown on the table, transfer 28 DEG C of 5~7d of light culture in pre-culture medium. The callus of preculture is transferred in continuing to cultivate 3~4 weeks on the screening and culturing medium containing Hyg and Cef, then by kanamycin-resistant callus tissue group Knit and transfer on the screening and culturing medium for only containing Hyg, screen 1~2 time.Take resistant calli to transfer on differential medium, 28 DEG C illumination differentiation.Differentiation seedling is transferred in root media, 28 DEG C of illumination cultivations 3~4 weeks, open culturing hardening 7d or so, most After be transplanted to crop field.The Transplantation of Regenerated Plantlets of acquisition uses hygromycin selection transformed plant after surviving.To positive transgenic plant root, Result (Fig. 2) the display OsSTRL2 promoters that stem, leaf and flower carry out GUS dyeing (activity analysis of beta-galactosidase) drive GUS is main to express in the tapetum of paddy rice little Hua flower pesticide and sporidiole, and this shows that OsSTRL2 promoters are that a flower pesticide is special The promoter of type.

The initiative of embodiment 2, male sterible series of rice

Osstrl2 rice male-sterile plants system is formulated by genetic engineering or other means

The coding region sequence of OsSTRL2 genes such as SEQ ID NO in the present embodiment：Shown in 2.The osstrl2 of the present embodiment Mutant material is, by spending No. 9 in conventional japonica rice kind, to be obtained by the sequence variations to OsSTRL2 genes.

Specifically, target site (the SEQ ID NO of OsSTRL2 genes are designed first using CRISPR/Cas9 technologies：9), so VK005-01-OsSTRL2 knockout carriers being built afterwards, recombinant vector being identified, the wherein expression of target site is started by OsU3 Son drives.Will be during knockout carrier imports and spends No. 9 genomes in wild rice by the rice transgenic method in embodiment 1. The Transplantation of Regenerated Plantlets of acquisition uses hygromycin selection transformed plant after surviving；Positive plant extracts blade STb gene, and centering spends No. 9 The target site of corresponding OsSTRL2 genes is through primer pair OsSTRL2-gRNA-seq-F (SEQ ID NO in genome：10) and OsSTRL2-gRNA-seq-R(SEQ ID NO：11) sequence verification is carried out after expanding, while investigating fertility table to transfer-gen plant Type, so as to verify OsSTRL2 gene functions.Result such as Fig. 3 and Biao 1, OsSTRL2 gene show as flower by the plant of successful knockout Medicine white is short and small, and pollen is drastically reduced and debility, and then obtains rice male-sterile plants system osstrl2.Can from above-mentioned analysis To find out, OsSTRL2 is gene necessary to paddy rice maintains normal male fertility, and lacking the gene can cause plant male not Educate, lowering the gene expression then reduces the male fertile of plant.

The OsSTRL2 transgenosis of table 1 knocks out phenotypes and genotype linkage analysis result of the T0 for plant：

Numeral represents the different fertility phenotype that different genotype plant is represented in table

Embodiment 3, the recovery of rice male-sterile plants system osstrl2 fertility are transgenic function complementation

No. 9 DNA of kind are spent with normal wild type paddy rice as template, through primer pair OsSTRL2-COM-F (SEQ ID NO：12) with OsSTRL2-COM-R (SEQ ID NO：13) fragment SEQ ID NO are obtained after expanding：14, the fragment is cloned into On carrier pCMBIYA1300, P1300-OsSTRL2-COM is successfully built.Similarly, by the Transgenic Rice in embodiment 1 Without tide in BC1F2 offsprings of the method by carrier P1300-OsSTRL2-COM importings by rice male-sterile plants system osstrl2 Chloramphenicol resistance but in the callus of the Immature-base culture of male sterility phenotype.The Transplantation of Regenerated Plantlets of acquisition is mould with tide after surviving Element screening transformed plant.Transgenosis T0 to positive plant observes rice fertility for plant, and investigation result (Fig. 4) display turns The fertility of the plant of OsSTRL2 complementation expressing genes is recovered, and becomes normally educate, and shows the expression of OsSTRL2 genes Be paddy rice maintain normal male fertility necessary to.

In sum, the present invention is obtained by controlling rice male reproductive development related gene OsSTRL2 and its encoding proteins The abnormal variant of rice male reproductive development is obtained, control rice male reproductive development and fertility is realized；The water that the present invention is obtained Rice mutant in vegetative growth stage and source parent's no significant difference, into after generative growth phase, send out by male reproductive organ Educate exception, pollen abortion and cause plant infertility, there is highly important application in agricultural production.

Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention In the protection domain that profit is required.

