生防因子草酸脱羧酶对木霉重寄生灰霉菌的影响机制研究

Oxalic acid (OA) is an important pathogenic factor of Botrytis cinerea. Trichoderma can degrade OA to prevent B. cinerea from infecting plant tissues utilizing the action of oxalate decarboxylase (OXDC). When Trichoderma antagonized B. cinerea, both OXDC and mycoparasitism-related hydrolase coding genes were up-regulated. After Trichoderma OXDC gene was knocked out, the mycoparasitism of the mutant strain was inhibited, suggesting that the mycoparasitism was affected by OXDC, but its mechanism is still unknown. In order to reveal the genes and pathways that OXDC affected in mycoparasitism, this project takes an efficient biocontrol agent, T. afroharzianum LTR-2, as the research object, and establishes an analysis system of the mycoparasitism. OXDC gene deletion and overexpression mutant strains will be constructed, and their transcriptome expression patterns during myco-parasitizing B. cinereal will be analyzed using RNA-seq. Differentially expressed genes (DEGs) related to mycoparasitism will be obtained, and the DEGs will be knocked out, knocked down, and overexpressed to identify their functions through analyzing phenotype and bio-control characteristics. Finally, the mechanism by which OXDC affects Trichoderma myco-parasitizing B. cinerea is expected. Through this study, new knowledge regarding to the mechanism of Trichoderma inhibiting B. cinerea will be acquired, and the theoretical basis of Trichoderma bio-controlling grey mould will be expanded.

草酸是灰霉菌侵染植物的重要致病因子，木霉可利用草酸脱羧酶（OXDC）降解草酸，阻止灰霉菌侵染植物。木霉拮抗灰霉菌时，OXDC和重寄生相关的细胞壁水解酶编码基因同时上调表达。将木霉OXDC基因敲除后，突变株重寄生作用被抑制，表明木霉重寄生灰霉菌受OXDC的影响，但其机制尚未可知。为了揭示OXDC通过哪些基因和途径作用影响了重寄生，本项目以一株高效生防木霉Trichoderma afroharzianum LTR-2为研究对象，建立木霉重寄生灰霉菌分析体系，构建OXDC基因缺失和过表达突变株，利用RNA-seq分析OXDC突变株重寄生灰霉菌的转录组表达模式，获得差异表达的重寄生相关基因。进一步针对差异基因构建缺失（弱化）和过表达突变株，通过分析表型和生防特性明确差异基因功能，揭示OXDC对木霉重寄生灰霉菌的影响机制。通过本研究将获得对木霉拮抗灰霉菌机制的新认识，拓展木霉防治灰霉病的理论基础。

灰霉可侵染包括常见经济作物在内的1400余种植物，引起的灰霉病每年造成严重减产。木霉生防菌株对于灰霉具有显著的拮抗作用并可缓解植物灰霉病。非洲哈茨木霉可利用其草酸脱羧酶（OXDC）降解灰霉分泌至侵染部位环境中的草酸（OA），并且其受OA诱导后OXDC基因和重寄生相关的细胞壁水解酶基因均上调表达。项目以揭示木霉OXDC对重寄生灰霉的影响机制为目的开展。在优化的对峙培养条件下获得接触前和接触后OXDC和CWDE基因表达的关联性特征。构建了OXDC基因缺失和过表达突变株，分析表明突变株对OXDC活性、OA降解能力、对灰霉拮抗和防病效果方面均有显著影响。通过RNA-seq技术分析突变株与出发株受OA诱导和对峙灰霉在转录组水平上的基因表达模式差异，并进行RT-qPCR验证，分析获得与OXDC基因表达模式相关性较高的重寄生相关的差异基因。构建了差异基因缺失/回复突变和过表达突变株，分析表明突变株对几丁质酶（CHIT）活性、对灰霉拮抗和防病效果方面均有显著影响。基于基因序列比对和表型分析鉴定为非洲哈茨木霉中的CHIT基因。基于上述结果总结非洲哈茨木霉的OXDC通过调控CHIT对重寄生灰霉的影响机制假说。项目另外还分析了木霉降解草酸对植物系统抗性的影响。本项目为更全面的了解木霉拮抗灰霉早期的生防机制提供基础数据，为科学筛选针对灰霉的木霉生防菌提供理论基础。