一条全新的MPK-WRKY途径调控油菜防御核盘菌的分子机制解析

Oilseed rape (Brassica napus L.) is one of the important oilcrops in China. Sclerotinia stem rot caused by Sclerotinia sclerotiorum frequently affects the yielding and quality of oilseed rape seeds. Therefore, it is essential to study and elucidate the physiological and molecular mechanisms of interaction between oilseed rape and S. sclerotiorum as well as to mine those genes that modulate the tolerance against S. sclerotiorum and explore their signal transduction networks. Mitogen-activated protein kinases (MAPKs/MPKs) and WRKY transcription factors are two classes of important proteins that regulate abiotic and biotic stresses in plants. Our previous studies identitified a bunch of MAPK and WRKY genes that were regulated by S. sclerotiorum in oilseed rape through microarray profilings and identified a wrky mutant that is more tolerant of S. sclerotiorum infection and therefore named it wts1 (wrky tolerant of S.sclerotiorum 1). We further identified that MAPK1 interacted with WTS1 protein in a yeast two-hybrid screening. In this proposal, we plan to characterize this MPK-WRKY signaling pathway in order to understand the implicated defense response and molecular mechanims through gene silencing and knock-out, overexpression, and also construction of constitutive active (CA) MAPK1, microarray identification of the target genes of this pathway. Through this proposal, we expect to elucidate a pathway mediated by MPK1-WTS1 in oilseed rape and also to lay a solid foundation for improving the tolerance of oilseed rape against S. sclerotiorum through modulating the expression level of these important genes.

油菜是我国重要的油料作物之一，核盘菌（Sclerotinia sclerotiorum）引起的菌核病严重危害油菜的产量与品质，研究油菜与核盘菌相互作用分子机制，挖掘其耐受核盘菌侵染的基因并阐明它们作用机制很关键。MAPK与WRKY是植物重要的调控逆境应答的蛋白。我们研究表明核盘菌能调控油菜多个MAPK激酶与WRKY转录因子的表达且wrky tolerant of Sclerotinia (wts1)突变体耐受核盘菌的侵染，还发现一个全新MAPK成员(MAPK1)与WTS1互作，但该MPK1-WTS1调控的抗病反应还未见报道。因此，本项目拟在此研究基础上利用基因沉默、过表达、激酶的组成型活化及靶基因筛选等方法，深入研究WTS1介导的防御信号转导途径及MPK1磷酸化活化WTS1的调控机理，阐明MPK1-WTS1在调控油菜防卫反应中的作用机制，为通过调控关键基因的表达提高油菜抗病性奠定理论基础。

油菜是我国重要的油料作物之一，其生长发育（包括衰老）受到多种内外环境的影响，逆境严重影响油菜的产量与品质。虽然油菜与拟南芥亲缘关系比较密切，但是其基因组经过长期的进化与加倍，具有非常复杂的一面。活性氧（ROS）在植物的逆境应答及生长发育（包括衰老）过程中，起着非常重要的调控作用。而活性氧的动态平衡受到植物自身的严密调控。MPK-WRKY级联途径是植物中保守的、调控逆境应答与激素信号转导的通路。但是，关于这条通路在油菜抗逆、抗病以及ROS信号转导作用中的研究报道寥寥。在前期研究中，我们发现油菜的WTS1的功能类似拟南芥中直系同源基因的功能，即正调控叶片衰老。我们继而发现WTS1促进衰老与ROS信号通路有关，但是具体的分子作用机制不清楚。而且，即使在拟南芥中，WTS1的直接上游MAPK蛋白尚未鉴定，而我们前期工作中发现油菜的MPK与BnaWTS1互作。 在本项目中，我们主要以油菜为研究试材，采用遗传与分子生物学等技术，筛选及鉴定了其作用机制特别是直接靶标。因此，我们希望能挖掘并解析油菜的叶片衰老调控机制。研究结果表明：通过烟草叶片的瞬时表达体系比起拟南芥同源基因WTS1来说，新发现油菜的WTS1能诱导ROS的产生及细胞死亡表现在：过表达BnaMTS1能够造成ROS累积、MDA含量增加、离子渗漏及DNA断裂。进而我们筛选到BnaWTS1能够诱导下游基因RbohD与F的表达，而且通过荧光素酶报告系统发现了BnaWTS1能够转录激活这两个基因的转录；最重要地是，用EMSA直接验证了BnaWTS1能够结合到这两个基因的启动子区域。因此我们的结论是BnaWTS1能够通过调控RbohD与F的表达调控ROS的累积，进而导致植物的叶片早衰。因为RbohD与F能够通过调控ROS累积影响植物的抗病性，因此，我们也正在测试过表达BanWTS1的拟南芥对腐生性真菌核盘菌（Sclerotinia sclerotiorum）及丁香假假单胞菌(Pseudomonas syringae)的抗性。总之，本项目的在前人的研究结果结果基础上继续推动了其BnaWTS1的下游作用机制是通过RbohD与F来调控ROS的产生进而促进衰老，对完善研究植物的衰老作用机制奠定基础。.