百脉根离子通道蛋白pollux及其与翻译延伸因子ef1a相互作用的分析-analysis of the interaction between pulse root ion channel protein pollux and translation elongation factor ef1a.docx

百脉根离子通道蛋白POLLUX及其与翻译延伸因子EFIA相互作用的研究摘要根瘤菌与豆科植物相互作用早期信号转导成为近年来共生体系研究的热点问题。共 生信号转导研究中，苜蓿和百脉根两种模式植物部分关键信号转导基因相继克隆。对这 些关键基因功能和作用机制研究，揭示其相互调控因素，有利于阐明共生信号转导的分 子机制。酵母双杂交技术为搜寻信号转导基因调控因子提供了一条有利途径。离子通道蛋白POLLUX是共生信号途径上游的关键功能基因，其突变后，百脉根 除不能结瘤外，生理上失去了钙离子振荡表型。本实验以以百脉根POLLUX膜内区段 (C．Pollux)为模板，设计引物构建了诱饵质粒pGBKT7．C-Pollux。以pGBKT7．C-Pollux为诱饵，筛选百脉根酵母双杂交文库，获得翻译延伸因子EFlA等多个与POLLUX膜内区段相互作用的克隆子。酵母回转实验、测序及NCBI-BLAST分析，表明翻译延伸 因子EFlA与离子通道蛋白POLLUX膜内区段(C．POLLUX)相互作用。依据百脉根数据库BLAST分析得到的序列，设计eDNA引物，RT-PCR扩增得到 翻译延伸因子EFlA全长eDNA。表达翻译延伸因子EFlA及其两个截短区段ED219、 ED267和离子通道蛋白C．POLLUX，并利用相应琼脂糖凝胶珠分离纯化目的蛋白。通 过Pull．down和Western Blotting实验证明离子通道蛋白C．POLLUC与翻译延伸因子 EFIA存在相互作用。利用小样mating方法，检验其他关键共生信号转导基因与翻译延 伸因子在酵母体内相互作用，表明结瘤因子受体NFRl，共生受体激酶SYMRK和离子通 道蛋白CASTOR与EFlA在酵母体内存在相互作用，而结瘤因子受体NFR5、钙和钙调 素结合蛋白CCaMK与翻译延伸因子EFlA在酵母体内不能够相互作用。关键词：百脉根：离子通道蛋白；酵母双杂交：翻译延伸因子华中农业大学2011届硕士研究生学位论文AbstractThe early signaling transduetion of symbiosis system in which the Rhizobium iIlterac血g legume plants with each others has been most focused on by global researchers．Multi of signaling key genes has been cloned from the two model plants，Medicago truncatula and Lotusjaponicus．To confirm the function and action mechanism,and how to inter-regulate of these genes，are useful for understanding the molecular mechanism of the symbiosis system． It’S a useful tool utilizing Yeast two-hybrid assay to find the regulation factor for thesignaling genes．Cation channel protein,POLLUX had function as an important role in the upstream symbiotic signaling net．The pollux mutant,could not form nodules and Was lack of calcium spiking phenotypc，physieally．We constructed BD-C-Pollux bait plasmid，and used it toscreen the Lotus Yeast two-hybrid eDNA library．There were lots of clones including thetranslation elongation factor 1 A acquired from library interacting、析m POLLUX．The positiveclones were identified by mating in yeast again,then sequenced and searched by programBLAST on NCBI net．We confirmed that the translation elongation factor 1A(EF IA)interacted with C—POLL