CN102876711A - Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line - Google Patents
Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line Download PDFInfo
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Abstract
The invention discloses a cultivation method of a rice engineering maintainer line and a method for applying the cultivated rice engineering maintainer line to breeding of a rice genic male sterile line. The cultivation method comprises the following steps: firstly, positioning and cloning a male sterile gene s of the rice genic male sterile line and a fertile gene S of a male sterile line source parent through a map position cloning method; connecting a color marker gene C and the fertile gene S to the same dual expression vector to enable the color marker gene C and the fertile gene S to be subjected to linkage inheritance and coseparation after being introduced into a plant; transferring the expression vector in the genic male sterile line by a trangenosis method to obtain a fertility restorer strain; and then carrying out back-crossing on the fertility restorer strain and the genic male sterile line to obtain the engineering maintainer line. The cultivation method can be applied to breeding of the rice genic male sterile line, and can simplify the breeding process of the genic male line greatly.
Description
Technical field
The present invention relates to the breeding technology field of crop sterile line, be specifically related to a kind of propagation method of self-pollination class crop male sterile line.
Background technology
Utilize the male infertility of plant to cultivate male sterile line, produce in a large number cenospecies by this genetic tool again, can make many crops particularly the hybrid vigour of self pollination crop be able in the production application.
According to three type theories, the male sterile of plant is divided into cytoplasmic male sterility, nuclear male sterility and three kinds of hereditary forms of nucleo-cytoplasmic interaction type male sterile.
Cytoplasmic male sterility refers to that sterile proterties is controlled by plasmone only, and is irrelevant with nucleus, and the simple sterile proterties of being controlled by tenuigenin is owing to can not find restorer, so also do not have application example on producing.
Nuclear male sterility refers to that sterile proterties only is controlled by cell nucleus gene, and is irrelevant with tenuigenin, is divided into dominant genic male sterile and recessive cytoblast sterile.The nuclear of having found at present is sterile to be recessive cytoblast sterile mostly.
The male sterile of nucleo-cytoplasmic interaction type is by plasmone and cell nucleus gene co-controlling, and existing maintenance line is kept its sterility, can recover F under the help of restorer again
1The fertility of cross-fertilize seed, such sterile line and maintenance line, restorer form three series mating, are to produce at present classical, the most ripe method, i.e. three series.But the procedure of breeding of three series and production link are complicated, so that the cycle of the new combination of seed selection is long, efficient is low, the popularization link is many, speed is slow, simultaneously high, the price of seed costs.
In the nuclear male sterility type, whether recessive cytoblast sterile is responsive to environmental factor according to sterility, and it is sterile to be divided into again the sterile and common nuclear of environmental sensitivity nuclear.
Environmental sensitivity nuclear is sterile, mainly refer at present photoperiod-temperature sensitive genie male-sterile line, its fertility was controlled by genic male sterile gene both, was subjected to again ambient light according to the regulation and control of length and temperature height, at certain developmental stage, long day, high temperature cause sterile, short day, low temperature cause and can educate, and have the feature of fertility conversion, can one be dual-purpose, ratio three is to reduce one to be, also the namely what is often called bilinear method.Photo-thermo-sensitive genetic male sterile line except do not need maintenance line, breed simple and easy and efficient higher, also have: it is wide 1) to recover spectrum, and nearly all normal kind with subspecies can both make its fertility restorer normal; 2) genetic behavior is simple, and sterility is controlled by 1～2 pair of recessive gene, and transformation and stable is conducive to cultivate polytype sterile line easily.But also should be pointed out that since the fertility of photo-thermo-sensitive genetic male sterile line be subjected to ambient light according to and temperature adjusting, if illumination and temperature Change be not in span of control in the sterile line propagation process, will cause the photo-thermo-sensitive genetic male sterile line breeding underproduction even failure.
