大豆抗疫病新基因RpsX精细定位，克隆及功能分析

The applicant identified a broad-spectrum resistance gene RpsX to Phytophthora sojae in the variety Xiu94-11, and initially mapped it on chromosome 3, which is not containing the typical resistance gene with NBS-LRR structure, suggesting that RpsX may be a new type of Rps gene. This project intends to finely map RpsX by derived large mapping population. Candidate genes were screened by comparing gene sequences between parents in the finely mapped interval. RpsX function-related genes were identified by tissue differential expression and Phytophthora–deduced differential expression analysis of candidate genes. The RpsX function-related genes were cloned and subcellularly localized, the RpsX gene function was verified and elucidated by the transgenic method and the CRISPR/Cas9-based RpsX functional gene knockout technique, and the functional markers of RpsX were developed. This project is expected to realize the new type excavation and functional identification of Rps gene, and it is of great significance to disclosure of its corresponding resistance mechanism. The functional markers based on RpsX can lay a foundation for molecular breeding against Phytophthora root rot and accelerate the breeding process.

申请人前期在大豆品种岫94-11中鉴定了一个对大豆疫病具有广谱抗性的基因RpsX，并初步作图在大豆3号染色体上一个不含有NBS-LRR抗病结构基因的区域，推测可能是一种新的抗疫病基因类型。本项目拟通过大群体精细定位RpsX，比对精细定位区间内基因在亲本间的序列差异，筛选候选基因。通过候选基因的组织差异表达和疫霉诱导差异表达分析确定RpsX功能相关基因。对RpsX功能相关基因进行克隆及亚细胞定位，通过将RpsX导入感病植株的功能验证和基于CRISPR/Cas9系统的RpsX功能基因敲除技术阐明RpsX基因功能，并开发RpsX的功能标记。本项目的研究有望实现大豆抗疫病基因新类型发掘和功能鉴定，对其相应抗性机制的解析具有重要意义，并且RpsX功能标记的开发能够为抗疫病分子育种选择奠定基础，加快育种进程。

大豆疫病是大豆生产中的一种毁灭性病害，该病害由大豆疫霉引起，目前利用抗病品种对该病害进行防治是最为有效的手段。但由于大豆疫霉的治病型复杂，毒力变化大，使得大豆中的抗病性很容易丧失，因此为有效的防治该病害需要不断挖掘新的抗病基因，应用于大豆育种中。项目组在大豆种质资源中筛选获得一个对中国大豆疫霉种群具有广谱抗性的一个抗疫病新基因RpX，位于大豆的3号染色体上。序列分析证实，由不等交换引起的144-bp插入导致候选基因中有两个额外的LRR编码片段。基于144-bp插入开发的标记用于分析遗传群体和种质，并证明可用于识别RpsX和RpsQ等位基因，并对候选基因记性转基因验证以及转录调控网络的构建。该研究表明LRR结构域中LRR单位的数量可能对大豆的大豆疫霉抗性十分重要重要。