SEQUENCE LISTING

<110>Sichuan Agricultural University

<120>A kind of rice male sterility changing gene OsSTRL2 and its application

<130> 2017

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 1293

<212> DNA

<213>OsSTRL2 genes

<400> 1

atggaagaga agaagcagca gcagcagcgt ccacagagag ggcgcgatgg catcctgcag 60

tatccgcacc ttttcttcgc ggcgctggcg ctggccctgc tcctcaccga cccgttccac 120

ctcggcccgc tcgccggggt ggactaccgg ccggtgaggc acgagctggc gccgtaccgc 180

gaggtgatgg cgcggtggcc gcgggacaac ggcagccggc tcaggcacgg caggctggag 240

ttcgtcggac aggtgttcgg gccggagtcc atcgagttcg accgccacgg ccgcggcccc 300

tacgccggcc tcgccgacgg ccgcgtcgtg cggtggatgg gggaggacgc cgggtgggag 360

acgttcgccg tcatgagccc tgactggtcg gagaaagttt gtgccaatgg ggtggagtcg 420

acgacgaaga agcagcacga gatggagcga cggtgcggcc ggcctctcgg gctgaggttt 480

cacggcgaga ccggcgagct ctacgtcgcc gacgcatact acgggctcat gtccgtcggt 540

ccgaacggcg gggtggcgac ctctctcgcg agagaagtcg gcgggagccc ggtcaacttc 600

gcgaacgacc tcgacatcca ccgcaacggc tccgtgttct tcaccgacac gagcacgaga 660

tacaacagaa aggatcatct gaacgttctg ctagaaggtg aaggcacagg gaggctgctc 720

agatatgacc cagaaaccaa agctgcccat gtcgtgctga gcgggctggt cttcccgaat 780

ggcgtgcaga tttctgacga ccagcagttc ctcctcttct ccgaaacaac aaactgcagg 840

ataatgcggt actggctgga agggccaaga gccgggcagg tggaggtgtt cgccgacctg 900

ccggggttcc cggacaacgt gcgactgagc agcggcggcg gcggcggacg gttctgggtg 960

gcgatcgact gctgcaggac ggcggcgcag gaggtgttcg ccaagcggcc gtggctgcga 1020

acgctctact tcaagctgcc cctgacgatg cggacgctgg ggaagatggt cagcatgcgg 1080

atgcacaccc tcgtcgcgct cctcgacggc gaaggggacg tcgtcgaggt gctcgaggac 1140

cggggcggcg aggtgatgcg gctggtgagc gaggtgaggg aggtggggcg caagctgtgg 1200

atcggcaccg tggctcataa ccacatcgcc acgatccctt acccgttgga agagcagagt 1260

agcagcagca gcagcaacgt gcttggtgat tga 1293

<210> 2

<211> 430

<212> PRT

<213>The encoding proteins of OsSTRL2 genes

<400> 2

Met Glu Glu Lys Lys Gln Gln Gln Gln Arg Pro Gln Arg Gly Arg Asp

1 5 10 15

Gly Ile Leu Gln Tyr Pro His Leu Phe Phe Ala Ala Leu Ala Leu Ala

20 25 30

Leu Leu Leu Thr Asp Pro Phe His Leu Gly Pro Leu Ala Gly Val Asp

35 40 45

Tyr Arg Pro Val Arg His Glu Leu Ala Pro Tyr Arg Glu Val Met Ala

50 55 60

Arg Trp Pro Arg Asp Asn Gly Ser Arg Leu Arg His Gly Arg Leu Glu

65 70 75 80

Phe Val Gly Gln Val Phe Gly Pro Glu Ser Ile Glu Phe Asp Arg His

85 90 95

Gly Arg Gly Pro Tyr Ala Gly Leu Ala Asp Gly Arg Val Val Arg Trp

100 105 110

Met Gly Glu Asp Ala Gly Trp Glu Thr Phe Ala Val Met Ser Pro Asp

115 120 125

Trp Ser Glu Lys Val Cys Ala Asn Gly Val Glu Ser Thr Thr Lys Lys

130 135 140

Gln His Glu Met Glu Arg Arg Cys Gly Arg Pro Leu Gly Leu Arg Phe

145 150 155 160

His Gly Glu Thr Gly Glu Leu Tyr Val Ala Asp Ala Tyr Tyr Gly Leu

165 170 175

Met Ser Val Gly Pro Asn Gly Gly Val Ala Thr Ser Leu Ala Arg Glu

180 185 190

Val Gly Gly Ser Pro Val Asn Phe Ala Asn Asp Leu Asp Ile His Arg

195 200 205

Asn Gly Ser Val Phe Phe Thr Asp Thr Ser Thr Arg Tyr Asn Arg Lys

210 215 220

Asp His Leu Asn Val Leu Leu Glu Gly Glu Gly Thr Gly Arg Leu Leu

225 230 235 240

Arg Tyr Asp Pro Glu Thr Lys Ala Ala His Val Val Leu Ser Gly Leu

245 250 255

Val Phe Pro Asn Gly Val Gln Ile Ser Asp Asp Gln Gln Phe Leu Leu

260 265 270

