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Method for creating rice engineering maintainer lines preventive against gene flow and application of rice engineering maintainer lines

A technology of engineering maintenance line and gene drift, applied in genetic engineering, application, plant genetic improvement and other directions, can solve the problems of pollen escape, uncertainty of target gene expression, limitation of transformation receptor materials, etc., to achieve convenient operation and avoid transgenic Ecological security risk, low cost effect

Inactive Publication Date: 2015-11-18
HUNAN HYBRID RICE RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another object of the present invention is to provide a method for creating a rice engineering maintainer line that prevents gene drift. The present invention integrates an exogenous reporter gene and a pollen lethal gene into the chromosome of wild-type rice through the CRISPR / Cas9 system to solve the problem of transformation problems. The problem of limited body material, the uncertainty of target gene expression caused by random integration sites of previous transgenes, and the problems of pollen escape and transgenic ecological risks are avoided

Method used

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  • Method for creating rice engineering maintainer lines preventive against gene flow and application of rice engineering maintainer lines
  • Method for creating rice engineering maintainer lines preventive against gene flow and application of rice engineering maintainer lines
  • Method for creating rice engineering maintainer lines preventive against gene flow and application of rice engineering maintainer lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Various rice tissue culture medium formulations

[0094] Induction medium NB: N6 large amount of salt, B5 trace salt, N6 iron salt, B5 vitamin, proline 0.5g / L, hydrolyzed casein 0.3g / L, BA0.1mg / L, sucrose 33.5g / L, agar powder 8.5g / L, adjust pH6.0

[0095] Subculture medium J3: MS massive salt, 10 times B5 trace salt, J3 iron salt FeSO 4 ·7H 2 O4 1.8mg / L, Na 2 EDTA55.9mg / L, DL vitamins (glycine 2.0mg / L, thiamine hydrochloride 1.0mg / L, pyridoxine hydrochloride 1.0mg / L, niacin 1.0mg / L, inositol 100mg / L), glutamine Ammonia 0.3g / L, proline 0.5g / L, 2,4-D2.5mg / L, maltose 30g / L, agar powder 8.5g / L, adjust pH6.0

[0096] Co-culture medium NBM: N6 large amount of salt, B5 trace salt, N6 iron salt, B5 vitamin, hydrolyzed casein 0.8g / L, 2,4-D2.5mg / L, maltose 30g / L, agar powder 8.5g / L, Acetosyringone 0.1mM, adjust pH5.6

[0097] Screening medium J3S: subculture medium J3, cephalosporin 500mg / L, carbapenicillin 400mg / L, hygromycin 50mg / L

[0098] Pre-differentiation medium Y: ...

Embodiment 2

[0120] Cloning and Ligation of Rice Gt1 Promoter

[0121] Using rice genomic DNA as a template, using primers GT1-F: 5-aaAAGCTTCACCCTCAATATTGG-3 (containing HindIII restriction site) (SEQ ID NO.5), GT1-R: 5-aaGGATCCGTTGTTGTAGGACTAATG-3 (containing BamHI restriction site) ( SEQ ID NO.6), obtain the gene sequence of the Gt1 promoter by PCR cloning and amplification, the base sequence of the Gt1 promoter is shown in SEQ ID NO.7, wherein,

[0122] The PCR amplification system and procedures are as follows:

[0123]

[0124]

[0125]PCR amplification reaction program: pre-denaturation at 94°C for 5 min; then denaturation at 94°C for 40 s; annealing at 52°C for 40 s; extension at 72°C for 2 min, a total of 35 reaction cycles were performed, and finally 10 min at 72°C.

[0126] The PCR results were detected by electrophoresis, and the target fragment was recovered and ligated with the pMD18-T cloning vector at 16°C. The ligation product was transformed into competent cells Ec...

Embodiment 3

[0143] The targeting vector recombinant pP1C.1 and the donor vector pSB130M-DsRed-ZmAA1 were co-transformed into wild-type rice (genotype AA) by Agrobacterium-mediated transgenic method.

[0144] Agrobacterium-mediated transformation

[0145] Induction and subculture of rice callus

[0146] Select healthy grains and peel off the glumes, and place them in an incubator at 37°C overnight. After the seeds were taken out, put them into a sterilized Erlenmeyer flask, sterilize the surface with ethanol with a volume fraction of 75% for 5 minutes, rinse with sterile water once, disinfect with 0.1% HgCl for 12 minutes, rinse with sterile water for 5 times, and then put Put sodium hypochlorite stock solution for disinfection for 40 minutes, rinse with sterile water for 5 times, dry on sterilized filter paper, and then inoculate on induction medium (NB), and let half of the embryo contact the medium. 20 capsules per dish, cultured in the dark at 25-26°C to induce callus. After 20 days...

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Abstract

The invention discloses a method for creating rice engineering maintainer lines preventive against gene flow. The method includes the steps of 1, site-specifically inserting an exogenous reporter gene and a pollen lethal gene into a flanking sequence of a fertility control gene of a wild rice chromosome so as to obtain a transgenic line containing the exogenous reporter gene and the pollen lethal gene, wherein the exogenous reporter gene, the pollen lethal gene and the fertility control gene are able to closely link and inherit; 2, hybridizing wild rice and homozygous recessive rice common genic male sterile lines to obtain F1-generation plants, hybridizing the F1-generation plants as male parents to the transgenic line to obtain a generation F2, screening the generation F2 to obtain hybrid lines containing the exogenous reporter gene, namely rice engineering maintainer lines required, wherein the homozygous recessive rice common genic male sterile lines are mutant lines with the sterility control gene. The invention further discloses application of the rice engineering maintainer lines preventive against gene flow in rice breeding.

Description

technical field [0001] The invention relates to a method for creating a rice engineering maintainer line for preventing gene drift and its application. Background technique [0002] The utilization of rice heterosis is the most effective way to increase rice yield. The low utilization rate of germplasm resources of "three-line method" hybrid rice and the safety constraints of "two-line method" hybrid rice hybrid seed production are important factors affecting the further expansion of hybrid rice planting area. Inventing new breeding techniques and developing rice heterosis utilization methods with high utilization rate of germplasm resources and safe hybrid seed production are the development direction of hybrid rice. Ordinary recessive genital sterile materials have stable sterility, safe hybrid seed production, and are easy to prepare high-yield, high-quality, multi-resistant combinations; the common disadvantage is that it is impossible to realize mass propagation of ste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29C12N15/65A01H1/02A01H5/00
Inventor 宋书锋李新奇李莉张大兵符习勤袁定阳袁隆平
Owner HUNAN HYBRID RICE RES CENT
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