【作者】 吕琴；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 环境工程， 2006， 博士

【摘要】 稻瘟病是一种严重危害水稻的毁灭性病害，其病原菌为子囊菌Magnaporthe grisea。稻瘟病菌也是一种研究病原真菌-寄主植物相互作用的模式生物。研究稻瘟病菌致病的分子机理不仅对于了解病原真菌-寄主的互作机理，而且对于开发新的杀菌剂具有重要的促进作用。稻瘟病菌致病相关基因的克隆和功能分析是研究稻瘟病菌致病机制的一种快速且有效的途径。非组蛋白基因6(mnh6)和Ⅲ型膜整合蛋白(mtp1)是稻瘟病菌的两个功能未知的基因。目前为止，还没有在稻瘟病菌或其他丝状真菌中研究过mnh6和mtp1基因或其同源基因的功能。本文克隆了mnh6和mtp1基因，通过DNA同源置换分别敲除了这2个基因，并分析了基因缺失引起的稻瘟病菌表型和致病性变化，确定了这2个基因在稻瘟病菌生长、发育和致病过程中的部分功能。主要研究结果如下：1．利用长距离PCR从稻瘟病菌附着胞cDNA文库中克隆了mnh6和mtp1基因的cDNA序列，并从基因组DNA中克隆了mnh6和mtp1基因的全长DNA序列。2．使用多种分析软件，分析和预测了MNH6和MTP1蛋白的结构和功能。MNH6为非组蛋白(DNA结合蛋白)，属于HMGB蛋白家属。MTP1为一种Ⅲ型膜整合蛋白，具有8个跨膜区，可能结合在细胞膜上。3．通过构建基因敲除载体和原生质体转化，分别获得了mnh6缺失或mtp1缺失的基因缺失突变子，并从DNA水平和RNA水平进行了验证。4．突变子△mnh6的菌落形态不同于野生型GUYl1，菌落呈灰白色，菌丝稀疏，生长速度变慢，产孢菌丝细胞壁的溶剂疏水性消失，菌丝细胞壁容易被细胞壁降解酶降解，产孢减少，孢子形态变得细长，孢子的附着胞形成率降低，附着胞的膨压降低，附着胞对寄主植物表面的穿透能力减弱，侵入后生长速度减慢，喷雾接种后对大麦和水稻C039的致病性消失。mnh6基因重新引入△mnh6后，突变子的表型恢复。5．MNH6-GFP融合蛋白主要在细胞核内表达，说明MNH6蛋白的作用位点主要在细胞核内。MNH6在稻瘟病菌致病循环过程中的各个发育时期具有重要作用，如菌丝生长，产孢，附着胞形成，穿透，侵入生长等时期，表明MNH6是稻瘟病菌致病性和完成侵染循环所必需的。6．突变子△mtp1的菌落形态类似于野生型Guyl1，但菌丝颜色较黑，产孢减少，孢子萌发延迟，附着胞形成延迟，离体接种发现突变子对大麦和水稻C039的致病性与野生型一致。突变子△mtp1在各种培养基上的生长速度与野生型没有差异，两者附着胞膨压也没有区别。7．mtp1启动子指导的GFP蛋白主要在稻瘟病菌的菌丝和孢子中表达，附着胞中表达量较少。mtp1基因对于稻瘟病菌的生长没有明显的作用，但对于稻瘟病菌的产孢、孢子萌发具有重要的作用。它对于稻瘟病菌的致病性是非必需的。更多还原

【Abstract】 Rice blast is a severe disease to harm rice and the rice blast fungus is a well-known ascomycete Magnaporthe grisea. This fungal pathogen has been used as a primary model for elucidating various aspects of the pathogen-host interaction with its host. And the results to research the molecular basis of this disease not only help us to understand the pathogen-host plant interaction, but also promote to exploit new bactericide. Identification of pathogenicity-relative genes is a quick and effective step to study the molecular basis of this disease. A nonhistone 6 (MNH6) and a type-III integral membrane protein (MTP1) are two unknown proteins in functions of rice blast fungus. Until now genes, mnh6 and mtp1, or their homologous proteins were studied in rice blast fungus or other filamentous fungi. In this paper, mnh6 and mtp1 genes in M. grisea were cloned and their roles in growth, development and pathogenicity were partly analyzed and identified by targeted gene replacement.The results are showed as following:1. cDNA fragments of mnh6 and mtp1 genes were cloned from M. grisea appressorium cDNA library and full length DNA fragments of these genes were cloned from genomic DNA by LD-PCR. 2. The structures and functions of MNH6 and MTP1 were analyzed and predicted by using several biological soft wares. MNH6 is a nonhistone (DNA combining protein), belonging to HMGB protein family. MTP1 is a type-III integral membrane protein, having eight transmembrane domains.3. Knockout mutants of mnh6 or mtp1 were obtained separately by gene replacement vector construction and fungal transformation. And knockout mutants were identified in DNA and RNA level using PCR, Southern Blot, RT-PCR, and real-time RT-PCR.4. Null mutantΔmnh6 produced appeared offwhite hyphae, showed reduction in growth, conidiation and appressorium formation, and also showed reduction in appressorium turgor pressure. And the hyphae were more readily wettable by a solution containing SDS and EDTA and more easily digested by fungal cell-wall-digesting enzymes. The appressoria produced byΔmnh6 mutants showed diminished host penetration and the infectious hyphae ofΔmnh6 mutants showed reduced in planta growth. Targeted deletion of mnh6 resulted in the greatly reduced virulence capacity to barley and rice CO39 in infection assays.5. MNH6-GFP fusion protein was observed mainly in nucleolus. And this implied MNH6 protein mainly functions in nucleolus. mnh6 plays significant roles in the all-different development events of the fungus globally during its disease cycle such as vegetative growth, conidiation, appressorium formation, penetration, infectious growth and conidiation in diseased tissues in M. grisea. The pleiotropic effects on fungal morphogenesis exhibited byΔmnh6 mutant suggested that mnh6 is required for effective pathogenicity and completing the disease cycle of rice blast fungus.6. Null mutantΔmtp1 showed reduction in conidiation, delayed in germination and appressorium formation, and its hyphae showed blacker than wild type strain. But mutants showed no differences in growth, appressorium turgor, and virulence capacity to barley and rice CO39.7. GFP controlled by mtp1 promotor was expressed mainly in hyphae and spores, less expressed in appressoria. mtp1 has no visible effects on the growth of rive blast fungus. However, it has significant effects on conidiation, conidium germination of the fungus. And, mtp1 is dispensable for pathogenicity of M. grisea.更多还原