Gene Disruption Dand Characterization Of BcChs? In Botrytis Cinerea

Botrytis cinerea appeared in a wide range of plants causing considerable economic losses to plant production all over the world.In our previous study,the Botrytis cinerea BcChsV gene(Bcchs5)disruption mutant was demonstrated 31%increasing in its chitin content,but the growth and pathogenicity was not affected.Fungal chitin synthase of classes V and VI(or ?),which contain an additional N-terminal myosin motor domain,have been shown to play important roles in pathogenesis.So we will carry out experiments on characterization of BcChs? in Botrytis cinerea.In this study,we constructed BcChs? knockout binary vector pCAMBIA-Bar-chs6 by the Gateway system,through Agrobacterium tumefaciens mediated transformation,obtained 5 stabilized mutants from Botrytis cinerea B05.10.Putative transformants were analysed by PCR and RT-PCR analysis,the results showed that Bcchs6-7 and Bcchs6-8 were the real mutants.In next study,we characterize Bcchs6 mutants via many experiments compare with the wild-type B.cinerea.The radial growth of Bcchs6 mutant exhibited small colonies with very little aerial mycelia.Besides,Bcchs6 mutant failed to form sclerotia on PDA medium.Bcchs6 mutant is hypersensitive to the change of osmotic pressure like cell wall and cell membrane disturbing agents.The chitin content of Bcchs6 mutant was increased 45.5%,and pathogenicity assays on tomato leaves indicated that they were significantly reduced in their ability to cause disease.In addition,the gene complementation of Bcchs6 mutant was studied.We constructed binary vector pCAMBIA-Bar-BcChs? through the recombinant plasmid pETHG-BcChs?,through Agrobacterium tumefaciens mediated transformation,obtained 3 gene complementation strain.It was found that gene complementation strain restore most of characteristics as wild-type.Analysis of them also showed that Bcchs6 is necessary for proper hyphal growth and pathogenicity of B.cinerea on tomato leaves.