CN101491211A - Full holding method based on plant dominant genic male sterility - Google Patents
Full holding method based on plant dominant genic male sterility Download PDFInfo
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- CN101491211A CN101491211A CNA2009101032564A CN200910103256A CN101491211A CN 101491211 A CN101491211 A CN 101491211A CN A2009101032564 A CNA2009101032564 A CN A2009101032564A CN 200910103256 A CN200910103256 A CN 200910103256A CN 101491211 A CN101491211 A CN 101491211A
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Abstract
The invention relates to a full maintenance method based on dominant genic male sterile line in a plant. Through the alternate use of a constructed engineering maintainer line A and an engineering maintainer line B, the 100 percent maintenance of the dominant genic male sterility can be simply realized. The method is suitable for the full maintenance of a natural dominant genic male sterile line and an engineering genic male sterile line. The method solves the problem that the production of the hybrid seed through the dominant genic male sterile line needs the artificial removal of a fertile plant in a female parent row; in addition, the full maintenance of the sterile line has to depend on the alternate use of the engineering maintainer line A and the engineering maintainer line B. The characteristic greatly reduces the possibility of the parent material leakage of a corresponding improved breed.
Description
Technical field
The present invention relates to field of plant variety breeding technology, particularly relate to a kind of full holding method of plant dominant genic male sterility.
Background technology
At present, in heterosis utilization, utilize the male sterile line production first generation of hybrid to be still economy, valid approach the most.Therefore, seek, find that excellent male sterile material is all plant breeders' a dream.Wherein, dominant genic male sterile is because hereditary pattern is simple, and sterile The Characters is stable, and sterile proterties is easy to shift, and has huge using value aspect heterosis breeding.So far, finding the dominant genic male sterile material that 20 many cases are natural on the important crops of kind surplus wheat, paddy rice, millet, rape, soybean, wild cabbage, the Chinese cabbage etc. 10.In addition, the dominant genic male sterile germ plasm resource of creating by genetic engineering means is as an approach easily and efficiently, also paid much attention to and commercial applications by vast breeding man.
No matter be natural dominant genic male sterility or gene engineering dominant genic male sterility, the genetic characteristics of its sterile proterties determined after the hybridization of dominant genic male sterility and maintenance line, will occur 50% sterile plant and 50% fertile plant in the filial generation colony.Utilizing the natural dominant genic male sterility production of hybrid seeds, generally is when plant blossom, by artificial means to female parent the educated strain in capable pursue strain and identify and pull out.The problem that this approach faces in actual applications is, the crops that the different plants flowering time successively differs in, the colony many concerning unit production of hybrid seeds area kind plant numbers such as wheat, paddy rice, millets, no matter carry out hybrid seed production still be that the seed production purity angle is considered from cost by manually pulling out the means that can educate strain, all unrealistic.And concerning the gene engineering dominant genic male sterility, means commonly used are to use weed killer herbicide to kill except that educating strain.The problem that this method exists is may bring potential environmental ecology risk after using weed killer herbicide, and the effect of removing extremely that can educate strain is subject to the influence of application process, applying environment weather conditions etc. and causes slipping through the net of fertile plant.Therefore, to tie up to the biggest problem of using in the heterosis breeding be how to realize the maintenance fully of male sterile line to dominant genic male sterile.And up to the present, about how realizing that 100% of dominant genic male sterile remains on both at home and abroad still effectively solved.
Summary of the invention
The object of the present invention is to provide a kind of method that realizes that plant dominant genic male sterility 100% keeps, emphasis is to solve to utilize dominant genic male sterility to produce the problem that hybrid seed need manually be pulled out fertile plant in the maternal row.
Because dominant genic male sterility is with after corresponding maintenance line is hybridized, from the seed that sterile strain is gathered, have 50% will become fertile plant, therefore, technical thought of the present invention is by setting up engineering maintenance line A and engineering maintenance line B, hybridize with dominant genic male sterility in turn with engineering maintenance line A and engineering maintenance line B, all educated strains of not carrying male sterile gene in the filial generation are caused death and the maintenance fully of realization dominant genic male sterility.Barnase albumen from bacillus amyloliquefaciens has extremely strong cytotoxicity, is widely used in the establishment of male sterility line of plants, the raising of disease resistance and the generation of stenospermocarpy etc.Barnase albumen is located the detachable N of being end (1-35 amino acids) and C end (36-110 amino acids) two peptide chains at the 36th Val (valine), and two peptide chains after breaking all do not have any cytotoxicity when single expression.