Phe Ser Glu Thr Thr Asn Cys Arg Ile Met Arg Tyr Trp Leu Glu Gly

275 280 285

Pro Arg Ala Gly Gln Val Glu Val Phe Ala Asp Leu Pro Gly Phe Pro

290 295 300

Asp Asn Val Arg Leu Ser Ser Gly Gly Gly Gly Gly Arg Phe Trp Val

305 310 315 320

Ala Ile Asp Cys Cys Arg Thr Ala Ala Gln Glu Val Phe Ala Lys Arg

325 330 335

Pro Trp Leu Arg Thr Leu Tyr Phe Lys Leu Pro Leu Thr Met Arg Thr

340 345 350

Leu Gly Lys Met Val Ser Met Arg Met His Thr Leu Val Ala Leu Leu

355 360 365

Asp Gly Glu Gly Asp Val Val Glu Val Leu Glu Asp Arg Gly Gly Glu

370 375 380

Val Met Arg Leu Val Ser Glu Val Arg Glu Val Gly Arg Lys Leu Trp

385 390 395 400

Ile Gly Thr Val Ala His Asn His Ile Ala Thr Ile Pro Tyr Pro Leu

405 410 415

Glu Glu Gln Ser Ser Ser Ser Ser Ser Asn Val Leu Gly Asp

420 425 430

<210> 3

<211> 2091

<212> DNA

<213>OsSTRL2 genes

<400> 3

aagttaagat caccgtctcc accaattccg tctttcctct gcgtgcaaat tccgtcttcc 60

ctcgctcctg atctccatgg aagagaagaa gcagcagcag cagcgtccac agagagggcg 120

cgatggcatc ctgcagtatc cgcacctttt cttcgcggcg ctggcgctgg ccctgctcct 180

caccgacccg ttccacctcg gcccgctcgc cggggtggac taccggccgg tgaggcacga 240

gctggcgccg taccgcgagg tgatggcgcg gtggccgcgg gacaacggca gccggctcag 300

gcacggcagg ctggagttcg tcggacaggt gttcgggccg gagtccatcg agttcgaccg 360

ccacggccgc ggcccctacg ccggcctcgc cgacggccgc gtcgtgcggt ggatggggga 420

ggacgccggg tgggagacgt tcgccgtcat gagccctgac tggtaacgaa cacctcgcct 480

gcattttgct ctcgccctcc acgaaaacac ctctcgtagc agtgtacaat tacgtgttct 540

tatattgcaa aaaaaggtcg gagaaagttt gtgccaatgg ggtggagtcg acgacgaaga 600

agcagcacga gatggagcga cggtgcggcc ggcctctcgg gctgaggttt cacggcgaga 660

ccggcgagct ctacgtcgcc gacgcatact acgggctcat gtccgtcggt ccgaacggcg 720

gggtggcgac ctctctcgcg agagaagtcg gcgggagccc ggtcaacttc gcgaacgacc 780

tcgacatcca ccgcaacggc tccgtgttct tcaccgacac gagcacgaga tacaacagaa 840

agtgtgcagc tgcagtatca ctctcttcag ttgtatcgat tctctatttc cttctatcgt 900

tcaagatttt ctgattagaa tcagttgtgc agggatcatc tgaacgttct gctagaaggt 960

gaaggcacag ggaggctgct cagatatgac ccagaaacca aagctgccca tgtcgtgctg 1020

agcgggctgg tcttcccgaa tggcgtgcag atttctgacg accagcagtt cctcctcttc 1080

tccgaaacaa caaactgcag gtgaaatggc acaagctttc acaggttctg aaaatactaa 1140

aggttaaaca agattcagaa ttgattaaca ttgcacgcat atgctgttct aggataatgc 1200

ggtactggct ggaagggcca agagccgggc aggtggaggt gttcgccgac ctgccggggt 1260

tcccggacaa cgtgcgactg agcagcggcg gcggcggcgg acggttctgg gtggcgatcg 1320

actgctgcag gacggcggcg caggaggtgt tcgccaagcg gccgtggctg cgaacgctct 1380

acttcaagct gcccctgacg atgcggacgc tggggaagat ggtcagcatg cggatgcaca 1440

ccctcgtcgc gctcctcgac ggcgaagggg acgtcgtcga ggtgctcgag gaccggggcg 1500

gcgaggtgat gcggctggtg agcgaggtga gggaggtggg gcgcaagctg tggatcggca 1560

ccgtggctca taaccacatc gccacgatcc cttacccgtt ggaagagcag agtagcagca 1620

gcagcagcaa cgtgcttggt gattgatact ttgataggct ggttttagca gcaacaaagg 1680

tgtactagtt gatgtattgt ttgtgtttgc cgggccatca tagaaagtgc ctggtgatct 1740

ctgggacttg atggcaaatg ttgggcaaat tgtgatcgaa taagattagt actagagtta 1800

tcgtgtaata aggacatgca tggactacca tgtatttcat gttatgacgc tcctaagagc 1860

cacagaccac agcgatggta ttagacccct tctcagaatg gttctgctca ttttcggctt 1920

cgatcgtggt acgcgttcgt gtcttcgtgt gatcggaaaa aaaatatttg ccgtttacaa 1980

gtgatagttt ttcagtggat gtaatttgta cgaaatacca tcgtacaaac gtttgtactt 2040

