拟南芥CJP1蛋白参与茉莉素调控的诱导型抗性的分子机理研究

Jasmonates (JAs) is a vital defense signal to resist the biotic stress and also an essential growth regulator mediated diverse growth and developmental processes. Recently, JA-regulated biotic resistance have been progressively studied: JA activated Ethylene-Responsive Element Binding Factor 1 (ERF) branch and MYC2 branch through the Coronatine Insensitive 1 (COI1) / Jasmonate ZIM-domain Proteins (JAZs)-dependent manner to elevate defense against pathogen infection and insect attack respectively. However, the JA-signaling components that control JA-regulated biotic resistance independent of developmental responses remain to be identified. .Previously, we identified several COI1-dependent JA-regulated proteins using Two-Dimensional Fluorescence Differential Gel Electrophoresis (2-D DIGE) in Arabidopsis, and selected one protein COI1-dependent JA-induced Protein 1（CJP1）for preliminary research. Over-expression and silencing of the CJP1 gene enhanced and reduced the JA-regulated dual defense responses against the necrotrophic pathogen and herbivorous insect respectively, but did not severely alter JA-mediated growth and developmental processes. These findings indicate that CJP1, a new component in JA signal pathway, might play as a positive regulator in plant defense but not a detectable role in plant development. .In this study, genetic, physiological, cytological, biochemical and molecular approaches will be used to reveal the function of CJP1 in JA-regulated biotic resistance and the position of CJP1 in JA signal pathway, therefore demonstrating the molecular mechanism of CJP1 by which plants integrate JA signal to protect themselves from insect attack and pathogen infection. The results of this study would provide new insights into the further understanding of plant stress responses, and might give new ideas for plant molecular breeding in cultivating new varieties with high resistance.

茉莉素调控植株对病原菌侵染和昆虫咬噬等生物胁迫的诱导型抗性；同时，也调节植物的生长发育进程。近年来，茉莉素调控的诱导型抗性机理研究正在深入展开。但是，对茉莉素信号途径中同时提高植物抗病和抗虫性，又不显著影响生长发育的蛋白知之不多。.前期工作中，我们采用2-D DIGE技术鉴定得到多个拟南芥中COI1依赖的茉莉素调控的蛋白。对其中一个COI1依赖的茉莉素诱导表达的蛋白CJP1的初步研究表明：CJP1是一个新的茉莉素信号途径中特异性诱导抗病抗虫双重防御反应的激发子。.本项目中，我们将利用遗传学、生理学、细胞学、生物化学和分子生物学等方法，探讨CJP1在茉莉素介导的抗病和抗虫反应中的作用机制，明确CJP1在茉莉素信号途径中的位置，从而阐明CJP1参与茉莉素调控的抵御生物胁迫的诱导型抗性的分子机理。本项目的实施和完成，将拓展对植物抵御生物胁迫抗性机理的的认识，为抗逆分子育种提供新的理论与技术途径。

植物细胞壁作为细胞和环境间的动态界面，既可保护和支持细胞，又可感知刺激产生信号进而介导细胞对外界环境的反应。灰霉菌是一种广寄主性的腐生型真菌，侵染植物后，可引发多种细胞壁降解酶和细胞壁降解产物的积累。上述成分可被不同膜受体蛋白感知，通过以茉莉素为代表的激素信号系统和MAPK级联传导等传递信号，并诱导植物的抗病性。.拟南芥类受体激酶CrRLK1L蛋白家族中多个成员可通过感受细胞壁的完整性信号影响营养器官和生殖器官发育的不同进程。本项目的目标蛋白THE1是CrRLK1L蛋白家族中的一员，可通过感知细胞壁纤维素的缺失信号调控暗中幼苗的伸长和成熟叶片的扩展。然而，THE1是否可感受灰霉菌侵染引发的细胞壁损伤信号调控防御反应以及参与其中的信号分子仍不清楚。.首先，本项目明确了茉莉素以COI1依赖的方式诱导THE1基因的表达。随后，本项目综合利用生理学、分子生物学和细胞生物学等多种方法对THE1过表达植株和突变体the1植株的灰霉菌抗性进行了深入研究。结果表明：THE1正调控拟南芥对灰霉菌的抗性。进而，通过酵母双杂交和BiFC方法证明THE1与鸟苷酸交换因子GEF4在体内和体外均发生相互作用。接下来，本项目在生理、分子和细胞多个层面对GEF4过表达植株和突变体gef4植株的灰霉菌抗性进行了研究。结果表明：GEF4同样正调控拟南芥对灰霉菌的抗性。为了探究THE1及其互作蛋白GEF4调控灰霉菌抗性的遗传上下游关系，本项目对突变体the1和GEF4过表达杂交植株以及突变体gef4和THE1过表达杂交植株的灰霉菌抗性进行了分析，发现THE1通过GEF4信号通路正调控拟南芥对灰霉菌的抗性。对突变体rop11抗病性分析表明，THE1很可能通过GEF4-ROP11途径调控植物的防御反应。另外，本项目通过转录组测序鉴定了多个THE1信号下游的转录因子、次生代谢催化酶和抗病蛋白等的编码基因，这些基因很可能位于THE1-GEF4-ROP11信号通路下游调控拟南芥对的灰霉菌抗性。其中，部分基因与以JA为代表的植物激素信号通路相关。.综上所述，THE1可通过GEF4-ROP11信号通路调控多个抗病相关基因从而正调控拟南芥对灰霉菌的抗性。这些结果将加深对THE1调控的诱导型抗病机制的理解，拓展对植物抵御生物胁迫抗性机理的认识，为抗逆分子育种提供新的理论与技术途径。