棉花黄萎病菌几丁质脱乙酰酶基因的克隆与功能分析

Chitin is a major component of fungal cell wall. Fungal cell wall can be damaged when chitin is hydrolyzed by chitinase existed in plant to chitin oligosaccharides. Chitin oligosaccharides are a representative general elicitor inducing defense responses in a wide range of plant cells. Some research results have showed that Chitin deacetylase (CDA) from fungi could modify chitin and allow hyphae to penetrate into plants and escape from plant defense mechanisms. In this project, we will explore the gene function of Chitin deacetylase (CDA) from Verticillium dahliae Kleb causing cotton wilt disease. Three CDA genes of V. dahliae (VdCDA) will be cloned and sequenced; a targeted gene replacement strategy based on homologous recombination will be adopted to generate gene deletion mutant strains including single-deletion strains and dual-deletion strains. Wild type strain served as the control, all kinds of deletion strains will be used to perform a series of assays to determine the biological characterization and biochemistry traits of VdCDA genes, to conform relationship between VdCDA genes and pathogenicity, to analyze the influence of VdCDA to host response. These results will help us to elucidate the functions of the three VdCDA genes when combine with the results of subcellular location assay, expression profile analysis and secreted activity detection of protein encoded by VdCDA genes. Based on these research results, it will be helpful for us to study and understand biological significance of chitin metabolism, to explore a new type of effective fungicide or create a new type of method to control cotton wilt disease.

几丁质是真菌细胞壁的主要组成成分，真菌侵染植物时，植物中的几丁质酶水解几丁质破坏真菌细胞壁结构的完整性，同时产生几丁质寡糖作为激发子激活植物防卫反应。已有研究表明，几丁质脱乙酰化酶（CDA）对几丁质的去乙酰化修饰作用是病原真菌逃避寄主天然防御反应的重要机制。本项目以棉花黄萎病菌为研究对象，对大丽轮枝菌编码几丁质脱乙酰酶的3个基因（VdCDA）进行克隆和结构分析；构建针对3个基因的单基因和双基因敲除突变体，通过对敲除突变体的生物学测定、生化性状测定、致病性鉴定及寄主抗性反应的测定，分析VdCDAs基因表达所决定的生化特性，VdCDAs基因与病原物生物学性状、致病性及寄主抗性反应的相关性，结合VdCDAs基因编码蛋白的分泌性测定、亚细胞定位及VdCDAs基因表达谱分析，以期明确棉花黄萎病菌VdCDAs基因的生物学功能，为棉花抗黄萎病分子育种提供候选基因及开发新型杀菌剂提供靶标奠定理论基础。

几丁质是真菌细胞壁的主要组成成分，真菌侵染植物时，植物中的几丁质酶水解几丁质破坏真菌细胞壁结构的完整性，同时产生几丁质寡糖作为激发子激活植物防卫反应。已有研究表明，几丁质脱乙酰化酶（CDA）对几丁质的去乙酰化修饰作用是病原真菌抵御寄主天然防御反应的重要机制。本研究从棉花黄萎病菌中克隆到3个几丁质脱乙酰酶基因（VdCDA1、VdCDA2、VdCDA3）和1个聚多糖脱乙酰酶基因（VdPDA1），序列分析表明，这4个基因编码的蛋白均具有PDA家族共有的5个保守结构域，可能具有PDA1家族蛋白的酶活性和功能特征。对VdCDAs及VdPDA1的基因功能进行分析，结果显示VdCDA1、VdCDA2和VdCDA3基因是大丽轮枝菌微菌核形成的关键基因；VdCDA1基因敲除突变体和VdCDA3基因敲除突变体均导致大丽轮枝菌产孢梗减少、产孢量下降，表明VdCDA1和VdCDA3基因影响大丽轮枝菌的产孢量；VdCDA1、VdCDA2、VdCDA3和VdPDA1基因分别敲除均导致大丽轮枝菌对棉花的致病力明显减弱，表明这4个基因都是棉花黄萎病菌致病关键基因；分泌性测定结果表明，VdCDA1、VdCDA2、VdCDA3和VdPDA1蛋白均为分泌蛋白；纯化的VdPDA1蛋白具有几丁质脱乙酰酶活性，可将含不同数目GlcNAc的几丁质寡糖进行去乙酰化修饰；VdPDA1基因敲除不影响大丽轮枝菌侵入寄主的能力，将VdPDA1蛋白进行瞬时表达，不会在烟草或棉花叶片上诱导过敏性坏死，表明VdPDA1蛋白不具有PAMP分子的作用；VdPDA1蛋白不能保护真菌菌丝免受寄主几丁质酶的水解，VdPDA1蛋白介导的几丁质脱乙酰化作用不能激发ROS的产生，不能使寄主中的MAPK磷酸化，也不能抑制抗性相关基因的表达，表明VdPDA1蛋白介导的几丁质脱乙酰化作用能够阻碍几丁质寡糖触发的植物免疫。