番茄抗叶霉病基因Cf-12的克隆、功能验证及抗病应答机制的研究

The tomato is the world's major vegetable crops.Tomato leaf mold disease, caused by biotrophic fungus C. fulvum, is a serious disease of Solanum lycopersicum (tomato). This disease can reduce both fruit yield and quality, and sometimes even kill the tomato plants. C. fulvum has many physiological races, and new physiological races can develop rapidly , making it difficult to manage this disease. An efficient method to control leaf mold outbreaks is breeding to obtain resistant cultivars. Recently, tomato varieties applicated for resistance breeding mainly contain Cf-5 and Cf-9 resistance genes, but these rasistance genes were over-comed by new pathogen physiologies. The Cf-12 gene, which has been shown to confer resistance to this disease in the field, can serve as a new candidate gene in breeding. Fine mapping, cloning and functional verification of Cf-12 gene, will be carried out based on the previous data from study of gene mapping and tomato material resequencing. And for the purpose of understanding the resistance response mechanism of Cf/Avr interaction, the differentially expressed genes in Cf-12-mediated resistance response will be studied by transcriptome sequencing method. The results will provide us important gene material and theoretical basis for the breeding work in the future.

番茄是世界上的一种重要蔬菜作物。番茄叶霉病是我国乃至世界番茄生产的主要病害，给番茄生产造成了巨大损失，番茄叶霉病原菌C. Fulvum是生理小种分化很快的一种真菌，目前我国生产上应用的主要是含Cf-5和Cf-9抗病基因的番茄品种，近年来我国部分地区生产上已经出现了含Cf-5和Cf-9抗病基因的番茄品种被侵染的现象。Cf-12抗病基因在反复的抗病性鉴定中对多个生理小种表现出了抗病性，具有很高的潜在应用价值。本项目组在前期的分子标记研究和番茄材料高通量测序数据的研究基础上，拟对Cf-12基因进行精细定位、基因克隆、功能验证、抗病基因专化性演变、抗病反应相关基因的功能以及Cf/Avr互作机理进行研究，并从分子水平上揭示Cf基因的抗病机制，旨在为今后的抗病育种工作提供宝贵的基因资源和理论基础。

通过多年的、多点的、多地的抗病性鉴定，CGN7495材料具有优良的C. fulvum 抗性，由Kanwar[196]的鉴定结果得知该材料含有Cf-12基因，但对于该基因的结构、功能等信息知之甚少。因此，本试验主要以番茄叶霉病新抗性候选基因Cf-12的挖掘和筛选以及其抗性应答机制为主要研究重点，主要研究内容如下：.1）通过对抗性材料的电子显微镜观察，明确Cf-12基因的抗病性组织学特征；.2）通过构建含Cf-12基因材料的6世代群体，明确Cf-12基因的抗性遗传规律；.3）通过父母本重测序及F2代抗、感群体SLAF简化测序，对所获得的多态性SNP位点进行ED和SNP-index关联分析的方法筛选Cf-12候选基因；.4）通过Swiss-Model、SOPMA等方法对候选Cf-12基因进行多生物信息学分析，并根据Cf-12候选基因CDS区核苷酸序列（Nucleotide）以及氨基酸（AA）序列探讨其与Hcr2s（Homologs of C. fulvum resistance gene Cf-2）、Hcr9s（Homologs of C. fulvum resistance gene Cf-9）基因家族的进化关系以及蛋白结构上存在的差异；. 通过转录组学分析初步明确Cf-12对C. fulvum的抗性应答机制，并采用qRT-PCR的表达模式分析以及VIGS（Virus-induced gene silenced）的方法验证候选基因。.将抗 C. fulvum 的番茄 Cf-12 基因克隆，明确其分子结构与功能，揭示了不同 Cf 基因间的进化关系及异同；揭示番茄 Cf-12 基因介导的抗性反应中相关应答基因的功能，明确这些基因在调控网络中的作用和相对位置，为抗病育种提供了理论基础。