【作者】 胡晓晶；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 硕士

【摘要】 根瘤菌与豆科植物能特异地形成共生体-根瘤。这种共生关系的建立涉及到低等微生物与其宿主植物之间复杂的信号识别、基因表达调控、分子间相互作用等过程。百脉根是研究共生固氮体系的模式豆科植物之一,对其根瘤菌结瘤因子信号转导途径和作用机制的研究,将有助于揭示根瘤菌与豆科植物共生关系建立和共生体形成的奥秘。百脉根结瘤信号通路NSP1和NSP2是根瘤菌共生信号转导途径中关键的信号蛋白,都属于GRAS家族转录因子,位于CCaMK (Ca2+/calmodulin-dependent protein kinase)下游,并受其调控。本研究的侧重点在于探究NSP1与NSP2间是否存在相互作用,相互作用区段以及检测NSP1基因是否具有结合启动子的能力以及行使此功能的区段,进而揭示NSP1、NSP2在结瘤因子信号传导途径的调控机制。其研究结果如下：1.构建了一系列nsp1和nsp2缺失突变体,利用酵母双杂交系统研究了百脉根结瘤信号通路NSP1和NSP2蛋白间的相互作用。结果表明NSP1具有与NSP2相互作用的功能,而且NSP2单独的LHR1结构域(105-186氨基酸)则能与NSP1相互作用,说明NSP1与NSP2在行使功能时可能形成异源二聚体。2.构建了一系列在大肠杆菌中表达的缺失突变体,并表达和纯化缺失不同结构域的截短蛋白,利用pull-down和western印迹转移技术进行了体外蛋白质相互作用。结果表明,NSP1缺失LHR1、VHIID两个结构域后仍具有与NSP2蛋白相互作用的功能,而缺失LHRII区段后则不能相互作用,说明NSP1的LHRII区段是两蛋白间相互作用所必需的。3.利用凝胶阻滞(EMSA)实验,初步检测了NSP1是否结合启始结瘤基因NIN的启动子的功能,但没有得到理想的实验结果,其原因可能是所用的DNA片段太长(500bp),在EMSA凝胶上只见到一些弥散的DNA带,因此该结果需要进一步证实。更多还原

【Abstract】 The leguminous plants can specifically establish a symbiosis "nodule" with rhizobia. The establishment of the symbiotic relationship is involved in the complex process incloding signal perception, gene expression and regulation, the interactions of intermolecules between microbes and host plants. Lotus japonicus is one of the model legumes plants for studying the system on the symbiotic nitrogen fixation. The research of the Nod factors signaling pathway and the mechanism of intermolecular interaction in Lotus japonicus will uncover the secret about the establishment of symbiotic relationship and the formation of the symbiosis.In Lotus japonicus, the proteins of NSP1 (Nodulation Signaling Pathway 1) and NSP2 are belonged to the GRAS family transcription factors, which are the pivotal signal proteins in the symbiotic signaling pathway, and in the downstream of the CCaMK. In this study, we focus on the protein-protein interactions between NSP1 and NSP2, characterization of interaction domain, and identification of the role of the NSP1 binding to the promoter of taget genes. The results are as follows:1. Constructing a series of deletion mutants of NSP1 and NSP2, we assessed the interactions between NSP1 and NSP2 using yeast two-hybrid approach. The results indicate that NSP1 can interact with NSP2 and the LHRI domain alone (105-186aa) of NSP2 is necessary for the interaction. This suggests that NSP1 and NSP2 may form heteropolymers for performing Nod factors signaling in L. japonicus.2. A series of full and truncated proteins of NSP1 and NSP2 were expressed and purfitied from E. coli, which contains different domains. The protein-protein interactions in vitro were performed using pull-down and detected with western blotting. The results showed that the NSP1 in the absence of LHR1 and VHIID could be interacted with NSP2, and the interaction was abolished without the domain of the LHRII. This reveals that the LHRII of NSP1 is involved in the interaction between NSP1 and NSP2 in L. japonicus.3. Using the electrophoretic mobility shift analysis (EMSA), we tried to test whether NSP1 could directly bind to the NIN gene promoter which is a initial nodulation gene. The preliminarily results just showed some DNA smear on the EMSA gel and need to be further demonstrated.更多还原