CN102876711B - 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
- Publication number
- CN102876711B CN102876711B CN201210426678.7A CN201210426678A CN102876711B CN 102876711 B CN102876711 B CN 102876711B CN 201210426678 A CN201210426678 A CN 201210426678A CN 102876711 B CN102876711 B CN 102876711B
- Authority
- CN
- China
- Prior art keywords
- gene
- sterile
- line
- paddy rice
- common line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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, then produce in a large number cenospecies by this genetic tool, can make many crops particularly the hybrid vigour of self pollination crop be able to application on 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, irrelevant with nucleus, and the sterile proterties of being controlled by tenuigenin is merely owing to can not find restorer, so also there is no application example on producing.
Nuclear male sterility refers to that sterile proterties is only controlled by cell nucleus gene, irrelevant with tenuigenin, is divided into dominant genic male sterile and recessive cytoblast sterile.The current core of having found is sterile is 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 under the help of restorer, recover F 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 more complicated, so that the cycle of the new combination of seed selection is long, efficiency is low, popularization link is many, speed is slow, and while seed costs is high, price.
In nuclear male sterility type, whether recessive cytoblast sterile is responsive to environmental factor according to sterility, is divided into again the sterile and common core of environmental sensitivity nuclear sterile.
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 subject 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 causes educating, and has the feature of fertility conversion, can one be dual-purpose, ratio three is to reduce one to be, also namely what is often called bilinear method.Photo-thermo-sensitive genetic male sterile line except not needing maintenance line, breed simple and easy and efficiency higher, also have: 1) recover spectrum wide, the normal kind of nearly all same subspecies can make its fertility restorer normal; 2) genetic behavior is simple, and sterility is controlled by 1～2 pair of recessive gene, and easily transformation and stable, is conducive to cultivate polytype sterile line.But also it should be pointed out that fertility due to photo-thermo-sensitive genetic male sterile line be subject to ambient light according to and temperature adjusting, if illumination and temperature Change be not in span of control in sterile line propagation process, will cause the photo-thermo-sensitive genetic male sterile line breeding underproduction even failed.
The sterile impact that is not generally subject to the outside atmosphere factor of common core, 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 nucleo-cytoplasmic interaction type is sterile, common core is sterile has following characteristics: 1) owing to being only subject to the control of a pair of recessive nuclear gene, and fertility is not affected by environment, easily selects sterile rate and sterile strain and be all 100% common line with genic sterile; 2) the normal kind of fertility is all its restorer, recovers spectrum and extensive, and combo freely makes 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 on existing hybrid rice basis, realize more high yield target.Therefore, common core is sterile 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, fail temporarily large-area applications on producing.
At present, common line with genic sterile mainly adopts to backcross and raises up seed with vegetative propagation, and for example, CN102121052A Chinese patent literature has been recorded and utilized molecular marker assisted selection by backcrossing to formulate and breed rice recessive line with genic sterile.In CN101946715A Chinese patent literature, recorded and utilized microspores culture and asexual clone technology seed selection and propagating cabbage type rape recessive gms line.In addition, have the breeding of can also regenerating of the common line with genic sterile of bibliographical information paddy rice, 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 need 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 to 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; Rice Engineering maintenance line product after cultivation can mechanize, mass-producing is 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, comprises the following steps:
(1) prepare the common line with genic sterile of paddy rice that genotype is ss, the sterile gene s(that the common line with genic sterile of described paddy rice was located and cloned to the method for employing map based cloning compares prediction sterile gene function by homology, and by gene knockout and mistake expression technology, verifies the function of 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 can recover the educated gene of the common line with genic sterile fertility of described paddy rice; Sterile gene s and can educate between gene S the difference that generally only has a base or several bases can be also disappearance or the insertion of a DNA fragmentation; Can educate gene S and take himself promoters driven (sequence of the front 2kb that promotor is coding region);
(3) color mark gene C and above-mentioned educated gene S are connected on same binary expression vector, make 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%, color mark gene C is separated by 250 * 10 with the physical distance that can educate gene S
3base, does not have interval base in the middle of C and S in 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) by described binary expression vector pS
cproceed in the described common line with genic sterile of paddy rice, by PCR, screen the fertility restorer strain (T obtaining with the common line with genic sterile of paddy rice of color mark gene C
0for transgenic line); In the fertility restorer strain that the present invention cultivates, because importing has, can educate gene S, be dominant and can educate gene S to sterile gene s, so T
0for transgenic line and offspring's strain thereof, will recover fertility; Again due to 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 by linkage inheritance, therefore gather in the crops after this fertility restorer strain seed, can be according to setting percentage and color mark auxiliary PCR detected result, by self propagated constantly (generally by the self propagated in 2～5 generations), can obtain stable fertility, setting percentage is high and (by test cross, verifies that its genotype is S with the homozygous lines of color mark gene C
cs
c);
(5) by described homozygous lines, (genotype is S
cs
c) backcross with common line with genic sterile (ss), according to whether fluorescing or PCR detects and determines whether backcross progeny carries color mark gene C, and by the copy number of southern blot detection backcross progeny color mark gene, obtain engineering maintenance line (the genotype sS that single copy inserts
c).
