Method for efficiently screening out non-transgenic mutants in agrobacterium tumefacien mediated gene editing

An Agrobacterium-mediated, gene-editing technology, which is applied in genetic engineering, biochemical equipment and methods, and the preparation of test samples, can solve the problems of high technical requirements and low screening efficiency of non-transgenic mutants, and the method is fast Simple, efficient, and popular

Active Publication Date: 2019-12-31
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for efficiently screening non-transgenic mutants in Agrobacterium-medi...

Method used

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  • Method for efficiently screening out non-transgenic mutants in agrobacterium tumefacien mediated gene editing
  • Method for efficiently screening out non-transgenic mutants in agrobacterium tumefacien mediated gene editing

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Embodiment 1

[0024] A method for efficient screening of non-transgenic mutants in Agrobacterium-mediated gene editing, such as figure 1 shown, including the following steps:

[0025] (1) On the T-DNA of the Crispr-cas9-PDS carrier, connect the GUS reporter gene activated by the CaMV 35S promoter, and transfer it into (Yu Yunzhou, Du Juan, Ji Jing. Research on freeze-thaw method of recombinant plasmid introduced into Agrobacterium tumefaciens[ J]. Journal of Jilin Agricultural University, 2003, 25(3): 257-259.) Agrobacterium EH105;

[0026] (2) Infect common tobacco leaves with leaf disc method (Tang Xianbing. Construction of dehydrin gene BDN1 binary expression vector and transformation of tobacco with Agrobacterium-mediated leaf disc method[D]. Capital Normal University, 2001.) to infect common tobacco leaves, OD Value = 0.6 EH105 bacteria solution for 15 minutes, blot the bacteria solution with sterilized filter paper, and inoculate until adding 20 mg·L -1 co-cultured on the MS solid m...

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Abstract

The invention discloses a method for efficiently screening out non-transgenic mutants in agrobacterium tumefacien mediated gene editing, which comprises the following steps: (1) connecting a CaMV 35Spromoter started GUS reporter gene to a traditional gene editing vector, and transferring the vector into an agrobacterium tumefacien EH105; (2) infecting common tobacco leaves with the agrobacteriumtumefacien EH105 transferred into the vector by using a leaf disc method, inoculating the infected leaves onto an MS solid culture medium containing acetosyringone, and co-culturing the leaves for 2 days at 25 DEG C in a dark environment; (3) inoculating a co-cultured explant onto a regeneration culture medium without screening pressure, and replacing the regeneration culture medium with a same fresh regeneration culture medium every 20 days; and (4) inoculating regenerated albino buds onto an MS culture medium containing Timentin, and carrying out sampling for GUS dyeing when albino seedlingsgrow to 3-4 leaves, wherein the albino seedlings without GUS signals are candidate non-transgenic mutants. According to the invention, transgenic mutants can be removed and non-transgenic mutant canbe obtained rapidly, and the method is rapid, convenient, simpler and more practical.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently screening non-transgenic mutants in Agrobacterium-mediated gene editing. Background technique [0002] The traditional Crispr-cas9 technology is to insert the T-DNA with the Crispr system into the recipient genome, and after transcription and translation, use the translated Cas9 protein to edit the target under the guidance of sgRNA. At the same time of editing, the recipient genome is transferred into the Crispr system, which still belongs to the concept of transgene. [0003] It has been confirmed that the T-DNA mediated by Agrobacterium has been translated when it is transferred into the nucleus and not integrated into the genome. It is expressed transiently, and the amount of transient expression is much greater than that of stable expression. Based on this theory, after the T-DNA is transferred into the nucleus, the transiently expressed and...

Claims

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Application Information

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IPC IPC(8): G01N27/447G01N1/28G01N1/30C12N15/82C12N15/65
CPCG01N27/447G01N1/28G01N1/30C12N15/8218C12N15/65
Inventor 陈龙正刘静宁宇
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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