Construction method and application of rice engineering maintainer line

A technology of engineering maintenance system and construction method, which can be applied in the fields of application, botanical equipment and method, introduction of foreign genetic material using vectors, etc. simple effect

Inactive Publication Date: 2015-08-19
HUNAN HYBRID RICE RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Another object of the present invention is to provide a method for constructing a rice engineering maintainer line. The present invention integrates the exogenous reporter gene into the chromosome of wild-type rice through the CRISPR / Cas9 system, which solves the problem of limited transformation recipient materials

Method used

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  • Construction method and application of rice engineering maintainer line
  • Construction method and application of rice engineering maintainer line
  • Construction method and application of rice engineering maintainer line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Various rice tissue culture medium formulations

[0080] 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, adjusted to pH 6.0

[0081] Subculture medium J3: MS massive salt, 10 times B5 trace salt, J3 iron salt FeSO4 ·7H 2 O 41.8mg / 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-D 2.5mg / L, maltose 30g / L, agar powder 8.5g / L, adjust pH 6.0

[0082] 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

[0083] Screening medium J3S: subculture medium J3, cephalosporin 500mg / L, carbapenicillin 400mg / L, hygromycin 50mg / L

[0084] Pre-differentiation me...

Embodiment 2

[0106] Cloning and Ligation of Rice Gt1 Promoter

[0107] Using rice genomic DNA as a template, using primers GT1-F: 5-aaAAGCTTCACCCTCAATATTGG-3 (containing Hind III restriction site) (SEQ ID NO: 5), GT1-R: 5-aaGGATCCGTTGTTGTAGGACTAATG-3 (containing BamH I restriction site site) (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,

[0108] The PCR amplification system and procedures are as follows:

[0109]

[0110] 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.

[0111] The PCR results were detected by electrophoresis, and the target fragment was recovered and ligated with the pMD18-T cloning vector (TaKaRa) at 16°C. The ligation product was transformed into co...

Embodiment 3

[0119] The targeting vector recombinant pP1C.1 and the donor vector pSB130M-Gt1-DsRed-CaMVTerm were co-transformed into wild-type rice (genotype AA) by Agrobacterium-mediated transgenic method.

[0120] Agrobacterium-mediated transformation

[0121] Induction and subculture of rice callus

[0122] 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 ...

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Abstract

The invention discloses a construction method of a rice engineering maintainer line. The method comprises the following steps: 1, integrating an exogenous reporter gene to the chromosome of wild type rice in a site-specific integration manner to obtain a transgenic line containing the exogenous reporter gene, and then executing the step 2, wherein the exogenous reporter gene and a wild type fertility regulatory gene can be subjected to close linkage inheritance; 2, hybridizing the transgenic line obtained in the step 1 with a homozygous recessive rice common nuclear male sterile line, and screening a strain containing the exogenous reporter gene as the needed rice engineering maintainer line, wherein the homozygous recessive rice common nuclear male sterile line is a fertility regulatory gene mutant strain. The invention further discloses application of the rice engineering maintainer line to rice breeding. By integrating the exogenous reporter gene to the chromosome of the wild type rice in the site-specific integration manner, the problem of limitation on transformation of a receptor material is solved. The rice engineering maintainer line is available for rice common recessive nuclear male sterile line propagation.

Description

technical field [0001] The invention relates to a method for constructing a rice engineering maintainer line 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 sterile line seeds. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65A01H1/02A01H5/00
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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