In 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 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 is only subject to the control of a pair of recessive nuclear gene, and fertility is not affected by environment, therefore more easily selects sterile rate and sterile strain and be all 100% common line with genic sterile.
Above-mentioned method of cultivation, in described step (3), described binary expression vector is preferably plant binary expression vector, is specifically preferably any one (being particularly preferably pCAMBIA1300 or pCAMBIA1390) in pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301, pBI121.
Above-mentioned method of cultivation, in described step (3), color mark gene C can be expressed in seed, makes seed with color mark, so that look is selected the seed with this color mark gene in subsequent applications.Described color mark gene is preferably 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
eGFPor blue fluorescent protein gene (GenBank:JX445134.1)
eBFP(GenBank:EF030494.1), red fluorescent protein gene more preferably wherein
dsRedor green fluorescence protein gene
eGFP.Expressive site according to described color mark gene C in seed, can select suitable specific expressing promoter, and for example, color mark gene C is expressed and just selected endosperm specificity expression promoter at endosperm, and endosperm specificity promoter has: P
gt1and P (GenBank:EU264103.1)
gluB1(GenBank:AY427569.1 or GenBank:JN389781.1).
In 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 on NCBI according to accession number, the primer in described step (2) is according to corresponding gene sequences design).
As a total technical conceive, the present invention also provides the application in the common line with genic sterile breeding of paddy rice of engineering maintenance line that a kind of above-mentioned method of cultivation obtains, and 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 by be divided into from educated gene S and color mark gene C import after 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 color mark gene C and can educate gene S; While breeding common line with genic sterile, the common line with genic sterile hybridization of the paddy rice that is ss by this project maintenance line and genotype obtains the common line with genic sterile seed of engineering maintenance line seed and paddy rice (ratio of the two is generally 1: 1 left and right) in filial generation simultaneously; Now, color mark gene C, under seed-specific expression promoters driven, is expressed specifically in engineering maintenance line seed, by color selector, can by described engineering maintenance line seed separation out, according to colour sorting genotype out, be sS
cengineering maintenance line seed, the common line with genic sterile hybridization of paddy rice that can to continue with genotype be ss, 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, not containing transgene component, can, with restorer hybridization for hybrid seeding, can be also sS with genotype
cheterozygosis seed hybridization, for breeding and the continuity of self material.
Compared with prior art, the invention has the advantages that:
(1) by transgenosis means initiative engineering maintenance line, breed the common line with genic sterile of paddy rice, the common line with genic sterile of paddy rice that breeding obtains is not containing transgene component; And the normal kind of fertility is all the restorer of the common line with genic sterile of this paddy rice, recover spectrum extremely extensive, combo freely, increases the probability of seed selection fine combination; Can open up subspecies indica and japonica hybrid advantage frontier immediately following the seed selection paces of conventional Rice, make rice yield on existing hybrid rice basis, realize more high yield target;
(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 than bilinear method many an engineering maintenance line, this project maintenance line does not need extra breeding, in breeding paddy rice common line with genic sterile, can select engineering maintenance line by machine look;
(3) intelligent sorting, easy to operate: to adopt machine intelligence sorting to replace Artificial Control, for the extensive mechanize production of hybrid seeds provides possibility.