The sterile impact that generally is not subjected to the outside atmosphere factor of common nuclear, no matter how envrionment conditions changes, always show as sterilely, this sterile type is more common at occurring in nature.Compare with photoperiod-temperature sensitive genie male-sterile line with the nucleo-cytoplasmic interaction type is sterile, common nuclear is sterile to have following characteristics: 1) owing to only be subjected to the control of a pair of recessive nuclear gene, and fertility is not affected by environment, selects easily sterile rate and sterile strain and all be 100% common line with genic sterile; 2) the normal kind of fertility all is its restorer, recovers spectrum and extensively, combo freely so that the probability of seed selection fine combination increase; 3) can open up subspecies indica and japonica hybrid advantage frontier immediately following the seed selection paces of Conventional Rice, make rice yield realize more high yield target on existing hybrid rice basis.Therefore, common nuclear is sterile is a kind of desirable sterile material.But because such sterile line do not have corresponding maintenance line, and self can not carry out the fertility conversion, breed very difficultly, fail temporarily large-area applications in production.
At present, common line with genic sterile mainly adopts to backcross and raises up seed with vegetative propagation, and for example, the CN102121052A Chinese patent literature has been put down in writing and utilized molecular marker assisted selection by backcrossing to formulate and breed the rice recessive line with genic sterile.Put down in writing in the CN101946715A Chinese patent literature and utilized microspores culture and asexual clone technology seed selection and propagating cabbage type rape recessive gms line.In addition, the breeding of can also regenerating of the common line with genic sterile of bibliographical information paddy rice is arranged, the common line with genic sterile of cotton can cottage propagation.By aforesaid method, researcher can be bred and be obtained a small amount of common line with genic sterile for scientific research and experiment, but the common sterile line quantity that breeding obtains is rare, and needs to spend a large amount of manpower and materials.If can solve the problem of common line with genic sterile large-scale breeding, this type large-scale application of common line with genic sterile in hybrid seeding, will greatly be discharged the heterotic potentiality of utilizing.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art; a kind of method of cultivation of Rice Engineering maintenance line is provided and cultivates the application of rear product in the common line with genic sterile breeding of paddy rice; but the Rice Engineering maintenance line product mechanize after the cultivation, mass-producing are applied to the crossbreeding of the common line with genic sterile of paddy rice, and can make the step of crossbreeding process simple, easy to operate.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is the method for cultivation of the engineering maintenance line of the common line with genic sterile of a kind of paddy rice, may further comprise the steps:
(1) preparing genotype is the common line with genic sterile of paddy rice of ss, the sterile gene s(that adopts the method location of map based cloning and clone the common line with genic sterile of described paddy rice is by homology comparison prediction sterile gene function, and by gene knockout and cross the function that expression technology is verified this sterile gene s);
(2) according to the primers of above-mentioned sterile gene, clone the common line with genic sterile source parent's of above-mentioned paddy rice (genotype is SS) allelotrope, be wild type gene S, because sterile gene s obtains from wild type gene S sudden change, therefore, this wild type gene S namely can recover the educated gene of the common line with genic sterile fertility of described paddy rice; Sterile gene s and can educate between the gene S difference that a base or several bases are generally only arranged also can be disappearance or the insertion of a dna fragmentation; Can educate gene S take himself promoters driven (promotor is as the sequence of the front 2kb of coding region);
(3) color mark gene C and above-mentioned educated gene S are connected on the same binary expression vector, make the color mark gene C with can educate gene S can linkage inheritance after importing plant, be divided into from (in paddy rice, if chain rate is 99%, then the color mark gene C is separated by 250 * 10 with the physical distance that can educate gene S
3Base does not have the interval base in the middle of C and the S among the present invention, and chain is 100%, both be divided into from), obtain binary expression vector pS
C
(4) adopt existing transgenic method (being preferably agrobacterium-mediated transformation or via Particle Bombardment Transformation method) with described binary expression vector pS
CChange in the common line with genic sterile of described paddy rice, screen the fertility restorer strain (T that obtains with the common line with genic sterile of paddy rice of color mark gene C by PCR
0For transgenic line); Can educate gene S because importing has in the fertility restorer strain that the present invention cultivates, be dominant and can educate gene S to sterile gene s, so T
0To recover fertility for transgenic line and offspring's strain thereof; Again since the color mark gene C with can educate gene S and be divided into from, T
0For the fertility shape of transgenic line and offspring's strain thereof and color mark with linkage inheritance, therefore after gathering in the crops this fertility restorer strain seed, can be according to setting percentage and color mark and auxiliary PCR detected result, by self propagated constantly (generally the self propagated by 2～5 generations get final product), can obtain stable fertility, setting percentage height and (verify that by test cross its genotype is S with the homozygous lines of color mark gene C
CS
C);
(5) (genotype is S with described homozygous lines
CS
C) backcross with common line with genic sterile (ss), according to whether fluorescing or PCR detects and determines whether backcross progeny carries the color mark gene C, and by the copy number that southern blot detects backcross progeny color mark gene, obtain engineering maintenance line (the genotype sS that single copy inserts
C).
