CN102870670B - Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice - Google Patents
Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice Download PDFInfo
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Abstract
The invention discloses a universal type breeding method for a rice engineering maintainer line. The method comprises the following steps: establishing a dual-element expression vector of color marker gene C; shifting the vector into a type with the genetype of SS through a transgenic method to obtain transgenic strains; obtaining isozygoty strains through selfing propagation; analyzing the insertion site of gene C of each isozygoty strain and obtaining an engineering maintainer line candidate bank through screening and establishment; and crossbreeding ordinary nucleic male sterility lines ofrice with the genetype of SS with the isozygoty strains in the candidate bank to obtain F1, obtaining F2 through selfing propagation of F1, breeding seeds with no color in the F2, inspecting sterile stains in each group of F2 stain after breeding, and screening the seeds of F1, where the sterile stains accounting for more than 98% of the F2 come from to take the seeds as the engineering maintainer line. The breeding method provided by the invention has the advantages of short period, strong universality, high efficiency and the like; and the obtained engineering maintainer line can be appliedto mechanized and large-scale propagation of ordinary nucleic male sterility lines of rice.
Description
Technical field
The invention belongs to the breeding technology field of crop sterile line, be specifically related to the propagation method of a kind of self-pollination class crop (for example paddy rice, wheat etc.) male sterile line.
Background technology
Utilize the male infertility of plant to cultivate male sterile line, come the mass production cenospecies by this genetic tool again, can make many crops particularly the hybrid vigour of self pollination crop be able to use producing.
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, recovers system by the sterile proterties of tenuigenin control owing to can not find merely, 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 controlled jointly by plasmone and cell nucleus gene, existing maintenance line is kept its sterility, can under the help that recovers system, recover the fertility of F1 cross-fertilize seed again, such sterile line and maintenance line, recovery system form three series mating, be to produce classical, the most ripe method, i.e. three series at present.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, promote that link is many, speed is slow, and while seed costs height, price are expensive.
In the nuclear male sterility type, whether to the environmental factor sensitivity, it is sterile to be divided into the sterile and common nuclear of environmental sensitivity nuclear again according to sterility for recessive cytoblast sterile.
Environmental sensitivity nuclear is sterile, mainly refer to photoperiod-temperature sensitive genie male-sterile line at present, its fertility was controlled by genic male sterile gene both, was subjected to ambient light according to the regulation and control of length and temperature height, at certain developmental stage again, 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 bilinear method of namely often saying.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 influence 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 sterile rate and sterile strain easily and all be 100% common line with genic sterile; 2) the normal kind of fertility all is its recovery system, recovers spectrum and extensive, and combo freely makes the probability of seed selection fine combination heighten; 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 line with genic sterile source is abundant, seed selection is simple relatively, stable fertility is easy to recover, and is a kind of desirable sterile material, but because such sterile line does not have corresponding maintenance line, and self can not carry out fertility conversion, breed very difficultly, this becomes the key factor of its scale operation utilization of restriction.
Present stage, the focus of studying common line with genic sterile concentrates on the location and clone of genic male sterile gene, a kind of " albumen coded sequence of control rice tapetum degradation " for example disclosed in the CN1884541A Chinese patent literature (application number is 200610027399.8), wherein, magnify the soldier and wait the common genic male sterile gene that obtains a paddy rice by map based cloning
TDR, the albumen that biological function explore is found this genes encoding is controlled the fertility of pollen by the control rice tapetum degradation.At the large-scale breeding of common line with genic sterile and be applied to aspect the actual production, rare report in the existing document, present common line with genic sterile mainly adopts to backcross and raises up seed with vegetative propagation, for example, the CN102121052A Chinese patent literature has been put down in writing and has been utilized molecular marker assisted selection by backcrossing to formulate and breed the paddy rice recessive gms line.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, have the common line with genic sterile of bibliographical information paddy rice to breed by regeneration, the common line with genic sterile of cotton can be bred by cuttage.By aforesaid method, researcher can be bred and be obtained a spot of common line with genic sterile for scientific research and experiment, but the common sterile line quantity rareness that breeding obtains, and need the cost lot of manpower and material resources.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.
