CN109566396A - A kind of plant hybridization system and application - Google Patents

Abstract

The invention discloses a kind of plant hybridization system and application, the plant hybridization system is made of sterile line, holding system and restorer；TWSD carrier is contained in the holding system, expression cassette of the TWSD carrier at least while containing 5 close linkages: herbicide resistance gene expression cassette, the silencing of plant endogenous anti-herbicide gene or knockout expression cassette, make maintainer seed have the expression cassette of macroscopic color or significant difference in shape compared with male-sterile seed, lead to the expression cassette of the pollen sterility containing above-mentioned expression cassette at sterile line fertility restorer expression cassette.The present invention hybridizes the maximum improvement of system, technology and the lethal technology of transgenic pollen are killed by color combining or shape tag technology, herbicide selective, greatly ensure entirely to hybridize the reliability and stability of system, Multiple techniques ensure the purity of hybrid seed, and average purity is increased to 98% from 95%.

Description

A kind of plant hybridization system and application

(1) technical field

The present invention relates to a kind of plant hybridization system and applications.

(2) background technique

The utilization of heterosis, hybrid vigor brings a rapid development to the volume increase of grain.Currently with the way of heterosis, hybrid vigor Diameter include artificial emasculation hybrid seeding, chemical emasculation hybrid seeding, using self-affinity hybrid seeding, utilize male sterility System.Male sterility ties up to extremely widespread in the application of modern hybrid seeding, generally uses three systems, two field method, i.e., sterile line, restorer, Keep system, parent propagation field, hybrid seeding field.And production of hybrid seeds formality simplifies, and hybrid seed yield is higher, to reduce seed production Cost.The sterile line in three line for hybrid seed production applied at present is mainly nucleo-cytoplasmic interreaction male sterility, and such sterility compares It is easy to get three systems.It is widely applied in the hybrid seeding of the field crops such as corn, jowar, wheat, rice.However, wanting Obtaining the sterile line with application value, holding system and restorer is an extremely cumbersome, large order.Pass through genetic engineering It is not only more traditional mostly time saving, laborsaving for breeding that means obtain male sterile line, restorer, but also is easy to keep that parent's is excellent Character has huge application prospect.Nineteen ninety, Mariani (Mariani, Debeuckeleer et al.1990) etc. are utilized The conversion of TA29-Barnase mosaic gene obtains sterile tobacco and the rape of transgenosis in the world for the first time；1992, he Further through the conversion of TA29-Barstar gene obtain above-mentioned sterile tobacco and rape restorer (Mariani C, Gossele V,De Beuckeleer M,et al.1992.).Hereafter, Reynaerts (Reynaerts A, Van de Wiele H, De Sutter G, et al.Scientia Horticulturae, 1993,55 (1): 125-139.) etc. again in succession Obtain sterile cabbage, the lettuce etc. of transgenosis.

A kind of male sterility system (TSS) has been invented by Pioneer Electronic Corp., the U.S..This insect sterile technique is opened based on anther-specific Mover mediated dna adenine methylase gene (dam) is expressed in plant, upsets the development of normal pollen, leads to male not It educates.The expression cassette and selection markers PAT (phosphonic acids serine acetyltransferase) gene expression frame close linkage of dam gene.PAT base Because assigning the characteristic of Plant Tolerance glufosinate-ammonium.When the TSS transgenosis sterile line for thinking same background hybridizes with nontransgenic plants, receive In the seed obtained 50% be transgenosis male sterile line, 50% is non-transgenic fertile line.It, can be with when producing cenospecies By spraying glufosinate-ammonium, 50% non-transgenic fertile line is killed, what is stayed in this way is all transgenosis sterile line, can be with father This hybridization obtains cenospecies.Same 50% is non-transgenic in above-mentioned cenospecies, and 50% is transgenosis.But in field planting The pollen that 50% nontransgenic plants generate provides pollen enough for all plant.

Above-mentioned method and TSS method based on barnase gene requires biggish increase cost, and produces and apply Also extremely complex, for example method based on barnase gene needs to carry out backcross transformation to different Parents respectively, and most Contain two kinds of foreign proteins of barnase and barstar in finished product, needs to carry out a large amount of safety evaluation work；And TSS method is raw Producing half in the final products obtained is transgenic seed, and half is non-transgenic seed.Therefore, either TSS is still based on The method of barnase is not all commercialized in hybrid corn seed production again and widely applies.

Before general ten years, Pioneer Electronic Corp., the U.S. has developed a kind of seed production technique (Seed Production Technology/SPT).Male sterile gene Ms45 and color mark technology of this technology based on corn itself, that is, exist Ms45 is mutated the DNA fragmentation that 3 expression cassette close linkage is imported in sterile line, and 3 expression cassettes are special in endosperm respectively Property expression red protein expression cassette, restore the normal Ms45 gene expression frame of fertility and kill containing above-mentioned two expression cassette The expression cassette of pollen.The above-mentioned plant containing SPT segment generates non-transgenic pollen, and 50% is red in the seed of acquisition Color, fertile, 50% is normal color, male sterile.Therefore, male-sterile seed can be used to breeding male sterile lines seed or For producing cenospecies, and red SPT seed can be used to breed SPT seed or for providing pollen for sterile line, raw Produce male-sterile seed.

The advantages of above method, is available with SPT method to produce non-transgenic corn, the disadvantage is that being screened by machine Red seed has certain probability leakage sieve, may be mixed into SPT seed in such male-sterile seed, cause to produce impure Cenospecies, bring great potential risk to large-scale production.Just because of this risk, results in SPT method and do not advise greatly The application of mould.

Selectively killing technology is the set genetically modified crops method of controlling security of exploitation recent years.It expresses in rice resistance to By the EPSPS gene of glyphosate, and the gene C YP81A6 for being resistant to Bentazon to rice itself carries out RNAi, and such transgenosis is made Object can be killed by Bentazon, while plant (Lin, C., the et of glyphosate can not be tolerated by spraying glyphosate removal al."A Built-In Strategy for Containment of Transgenic Plants:Creation of Selectively Terminable Transgenic Rice."Plos One3.3(2008):e1818.).The table in corn Up to the EPSPS gene of tolerance glyphosate, and the gene C YP81A9 that nicosulfuron is resistant to it carries out RNAi, such transgenosis Crop can be killed by nicosulfuron, while can use glyphosate removal of impurities (Li, Jing, et al., 2013, Plos One 8 (12):e81645.)。

(3) summary of the invention

It is an object of the present invention to provide a kind of plant hybridization system and its application, which combines the color or shape of seed Shape labelling technique and herbicide selective kill technology, solve the problems, such as maximum in a crossbreeding system, i.e. production of hybrid seeds body The stability problem of system.Color or shape tag is used alone, an error rate is had during machine choice, is caused not Educate be in be mixed into fertile seed, it is impure so as to cause the production of hybrid seeds.And selectively killing technology of the invention is combined, spray can be passed through It applies conventional herbicide and kills possible mixed fertile plant, to eliminate pollution.Selectively killing technology is used alone, without making With color or shape tag, there is also the risks of pollution while the waste for causing seed.Therefore, the present invention provides one Kind color or shape tag and herbicide selective kill the double control technology combined, can ensure to hybridize to greatest extent The high-purity and stability of seed production system (seed production purity is increased to 98% from 95%).

The technical solution adopted by the present invention is that:

The present invention provides a kind of plant hybridization system, and the plant hybridization system is by sterile line, holding system and restorer group At；The sterile line is that cell nucleus gene mutation obtains, which is that plant is formed necessary to fertile pollen；It is described System is kept to contain TWSD carrier, expression cassette of the TWSD carrier at least while containing 5 close linkages: (1) Herbicid resistant Gene expression frame is made of " constitutive promoter-anti-herbicide gene-terminator "；(2) plant endogenous anti-herbicide gene Silencing knocks out expression cassette, is made of " constitutive promoter-hairpin RNA-terminator "；(3) sterile line fertility restorer expression cassette, It is made of " promoter-recovery infertility line fertility gene-terminator "；(4) make maintainer seed compared with male-sterile seed The expression cassette of significant difference with macroscopic color perhaps in shape is by " specificity promoter-leads to color or shape The gene-terminator of shape difference " is constituted；(5) expression cassette for leading to the pollen sterility containing above-mentioned expression cassette, by " pollen-specific Property promoter-Pollen sterility gene-terminator " constitute, while above-mentioned holding system can be used to breeding male sterile lines and keep system；Institute Stating restorer is any one with the good self-mating system of sterile line coordinate force.

Further, the herbicide for keeping system that there are one or more non-transgenic plants itself not tolerate is (following Referred to as " herbicide A ") resistance simultaneously can be killed by the herbicide (hereinafter referred to as " herbicide B ") of non-transgenic plant self tolerance It goes out, maintainer seed is compared with male-sterile seed with the significant difference in macroscopic color or character；The recovery System is any one with the good self-mating system of sterile line coordinate force.

