大丽轮枝菌细胞色素P450基因（VdCYP3）致病功能初步研究

Verticillium dahliae is one of most important soilborne pathogenic fungus causing Verticillium wilt diseases in many crops. Current research about the pathogenic mechanism is mainly focused on the effectors, transcription factors, cell wall degradation enzymes, etc. In previous study, a mutant which the pathogenicity and the virulence of the secondary metabolites significantly decreased was acquired by the rapid screening from Verticillium dahliae T-DNA random insertion mutants. Insertion location and gene knockout experiments demonstrated that the phenotype of this mutant was caused by the function loss of cytochrome P450 gene VdCYP3. However, there is no report about cytochrome P450 genes participated in the infection progress of Verticillium dahliae by regulating the secondary metabolites. In this program, the characterization of VdCYP3 will be demonstrated by gene expression, subcellular localization, and gene polymorphism. The pathogenic function of VdCYP3 will be illustrated by gene complementation, overexpression, and pathogenic identification. Furthermore, the role of VdCYP3 in the regulation of secondary metabolism and the production of secondary metabolites will be identified by transcriptome, physiological and biochemical analysis. After all, this research will provide theoretical foundations for the research of pathogenic function of cytochrome P450 in Verticillium dahliae and the pathogenic mechanism of Verticillium dahliae infecting host by regulating the biosynthesis of secondary metabolites.

大丽轮枝菌是引起多种重要农作物产生黄萎病的土传性病原真菌，当前针对其致病机理的研究主要聚焦于效应子、转录因子、细胞壁降解酶等致病因子。本项目前期通过大丽轮枝菌T-DNA随机插入突变体筛选获得1个致病力及其次级代谢物毒性显著下降的突变体，基因定位和基因敲除实验证明细胞色素P450基因（VdCYP3）是引起该突变体致病力显著下降的靶标基因，该类基因及其调控次级代谢物参与大丽轮枝菌侵染寄主的致病机理未见报道。在此基础上，本项目拟通过基因表达、亚细胞定位、基因变异等解析基因的基本特性；应用基因功能互补、异源过表达和致病性鉴定明确基因参与侵染寄主的致病功能；利用转录组和次级代谢物生理生化测定阐明基因调控次级代谢物合成的过程及其对次级代谢物合成的影响。完成上述研究将为大丽轮枝菌细胞色素P450家族基因的致病功能及其调控次级代谢合成参与侵染寄主的致病机理研究提供理论依据。

大丽轮枝菌是引起棉花、番茄、马铃薯、辣椒、草莓和西瓜等重要农作物产生黄萎病的病原真菌，每年给农业生产带来巨大损失，难以防治。细胞色素P450家族基因通常通过参与次级代谢物的合成和调控次级代谢物的毒性而影响病原菌的致病功能，该类基因在大丽轮枝菌中未见报道。本项目以前期通过大丽轮枝菌致病相关突变体的筛选和基因定位发现的致病关键细胞色素P450基因VdCYP1 (项目申请时命名为VdCYP3)为研究对象，开展了其致病功能及其调控次级代谢物合成的机理研究，取得了如下阶段性结果：1）通过基因VdCYP1表达特性、亚细胞定位观察和多态性分析，发现基因VdCYP1在侵染寄主早期被显著诱导上调表达，发挥催化功能部位集中在细胞膜上，且该基因变异与其致病力差异不呈显著相关性；2）通过基因VdCYP1敲除突变株表型分析、基因VdCYP1回补载体的构建、转化和致病功能鉴定，进一步明确VdCYP1是大丽轮枝菌的重要致病因子，但是该基因不参与大丽轮枝菌的基础生长发育和碳源利用过程；3）通过转录组分析、代谢途径分析和代谢产物筛选鉴定，推测VdCYP1 通过调控有毒次级代谢组分磺胺醋酰（SFA）合成，进而影响大丽轮枝菌的致病过程。上述研究为大丽轮枝菌细胞色素P450家族基因的致病功能及参与次级代谢合成的研究提供了理论依据，丰富了大丽轮枝菌致病机理的基础理论研究。