白菜BrRLP1.1基因介导霜霉病抗性的分子机制

Downy mildew is prevalent widely and fast throughout the country，and causes heavy damage to the production and quality of Brassica rapa species, especially to fast-growing seedling Chinese cabbage and pak choi. We first carried out the research on mining downy mildew resistance gene, and have cloned the first downy mildew resistance gene BrSTK1 in Chinese cabbage. In present study, we identified a new candidate gene BrRLP1.1 located on chromosome A01 by using genome resequencing of 302 advanced lines and genome wide association analysis. BrRLP1.1 encoding a receptor like protein. The extracellular receptor domain with or without of 2 leucine rich repeats maybe the key node for BrRLP1.1. We aim to confirm that BrRLP1.1 is the critical gene confers the immune response of downy mildew by using transgenic validation and expression pattern analysis. To investigate the functional sites sensing the external factors, we will construct the protein vector with domain replacement and carry out transient protein expression in tobacco. Moreover, the downstream signal transduction pathway and related genes will be further explored by transcriptome sequencing, and the interacting protein of BrRLP1.1 will be identified by yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation experiments to construct a molecular regulation network. The results have important significance for revealing the molecular mechanism of downy mildew resistance, and will provide theoretical basis & new gene source for the molecular breeding of disease resistance varieties in Brassica rapa.

霜霉病流行范围广、发病速度快，严重影响了白菜（特别是苗用白菜和小白菜等速生叶菜）的产量和品质。本课题组在国内率先开展了抗霜霉病基因挖掘研究，克隆了首个霜霉病抗性基因BrSTK1。在此基础上，利用302份种质的重测序和全基因组关联分析，在A01染色体上鉴定了一个新的、具有更高抗性的抗霜霉病候选基因BrRLP1.1，该基因编码类受体蛋白，抗感等位基因受体结构域相差2个富含亮氨酸重复单元，推测这是导致表型差异的主要原因。本项目拟通过转基因确证BrRLP1.1是介导霜霉病抗性的关键基因；构建结构域置换蛋白载体，借助烟草瞬时转化系统，鉴定胞外结构域识别病原菌信号分子的关键位点；基于转录组测序鉴定下游信号传导途径及其相关应答基因；运用酵母双杂等技术鉴定与BrRLP1.1互作的蛋白，构建BrRLP1.1介导霜霉免疫反应的分子调控网络。研究结果将为解析白菜抗霜霉病分子机制和抗病分子育种提供理论基础。

霜霉病流行范围广、发病速度快，严重影响了白菜的产量和品质，挖掘抗病基因、培育抗病品种是保障白菜安全生产的最有效途径。项目通过关联分析以及候选基因序列变异、表达分析、单体型分析、分离群体标记检测和白菜子叶瞬时表达实验等分析，表明该区间内的基因BrRLP1.1是控制霜霉病抗性的候选基因，位于其胞外结构域的第309位的SNP突变是导致抗感表型差异的主要原因；根据BrRLP1.1基因在抗感材料中的序列变异，开发了与之紧密连锁的InDel标记；构建了过表达和CRISPR载体，转化获得了白菜转基因植株，结果显示感病材料中过表达BrRLP1.1能显著增强霜霉病抗性。利用酵母双杂交筛选到与BrRLP1.1互作的蛋白BrPBS1和BrMDHAR1，进一步利用双荧光素酶互补实验确证了BrRLP1.1与BrMDHAR1的互作。此外，项目首次在全基因组分析了响应白菜霜霉病的差异表达基因和长链非编码RNA（lncRNA），鉴定了系列参与抗病反应的关键lncRNA及其靶基因，进一步的研究发现MAPK信号途径基因BrMAPK15及其反义lncRNAMSTRG.19915在白菜霜霉病抗性反应中发挥重要作用。根据以上研究结果，我们推测，BrRLP1.1可能直接或间接通过BrPBS1激活胞内MAPK级联信号途径，LnRNA MSTRG.19915等参与了MAPK信号的调控；同时，BrRLP1.1还可以直接结合BrMDHAR1，调控抗病反应中ROS的平衡。由此证明，BrRLP1.1可以通过多种途径参与白菜抗霜霉病免疫反应，这些研究结果为今后对大白菜抗霜霉病分子机理的深入研究奠定了基础。