Sterile line of the present invention causes the gene of infertility to be reproductive tissuespecific expressing gene after mutating, most may be used It can be anther or pollen specific expressing gene.Providing in the present invention causes the gene of infertility to include corn after mutating Ms45 (Unger E, Cigan A M, Trimnell M, et al., 2002, Transgenic Research, 11 (5): 455-465), corn Zm13 (Hamilton D A, Roy M, Rueda J, et al., 1992, Plant molecular Biology, 18 (2): 211-218), the PS1 of rice (Zou J T, Zhan X Y, Wu H M, et al., 1994, American Journal of botany, 552-561) and gene with similar functions.

Herbicide resistance gene expression cassette of the present invention, which assigns, to be kept being one or more herbicide A resistances, by leading Enter a Bar gene to realize, is made of " constitutive promoter-anti-herbicide gene-terminator ", composing type starting Son can be cauliflower mosaic virus (CaMV) 35S promoter p35S (Odell, Joan T., Ferenc Nagy, and Nam- Hai Chua, 1985, Nature, 810-812), Actin1 promoter pAct1 (McElroy D, the Zhang W, Cao of rice J, et al., 1990, The Plant Cell, 2 (2): 163-171.), corn Ubiquitin promoter pUBI (Christensen A H,Sharrock R A,Quail P H,1992,Plant molecular biology,18(4): 675-689) and its promoter with expression patterns；Anti-herbicide gene can be Antiglyphosate gene CP4-EPSPS (Monsanto Company), G1174 (Chinese patent: 2011100093290), anti-glufosinate gene Bar (Thompson C J, Movva N R, Tizard R, et al, 1987, The EMBO journal, 6 (9): 2519.) and its gene with similar functions.It is excellent Selecting herbicide tolerant gene expression frame is glufosinate-ammonium tolerance gene bar expression cassette DNA fragmentation pUBI-bar-ter, sequence such as SEQ Shown in ID No.2.

The silencing of plant endogenous anti-herbicide gene of the present invention knocks out expression cassette as rnai expression frame, assigns The function of keeping system to be killed by herbicide B is realized by silencing or the plant endogenous Bar gene of knockout, by " composing type Promoter-hairpin RNA-terminator " is constituted, preferably CYP81A9 gene RNAi segment (nucleotide sequence such as SEQ ID No.1 institute Show) or CYP81A6 gene RNAi segment (nucleotide sequence is as shown in SEQ ID No.7).

Sterile line fertility restorer expression cassette of the present invention is by " promoter-recovery infertility line fertility gene-terminator " It constitutes, is realized by importing a fertile gene, it includes Ms45, Zm13 of corn, rice that the gene of infertility is caused after mutation PS1 and gene (Reema Khurana, Sanjay Kapoor, and Akhilesh with similar functions K.Tyagi.2012, Critical Reviews in Plant Sciences 31 (5): 359-390.).Promoter can be The promoter for restoring the gene itself of sterile line fertility, is also possible to other male tissue specific expressing promoters, preferably beautiful The p5126 of rice, the preferred p5126-Ms45-ter of sterile line fertility restorer expression cassette, sequence is as shown in SEQ ID No.3.

The expression cassette of the present invention for making pollen sterility is by " pollen specific promoter-Pollen sterility gene-terminator " It constitutes, is realized by importing the gene that one inactivates pollen.Pollen specific promoter includes 5126 promoters of corn (United States Patent (USP): US8257930B2), Zm13 promoter (Hamilton D A, Roy M, Rueda J, et al., 1992, Plant molecular biology, 18 (2): 211-218), PS1 promoter (Zou J T, Zhan X Y, the Wu H of rice M, et al., 1994, American journal of botany, 552-561), TA29 promoter (the Koltunow A of tobacco M,Truettner J,Cox K H,et al.,1990,The Plant Cell,1990,2(12):1201-1224)、NTP303 Promoter (Weterings K, Schrauwen J, Wullems G, et al., 1995, The Plant Journal, 8 (1): (Reema Khurana, Sanjay Kapoor, and Akhilesh K.Tyagi. " the Anthology of such as 55-63) Anther/Pollen-Specific Promoters and Transcription Factors."Critical Reviews in Plant Sciences 31.5(2012):359-390.).Pollen sterility gene includes any prevention or destruction pollen development To cause pollen development to be obstructed, depauperation or be disproportionated, and then make gene of the pollen without fertility.These gene packets Include amylase gene, protease gene, RNA enzyme gene etc..It is preferred that the cornstarch enzyme for inactivating the pollen containing TWSD segment Specific expressed frame pPg47-AAI-ter, sequence is as shown in SEQ ID No.4.

It is of the present invention so that maintainer seed has in macroscopic color or character compared with male-sterile seed Significant difference expression cassette, realized by importing foreign gene or regulating and controlling the expression of plant endogenous genes, including make to plant Son becomes red red fluorescent protein gene expression frame E35s-LTP2-DsRED-ter, and sequence is as shown in SEQ ID No.5； And lead to (Taliercio, E.W, the et that corn seed becomes smaller using cell wall-bound invertase 2 (InCW2) in interference corn Al.1999, Journal of Plant Physiology155 (2): 197-204.) expression cassette InCW2-RNAi, sequence such as SEQ Shown in ID No.6.

Restorer of the present invention is preferably corn prosperous 72 or rice 9311.

The present invention also provides a kind of application method of plant hybridization system, i.e., the sterile line provided through the invention is kept System and restorer, three series mating carry out preparing hybrid kind, and cenospecies is the fertile non-transgenic seed without containing TWSD segment, tool Body is as follows:

(1) keep the expansion of system numerous: the holding system of sowing segment containing TWSD carries out self-pollination, due to containing in TWSD segment 50% pollen containing TWSD segment for having an expression cassette for leading to pollen sterility, therefore system being kept to generate be it is sterile, in addition 50% is the fertile pollen without TWSD segment, distinguishes TWSD transgenosis by the special color of transgenic seed or shape Seed and non-transgenic seed；Wherein, TWSD transgenic seed can be used for continuing expanding numerous holding system, can be used for expanding it is numerous not Educating is seed；Non-transgenic seed is sterile line, can be used for numerous or cenospecies the preparation of expansion of sterile line；Non-transgenic kind Possible mixed TWSD transgenic seed in son can harvest segment containing TWSD by removing when the normal use herbicide B of field Holding system.

(2) expansion of sterile line is numerous: by the holding system of sterile line and the segment containing TWSD with suitable proportion (the preferably seed of 5:2 Amount or line number) interval plantation, keep system to generate two kinds of pollen, half contains the TWSD segment for enabling pollen inactivation expression cassette, infertility； The other half is free of the pollen of TWSD segment, fertile；Removing while by sterile line herbicide spraying B weeding may be mixed TWSD seed, then allow holding system to sterile line plant pollination after, the seed generated on sterile line plant be all non-transgenic not It educates and is, obtain the male-sterile seed for being free of TWSD segment；

(3) cenospecies: sterile line and restorer interval plantation of the step (2) without TWSD segment is prepared, it can in sterile line It is miscellaneous that the existing extremely a other maintainer plant containing TWSD segment of energy can spray selective herbicide killing in the V3-V5 phase It is killed while careless；Restorer gives the sterile line without TWSD segment to pollinate, and the seed of harvest is to be free of TWSD segment Fertile cenospecies.

Plant hybridization system in the present invention can be used for monocotyledon, including corn, rice, wheat, barley and height Fine strain of millet etc. and dicotyledon, including soybean, rape, cotton and tobacco etc..

Compared with prior art, the beneficial effects are mainly reflected as follows: the purity of seed directly affects crop most Whole yield, so the purity for improving hybrid seed is very crucial.Compared with current existing hybridization system, the present invention hybridizes system Maximum improvement is to kill technology by color combining or shape tag technology, herbicide selective and transgenic pollen causes Dead technology greatly ensures entirely to hybridize the reliability and stability of system, and Multiple techniques ensure the purity of hybrid seed, average Purity is increased to 98% from 95%.

(4) Detailed description of the invention

The T-DNA structural schematic diagram of Fig. 1: TWSD carrier.CYP81 gene RNAi indicates corn C YP81A9 or CYP81A6 Gene RNAi frame；Herbicide tolerance genes indicate glyphosate tolerance gene C P4, glufosinate-ammonium tolerance gene bar or flazasulfuron The gene expression frames such as tolerance gene 3X-1；Restoring gene refers to that Ms45 and RTS etc. can restore due to plant autogene The gene expression frame of male sterile fertility caused by being mutated；Pollen lethal gene indicates the specific expressed starch in pollen The genes such as enzyme, lipase, protease and RNA enzyme so as to cause pollen abortion expression cassette；Color gene refers in seed endosperm In specific expressed red fluorescent protein or other convenient for identification color expression cassette.RB is right margin, and LB is left margin.

The T-DNA structural schematic diagram of Fig. 2: TWSDS carrier.CYP81 gene RNAi indicate corn C YP81A9 or CYP81A6 gene RNAi frame；Herbicide tolerance genes indicate glyphosate tolerance gene C P4, glufosinate-ammonium tolerance gene bar or pyridine The gene expression frames such as Sulfometuron Methyl tolerance gene 3X-1；Restoring gene refers to that Ms45 and RTS etc. can restore due to plant certainly The gene expression frame of male sterile fertility caused by body gene mutation；Pollen lethal gene indicates specific expressed in pollen The genes such as amylase, lipase, protease and RNA enzyme so as to cause pollen abortion expression cassette；InCW2 gene RNAi indicates InCW2 gene RNAi expression cassette.RB is right margin, and LB is left margin.

