Establishment Of Agrobacterium-mediated Transformation System And Construction Of T-DNA Insertional Mutant Library For Hypomyces Perniciosu

Wet bubble disease is a fungal disease in Agaricus bisporus caused by Hypomyces perniciosa.With the increasing cultivation,the incidence of the disease has increased significantly,especially for the major producing areas in China.There is no effective preventive measures for this disease.So,it is urgent to study about the pathogenesis mechanisms of wet bubble disease,especially for the disease-causing genes.An efficient and stable Agrobacterium-mediated genetic transformation system and mutant library construction are the foundation and effective means to excavate and study pathogenicity related genes,but there is not any study about these.Therefore,we used Hypomyces perniciosa strain WH001 as the research material,established and optimized genetic transformation system mediated by Agrobacterium tumefaciens and used this technology to construct T-DNA insertion mutation library for Hypomyces perniciosa.We acquired a large number of phenotype changed and risk of disease reduced mutant pool.This study also laid a foundation on excavating unknown gene functions of Hypomyces perniciosa,analyzing biological characters,and discussing pathogenic molecular mechanism.The gene functions of Hypomyces perniciosa can be more perfect.Main finding is as below:(1)The freeze-thaw method was applied in the paper to put plasmid DNA of pCAMBIA1302 into Agrobacterium tumefaciens EHA105.PCR identification was used to acquire positive transformant.Agrobacterium engineering bacterium With GFP fluorescent protein was constructed successfully.With the receptor of WH001,genetic transformation technology(ATMT)mediated by Agrobacterium tumefaciens was used for the first time to transform it successfully.In addition,the author discussed primary factors of affecting transformation efficiency and established the high-efficient and stable ATMT transformation system.Results show that the concentration of hygromycin sensibility of Hypomyces perniciosa was 250ng/L.Mycelium pellet was the receptor and hygromycin was the selection marker.HPS gene and CaMV 35 S promoter were used to do PCR identification on the acquired transformant to acquire lots of positive transformants.Fluorescin imager in vivo detected that some transformants generated fluorescence,indicating that GFP gene was expressed in positive transformants.The author further optimized genetic transformation system.When agrobacterium infection concentration OD600 nm was 0.8 and infection time was 45 min,AS concentration in the medium was 1mg/ml.The cultivation time was 2 days.Transformation efficiency reached the maximum—8.78%.These results have laid a good foundation on studying virulence gene functions of WH001.(2)In the study,freeze-thaw method transferred binary vector pBHt1 to Agrobacterium tumefaciens AGL-1.Agrobacterium-mediated method was used to construct T-DNA insertion mutation library of WH001 strain With high virulence.A total of 964 transformants were obtained.11 types of transformants With stable resistance Were selected to do PCR identification and morphological observation.PCR identification results of hygromycin phosphotransferase(hph)showed that T-DNA has been imported WH001 chromosome.Morphological identification results showed that compared with WH001,types of strains reduced growth rate;types of strains increASed growth rate;types of strains became light yellow;types of strains became White;types of strains generated purple pigment;types of strains generated pink pigment;types of strains became radial;types of strains aerial hyphae;types of strains reduced virulence;types of strains reduced sporulation quantity.AS a result,Hypomyces perniciosa mutant strains With reduction of phenotype and virulence were acquired and laid a foundation on using TAIL-PCR technology to identify accurate positioning of insertion sites,and excavating important gene functions and pathogenic molecular mechanism of WH001.