甘蓝型油菜SPL基因介导ROS和SA途径提高菌核病抗性的分子机制

Sclerotinia sclerotiorum causes serious yield loss and quality reduction in rapeseed. Understanding the molecular mechanism of Sclerotinia sclerotiorum resistance is the precondition to develop disease-resistant varieties in rapeseed and guarantee the food and edible oil security in our country. In previous study, we identified a rapeseed spl mutant with significantly increased resistance to Sclerotinia sclerotiorum, which also shows over-accumulation of H2O2, higher antioxidase activity, and reduced oxalic acid sensitivity during seed germination. Besides, salicylic acid (SA) biosynthesis and signaling pathway genes were also activated, leading to significant increase of salicylic acid (SA) content. Fine mapping results revealed that SPL may encode a receptor like cytoplasmic kinase (RLCK) protein with unknown function, and its relatively down-regulated expression level is significantly positively correlated with Sclerotinia sclerotiorum resistance. To unravel the molecular basis of SPL functions, we propose to assess the spatio-temporal and induced expression patterns of SPL gene and subcellular location of SPL protein; to identify SPL interacting substrate proteins and their and their key phosphorylation sites, and construct a phosphorylation signaling network underlying disease resistance based on results of protein inter-actome and quantitative phosphoproteomics; to uncover the molecular mechanism of Sclerotinia sclerotiorum resistance enhanced by reactive oxygen species (ROS) and SA pathways, which regulated by interaction and phosphorylation between SPL and its interacting partners, according to comparison between single and double mutants in Arabidopsis thaliana, and that of phenotypes, physiological indexes, and transcriptomes among the complementary, overexpression and RNAi transgenic rapeseed lines. Our researches will provide insight into further understanding the plant immune signal transduction regulation network, lay a theoretical foundation for disease-resistant breeding in rapeseed and its relatives.

菌核病严重影响油菜产量和品质。理解菌核病抗病分子机制，是油菜抗病育种和保障我国粮油安全的需要。前期研究发现，菌核病抗性提高的spl突变体中H2O2异常积累，抗氧化酶活性升高、种子萌发的草酸敏感性降低、水杨酸途径基因激活，水杨酸含量增加。精细定位发现SPL可能为未知功能的类受体胞质激酶，菌核病诱导表达下调水平与菌核病抗性显著正相关。本项目中，我们将分析SPL的时空、诱导表达模式和亚细胞定位；以蛋白互作组和磷酸化蛋白组鉴定SPL互作底物蛋白及其关键磷酸化位点，构建以SPL为核心的磷酸化抗病信号转导网络；比较拟南芥单/双突变体，互补、过表达和RNAi等油菜植株的表型、生理指标和转录组，揭示SPL与底物蛋白互作和磷酸化修饰介导活性氧和水杨酸途径，以提高油菜菌核病抗性的分子机制。研究结果有助于完善植物免疫防御信号转导网络，为油菜及其近缘物种的抗病育种提供理论依据。

油菜(Brassica napus)是世界上最重要的油料作物之一，广泛种植于我国长江中上游流域。核盘菌病害是油菜生产主要的限制因素，虽然在拟南芥中的免疫调控机制已取得了显著的突破，但由于油菜基因组的复杂性，油菜抗病机制的研究还亟待加强。项目组前期从油菜自交系ZS11 EMS诱变群体中鉴定了一个抗菌核病和光依赖的类病斑突变体spl，通过RNA-seq和bulked segregant analysis定位确定了BnaC01.SPL基因为突变基因，该基因编码未知功能的RLCK VIb型蛋白。在本项目资助下，我们明确了BnaC01.SPL调节油菜菌核病防御反应的分子机制，开展的工作如下：首先，利用RNA-seq技术分析了BnaC01.SPL在不同胁迫处理下的表达变化，并通过GUS组织化学染色分析BnaC01.SPL的基因表达模式。其次，我们构建了BnaC01.SPL过表达、干扰和CRISPR编辑植物表达载体并转化ZS11，确定BnaC01.SPL蛋白通过在质膜结构的表达影响油菜菌核病抗性；通过转基因植株表型观察及生理指标的测定，我们发现BnaC01.SPL过表达会提高植株体内水杨酸与脱落酸激素含量，延缓植株生长发育；进一步qRT-PCR和遗传分析证实，BnaC01.SPL在油菜中过表达会显著提升水杨酸合成关键基因的表达，进而提高水杨酸响应病程关键基因NPR1的表达水平。最后，通过在拟南芥中的遗传学实验分析，证实了BnaC01.SPL与SA途径的上下游调控关系，推测SA的合成与转导能促进BnaC01.SPL的表达水平。通过酵母单杂、酵母双杂以及IP-MS实验，我们筛选到BnaC01.SPL上游调控转录因子NAC83和互作蛋白BnaC08.LHCB6，为后续解析BnaC01.SPL调控植株核盘菌抗性的分子机制奠定了基础。

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