灰葡萄孢菌一个新的产孢和致病相关基因的鉴定和功能分析

Botrytis cinerea is a typical necrotrphic plant pathogenic fungus,which infects over 200 cultivated plant species and causes grey mold with significant economic losses.In our previous study,a library of T-DNA insertion mutants of B.cinerea was generated through Agrobacterium-mediated transformation.One of these mutants shows a significant decrease in sporulation and complete loss of virulence in tomato.The genome of this mutant contains a single T-DNA copy, analysis of its flanking sequence revealed an ORF encoding a hypothetical protein predicted to contain up to six transmembrane structure. This protein shares no homology with any other known protein. In this proposal, we will investigate the biological function of this novel gene.This proposed project will carry out the following research tasks:Cloning of the full-length cDNA,genetic complement of mutant, screening of interacting protein by using the method of yeast two-hybrid and co-immunoprecipitation, overexpression of the gene of interest, analysis of gene spatiotemporal expression pattern, preparation of recmobinant protein and biochemical characterization, subcellular localization and cytological observation on B.cinerea mutant strain invasion in tomato.The results of this project will improve our understanding of the molecular mechanisms underlying the growth and development of this fungus and its pathogenicity in plants.

灰葡萄孢菌（Botrytis cinerea）是一种典型的死体营养型病原真菌，引起200多种植物的灰霉病，造成严重的经济损失。前期我们利用农杆菌介导法构建了该菌的T-DNA插入突变体库，从中筛选出了一个产孢能力显著减弱且对番茄的致病力丧失的突变体。该突变体中T-DNA为单拷贝插入，插入位点为一预测的含6次跨膜结构的膜蛋白，经Blast分析未发现有已知功能的同源基因，是一个未知功能的新基因。本研究拟对该基因的功能进行鉴定，分析其在灰葡萄孢产孢和致病性中所起的作用及机制。内容包括该基因全长cDNA的克隆、突变体遗传互补、利用酵母双杂交和免疫共沉淀技术查找互作蛋白、基因超表达菌株构建、基因的时空表达分析、重组蛋白制备及其生化分析、目标蛋白的亚细胞定位以及突变体侵染番茄过程的细胞学观察等。根据这些结果，分析该基因功能以及参与的生化或信号途径，以加深对灰葡萄孢生长发育和致病分子机制的认识。

灰葡萄孢菌（Botrytis cinerea）是一种典型的死体营养型病原真菌，引起200多种植物的灰霉病，造成严重的经济损失。本项目主要研究了灰葡萄孢的致病相关基因。在对利用农杆菌介导法构建的该菌的T-DNA插入突变体库的筛选后，从中筛选出的一个产孢能力减弱且对番茄丧失致病力的突变体进行了研究，该突变体中T-DNA为单拷贝插入，插入位点为一预测的跨膜蛋白，初步分析为与酵母的囊泡分泌系统基因sec5有一定的同源性，是一个未知功能的新基因。本研究对该基因的功能进行了鉴定，对其在灰葡萄孢生物学和致病性中所起的作用及机制进行了探讨。发现该基因参与了灰葡萄孢菌的囊泡系统的形成，该基因的缺失影响了物质的运输能力，导致囊泡系统功能缺陷，对菌丝生长、产孢、致病性都产生影响。