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Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method and its application

A technology of Agrobacterium tumefaciens and genes, applied in biochemical equipment and methods, viruses/bacteriophages, DNA/RNA fragments, etc., can solve problems such as inability to accurately insert specific sites

Active Publication Date: 2021-05-18
GUANGXI UNIV
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Problems solved by technology

[0006] So far, effective genetic manipulation cannot be carried out by transforming protoplasts in S. cane whip. It can only rely on Agrobacterium-mediated transformation to introduce exogenous DNA fragments, and Agrobacterium-mediated transformation of exogenous DNA fragments It is random and cannot be precisely inserted at a specific site

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  • Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method and its application
  • Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method and its application
  • Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method and its application

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[0025]The Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method of the present invention is through the endogenous U6 gene promoter (Ss PU6, the base sequence of SEQ.ID.No.17; Ss U6RNA, the base sequence of SEQ.ID.No.18) was fused with the target sequence of the inserted site and the sgRNA sequence by Overlapping PCR, and recombined with the endogenous gapd gene of Ustilago sativa by In-fusion technology In the binary vector of the Cas9 gene and hygromycin resistance gene driven by the subunit; then Agrobacterium tumefaciens carrying the binary vector mediated the transformation of Ustilago sativa so as to achieve the site-directed insertion of the Ustilago sativa genome .

[0026] In order to further clarify the above inventive concept, the sugarcane smut pheromone gene mfa2 is taken as an example for illustration. The experimental methods in the following examples, unless otherwise specified, are conventional methods; the experi...

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Abstract

The invention discloses a gene-directed insertion inactivation method mediated by Agrobacterium tumefaciens and CRISPR-Cas9, using the endogenous snRNA promoter of Ustilago sativa (U6 gene promoter of Ustilago saccharum) to drive the sgRNA expression cassette , the CRISPR‑Cas9 system was integrated with the Agrobacterium tumefaciens T‑plasmid to construct an Agrobacterium tumefaciens mediated site-directed insertion vector inactivation system for the Ustilago sclerotinum gene using hygromycin as a resistance selection marker; the target gene The specific recognition sequence of the sgRNA expression cassette was cloned into the sgRNA expression cassette, which was used to transform the basidiospores of Ustilago sativa, so that the jumpable DNA fragment of the vector system was accurately inserted into the target gene target sequence of Ustilago sativa, and the purpose of destroying gene function was achieved. The invention provides an important tool for studying the functional genomics of the sugarcane whip smut, the system has high efficiency and accuracy, and is convenient for carrying out gene function research in the sugarcane whip smut.

Description

technical field [0001] The invention belongs to a molecular tool and an experimental system for fungal gene knockout in the field of microbial genetic engineering, and in particular relates to a gene-directed insertion inactivation method mediated by Agrobacterium tumefaciens and CRISPR-Cas9 and its application. Background technique [0002] Sugarcane whip smut (Sporisorium scitamineum) is the pathogenic bacterium of sugarcane smut, which belongs to Basidiomycotina smut. The sugarcane smut caused by it is a global disease, which has caused great harm to sugarcane production in my country. There are three distinct stages in the life cycle of Ustilago sativa: yeast-like haploid, dikaryotic hyphae, and diploid teliospores. [0003] Gene inactivation technology is a genetic engineering technology that changes the specific gene sequence by changing the genetic genes of organisms, resulting in gene inactivation and loss of function, and then deduces the biological function of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/80
CPCC07K14/37C12N15/80C12N2800/30
Inventor 陈保善卢姗吕哲姚颜萍沈笑瑞蔡晓薇
Owner GUANGXI UNIV
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