钙离子介导拟南芥病原菌感受相关基因EIC1的功能研究

Calcium (Ca2+) signals have been implicated in regulating many aspects of plant growth and responses to the environment. However, how plants perceive pathogene and induce increase of cytosolic Ca2+ concentration ([Ca2+]i) is still unknown. Preliminary study shows one of the earliest signaling events after pathogen perception is a rapid change in the [Ca2+]i and concomitant membrane depolarization. Based on this, we made the EMS mutant pool in which Arabidopsis thaliana constitutively expressing photoprotein APOAEQUORIN which sensitive to Ca2+ and try to find the mutants defects in early calcium signaling in response to flg22 induced biotic stress. During screening we identified five candidates that defact in flg22 induced [Ca2+]i increase and cloned gene EIC1. Preliminary bioinformatics analysis showed that the protein containing transmembrane domains. Its homologous genes have been proved to be related to multiple drug resistance in animals, and functions in plants have rarely been reported. According to the existing research basis, EIC1 is likely to be the Ca2 + channel protein induced by the pathogen we are looking forIn this research Moreover, we will analysis the EIC1 gene founction via electrophysiological techniques, Cameleon techniques and so on. The outcome of this project will demonstrate EIC1 is a component of biotic stresses induced Ca2+ channel and further elucidate the molecular mechanism of plant resistance to adversity. The results of this project provide new ideas for the study of plant disease resistance.

钙信号是植物响应环境胁迫早期复杂信号通路的重要组分，对植物生长和环境响应都起到调节作用。然而，植物是如何感知病原菌并且诱导胞内[Ca2+]i升高以及植物免疫相关的Ca2+离子通道都还不清楚。基于此，本项目拟对持续表达水母荧光蛋白APOAEQUORIN的EMS诱变突变体进行筛选，寻找病原菌flg22诱导[Ca2+]i浓度升高缺失的突变体，克隆相关基因并研究其功能。目前克隆到ABC（ATP binding cassette）转运蛋白家族成员EIC1，该蛋白具有跨膜结构域,在植物中的功能鲜有报道。根据现有研究，EIC1很有可能是我们所要寻找的病原菌诱导的Ca2+通道蛋白。我们将以EIC1为研究对象，拟采用电生理及Cameleon显影技术等研究方法，对EIC1基因功能进行深入研究，探索生物胁迫如何诱导钙离子通道及传递钙信号这一科学问题，阐明钙离子介导植物感受生物胁迫并启动下游防御的分子机制。

本研究在已筛选获得的五个表型较好的生物胁迫诱导钙离子信号缺失候选突变体株系，命名为eic1，eic2，eic3，eic4，eic5（defect in elicitors-induced [Ca2+]i change）中克隆到其中两个在flg22处理下钙信号明显减弱的基因。在此基础上对其中EIC1基因展开深入研究，研究发现EIC1基因可以互补eic1突变体在flg22诱导下钙信号缺失的表型，说明EIC1基因与此表型直接相关。在此基础上，通过实验证明EIC1基因参与植物的抗生物胁迫过程，且具有特异性。并且细胞亚定位显示EIC1基因位于细胞膜上，转染HEK293细胞和非洲爪蛙卵母细胞中检测EIC1基因，发现2uM flg22处理时，胞内钙离子浓度较处理前明显上升。且检测抗病相关基因表达发现在病原菌入侵后和野生型有明显差异。因此，我们推测EIC1是植物钙离子介导的生物胁迫的离子通道及其相关蛋白。