In the above-mentioned method of cultivation, the common line with genic sterile of described paddy rice can be to obtain by hereditary and selection, physical chemistry mutagenesis, genetically engineered and other various means or regular approach, preferred physical chemistry mutafacient system.The sterile proterties of the common line with genic sterile of described paddy rice is the recessive character by Dominant gene, and the described gene S that educates is dominant to sterile gene s, and sterile proterties is not affected by external environmental condition.In preferred scheme, because sterile proterties only is subjected to the control of a pair of recessive nuclear gene, and fertility is not affected by environment, and therefore easier to select sterile rate and sterile strain all be 100% common line with genic sterile.
Above-mentioned method of cultivation, in the described step (3), described binary expression vector is preferably plant binary expression vector, specifically is preferably any one (being particularly preferably pCAMBIA1300 or pCAMBIA1390) among pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301, the pBI121.
Above-mentioned method of cultivation, in the described step (3), the color mark gene C can be expressed in seed, makes seed with color mark, so that look is selected seed with this color mark gene in the subsequent applications.Described color mark gene is preferably the red fluorescent protein gene
DsRed(GenBank:DQ493888.2), red fluorescent protein marker gene
RFP(GenBank:GQ495894.1), green fluorescence protein gene
GFP(GenBank:AF286456.1), green fluorescence protein gene
EGFP(GenBank:JX445134.1) or the blue fluorescent protein gene
EBFP(GenBank:EF030494.1), red fluorescent protein gene more preferably wherein
DsRedOr green fluorescence protein gene
EGFPAccording to the expressive site of described color mark gene C in seed, can select suitable specific expressing promoter, for example, the color mark gene C is expressed at endosperm and is just selected endosperm specificity expression promoter, and endosperm specificity promoter has: P
Gt1(GenBank:EU264103.1) and P
GluB1(GenBank:AY427569.1 or GenBank:JN389781.1).
In the above-mentioned method of cultivation, described sterile gene s is preferably
Msp1,
Pair1,
Pair2,
Zep1,
Mel1,
Pss1,
Tdr,
Udt1,
Gamyb4,
Ptc1,
Api5,
Wda1,
Cyp704B2,
Dpw,
Mads3,
Osc6,
Rip1,
CsaOr
Aid1, the described gene S that educates is corresponding paddy rice wild type gene (can obtain corresponding gene sequences at NCBI according to accession number, design gets final product the primer in the described step (2) according to corresponding gene sequences).
As a total technical conceive, the application of engineering maintenance line in the common line with genic sterile breeding of paddy rice that the present invention also provides a kind of above-mentioned method of cultivation to obtain, it is to be sS with single copy color mark gene C and genotype that described engineering keeps
CStrain, this project maintenance line be by will be divided into from educated gene S and after the color mark gene C imports common line with genic sterile acceptor (ss), with common line with genic sterile (ss) backcross breeding gained, therefore genetic background and the common line with genic sterile of paddy rice (ss) of described engineering maintenance line are basically identical, unique difference be to have inserted in the genome of engineering maintenance line be divided into from the color mark gene C and can educate gene S; When breeding common line with genic sterile, be the common line with genic sterile hybridization of paddy rice of ss with this project maintenance line and genotype, in filial generation, obtain simultaneously the common line with genic sterile seed of engineering maintenance line seed and paddy rice (ratio of the two is generally about 1: 1); At this moment, the color mark gene C is expressed in engineering maintenance line seed under the seed-specific expression promoters driven specifically, can with described engineering maintenance line seed separation out, be sS according to colour sorting genotype out by color selector
CEngineering maintenance line seed, can continue with genotype is the common line with genic sterile hybridization of the paddy rice of ss, the breeding genotype is sS
CHeterozygosis seed and the genotype common line with genic sterile seed that is ss; Remaining seed is the common line with genic sterile seed of paddy rice that genotype is ss, does not contain transgene component, can be used for hybrid seeding with restorer hybridization, can be sS with genotype also
CHeterozygosis seed hybridization, be used for breeding and the continuity of self material.
