Near-in-situ Complementation Method for Site-directed Insertion Mutation of Fungal Genes

A gene and mutant technology, applied in the field of near-in-situ complementation of fungal gene-directed insertion mutation, can solve problems such as impact, inability to verify gene complementation, and inability to achieve accurate recovery

CN107043778BActive Publication Date: 2021-08-17GUANGXI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2021-08-17

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Abstract

The invention discloses a near-in-situ complementation method for site-directed insertion mutation of fungal genes, in which a modified complementary gene segment and a U6 promoter drive an sgRNA expression cassette carrying a specific recognition sequence of a resistance gene and a T-plasmid of Agrobacterium tumefaciens Integrate and construct Agrobacterium tumefaciens-mediated pathogenic fungal gene site-directed complementation vector with nourthricin as resistance selection marker; use the complementary vector to transform spores of pathogenic fungal gene insertion inactivation mutants, relying on the mutants carried The Cas9 cuts the target site, so that the complementary gene fragment can be accurately inserted into the mutant's resistance gene target sequence. The inventors applied this method to the smut Δmfa2, prf or g827 mutant strains of sugarcane whip smut to achieve accurate site-specific insertion of complementary DNA fragments and restore the function of the target gene. Genomics provides important tools.
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Description

technical field

[0001] The invention belongs to a molecular tool and an experimental system for fungal gene knockout and complementation in the field of microbial genetic engineering, and in particular relates to a near-in-situ complementation method for site-directed insertion mutation of fungal genes. Background technique

[0002] 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 gene. Gene complementation is a technique for artificially introducing the missing gene into a gene deletion mutant so as to restore the function of the gene. Gene inactivation and gene complementation are widely used in the study of gene function.

[0003] Agrobacterium tumefaciens (Agrobac terium tumefaciens) is a Gram-negative Agrobacterium, under natural conditions, it can infect plant cells at t...

Examples

Embodiment Construction

[0034] The near-in-situ complementation method of fungal gene site-directed insertion mutation of the present invention is to express the complementary gene fragment and the U6 promoter that drive the expression of sgRNA carrying the specific recognition sequence of the resistance gene after the base has been changed at the target site but the amino acid sequence is unchanged The cassette is integrated with the T-plasmid of Agrobacterium tumefaciens, and the Agrobacterium tumefaciens-mediated site-directed complementation vector of pathogenic fungal genes is constructed with nourthricin as a resistance selection marker; the complementary vector is used to transform the insertional inactivation mutant of pathogenic fungal genes The spores rely on the Cas9 carried in the gene insertion inactivation mutant to cut the target site, so as to realize the accurate insertion of the complementary gene fragment into the resistance gene target sequence of the mutant. Among them, the comple...