Because increasing research institution and researcher are recognized utility value and the potentiality that common line with genic sterile is huge, in the recent period many research institutions have strengthened research input and research dynamics on the common line with genic sterile large-scale breeding problem, and obtained gratifying progress, for example Hunan Research Centre for Hybrid Rice is by locating and cloning sterile gene s and can educate gene S, structure engineering maintenance line is bred common line with genic sterile and has been obtained success, but this method need be cloned sterile gene s and can be educated gene S, strengthen the difficulty that the engineering maintenance line makes up, and prolonged the progress that the engineering maintenance line makes up greatly.Therefore, explore a kind of breeding and method for creating of common line with genic sterile of simple and highly versatile, will be significant for advancing common line with genic sterile large-scale application in producing and further discharging the heterotic potentiality aspect that utilizes.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art; the method of cultivation of the universal paddy rice engineering maintenance line that a kind of step is simple, easy to operate, the cycle is short, efficient is high is provided; also correspondingly provide the application of a kind of universal paddy rice engineering maintenance line in the breeding of the common line with genic sterile of paddy rice; this application can realize the mechanize of the common line with genic sterile of paddy rice, the crossbreeding of mass-producing, and can accelerate the process that common line with genic sterile is used to realization from seed selection greatly.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of method of cultivation of universal paddy rice engineering maintenance line, may further comprise the steps:
(1) preparing genotype is the common line with genic sterile of paddy rice of ss, and s represents to control the sterile gene of the sterile proterties of the common line with genic sterile of paddy rice;
(2) the binary expression vector pC of structure color mark gene C;
(3) adopt existing transgenic method (for example agrobacterium-mediated transformation or particle gun conversion method) with among the source parent or the normal kind of fertility of the described binary expression vector pC transgene type common line with genic sterile of above-mentioned paddy rice that is SS (S represents the educated gene S to sterile gene s dominance), according to color mark gene design primer, obtain having the T of color mark gene C by the PCR screening
0For transgenic line; Again with T
0Obtain to have single homozygous lines that copies the color mark gene for transgenic line by continuous self propagated, its genotype is that SSCC(has verified its genotype by test cross);
(4) the insertion site (insert site analytical procedure generally be adopt conventional joint method or TAIL-PCR method) of color mark gene C in rice genome in each homozygous lines of obtaining of analytical procedure (3), and according to the on position of color mark gene C at rice genome, filter out the homozygous lines group storehouse of a series of single copies, make the rice genome in the homozygous lines group storehouse every 0 ~ 2cM a color mark gene C just be arranged, this homozygous lines group storehouse has namely constituted an engineering maintenance line candidate storehouse;
(5) each homozygous lines (SSCC) in the middle engineering maintenance line candidate storehouse of the common line with genic sterile of paddy rice (ss) in the step (1) and step (4) is hybridized, obtain F
1The heterozygosis seed, F
1A heterozygosis seed self propagated generation obtains the F of several genes type
2Seed selects technology to reject F earlier by look then
2The seed that has the color mark gene C in the seed is not respectively organized F with the color mark gene C with residue
2Seed is cultivated respectively, obtains the corresponding F that respectively organizes
2The aforementioned F that respectively organizes investigates in strain system
2Sterile strain number in the strain system filters out the F that accounting 〉=98% is counted in sterile strain
2Strain system group, and with this F
2The F in strain system group source
1(genotype is s to the heterozygosis seed as the engineering maintenance line of the common line with genic sterile of described paddy rice
cS
C).
In the method for cultivation of the invention described above, genotype is s
cS
CEngineering maintenance line heterozygosis seed be according to the color mark gene C with can educate gene S at F
2Genetic linkage analysis conclusive evidence in generation obtains, and it is based on following ultimate principle: each strain cross in the common line with genic sterile of paddy rice (ss) and the engineering maintenance line candidate storehouse obtains F
1The heterozygosis seed, F
1A heterozygosis seed self propagated generation obtains F
2Seed will not carry the F of color mark gene by the look choosing
2After going down, the seed kind obtains F
2Colony, investigation F
2The sterile strain number of colony if sterile strain is counted accounting when being 1/4 left and right sides, is then analyzed and is judged color mark C and can educate gene S and be positioned on the coloured differently body; If sterile strain is counted accounting greater than 1/4 and less than 98% o'clock, then color mark C with can educate gene S and be positioned on the same karyomit(e), and color mark C and the recombination fraction that can educate gene S are between 1% to 50%; If sterile strain is counted accounting more than 98%, then can judge color mark C and can educate gene S and be positioned on the same karyomit(e), and therefore color mark C and the recombination fraction that can educate gene S, stay and select sterile strain to count accounting at the F more than 98% below 1%
2The F in source
1Seed is the engineering maintenance line that the present invention obtains.
