Agrobacterium-mediated Leptosphaeria biglobosa genetic transformation method

Abstract

The invention provides an Agrobacterium-mediated leptosphaeria biglobosa genetic transformation method, which belongs to the technical field of genetic transformation of plant pathogenic fungi. The method comprises the following steps: 1) inoculating agrobacterium containing plasmid pCHs-GFP in a LB liquid medium for culture, and then transferring the agrobacterium into a CM liquid medium containing 150-250 [mu]g / mL acetosyringone to obtain agrobacterium bacterial fluid; 2) collecting the conidiospore of the leptosphaeria biglobosa, and mixing the conidiospore with sterile water to prepare a conidiospore suspension having a concentration of (0.5-9)*10<6> / mL; and 3) mixing the agrobacteriumbacteria liquid with the conidiospore suspension, co-cultivating the material for 3-4 days, transferring the material to a screening medium, and screening and culturing the material for 7-15 days to obtain a transformant; The method has high transformation efficiency, low copy and genetic stability.

Description

technical field [0001] The invention belongs to the technical field of genetic transformation of plant pathogenic fungi, and in particular relates to a method for genetic transformation of Rapeseed blackleg fungus mediated by Agrobacterium. Background technique [0002] Rapeseed blackleg (Phoma stem canker) is currently one of the important factors restricting the healthy development of rapeseed industry in many countries. Studies have shown that in many rapeseed producing areas where black shank is endemic in the world, the pathogenic compound species Leptosphaeria maculans and Leptosphaeria biglobosa co-exist and infect host crops alone or together. L.maculans can cause rot at the base of rapeseed stems and cause severe yields loss. The spread and evolution of rapeseed black shank in various countries in the world showed that L.biglobosa was gradually replaced by L.maculans. In 1999, the pathogenic bacteria L.biglobosa was isolated and obtained for the first time in my c...

AI Technical Summary

Problems solved by technology

At present, the widely used fungal transformation methods include plasmid co-transformation, electric shock transformation, gene gun, restriction endonuclease-mediated transformation (REMI) and Agrobacterium-mediated transformation, but plasmid co-transformation, electric shock transformation, gene gun method, restriction Transformation methods such as endonuclease-mediated transformation are prone to problems such as unstable transformants and low transformation efficiency due to the cumbersome operation and poor repeatability of the protoplast preparation and regeneration process.

Although electroporation and biolistic methods can also use spores and mycelia as receptors, which simplifies the operation steps, the transformation efficiency is low, and the genetic stability of transformants is poor.

Images