Compared with prior art, the invention has the advantages that:
(1) breed the common line with genic sterile of paddy rice by transgenosis means initiative engineering maintenance line, the common line with genic sterile of paddy rice that breeding obtains does not contain transgene component; And the normal kind of fertility all is the restorer of the common line with genic sterile of this paddy rice, and it is extremely extensive to recover spectrum, and combo is free, so that the probability of seed selection fine combination increases; Can open up subspecies indica and japonica hybrid advantage frontier immediately following the seed selection paces of Conventional Rice, make rice yield realize more high yield target on existing hybrid rice basis;
(2) reproductive process is simple, and reproductive efficiency is high: sterile line propagation is not restricted by envrionment conditions, and seed purity is high; Although Duoed an engineering maintenance line than bilinear method, this project maintenance line does not need extra breeding, in the common line with genic sterile of breeding paddy rice, can select the engineering maintenance line by the machine look;
(3) intelligent sorting, easy to operate: as to adopt the machine intelligence sorting to replace Artificial Control, for the extensive mechanize production of hybrid seeds provides possibility.
Description of drawings
Fig. 1 is the process flow sheet of common line with genic sterile breeding in the embodiment of the invention.
Fig. 2 is the endosperm-specific red fluorescent protein expression vector p that makes up in the embodiment of the invention
TDR DsRed
Fig. 3 be in the embodiment of the invention southern blot detect 4 individual plants that isozygoty (
TDR DsRed /
TDR DsRed ) with the insertion copy number of common line with genic sterile (ss) backcross progeny, wherein, 1 is Marker; 2 and 4 is that single copy inserts; 5 is that two copies insert; 3 is the amixia signal.
Embodiment
The invention will be further described with concrete preferred embodiment below in conjunction with Figure of description, but protection domain not thereby limiting the invention.
Embodiment:
Paddy rice
TDRThe degraded of the albumen control paddy rice suede adhesion coating of (Tapetum Degradation Retardation) genes encoding is the important factor in rice tapetum growth and the degrading and regulating network.If
TDRGene is undergone mutation, and Rice Anther suede adhesion coating will be degenerated, and cause the paddy pollen abortion, produces the common line with genic sterile of a kind of paddy rice (referring to the CN1884541A Chinese patent literature).
The application of the engineering maintenance line of the common line with genic sterile of a kind of paddy rice of the present invention in the common line with genic sterile breeding of paddy rice, it is above-mentioned that the concrete object of its application is present embodiment
TdrThe common line with genic sterile of gene mutation body specifically may further comprise the steps (technical process can referring to Fig. 1):
1.
60The Co-gamma-ray and mutagenesis obtains a strain paddy rice sterile mutant, shows this sterile proterties by a recessive single-gene (being sterile gene) control according to genetic analysis, and (genotype is to belong to the common line with genic sterile of a kind of paddy rice
Tdr/tdr).We also can buy the common nuclear sterile mutant of paddy rice, network address: http://signal.salk.edu/cgi-bin/RiceGE from mutant library RiceGE; The common genic male sterile gene applicable to the inventive method is as shown in table 1 below:
Table 1: the common nuclear sterile mutant tabulation of paddy rice
| Genic male sterile gene | The albumen of corresponding fertile gene coding | Corresponding fertile gene function | The NCBI accession number | RiceGE mutant subscription number |
| msp1 | LRR receptoroid kinases LRRkinas | The sporule early development | AB103395.1 | PFG_3A-07135.R |
| pair1 | The Coiled-coil domain protein | Homologous chromosomes joint conference | AB158462.1 | FL009505 |
| pair2 | The HORMA domain protein | Homologous chromosomes joint conference | AB109238.1 | RMD_TTL-04Z11KJ91 |
| zep1 | The Coiled-coil domain protein | Meiophase, synaptonemal complex formed | GU479042.1 | PFG_1B-18520.U |