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, preferably, the sterile proterties of the common line with genic sterile of described paddy rice is the recessive cytoblast sterile proterties by single-gene control, described to educate the sterile gene s of gene S be dominance, and sterile proterties is not influenced 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 (2), described binary expression vector pC specifically is preferably any one (being preferably pCAMBIA1300 or pCAMBIA1390 especially) among pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301, the pBI121.
Above-mentioned method of cultivation, in the described step (2), the color mark gene C can be expressed in seed, makes seed have color mark, so that look is selected the seed that has this color mark gene in the subsequent applications.Described color mark gene is preferably the red fluorescent protein gene
DsRed, the red fluorescence marker gene
RFP, green fluorescence protein gene
GFP, green fluorescence protein gene
EGFPOr blue fluorescent protein gene
EBFP, 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.
As a total technical conceive, the application of universal paddy rice engineering maintenance line in the common line with genic sterile breeding of paddy rice that the present invention also provides a kind of aforesaid method to cultivate, it is to have the color mark gene C and genotype is s that described engineering keeps
cS
CThe heterozygosis seed, the genetic background of this project maintenance line is consistent with the common line with genic sterile of paddy rice (ss), unique difference is to have the color mark gene C in the genome of engineering maintenance line and can educate gene S, and the color mark gene C in the engineering maintenance line with can educate gene S close linkage (verifying its genotype by test cross), when breeding application, the common line with genic sterile of described paddy rice that earlier with described engineering maintenance line and genotype is ss is hybridized, in filial generation, obtain the common line with genic sterile seed of engineering maintenance line seed and paddy rice (ratio of the two was generally 1: 1) simultaneously, at this moment, the color mark gene C is under engineering maintenance line seed-specific expression promoters driven, in engineering maintenance line seed, express specifically, described engineering maintenance line seed separation can be come out by color selector, the genotype of coming out by colour sorting is s
cS
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 s
cS
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 that hybridization is used for hybrid seeding with recovering, and can be s with genotype also
cS
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 recovery system of the common line with genic sterile of this paddy rice, and it is extremely extensive to recover spectrum, and the combo freedom makes the probability of seed selection fine combination heighten; 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, the reproductive efficiency height: sterile line propagation is not restricted by envrionment conditions, the seed purity height; Though 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; The more important thing is, do not need to clone in addition sterile gene s and can educate gene S, simplify and accelerated the structure of engineering maintenance line greatly; On transformation receptor, also be further optimized, become conversion fertile line acceptor by conversion sterile line acceptor and make up the engineering maintenance line, further reduced the construction cost that transforms difficulty and engineering maintenance line;
(3) versatility improves greatly: for the common line with genic sterile in any sterile site, can from engineering maintenance line candidate storehouse, look for a strain system to make the genetic distance≤1cM in color mark gene C the educated site corresponding with sterile site, therefore, method of the present invention improves the versatility of engineering maintenance line greatly;
(4) intelligent sorting, easy to operate: as to replace Artificial Control by the machine intelligence sorting, 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 crossbreeding in the embodiment of the invention.
The endosperm-specific green fluorescence protein expression carrier pEGt of Fig. 2 for making up in the embodiment of the invention.
Fig. 3 is the screening process figure of engineering maintenance line among the present invention.
Fig. 4 is the impact analysis figure of gene recombination in engineering maintenance line among the present invention and the common line with genic sterile hybrid seeding.
Embodiment
Below in conjunction with Figure of description and concrete preferred embodiment the present invention is further described, but protection domain not thereby limiting the invention.
Embodiment 1:
The application of a kind of universal paddy rice engineering maintenance line of the present invention in the common line with genic sterile crossbreeding of paddy rice, the concrete object of its application is the common line with genic sterile of paddy rice T98B, specifically may further comprise the steps (technical process can referring to Fig. 1):
1. the common line with genic sterile of seed selection:
60The Co-gamma-ray and mutagenesis obtains the sterile mutant of a strain paddy rice from paddy rice T98B, show this sterile proterties by a recessive single-gene (being sterile gene) control by genetic analysis, and (genotype is to belong to the common line with genic sterile of a kind of paddy rice
Ss).We also can buy the sterile mutant of the common nuclear 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 sterile mutant tabulation of the common nuclear of paddy rice
| Genic male sterile gene | Corresponding fertile gene encoded protein | 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 apoptosis protein 5 | Postpone tapetum degradation | NM_001053191.1 | PFG_2C-50137.R |
| wda1 | The carbon lyase | Synthetic and the extine formation of lipid | 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. structure binary expression vector: be skeleton carrier with pCAMBIA1300, make up the color mark gene
EGFPBinary expression vector, the color mark gene
EGFPBy endosperm specificity promoter P
Gt1Drive (referring to Fig. 2).
