致病力丧失的大丽轮枝菌突变体V07_581致病相关基因的鉴定及其功能分析

Verticillium dahliae can cause Verticillium wilt on many economic and horticultural crops.Because the pathogenicity mechanism of V. dahliae is largely unknown, there are no effecetive management strategies for controlling wilt diseases by now. To explicit the pathogenicity mechanism, by using the Agrobacterium tumefaciens-mediated transformation (ATMT), we constructed the T-DNA insertional mutant libraries of V. dahliae and obtained T-DNA insertional mutants. Based on these results, we will choose the mutant V07_581 which has full defects in the pathogenicity, using the method of plasmid rescue to clone the flanking sequence of the T-DNA insertional site of V07_581, and obtain the full lenth of its pathogenicity gene. Combined with the genome sequence database of V. daliae and the method of gene knockout and complementation, the pathogenecity gene will be identified. By constructing site-directed mutants,part of the gene knockout mutants,gene knockout mutants and their corresponding complemented mutants, we will study the effect of different domains of pathogenecity gene on the biological characteristics and pathogenecity of V. dahliae.The gene expression characteristics and cellular localization of gene expression products will be studied via assay of Realtime PCR and GFP fusion expression.These results will play an important role on clarifying the pathogenecity mechanism of Verticillium wilt, providing new ideas for molecular breeding and the targets of the low toxic new fungicides.

大丽轮枝菌严重危害多种重要经济作物和园艺作物。然而目前对其侵染和致病的分子机理仍所知有限，缺乏科学有效的防治方法。为了进一步明确该病原菌的致病机制，申请人构建了农杆菌介导的大丽轮枝菌遗传转化体系及其T-DNA插入突变体库。选取致病力完全丧失的突变体V07_581为研究对象，利用质粒拯救等技术克隆其T-DNA插入位点的侧翼序列，获得致病相关基因的全长序列，结合大丽轮枝菌基因组数据库和基因敲除回补方法，鉴定其致病相关基因；分别构建并获得致病相关基因定点突变、部分基因敲除、全基因敲除的不同突变体和回复突变体，分析致病相关基因不同功能域对大丽轮枝菌生物学特性和致病性的影响；采用Realtime PCR、GFP融合表达技术，研究致病相关基因的实时表达特征及其表达产物的亚细胞定位。预期的研究结果对揭示大丽轮枝菌的致病机制具有重要意义，为抗病育种的选育和新型杀菌剂的研发提供新的思路和靶标。