细胞壁相关受体激酶BrWAK1介导白菜霜霉病抗性的分子机制研究

Downy mildew severely damages the yield and quality of Brassica rapa species, especially to fast-growing seedling Chinese cabbage and non-heading cabbage. Resistant genes identification and sustainable resistant breeding are the most effective way for brassica safe and efficient production. We first carried out the research on mining and utilizing downy mildew resistance genes, and have first cloned the major resistant QTL-BraDM by fine mapping. Subsequently, we identified the candidate resistant gene, which encodes a wall-associated kinase and named as BrWAK1, through sequence variation, expression analysis and transient transformation. Dramatic variations in extracellular signal perception domain (GUB and EGF) were found between resistant and susceptive parents, which should be the reason for different signal perception and disease resistant ability. In this study, we aim to firstly confirm BrWAK1 play an important role to mediate Brassica downy mildew resistance by transgenic and CRISPR technology. Then chimeric receptors containing the extracellular domain of BrWAK1 and kinase domain of Arabidopsis BRI1 were applied to research the function of BrWAK1’s extracellular domain and identify pathogenic signal molecule. To identify the interaction protein of BrWAK1, co-immunoprecipitation (Co-IP) combining MS technology will be carried out. Moreover, the downstream signal transduction pathway and molecular regulation network will be further explored by transcriptome sequencing and hormone concentration detection. The results have important significance for revealing the molecular mechanism of downy mildew resistance, and will provide theoretical basis for the molecular breeding of disease resistance varieties in Brassica rapa.

霜霉病严重影响了白菜特别是苗用白菜和不结球白菜的产量和品质，发掘抗病基因、培育持久抗病品中是白菜安全高效生产的有效途径。本课题组在国内率先开展了白菜抗霜霉病基因挖掘与利用研究，精细定位了首个抗病主效QTL-BraDM，并鉴定了抗病候选基因BrWAK1，该基因编码细胞壁相关受体激酶，抗感等位基因的胞外结构域（GUB和EGF）差异明显，推测这是导致抗病信号识别功能变异、抗感表型差异的主要原因。本项目拟通过转基因和CRISPR技术确证BrWAK1是介导白菜霜霉病抗性的关键功能基因；构建BrWAK1胞外结构域与BRI1激酶结构域的嵌合载体，通过瞬时转化鉴定BrWAK1胞外结构域的功能及其识别的病原菌信号分子；利用Co-IP结合质谱分析鉴定BrWAK1的互作蛋白；通过转录组测序及激素含量测定等对BrWAK1介导的基因调控网络进行研究。项目结果将为揭示白菜抗霜霉病的分子机制和抗病分子育种提供理论依据。

植物细胞壁是抵御物理损伤和病原体攻击的第一道防线。细胞壁相关激酶（WAK，wall-associated kinase）能够感知细胞壁基质的变化，并将信号转化到细胞质中激活植物发育和防御反应。白菜霜霉病可导致大白菜（Brassica rapa L.ssp.pekinensis）产量的巨大损失。本项目中，我们利用抗病自交系T12-19和感病系91-112构建的双单倍体（DH）群体，将主效QTL位点定位到A08染色体中，并在20 kb抗病区间中鉴定了抗病候选基因BrWAK1。我们发现，水杨酸和病原体接种都可诱导BrWAK1的表达，并且在91-112中过表达BrWAK1可显著增强抗病性，而在T12-19中BrWAK1的基因编辑会增加其感病性。除此之外，我们发现BrWAK1的胞外GUB结构域的变异是造成T12-19抗病性的主要原因，并鉴定到BrWAK1可以与BrBAK1互作，激活下游MAPK信号途径，从而触发防御反应。本项目研究结果中，BrWAK1是第一个被鉴定和完全表征的赋予大白菜抗病性的WAK基因，并且，BrWAK1的过表达和基因编辑对植物生物量没有显著影响，该结果将大大加快大白菜抗霜霉病的育种进程。