3. transform the source parent of common line with genic sterile: adopting agrobacterium-mediated transformation is in the genome of source parent paddy rice T98B of the common line with genic sterile of above-mentioned paddy rice of SS with above-mentioned binary expression vector transgene type, if the total genetic distance of this genome is P, according to the chain rule of reorganization, recombination fraction is 1% o'clock, genetic distance is 1cM, so the color mark gene
EGFPBe inserted in left side 1cM or the right 1cM that can educate gene S, the two gene recombination rates that can both obtain are 1% strain system, so only need P/2 T in theory
0In generation,, single transformant that copies can obtain a strain color mark gene
EGFPBe 99% strain system with educating the chain frequency of gene S; Because the total genetic distance of paddy rice is about 1750cM, if obtain a strain color mark gene
EGFPBe 99% strain system with educating the chain frequency of gene S, then need 875 T in theory
0Transformant for single copy; In the present embodiment, obtain have 3133 to have the color mark gene altogether
EGFPT
0For transgenic line; Behind the results seed, have single homozygous lines that copy the color mark gene by 1546 of self propagated 6 generations acquisitions, genotype is SS/
EGFPEGFP
4. inserting the site analyzes: according to 1546 that obtain in the flanking sequence analytical procedure 3 insertion sites that have the homozygous lines of single copy color mark gene, the insertion site of these 1546 homozygous lines relatively is evenly distributed in the rice genome, inserting the genetic distance of site between rice genome is 0.1cM～1.93cM, and also namely list copies the color mark gene
EGFPBetween genetic distance be 0.1cM～1.93cM, meet engineering maintenance line candidate storehouse has a color mark gene every 0～2.0cM standard.
5. genetic linkage analysis: with 1546 homozygous lines (SS/ that have the color mark gene in the common line with genic sterile (ss) of above-mentioned paddy rice T98B and the step 4
EGFPEGFP) hybridization, obtain 1433 F
1The heterozygosis seed, a heterozygosis seed self propagated generation obtains F
2Seed sub-elects F by the look choosing
2In carry the fluorescent mark gene
EGFPSeed, do not carry the fluorescent mark gene from remainder
EGFPF
2Respectively select the kind of 50 left and right sides full grains in the seed and go down afterwards to obtain 1433 F
2Colony, investigation F
2The sterile strain number of colony.At 1433 F
2Have the sterile strain ratio of two colonies to have only a colony greater than 98% near 98%(in the colony, another one is 96.08%), be respectively 98.04% and 96.08%, enlarge this two F
2Colony is to 1000 strains and investigate the ratio of sterile strain, and the ratio of sterile strain still is respectively 98% and 96%, and wherein sterile strain ratio is 98% F
2The F that colony originates
1Color mark gene and the chain frequency that can educate gene meet the structure requirement of engineering maintenance line greater than 99% in the heterozygosis strain system, stay to be elected to be to be the engineering maintenance line, and genotype is designated as s
Egfp S
EGFP
In the step 5 of present embodiment, the screening process of engineering maintenance line earlier with each strain cross in common line with genic sterile (ss) and the engineering maintenance line candidate storehouse, obtains F as shown in Figure 3
1Heterozygosis seed (s
Egfp S
EGFP ), a heterozygosis seed self propagated generation obtains F
2Seed is obtaining F
2Nine kinds of genotype are arranged in the seed, be respectively SSCC, SsCc, SSCc, SsCC, ssCc, ssCC, sscc, Sscc, SScc(wherein C namely refer to
EGFP) (referring to Fig. 3), the six kinds of genotypic F in front
2Seed can sorting be rejected (part that is black box choosing among Fig. 3) by look choosing, remaining three kinds genotypic not with the F of color mark gene
2After going down, the seed kind obtains respectively to organize F
2Colony, F is respectively organized in investigation
2Accounting is counted in the sterile strain of colony, if sterile strain is counted accounting near 1/4, then judges the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene S and be positioned on the coloured differently body; If sterile strain is counted accounting between 25% to 98%, then judge the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene S and be positioned on the same karyomit(e), and color mark
EGFPAnd can educate the recombination fraction of gene S between 1% to 50%; If sterile strain is counted accounting more than 98%, then judge the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene S and be positioned on the same karyomit(e), and color mark
EGFPWith the recombination fraction that can educate gene S below 1%.Therefore, stay and select sterile strain to count accounting at the F more than 98%
2The F that originates
1The heterozygosis seed is present embodiment and requires the engineering maintenance line that obtains.Therefore genotype is s among the present invention
Egfp S
EGFP The heterozygosis seed be according to the color mark gene
EGFPWith can educate gene S at F
2Genetic linkage analysis in generation obtains.