Fig. 3: holding is the route map of transformation.Ms:ms indicates homozygous sterile line.

Fig. 4: system is kept to expand numerous schematic diagram.

Fig. 5: self-mating system expands numerous schematic diagram.

Fig. 6: the schematic diagram of cenospecies preparation.

(5) specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:

Without departing from the spirit and substance of the present invention, modification the method for the present invention, step or condition made Or replacement all belongs to the scope of the present invention.

Embodiment 1, TWSD vector construction

In order to construct corn TWSD carrier, it is respectively synthesized following DNA fragmentation: 1, CYP81A9 gene RNAi segment (i.e. plant The silencing of endogenous anti-herbicide gene knocks out expression cassette), nucleotide sequence is as shown in SEQ ID No.1, the distribution of sequence both sides XhoI restriction endonuclease sites are set；2, glufosinate-ammonium tolerance gene bar expression cassette (i.e. herbicide resistance gene expression cassette) DNA Segment pUBI-bar-ter, for nucleotide sequence as shown in SEQ ID No.2, the site KpnI is respectively set in sequence both ends；3, fertility Expression cassette (i.e. sterile line fertility restorer expression cassette) p5126-Ms45-ter of related gene Ms45, sequence such as SEQ ID No.3 Shown, the site HpaI is respectively set in sequence both ends；4, the cornstarch enzyme spcificity table for inactivating the pollen containing TWSD segment Up to frame (expression cassette for leading to the pollen sterility containing above-mentioned expression cassette) pPg47-AAI-ter, sequence such as SEQ ID No.4 institute Show, the site EcoRI and HindIII is respectively set in the end of sequence 5 ' and 3 ' ends；5, corn seed is made to become red red fluorescence egg White gene expression frame (i.e. so that maintainer seed compared with male-sterile seed with the significant difference in macroscopic color Expression cassette) E35s-LTP2-DsRED-ter, for sequence as shown in SEQ ID No.5, the site HindIII is respectively set in sequence both ends； 6, the InCW2 gene RNAi expression cassette for making corn seed become smaller is (i.e. so that maintainer seed has meat compared with male-sterile seed The expression cassette of the visible significant difference in shape of eye) InCW2-RNAi, sequence is as shown in SEQ ID No.6, sequence both ends point It She Zhi not the site HindIII.Meanwhile in order to construct the TWSD carrier of rice, CYP81A6 gene RNAi segment is synthesized, sequence is such as Shown in SEQ ID No.7, sequence both sides distribution setting XhoI restriction endonuclease sites.

In order to obtain the TWSD carrier for making maintainer seed become red, based on binary vector pCambia1300, such as It is upper described, hygromycin gene hptII therein is replaced into CYP81A9 gene RNAi segment.Obtain pCambia1300- The carrier of p35S-CYP81A9RNAi.Then artificial synthesized pUBI-bar-ter is cloned into using restriction enzyme site KpnI In pCambia1300-p35S-CYP81A9RNAi, carrier pCambia1300-p35S-CYP81A9RNAi-pUBI-bar is obtained. Recycle restriction enzyme site HpaI that p5126-Ms45-ter segment is connected into carrier pCambia1300-p35S-CYP81A9RNAi- In pUBI-bar, carrier pCambia1300-p35S-CYP81A9RNAi-pUBI-bar-p5126-Ms45 is obtained.Then, it utilizes PPg47-AAI-ter segment is connected into carrier pCambia1300-p35S-CYP81A9RNAi-pUBI- by EcoRI and HindII In bar-p5126-Ms45, carrier pCambia1300-p35S-CYP81A9RNAi-pUBI-bar-p5126-Ms45- is obtained PPg47-Zm-AAI carrier.Finally, artificial synthesized E35s-LTP2-DsRED-ter is connected into carrier by HindII In pCambia1300-p35S-CYP81A9RNAi-pUBI-bar-p5126-Ms45-pPg47-Z m-AAI, carrier is obtained pCambia1300-p35S-CYP81A9RNAi-pUBI-bar-p5126-Ms45-pPg47-Zm-AAI-E35s-LTP2- DsRED, this carrier name are as follows: the structure of ZmTWSD, the T-DNA sequence in the carrier are as shown in Figure 1.

In order to obtain the TWSD carrier for making maintainer seed become smaller, by carrier pCambia1300-p35S- E35s-LTP2- in CYP81A9RNAi-pUBI-bar-p5126-Ms45-pPg47-Zm-AAI-E35s-LTP2-D sRED DsRED-ter segment is substituted for artificial synthesized InCW2-RNAi segment.It is final to obtain carrier pCambia1300-p35S- 2 RNAi-ter of CYP81A9RNAi-pUBI-bar-p5126-Ms45-pPg47-Zm-AAI-pInCW2-InCW, this carrier life Entitled ZmTWSDS.The structure of T-DNA sequence in the carrier is as shown in Figure 2.

Maintainer seed is set to become red rice TWSD carrier construction method similar with its method on corn, uniquely Difference be that the CYP81A9RNAi segment in the first step among XhoI restriction enzyme site is replaced with CYP81A6RNAi segment.Finally The carrier of acquisition is pCambia1300-p35S-CYP81A9RNAi-pUBI-bar-pRTS-RTS-pPg47-Zm- AAI-E35s- LTP2-DsRED-ter, this carrier name are as follows: the structure of OsTWSD, the T-DNA sequence in the carrier are as shown in Figure 1.

Finally, above-mentioned T-DNA plasmid is transferred in Agrobacterium LB4404 by the method that electricity turns, by containing 15 μ g/ml The YEP solid medium of the kanamycins of tetracycline and 50 μ g/mL filters out positive colony, and protects bacterium, is used for next plant Object conversion.

Embodiment 2, corn transformation

Comparative maturity, such as Frame etc. describe the method using Agrobacterium-mediated Transformation corn to the method for transformation of corn (Frame et al.,(2002)Plant Physiol,129:13-22).The agriculture of ZmTWSD containing carrier and ZmTWSDS are taken respectively Bacillus (i.e. the Agrobacterium of the carrier containing T-DNA) draws plate, chooses single colonie inoculation, prepares conversion and uses Agrobacterium.It takes after pollinating 8-10 days Ms45 gene lacks functionality mutant Hi-II corncob.Collect all immature embryos (size 1.0-1.5mm).It will contain The Agrobacterium and immature embryo for having T-DNA carrier co-culture 2-3 days (22 DEG C).It shifts on immature embryo to calli induction media (containing the Timentin of 200mg/L in culture medium, for killing Agrobacterium, referring to (Frame et al., (2002) Plant Physiol, 129:13-22)), 28 DEG C dark culture 10-14 days.All callus are gone into the sieve with final concentration 2mM glufosinate-ammonium Select on culture medium (Frame et al., (2002) Plant Physiol, 129:13-22), 28 DEG C dark culture 2-3 weeks.Transfer institute On some tissues to the screening and culturing medium of the fresh glufosinate-ammonium of 2mM containing final concentration, 28 DEG C dark culture 2-3 weeks.Then, all sieves are shifted In the embryonal connective tissue to regeneration culture medium survived after choosing (Frame et al., (2002) Plant Physiol, 129:13-22), 28 DEG C dark culture 10-14 days, one strain of every ware.It shifts in embryonal connective tissue to fresh regeneration culture medium, 26 DEG C of illumination cultivations 10-14 days.(Frame et al., (2002) Plant are shifted on all full-grown plants to root media Physiol, 129:13-22), 26 DEG C of illumination cultivations are complete until root development, are then transplanted to single plant culture in greenhouse, and detection turns The antiweed ability of gene corn.With the 18% glufosinate-ammoniumaqua aqua sprinkling of 200 times of Bayer of dilution, rear blade turns to be yellow within 7 days, Withered is feminine gender；It is positive plant as spray clear water control growing way, obtains ZmTWSD containing carrier and ZmTWSDS respectively and turn Gene corn strain, is denoted as ZmTWSD and ZmTWSDS, is as holding.

The screening of embodiment 3, corn TWSD strain

1, by backcross transformation obtain nuclear gene mutation sterile line (refer to Chinese patent application, application number: 201610748791.5).We obtain the female parent of commercial varieties " Zheng Dan 958 " " Zheng 58 " (Z58) and commercial varieties are first The Ms45 gene mutation sterile line of jade 335 maternal (PH6WC).

