Construction method of Curvularia lunata mutated bacterial strain

Abstract

The invention relates to a method for constructing a mutant strain of maize curvularia leaf spot fungus. The curvularia lunata strain is picked up to be subject to the intermediate culture to prepare the protoplast suspending liquid; colon bacillus strain in cold-preservation containing plasmids is picked up to be subject to the inoculation culture to be extracted plasmid DNA and prepare the enzyme-plasmid mixing liquid; the protoplast suspending liquid and the enzyme-plasmid mixing liquid are evenly mixed and subject to the culture and the filtration to obtain mutant, and the mutant is cultured on PDAs with hygromycin and without hygromycin, and is filtered to obtain the stable mutant strain of maize curvularia leaf spot fungus. The method adopts the restriction Enzyme-mediated gene integration technology to mix the plasmid DNA and the prepared trichoderma protoplast and makes the exogenous linear plasmid fragments inserted in the genome to induce the insertion mutation under the action of the restriction Enzyme-mediated. The mutant strain obtained by the method provides abundant filtrating bacteria sources for the filtration of the weak pathogenic curvularia lunata strain, and can be used to the cloning of genes related to the maize curvularia lunata pathogenicity and the research on the pathogenesis mechanism of the maize curvularia lunata strain.

Description

technical field [0001] The present invention relates to a method for constructing a mutant strain of Curvularia maize leaf spot, in particular to a method for obtaining a mutant strain of Curvularia maize leaf spot through a restriction endonuclease-mediated gene integration technique, which is used for corn bending Screening of weakly pathogenic strains of Phytophthora spp. Background technique [0002] Curvularia Leaf Spot, caused by Curvularia lunata (Wakker) Boed, has caused major damage in most countries of the world. In 1996, the disease caused severe maize loss in China, and 40% of the maize production areas were infected by the disease. During the disease investigation in Liaoning Province in 1998, albino mutant strains of Curvularia maize leaf spot were found, and the pathogenicity of these albino strains was weaker than that of ordinary black strains. These albino strains and wild strains were cultured in PDA medium at 28°C for one week at the same time, and the ...

AI Technical Summary

Problems solved by technology

[0003] The traditional method of selecting disease-resistant varieties to control the occurrence of the disease is an effective way to control the occurrence of the disease, but it is difficult to achieve, because the selection and breeding of a disease-resistant variety often takes a long time, and many studies at home and abroad have proved that The pathogenic bacteria themselves have the phenomenon of pathogenic differentiation, which brings difficulties to the selection of disease-resistant varieties

On the other hand, existing studies have shown that when a disease-resistant variety is planted on a large scale in the same area for many years, it often loses its original disease-resistant ability, which is the result of the variation of the pathogenic strain race.

Current research focuses on the pathogenic bacteria's occurrence regularity, identification of variety resource resistance, and pathogen prevalence. However, there is a lack of in-depth research on the pathogenic differentiation of this pathogenic bacteria.

Although some isoenzymes and DNA polymorphism analysis in the previous work can reflect their relationship with pathogenic differentiation to a certain extent, DNA polymorphism itself cannot fully reflect which functional factors regulate pathogenic bacteria in which way. pathogenic differentiation of

[0004] At present, the method of creating mutations is mainly based on physical and chemical mutagenesis. This method has low mutagenesis efficiency and needs to screen mutants from a large number of strains through a single clone. Cloning mutant genes by methods such as maps is labor-intensive and time-consuming