| mel1 | ARGONAUTE (AGO) family protein | The premeiotic cell fission of sexual cell | AB297928.1 | RMD_03Z11CW42-2 |
| pss1 | The Kinesin family protein | Microgamete reduction division dynamic change | BK007977.1 | T07702T |
| tdr | The bHLH transcription factor | Tapetum degradation | AK106761.1 | PFG_3A-08673.R |
| udt1 | The bHLH transcription factor | Tapetum degradation | AY953870.1 | PFG_3D-00330.R |
| gamyb4 | Myb transcription factor | Aleurone layer and pollen sac are grown | NM_001051127.1 | PFG_1E-03950.R |
| ptc1 | The PHD-finger transcription factor | Tapetum and pollen granule are grown | GU597363.1 | RMD_02Z15CL49 |
| api5 | Inhibitor of |
Postpone tapetum degradation | NM_001053191.1 | PFG_2C-50137.R |
| wda1 | The carbon lyase | Lipid synthesis and extine form | AK100751.1 | PFG_2D-01704.R |
| cyp704B2 | Cytochrome P450 gene family | Pollen sac and extine are grown | NM_001055627.2 | T07707T |
| dpw | The lipid acid reductase enzyme | Pollen sac and extine are grown | NM_001055618.1 | RMD_05NPBMB39 |
| mads3 | Homoeosis C class transcription factor | Pollen sac is grown and pollen development late period | AK108568.1 | FL059810 |
| osc6 | Fat transfer family albumen | Liposome and extine are grown | NM_001074690.1 | M0033824 |
| rip1 | The WD40 domain protein | Pollen maturation and sprouting | DQ491004.1 | PFG_1D-01918.L |
| csa | Myb transcription factor | The distribution of pollen and pollen sac sugar | NM_001049255 | PFG_K-05711 |
| aid1 | Myb transcription factor | The pollen sac cracking | AY429017.1 | T25683T |
2. the above-mentioned sterile gene of map based cloning, homology compare of analysis find this mutant be by
TDRThe gene generation of undergoing mutation, mutated genes
TdrWith wild type gene
TDRBetween only have the difference of a base.
3. according to above-mentioned
TdrThe following primer of the sequences Design of gene:
TDR-F:ATGGGAAGAGGAGACCACCTGCTGA；
TDR-R: TCAATCAAACGCGAGGTAATGCAGG；
Clone the common line with genic sterile of above-mentioned paddy rice source parent's allelotrope, i.e. wild type gene according to the primer of design
TDR, because sterile gene
TdrFrom wild type gene
TDRSudden change obtains, therefore, and this wild type gene
TDRNamely can recover the educated gene of the common line with genic sterile fertility of present embodiment paddy rice; Wild type gene
TDRWith himself promoters driven.
4. with the red fluorescent protein gene
DsRedWith above-mentioned
TDRGene is connected on the same binary expression vector, and the skeleton carrier that present embodiment is selected is pCAMBIA1300, makes the red fluorescent protein gene
DsRedWith can educate gene
TDRCan linkage inheritance after importing plant, be divided into from, obtain binary expression vector p
TDR DsRed (referring to Fig. 2).
5. adopt agrobacterium-mediated transformation (or via Particle Bombardment Transformation method) with described binary expression vector p
TDR DsRed Change in the common line with genic sterile of above-mentioned paddy rice, according to
DsRedPrimers DsRed-F:CAACACCGTGAAGCTGAAGG; DsRed-R:CTACAGGAACAGGTGGTGGC is to T
0Carry out PCR for transgenic line and detect, screening obtains 17 strains with the red fluorescent protein gene
DsRedThe fertility restorer strain (T of the common line with genic sterile of paddy rice
0For transgenic line); Because having, importing can educate gene in the 17 strain fertility restorer strains that present embodiment is cultivated
TDR, and can educate gene
TDRTo sterile gene
TdrFor dominant, so this 17 strain T
0To recover fertility for transgenic line and offspring's strain thereof; Again because the red fluorescent protein gene
DsRedWith can educate gene
TDRBe divided into from, 17 strain T
0For the fertility shape of transgenic line and offspring's strain thereof and color mark with linkage inheritance, therefore after gathering in the crops this fertility restorer strain seed, by self propagated (present embodiment has adopted the self propagated in 5 generations), can educate the method that individual plant sowing, rubescent look fluorescent seeds are reserved seed for planting, obtain 4 stable fertilities, setting percentage is high and with the red fluorescent protein gene
DsRedHomozygous lines (genotype is
TDR DsRed / TDR DsRed ).