tttcatcata gtcattagcc tttcatgaat agtactcaca tttgtaaggc c 2091

<210> 4

<211> 43

<212> DNA

<213>Artificial sequence

<400> 4

accagtctct ctctcaagct tatggaagag aagaagcagc agc 43

<210> 5

<211> 41

<212> DNA

<213>Artificial sequence

<400> 5

gctcaccata ctagtggatc catcaccaag cacgttgctg c 41

<210> 6

<211> 2141

<212> DNA

<213>OsSTRL2 genes

<400> 6

ttacctctct cgacgtaagc catggtcgcg cccctcggat cgccctagcc caccctctgc 60

gatggaaaga actacaatga taagattttc gctcgaggcg agaagaatta agaaggggaa 120

accgaaggag aagggattcg cgacattctt ccttgcgcgt ggagagacta agagaccggt 180

gagagggtcg cgggccttgc gtcttttatt ggccattgac tcgtagaccc gaagttttcg 240

ttgaggtatg gggcccaatg tgtcagtgac acgaattgtt ggcctgcatg ccggtgagga 300

aagagcatcg gcaggaagaa ggtgccacgt caattgtgct tttgtgagcc gttggattaa 360

ggatgtgggt tcgagatgaa ttcaaaaggt caaataatct tccggctgtg gcgctctcta 420

agtctcacct ttctatggtc aaataatgca ttagatagag aagatagtat aaggtttagg 480

gcatgttcac tttgataccg ttttcaacct taccaaattt tggtaaaatt gttaaaaaaa 540

agttgctaca tttagtttga tgccaaattt tagtaactat ataagaaatc attaactata 600

taagaaaaag gttctttttg gcatcaaaat gaataggccc ttatagtata tggtggtcat 660

caattctaat tttatccact cgtctactta ttaattaaga gataacatat ataatatatt 720

gcttttaata tacttactct gatatgctat actaattttt ctctatttgt tttcacttaa 780

tttattggtc tcttctctcg gattcctata agtttgtagt tttgtgtaga gattgtcctt 840

tcgtctcttc ctctctctct ctctctctct ctttctttct ctattccatc ttatcacatg 900

atcctacatg acatcttata gctaagagat ccatagtagg ttgagttcgg gaggcaaaca 960

aagaaagcga ttagagcgat attaatcaag tatcagtcta aaaaagcttg gaaaatggat 1020

taatatgatt tttaaagttt ttttatatag aaaacttttg taaaaaaacg tactttagca 1080

gtttgaaaag cgtgtgcgaa aaaaagatga aggtgggtag gaaaagaagg gagaataaca 1140

cagcataagc atgttttacc cctcgactat taactcttga ttaaggaaca tggtaaaatt 1200

tataccacta agttatcgaa accatatata aatgactttt aagataatct ccggtttttt 1260

ttaatatgaa acgtcattga ttttttaaga tatatttaac cattttcaaa cctaagaggt 1320

ggtttatttt ccctcaagag aacgcgccgt tattttttta cgtcacctaa aaaacataat 1380

tttttggaaa aaaatatatg aaaagacaga tgaatatatg atttttttaa aatacagatt 1440

caaactttac ttacacaaat agaaacaaaa aacaataaat ttgcttatga atggtcaaat 1500

tcattatttt tatttgtacc catgtaagtc aaatttaaac ttcatggtca ttgatatatt 1560

tataaaaaat agttttttag ttgaatttta aagtaacaaa aaaaacaaag ggacaaatag 1620

acttccctat aacccaacgc atgctcaacc acagtaaatg ttttagctta gacttagagt 1680

tagttttctt gcaacatcga cacaccattg cctgtgtctt gtgatttttc ctgcacgttt 1740

agaagggtgg catcgattga acttctggac acttggagtt cttccttctt cgtgatgcac 1800

cttttgcttt acagcgctag caatggccat ggtcagggcc ctcagccctg gccgggttcg 1860

gttacatgca tcgtgatatg cttgttgacc tgtgcatctt gcaccgtcat ccaagcaatg 1920

caaaacatgc aaatccccac ttcgaaagca caagattcct tggctattcc gaaccaacag 1980

aacacctact cccaaacaat cacgctgact catgcaacct ccatgcatcg aagatatatt 2040

ttcgctgcaa aggcattaag aatttaagtt aagatcaccg tctccaccaa ttccgtcttt 2100

cctctgcgtg caaattccgt cttccctcgc tcctgatctc c 2141

<210> 7

<211> 41

<212> DNA

<213>Artificial sequence

<400> 7

gagatctaca gcgctaagct tttacctctc tcgacgtaag c 41

<210> 8

<211> 40

<212> DNA

<213>Artificial sequence

<400> 8

ggactgacca cccggggatc cggagatcag gagcgaggga 40

<210> 9

<211> 23

<212> DNA

