甘蓝型油菜MKK9基因在菌核病抗性中的功能分析

Sclerotinia stem rot (SSR) is the most devastating disease of rapeseed (Brassica napus). Breeding and cultivation of resistant varieties are the most efficient and economic approach to the control of SSR in rapeseed. However, the molecular mechanism of SSR resistance is still poorly understood. We have performed comparative transcriptomic analysis to understand the differential defense response to S. sclerotiorum in a resistant line and a susceptible line of B. napus (J964，J902), and found that resistance differences between the resistant and susceptible lines are associated with the intensity of defense responses. Mitogen-activated protein kinase (MAPK) signaling cascades play pivotal roles in signal transduction of immune responses. Among the MAPK signaling cascades, we found MKK9 was more intensely induced in the resistant line than in the susceptible line. It might play important roles in SSR resistance. We plan to investigate the contribution of MKK9 to the SSR resistance by gene over-expression and knock-out, and confirm the downstream pathway which regulates by MKK9 by comparing the changes of hormone content, camalexin content, defense-related enzyme activity and transcriptomic before and after inoculation. MAPK signaling cascades in which MKK9 involve will be verified by yeast two hybrid system, bimolecular fluorescence complementation and kinase activity assay. Finally, we will explore the function of MKK9 in resistance to SSR in B. napus through the research above. The study will provide new insights on the molecular mechanism of the SSR resistance, as well as valuable gene resource for the molecular breeding of SSR resistance in rapeseed.

菌核病是制约我国油菜生产最主要的病害。培育抗病品种是防治菌核病最经济有效的途径，但我们对油菜抗菌核病的分子机制还了解较少。申请人前期对菌核病抗性差异明显的2个油菜纯系J964和J902进行比较转录组测序，发现抗感差异与抗病反应的剧烈程度有关，其中在抗病信号传导中起关键作用的MAPK信号级联中的MKK9在抗病材料中诱导表达更加剧烈，很可能对菌核病抗性起重要作用。本项目拟在此基础上分别对该基因进行过表达和敲除，确定其对菌核病抗性的作用。通过对这些材料在正常及接菌条件下的激素含量、植物抗毒素含量、与防卫相关的酶活性、转录组变化等分析来确定MKK9调控的下游途径；通过酵母双杂、双分子荧光互补和激酶活性测定确定抗菌核病反应中MKK9参与的MAPK级联。通过上述研究探索MKK9在抗菌核病反应中的生物学功能，为揭示油菜抗菌核病的分子机理奠定基础，对有效开展油菜菌核病抗性育种具有重要的理论意义和应用价值。

油菜菌核病是由核盘菌（Sclerotinia sclerotiorum (Lib.) de Bary）引起的真菌性病害，每年导致油菜产量损失10%-20%，是制约我国油菜生产最主要的病害。培育抗（耐）病油菜品种是防治油菜菌核病最为经济有效的途径。但我们对抗菌核病的分子机制还了解较少。因此鉴定参与抗菌核病的一些关键基因，并对其进行功能分析，解析油菜抗菌核病的分子调控网络，为揭示油菜抗菌核病的分子机理奠定基础，对有效开展油菜菌核病抗性育种具有重要的理论意义和应用价值。本研究发现BnMKK9受到核盘菌侵染和JA处理的诱导表达。在油菜中过表达BnMKK9DD（激酶持续激活状态）可显著增强油菜菌核病抗性，而拟南芥该基因敲除突变体的菌核病抗性减弱。在烟草中瞬时表达BnMKK9引起H2O2积累，产生过敏性细胞死亡，且瞬时表达BnMKK9DD的烟草对菌核病抗性增强，因此在油菜中过表达BnMKK9DD提高菌核病抗性可能与增加H2O2的积累有关。在拟南芥中诱导表达BnMKK9DD后，拟南芥对核盘菌的抗性显著增强，进一步发现是由于诱导表达BnMKK9DD后，拟南芥抗病相关基因（PDF1.2、PR3和PR4）显著诱导表达，JA和Eth信号转导途径被激活。通过酵母双杂交和Pull-Down发现BnMKK9和BnMPK18互作，且BnMPK18在核盘菌侵染后也诱导表达。进一步发现BnMPK18上与BnMKK9互作结构域为aa 390—437。此外我们发现过表达BnMPK18也显著增强油菜菌核病抗性，而拟南芥mpk18菌核病抗性减弱。因此我们的研究发现BnMKK9-BnMPK18信号级联在油菜抗菌核病反应中起到重要作用。BnMKK9-BnMPK18激活了抗病反应，包括JA/Eth信号转导、抗病相关基因的表达、H2O2积累以及过敏性细胞死亡，从而产生抗病性。课题实施期间发表SCI论文3篇，中文核心期刊论文3篇，申请国家发明专利1 项，培养博士研究生2名，硕士研究生2名。