Accompanying drawing explanation
Fig. 1 is the process flow sheet of common line with genic sterile breeding in the embodiment of the present invention.
Fig. 2 is the endosperm-specific red fluorescent protein expression vector p building in the embodiment of the present invention
tDR dsRed .
Fig. 3 be in the embodiment of the present 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 amixia signal.
Embodiment
Below in conjunction with Figure of description, the invention will be further described with concrete preferred embodiment, but protection domain not thereby limiting the invention.
embodiment:
Paddy rice
tDRthe albumen of (Tapetum Degradation Retardation) genes encoding is controlled the degraded of paddy rice suede adhesion coating, is the important factor in rice tapetum growth and degrading and regulating network.If
tDRgene is undergone mutation, and Rice Anther suede adhesion coating will be degenerated, and cause paddy pollen abortion, produces the common line with genic sterile of a kind of paddy rice (referring to CN1884541A Chinese patent literature).
The application of the engineering maintenance line of the common line with genic sterile 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 the present embodiment
tdrthe common line with genic sterile of gene mutation body, specifically comprises the following steps (technical process can referring to Fig. 1):
1.
60co-gamma-ray and mutagenesis, obtains a strain paddy rice sterile mutant, according to genetic analysis, shows that this sterile proterties is by a recessive single-gene (being sterile gene) control, 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 core 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 core sterile mutant of paddy rice list
| Genic male sterile gene | The albumen of corresponding fertile gene coding | Corresponding fertile gene function | NCBI accession number | RiceGE mutant subscription number |
| msp1 | LRR receptoroid kinases LRRkinas | Sporule early development | AB103395.1 | PFG_3A-07135.R |
| pair1 | Coiled-coil domain protein | Homologous chromosomes joint conference | AB158462.1 | FL009505 |
| pair2 | HORMA domain protein | Homologous chromosomes joint conference | AB109238.1 | RMD_TTL-04Z11KJ91 |
| zep1 | 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 | Kinesin family protein | Microgamete reduction division dynamic change | BK007977.1 | T07702T |
| tdr | BHLH transcription factor | Tapetum degradation | AK106761.1 | PFG_3A-08673.R |
| udt1 | 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 | 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 | 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 | 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 | 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 | 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 sequences Design of gene:
TDR-F:ATGGGAAGAGGAGACCACCTGCTGA；
TDR-R: TCAATCAAACGCGAGGTAATGCAGG；
According to the primer of design, clone the common line with genic sterile of above-mentioned paddy rice source parent's allelotrope, i.e. wild type gene
tDR, due to sterile gene
tdrfrom wild type gene
tDRsudden change obtains, therefore, and this wild type gene
tDRcan recover the educated gene of the common line with genic sterile fertility of the present embodiment paddy rice; Wild type gene
tDRwith himself promoters driven.
4. by red fluorescent protein gene
dsRedwith above-mentioned
tDRgene is connected on same binary expression vector, and the skeleton carrier that the present embodiment is selected is pCAMBIA1300, makes 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) by described binary expression vector p
tDR dsRed proceed in the common line with genic sterile of above-mentioned paddy rice, according to
dsRedprimers DsRed-F:CAACACCGTGAAGCTGAAGG; DsRed-R:CTACAGGAACAGGTGGTGGC is to T
0for transgenic line, carry out PCR detection, screening obtains 17 strains with red fluorescent protein gene
dsRedthe fertility restorer strain (T of the common line with genic sterile of paddy rice
0for transgenic line); In the 17 strain fertility restorer strains that the present embodiment is cultivated, because having, importing can educate gene
tDR, and can educate gene
tDRto sterile gene
tdrfor dominant, so this 17 strain T
0for transgenic line and offspring's strain thereof, will recover fertility; Again due to 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 by linkage inheritance, therefore gather in the crops after this fertility restorer strain seed, by self propagated (the 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 red fluorescent protein gene
dsRedhomozygous lines (genotype is
tDR dsRed / TDR dsRed ).