With the common line with genic sterile of the paddy rice in the present embodiment above-mentioned steps 1 (
Tdr/tdr) as 4 homozygous lines with the red fluorescence marker gene in recurrent parent and the above-mentioned steps 5 (
TDR DsRed / TDR DsRed ) backcross, staying the seed that selects rubescent look fluorescence in 4 individual plants, and pass through the copy number that southern blot detects the color mark gene of 4 individual plant backcross progenies, the 2 pnca gene types that obtain are
Tdr/TDR DsRed Single copy insert strain (referring to Fig. 3), with genotype be
Tdr/TDR DsRed The pollen artificial pollination that produces of strain to the common line with genic sterile of the above-mentioned paddy rice of 5 strains (
Tdr/tdr) column cap carry out test cross, this 5 strain sterile mutant can both be normally solid, average setting percentage is 94.3%, this explanation genotype is
Tdr/TDR DsRed The heterozygosis seed can recover the common line with genic sterile of present embodiment paddy rice (
Tdr/tdr) fertility, so genotype is
Tdr/TDR DsRed The heterozygosis seed can be described as
TdrThe engineering maintenance line of the common line with genic sterile of present embodiment paddy rice of sudden change.
7. divide individual plant to gather in the crops above-mentioned 5 pnca gene types to be
Tdr/tdrThe solid gained seed of the common line with genic sterile test cross of paddy rice, select each single-strain seed according to fluorescence color, through card square analysis, the seed rate of the seed of issue of bidding documents note red fluorescence and not issue of bidding documents note fluorescence is 1: 1(sees the following form 2), the seed cdna type of issue of bidding documents note red fluorescence is
Tdr/TDR DsRed , the seed cdna type of not issue of bidding documents note fluorescence is
Tdr/tdr, therefore, genotype is
Tdr/TDR DsRed Seed can continue on for the next time breeding of the common line with genic sterile of paddy rice as the engineering maintenance line, and genotype is
Tdr/tdrSeed then use the seed of the required common line with genic sterile of paddy rice that makes for present embodiment.
Table 2: seed fluorescence statistic analysis result
| Sterile strain numbering | Individual plant is grain number (grain) effectively | Rubescent look fluorescence (grain) | Not rubescent look fluorescence (grain) | Fluoresce: do not fluoresce | X 2 1∶1 |
| S1 | 1346 | 663 | 683 | 1∶1.003 | 0.268 |
| S2 | 1289 | 631 | 658 | 1∶1.014 | 0.524 |
| S3 | 1203 | 589 | 614 | 1∶1.005 | 0.479 |
| S4 | 1158 | 570 | 588 | 1∶1.021 | 0.35 |
| S5 | 1123 | 552 | 571 | 1∶0.984 | 0.289 |
X
2﹤ P=3.841 meets 1: 1 ratio.
674 not fluorescent seeds that obtain with sterile strain S1 breeding in the above-mentioned application are as the engineering maintenance line of the common line with genic sterile of present embodiment paddy rice and present embodiment
Tdr/TDR DsRed Carry out small-scale hybrid seeding.Select 500 normal seed germination seedling of full grains color from 674 not fluorescent seeds after, transplant 400 strains to the land for growing field crops, 50 row are planted, as female parent by per 8 strain delegation.Transplant 2 row engineering maintenance lines as male parent around maternal.Divide seedling stage individual plant to get maternal blade, carry DNA after, use
DsRedThe primer DsRed-F:CAACACCGTGAAGCTGAAGG of gene order design; DsRed-R:CTACAGGAACAGGTGGTGGC carries out PCR and detects, and 400 strain female parents all do not detect the purpose band of 556bp, and the engineering maintenance line can detect the purpose band of 556bp, and this explanation issue of bidding documents note fluorescence and genotype are
Tdr/TDR DsRed Engineering maintenance line seed is corresponding, and not issue of bidding documents note fluorescence then with genotype is
Tdr/tdrThe seed of common line with genic sterile corresponding.Before the heading flowering, with plastics film isolation production of hybrid seeds field, prevent from altering powder.Behind the Grain Ripening, dry and weigh, 400 strain female parents obtain the 13.63kg seed altogether.Obtain the fluorescigenic seed of 6.95kg after the look choosing separates, can be used as the common line with genic sterile that the engineering maintenance line is used for breeding lower season.The remaining not fluorescent seed of 6.68kg namely is that genotype is
Tdr/tdrThe line with genic sterile seed.As seen, according to the inventive method, obtain the above-mentioned common line with genic sterile seed of 6.68kg with 400 common line with genic sterile seeds of paddy rice with regard to breeding.