Intein is the one section amino acid sequence that is present in protein translation product (precursor protein) reading frame, changes in the process of maturation protein at precursor protein, and by trans-splicing, intein is sheared release and is free on outside the maturation protein.This trans-splicing process does not need the participation of specific cellular environment and any co-factor, and can carry out external.From the N end of the Ssp DnaE intein of Synechocystis sp.PCC6803 (cytoalgae) (123 amino acid residues: Int-N) and the C end (36 amino acid residues: Int-C) montage domain code area by the genome sequence of 750Kb separately.But these two the protein translation products that contain intein N end (DnaE-N/Int-N) and C end (Int-C/DnaE-C) montage domain respectively still can be finished the complete DnaE albumen of being spliced to form of extein (exteins) DnaE-N and DnaE-C by Int-N and the intersegmental identification of Int-C intein sheet.Based on this characteristic, aspects such as Ssp DnaE intein detects at protein interaction, the structure of cyclisation albumen and enzyme reconstruction have obtained using widely.The present invention promptly is an above-mentioned functions of utilizing Ssp DnaE intein to be had, Barnase albumen n end peptide chain after will breaking in the fertile plant after dominant genic male sterility keeps and C end peptide chain reconnect the Barnase albumen into wild type, cause fertile plant to grow and be obstructed and death, make dominant genic male sterility keep back colony to show 100% male sterile thus.
The present invention realizes by following step successively:
(1) the N end BR-N of Barnase toxalbumin and the N end Int-N of Ssp DnaE intein are merged, obtain mosaic gene, mosaic gene obtains engineering maintenance line A after transforming maintenance line under the driving of constructive expression's promotor CaMV35S, its homozygous genotype is BR-N/Int-N::BR-N/Int-N, and self propagated keeps;
(2) the C end Int-C of Ssp DnaE intein and the C end BR-C of Barnase toxalbumin are merged, obtain mosaic gene, mosaic gene obtains engineering maintenance line B after importing the same maintenance line of step (1) under the driving of CaMV35S promotor, its homozygous genotype is Int-C/BR-C::Int-C/BR-C, and self propagated keeps;
(3) with engineering maintenance line A and the hybridization of dominant genic male sterile plant, the intragroup sterile plant genotype of filial generation is MS::BR-N/Int-N, this sterile plant hybridizes with engineering maintenance line B again, sterile plant and two kinds of individualities of fertile plant appear in filial generation, and genotype is respectively MS::Int-C/BR-C and BR-N/Int-N::Int-C/BR-C; BR-N/Int-N::Int-C/BR-C genotype wherein is individual has recovered the 26S Proteasome Structure and Function of Barnase wild type toxalbumin because the montage of Int-N and Int-C intein reconnects BR-N and BR-C polypeptide, causing these can educate individuality can not normal development and cause death, only remaining genotype is the sterile plant of MS::Int-C/BR-C in the colony, and colony shows as sterile fully;
(4) sterile plant MS::Int-C/BR-C is hybridized maintenance fully with engineering maintenance line A again;
(5) hybridize with the male sterile line after engineering maintenance line A and engineering maintenance line B and the previous generation maintenance in turn, realize that all risk insurance of dominant genic male sterility is held.
In said method step (1), (2):
The N end 1-35 amino acids of BR-N:Barnase toxalbumin;
The C end 36-110 amino acids of BR-C:Barnase toxalbumin;
123 amino acid residues of Int-N:Ssp DnaE intein N end;
Int-C:Ssp DnaE intein C holds 36 amino acid residues;
CaMV35S: cauliflower mosaic virus promoter;
MS is a sterile gene in the step (3).
Barnase gene coded sequence, Ssp DnaE intein coded sequence, CaMV35S promoter sequence related in the said method are known information, all can freely consult in the disclosed intein database of Genebank database and NEB company, employed plant genetic transformation technology is a known technology.
Advantage of the present invention:
(1) by the using in turn of constructed engineering maintenance line A of the present invention and engineering maintenance line B, can realize easily that 100% of dominant genic male sterile keeps.This invention is fit to the maintenance fully of natural dominant genic male sterility and engineering caryon sterile line.
