Cloning And Functional Analysis Of VdLac In Verticillium Dahliae

Verticillium dahliae Kleb.is a typical soil-borne pathogenic fungus,and can cause Verticillium wilt on many plant species.Microsclerotia of V.dahliae are main dormant structure that enable V.dahliae to survive up to 14 years in the absence of a host.It also have strong resistance.Melanins are pigment formed by phenolic or indole compounds,also are hydrophobic biopolymers with negatively charge.Melain has a protective role in the host and in the environment.Laccases are the key enzymes of fungal melanin synthesis pathway and play important roles in fungal life history.In this study,the functions of VdLac gene were analyzed by gene knocked out and complemented.The results showed as following:1.According to the published VdLs.17 genome sequence on NCBI,VdLac gene in JY was cloned,and the VdLac complete DNA and cDNA sequences were 1978 bp and 1713 bp in size,respectively,encoding a protein with 570 amino acid.2.VdLac gene was knocked out using Agrobacterium tumifaciens-mediated transformation(ATMT).Four positive knockout mutants were obstained by hygromycin resistance screening and PCR detection.3.VdLac gene was complemented in deletion mutants by PEG-mediated protoplasts transformation.One transformant was obstain by G418 resistance and PCR detection.4.In order to characterize the VdLac function,a series of biological functions were analyzed in deletion and complementation mutants.The results showed that,the deletion mutants were more sensitive to nitrosative stress,and could produce laccase on CM plates,but the production was reduced.The results indicated that VdLac gene was not required for vegetative growth,conidia production,pathogenicity and melanin synthesis.