<213>OsSTRL2 genes

<400> 9

ggaacgggtc ggtgaggagc agg 23

<210> 10

<211> 18

<212> DNA

<213>Artificial sequence

<400> 10

tcttccctcg ctcctgat 18

<210> 11

<211> 18

<212> DNA

<213>Artificial sequence

<400> 11

tggcacaaac tttctccg 18

<210> 12

<211> 41

<212> DNA

<213>Artificial sequence

<400> 12

acgacggcca gtgccaagct tttacctctc tcgacgtaag c 41

<210> 13

<211> 42

<212> DNA

<213>Artificial sequence

<400> 13

tatgaccatg attacgaatt ctgacttact cggttagctc gt 42

<210> 14

<211> 4561

<212> DNA

<213>OsSTRL2 genes

<400> 14

ttacctctct cgacgtaagc catggtcgcg cccctcggat cgccctagcc caccctctgc 60

gatggaaaga actacaatga taagattttc gctcgaggcg agaagaatta agaaggggaa 120

accgaaggag aagggattcg cgacattctt ccttgcgcgt ggagagacta agagaccggt 180

gagagggtcg cgggccttgc gtcttttatt ggccattgac tcgtagaccc gaagttttcg 240

ttgaggtatg gggcccaatg tgtcagtgac acgaattgtt ggcctgcatg ccggtgagga 300

aagagcatcg gcaggaagaa ggtgccacgt caattgtgct tttgtgagcc gttggattaa 360

ggatgtgggt tcgagatgaa ttcaaaaggt caaataatct tccggctgtg gcgctctcta 420

agtctcacct ttctatggtc aaataatgca ttagatagag aagatagtat aaggtttagg 480

gcatgttcac tttgataccg ttttcaacct taccaaattt tggtaaaatt gttaaaaaaa 540

agttgctaca tttagtttga tgccaaattt tagtaactat ataagaaatc attaactata 600

taagaaaaag gttctttttg gcatcaaaat gaataggccc ttatagtata tggtggtcat 660

caattctaat tttatccact cgtctactta ttaattaaga gataacatat ataatatatt 720

gcttttaata tacttactct gatatgctat actaattttt ctctatttgt tttcacttaa 780

tttattggtc tcttctctcg gattcctata agtttgtagt tttgtgtaga gattgtcctt 840

tcgtctcttc ctctctctct ctctctctct ctttctttct ctattccatc ttatcacatg 900

atcctacatg acatcttata gctaagagat ccatagtagg ttgagttcgg gaggcaaaca 960

aagaaagcga ttagagcgat attaatcaag tatcagtcta aaaaagcttg gaaaatggat 1020

taatatgatt tttaaagttt ttttatatag aaaacttttg taaaaaaacg tactttagca 1080

gtttgaaaag cgtgtgcgaa aaaaagatga aggtgggtag gaaaagaagg gagaataaca 1140

cagcataagc atgttttacc cctcgactat taactcttga ttaaggaaca tggtaaaatt 1200

tataccacta agttatcgaa accatatata aatgactttt aagataatct ccggtttttt 1260

ttaatatgaa acgtcattga ttttttaaga tatatttaac cattttcaaa cctaagaggt 1320

ggtttatttt ccctcaagag aacgcgccgt tattttttta cgtcacctaa aaaacataat 1380

tttttggaaa aaaatatatg aaaagacaga tgaatatatg atttttttaa aatacagatt 1440

caaactttac ttacacaaat agaaacaaaa aacaataaat ttgcttatga atggtcaaat 1500

tcattatttt tatttgtacc catgtaagtc aaatttaaac ttcatggtca ttgatatatt 1560

tataaaaaat agttttttag ttgaatttta aagtaacaaa aaaaacaaag ggacaaatag 1620

acttccctat aacccaacgc atgctcaacc acagtaaatg ttttagctta gacttagagt 1680

tagttttctt gcaacatcga cacaccattg cctgtgtctt gtgatttttc ctgcacgttt 1740

agaagggtgg catcgattga acttctggac acttggagtt cttccttctt cgtgatgcac 1800

cttttgcttt acagcgctag caatggccat ggtcagggcc ctcagccctg gccgggttcg 1860

gttacatgca tcgtgatatg cttgttgacc tgtgcatctt gcaccgtcat ccaagcaatg 1920

caaaacatgc aaatccccac ttcgaaagca caagattcct tggctattcc gaaccaacag 1980

aacacctact cccaaacaat cacgctgact catgcaacct ccatgcatcg aagatatatt 2040

ttcgctgcaa aggcattaag aatttaagtt aagatcaccg tctccaccaa ttccgtcttt 2100

cctctgcgtg caaattccgt cttccctcgc tcctgatctc catggaagag aagaagcagc 2160

agcagcagcg tccacagaga gggcgcgatg gcatcctgca gtatccgcac cttttcttcg 2220

