Construction method of cabbage type rape gene mutant PTG8 and application of construction method

A technology of Brassica napus, construction method, applied in the field of genetic engineering of genetic crops

Active Publication Date: 2019-09-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the technology that the mutation of C5 copy of FAD2 gene and the simultaneous mutation of both copies of A5&C5 can increase the content of oleic acid.

Method used

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  • Construction method of cabbage type rape gene mutant PTG8 and application of construction method
  • Construction method of cabbage type rape gene mutant PTG8 and application of construction method
  • Construction method of cabbage type rape gene mutant PTG8 and application of construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Agrobacterium-mediated screening and verification of high-efficiency genetic transformation receptors for winter rapeseed and establishment and improvement of supporting transformation procedures

[0032] 1. Identification of explant regeneration ability of different strains

[0033] Under the same conditions, the callus induction rate and bud regeneration rate of hypocotyl explants of 13 different lines were quite different, the highest callus induction rate was over 90%, and the lowest was less than 1%. In general, materials with higher callus induction rate also had higher shoot regeneration rate. According to the performance of callus induction rate and bud regeneration rate of each genotype in tissue culture, three varieties (lines) ZP1, Huashuang 5 (bred variety) and Zhongshuang 9 (bred variety) were selected from the above 13 materials. ) for further genetic transformation efficiency analysis.

[0034] 2. Comparison of transformation efficiency of dif...

Embodiment 2

[0053] The acquisition of embodiment 2 mutant material

[0054] S1: Screen target sequences and design primers.

[0055] The nucleotide sequences of Brassica napus BnaC.FAD2.a and BnaA.FAD2.a genes were obtained from NCBI database, see SEQ ID NO:3 and SEQ ID NO:4 respectively. Apply the online software CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) to design a 20bp target sequence, and screen out +424~443bp, 578~597bp two base sites The target sequences are respectively: CGACGCCACCATTCCAACACtgg, see SEQ ID NO:5; ACTTAGCCTTCAACGTCTCGgga, see SEQ ID NO:6. In order to obtain the complete sgRNA sequence, design the forward primer and reverse primer of the target sequence according to the requirements, as shown in Table 1 below.

[0056] Table 1 Target primers

[0057]

[0058] S2: Construction of the dual-target gene editing vector BnPTG8 vector.

[0059] ①Obtain the complete sgRNA sequence by PCR reaction. The PCR system is shown in Table 2, and the primers used...

Embodiment 3

[0146] Example 3 Determination of fatty acid composition in seeds of PTG8-46 and PTG8-116 homozygous mutant plants

[0147] After the mutant plants were obtained in Example 2, the homozygous mutant plants were obtained by selfing.

[0148] The fatty acid composition of mutant seeds was determined as follows.

[0149] Sample preparation:

[0150] 1) Two strains of PTG8-46 and PTG8-116 homozygous mutants, select 3 individual plants for each strain, randomly select about 25 plump seeds for each individual plant, and grind them into powder with a mortar Into a 10mL test tube, the wild-type ZP1 seeds were selected as above.

[0151] 2) Add 1mL of anhydrous diethyl ether: petroleum ether (1:1) mixture and an equal volume of potassium hydroxide-methanol solution (0.5 mol / L) (500ml formaldehyde + 11.2g potassium hydroxide), and let stand at room temperature for 60min.

[0152] 3) Add ultrapure water to 10mL, let stand for 10min, take 500μL-600μL into the sample bottle, and wait for...

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Abstract

The invention belongs to the technical field of genetic crop gene engineering, and particularly relates to a construction method of cabbage type rape gene mutant PTG8 and an application of the construction method. The technical scheme includes that the construction method includes the steps: designing and screening target sequence of two base sites according to gene nucleotide sequences of cabbage type rape BnaC.FAD2.a and BnaA.FAD2.a, and designing primers according to the target sequence; constructing a double-target gene editing BnPTG8 vector; performing genetic transformation on the BnPTG8 vector and acceptor materials to obtain the gene mutant. According to the construction method, a plurality of different sources of cabbage type winter rape strains are screened, an efficient and stable transformation gene acceptor material ZP1 is acquired and accurately edited to obtain a BnaC.FAD2.a single-gene mutant and a double-gene mutant of the BnaC.FAD2.a and the BnaA.FAD2.a, the foundation is laid for building of an efficient winter rape genetic transformation technology platform, and a new germplasm resource is provided for improvement of cabbage type rape fatty acid.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of genetic crops, and in particular relates to a method for constructing a gene mutant PTG8 of Brassica napus and its application. Background technique [0002] The genetic transformation of Brassica napus can be realized through many ways, among which the genetic transformation mediated by Agrobacterium tumefaciens is currently the most commonly used method with the highest transformation efficiency. However, Agrobacterium-mediated transformation methods are affected by many factors. Winter rapeseed is the main type of rape cultivated in my country, but the genetic transformation efficiency of winter rapeseed is low, and there is also a lack of efficient and stable genotypes. Therefore, it is of great application value to screen the transformation genotypes of Brassica napus with excellent agronomic traits and high transformation efficiency, and to establish a complete and efficient ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/53A01H5/00A01H6/20
CPCC12N9/001C12N15/8213C12N15/8247C12N2810/10C12Y103/01035
Inventor 周永明黄会斌范楚川崔婷婷
Owner HUAZHONG AGRI UNIV
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