Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification

A soybean and system technology, applied in the field of plant molecular biology, can solve the problems of ZFNs system complexity, time-consuming efficiency, etc., and achieve the effect of high mutation rate and simple system

Inactive Publication Date: 2015-03-25
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first purpose of the present invention is to provide the construction of a soybean CRISPR/Cas9 system, which solves the pr

Method used

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  • Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification
  • Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification
  • Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification

Examples

Experimental program
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Effect test

Embodiment 1

[0037] This example is used to illustrate the construction process of soybean CRISPR / Cas9 system.

[0038] (1) Pick a single colony containing the pCambia3301 plasmid, inoculate it in 50ml LB liquid medium, add kanapenicillin to the medium to a final concentration of 100ng / ml, and culture overnight in a shaker at 37°C and 200 rpm.

[0039](2) Collect colonies, extract pCambia3301 plasmid by alkaline lysis, measure the plasmid concentration by spectrophotometer, and adjust the concentration to 200ng / ul.

[0040] (3) Take 5ug of the pCambia3301 plasmid in step (2), and use NcoI and PmlI enzymes (Fermentas) to double-digest overnight.

[0041] (4) The digested product was electrophoresed on a 0.8% agarose gel, and the large DNA fragment after double digestion of the pCambia3301 plasmid was recovered with a gel purification recovery kit (Tiangen). The concentration of the recovered pCambia3301 large fragment DNA was measured by a spectrophotometer, and the concentration was adjus...

Embodiment 2

[0063] This example is used to illustrate the functional verification of soybean CRISPR / Cas9 system for soybean gene modification.

[0064] Three soybean genes Glyma06g14180, Glyma08g02290 and Glyma12g37050 were selected as research objects, and the primer synthesis sequences are shown in Table 1:

[0065] Table 1: Design of primers for soybean target genes

[0066]

[0067] Since the CRISPR / Cas9 system is constructed based on the core sequence designed for the soybean target gene, the core sequence in the CRISPR / Cas9 system can identify the soybean target gene, thereby providing a location for the modification and editing function of cas9. In Table 1, the italics are the enzyme cleavage site, the bold is the PAM sequence, namely NGG, cas9 cuts from 3-5bp upstream of NGG, so the cleavage happens exactly at the enzyme cleavage site, which provides convenience for later verification experiments Conditions, that is, by using enzyme digestion test, it can be seen whether a mut...

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Abstract

The invention relates to construction of a soybean CRISPR/Cas9 system. The construction disclosed by the invention is mainly characterized in that a core sequence is designed according to a target gene, and the core sequence is constructed into an expression carrier with cas9. The soybean CRISPR/Cas9 system utilizes the core sequence to identify the target gene, the cas9 shears the target gene, and various mutations can be generated in the process of repairing a broken chain by an organism. The invention also provides application of the soybean CRISPR/Cas9 system in soybean gene modification. With the adoption of the construction and the application provided by the invention, the constructed soybean CRISPR/Cas9 system is simple, the soybean gene can be rapidly and efficiently modified, and the problems of the traditional ZFNs system is too complex, wastes time and is low in efficiency are overcome.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and relates to a site-directed mutation system, in particular to the construction of a soybean CRISPR / Cas9 system and its application in soybean gene modification. Background technique [0002] Site-specific modification of genes is one of the most important methods in the field of biological research. With the development of science, more and more silencing techniques are developing rapidly. From classical MES random mutagenesis, T-DNA or transposon insertional inactivation to zinc finger structures (ZFs) and transcription activator-like effector nucleases (TALENs) site-directed mutagenesis, these techniques have greatly facilitated the study of gene function. process. However, zinc finger ribonuclease (ZFN) and transcription activator-like effector nuclease (TALENs) technologies need to design specific endonucleases for each target gene, and the construction process is cumbersome, which...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 胡正孙现军宋国华姜奇彦张辉
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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