cggcgctggc gctggccctg ctcctcaccg acccgttcca cctcggcccg ctcgccgggg 2280

tggactaccg gccggtgagg cacgagctgg cgccgtaccg cgaggtgatg gcgcggtggc 2340

cgcgggacaa cggcagccgg ctcaggcacg gcaggctgga gttcgtcgga caggtgttcg 2400

ggccggagtc catcgagttc gaccgccacg gccgcggccc ctacgccggc ctcgccgacg 2460

gccgcgtcgt gcggtggatg ggggaggacg ccgggtggga gacgttcgcc gtcatgagcc 2520

ctgactggta acgaacacct cgcctgcatt ttgctctcgc cctccacgaa aacacctctc 2580

gtagcagtgt acaattacgt gttcttatat tgcaaaaaaa ggtcggagaa agtttgtgcc 2640

aatggggtgg agtcgacgac gaagaagcag cacgagatgg agcgacggtg cggccggcct 2700

ctcgggctga ggtttcacgg cgagaccggc gagctctacg tcgccgacgc atactacggg 2760

ctcatgtccg tcggtccgaa cggcggggtg gcgacctctc tcgcgagaga agtcggcggg 2820

agcccggtca acttcgcgaa cgacctcgac atccaccgca acggctccgt gttcttcacc 2880

gacacgagca cgagatacaa cagaaagtgt gcagctgcag tatcactctc ttcagttgta 2940

tcgattctct atttccttct atcgttcaag attttctgat tagaatcagt tgtgcaggga 3000

tcatctgaac gttctgctag aaggtgaagg cacagggagg ctgctcagat atgacccaga 3060

aaccaaagct gcccatgtcg tgctgagcgg gctggtcttc ccgaatggcg tgcagatttc 3120

tgacgaccag cagttcctcc tcttctccga aacaacaaac tgcaggtgaa atggcacaag 3180

ctttcacagg ttctgaaaat actaaaggtt aaacaagatt cagaattgat taacattgca 3240

cgcatatgct gttctaggat aatgcggtac tggctggaag ggccaagagc cgggcaggtg 3300

gaggtgttcg ccgacctgcc ggggttcccg gacaacgtgc gactgagcag cggcggcggc 3360

ggcggacggt tctgggtggc gatcgactgc tgcaggacgg cggcgcagga ggtgttcgcc 3420

aagcggccgt ggctgcgaac gctctacttc aagctgcccc tgacgatgcg gacgctgggg 3480

aagatggtca gcatgcggat gcacaccctc gtcgcgctcc tcgacggcga aggggacgtc 3540

gtcgaggtgc tcgaggaccg gggcggcgag gtgatgcggc tggtgagcga ggtgagggag 3600

gtggggcgca agctgtggat cggcaccgtg gctcataacc acatcgccac gatcccttac 3660

ccgttggaag agcagagtag cagcagcagc agcaacgtgc ttggtgattg atactttgat 3720

aggctggttt tagcagcaac aaaggtgtac tagttgatgt attgtttgtg tttgccgggc 3780

catcatagaa agtgcctggt gatctctggg acttgatggc aaatgttggg caaattgtga 3840

tcgaataaga ttagtactag agttatcgtg taataaggac atgcatggac taccatgtat 3900

ttcatgttat gacgctccta agagccacag accacagcga tggtattaga ccccttctca 3960

gaatggttct gctcattttc ggcttcgatc gtggtacgcg ttcgtgtctt cgtgtgatcg 4020

gaaaaaaaat atttgccgtt tacaagtgat agtttttcag tggatgtaat ttgtacgaaa 4080

taccatcgta caaacgtttg tactttttca tcatagtcat tagcctttca tgaatagtac 4140

tcacatttgt aaggccgaag gtgttgtcct ataaagaaaa aaaaatgtac cagtaagtag 4200

ggtggctaat gagccaactc ggctcggctc gtaaaaactc gttaaattaa caagctagct 4260

tggcttgact cattaacata ataagtaaaa aactgaactt gactcgactc gttaaaaagt 4320

ctgagttggc tcattaaact cactaaactc gttgcaccaa aatacatgat ttaaaattca 4380

tatacaatat aatttgttgg cctatgacca aagattacga ttgacatgtt atgcatcatg 4440

actcataaga ttgtgctaca atggataatt atgaatttac gacctttaca cctgtaaata 4500

tatagacaat taacttctat tttaatgcat cacgagctca acgagctaac cgagtaagtc 4560

a 4561

<210> 15

<211> 1285

<212> DNA

<213>Rice male sterility mutant

<400> 15

atggaagaga agaagcagca gcagcagcgt ccacagagag ggcgcgatgg catcctgcag 60

tatccgcacc ttttcttcgc ggcgctggcg ctggccctgc tacctcaccg acccgttcca 120

cctcggcccg ctcgccgggg tggactaccg gccggtgagg cacgagctgg cgccgtaccg 180

cgaggtgatg gcgcggtggc cgcgggacaa cggcagccgg ctcaggcacg gcaggctgga 240

gttcgtcgga gaggtgttcg ggccggagtc catcgagttc gaccgccacg gccgcggccc 300

