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Method for obtaining yellow seed germplasm of brassica napus based on CRISPR/Cas9 technology

A cabbage type rapeseed technology and technology, applied in the field of genetic engineering, can solve the problems of high labor intensity, prone to trait separation, long time required for breeding, etc., and achieve the effect of enriching knowledge and broad application prospects.

Pending Publication Date: 2020-02-11
YANGZHOU UNIV
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Problems solved by technology

[0004] Traditional hybrid breeding has made great contributions to improving the quality and yield of rapeseed in my country, but there are still many restrictive factors, such as long time required for breeding, high labor intensity, and can only be carried out with the same species or close relatives. Species hybridization and trait segregation after hybridization are prone to problems, but today's plant genome engineering technology provides more and better options to solve these problems

Method used

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  • Method for obtaining yellow seed germplasm of brassica napus based on CRISPR/Cas9 technology
  • Method for obtaining yellow seed germplasm of brassica napus based on CRISPR/Cas9 technology
  • Method for obtaining yellow seed germplasm of brassica napus based on CRISPR/Cas9 technology

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Embodiment 1

[0034] A method for specifically knocking out the BnMYB123 gene of Brassica napus using the CRISPR-Cas9 system, the method includes the following steps:

[0035] (1) A target site was designed in the conserved region of the two copies of the BnMYB123 gene (BnaA.MYB123 and BnaC.MYB123). The sequence of the target site is shown in SEQ ID NO. 3, and the sgRNA is located at the second of the two copies of the BnMYB123 gene. On the second exon, such as Figure one Shown

[0036] (2) Design and synthesize a pair of single-stranded oligo DNA sequences according to the target site sequence, the sequences of which are shown in SEQ ID NO. 4 and SEQ ID NO. 5 respectively;

[0037] (3) Obtain the sgRNA expression cassette by cutting and joining method;

[0038] (4) Connect the sgRNA expression cassette to the CRISPR-Cas9 vector, and then transform E. coli Trans5α to obtain the plant expression vector BnMYB123-Cas9;

[0039] (5) The BnMYB123-Cas9 vector was transformed into the Brassica napus recep...

Embodiment 2

[0044] Design and synthesis of knockout target sites

[0045] 1. Principles of target site selection:

[0046] (1) The length of the target sequence is 20bp. The 3 bases downstream of the sequence are NGG;

[0047] (2) The high GC% of the target sequence can improve the efficiency of shooting, so the target should preferably contain 11-14 C / G;

[0048] (3) Use the target site sequence to perform blast analysis on the rape genome to avoid off-target.

[0049] 2. Via the online website CRISPR-P( http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR ), a target site was designed in the conserved region of the two copies of BnMYB123 gene (the target site position is as figure 1 Shown), sgRNA is located on the second exon of two copies of BnMYB123 gene, as Figure one Shown.

Embodiment 3

[0051] Construction of knockout vector

[0052] The construction of knockout vector adopts the modified vector pYLCRISPR / Cas9-DH, and the vector construction method is as follows:

[0053] 1. First, synthesize the necessary primers according to the vector construction method as shown in Table 1.

[0054] Table 1 Primers necessary for vector construction

[0055]

[0056] 2. The formation of double-stranded target sequence

[0057] Dissolve the forward and reverse target sequence with TE solution into 100μM mother liquor; take 1μL each of the forward target sequence (SEQ IDNO.4) and reverse target sequence (SEQ ID NO.5) solution, add 98μl 0.5×TE solution and mix well , Prepared into a 1μM working solution; take 10μL of the above mixture into a PCR tube, place it in a PCR machine, heat at 90°C for 30sec, move to room temperature and cool to complete annealing to form a double-stranded target sequence.

[0058] 3. Restriction digestion and linking of gRNA expression cassette

[0059] (1) Th...

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Abstract

The present invention belongs to the technical field of gene engineering and relates to a method for obtaining yellow seed germplasm of brassica napus based on a CRISPR / Cas9 technology. A CRISPR / Cas9system is used for carrying out gene editing on a brassica napus BnMYB123 gene, so that the yellow seed brassica napus with BnMYB123 gene function deletion is obtained. The BnMYB123 has two copies inthe brassica napus, gene coding region sequences of the BnMYB123 are shown as SEQ ID NO:1 and SEQ ID NO:2, and the sequence length is 786 bp. The BnMYB123 can regulate and control synthesis and accumulation of isorhamnetin and epicatechin, thereby regulating and controlling seed coat color of the brassica napus. The method provides a brand-new way for breeding work of the yellow seed germplasm andprovides a theoretical basis for development of the brassica napus yellow seed germplasm.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for obtaining yellow rapeseed germplasm of Brassica napus based on CRISPR / Cas9 technology, and specifically relates to the use of CRISPR / Cas9 system to perform gene editing on Brassica napus BnMYB123, thereby changing the species of Brassica napus The color of the skin, the yellow seed germplasm of Brassica napus was obtained. Background technique [0002] Brassica napus L. (Brassica napus L., 2n=38, AACC) is a Brassica genus of the cruciferous family. It is the second largest oil crop in the world after soybeans. It is widely cultivated all over the world. In view of the important economic status of rape and people's demand for vegetable oil, how to increase the oil content of Brassica napus and improve the quality of rapeseed oil and meal has become an important goal of rape breeding. Compared with brown-seed rapeseed rape, yellow-seed cabbage rape has many advan...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/113C12N15/29A01H5/10A01H6/20
CPCC07K14/415C12N15/113C12N15/8218C12N15/825C12N15/8267C12N2310/20
Inventor 谢涛王幼平蒋金金陈鑫戎浩郭图丽蒋波
Owner YANGZHOU UNIV
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