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SgRNA for editing non-heading Chinese cabbage CENH3 gene and CRISPR/Cas9 vector and application thereof

A head cabbage and gene technology, applied in the field of sgRNA and its CRISPR/Cas9 vector, can solve the problems of low haploid induction efficiency and long period, and achieve the effects of short experimental period, simple operation and easy operation.

Active Publication Date: 2021-10-26
NANJING AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the haploid breeding of non-heading Chinese cabbage mainly relies on plant tissue culture technology to induce haploid plants, and then obtain double haploids through chromosome group doubling, but tissue culture is not suitable for haploid induction of non-heading Chinese cabbage varieties. Low efficiency and long cycle

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  • SgRNA for editing non-heading Chinese cabbage CENH3 gene and CRISPR/Cas9 vector and application thereof
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  • SgRNA for editing non-heading Chinese cabbage CENH3 gene and CRISPR/Cas9 vector and application thereof

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Experimental program
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Embodiment 1

[0051] The test material is non-heading Chinese cabbage variety 'Sijiu Caixin'

[0052] 1. Design of sgRNA and construction of carrier Brcenh3-Cas9

[0053] 1.1 Design and screening of sgRNA in non-heading Chinese cabbage CENH3 gene

[0054] Use CRISPR-p2.0 (http: / / crispr.hzau.edu.cn) online software to analyze the target site of the non-heading Chinese cabbage CENH3 (NC_024803.2) gene sequence downloaded from NCBI, preferably in the exon Sequences with a GC content of not less than 50% and close to the 5' end of the gene coding region are used as target sites; the candidate sg RNA is compared with the genome sequence of Chinese cabbage to minimize the pairing of sg RNA and predicted off-target site sequences The number of bases, the nucleotide sequences of the sgRNA selected at last are:

[0055] sgRNA1: 5'-AGAAGAGGAGGCAGCCAAGG-3' (SEQ ID NO.1)

[0056] sgRNA2: 5'-GACTGCTTCATCTTCGGCGG-3' (SEQ ID NO.2)

[0057] 1.2 Construction of Brcenh3-sgRNA fragment

[0058] The first...

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Abstract

The invention discloses sgRNA for editing a non-heading Chinese cabbage CENH3 gene and a CRISPR / Cas9 vector and application thereof. According to the invention, the sgRNA of the non-heading Chinese cabbage CENH3 gene is designed, the CRISPR / Cas9 gene editing vector is constructed, the CRISPR / Cas9 vector connected with a target DNA fragment is transferred into a non-heading Chinese cabbage genome, and site-directed mutation of the non-heading Chinese cabbage CENH3 gene is achieved through gene editing. According to the invention, the endogenous gene CENH3 of the non-heading Chinese cabbage is edited by combining a pollen nano magnetic transformation technology with a CRISPR / Cas9 technology, a CENH3 haploid induction system is obtained through plant resistance screening, PCR detection and gene sequencing screening, and a foundation is laid for obtaining haploids by utilizing a CENH3-mediated chromosome elimination mechanism in non-heading Chinese cabbage double haploid breeding.

Description

technical field [0001] The invention relates to the field of plant transgenic technology and the field of crop genetics and breeding, in particular to sgRNA for editing the CENH3 gene of non-heading Chinese cabbage and its CRISPR / Cas9 vector and application. Background technique [0002] Non-heading Chinese cabbage (Brassica rapa ssp.chinensis) is a cross-pollinated crop with significant heterosis. The key to utilizing plant heterosis is to select homozygous inbred lines with homozygous genotypes, consistent traits, and simple genetic basis. In cruciferous crops, 7-8 generations of continuous selfing are required to obtain homozygous lines by traditional breeding methods. If haploid plants are obtained, DH (doubled haploid) pure lines can be obtained in one generation after chromosome doubling, which speeds up the breeding process. At present, the haploid breeding of non-heading Chinese cabbage mainly relies on plant tissue culture technology to induce haploid plants, and t...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/55C12N15/29A01H5/02A01H5/00A01H6/20
CPCC12N15/8218C12N15/8207C12N15/8287C07K14/415C12N9/22
Inventor 张昌伟潘尧铧肖栋任一鸣刘同坤侯喜林李英胡春梅王建军
Owner NANJING AGRICULTURAL UNIVERSITY