2, Transgenic corn lines (ZmTWSD) selfing for the ZmTWSD containing conversion carrier for obtaining embodiment 2, will obtain 218 ZmTWSD transgenosis T0 for plantlet of transplant into greenhouse, the Z58 sterile line of step 1 is carried out respectively with these pollen Pollination, harvest T0 is for seed, and about 50% is the Z58 sterile line transgenic seed containing ZmTWSD segment, and in addition about 50% is not Z58 sterile line non-transgenic seed containing ZmTWSD segment.It then is maternal carry out with the Z58 sterile line of the segment containing ZmTWSD 5-6 picks out the near-isogenic line of the Z58 sterile line background of ZmTWSD transgenic strain, is screened out from it for continuous backcross 85 about 50% pollen is fertile, and the endosperm of 50% pollen sterility is that red list copies, insertion point is stablized, clear, The suitable strain of destination protein expression quantity.The glufosinate resistance of these near-isogenic lines and nicosulfuron resistance are compared again Compared with analysis, we screen 35 glufosinate resistances well and the plant sensitive to nicosulfuron from above-mentioned 85 ZmTWSD plant Strain.Finally, according to plant height, growth potential, yield, breeding time, growth cycle, the statistic analysis result of the characters such as setting percentage, sieve The holding system of 5 Z58 sterile line backgrounds is selected, study on the industrialization is used for.

3, the Transgenic corn lines (ZmTWSDS) that 2 method of embodiment obtains the ZmTWSDS containing conversion carrier are selfed, it will Obtain 193 ZmTWSDS transgenosis T0 for plantlet of transplant into greenhouse, with these pollen respectively to the Z58 sterile line of step 1 It pollinating, T0 is for seed for harvest, and about 50% is the Z58 sterile line transgenic seed containing ZmTWSDS segment, and in addition about 50% For the Z58 sterile line non-transgenic seed without containing ZmTWSDS segment.Then with the Z58 sterile line containing ZmTWSDS be it is maternal into Row 5-6 picks out the near-isogenic line of the Z58 sterile line background of ZmTWSDS transgenic strain for continuous backcross.From above-mentioned product Filter out that 69 about 50% pollen is fertile in system, single copy of 50% pollen sterility, insertion point are stablized, clear, mesh The suitable strain of expressing quantity.The glufosinate resistance of these near-isogenic lines and nicosulfuron resistance are compared again Analysis, we screen 30 glufosinate resistances well and the plant sensitive to nicosulfuron from above-mentioned 69 ZmTWSDS plant Strain.Finally, according to plant height, growth potential, yield, breeding time, growth cycle, the statistic analysis result of the characters such as setting percentage, sieve The holding system of 5 Z58 sterile line backgrounds is selected, study on the industrialization is used for.

Embodiment 4, corn keep the transformation of system

In order to the transformation of TWSD segment into other sterile lines, be needed turn keeping system to carry out backcrossing with PH6WC sterile line It educates.It is female parent with PH6WC sterile line, the holding system for the Z58 sterile line background that embodiment 3 is screened is to return after male parent carries out 4-6 generation It hands over, is selfed twice after the completion of marking confirmation transformation by High Density Molecular, finally obtains stable PH6WC sterile line The TWSD of background keeps system (Fig. 3).

Embodiment 5, corn keep the expansion of system numerous

The maintainer seed of the Z58 background prepared in sowing embodiment 3,3-4 seeds are sowed in every hole, then to the V3 phase Maintainer plant sprays the diluted guarantor's examination of 1:200 up to (Beyer Co., Ltd), and killing mixed may turn base without the non-of TWSD segment Because of plant, maintainer plant is allowed to be selfed, harvests maintainer seed.There is 50% transgenosis kind in these maintainer seeds of harvest The sub non-transgenic seed with 50%.If it is TWSD carrier strain, the seed containing TWSD segment is red, is free of TWSD piece The seed of section is normal corn color, wherein red transgenic seed can be used for keeping being that expansion is numerous or sterile line expansion is numerous, The non-transgenic seed of normal color is male-sterile seed, and the expansion that can be used for sterile line is numerous or cenospecies prepares (Fig. 4).Such as Fruit is TWSDS carrier strain, and the seed endosperm containing TWSD segment becomes smaller and seed is caused to become smaller, the seed without TWSD segment Normal in size, wherein the transgenic seed of seedlet can be used for keeping be expand numerous or sterile line expand it is numerous, normal size it is non- Transgenic seed is male-sterile seed, and the expansion that can be used for sterile line is numerous or cenospecies prepares (Fig. 4).

Embodiment 6, the expansion of corn sterile line are numerous

The maintainer seed and Z58 male-sterile seed for the Z58 background that 3 method of embodiment is obtained are mixed with the ratio of 2:5 It sows afterwards or with the line number of 2:5 than sowing, all male-sterile seeds in the seed of harvest on sterile line plant, and Z58 Having half in the maintainer seed of background is male-sterile seed, the other half is TWSD seed.If kept on system and sterile plant Seed it is mixed receive if, final 6/7 is male-sterile seed, and 1/7 is TWSD seed (Fig. 5).If it is TWSD carrier strain, contain The seed of TWSD segment is red, and the seed without TWSD segment is normal corn color, and 1/7 TWSD seed can pass through face It color discrimination and screens.If it is TWSDS carrier strain, the seed endosperm containing TWSD segment becomes smaller and seed is caused to become smaller, The normal in size of seed without TWSD segment, 1/7 TWSD seed by size discrimination and can screen.If kept If seed in system and sterile plant separately harvests, all male-sterile seeds in the seed of harvest on sterile line plant, And having half in TWSD maintainer seed is male-sterile seed, the other half is TWSD seed.

The application of embodiment 7, corn hybridization system

The male-sterile seed of Z58 background prepared by embodiment 6 is maternal and Z58 male parent-prosperous 72 or other cooperations The high male parent of power is with the line number of 5:2 than sowing.Then, the nicosulfuron for spraying 40g/ha to the sterile line plant of V3-V5 phase removes Grass, while removal of impurities can also be played the role of, killing may the mixed transgenic plant not containing TWSD segment in sterile line. After Parent hybridization, the seed on female parent is cenospecies, these hybrid seeds are fertile non-turn without containing TWSD segment Gene seed can be used for later period sale and Production of Large Fields (Fig. 6).

Embodiment 8, rice conversion:

By backcross transformation obtain nuclear gene mutation sterile line (refer to Chinese patent, application number: 201610748791.5).We obtain the RTS gene mutation sterile lines of commercial varieties " elegant water 134 " and " 9311 ".

The preparation method of transgenic paddy rice is using the prior art (Lu Xiongbin, Gong ancestral an ancient egg-shaped, holed wind instrument (1998) life science 10:125- 131；Liu Fan etc. (2003) Molecular Plant Breeding 1:108-115).The RTS gene lacks functionality mutant for choosing mature and plump is " elegant Water -134 " seed decladding, induction generate callus as converting material.OsTWSD Agrobacterium is taken to draw plate.Single colonie inoculation is chosen, Prepare conversion and uses Agrobacterium.Callus to be transformed is put into the Agrobacterium bacterium solution that OD is 0.6 or so (Agrobacterium bacterium solution Preparation: by Agrobacterium inoculation to culture medium, culture to OD is 0.6 or so；Culture medium composition: 3g/L K₂HPO₄、1g/ LNaH₂PO₄、1g/LNH₄Cl、0.3g/L MgSO₄·7H₂O、0.15g/L KCl、0.01g/L CaCl₂、0.0025g/L FeSO₄·7H₂O, 5g/L sucrose, 20mg/L acetosyringone, solvent are water, pH=5.8), allow Agrobacterium to be integrated to callus Then callus is transferred to co-culture medium (MS+2mg/L2,4-D+30g/L glucose+30g/L sucrose+3g/ by surface L agar (sigma 7921)+20mg/L acetosyringone) in, it co-cultures 2-3 days.Callus after being converted with aseptic water washing turns Move on to screening and culturing medium (MS+2mg/L 2,4-D+30g/L sucrose+3g/L agar (sigma 7921)+20mg/L acetosyringone + 2mM glufosinate-ammonium (Sigma)) on, screening and culturing two months (intermediate subculture is primary).After screening, the good callus of growth vigor Be transferred to pre- differential medium (MS+0.1g/L inositol+5mg/L ABA+1mg/L NAA+5mg/L 6-BA+20g/L sorbierite+ 30g/L sucrose+2.5g/L gelrite) on cultivate 20 days or so, then callus break up in advance is moved on to break up and be cultivated On base, the daily differentiation of illumination in 14 hours germination.After 2-3 weeks, resistance regeneration plant is transferred to root media (1/2MS+ 0.2mg/L NAA+20g/L sucrose+2.5g/L gelrite) on strengthening seedling and rooting, finally by regeneration plant wash away agar transplanting in Greenhouse selects that yield is high, seed is big or biomass is high etc. and can be improved the transgenic line of rice yield, cultivates new varieties. The transgenic rice plant containing above-mentioned conversion carrier He the empty carrier for containing only riddled basins EPSPS is obtained respectively.

The screening of embodiment 9, OsTWSD strain

It, will by transgenic rice plant (the being named as OsTWSD) plantation for the OsTWSD containing conversion carrier that embodiment 8 obtains The 356 OsTWSD transgenosis T0 obtained harvest T0 for seed for plantlet of transplant to Transgenic studies Tanaka.Then, T0 is sowed For seed, it is screened out from it that 129 about 50% pollen is fertile, single copy of 50% pollen sterility, insertion point are stablized, Clearly, the suitable strain of destination protein expression quantity.Glufosinate resistance and Bentazon resistance to above-mentioned strain are compared analysis, We are from screening good and sensitive to the Bentazon plant of 62 glufosinate resistances.Finally, according to plant height, growth potential, point Tiller, yield, breeding time, growth cycle, the statistic analysis result of the characters such as setting percentage filter out 5 " elegant water -134 " backgrounds System is kept, study on the industrialization is used for.

