Mutants Coustruction Of Botrytis Cinerea And Gene Cloning

Botrytis cinerea causes grey mold in a wide range of plants and leadsto the significant economic loss. In this study, the method ofAgrobacterium tumefaciens mediated transformation of B.cinerea wasestablished. We coustructed pCAMBIA1390 vector into A.tumefaciens.Thevector carrys the hygromycin B resistance gene as a screening marker.Aftertransformation of B.cinerea spores, a T-DNA insertion mutant library wascreated. Among these mutants, important phenotypes including reductionin growth rate, loss or reduction in conidiation and loss of pathogenicitywere screened out.At first we screened 27 genetic stability mutant strains, according totransformant culture medium in PSA with wild-type B.cinerea, as control.Seven B.cinerea mutant strains with changes in the growth rate, sporeproduction capacity and capability to resistance drug were selected forfurther study. T-DNA Flanking sequence in these transformants wasanalysised with TAIL-PCR. Then, the T-DNA integration sites were testedusing PCR with gene specific primers.The T-DNA flanking sequence of the mutants’molecular technology to Prove. RT-PCRBecause of the insertion of T-DNA in the No.7 mutant, the phenotypeof this mutant was different from the wild type B.cinerea strain.Itsperformances include: slow growth rate, reduced sporulation capacity,sparse mycelium and the capacity of pathogenicity on tomato decreased.After HiTAIL-PCR cloning and analysis of this sequence, we designed aseries of specific primers and carried out PCR detection for confirmation ofT-DNA integration site. Meanwhile, using RT-PCR the gene expressionwas analysised and the loss of gene expression indicates that this gene wasinactivated. After gene Blast, we found this gene corresponding to theT-DNA insertion site has unknown function. The tolerance of mutant toosmotic regulators and fungicides were further studied. With thehygromycin B and cultured 15d the mycelium covered more than half ofthe plate, but could not covered full of the plate. The pathogenicity ofmutant No.7 on tomato leaf has no change. Under the stress of NaCl andfungicide procymidone,the similar phenotype was observed from thewildtype and mutant No.7.