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

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.

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...

AI Technical Summary

Problems solved by technology

[0005] So far, in many pathogenic fungi, it is still impossible to carry out effective genetic manipulation by transforming protoplasts, and can only rely on Agrobacterium-mediated transformation to introduce foreign DNA fragments, and Agrobacterium-mediated transformation of foreign DNA fragments has a random Sexuality, unable to perform precise insertion at a specific site

Moreover, in the verification of gene complementation after gene knockout, some species cannot perform gene complementation verification due to the lack of corresponding gene complementation technology; at present, the method of randomly inserting complementary gene fragments is adopted in pathogenic fungi When a complementary fragment is randomly inserted into the DNA coding region of another functional gene, it will affect the function of other genes and cannot achieve the purpose of accurately restoring the function of the knocked-out gene to study the function of the gene

Images