Embodiment 10, rice keep the transformation of system

For " elegant water -134 " background that the transformation of TWSD segment into other sterile lines, is needed embodiment 9 to obtain System is kept to be hybridized and be returned with 9311 sterile line of rice strain.It is that female parent passes through after backcrossing 4-6 generation with 9311 sterile lines It is selfed twice after the completion of High Density Molecular label confirmation transformation, finally obtains 9311 stable sterile line backgrounds TWSD keeps system (Fig. 3).

Embodiment 11, rice keep the expansion of system numerous

The maintainer seed of embodiment 9 " elegant water -134 " background is sowed, then to after planting about 20 days, i.e. one heart stage of two leaf Maintainer plant spray 1:200 diluted guarantor examination up to (Beyer Co., Ltd), killing may mixed non-turn without TWSD segment Gene plant, then maintainer plant is allowed to be selfed, harvest maintainer seed.There is 50% transgenosis kind in the maintainer seed of harvest The sub non-transgenic seed with 50%.Transgenic seed containing TWSD segment is red, and the seed without TWSD segment is positive Ordinary water rice color, wherein red transgenic seed can be used for keeping be expand numerous or sterile line expand it is numerous, normal color it is non- Transgenic seed is male-sterile seed, and the expansion that can be used for sterile line is numerous or cenospecies prepares (Fig. 4).

Embodiment 12, the expansion of rice sterile line are numerous

The maintainer seed of " elegant water -134 " background and " elegant water -134 " male-sterile seed with the line number of 2:5 than interval kind It plants.It allows maintainer plant to generate pollen and gives sterile line plant pollination.The seed of sterile line plant is harvested, these seeds are all infertility It is seed, the expansion of the preparation and sterile line that can be used for cenospecies is numerous (Fig. 5).

Embodiment 13, paddy rice cross breeding system and its application

Sow the male-sterile seed and " 9311 " seed of embodiment 12 " elegant water -134 " background.Then to after planting about 20 It, i.e. the rice shoot of one heart stage of two leaf sprays the selective herbicide Bentazon of field normal use concentration for weeding, simultaneously also It can play the role of removal of impurities, killing may the mixed transgenic plant containing TWSD segment." elegant water -134 " background Male-sterile seed and " 9311 " are planted with the proportional spacing of 5:2.It allows " 9311 " plant to generate pollen and gives sterile line plant pollination, receive Obtain the seed of sterile line plant, as cenospecies (Fig. 6).

Embodiment 14, TWSD hybridization system are higher than the seed production purity of the hybridization system of no selectively killing technology

As control, method as described in example 1 above, we construct the carrier of no selectively killing technology PCambia1300-pUBI-bar-p5126-Ms45-pPg47-Zm-AAI-E35s-LTP2-D sRED, and obtain corresponding guarantor Holding is plant.We hybridize the seed production purity of system to the hybridization system of no selectively killing technology with the TWSD in the present invention It compares.

Hybridization system	Seed production purity
		TWSD technology	98.57±3.36
CK1	95.27±2.77

CK1 is only color identification without the hybridization system of selectively killing technology.Purity is the flat of 3 years test results Means standard deviation.Annual test area is 667m², corn field planting density is 5000 plants/acre.

Embodiment 15, TWSD hybridization system are higher than the seed production purity of no color or the hybridization system of shape tag

As control, method as described in example 1 above, we construct the carrier of no selectively killing technology PCambia1300-p35S-CYP81A9RNAi-pUBI-bar-p5126-Ms45-pPg47-Z m-AAI-E35s, and obtain phase The maintainer plant answered.We hybridize the system of system to the hybridization system of no selectively killing technology with the TWSD in the present invention Kind purity compares.

Hybridization system	Seed production purity
		TWSD technology	98.57±3.36
CK2	96.61±2.91

CK2 is only selectively killing technology without color or the hybridization system of shape identification technology.Purity is 3 years The mean+SD of test result.Annual test area is 667m², corn field planting density is 5000 plants/acre.

Sequence table

<110>Zhejiang University

<120>a kind of plant hybridization system and application

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 672

<212> DNA

<213>unknown (Unknown)

<400> 1

ctcgagaacc accaactcgc gatccatcga tttgcagaac accacccaac agccagacca 60

tcactcgatc ggtagacatg gataaggcct acatcgccgc cctctccgcc gccgccctct 120

tcttgctcca ctacctcctg ggccgccggg ccggcggcga gggcaaggcc aaggccaagg 180

gctcgcggcg gcggctcccg ccgagccctc cggcgatccc gttcctgggc cacctccacc 240

tcgtcaaggc cccgttccac ggggcgctgg cccgcctcgc ggcgcgccac ggcccggtgt 300

tctccatgcg cctggggacc cggcgcgccg tggtcgtgtc gtcgccggac tgcgccaggg 360

agtgcttgga gaacaccggg ccgtggcgcg ccgcgaggcg ggccagcgcc ccgtggaacg 420

gggccttgac gaggtggagg tggcccagga acgggatcgc cggagggctc ggcgggagcc 480

gccgccgcga gcccttggcc ttggccttgc cctcgccgcc ggcccggcgg cccaggaggt 540

agtggagcaa gaagagggcg gcggcggaga gggcggcgat gtaggcctta tccatgtcta 600

ccgatcgagt gatggtctgg ctgttgggtg gtgttctgca aatcgatgga tcgcgagttg 660

gtggttctcg ag 672

<210> 2

<211> 2952

<212> DNA

<213>unknown (Unknown)

<400> 2

ggtaccgtta accgcccggg cgtgcagcgt gacccggtcg tgcccctctc tagagataat 60

gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg 120

aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa taatataatc 180

tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg 240

tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc 300

atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt 360

agtacatcca tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc 420

tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta 480

tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc 540

tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc 600

tgttaaacgc cgtcgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg 660

ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt 720

tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc 780

agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcacggc agctacgggg 840

gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata aatagacacc 900

ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca cacaaccaga 960

tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc 1020

ccccccctct ctaccttctc tagatcggcg ttccggtcca tggttagggc ccggtagttc 1080

tacttctgtt catgtttgtg ttagatccgt gtttgtgtta gatccgtgct gctagcgttc 1140

gtacacggat gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc 1200

tttggggaat cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt 1260

tttgtttcgt tgcatagggt ttggtttgcc cttttccttt atttcaatat atgccgtgca 1320

cttgtttgtc gggtcatctt ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg 1380

gttgggcggt cgttctagat cggagtagaa ttgctgtttc aaactacctg gtggatttat 1440

taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg 1500

atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac 1560

agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc 1620

gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac 1680

tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc 1740

taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca 1800

gcatctattc atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt 1860

ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt 1920

tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct 1980

caccctgttg tttggtgtta cttctgcagg tcgactctag aggatcctaa agcaagcaag 2040

agcaataaac aatgagccca gaacgacgcc cggccgacat ccgccgtgcc accgaggcgg 2100

acatgccggc ggtctgcacc atcgtcaacc actacatcga gacaagcacg gtcaacttcc 2160

gtaccgagcc gcaggaaccg caggagtgga cggacgacct cgtccgtctg cgggagcggt 2220

atccctggct cgtcgccgag gtggacggcg aggtcgccgg catcgcctac gcgggcccct 2280

ggaaggcacg caacgcctac gactggacgg ccgagtcgac cgtgtacgtc tccccccgcc 2340

accagcggac gggactgggc tccacgctct acacccacct gctgaagtcc ctggaggcac 2400

agggcttcaa gagcgtggtc gctgtcatcg ggctgcccaa cgacccgagc gtgcgcatgc 2460

acgaggcgct cggatatgcc ccccgcggca tgctgcgggc ggccggcttc aagcacggga 2520

actggcatga cgtgggtttc tggcagctgg acttcagcct gcccgtaccg ccccgtccgg 2580

tcctgcccgt caccgagatt tgactcgagc tgcgaggagt ctggtggcaa ctaagccgtc 2640

atcgtcatat atagcctcgt ttaattgttc atctctgatt cgatgatgtc tcccaccttg 2700

tttcgtgtgt tcccagtttg ttcatcgtct tttgatttta ccggccgtgc tctgcttttg 2760

tttttgtttc acctgatctc tctctgactt gatgtaagag tggtatctgc tacgactata 2820

tgttgttggg tgaggcatat gtgaatgaaa tctatgaaag ctccggctat atatatttat 2880

acaaagggta tgagatggat gtgaatccta gagcatatgt gtccaacaat caattcgtcg 2940

agggggaggt ac 2952

<210> 3

<211> 2177

<212> DNA

<213>unknown (Unknown)