By 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 red fluorescence marker gene in recurrent parent and above-mentioned steps 5 (
tDR dsRed / TDR dsRed ) backcross, staying and select the seed of sending out red fluorescence in 4 individual plants, and by southern blot, detect the copy number of 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), by 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 strain (
tdr/tdr) column cap carry out test cross, this 5 strain sterile mutant can be normally solid, average setting percentage is 94.3%, this explanation genotype is
tdr/TDR dsRed heterozygosis seed can recover the common line with genic sterile of the present embodiment paddy rice (
tdr/tdr) fertility, so genotype is
tdr/TDR dsRed heterozygosis seed can be described as
tdrthe engineering maintenance line of the common line with genic sterile of the present embodiment paddy rice of sudden change.
7. a minute individual plant is gathered in the crops above-mentioned 5 pnca gene types and is
tdr/tdrthe solid gained seed of the common line with genic sterile test cross of paddy rice, according to fluorescence color, select each single-strain seed, through card square analysis, the seed of issue of bidding documents note red fluorescence and the seed rate of not sending out mark fluorescent are 1: 1(sees the following form 2), issue of bidding documents remembers that the seed cdna type of red fluorescence is
tdr/TDR dsRed , the seed cdna type of not sending out mark fluorescent is
tdr/tdr, therefore, genotype is
tdr/TDR dsRed seed as engineering maintenance line, can continue on for the breeding of the common line with genic sterile of paddy rice next time, and genotype is
tdr/tdrseed be the seed of the required common line with genic sterile of paddy rice making of the present embodiment application.
Table 2: seed fluorescence statistic analysis result
| Sterile strain numbering | Individual plant is grain number (grain) effectively | Send out red fluorescence (grain) | Do not send out red fluorescence (grain) | Fluoresce: do not fluoresce | |
| 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 the ratio of 1: 1.
Engineering maintenance line with 674 not fluorescent seeds that in above-mentioned application, sterile strain S1 breeding obtains as the common line with genic sterile of the present embodiment paddy rice and the present embodiment
tdr/TDR dsRed carry out small-scale hybrid seeding.From 674 not fluorescent seeds, select after 500 normal seed germination seedling of full grains color, transplant 400 strains to land for growing field crops, every 8 strain a line, plant 50 row, as female parent.Maternal surrounding is transplanted 2 row engineering maintenance lines as male parent.Divide seedling stage individual plant to get maternal blade, carry after DNA, use
dsRedthe primer DsRed-F:CAACACCGTGAAGCTGAAGG of gene order design; DsRed-R:CTACAGGAACAGGTGGTGGC carries out PCR detection, and 400 strain female parents all do not detect the object band of 556bp, and engineering maintenance line can detect the object band of 556bp, and mark fluorescent is sent out in this explanation and genotype is
tdr/TDR dsRed engineering maintenance line seed is corresponding, does not send out mark fluorescent and with genotype is
tdr/tdrthe seed of common line with genic sterile corresponding.Before heading flowering, by plastics film isolation production of hybrid seeds field, prevent from altering powder.After Grain Ripening, dry and weigh, 400 strain female parents obtain 13.63kg seed altogether.After look choosing is separated, obtain the fluorescigenic seed of 6.95kg, can be used as engineering maintenance line for breeding the common line with genic sterile in lower season.The remaining not fluorescent seed of 6.68kg is that genotype is
tdr/tdrline with genic sterile seed.Visible, according to the inventive method, with 400 common line with genic sterile seeds of paddy rice, just can breed and obtain the above-mentioned common line with genic sterile seed of 6.68kg.