6. breeding is used: be s with genotype in the step 5
Egfp S
EGFP Engineering maintenance line heterozygosis seed and the common line with genic sterile hybridization of the genotype above-mentioned paddy rice T98B that is ss because color mark gene in the engineering maintenance line
EGFPWith the genetic distance≤1cM that can educate gene S, so have≤1% recombination probability, as shown in Figure 4, the offspring can produce two kinds of recons, genotype be ssCc and Sscc(wherein C namely refer to
EGFP), its accounting is all below 0.5%, and genotype is s
Egfp S
EGFP The engineering maintenance line and the accounting of the genotype common line with genic sterile that is ss all more than 49.5%.Be that recon and the genotype of ssCc is s by look choosing with genotype
Egfp S
EGFP The engineering maintenance line sort out, the common line with genic sterile and the genotype that stay genotype and be ss are the recon of Sscc, as seen breeding purity 〉=99%(of obtaining common line with genic sterile is according to the purity requirement of hybrid rice seeds quality grading standard GB 4404.1-1996 regulation: original seed level parent 〉=99.9%, breeding level parent 〉=99.0%, so P/2 T among the present invention
0Can obtain desirable engineering maintenance line for transformant, by this project maintenance line breeding male sterile lines, its purity can reach breeding level parent's purity requirement), can further genotype be removed for the Sscc recon by removal of impurities.And the recon genotype that the look despecking removes is ssCc, because this recon is sterile, itself and common line with genic sterile ss hybridization can not be solid, so this recon can not influence engineering maintenance line s
Egfp S
EGFP Continue on for the breeding of common line with genic sterile.
In the present embodiment, genotype is s
Egfp S
EGFP Engineering maintenance line and the common line with genic sterile hybridization that is ss of 3 pnca gene types, the common line with genic sterile of 3 strains can both be normally solid, average setting percentage is 95.3%.Divide individual plant to gather in the crops 3 strain seeds that common line with genic sterile is tied, the color selector device is s with genotype
Egfp S
EGFP Engineering maintenance line seed separation come out, the seed that stays namely is that genotype is the line with genic sterile seed of ss, through card square analysis, the seed rate of the seed of issue of bidding documents note fluorescence and not issue of bidding documents note fluorescence is that 1 ︰ 1(sees the following form 2), wherein not fluorescent seed is for will breed the common line with genic sterile seed that obtains.
Table 2: seed fluorescence statistic analysis result
| Sterile strain numbering | Individual plant is grain number (grain) effectively | (grain) fluoresces | (grain) do not fluoresce | X 2?（1︰1） |
| T98BS1 | 884 | 451 | 433 | 0.327 |
| T98BS2 | 841 | 428 | 413 | 0.233 |
| T98BS3 | 793 | 407 | 386 | 0.504 |
X
2﹤ P=3.841 meets the ratio of 1 ︰ 1.
Embodiment 2:
The application of a kind of universal paddy rice engineering maintenance line of the present invention in the common line with genic sterile crossbreeding of paddy rice, the concrete object of its application is paddy rice
CSAThe common line with genic sterile of transgenation specifically may further comprise the steps (technical process can referring to Fig. 1):
1. choose common line with genic sterile:
CSAMyb transcription factor of expressing tapetal cell of genes encoding is a crucial regulative transcription factor of pollen development, and it is sterile that its mutant shows as common nuclear, and genotype is designated as
Csa/
CsaIt is fine to buy paddy rice Japan from mutant library RiceGE
CsaGene mutation body, network address: http://signal.salk.edu/cgi-bin/RiceGE, the subscription number ginseng sees the above table 1.
2. structure binary expression vector: be skeleton carrier with pCAMBIA1300, make up the color mark gene
EGFPBinary expression vector, the color mark gene
EGFPBy endosperm specificity promoter P
Gt1Drive (referring to Fig. 2).
