【作者】 代广中；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2011， 硕士

【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EF1A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EF1A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cDNA引物,RT-PCR扩增得到翻译延伸因子EF1A全长cDNA。表达翻译延伸因子EF1A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EF1A存在相互作用。利用小样rnating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NFR1,共生受体激酶SYMRK和离子通道蛋白CASTOR与EF1A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EF1A在酵母体内不能够相互作用。更多还原

【Abstract】 The early signaling transduction of symbiosis system in which the Rhizobium interacting 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 Lotus japonicus. 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 the signaling 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 phenotype, physically. We constructed BD-C-Pollux bait plasmid, and used it to screen the Lotus Yeast two-hybrid cDNA library. There were lots of clones including the translation elongation factor 1A acquired from library interacting with POLLUX. The positive clones were identified by mating in yeast again, then sequenced and searched by program BLAST on NCBI net. We confirmed that the translation elongation factor 1A (EF1A) interacted with C-POLLUX.Then we designed the primers of EF1A according to the sequence on the database of Lotus japonicus, isolated the total RNA, obtained of the translation elongation factor 1A full length cDNA by RT-PCR. To express the protein in E.coli, the fusion tag plasmid were constructed, such as pET28a-EF1A, pET28a-ED219, pET28a-ED267, pGEX-6p1-C-Pollux, and then the fusion proteins were purified with the suitable beads. Pull down and Western blotting assays confirmed the interaction between C-POLLUX and EF1A in vitro. And further more EF1A interacting with other signaling genes were checked by small scale mating in yeast cell, the results were including that NFR1, SYMRK and CASTOR interacted with EF1A in yeast cells while NFR5 or CCaMK did not.更多还原