<110> Hunan Research Centre for Hybrid Rice
The method of cultivation of <120> 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 (8)
1. a method of cultivation for Rice Engineering maintenance line, comprises the following steps:
(1) prepare the common line with genic sterile of paddy rice that genotype is ss, by adopting the method for map based cloning to locate and clone the sterile gene s of the common line with genic sterile of described paddy rice;
(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; Can educate gene S with himself promoters driven, the sequence of the front 2kb that promotor is coding region;
(3) color mark gene C and above-mentioned educated gene S are connected on same binary expression vector, make color mark gene C and can educate gene S energy linkage inheritance after importing plant, and color mark gene C does not have interval base in the middle of gene S with can educate, chain is 100%, both be divided into from, obtain binary expression vector pS
c;
(4) adopt existing transgenic method by described binary expression vector pS
cproceed in the described common line with genic sterile of paddy rice, finally obtain the fertility restorer strain S of the common line with genic sterile of paddy rice
cs
c;
(5) homozygous lines of described fertility restorer strain and common line with genic sterile ss are backcrossed, acquisition genotype is sS
cthe strain that single copy inserts, is engineering maintenance line;
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.
2. method of cultivation according to claim 1, is characterized in that: in described step (3), described binary expression vector is plant binary expression vector, is specially pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301 or pBI121.
3. method of cultivation according to claim 2, is characterized in that: described plant binary expression vector is pCAMBIA1300 or pCAMBIA1390.
4. method of cultivation according to claim 1, is characterized in that: in described step (3), described color mark gene is red fluorescent protein gene
dsRed, red fluorescent protein marker gene
rFP, green fluorescence protein gene
gFP, green fluorescence protein gene
eGFPor blue fluorescent protein gene
eBFP.
5. method of cultivation according to claim 4, is characterized in that: described color mark gene is red fluorescent protein gene
dsRedor green fluorescence protein gene
eGFP.
6. according to the method for cultivation described in any one in claim 1～5, it 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.
7. according to the method for cultivation described in any one in claim 1～5, it is characterized in that: the transgenic method in described step (4) is agrobacterium-mediated transformation or via Particle Bombardment Transformation method.
8. the application of the engineering maintenance line that method of cultivation obtains as described in any one in claim 1～7 in the common line with genic sterile breeding of paddy rice, 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 color mark gene C with can educate gene S, when the common line with genic sterile of breeding paddy rice, the common line with genic sterile hybridization of the paddy rice that is ss by this project maintenance line and genotype, in filial generation, obtain the common line with genic sterile seed of engineering maintenance line seed and paddy rice simultaneously, again by color selector by described engineering maintenance line seed separation out, remaining seed is the common line with genic sterile seed of paddy rice that genotype is ss.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210426678.7A CN102876711B (en) | 2012-10-31 | 2012-10-31 | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210426678.7A CN102876711B (en) | 2012-10-31 | 2012-10-31 | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102876711A CN102876711A (en) | 2013-01-16 |
| CN102876711B true CN102876711B (en) | 2014-03-05 |
Family
ID=47478226
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210426678.7A Active CN102876711B (en) | 2012-10-31 | 2012-10-31 | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102876711B (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805630A (en) * | 2012-11-12 | 2014-05-21 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Novel plant fertility regulation structure and application thereof |
| CN104004775B (en) * | 2013-02-26 | 2018-08-28 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | One sterility changing gene and its application |
| WO2014154141A1 (en) * | 2013-03-29 | 2014-10-02 | 湖南杂交水稻研究中心 | Mechanized seed production method using female-sterile hybrid rice plants |
| CN105660361A (en) * | 2016-01-11 | 2016-06-15 | 湖南杂交水稻研究中心 | Breeding method of rice common genic male sterile line |
| CN106544358A (en) * | 2016-11-25 | 2017-03-29 | 湖南杂交水稻研究中心 | A kind of propagation method of the common line with genic sterile of Oryza sativa L. |
| 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 |