3. transform the source parent of common line with genic sterile: adopt agrobacterium-mediated transformation with above-mentioned binary expression vector transgene type to be
CSA/
CSAThe fine genome of source parent paddy rice Japan of the common line with genic sterile of above-mentioned paddy rice in, establishing the total genetic distance of this genome is P, according to the chain rule of reorganization, recombination fraction is 1% o'clock, genetic distance is 1cM, so the color mark gene
EGFPBe inserted in and educate gene
CSALeft side 1cM or the right 1cM, the two gene recombination rates that can both obtain are 1% strain system, so only need P/2 T in theory
0In generation,, single transformant that copies can obtain a strain color mark gene
EGFPWith can educate gene
CSAChain frequency is 99% strain system; Because the total genetic distance of paddy rice is about 1750cM, if obtain a strain color mark gene
EGFPWith can educate gene
CSAChain frequency is 99% strain system, then needs 875 T in theory
0Transformant for single copy; In the present embodiment, obtain have 3631 to have the color mark gene altogether
EGFPT
0For transgenic line; Behind the results seed, have single homozygous lines that copy the color mark gene by 1732 of self propagated 6 generations acquisitions, genotype is
CSACSA/
EGFPEGFP
4. inserting the site analyzes: according to 1546 that obtain in the flanking sequence analytical procedure 3 insertion sites that have the homozygous lines of single copy color mark gene, the insertion site of these 1732 homozygous lines relatively is evenly distributed in the rice genome, inserting the genetic distance of site between rice genome is 0.1cM～1.96cM, and also namely list copies the color mark gene
EGFPBetween genetic distance be 0.1cM～1.96cM, meet engineering maintenance line candidate storehouse has a color mark gene every 0～2cM standard.
5. genetic linkage analysis: the common line with genic sterile that above-mentioned paddy rice Japan is fine (
Csa/
Csa) with step 4 in 1732 homozygous lines that have a color mark gene (
CSACSA/
EGFPEGFP) hybridization, obtain 1546 F
1The heterozygosis seed, a heterozygosis seed self propagated generation obtains F
2Seed sub-elects F by the look choosing
2In carry the fluorescent mark gene
EGFPSeed, do not carry the fluorescent mark gene from remainder
EGFPF
2Respectively select the kind of 50 left and right sides full grains in the seed and go down afterwards to obtain 1546 F
2Colony, investigation F
2The sterile strain number of colony.At 1546 F
2The sterile strain ratio of a colony being arranged greater than 98% in the colony, is 98.69%, enlarges this F
2Colony is to 1000 strains and investigate the ratio of sterile strain, and the ratio of sterile strain illustrates this F still greater than 98%
2The F that colony originates
1Color mark gene and the chain frequency that can educate gene meet the structure requirement of engineering maintenance line greater than 99% in the heterozygosis strain system, stay to be elected to be to be the engineering maintenance line, and genotype is designated as
Csa Egfp CSA EGFP
In the step 5 of present embodiment, the screening process of engineering maintenance line as shown in Figure 3, earlier with common line with genic sterile (
Csa/
Csa) with engineering maintenance line candidate storehouse in each strain cross, obtain F
1The heterozygosis seed (
Csa Egfp CSA EGFP ), a heterozygosis seed self propagated generation obtains F
2Seed is obtaining F
2Nine kinds of genotype are arranged in the seed, be respectively wherein s acute pyogenic infection of finger tip of SSCC, SsCc, SSCc, SsCC, ssCc, ssCC, sscc, Sscc, SScc(
Csa, the S acute pyogenic infection of finger tip
CSA, the c acute pyogenic infection of finger tip
Egfp, C refers to
EGFP) (referring to Fig. 3), the six kinds of genotypic F in front
2Seed can sorting be rejected (part that is black box choosing among Fig. 3) by look choosing, remaining three kinds genotypic not with the F of color mark gene
2After going down, the seed kind obtains respectively to organize F
2Colony, F is respectively organized in investigation
2Accounting is counted in the sterile strain of colony, if sterile strain is counted accounting near 1/4, then judges the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene S and be positioned on the coloured differently body; If sterile strain is counted accounting between 25% to 98%, then judge the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene
CSABe positioned on the same karyomit(e), and color mark
EGFPWith can educate gene
CSARecombination fraction between 1% to 50%; If sterile strain is counted accounting more than 98%, then judge the F in its corresponding source
1The color mark of heterozygosis seed
EGFPWith can educate gene
CSABe positioned on the same karyomit(e), and color mark
EGFPWith can educate gene
CSARecombination fraction below 1%.Therefore, stay and select sterile strain to count accounting at the F more than 98%
2The F that originates
1The heterozygosis seed is present embodiment and requires the engineering maintenance line that obtains.Therefore genotype is among the present invention
Csa Egfp CSA EGFP The heterozygosis seed be according to the color mark gene
EGFPWith can educate gene
CSAAt F
2Genetic linkage analysis in generation obtains.
