辣椒疫病小种专化抗病候选基因的克隆及其功能鉴定

Phytophthora blight is a devastating pathogen of capsicum and is caused by the oomycete plant pathogen Phytophthora capsici which has differentiated in several distinct physiologic races. Cloning and characterization of Phytophthora capsici resistance candidate genes (RCG) from race-specific resistance capsicum (RSRC) is an unsolved scientific issue. Our research group has collected and bred dozens of RSRC, and identified & preserved Phytophthora capsici race of ph1, ph2, ph3 and ph4 since 2006. Cv.A56 used as tested material in this project is one of RSRC and it resists to race ph2 of P. capsici, but it is susceptible to race ph3 of P. capsici.Techniques of RNA-seq, quantitative protein profiling using two-dimensional gel electrophoresis and mass spectrometric identification can be applied to analyze the differences of disease resistance gene expression of both mRNA and protein levels after cv. A56 is inoculated the race between ph2 and ph3. The relationship of the gene expression between mRNA and protein levels will be found and the candidate genes can be obtained. Secondly, candidate gene expression profile can be analyzed and validated by the method of reverse transcriptase quantitative real-time PCR (RT-qPCR), and functional characterization of candidate gene expression is tested by TRV-VIGS( tobacco rattle virus-virus induced gene silencing) technique. Finally, transgenic capsicum plants could be obtained by Agrobacterium -mediated transformation, and the disease resistance of transgenic plants will be evaluated and the functional characterization of candidate gene will be clarified. The goal of this project is to definitude RSRC genes and its function so that we can understand the molecular mechanism of RSRC.

辣椒疫病是辣椒产业上极具危害性的一种病害，其病原菌已有生理小种分化。克隆及鉴定其抗病候选基因，明确其功能是尚未解决的科学问题。前期项目组搜集与创制了数十份辣椒疫病小种专化抗源，鉴定和保存我国辣椒疫病生理小种ph1、ph2、ph3和ph4。本项目拟以接种疫病非亲和小种ph2（A56对其抗病）与亲和小种ph3（A56对其感病）的小种专化抗源纯系A56为试材，采用转录组测序技术以及双向凝胶电泳蛋白质分离技术与质谱鉴定技术，克隆与分析A56接种非亲和小种与亲和小种后差异表达基因与差异蛋白质，寻找它们彼此间基因表达的关系，筛选候选基因；然后，以RT-qPCR技术分析其表达谱，再以VIGS技术快速鉴定其功能；最后，以农杆菌介导的遗传转化方法转化辣椒，并对转基因株系进行抗病性鉴定以及对候选基因进行功能分析。旨在明确辣椒疫病小种专化抗病候选基因与其功能，为揭示辣椒疫病小种专化抗病性分子机理提供依据。

辣椒疫病是辣椒产业上极具危害性的一种病害。辣椒对疫病的抗性表现为非小种专化抗性和小种专化抗性共存。前者由多个微效基因控制，利用难度大。后者是由单基因或主效基因控制的抗病性，抗病效能高。项目组搜集与创制了一批辣椒疫病小种专化抗源，开展了辣椒对不同疫病病原菌表现亲和抗性与非亲和抗性的研究，鉴定出我国辣椒疫病病原菌不同生理小种。在此基础上，采用转录组测序技术以及双向凝胶电泳蛋白质分离技术与质谱鉴定技术，筛选和克隆了CaRGA1、CaRGA2、CaPTI1、CaPOD、CaTF、CaSBP11、CaHIR4、CaSBP12、CaMYB、CaPIP1-1、CaCP、CaPAO、CaRCCR、CaBPM4、CaDIR7等15个辣椒小种专化抗性候选基因。利用RT-qPCR、VIGS、超表达与基因敲除等技术对其表达特性与功能进行了鉴定，结果表明这些基因可分为抗性关键基因、抗性重要相关基因和抗性相关基因，其中CaRGA1、CaRGA2和CaPTI1是辣椒疫病专化抗性的关键基因；CaPOD、CaTF、CaSBP11、CaHIR4、CaSBP12、CaMYB为抗性重要相关基因；CaPIP1-1、CaCP、CaPAO、CaRCCR、CaBPM4、CaDIR7是抗性相关基因。基因表达信号通路分析认为，CaRGA1和CaRGA2基因的表达通过茉莉酸信号途径，CaPTI1基因的表达通过乙烯、水杨酸和茉莉酸信号途径调控辣椒对疫病的专化型抗性。这些研究结果对于揭示辣椒疫病小种专化抗病性分子机理提供了科学依据。