摘要
大丽轮枝菌(Verticillium dahliae)侵染引起的棉花黄萎病是棉花生产上的重要病害之一,而其致病的分子机制尚不清楚,为此,以强致病力落叶型黄萎病菌FGH2为材料,利用农杆菌介导转化技术构建棉花黄萎病菌T-DNA插入突变体库并进行致病缺陷突变体的筛选。当农杆菌与黄萎病菌分生孢子浓度比为250∶1、承载介质为NC膜、乙酰丁香酮浓度400μmol/L、共培养时间48h时,转化效率高达每106个分生孢子产生612.5个转化子。通过采用优化后的转化体系,构建了含1 200个T-DNA插入转化子的棉花黄萎病菌突变体库。利用苗期分生孢子蘸根法测定300个转化子在棉花幼苗上的致病力,获得3个致病力显著降低的突变体181、546、1009,其中546的微菌核形成能力丧失,1009的微菌核形成能力明显下降。黄萎病菌T-DNA插入突变体库的构建,为棉花黄萎病菌致病及微菌核形成机制的研究奠定了基础。
The fungus Verticillium dahliae causes cotton Verticillium wilt ,one of the most devas-tating diseases of cotton ,however ,the molecular basis of pathogenicity of V .dahliae is unclear .In this study ,Agrobacterium tumefaciens-mediated transformation（ATMT）was applied for inser-tional mutagenesis of V .dahliae FGH2 ,a highly virulent and defoliating strain .The collection of 1 200 T-DNA random insertion transformants of V .dahliae was generated ,using the optimized cocultivating conditions of V .dahliae conidia and A grobacterium（1∶250 ratio ）for 48 h ,with the AS concentration of 400 μmol/L and the nitrocellulose filter as the filter type in the co-cultiva-tion ,resulting in 612 .5 transformants per 106 conidia .T hree pathogenicity defective V .dahliae mutants were obtained through testing the pathogenicity of 300 T-DNA transformants on the cot-ton variety Yinshan 1 seedling by dipping roots with spore suspension ,including one T-DNA in-sertion mutant losing the ability for forming microsclerotia and one T-DNA insertion mutant de-fective of microsclerotia development capability .The T-DNA insertional mutagenesis library of V . dahliae lays a foundation for researching molecular mechanisms of pathogenicity and microsclero-tia development of V .dahliae .
出处
《河南农业科学》
CSCD
北大核心
2014年第1期69-73,83,共6页
Journal of Henan Agricultural Sciences
基金
国家自然科学基金项目(30700520)
河南省自然科学基金项目(2008A208015)
河南省教育厅科学技术研究重点项目(14A210025)
关键词
棉花黄萎病
大丽轮枝菌
农杆菌介导的遗传转化
T-DNA插入突变
致病性
cotton Verticillium wilt
Verticilliurn dahliae
Agrobacterium tume f aciens-mediated transformation
T-DNA insertional mutagenesis
pathogenicity