ctacgccggc ctcgccgacg gccgcgtcgt gcggtggatg ggggaggacg ccgggtggga 360

gacgttcgcc gtcatgagcc ctgactggtc ggagaaagtt tgtgccaatg gggtggagtc 420

gacgacgaag aagcagcacg agatggagcg acggtgcggc cggcctctcg ggctgaggtt 480

tcacggcgag accggcgagc tctacgtcgc cgacgcgtac tacgggctca tgtccgtcgg 540

tccgaacggc ggggtggcga cctctctcgc gagagaagtc ggcgggagcc cggtcaactt 600

cgcgaacgac ctcgacatcc accgcaacgg ctccgtgttc ttcaccgaca cgagcacgag 660

atacaacaga aaggatcatc tgaacgttct gctagaaggt gaaggcacag ggaggctgct 720

cagatatgac ccagaaacca aagctgccca tgtcgtgctg agcgggctgg tcttcccgaa 780

tggcgtgcag atttctgacg accagcagtt cctcctcttc tccgaaacaa caaactgcag 840

gataatgcgg tactggctgg aagggccaag agccgggcag gtggaggtgt tcgccgacct 900

gccggggttc ccggacaacg tgcgactgag cagcggcggc ggcggcggac ggttctgggt 960

ggcgatcgac tgctgcagga cggcggcgca ggaggtgttc gccaagcggc cgtggctgcg 1020

aacgctctac ttcaagctgc ccctgacgat gcggacgctg gggaagatgg tcagcatgcg 1080

gatgcacacc ctcgtcgcgc tcctcgacgg cgaaggggac gtcgtcgagg tgctcgagga 1140

ccggggcggc gaggtgatgc ggctggtgag cgaggtgagg gaggtggggc gcaagctgtg 1200

gatcggcacc gtggctcata accacatcgc cacgatccct tacccgttgg aagagcagag 1260

tagcagcaac gtgcttggtg attga 1285

<210> 16

<211> 127

<212> PRT

<213>Rice male sterility mutant

<400> 16

Met Glu Glu Lys Lys Gln Gln Gln Gln Arg Pro Gln Arg Gly Arg Asp

1 5 10 15

Gly Ile Leu Gln Tyr Pro His Leu Phe Phe Ala Ala Leu Ala Leu Ala

20 25 30

Leu Leu Pro His Arg Pro Val Pro Pro Arg Pro Ala Arg Arg Gly Gly

35 40 45

Leu Pro Ala Gly Glu Ala Arg Ala Gly Ala Val Pro Arg Gly Asp Gly

50 55 60

Ala Val Ala Ala Gly Gln Arg Gln Pro Ala Gln Ala Arg Gln Ala Gly

65 70 75 80

Val Arg Arg Arg Gly Val Arg Ala Gly Val His Arg Val Arg Pro Pro

85 90 95

Arg Pro Arg Pro Leu Arg Arg Pro Arg Arg Arg Pro Arg Arg Ala Val

100 105 110

Asp Gly Gly Gly Arg Arg Val Gly Asp Val Arg Arg His Glu Pro

115 120 125

<210> 17

<211> 386

<212> DNA

<213>Wild rice

<400> 17

atggaagaga agaagcagca gcagcagcgt ccacagagag ggcgcgatgg catcctgcag 60

tatccgcacc ttttcttcgc ggcgctggcg ctggccctgc tcctcaccga cccgttccac 120

ctcggcccgc tcgccggggt ggactaccgg ccggtgaggc acgagctggc gccgtaccgc 180

gaggtgatgg cgcggtggcc gcgggacaac ggcagccggc tcaggcacgg caggctggag 240

ttcgtcggac aggtgttcgg gccggagtcc atcgagttcg accgccacgg ccgcggcccc 300

tacgccggcc tcgccgacgg ccgcgtcgtg cggtggatgg gggaggacgc cgggtgggag 360

acgttcgccg tcatgagccc tgactg 386

<210> 18

<211> 387

<212> DNA

<213>Rice male sterility mutant

<400> 18

atggaagaga agaagcagca gcagcagcgt ccacagagag ggcgcgatgg catcctgcag 60

tatccgcacc ttttcttcgc ggcgctggcg ctggccctgc tacctcaccg acccgttcca 120

cctcggcccg ctcgccgggg tggactaccg gccggtgagg cacgagctgg cgccgtaccg 180

cgaggtgatg gcgcggtggc cgcgggacaa cggcagccgg ctcaggcacg gcaggctgga 240

gttcgtcgga caggtgttcg ggccggagtc catcgagttc gaccgccacg gccgcggccc 300

ctacgccggc ctcgccgacg gccgcgtcgt gcggtggatg ggggaggacg ccgggtggga 360

gacgttcgcc gtcatgagcc ctgactg 387

Claims (9)

1. a kind of rice male sterility changing gene OsSTRL2, it is characterised in that the DNA sequence dna of the gene is selected from following group One of sequence：

A () has SEQ ID NO:Nucleotide sequence shown in 1；Or,

(b) and above-mentioned SEQ ID NO:1 gene order has at least 90% sequence similarity, and the DNA sequences with identical function Row；

