大丽轮枝菌菌核型菌株T-DNA插入突变体库的构建及微菌核发育异常突变体的筛选

摘要

To illustrate the molecular mechanism underlying microsclerotial development in Verticillium dahliae, agrobacterium tumefaciens-mediated transformation ( ATMT) was applied for insertional mutagenesis of V. dahliae conidia. A T-DNA insertion mutant library of a sclerotium-type strain V08DF1 including 2 000 transformants were constructed. All of the 2 000 transformants cultured on two kinds of medium plates ( Czapek-Dox medium plate and Czapek-Dox plus cotton root extract medium plate, or Czapek-Dox medium plate and PDA medium plate) were observed for colony morphology. 130 mutants with significant difference in colony morphology from V08DF1 were selected, among which 14 showed abnormal mi-crosclerotia development. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that seven mutants out of baove 14 were single-copy T-DNA insertion, five were two-copy T-DNA insertion, and two were three-copy T-DNA insertion.%为阐明大丽轮枝菌微菌核形成发育的分子机理,利用农杆菌介导的遗传转化方法,建立了包含2000个转化子的大丽轮枝菌菌核型菌株V08DF1的T-DNA插入突变体库,并在查氏、查氏-根或PDA培养基上培养,观察各转化子的菌落形态。从中筛选出130个菌落特征与野生型菌株V08DF1有明显差异的突变体菌株,其中14个突变体菌株为微菌核发育受阻。对这14个微菌核发育异常的突变体菌株进行T-DNA插入的PCR验证,均能扩增到潮霉素抗性基因,Southern 杂交显示其中7个为单拷贝插入,5个为双拷贝插入,2个为三拷贝插入,表明T-DNA已经成功整合到这些突变株的基因组DNA 中。