大丽轮枝菌微菌核发育的分子机理研究

Verticillium dahliae is the primary causal agent of Verticillium wilt and is able to infect many important crops. Because the microsclerotia are the most important structures of the pathogen for survival and causing initial infections, the microsclerotia are regarded as one of the direct targets of control of this disease. But the mechanism of microsclerotial formation and development is unknown. A promoter trap vector carrying GFP gene was used to optimize the Agrobacterium tumefaciens-mediated transformation (ATMT) of V. dahliae. The T-DNA insertion mutant libraries of a sclerotium type, a hyphal type and a intermediate type strain were constructed. Based on these results, in this project we will screen more single copy T-DNA integration mutants those microsclerotial phenotype changed. Combined with the genome sequence database of V. dahliae, microsclerotial development related genes will be cloned and complemented. Then the molecular mechanisms of microsclerotial development related genes will be clarified through the gene function prediction via the nucleic acid sequences analysis and amino acid sequences analysis, studying on the gene expression characteristics when the mutants and wild strains will be cultured in different culture conditions via assay of gene expression level and cellular localization of gene expression products, analysis the relationship between the virulence and microsclerotial development via pathogenicity assays. These results will play an important role on study on occurrence regularity and SPM of Verticillium wilt and provide the targets of the low toxic new fungicide. In addition, this project will help to announcing the law of population genetic variation of microsclerotia development in V. dahliae.

大丽轮枝菌是引起多种重要作物黄萎病的主要土传病原真菌，微菌核是其在土壤中的主要存活结构和初侵染源，也是防治的直接靶标。但微菌核形成发育的机制尚不清楚。本项目在农杆菌介导的双向启动子诱捕载体插入的大丽轮枝菌菌核型、中间型和菌丝型菌株的3个突变体库的基础上，筛选出单拷贝插入的微菌核发育受影响的突变菌株。结合大丽轮枝菌基因组数据库，从这些突变菌株中克隆参与调控微菌核形成发育的基因并进行回补验证。并且通过核酸序列和氨基酸序列的比较分析进行基因功能预测，通过对突变菌株和野生菌株在不同培养条件下进行相关基因实时表达特性分析和表达产物的细胞定位进行表达特性研究，以及通过致病性鉴定分析微菌核发育和致病性的关系，从而阐明微菌核发育相关基因的作用机制。本研究对明确黄萎病流行规律和制定防治措施具重要意义，为开发新型、低毒、环境友好的杀菌剂提供靶标。同时，也将有助于揭示大丽轮枝菌微菌核发育的群体遗传变异规律。

大丽轮枝菌是引起多种重要作物黄萎病的主要土传病原真菌，微菌核是其在土壤中的主要存活结构和初侵染源，也是防治的直接靶标。但微菌核形成发育的机制尚不清楚。通过本项目的研究，用已经建立的农杆菌介导的转化（ATMT）方法，扩建了棉花黄萎病菌T-DNA 插入突变体库，包括大丽轮枝菌菌核型、中间型和菌丝型菌株的3 个突变体库，规模达到9000个突变体。从中筛选获得微菌核发育显著异常的突变体15个，进一步通过Southern杂交，从微菌核发育显著异常的突变体中筛选出7个单拷贝插入的突变体，用Tail PCR方法结合大丽轮枝菌基因组数据库进行比对，明确了其中5个单拷贝突变体T-DNA插入到基因组上的位点。对其中1个单拷贝插入的突变体2H3进行了敲除和回补验证，明确基因VDAG_ 00190.1与微菌核发育有关。通过RACE技术，克隆了VDAG_ 00190.1基因的全长cDNA序列，该基因编码2189个氨基酸组成的conidial yellow pigment biosynthesis polyketide synthase。另外，从V07df2 T-DNA插入突变体库中筛选到致病力显著降低单拷贝插入突变体6C4，发现该突变体中VdNUC-2被破坏。通过RACE技术，克隆了VdNUC-2基因的全长cDNA序列。该基因编码1018个氨基酸组成的蛋白，已有报道该基因是细胞磷酸盐吸收调控的关键基因。该基因突变体在低磷酸盐培养条件下微菌核及分生孢子数量显著减少。通过对其信号途径下游的控制磷酸盐吸收的关键转录因子VdNUC-1的功能分析，以及体外磷酸盐的回补实验，证实VdNUC-2基因分别独立控制致病过程和细胞内磷酸盐平衡过程。亚细胞定位试验表明，该基因在细胞质和细胞核中均有表达。此外，对菌丝型大丽轮枝菌蛋白激酶A的催化亚基基因VdPKAC1进行了功能研究，揭示了其在菌丝生长、微菌核发育、产孢以及调控致病力方面的作用。本研究已经发表研究论文5篇，其中SCI论文2篇，国内核心期刊论文3篇；培养硕士研究生1名。对明确黄萎病流行规律和制定防治措施具重要意义，为开发新型、低毒、环境友好的杀菌剂提供靶标。