【作者】 周平；

【导师】 刘榜；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 哺乳动物主要组织相容性复合体-Ⅲ(MHC-Ⅲ)涉及免疫、自体免疫及常见复杂疾病的易感性的特点已经在多种群体中被广泛研究。MHC-Ⅲ区域包含各种不同类型的基因,虽然其中大多具有较早的起源,但有些似乎只存在于脊椎动物中。MHC-Ⅲ区域包含诸如补体成份、类固醇-21-羟化酶、热激蛋白、肿瘤坏死因子等编码基因以及一些功能尚不明确的基因,其中包括本研究所涉及的G4基因。本研究从蛋白序列及结构特性、基因表达模式、蛋白分子进化、亚细胞定位、蛋白间相互作用等角度对G4蛋白进行研究,取得了如下结果:1.生物信息学分析发现,G4蛋白在人、黑猩猩、猕猴、小鼠、褐鼠、牛、猪、马、短尾负鼠、鸭嘴兽等物种的序列同源性高达68.09%;主要包含3个高度保守的区段,分别对应小鼠G4蛋白序列的氨基端1-18aa区段、富含Ser的34-52aa区段以及靠近羧基端的173-260aa区段;小鼠G4蛋白不具有明显可辨的N-端信号肽序列;小鼠G4蛋白大致分为两个结构域,即氨基端的亲水球状结构域和羧基端的疏水跨膜结构域。2.利用半定量RT-PCR技术对猪及小鼠G4基因的组织表达分布进行了检测,发现G4基因在所检测的组织中广泛表达,同时检测到小鼠G4基因在脊髓中具有相对较高的表达量;3.在BHK-21细胞中瞬时转染G4蛋白表达载体后,经CCK-8分析显示,野生型G4蛋白对BHK-21细胞增殖无影响,G123A和Y167F位点的单突变型能降低BHK-21细胞的增殖速率,而G123A和Y167F位点的双突变却使BHK-21细胞增殖的速率出现回升,甚至超过野生型的表现;4.对外源表达的猪G4蛋白在PK-15细胞中的亚细胞定位进行分析发现,G4-GFP融合蛋白存在于细胞质中,荧光信号在胞质中的分布具有明显的颗粒感;进一步用红色荧光融合表达载体在BHK-21细胞中对小鼠的G4蛋白亚细胞定位进行分析发现,小鼠G4蛋白定位于线粒体上;N-末端18个氨基酸残基的缺失不影响小鼠G4蛋白的亚细胞定位;5.免疫共沉淀分析显示,在HEK-293细胞中外源表达G4蛋白和FGFR3后,在未经FGFs刺激的情况下未能检测到G4蛋白与FGFR3之间存在相互作用;本研究在实验结果、文献及生物信息学分析的基础上,提出了以下几点认识:1.通过分析G4基因及与G4蛋白有关的五种蛋白(FGFR3、GSK-3、CK1、CK2和CaMKⅡ)的组织表达分布,提出G4基因可能在神经系统中扮演较为重要的角色;2.通过对小鼠G4蛋白两个氨基酸残基位点的变异分析,结合G4蛋白的分子进化,认为蛋白序列中若干非中性或近中性的点突变可能整合为一段中性的进化过程;3.综合G4蛋白序列及结构特征、亚细胞定位结果及其与FGFR3的相互作用,认为G4蛋白通过其羧基端的疏水跨膜区域定位于线粒体的外膜上;4.根据G4蛋白的亚细胞定位以及FGFR3信号途径的特点,提出G4蛋白与FGFR3的相互作用发生在FGFR3受到激活并内化之后,FGFR3以内吞小泡的形式靠近线粒体进而与线粒体外膜上的G4蛋白发生相互作用。更多还原

【Abstract】 The mammalian Major Histocompatibility Complex-Ⅲ(MHC-Ⅲ) has been widely studied on the characteristics related to immunity,autoimmunity,and susceptibility to common complex diseases.Many different types of genes have been discovered in MHC-Ⅲ,although the majority of the genes can be traceable in ancient time,some of them seems to be confined to vertebrate.MHC-Ⅲcontains genes for complement components,steroid 21-hydroxylase,heatshock proteins,tumor necrosis factor,and a number of genes with unknown function,such as G4 gene studied in this research.Several aspects,such as protein sequence and structure characteristics,gene expression profile,molecular evolution of proteins,protein subeellular localization,and interaction of proteins have been considered in this study of G4 protein.The findings show that:1.Mouse G4 protein shares 68.09%with human,chimpanzee,macaque,brown rat, cattle,pig,horse,opossum and platypus et al.;There are three high conservative regions in G4 protein sequence,N-terminal 1-18aa region,Ser-rich 34-52aa region and near C-terminal 173-260aa region;Signal peptide has not been discovered within the N-terminal sequence of mouse G4 protein;There are roughly two structural domains in the mouse G4 protein,the N-terminal hydrophilic globular domain and the C-terminal hydrophobic trans-membrane domain.2.RT-PCR showed that G4 is widely expressed in the detected tissues,and the expression of mouse G4 gene in spinal cord is relatively high.3.The impacts of G4 protein on the cell proliferation in BHK-21 was evaluated by Cell Counting Kit-8 assay.The results showed that the cell proliferation rate does not influenced by the wild type G4 protein,but reduced by the G123A or Y167F single site mutation types,whereas promoted by the G 123A/Y 167F double site mutation type.4.Subcellular localization of pig G4 protein exogenous expressed in PK-15 cell-line has been analyzed.It suggested that G4-GFP fusion protein is located in the cell cytoplasm,in which the fluorescent signal is presented like a granular image.Further studies on mouse G4 protein subeellular localization have been performed in BHK-21 cell-line with RFP fusion expression plasmid.It has been discovered that mouse G4 protein is located on the mitoehondria.The subcellular localization of mouse G4 protein can not be influenced by the truncation of its N-terminal 1-18aa region.5.The interaction between G4 protein and FGFR3,exogenous expressed in HEK-293 cell-line,has not been detected with co-immunoprecipitation under no FGFs stimulation.Based on the experimental findings,cited references and bioinformatics analysis, several views have been presented as follows:1.G4 gene may play an important role in the nervous system according to the expression profiles of G4 gene and other five proteins relate to G4 protein,including FGFR3,GSK-3,CK1,CK2 and CaMKⅡ.2.Several non-neutral or near neutral mutations in the protein sequence may be integrated as a neutral process of molecular evolution,based on two residues mutations analysis of the mouse G4 protein and the protein molecular evolution.3.Integrating the protein sequence and structure characteristics,subcellular localization,and interaction with FGFR3,G4 protein is indicated to be located on the outer mitoehondrial membrane through its C-terminal hydrophobie trans-membrane domain.4.According to the G4 protein sub-cellular localization and the FGFR3 signaling profile,the interaction between G4 protein and FGFR3 is considered to happen after the internalization of FGFR3 which is activated.The activated FGFR3 approach the mitochondria in the form of endoeytie vesicle,and then interact with the G4 protein which is located on the outer mitochondrial membrane.更多还原