(2) male sterile line in this invention keep fully must rely on using in turn of engineering maintenance line A and two maintenance lines of engineering maintenance line B, these characteristics reduce the parent material of the corresponding improved variety possibility that leaks greatly.
Embodiment
Embodiment 1: all risk insurance of wild cabbage dominant genic male sterility is held
(1) the N end BR-N of Barnase toxalbumin and the N end Int-N of Ssp DnaE intein are merged acquisition BR-N/Int-N mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported the maintenance line of wild cabbage dominant genic male sterility, obtain BR-N/Int-N transgenosis pure lines after the selfing and as engineering maintenance line A;
(2) the C end Int-C of Ssp DnaE intein and the C end BR-C of Barnase toxalbumin are merged acquisition Int-C/BR-C mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported same wild cabbage dominant genic male sterility maintenance line in the step (1), selfing obtains Int-C/BR-C transgenosis pure lines and as engineering maintenance line B;
(3) breeding of engineering maintenance line A system and B system: engineering maintenance line A system and B are tied up to self propagated maintenance respectively in the isolated area.
MS::BR-N/Int-N sterile plant (reservation) and ms::BR-N/Int-N fertile plant (removal) appear in (4) engineering maintenance line A and wild cabbage dominant genic male sterile plant hybridization in the filial generation;
(5) let alone open pollination hybridization in engineering maintenance line B and the MS::BR-N/Int-N sterile plant isolated area, from sterile plant results genotype is MS::Int-C/BR-C sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death), and plantation afterwards colony shows 100% sterile;
(6) plant in 1: 2 ratio in engineering maintenance line A and the MS::Int-C/BR-C sterile plant isolated area, let alone open pollination hybridization when blooming, from sterile plant results genotype is MS::BR-N/Int-N sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death), and plantation afterwards colony shows 100% sterile;
(7) hybridize with the male sterile line after engineering maintenance line A and engineering maintenance line B and the previous generation maintenance in turn, realize that all risk insurance of wild cabbage dominant genic male sterility is held.
Embodiment 2: all risk insurance of paddy rice Pingxiang dominant genic male sterility is held
(1) Barnase toxalbumin N end BR-N and Ssp DnaE intein N end Int-N are merged acquisition BR-N/Int-N mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported the maintenance line of paddy rice Pingxiang dominant genic male sterility, obtain BR-N/Int-N transgenosis pure lines after the selfing and as engineering maintenance line A;
(2) the C end Int-C of Ssp DnaE intein and the C end BR-C of Barnase toxalbumin are merged acquisition Int-C/BR-C mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported the maintenance line of paddy rice Pingxiang dominant genic male sterility, selfing obtains Int-C/BR-C transgenosis pure lines and as engineering maintenance line B;
(3) breeding of engineering maintenance line A system and B system: engineering maintenance line A system and B are tied up to self propagated maintenance respectively in the isolated area;
MS::BR-N/Int-N sterile plant (reservation) and ms::BR-N/Int-N fertile plant (removal) appear in (4) engineering maintenance line A and paddy rice Pingxiang dominant genic male sterile plant hybridization in the filial generation;
(5) hybridization in engineering maintenance line B and the MS::BR-N/Int-N sterile plant isolated area, obtain MS::Int-C/BR-C sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death) from sterile plant, plantation afterwards colony shows 100% sterile;
(6) plant in 1: 2 ratio in engineering maintenance line A and the MS::Int-C/BR-C sterile plant isolated area, catch up with the powder mode to hybridize with bamboo pole when blooming, obtain MS::BR-N/Int-N sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death) from sterile plant, plantation afterwards colony shows 100% sterile;
(7) hybridize with the male sterile line after engineering maintenance line A and engineering maintenance line B and the previous generation maintenance in turn, realize that all risk insurance of paddy rice Pingxiang dominant genic male sterility is held.