6. breeding is used: with genotype in the step 5 be
Csa Egfp CSA EGFP Engineering maintenance line heterozygosis seed and genotype be
Csa/
CsaThe fine common line with genic sterile hybridization of above-mentioned paddy rice Japan because color mark gene in the engineering maintenance line
EGFPWith can educate gene
CSAGenetic distance≤1cM, so have≤1% recombination probability, as shown in Figure 4, the offspring can produce two kinds of recons, genotype is wherein s acute pyogenic infection of finger tip of ssCc and Sscc(
Csa, the S acute pyogenic infection of finger tip
CSA, the c acute pyogenic infection of finger tip
Egfp, C refers to
EGFP), its accounting is all below 0.5%, and genotype is
Csa Egfp CSA EGFP Engineering maintenance line and genotype be
Csa/
CsaThe accounting of common line with genic sterile all more than 49.5%.Be that recon and the genotype of ssCc is by look choosing with genotype
Csa Egfp CSA EGFP The engineering maintenance line sort out, stay genotype and be
Csa/
CsaCommon line with genic sterile and genotype be the recon of Sscc, as seen breeding purity 〉=99%(of obtaining common line with genic sterile is according to the purity requirement of hybrid rice seeds quality grading standard GB 4404.1-1996 regulation: original seed level parent 〉=99.9%, breeding level parent 〉=99.0%, so P/2 T among the present invention
0Can obtain desirable engineering maintenance line for transformant, by this project maintenance line breeding male sterile lines, its purity can reach breeding level parent's purity requirement), can further genotype be removed for the Sscc recon by removal of impurities.And the recon genotype that the look despecking removes is ssCc, because this recon is sterile, itself and common line with genic sterile ss hybridization can not be solid, so this recon can not influence the engineering maintenance line
Csa Egfp CSA EGFP Continue on for common line with genic sterile
Csa/
CsaBreeding.
In the present embodiment, genotype is
Csa Egfp CSA EGFP Engineering maintenance line and 6 pnca gene types be
Csa/
CsaThe hybridization of common line with genic sterile, the common line with genic sterile of 6 strains can both be normally solid, average setting percentage is 96.8%.Divide individual plant to gather in the crops 6 strain seeds that common line with genic sterile is tied, the color selector device with genotype is
Csa Egfp CSA EGFP Engineering maintenance line seed separation come out, the seed that stays namely is that genotype is
Csa/
CsaThe line with genic sterile seed, through card square analysis, the seed of issue of bidding documents note fluorescence and not issue of bidding documents remember that the seed rate of fluorescence is that 1 ︰ 1(sees the following form 3), wherein not fluorescent seed is for will breed the common line with genic sterile seed that obtains.
Table 3: seed fluorescence statistic analysis result
| Sterile strain numbering | Individual plant is grain number (grain) effectively | (grain) fluoresces | (grain) do not fluoresce | X 2 （1︰1） |
| cas1 | 910 | 463 | 447 | 0.247 |
| cas2 | 894 | 458 | 436 | 0.493 |
| cas3 | 923 | 469 | 454 | 0.212 |
| cas4 | 887 | 457 | 430 | 0.762 |
| cas5 | 903 | 464 | 439 | 0.637 |
| cas6 | 878 | 446 | 432 | 0.192 |
X
2﹤ P=3.841 meets the ratio of 1 ︰ 1.