C () under strict conditions can be with the DNA sequence dna of the DNA hybridization of sequence (a) described；

(d) DNA sequence dna complementary with any of the above-described sequence.

2. a kind of albumen that rice male sterility changing gene OsSTRL2 is encoded, it is characterised in that the amino acid sequence of the albumen Such as SEQ ID NO：Shown in 2.

3. expression cassette, expression vector or engineering bacteria containing the gene described in claim 1.

4. a kind of paddy pollen specifically expressing OsSTRL2 promoters, it is characterised in that the paddy pollen specifically expressing The DNA sequence dna of OsSTRL2 promoters and SEQ ID NO:DNA sequence dna shown in 6 has 80% or more than 90% homology；Or, The paddy pollen specifically expressing OsSTRL2 promoters are in SEQ ID NO:Added in DNA sequence dna shown in 6, replaced, inserted Or lack the mutant or allele or derivative of the generation of one or more nucleotides；Or, the paddy pollen is special Different expression OsSTRL2 promoters have and SEQ ID NO:The product of the DNA sequence dna hybridization shown in 6.

5. paddy pollen specifically expressing OsSTRL2 promoters according to claim 4, it is characterised in that the paddy rice Powder specifically expressing OsSTRL2 promoters are by SEQ ID NO:DNA sequence dna shown in 6 is constituted.

6. applications of a kind of rice male sterility changing gene OsSTRL2 in rice male sterility changing, it is characterised in that logical Cross mutation sterility changing genes of SEQ ID NO:1 obtains male sterile material.

7. a kind of application of mutant material, it is characterised in that the mutant material is malesterile mutants, the male is not Educate mutant be as caused by the mutation of nucleotide sequence, with male sterile performance, the nucleotide sequence such as SEQ ID NO:Shown in 1.

8. application according to claim 7, it is characterised in that malesterile mutants are prepared by the following：To educate Property controlling gene SEQ ID NO：1 is building up to promoter SEQ ID NO：6 downstream, so as to drive the sterility changing gene in flower Specifically expressing in powder, or by RNAi methods, build by SEQ ID NO：6 start can be with silence SEQ ID NO：1 base The RNAi carrier of cause, so as to obtain SEQ ID NO：The malesterile mutants of 1 gene.

9. it is a kind of recover male sterile plants fertility method, it is characterised in that comprise the following steps：

1) with normal fertility rice varieties DNA as template, obtained after being expanded using OsSTRL2-COM-F and OsSTRL2-COM-R Nucleotide sequence such as SEQ ID NO：Fragment shown in 14；The nucleotide sequence of the OsSTRL2-COM-F such as SEQ ID NO： Shown in 12, OsSTRL2-COM-R such as SEQ ID NO：Shown in 13；

2) fragment that will be expanded is cloned on carrier pCMBIYA1300, success carrier construction P1300-OsSTRL2-COM；

3) carrier P1300-OsSTRL2-COM is imported in the rice male-sterile plants system caused by OsSTRL2 is mutated；

4) Transplantation of Regenerated Plantlets for obtaining uses hygromycin selection transformed plant after surviving, and plant recovers to educate.