Embodiment 3: all risk insurance of tomato dna engineering dominant genic male sterility is held
(1) utilizes the transgenosis means that male sterile gene is imported tomato and obtain the gene engineering dominant genic male sterility;
(2) the N end BR-N of Barnase toxalbumin and the N end Int-N of Ssp DnaE intein are merged acquisition BR-N/Int-N mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported same tomato variety in the step (1), selfing obtains BR-N/Int-N transgenosis pure lines and as engineering maintenance line A;
(3) the C end Int-C of Ssp DnaE intein and the C end BR-C of Barnase toxalbumin are merged acquisition Int-C/BR-C mosaic gene, mosaic gene is with after the CaMV35S promotor is connected, utilize agrobacterium-mediated transformation that it is imported same tomato variety in the step (1), selfing obtains Int-C/BR-C transgenosis pure lines and as engineering maintenance line B;
(4) breeding of engineering maintenance line A system and B system: engineering maintenance line A system and B are tied up to self propagated maintenance respectively in the isolated area;
MS::BR-N/Int-N sterile plant (reservation) and ms::BR-N/Int-N fertile plant (removal) appear in (5) artificial hybridization when engineering maintenance line A and tomato dna engineering dominant genic male sterile plant blossom in the filial generation;
Artificial hybridization when (6) engineering maintenance line B and MS::BR-N/Int-N sterile plant bloom, obtain MS::Int-C/BR-C sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death) from sterile plant, plantation afterwards colony shows 100% sterile;
Artificial hybridization when (7) engineering maintenance line A and MS::Int-C/BR-C sterile plant bloom, obtain MS::BR-N/Int-N sterile plant seed and BR-N/Int-N::Int-C/BR-C fertile plant seed (causing death) from sterile plant, plantation afterwards colony shows 100% sterile;
(8) hybridize with the gene engineering dominant genic male sterility after engineering maintenance line A and B and the previous generation maintenance in turn, realize that all risk insurance of tomato dna engineering dominant genic male sterility is held.
Claims (1)
1, a kind of full holding method based on plant dominant genic male sterility is characterized in that realizing by following step successively:
(1) the N end BR-N of Barnase toxalbumin and the N end Int-N of Ssp DnaE intein are merged, obtain mosaic gene, mosaic gene obtains engineering maintenance line A after transforming maintenance line under the driving of constructive expression's promotor CaMV35S, its homozygous genotype is BR-N/Int-N::BR-N/Int-N, and self propagated keeps;
(2) the C end Int-C of Ssp DnaE intein and the C end BR-C of Barnase toxalbumin are merged, obtain mosaic gene, mosaic gene obtains engineering maintenance line B after importing the same maintenance line of step (1) under the driving of CaMV35S promotor, its homozygous genotype is Int-C/BR-C::Int-C/BR-C, and self propagated keeps;
(3) with engineering maintenance line A and the hybridization of dominant genic male sterile plant, the intragroup sterile plant genotype of filial generation is MS::BR-N/Int-N, this sterile plant hybridizes with engineering maintenance line B again, sterile plant and two kinds of individualities of fertile plant appear in filial generation, and genotype is respectively MS::Int-C/BR-C and BR-N/Int-N::Int-C/BR-C; BR-N/Int-N::Int-C/BR-C genotype wherein is individual has recovered the 26S Proteasome Structure and Function of Barnase wild type toxalbumin because the montage of Int-N and Int-C intein reconnects BR-N and BR-C polypeptide, causing these can educate individuality can not normal development and cause death, only remaining genotype is the sterile plant of MS::Int-C/BR-C in the colony, and colony shows as sterile fully;
(4) sterile plant MS::Int-C/BR-C is hybridized maintenance fully with engineering maintenance line A again;
(5) hybridize with the male sterile line after engineering maintenance line A and engineering maintenance line B and the previous generation maintenance in turn, realize that all risk insurance of dominant genic male sterility is held.
In said method step (1), (2):
The N end 1-35 amino acids of BR-N:Barnase toxalbumin;
The C end 36-110 amino acids of BR-C:Barnase toxalbumin;
123 amino acid residues of Int-N:Ssp DnaE intein N end;
Int-C:Ssp DnaE intein C holds 36 amino acid residues;
CaMV35S: cauliflower mosaic virus promoter;
MS is a sterile gene in the step (3)
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| CNA2009101032564A CN101491211A (en) | 2009-02-25 | 2009-02-25 | Full holding method based on plant dominant genic male sterility |
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| CNA2009101032564A CN101491211A (en) | 2009-02-25 | 2009-02-25 | Full holding method based on plant dominant genic male sterility |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013067928A1 (en) * | 2011-11-08 | 2013-05-16 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Construct for regulating fertility of plant pollens and usage thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013067928A1 (en) * | 2011-11-08 | 2013-05-16 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Construct for regulating fertility of plant pollens and usage thereof |
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Open date: 20090729 |