<400> 3

gttaacggta cccctatgat ttagaataat atacaaatat attacataaa aaatatatta 60

attgaattag tgttgtctaa tttataatta ttagaatgta attcaattcc aacgaaacaa 120

cggggcctta ggtttaatat cttccttaca ctgcgaaaat gttgttacac ttgccaaaaa 180

aaatcaatcg catatttacc ttacaaggac atattttagc aaaatgctat agacatgaat 240

ccaacgtaat caatagagtg agatttactg gtaaactacc aattgctcat ctgctcggta 300

ccaaccagcc tttcctatta ccatgcacat gttgcctctc aactgcagca tctttcaagc 360

cgtgagcaga catgttgcag atcgaagtaa ggtatatatg tgcatagtct cctaattctt 420

catcttcaac ctctagctga ttgatctctg gtatttacca ctctttcctt ccttccttcc 480

ttcaattcta aataccacaa atcaaagttg ctttgccatg gagaagagga acctgcagtg 540

gcggcgaggg cgtgatggca tcgtgcagta ccctcacctc ttcttcgcgg ccctggcgct 600

ggccctccta gtcgcggacc cgttcggcct cagtccgctg gccgaggtcg actaccggcc 660

ggtgaagcac gagctcgcgc cgtacgggga ggtcatgggc agctggccca gagacaatgc 720

cagccggctc aggcgcggga ggctggagtt cgtcggcgag gtgttcgggc cggagtctat 780

cgagttcgat ctccagggcc gcgggccgta cgccggcctc gccgacggcc gcgtcgtgcg 840

gtggatgggc gaggaggccg ggtgggagac gttcgccgtc atgaatcctg actggtcaga 900

agaagtctgt gccaatggag tgaactcaac gacgaggaag cagcacgaga aggaggagtt 960

ctgcggccgg ccgctcggcc tgaggttcca cggggagacc ggcgagctct acgtcgccga 1020

cgcgtactac ggtctcatgg tcgttggcca gagcggcggc gtggcgtcct ccgtcgcgag 1080

ggaagccgac ggggacccca tccggttcgc gaacgacctc gatgtgcaca ggaatggctc 1140

cgtattcttc actgacacga gcatgagata cagcagaaag gaccatctga acatcctgtt 1200

agaaggagaa ggcaccggga ggctgctcag gtatgatcca gaaacaagcg gtgtccatgt 1260

cgtgctcaag gggctggtgt tcccaaacgg cgtgcagatc tcagaggacc atcagtttct 1320

tctcttctcc gagacaacaa actgcaggat aatgaggtac tggctggaag gcccaagagc 1380

gggcgaggta gaggtgttcg cgaacctgcc gggcttcccc gacaacgtgc gctccaacgg 1440

caggggccag ttctgggtgg cgatcgactg ctgccggacg ccggcgcagg aggtgttcgc 1500

caagaggccg tggctccgga ccctgtactt caagttcccg ctgtcgctca aggtgctcac 1560

ttggaaggcc gccaggagga tgcacacggt gctcgcgctc ctcgacggcg aagggcgcgt 1620

cgtggaggtg ctcgaggacc ggggccacga ggtgatgaag ctggtgagcg aggtgcggga 1680

ggtgggccgc aagctgtgga tcggaaccgt ggcgcacaac cacatcgcca ccatccccta 1740

ccctttagag gactaaccat gatctatgct gtttcaatgc ctcctaatct gtgtacgtct 1800

ataaatgtct aatgcagtca ctggttgtaa tcttgtttgt gtttggcaaa ttggcataat 1860

aatggacaga ttcaatgggc attggtgctg tagtcgcatc acactaattg aatgggatca 1920

tgttgagctc tcactttgct acaatttgct ccagcttgta cggttgtacc ctcttgctcg 1980

tctatagtaa gggccatcta aaaaaaactc aaattagatc tgcaatacaa gtatgattgg 2040

gccgaatttg gattgtcacg ggtccgcgac cgcgaattgg gctcggtttg atttagccga 2100

catagtagtg accgacccga gccggcggcg agccaaaccg agcggacgcc gccatcacca 2160

tcgtggaatt cgttaac 2177

<210> 4

<211> 4676

<212> DNA

<213>unknown (Unknown)

