The creation method and application of rice engineering maintainer line preventing gene drift

A technology of engineering maintainer and gene drift, which is applied in the fields of genetic engineering, application, plant gene improvement, etc. It can solve the problems of transformation receptor material limitation, pollen escape, target gene expression uncertainty, etc., and achieves low cost and convenient operation , The effect of avoiding the risk of genetically modified ecological safety

Inactive Publication Date: 2018-07-06
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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  • The creation method and application of rice engineering maintainer line preventing gene drift
  • The creation method and application of rice engineering maintainer line preventing gene drift
  • The creation method and application of rice engineering maintainer line preventing gene drift

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Various rice tissue culture medium formulations

[0094] Induction medium NB: N6 massive salt, B5 trace salt, N6 iron salt, B5 vitamin, proline 0.5g / L, hydrolyzed casein 0.3g / L, BA 0.1mg / L, sucrose 33.5g / L, agar powder 8.5g / L, adjusted to pH 6.0

[0095] Subculture medium J3: MS massive salt, 10 times B5 trace salt, J3 iron salt FeSO 4 ·7H 2 O 41.8mg / L, Na 2 EDTA 55.9mg / 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-D 2.5mg / L, maltose 30g / L, agar powder 8.5g / L, adjust pH 6.0

[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-D 2.5mg / L, maltose 30g / L, agar powder 8.5g / L, Acetosyringone 0.1mM, adjust pH 5.6

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

[0098] Pre-differentiation medium...

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 BamH I restriction site point) (SEQ ID NO.6), the gene sequence of the Gt1 promoter is obtained by PCR cloning and amplification, and 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 comp...

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 a rice engineering maintainer line that prevents gene drift, comprising: step 1, inserting an exogenous reporter gene and a pollen lethal gene into the flanking sequence of a fertility regulation gene on a wild-type rice chromosome to obtain an exogenous A transgenic line of a reporter gene and a pollen lethal gene, wherein the exogenous reporter gene, the pollen lethal gene and the fertility regulation gene can be closely linked and inherited; The F1 generation plants are obtained by crossing the breeding lines, and then the F1 generation plants are used as male parents to cross the transgenic lines to obtain the F2 generation, and the heterozygous lines containing the exogenous reporter gene are selected from the F2 generation to be the required rice engineering maintainer lines. Wherein, the homozygous recessive common male sterility line of rice is a mutant line of a fertility regulation gene. The invention also discloses the application of the rice engineering maintainer line for preventing gene drift 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 Patents(China)
IPC IPC(8): C12N15/82C12N15/29C12N15/65A01H1/02A01H6/46
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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