High-efficiency preparation method for simultaneously preparing multiple member mutants of same gene family of cabbage mustard

A gene family and mutant technology, applied in the field of plant molecular biology, can solve the problems of manpower consumption, low mutation efficiency, and long time, so as to save costs, improve mutation efficiency, and reduce experiment difficulty and intensity.

Active Publication Date: 2018-06-15
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional methods of inducing mutants include spontaneous mutation, somatic clone mutation and physical and chemical induced mutants, all of which can only obtain individuals with single-gene mutations, and the workload is heavy, manpower and resources are consumed, the mutation efficiency is low, and the time is long

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  • High-efficiency preparation method for simultaneously preparing multiple member mutants of same gene family of cabbage mustard
  • High-efficiency preparation method for simultaneously preparing multiple member mutants of same gene family of cabbage mustard
  • High-efficiency preparation method for simultaneously preparing multiple member mutants of same gene family of cabbage mustard

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preparation example Construction

[0035] A method for efficiently preparing mutants of multiple members of the same kale gene family at the same time, comprising: selecting common target sequences of different members of the same kale gene family, and synthesizing a pair of complementary Oligo sequences according to the selected target sequences, Then carry out the ligation and transformation reaction to obtain the recombinant plasmid, which is then digested with the pCC plasmid to recover the target band, and then connect the two target bands to construct the CRISPR / Cas9 expression vector, and finally use the root Genetic transformation method mediated by Agrobacterium carcinoma to obtain transgenic mutant plants. , see the flow chart figure 1 .

[0036] For the preparation of mutants of multiple members of all gene families in kale, the method of the present invention can be used to prepare. Below we will describe the kale PDS gene family as an example. The specific process is as follows:

Embodiment 1

[0037] Example 1 Selection of Brassica oleracea PDSs gene target sequence

[0038] Because in the prior art, the method of knocking out multiple genes in kale plants by using the CRISPR / Cas9 system is mostly to select several different target sites to knock out one by one, which is time-consuming, laborious and costly. However, the present invention provides prerequisites for simultaneously knocking out multiple genes in plants in a subsequent transformation by screening the common target sequences of different members of the same gene family. Therefore, the screening of common target sequences of different members of the same gene family is very important.

[0039] The Kale PDS gene family has two members, BaPDS1 and BaPDS2. Through cloning, the full-length CDS of the two genes, BaPDS1 and BaPDS2, were obtained. Using CRISPR-P (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) online software to analyze the target sites of these two genes and screen the common target sequen...

Embodiment 2

[0042] Example 2 Construction of Kale CRISPR / Cas9 Expression Vector

[0043] The construction process of the kale CRISPR / Cas9 expression vector specifically includes the following steps:

[0044] 1. Target sequence annealing and annealing: According to the selected target sequence, synthesize a pair of complementary Oligo DNA sequences, and add enzyme cutting sites CACC and AAAC to their 5' ends, and then anneal and anneal to obtain Oligo DNA with sticky ends. DNA double-stranded sequence. Among them, the reaction procedure of annealing and renaturation is: denaturation at 95°C for 5 minutes, 30s at 1°C, cooling to 25°C, and storage at 4°C.

[0045] 2. Digestion of the pSG vector: the restriction endonuclease BbsI was used to digest the pSG vector. The digestion process was 37° C. for overnight reaction, followed by 65° C. for 20 minutes, and then the digested product was recovered.

[0046] 3. Ligation and transformation: Ligate the recovered enzyme-digested product with th...

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Abstract

The invention provides a high-efficiency preparation method for simultaneously preparing multiple member mutants of the same gene family of cabbage mustard. The preparation method comprises the following steps: according to the whole length of each member of the gene family of the cabbage mustard, analyzing a target site of each member, and screening a common target sequence; constructing a CRISPR / Cas9 expression vector according to the selected target sequence, then transforming into agrobacterium competent state, infecting explants, and then performing tissue culture on the explants to obtain mutant plants. Multiple genes of the same gene family in a cabbage mustard plant are simultaneously knocked out through once transformation, and a large number of mutant materials can be obtained through mutation of different members, so that the mutation efficiency is improved, the workload is saved, the working time is saved, the problems that in past gene family mutation, multiple vectors arerequired to be designed respectively and the vectors are required to be separately transformed so as to obtain different mono-mutant and multi-mutant materials, induction is long in time and high indifficulty and the like are solved, and thus the advantages of simultaneousness and high efficiency of the preparation method are embodied.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to an efficient preparation method for mutants of multiple members of the same gene family of kale at the same time. Background technique [0002] Molecular biology studies have found that many genes exist in the form of gene families. A gene family refers to a group of related genes with similar sequences in exons, which are produced by duplication and mutation of a common ancestral gene. Members of the same family are sometimes closely arranged together to form a gene cluster; more often, they are scattered in different parts of the same chromosome, or even located on different chromosomes, with different expression regulation modes. With the deepening of research, researchers have found that many genes related to important traits such as yield, quality, and resistance in crops exist in the form of gene families, and there are multiple members in crops...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/20
CPCC12N9/22C12N15/8205C12N15/8213
Inventor 孙勃张芬郑爱红汤浩茹薛生玲江雷雨袁巧江敏陈清张勇罗娅王小蓉王燕刘泽静李梦瑶
Owner SICHUAN AGRI UNIV
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