棉花黄萎病菌微菌核形成调控途径中关键基因的功能解析

Cotton wilt disease, caused by the phytopathogenic fungus Verticillium dahliae Kleb., is a destructive soil-borne disease threaten to cotton production. The dormant microsclerotia survived in soil are the primary infectious propagules. This project focuses on analysing gene function involved in microsclerotia formation and understanding its intrinsic molecular mechanisms in V. dahliae. In this project, single T-DNA insertional mutants with abnormal microsclerotia formation will be screened from the T-DNA insertional mutant library constructed previously. The flanking sequences of T-DNA insertional sites and the full sequences of mutant genes will be obtained by high-efficiency thermal asymmetric interlaced (TAIL) PCR,Southern blot techniques and bioinformatics. A series of assays will be performed including knockout，genetic complement and over expression of target genes in order to confirm the genes are involved in microsclerotia formation; subcellular location assay and expression profile analysis of relative proteins in order to elucidate biological significance of gene expression involved in microsclerotia formation; pathogenicity and biological characters tests of mutant strains in order to analyze the effect of microsclerotia on V. dahliae. Based on these research, it is helpful for us to study and understand mechanism of microsclerotia formation in Verticillium dahliae Kleb., to explore a new type of effective fungicide or create a new type of method to control cotton wilt disease.

棉花黄萎病是目前棉花生产中最具毁灭性的一种土传病害，主要以微菌核在土壤中长期存活、萌发侵染，是棉花黄萎病的主要初侵染来源。课题以微菌核为研究对象，重点关注微菌核形成的相关基因及其内在分子调控机制。本项目拟利用课题组在前期研究中建立的T-DNA插入突变体库，筛选微菌核形成异常的突变体进行研究，利用Southern杂交、TAIL-PCR技术和生物信息学方法获得单插入突变体的突变基因全长序列；通过基因功能的互补、敲除、和过表达实验，明确微菌核形成调控途径中的关键基因；通过对目标基因的结构分析、编码蛋白产物的亚细胞定位和表达谱的分析结合突变体生物学性状的观察初步明确目标基因表达的生物学意义。本项目的开展有助于解析棉花黄萎病菌微菌核形成的分子调控机理，为探索新型杀菌剂和建立防治黄萎病新策略奠定基础。

棉花黄萎病是目前棉花生产中最具毁灭性的一种土传病害，主要以微菌核在土壤中长期存活、萌发侵染，是棉花黄萎病的主要初侵染来源。课题以微菌核为研究对象，重点关注微菌核形成的内在分子调控机制。本项目利用课题组在前期研究中建立的T-DNA插入突变体库，筛选微菌核形成异常的单拷贝突变体进行研究。在项目执行期内，共鉴定4个跟微菌核形成密切相关的基因，分别是聚酮合酶基因（VdPKS1）、富含丝氨酸蛋白的基因（VdSRP1）、葡聚糖内切酶基因（VdEga1）、土传真菌中保守的未知功能的基因（VdSP1）。其中，VdEga1 和VdSP1的敲除突变体完全不能产生黑色微菌核，互补后可以恢复表型，说明这两个基因编码的蛋白是大丽轮枝菌微菌核形成所必需的。VdPKS1、VdSRP1敲除突变体部分影响了微菌核的形成，其中在VdSRP1的敲除突变体中，微菌核形成的数量明显减少，而VdPKS1的敲除突变体中，微菌核表面没有黑色素的附着，说明黑色素并不是微菌核形成所必需的。四个基因的敲除突变体都共同影响了大丽轮枝菌的致病力，穿透试验表明敲除突变体穿透玻璃纸并没有障碍，因此推测微菌核形成或者调控途径可能与致病途径有重合。本项目的开展有助于解析棉花黄萎病菌微菌核形成和萌发的分子调控机理，为探索新型杀菌剂和建立防治黄萎病新策略奠定基础。