Claims (8)
1. the method for cultivation of a universal paddy rice engineering maintenance line may further comprise the steps:
(1) preparing genotype is the common line with genic sterile of paddy rice of ss, and s represents to control the sterile gene of the sterile proterties of the common line with genic sterile of paddy rice;
(2) binary expression vector of structure color mark gene C;
(3) adopt among the source parent or the normal kind of fertility of existing transgenic method with the described binary expression vector transgene type common line with genic sterile of above-mentioned paddy rice that is SS, screening obtains having the T of color mark gene C
0For transgenic line; Again with T
0Obtain to have single homozygous lines that copies the color mark gene for transgenic line by continuous self propagated, its genotype is SSCC;
(4) the insertion site of color mark gene C in rice genome in each homozygous lines of obtaining of analytical procedure (3), and according to the on position of color mark gene C at rice genome, filter out the homozygous lines group storehouse of a series of single copies, make the rice genome in the homozygous lines group storehouse every 0 ~ 2cM a color mark gene C just be arranged, this homozygous lines group storehouse has namely constituted an engineering maintenance line candidate storehouse;
(5) each homozygous lines in the engineering maintenance line candidate storehouse in the common line with genic sterile of paddy rice in the step (1) and the step (4) is hybridized, obtain F
1The heterozygosis seed, F
1A heterozygosis seed self propagated generation obtains the F of several genes type
2Seed selects technology to reject F earlier by look then
2The seed that has the color mark gene C in the seed is not respectively organized F with the color mark gene C with residue
2Seed is cultivated respectively, obtains the corresponding F that respectively organizes
2The aforementioned F that respectively organizes investigates in strain system
2Sterile strain number in the strain system filters out the F that accounting 〉=98% is counted in sterile strain
2Strain system group, and with this F
2The F in strain system group source
1The heterozygosis seed is as the engineering maintenance line of the common line with genic sterile of described paddy rice.
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 cytoblast sterile proterties by single-gene control, can educate the sterile gene s of gene S is dominance, and sterile proterties is not influenced by external environmental condition.
3. method of cultivation according to claim 1 and 2, it is characterized in that: in the described step (2), described 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 binary expression vector is pCAMBIA1300 or pCAMBIA1390.
5. method of cultivation according to claim 1 and 2, it is characterized in that: in the described step (2), described color mark gene is the red fluorescence marker gene
DsRed, the red fluorescence 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. method of cultivation according to claim 1 and 2, it is characterized in that: sterile gene s is
Msp1,
Pair1,
Pair2,
Zep1,
Mel1,
Pss1,
Tdr,
Udt1,
Gamyb4,
Ptc1,
Api5,
Wda1,
Cyp704B2,
Dpw,
Mads3,
Osc6,
Rip1,
CsaOr
Aid1, can educate gene S and be corresponding paddy rice wild type gene.
8. one kind as the application of universal paddy rice engineering maintenance line in the common line with genic sterile breeding of this paddy rice that method of cultivation obtains as described in each in the claim 1～7, it is characterized in that: it is to have the color mark gene C and genotype is s that described engineering keeps
cS
CThe heterozygosis seed, in the described engineering maintenance line color mark gene C with can educate gene S close linkage, when breeding application, the common line with genic sterile of described paddy rice that earlier with described engineering maintenance line and genotype is ss is hybridized, in filial generation, obtain the common line with genic sterile seed of engineering maintenance line seed and paddy rice simultaneously, by color selector described engineering maintenance line seed separation is come out, remaining seed is the common line with genic sterile seed of paddy rice that genotype is ss again.
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| CN104846009B (en) * | 2015-05-18 | 2018-02-13 | 湖南杂交水稻研究中心 | A kind of construction method of Rice Engineering maintainer and its application |
| CN105063083B (en) * | 2015-07-16 | 2018-07-06 | 湖南杂交水稻研究中心 | Prevent method for creating and its application of the Rice Engineering maintainer of genetic drift |
| CN108243963A (en) * | 2017-12-18 | 2018-07-06 | 海南波莲水稻基因科技有限公司 | A kind of rice PTC1 deletion mutants body and its method for identifying molecules and application |
| CN108949814A (en) * | 2018-07-04 | 2018-12-07 | 青岛袁策集团有限公司 | A kind of breeding method of the transgenic paddy rice sterile line based on TDR gene |
| CN108949816A (en) * | 2018-07-04 | 2018-12-07 | 青岛袁策集团有限公司 | A kind of breeding method of the transgenic paddy rice sterile line based on PAIR1 gene |
| CN108949811A (en) * | 2018-07-04 | 2018-12-07 | 青岛袁策集团有限公司 | A kind of breeding method of the transgenic paddy rice sterile line based on C6 gene |
| CN108949815A (en) * | 2018-07-04 | 2018-12-07 | 青岛袁策集团有限公司 | A kind of breeding method of the transgenic paddy rice sterile line based on PTC1 gene |
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