Biological Function Study On Ergosterol Synthesis-Related Genes Erg4A,Erg4B And Erg4C Of Penicillium Expansum

Penicillium expansum is an important postharvest pathogen,which not only causes a variety of fruits to rot,but also produces patulin in fruits.Ergosterol is a unique component in fungal cytoplasmic membrane,and erg4 is a key gene involved in the final synthesis of ergosterol.However,the biological function of the gene is unknown in P.expansum.In this study,based on the bioinformatics analysis,subcellular localization and gene expression pattern analysis of erg4 family members,gene deletion mutants and complementation strains were obtained by gene knockout technology,then their phenotypes were analyzed.The effects of erg4 gene family on mycelial growth,conidial morphology,virulence,ergosterol synthesis,exogenous stress response and pathogenicity were clarified.The main results are as follows:1.There were three erg4 genes in P.expansum,which were erg4A,erg4B and erg4C,respectively.The full-length genes were 1476 bp,1619 bp and 1762 bp,respectively,encoding 491,496 and 531 amino acids,respectively.The encoded proteins are stable hydrophobic proteins with positive charge and belong to transmembrane proteins.2.The subcellular localization results showed that Erg4A,Erg4B and Erg4C proteins were located in the endoplasmic reticulum.RT-q PCR results showed that the expressions of erg4A,erg4B and erg4C were significantly different at different growth stages,solid and liquid,and dark and blue culture conditions.Among them,the response of erg4B was the most active.3.The gene deletion mutants?erg4A,?erg4B and?erg4C were obtained by homologous recombination,Agrobacterium-mediated transformation and RT-q PCR verification.Replenishment plasmids were inserted into knockout mutants through Agrobacterium-mediated transformation.After screening with G-418 resistant plates and PCR verification,the corresponding replenishment strains?erg4A-C,?erg4B-C and?erg4C-C were obtained.4.Phenotypic analysis results of wild type,mutant and complementary strains showed that erg4A,erg4B and erg4C deletion mutants had significantly lower sporulation ability,clavulin content and ergosterol accumulation than the wild type.In addition,the deletion of erg4B resulted in obvious defects in conidial morphology.In summary,there are three erg4 genes in P.expansum,and their sequence information and encoded protein structure have certain specificity.The Erg4 family proteins were only localized on the endoplasmic reticulum and catalyzed the final synthesis of ergosterol on the endoplasmic reticulum membrane.The erg4B plays a major role in different stages of mycelium growth and development,and can actively respond to changes in O₂ content,erg4C is not sensitive to blue light signal.In addition,erg4B can participate in the morphogenesis of conidia,and the synthesis of patulin and ergosterol is inhibited after the deletion of this gene.