OsCBSX3在水稻应答病虫害中的功能及分子机制研究

Two interconnected layers of plant immunity, termed pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), are employed by plants to defend thermselves against the attacks of various pathogens. A crucial step in these two layers of immunity is the transcriptional regulation of vast arrays of defense associated genes. In Arabidopsis thaliana the cluster of LURP (late upregulated in response to Hyaloperonospora arabidopsidis) genes was found to exhibit a coordinated transcriptional upregulation in response to pathogen infection. LURP genes play an important role downstream of PTI and ETI induction. Their functional characterization as well as clarification of the regulation of their expression wil be helpful for our understanding of mechanisms of plant immunity. In the present project, we will study the function and transcriptional regulation of a LURP member of rice, termed OsCBSX3, which was found previously to respond by transcript accumulation to Magnaporthe oryzae inoculation and Cnaphalocrocis medinalis attack. The function of OsCBSX3 in rice responses to Magnaporthe oryzae and Cnaphalocrocis medinalis attack will be characterized by its overexpression and RNAi-mediated silencing in transgenic rice plants. We will also examine its possilbe involvement in defense signaling pathways dependent on the phytohormones salicylic acid (SA), jasmonic acid (JA) or ethylene (ET) by analyzing transcript levels of marker genes of the respective pathways in these rice lines. In addition, a 5' deletion analysis of OsCBSX3 promoter will be carried out to identified regions or cis-elements responsible for the responsiveness of this gene to Magnaporthe oryzae, Cnaphalocrocis medinalis, SA, JA or ET. Based on this project, the function of OsCBSX3 in rice pathogen and pest resistance and the respeive underlying mechanisms will be clarified, which will be helpful for the genetic improvement of rice disease and pest resistance.

植物采用PTI和ETI两个彼此相互关联的抗病反应来对付入侵的病原菌，后期持续上调蛋白（LURP）是PTI和ETI下游信号通路中共享的一类蛋白，可能在植物抗病反应中起重要调节作用，鉴定其功能和剖析表达调控机制有助于植物抗病机制的阐释。本项目选择应答稻瘟病菌和稻纵卷叶螟的水稻LURP基因OsCBSX3，开展其功能及表达调控机制研究。项目通过OsCBSX3过表达和RNA沉默，分析水稻抗病和抗虫能力的变化以及对SA、JA和ET等信号途径相关基因表达的影响。通过分析其启动子系列缺失突变体的诱导活性，寻找启动子上应答稻瘟病、稻瘟病菌和外源SA、JA或ET处理的区域及相应的作用元件。实施本项目可望进一步明确OsCBSX3在水稻抗病和抗虫中的作用及其与SA、JA或ET信号通路之间的关系，揭示其应答病原菌持续上调的分子机制，为植物抗病和抗虫遗传改良提供帮助。

CBS结构域包含蛋白(CBS domain containing protein, CDCP)组成了植物中一类较大的家族，参与了植物体内的多种生物学过程。本项目克隆的水稻OsCBSX3全长639个碱基，编码213个氨基酸，序列分析表明包含有两个CBS结构域的蛋白。利用荧光定量PCR技术分析了该基因的表达特征，结果表明，在稻瘟病，稻纵卷叶螟，外源激素（SA和MeJA）处理条件下都表现为显著的上调表达。利用农杆菌在烟草叶片瞬间表达进行亚细胞定位分析，表明OsCBSX3-GFP融合蛋白定位于细胞膜上。利用T3代转基因株系进行了水稻的抗病或抗虫分析，结果表明OsCBSX3在转基因水稻中过表达可以提高对稻瘟病或稻纵卷叶螟的抗性。在接种稻瘟病菌5 d后，观察转基因株系和野生型水稻植株叶片的发病情况，结果显示转基因株系的病斑大小和病斑数目相比野生型都要小。利用荧光定量PCR技术分析了水稻叶片的稻瘟病菌DNA含量，发现两个转基因株系（#1和#3）比野生型稻瘟病菌DNA含量都要低。在接种30 d后，转基因株系比野生型表现出跟好的抗性状态。在抗虫分析方面，转基因水稻接种稻纵卷叶螟后的卷叶率明显低于野生型，损害级别也明显较低。OsCBSX3启动子1400bp区间范围内预测到CGTCA-motif，W box等多个应答逆境的元件。以OsCBSX3启动子诱导GUS的转基因水稻为材料，进行GUS活性分析，发现在稻瘟病或稻纵卷叶螟诱导下OsCBSX3启动子能够增强GUS基因表达。进一步对启动子缺失体的GUS活性分析表明，应答稻瘟病菌的最短片段是-191bp。该片段中的W box元件可能是应答稻瘟病菌的关键元件。