<400> 4

gaattcgtta acggacactg tctggtggca taccagacag tccggtgtgc cagatcaggg 60

cacccttcgg ttcctttgct cctttgcttt tgaaccctaa ctttgatcgt ttattggttt 120

gtgttgaacc tttatgcacc tgtggaatat ataatctaga acaaactagt tagtccaatc 180

atttgtgttg ggcattcaac caccaaaatt atttatagga aaaggttaaa ccttatttcc 240

ctttcaatct cccccttttt ggtgattgat gccaacacaa accaaagaaa atatataagt 300

gcagaattga actagtttgc ataaggtaag tgcataggtt acttagaatt aaatcaattt 360

atacttttac ttgatatgca tggttgcttt cttttatttt aacattttgg accacatttg 420

caccacttgt tttgtttttt gcaaatcttt ttggaaattc tttttcaaag tcttttgcaa 480

atagtcaaag gtatatgaat aagattgtaa gaagcatttt caagatttga aatttctccc 540

cctgtttcaa atgcttttcc tttgactaaa caaaactccc cctgaataaa attctcctct 600

tagctttcaa gagggtttta aatagatatc aattggaaat atatttagat gctaattttg 660

aaaatatacc aattgaaaat caacatacca atttgaaatt aaacatacca atttaaaaaa 720

tttcaaaaag tggtggtgcg gtccttttgc tttgggctta atatttctcc ccctttggca 780

ttaatcgcca aaaacggaga ctttgtgagc catttatact ttctccccat tggtaaatga 840

aatatgagtg aaagattata ccaaatttgg acagtgatgc ggagtgacgg cgaaggataa 900

acgataccgt tagagtggag tggaagcctt gtcttcgccg aagactccat ttccctttca 960

atctacgact tagcatagaa atacacttga aaacacatta gtcgtagcca cgaaagagat 1020

atgatcaaag gtatacaaat gagctatgtg tgtaatgttt caatcaaagt ttcgagaatc 1080

aagaatattt agctcattcc taagtttgct aaaggtttta tcatctaatg gtttggtaaa 1140

gatatcgact aattgttctt tggtgctaac ataagcaatc tcgatatcac ccctttgttg 1200

gtgatccctc aaaaagtgat accgaatgtc tatgtgctta gtgcggctgt gttcaacggg 1260

attatccgcc atgcagatag cactctcatt gtcacatagg agagggactt tgctcaattt 1320

gtagccatag tccctaaggt tttgcctcat ccaaagtaat tgcacacaac aatgtcctgc 1380

ggcaatatac ttggcttcgg cggtagaaag agctattgag ttttgtttct ttgaagtcca 1440

agacaccagg gatctcccta gaaactgaca agtccctgat gtgctcttcc tatcaatttt 1500

acaccctgcc caatcggcat ctgaatatcc tattaaatca aaggtggatc ccttggggta 1560

ccaaagacca aatttaggag tgtaaactaa atatctcatg attcttttca cggccctaag 1620

gtgaacttcc ttaggatcgg cttggaatct tgcacacatg catatagaaa gcatactatc 1680

tggtcgagat gcacataaat agagtaaaga tcctatcatc gaccggtata ccttttggtc 1740

tacggattta cctcccgtgt cgaggtcgag atgcccatta gttcccatgg gtgtcctgat 1800

gggcttggca tccttcattc caaacttgtt gagtatgtct tgaatgtact ttgtttggct 1860

gatgaaggtg ccatcttgga gttgcttgac ttgaaatcct agaaaatatt tcaacttccc 1920

catcatagac atctcgaatt tcggaatcat gatcctacta aactcttcac aagtagattt 1980

gttagtagac ccaaatataa tatcatcaac ataaatttgg catacaaaca aaacttttga 2040

aatggtttta gtaaagagag taggatcggc tttactgact ctgaagccat tagtgataag 2100

aaaatctctt aggcattcat accatgctgt tggggcttgc ttgagcccat aaagcgcctt 2160

tgagagttta taaacatggt tagggtactc actatcttca aagccgagag gttgctcaac 2220

atagacctat tcaccccatt tgatcacttt tttggtcctt caggatctaa tagttatgta 2280

taatttagag tctcttgttt aatggccaga tatttctaat taatctaaga atttatgata 2340

ttttttaatt ttttatcatg tctgatgaga attaacataa aggctcaatt gggtcctgaa 2400

ttaataatag agtgaaaatt aatccagagg ctctattaga accttcaatt agtaatacca 2460

agatatatat aagatagtag agtatagttt aaatgttggc attgttcatt ctttcttttg 2520

ttatttaatt tatgctttcc acggtggtta gtggttactt ctgaagggtc caaataatgc 2580

atgaagagtt tgaggacaag aagtctgccc taaaaatagc gatgcaaagg catggtgtcc 2640

aagccataca tatagcgcac taattttatc agcagaacaa tggtatttat aggtcctagt 2700

gcccaggcaa caagagacac gaataaagca tcgatcacga caccatggcg gcgacaatgg 2760

cagtgacgac gatggtgacg aggagcaagg agagctggtc gtcattgcag gtcccggcgg 2820

tggcattccc ttggaagcca cgaggtggca agaccggcgg cctcgagttc cctcgccggg 2880

cgatgttcgc cagcgtcggc ctcaacgtgt gcccgggcgt cccggcgggg cgcgacccgc 2940

gggagcccga tcccaaggtc gtccgggcgg cctgcggcct ggtccaggca caagtcctct 3000

tccaggggtt taactgggag tcgtgcaagc agcagggagg ctggtacaac aggctcaagg 3060

cccaggtcga cgacatcgcc aaggccggcg tcacgcacgt ctggctgcct ccaccctcgc 3120

actccgtctc gccacaaggc tacatgccag gccgcctata cgacctggac gcgtccaagt 3180

acggcacggc ggcggagctc aagtccctga tagcggcgtt ccacggcagg ggcgtgcagt 3240

gcgtggcgga catcgtcatc aaccaccggt gcgcggaaaa gaaggacgcg cgcggcgtgt 3300

actgcatctt cgagggcggg actcccgacg accgcctgga ctggggcccc gggatgatct 3360

gcagcgacga cacgcagtac tcggacggga cggggcaccg cgacacgggc gaggggttcg 3420

cggcggcgcc cgacatcgac cacctcaacc cgcgcgtgca gcgggagctc tccgcctggc 3480

tcaactggct caggtccgac gccgtggggt tcgacggctg gcgcctcgac ttcgccaagg 3540

gctactcgcc ggccgtcgcc agaatgtacg tggagagcac ggggccgccg agcttcgtcg 3600

tcgcggagat atggaactcg ctgagctaca gcggggacgg caagccggcg cccaaccagg 3660

accagtgccg gcaggagctg ctggactgga cgcgggccgt cggcgggccc gccatggcgt 3720

tcgacttccc caccaagggc ctgctgcagg cgggcgtgca gggggagctg tggcggctgc 3780

gcgacagctc cggcaacgcg gccggcctga tcgggtgggc gcccgagaag gccgtcacct 3840

tcgtcgacaa ccatgacacc gggtcgacgc agaagctctg gccgttccca tccgacaagg 3900

tcatgcaggg ctacgcctac atcctcaccc atccaggagt cccctgcatt ttctacgacc 3960

acatgttcga ctggaacctg aagcaggaga tatccacgct gtctgccatc agggcgcgga 4020

acggcatccg cgccgggagc aagctgcgga tcctcgtggc ggacgcggac gcgtacgtgg 4080

ccgtcgtcga cgagaaggtc atggtgaaga tcgggacaag gtacggcgtg agcagcgtgg 4140

tcccgtcgga tttccacccg gcggcgcacg gcaaggacta ctgcgtctgg gagaaagcga 4200

gcctccgcgt cccggcgggg cgccacctct agcagctcag attgctcagt cttgtgctgc 4260

attgcaaaca cagcagcacg acactgcata acgtcttttc cttgagatct gacaaagcag 4320

cattagtccg ttgatcggtg gaagaccact cgtcagtgtt gagttgaatg tttgatcaat 4380

aaaatacggc aatgctgtaa gggttgtttt ttatgccatt gataatacac tgtactgttc 4440

agttgttgaa ctctatttct tagccatgcc aagtgctttt cttattttga ataacattac 4500

agcaaaaagt tgaaagacaa aaaaaaaaac ccccgaacag agtgctttgg gtcccaagct 4560

actttagact gtgttcggcg ttccccctaa atttctcccc ctatatctca ctcacttgtc 4620

acatcagcgt tctctttccc ctatatctcc acgtcgacgc ggccgatcca agcttc 4676

<210> 5

<211> 2367

<212> DNA

<213>unknown (Unknown)

<400> 5

aagcttccca tggagtcaaa gattcaaata gaggacctaa cagaactcgc cgtaaagact 60

ggcgaacagt tcatacagag tctcttacga ctcaatgaca agaagaaaat cttcgtcaac 120

atggtggagc acgacacgct tgtctactcc aaaaatatca aagatacagt ctcagaagac 180

caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat 240

tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa 300

tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga cagtggtccc 360

aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 420

tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc acaatcccac 480

taagctgacc gaagctggcc gctctagaac tagtggatct cgatgtgtag tctacgagaa 540

gggttaaccg tctcttcgtg agaataaccg tggcctaaaa ataagccgat gaggataaat 600

aaaatgtggt ggtacagtac ttcaagaggt ttactcatca agaggatgct tttccgatga 660

gctctagtag tacatcggac ctcacatacc tccattgtgg tgaaatattt tgtgctcatt 720

tagtgatggg taaattttgt ttatgtcact ctaggttttg acatttcagt tttgccactc 780

ttaggttttg acaaataatt tccattccgc ggcaaaagca aaacaatttt attttacttt 840

taccactctt agctttcaca atgtatcaca aatgccactc tagaaattct gtttatgcca 900

cagaatgtga aaaaaaacac tcacttattt gaagccaagg tgttcatggc atggaaatgt 960

gacataaagt aacgttcgtg tataagaaaa aattgtactc ctcgtaacaa gagacggaaa 1020

catcatgaga caatcgcgtt tggaaggctt tgcatcacct ttggatgatg cgcatgaatg 1080

gagtcgtctg cttgctagcc ttcgcctacc gcccactgag tccgggcggc aactaccatc 1140

ggcgaacgac ccagctgacc tctaccgacc ggacttgaat gcgctacctt cgtcagcgac 1200

gatggccgcg tacgctggcg acgtgccccc gcatgcatgg cggcacatgg cgagctcaga 1260

ccgtgcgtgg ctggctacaa atacgtaccc cgtgagtgcc ctagctagaa acttacacct 1320

gcaactgcga gagcgagcgt gtgagtgtag ccgagtaacc atggcctcct ccgagaacgt 1380

catcaccgag ttcatgcgct tcaaggtgcg catggagggc accgtgaacg gccacgagtt 1440

cgagatcgag ggcgagggcg agggccgccc ctacgagggc cacaacaccg tgaagctgaa 1500

ggtgacgaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta 1560

cggctccaag gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt 1620

ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggcgaccgt 1680

gacccaggac tcctccctgc aggacggctg cttcatctac aaggtgaagt tcatcggcgt 1740

gaacttcccc tccgacggcc ccgtgatgca gaagaagacc atgggctggg aggcctccac 1800

cgagcgcctg tacccccgcg acggcgtgct gaagggcgag acccacaagg ccctgaagct 1860

gaaggacggc ggccactacc tggtggagtt caagtccatc tacatggcca agaagcccgt 1920

gcagctgccc ggctactact acgtggacgc caagctggac atcacctccc acaacgagga 1980

ctacaccatc gtggagcagt acgagcgcac cgagggccgc caccacctgt tcctgtagcg 2040

gcccatggac ctagacttgt ccatcttctg gattggccaa cttaattaat gtatgaaata 2100

aaaggatgca cacatagtga catgctaatc actataatgt gggcatcaaa gttgtgtgtt 2160

atgtgtaatt actagttatc tgaataaaag agaaagagat catccatatt tcttatccta 2220

aatgaatgtc acgtgtcttt ataattcttt gatgaaccag atgcatttca ttaaccaaat 2280

ccatatacat ataaatatta atcatatata attaatatca attgggttag caaaacaaat 2340

ctagtctagg tgtgttttgc aagcttg 2367

<210> 6

<211> 3012

<212> DNA

<213>unknown (Unknown)

