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Rop(Rho-related GTPase of plant)是植物中存在的一种特殊的小G蛋白。其与GTP(guanosine triphosphate)结合形成激活态,与GDP(guanosine diphosphate)结合形成失活态,并通过其激活态和失活态的转换启动和终止植物多种信号过程。Rop不同结合形式的转换受到一系列调控因子的调控。本研究以一个辣椒(Capsicum annuum L.)Rop蛋白(CaRop1)的组成型激活态CA-CaRop1为诱饵,利用ProQuestTM酵母双杂交体系,从辣椒幼苗猎物文库中分离获得一种Rop GTPase激活蛋白(Rop GTPase activating proteins,RopGAP)基因,将其命名为CaRopGAP3。生物信息学分析表明,CaRopGAP3全长1597bp,包含一个1437bp的开放阅读框,编码478个氨基酸,其氨基酸序列不仅含有3个保守的GAP结构域,其N端还含有CRIB(Cdc42/Rac-interactive binding motif)结构域,属于植物特有的一类RopGAP亚家族。酵母双杂交验证显示CaRopGAP3只能与组成型激活态(CA)的CA-CaRop1互作而不能与显性失活态(DN)的DN-CaRop1互作,且含CRIB结构域的N端对他们之间的互作没有明显的调控作用。亚细胞定位分析显示,CaRopGAP3主要分布于细胞膜上,且含CRIB结构的N端在其膜定位中起重要调节作用。荧光定量PCR分析显示,CaRopGAP3基因在辣椒幼叶中的表达量最高,约为幼根的17倍、成熟根和花器官的8倍。CaRopGAP3基因的这种结构及组织表达特点可能与其参与的特定信号路径密切相关。本研究为进一步解析辣椒CaRop1介导的信号调控机制提供基础数据。
Rop (Rho-related GTPase of plant) is a special small G protein present in plants. It binds to GTP (guanosine triphosphate) to form an inactive state, which binds to GDP (guanosine diphosphate) to form an inactive state and initiates and terminates a variety of signaling processes in plants by switching between its active and inactive states. Rop different combinations of conversion by a series of regulatory factors. In this study, we used the pro-yeast two-hybrid system CA-CaRop1, a constitutively activated form of Capsicum annuum L. Rop protein (Caop1), as a bait to isolate a Rop GTPase activating protein (Rop) GTPase activating proteins, RopGAP) gene, named it CaRopGAP3. Bioinformatics analysis showed that CaRopGAP3 is 1597 bp in length and contains a 1437 bp open reading frame encoding 478 amino acids. The amino acid sequence of CaRopGAP3 contains not only 3 conserved GAP domains but also CRIB (Cdc42 / Rac-interactive binding motif domain, belonging to plant-specific RopGAP subfamily. Yeast two-hybrid assays demonstrated that CaRopGAP3 interacts only with CA-CaRop1 in a constitutively activated state (CA) but not with DN-CaRopl in dominant-negative state (DN), and contains the N-terminus of the CRIB domain to them No obvious interaction between the role of regulation. Subcellular localization analysis showed that CaRopGAP3 mainly distributed on the cell membrane, and N-terminal of CRIB structure played an important regulatory role in membrane localization. Quantitative real-time PCR analysis showed that the CaRopGAP3 gene expressed the highest level in young leaves, about 17 times that of young roots and 8 times of mature roots and floral organs. This structure and tissue expression characteristics of CaRopGAP3 gene may be closely related to the specific signal pathways involved. This study provides the basic data for further analysis of CaRop1-mediated signaling regulation in pepper.