Construction method and application of eggplant CRISPR/Cas9 gene knock-out vector

A gene knockout and construction method technology, applied in the fields of application, horticultural methods, genetic engineering, etc., can solve problems such as the inability to obtain stable and non-exogenous gene insertion, and the inability to knock out genes at a fixed point

Inactive Publication Date: 2018-11-02
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Claims
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Problems solved by technology

[0006] The problem to be solved by the present invention is to provide an eggplant CRISPR/Cas9 gene knockout method aimed at the problems of inability to knock out genes at specifi

Method used

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  • Construction method and application of eggplant CRISPR/Cas9 gene knock-out vector
  • Construction method and application of eggplant CRISPR/Cas9 gene knock-out vector
  • Construction method and application of eggplant CRISPR/Cas9 gene knock-out vector

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Embodiment Construction

[0050] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0051] The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0052] The present invention will be further described below in conjunction with specific embodiments.

[0053] Example Obtaining and Identification of Eggplant SmWRKY26 (Sme2.5_01585.1_g00006.1) Gene Knockout Strain

[0054] 1. Selection of gRNA target site

[0055] According to the principle of CRISPR / Cas9 technology to design the target site, the target site of the present invention is designed on the first exon of the SmWRKY26 gene. See SEQ ID NO: 1, the bold part is the SmWRKY26 exon, and the underlined part is the gRNA target site sequence;

[0056] 2. Design and annealing of gRNA

[0057] 2.1 Taking SmWRKY26 genomic DNA SEQ ID NO:1 as a reference, design the primer sequence for amplifying gRNA, the sequence is as follow...

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Abstract

The invention relates to a construction method and application of an eggplant CRISPR/Cas9 gene knock-out vector, and belongs to the field of a biological technology. The method comprises the followingsteps: designing a target site gRNA by taking genome DNA sequence of an eggplant WRKY26 gene as reference; constructing an expression box of the target site gRNA and Cas9 protein; inserting the expression box of the gRNA and the Cas9 protein into a binary expression vector; transferring recombinant plasmid into an agrobacterium EHA105 strain, mediating by EHA105 and transferring eggplant seed leaves to obtain a genetic transformation plant, and performing PCR and sequencing verification to determine a mutation strain. An eggplant SmWRKY26 gene serves as an example, and the eggplant gene is subjected to geodesic mutation rapidly, simply and efficiently.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a construction method and application of an eggplant CRISPR / Cas9 gene editing vector. Background technique [0002] Eggplant is one of the important solanaceous vegetables. With the publication of eggplant genome data, research on eggplant gene function and modern molecular breeding of eggplant have been developed rapidly. For example, virus-induced gene silencing, RNAi, overexpression and other gene function research methods, as well as modern molecular breeding methods represented by molecular marker breeding, transgenic breeding and molecular design breeding occupy a certain position in eggplant research. However, these methods have some shortcomings: the obtained materials cannot be genetically stable, non-specific knockout, and long breeding cycle. [0003] Genome editing technology has become an important method for gene function research and precise molecular breeding. Commonly us...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113A01H4/00A01H5/00A01H6/82
CPCA01H4/00C12N15/113C12N15/8213C12N2310/10C12N2310/20
Inventor 杨艳庄勇周晓慧刘军
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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