<400> 6

aagcttcccc tccaacggcg aacatatttc cggctcgccc gagccctgcc ttcgctaaga 60

agcaaccctg actaaatcac cgcaccgacc gaccaaatcg caggagcatt taatgcaaag 120

gtggcctgac acctttatcc tgacgcgcgc cccccggcag agccgaagtg accgccgtca 180

cttcgccgct ccactgaccg gcctgacaga aggacagcgt cgcctgcgcc actctgactg 240

cagtgccact tgacagagtg agactgacag gcagtcaggc cctgccgaag gcaccatagg 300

aagctccgct tcgcccgacc cagggctcgg actcgggcta agtcccggaa gacggcgaac 360

tccgctccgc ccgacccagg gctcggactc gggctaagtc ccggaagacg gcgaactccg 420

ctccgcccga cccagggctc ggactcgggc taagtcccgg aagacggcga actccgctcc 480

gcccgaccca gggctcggac tcgggctcag ccccagaaga cgacgaactc cgcttcgccc 540

gacccagggc tcggactccg ccctggcctc agccgacggc ctccgcctcg cccgacccag 600

gggctcggac tcggcctcgg ccacggaaga cagactcgac ctcggcttcg gaggagcctc 660

cacatcgccc aacctagggc gcaggccagc cacgtcaaca ggaggcgcca tcatcaccct 720

accccgagct gactcgggcc gcagggaaca agaccggcgt cccatctggc tagctccgcc 780

agataggcaa tgatggcgcc ccgcatactc tgtaacgacg gcggctctca gcccccttac 840

ggaagcaaga ggacgtcagc aaggacccaa ccgctccgac agctgtccct ccgccaggct 900

ccatcgctcc tccgacggcc acgacatcac accagctggg tgccaaagtc tctccgtctg 960

ccacaacggc atgtacttag ggcgctagct ctcctccgct agacacgtag cactctgcta 1020

caccccccat tgtacacctg gatcctctcc ttacgcctat aaaaggaagg accagggccc 1080

tcttggagag ggttggccgc gcggggacga ggacgagaca ggcgctcgcc tggagccgct 1140

cgctccctct cccgcgtgga cgcttgtaac cccctactgc aagcgcaccc gaccggggcg 1200

cgggacgaac atgaaggccg cgggattccc acctctctca cgccggtctc cggccgcctc 1260

gctctccccc cttcgcgctc gccctcgcgc tcgacccatc tgggctgggg cacgcggcga 1320

cactcactcg tcggcccagg gaccccccgg tctcgaaacg tcgacacatt taatgcttaa 1380

tctaacaata attactttta tatatttgat cgaactttaa aagatttggc tcctcagaaa 1440

gatagacgat gtgcatttat tttagaaatg ataggatata tttaaggcga catatggaat 1500

agacaggttg ggatggagta gatatatata gattcatacg actaaagttg tgttcagcag 1560

atcatatata tagctatgca aaacactatt ttacactgta gatagcaata tttatgttgt 1620

ttagagagtg aattttgaaa taagaggtga gatagagaat gtgatatggc agctaagagc 1680

tagttttcaa caagtttgtt tctataatag tatagctaga tagacagaca cgtagctaaa 1740

atggtcaact gaccttcgta caaaaaaaaa ctttaatcca gagatatagc aatatatgct 1800

tataactcca gacctcagag tcaacccaca gatttttgta tattcgtcaa aaagatccac 1860

agatttgtgg tagagagtat gttttagtag tagtggtata tctcccttgt gggacgacga 1920

agcagccggt cagccggcgc tccggccggc cggccggcct gcgcctgcac agtacaaata 1980

gcaccccccg tcctccagtt ggctgcatcc ttctagcttc gatctctata tacctctgta 2040

tgtgagtgag gccaatgaga gccctcgtag tcgtaagttt cgcttcggcg tgcttgctgc 2100

tgctgttgca gctcgcagga gcgtcgcatg tcgtctacaa ctacaaggac ctcgaagccg 2160

aggctgctgc ggcgacggac caggtgccgc cgtccatcgt caaccccctg ctcaggacgg 2220

ggtacacttc cagcccccca agaactggat caatggtaat gtaaagctac taactaatcc 2280

accacccaac gtcgtttgaa ggtgatgtgt gtgttaagca tctcctgaaa tatataagag 2340

agcgaggcta gtaatcggct tgttgttcag ggttttggtt taccacttgt agcctcaact 2400

aatatagtta gtagctttac attaccattg atccagttct tggggggctg gaagtgtacc 2460

ccgtcctgag cagggggttg acgatggacg gcggcacctg gtccgtcgcc gcagcagcct 2520

cggcttcgag gtccttgtag ttgtagacga catgcgacgc tcctgcgagc tgcaacagca 2580

gcagcaagca cgccgaagcg aaacttacga ctacgagggc tctcattggc ctcactcaca 2640

tacagaggta tatagagatc gaagctagaa ggatgcagcc atagcggccc atggacctag 2700

acttgtccat cttctggatt ggccaactta attaatgtat gaaataaaag gatgcacaca 2760

tagtgacatg ctaatcacta taatgtgggc atcaaagttg tgtgttatgt gtaattacta 2820

gttatctgaa taaaagagaa agagatcatc catatttctt atcctaaatg aatgtcacgt 2880

gtctttataa ttctttgatg aaccagatgc atttcattaa ccaaatccat atacatataa 2940

atattaatca tatataatta atatcaattg ggttagcaaa acaaatctag tctaggtgtg 3000

ttttgcaagc tt 3012

<210> 7

<211> 553

<212> DNA

<213>unknown (Unknown)

<400> 7

ctcgagcagt gcaccagagt cacagaaaca catcacacat tcgtgagctc agcttagcca 60

tggataacgc ctacattatt gccattctct ctgtagctat cctcttcttg ctccactact 120

acctcctcgg ccgcggcaat ggcggggcgg cgcggctgcc gccgggtcca ccggccgtcc 180

cgatcctggg acacctccac ctcgtcaaga agccgatgca cgccaccatg tcccgcctcg 240

ccgagcggta cgggccggtg ttctcgctgc gcctcgggtc gcggcgcgcc gtggtggtgt 300

cgtcgccggg gtgcgccagg gagtgcttca ccgagatctg cttcttgacg aggtggaggt 360

gtcccaggat cgggacggcc ggtggacccg gcggcagccg cgccgccccg ccattgccgc 420

ggccgaggag gtagtagtgg agcaagaaga ggatagctac agagagaatg gcaataatgt 480

aggcgttatc catggctaag ctgagctcac gaatgtgtga tgtgtttctg tgactcctgg 540

tgcactgctc gag 553

Claims (10)

1. a kind of plant hybridization system, it is characterised in that the plant hybridization system is made of sterile line, holding system and restorer； The sterile line is that cell nucleus gene mutation obtains, and wherein cell nucleus gene is that plant is formed necessary to fertile pollen；It is described System is kept to contain TWSD carrier, expression cassette of the TWSD carrier at least while containing 5 close linkages: (1) Herbicid resistant Gene expression frame is made of " constitutive promoter-anti-herbicide gene-terminator "；(2) plant endogenous anti-herbicide gene Silencing knocks out expression cassette, is made of " constitutive promoter-hairpin RNA-terminator "；(3) sterile line fertility restorer expression cassette, It is made of " promoter-recovery infertility line fertility gene-terminator "；(4) make maintainer seed compared with male-sterile seed The expression cassette of significant difference with macroscopic color perhaps in shape is by " specificity promoter-leads to color or shape The gene-terminator of shape difference " is constituted；(5) expression cassette for leading to the pollen sterility containing above-mentioned expression cassette, by " pollen-specific Property promoter-Pollen sterility gene-terminator " constitute；The restorer is that any one is good certainly with sterile line coordinate force Hand over system.

2. plant hybridization system as described in claim 1, it is characterised in that in the sterile line cell nucleus gene be anther or Pollen specific expressing gene.

3. plant hybridization system as claimed in claim 2, it is characterised in that the different expression gene includes corn Ms45 base Cause, corn Zm13 gene, rice PS1 gene.

4. plant hybridization system as described in claim 1, it is characterised in that composing type in the herbicide resistance gene expression cassette Promoter is the Actin1 promoter pAct1 or corn of cauliflower mosaic virus (CaMV) 35S promoter p35S, rice Ubiquitin promoter pUBI；Anti-herbicide gene is Antiglyphosate gene CP4-EPSPS, G1174 or anti-glufosinate gene Bar。

5. plant hybridization system as described in claim 1, it is characterised in that the herbicide resistance gene expression cassette is nucleotide Sequence glufosinate-ammonium tolerance gene bar expression cassette as shown in SEQ ID No.2.

6. plant hybridization system as described in claim 1, it is characterised in that the silencing of the plant endogenous anti-herbicide gene or Knocking out expression cassette is nucleotide sequence CYP81A9 gene RNAi segment as shown in SEQ ID No.1 or nucleotide sequence such as SEQ CYP81A6 gene RNAi segment shown in ID No.7.

7. plant hybridization system as described in claim 1, it is characterised in that pollen-specific in the expression cassette for making pollen sterility Property promoter includes 5126 promoters of corn, Zm13 promoter, the PS1 promoter of rice, the TA29 promoter of tobacco or NTP303 promoter；Pollen sterility gene includes amylase gene, protease gene or RNA enzyme gene.

8. plant hybridization system as described in claim 1, it is characterised in that the sterile line fertility restorer expression cassette nucleotides sequence Column make the expression cassette nucleotide sequence of pollen sterility as shown in SEQ ID No.4 as shown in SEQ ID No.3.

9. plant hybridization system as described in claim 1, it is characterised in that described so that maintainer seed and male-sterile seed phase Than the expression cassette with the significant difference in macroscopic color or character, comprising: (1) seed is made to become red red Fluorescence protein gene expression cassette E35s-LTP2-DsRED-ter, nucleotide sequence is as shown in SEQ ID No.5；(2) seed becomes smaller Expression cassette InCW2-RNAi, nucleotide sequence is as shown in SEQ ID No.6.

10. the application of plant hybridization system described in a kind of claim 1, it is characterised in that the application method are as follows:

(1) keep the expansion of system numerous: the holding system of sowing segment containing TWSD carries out self-pollination, special by transgenic seed Color or shape distinguish transgenic seed and non-transgenic seed, collect the holding system of the segment containing TWSD；

(2) expansion of sterile line is numerous: the holding system of step (1) segment containing TWSD and the plantation of sterile line interval, by spraying to sterile line Spraying herbicide removes transgenic plant while weeding, then allow holding system to sterile line plant pollination after, sterile line plant The seed of upper generation is all non-transgenic sterile line, obtains the male-sterile seed for being free of TWSD segment；

(3) cenospecies: sterile line and restorer interval plantation of the step (2) without TWSD segment is prepared, to sterile line in V3-V5 Phase herbicide spraying kills transgenic plant while killing weeds；Restorer gives the sterile line without TWSD segment to pollinate, and receives The seed obtained is cenospecies.