大丽轮枝菌导致棉花落叶的致病基因鉴定及其机制解析

Cotton wilt disease caused by Vertcillium dahliae was one of the main reasons of cotton loss. Vertcillium dahliae defoliating strains make cotton leave defoliate and death. The Cotton wilt disease became more and more severely recently, but the defoliation gene of Vertcillium dahliae that induced the cotton leave defoliate remain unclear. In this research, we selected 5 defoliating strains and 5 nondefoliating strains for genome sequencing based on Pacbio and illumine method. Compared the genome of the two kind of strains, we found in defoliating strains exist large fragment insertion and got 29 candidate genes from this region. Based on the RNA-seq data of infection process and gene funcitional prediction, about 10 genes were selected for functional analysis at first step. Knock-out, complement and overexpression methods were used to identifying the defoliating genes. The function and biochemical activity of the genes were analyzed by transient expression in Nicotiana benthaminan and prokaryotic expression in bacterial. Subcellular localization of the genes during the infection process was observed by overexpression the genes fused with RFP. The defoliation mechanism was analyzed by identification of the targets of the genes in cotton plant by yeast-two hybrid and Co-IP method. The aim of this project is identification of the defoliation genes in V. dahlia and shed light on the management of the disease.

大丽轮枝菌落叶型菌系对棉花的危害日趋严重，但导致落叶的致病基因（即落叶基因）尚未明确。本研究对筛选出的5个落叶型和5个非落叶型菌系开展了Pack-bio和illumina测序，对比两种菌系，发现落叶型菌系基因组中存在大的插入片断，获得落叶型菌系特有候选基因29个。在此基础上，本项目拟利用侵染前后的RNA-seq数据分析候选基因的表达模式，结合基因功能预测，筛选出10个左右基因；将这些基因进行敲除、互补以及在非落叶型菌系中过表达，鉴定落叶基因；通过烟草瞬时表达以及原核表达体系检测该基因编码蛋白的生化活性，分析该基因的功能；将该基因与RFP融合在大丽轮枝菌中过表达，观察在侵染过程中的定位，明确干扰植物的部位；通过酵母双杂交等手段分离其在植物细胞中的靶标蛋白，解析其作用模式。本项目的目的是克隆大丽轮枝菌落叶基因并解析其作用机制，对棉花黄萎病的防控具有重要的理论意义。

棉花黄萎病俗称棉花“癌症”，对棉花生产具有极大危害性，每年减产10-15%，是棉花第一大病害, 其病原菌为大丽轮枝菌，该菌的落叶型菌系对棉花的危害日趋严重，但导致落叶的致病基因（即落叶基因）尚未明确，本研究针对该问题，通过对筛选出的5个落叶型和5个非落叶型菌系开展了Pack-bio和illumina测序，对比两种菌系，发现落叶型菌系基因组中存在大的插入片断，并获得了落叶型菌系特有候选基因29个。在此基础上，利用侵染前后的RNA-seq数据分析候选基因的表达模式，结合基因功能预测，筛选出了10个左右基因；将这些基因分别进行了敲除、互补以及在非落叶型菌系中过表达，鉴定到落叶基因CQ2_0028g06440a为关键的落叶型基因；该基因在落叶型菌株CQ2中有2个拷贝，在非落叶型菌株BP2，JR2，VdLs17中，与落叶型基因的相似性为90%。该基因预测有信号肽，在侵染过程中上调表达，在侵染过程中可分泌到胞外。通过烟草瞬时表达以及原核表达体系检测该基因编码蛋白的生化活性，分析了该基因的功能；将该基因与RFP融合在大丽轮枝菌中过表达，观察了其在侵染过程中的定位，明确干扰植物的部位在于细胞质内；通过酵母双杂交等手段分离其在植物细胞中的靶标蛋白，解析了其作用模式。本项目鉴定了落叶型菌系中的落叶基因并解析了其作用机制，为生产上控制该类菌系危害提供理论基础。