＜110〉Hunan Research Centre for Hybrid Rice
＜120〉method of cultivation of Rice Engineering maintenance line and the application in the common line with genic sterile breeding of paddy rice thereof
<160> 2
<210> 1
<211> 20bp
<212> DNA
＜213〉artificial sequence
<400> 1
caacaccgtg aagctgaagg 20
<210> 2
<211> 20bp
<212> DNA
＜213〉artificial sequence
<400> 2
ctacaggaac aggtggtggc 20
Claims (9)
1. the method for cultivation of a Rice Engineering maintenance line may further comprise the steps:
(1) preparing genotype is the common line with genic sterile of paddy rice of ss, locates and clone the sterile gene s of the common line with genic sterile of described paddy rice by the method that adopts map based cloning;
(2) according to the primers of above-mentioned sterile gene, clone the common line with genic sterile of above-mentioned paddy rice source parent's educated gene S, this can educate gene S can recover the common line with genic sterile fertility of described paddy rice;
(3) color mark gene C and above-mentioned educated gene S are connected on the same binary expression vector, make the color mark gene C with can educate gene S can linkage inheritance after importing plant, be divided into from, obtain binary expression vector pS
C
(4) adopt existing transgenic method with described binary expression vector pS
CChange in the common line with genic sterile of described paddy rice, obtain at last the fertility restorer strain S of the common line with genic sterile of paddy rice
CS
C
(5) homozygous lines and the common line with genic sterile ss with described fertility restorer strain backcrosses, and the acquisition genotype is sS
CThe strain that single copy inserts is the engineering maintenance line.
2. method of cultivation according to claim 1, it is characterized in that: the sterile proterties of the common line with genic sterile of described paddy rice is the recessive character by Dominant gene, the described gene S that educates is dominant to sterile gene s, and sterile proterties is not affected by external environmental condition.
3. method of cultivation according to claim 1, it is characterized in that: in the described step (3), described binary expression vector is plant binary expression vector, is specially pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301 or pBI121.
4. method of cultivation according to claim 3, it is characterized in that: described plant binary expression vector is pCAMBIA1300 or pCAMBIA1390.
5. method of cultivation according to claim 1, it is characterized in that: in the described step (3), described color mark gene is the red fluorescence marker gene
DsRed, the red fluorescent protein marker gene
RFP, green fluorescence protein gene
GFP, green fluorescence protein gene
EGFPOr blue fluorescent protein gene
EBFP
6. method of cultivation according to claim 5, it is characterized in that: described color mark gene is the red fluorescent protein gene
DsRedOr green fluorescence protein gene
EGFP
7. each described method of cultivation according to claim 2～6 is characterized in that: described sterile gene s is
Msp1,
Pair1,
Pair2,
Zep1,
Mel1,
Pss1,
Tdr,
Udt1,
Gamyb4,
Ptc1,
Api5,
Wda1,
Cyp704B2,
Dpw,
Mads3,
Osc6,
Rip1,
CsaOr
Aid1, the described gene S that educates is corresponding paddy rice wild type gene.
8. each described method of cultivation according to claim 2～6 is characterized in that: the transgenic method in the described step (4) is agrobacterium-mediated transformation or via Particle Bombardment Transformation method.
9. one kind such as the application of engineering maintenance line in the common line with genic sterile breeding of paddy rice that method of cultivation obtains as described in each in the claim 1～8, it is characterized in that: it is to be sS with single copy color mark gene C and genotype that described engineering keeps
CStrain, described engineering maintenance line be in the genome of the common line with genic sterile of described paddy rice, inserted be divided into from the color mark gene C with can educate gene S, when the common line with genic sterile of breeding paddy rice, be the common line with genic sterile hybridization of paddy rice of ss with this project maintenance line and genotype, in filial generation, obtain simultaneously the common line with genic sterile seed of engineering maintenance line seed and paddy rice, again by color selector with described engineering maintenance line seed separation out, remaining seed is the common line with genic sterile seed of paddy rice that genotype is ss.
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