枯萎病菌胁迫下的黑籽南瓜抗性基因表达差异及NBS类关键基因功能鉴定

项目名称： 枯萎病菌胁迫下的黑籽南瓜抗性基因表达差异及NBS类关键基因功能鉴定

项目编号： No.31460516

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 杨正安

作者单位： 云南农业大学

项目金额： 50万元

中文摘要： 枯萎病是影响我国瓜类品质和产量的重要病害,且该病抗性遗传资源缺乏，培育抗病新品种需要寻找更多的抗病基因资源。黑籽南瓜对瓜类枯萎病抗病力强，是筛选抗枯萎病基因的理想材料。该项目在前期成功筛选黑籽南瓜高抗枯萎病材料的基础上，采用基因组学结合转录组学分析的方法，拟开展以下研究，首先搜索黄瓜和甜瓜等基因组数据库中的NBS类基因保守序列并设计简并或特异引物，克隆黑籽南瓜抗病基因同源片段（RGA）；二是利用克隆的RGA对比转录组测序筛选到的黑籽南瓜与枯萎病菌互作的差异表达基因，缩小筛选范围，快速锁定NBS类关键差异表达基因；采用荧光定量PCR验证后，RACE技术获得全长基因；最后利用gateway技术构建关键基因的RNAi干涉载体和植物超表达载体，对黑籽南瓜高抗和感病材料进行遗传转化、最终确认基因功能。项目可解析黑籽南瓜与枯萎病菌二者之间非亲和互作的分子作用机制；同时为持久防控瓜类枯萎病提供基因资源。

中文关键词： 黑籽南瓜；抗枯萎病基因；NNBS-LRR；差异表达基因；功能鉴定

英文摘要： Fusarium wilt is an important disease that affects the quality and yield melons, and it lacks the resistance of genetic resources in China, if breeding disease-resistant varieties need to look for more resources of resistance genes. Cucurbita ficifolia has the strong resistance to the fusarium wilt of melons , and it is an ideal material to screen resistance to fusarium wilt genes . The project is on the basis of screening fusarium wilt -resistant materials of Cucurbita ficifolia successfully in early, using the method of genomics and transcriptome analysis to carry out the following research: First search the conserved sequences of NBS in the genome database of cucumber and melon, then design the degenerate or specific primers and clone the resistance gene analogs of Cucurbita ficifolia. Second, use the RGA cloned contrasting to the differentially expressed genes interacting with blight on Cucurbita ficifolia, micry the scope of screening, and target the key different expressed genes of NBS quickly; after the validation of quantitative PCR,use RACE technology to obtain full-length gene; Finally use the gateway technology to build the key genes of interference carrier and plant expression vector of RNAi , and transform the high resistant and infected materials for genetic, then confirm the function of genes in the end.The project can parse the molecular mechanism of the affinity interactions between Cucurbita ficifolia and fusarium wilt, at the same time provide gene resources for the prevention and control fusarium wilt of melons in the long time.

英文关键词： Cucurbita ficifolia Bauche;Fusarium wilt resistant gene;NBS-LRR;Differential expression gene;The identification of gene function