由土传真菌大丽轮枝菌引起的黄萎病给番茄生产造成了严重损失。前期应用RNA-seq技术分析了大丽轮枝菌侵染感病番茄Micro-Tom的转录组，发现了2041个差异表达基因，其中282个差异表达基因分布在87个KEGG通路(KEGG Pathway)中。本项目拟以植物与病菌互作相关通路中差异表达基因作为目标基因，对其进行序列查询和同源比对分析，确定候选基因，利用RACE方法获得全长。利用病毒诱导的基因沉默（VIGS）和超表达方法分别对候选上、下调基因进行功能分析，得到感病关键基因。对感病关键基因进行亚细胞定位，同时以感病关键基因为诱饵分别筛选大丽轮枝菌和番茄酵母双杂文库，从而分别获得与之互作的效应子和番茄蛋白基因序列，以期诠释番茄感病关键基因信号转导途径和番茄与黄萎病菌亲和互作的分子机制，为培育番茄抗病品种提供理论支撑和基因资源。

Verticillium wilt disease caused by soil-born Verticillium dahliae results in a great loss of tomato yield. RNA-seq method was previously used to analyze transcriptome of Micro-Tom tomato infected by V. dahlia. Two thousands and forty one differential expression genes were found in tomato expression profiles, among which 282 differential expression genes were assigned to 87 KEGG pathways. In order to determine the candidate genes, differential expression genes involved in plant-pathogen interaction analyzed by using homologous comparison will be selected as the target genes, the full-length sequences of which will be amplified by using rapid amplified cDNA ends (RACE) method. Virus induced gene silencing (VIGS) and overexpression methods will be used to analyze the function of the up-regulated and down-regulated candidate genes, respectively. To analyze the mechanisms of the susceptible key genes, subcellular localization will be determined by using transient transformation in onion epidermis, and V. dahliae and tomato yeast two-hybrid libraries are screened with the susceptible key genes as baits to acquire interaction target proteins, respectively. Gene sequences of V. dahliae effectors and tomato interaction proteins can also be obtained via the interaction target proteins. The present study will explain the role of the susceptible key genes in signal transduction pathways and the molecular mechanism of compatible interaction between tomato and Verticillium wilt fungus. It will also benefit to breed vascular wilt disease resistance cultivars and help to obtain gene resources for disease resistance breeding in tomato.

由土传真菌大丽轮枝菌引起的黄萎病给番茄生产造成了严重损失。对 RNA-seq技术得到的大丽轮枝菌侵染感病番茄Micro-Tom转录组进行分析，发现了1985个差异表达基因。根据上调基因Log2fold-change≥3.5和下调基因Log2fold-change≤－3.5的差异基因表达倍数，以及这些差异基因在NR、SwissProt、GO、KEGG和COG数据库里中注释与植病互作相关的基因，我们挑选出了126个（77个上调基因和49下调基因）主要候选感病基因；与此同时，利用RT-qPCR方法，我们对KEGG通路中植物与病菌互作和植物激素信号转导2个途径的48个差异表达基因进行了表达谱分析，筛选到JAZ节点上2个高表达的候选感病基因（Solyc12g009220.1、Solyc07g042170.2）。通过PCR高保真扩增方法，从番茄Micro-Tom中克隆了2个JAZ基因的全长序列。这2个基因都构建了基因敲除沉默的pCRISPER-in-one双元载体和基因过表达的pOE-3HA双元载体，并通过农杆菌介导已转化到Micro-Tom番茄中。随后，我们将用大丽轮枝菌感染转化后的番茄植株，鉴定其抗感表型。综上所述，这些实验结果为进一步深入研究并最终获得番茄黄萎病感病关键基因奠定很好的前期基础。