| 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 |
| CN108949817A (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 MTR1 gene |
| CN108841859A (en) * | 2018-07-04 | 2018-11-20 | 青岛袁策集团有限公司 | A kind of breeding method of the transgenic paddy rice sterile line based on MSP1 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 |
| CN109652353A (en) * | 2018-12-24 | 2019-04-19 | 青岛袁策集团有限公司 | A kind of construction method of engineered strain |
| CN110331161B (en) * | 2019-07-31 | 2021-04-02 | 湖南杂交水稻研究中心 | Method for improving color selection precision of rice genetic engineering genic male sterile line seeds by utilizing dominant black glume characters |
| CN111543308A (en) * | 2020-05-06 | 2020-08-18 | 中国农业科学院蜜蜂研究所 | Preparation method of pollen for pollination |
| CN111575313A (en) * | 2020-05-13 | 2020-08-25 | 江西省超级水稻研究发展中心 | Method for performing site-directed mutagenesis and detection on rice TDR gene by using CRISPR \ Cas9 system |
| CN112314429B (en) * | 2020-10-29 | 2023-03-28 | 海南波莲水稻基因科技有限公司 | Breeding method of rice nuclear male sterility maintainer line |
| CN113817768A (en) * | 2021-09-13 | 2021-12-21 | 湖南杂交水稻研究中心 | Method for improving rice temperature-sensitive sterile line, application and recombinant vector |
| CN114773442B (en) * | 2022-03-24 | 2023-03-24 | 中国农业科学院农业基因组研究所 | Rice male low-temperature-sensitive sterile line created based on AGO1d gene editing and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100529078C (en) * | 2006-06-08 | 2009-08-19 | 上海交通大学 | Protein coding sequence for controlling rice tapetum degradation |
-
2012
- 2012-10-31 CN CN201210426678.7A patent/CN102876711B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100529078C (en) * | 2006-06-08 | 2009-08-19 | 上海交通大学 | Protein coding sequence for controlling rice tapetum degradation |
Non-Patent Citations (4)
| Title |
|---|
| Li N et al.The Rice Tapetum Degeneration Retardation Gene Is Required for Tapetum Degradation and Anther Development.《The plant Cell》.2006,第18卷(第11期),2999-3014. |
| The Rice Tapetum Degeneration Retardation Gene Is Required for Tapetum Degradation and Anther Development;Li N et al;《The plant Cell》;20061130;第18卷(第11期);2999-3014 * |
| 李宏业等.雄性不育及育性恢复基因表达载体的构建.《热带作物学报》.1999,第20卷(第2期),53-56. |
| 雄性不育及育性恢复基因表达载体的构建;李宏业等;《热带作物学报》;19990630;第20卷(第2期);53-56 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102876711A (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102876711B (en) | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line | |
| CN106544358A (en) | A kind of propagation method of the common line with genic sterile of Oryza sativa L. | |
| CN102870670B (en) | Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice | |
| WO2014187312A1 (en) | Establishment of maintainer plant line and sterile line and use thereof | |
| WO2020248969A1 (en) | Male sterility maintainer line plant and use thereof | |
| WO2015035951A1 (en) | Use of genic male sterility gene and mutation thereof in hybridization | |
| Yang et al. | Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis | |
| WO2021104220A1 (en) | Expression regulation of pollen competitiveness genes stk1; 2 and application thereof in improving efficiency of propogating plant nucleus male-sterile line | |
| CN102960234B (en) | High-efficiency seed labeling method for propagation of plant male sterile line | |
| Xia et al. | A method for mechanized hybrid rice seed production using female sterile rice | |
| Farinati et al. | Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications | |
| CN102965391B (en) | High-efficiency seed labeling method for propagation of plant male sterile line | |
| Zhou et al. | Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production | |
| CN115918523B (en) | Intelligent sterile line propagation method of millet independent of seed sorting machine | |
| Kumar | Male sterility in vegetables | |
| CN102321644B (en) | Control gene of rice panicle pedicel length and application thereof | |
| WO2022042620A1 (en) | Method for propagating sporophyte recessive nuclear male sterile | |
| CN106318923B (en) | The protein and its gene of a kind of High Temperature Stress down regulation Development of Chloroplasts and application | |
| CN102726285A (en) | Preparation method, seed propageation method and application of seeds of rice male sterile line | |
| CN112195269B (en) | Molecular marker related to rice nuclear male sterility phenotype and application thereof | |
| CN100401878C (en) | Method for improving breeding benefit of three-line hybrid wheat | |
| CN102321633B (en) | Pleiotropic gene for controlling vegetative growth and development of floral organs of rice and application thereof | |
| Li et al. | Construction of a novel female sterility system for hybrid rice | |
| Davies et al. | Development of an activation tagging system for maize | |
| CN106916845B (en) | Method for creating cytoplasmic male sterile line by using kenaf transgenosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |