彩鲫Ran基因全长cDNA的克隆及其组织表达特异性分析

Full Length cDNA Cloning and Tissue Expression Specificity of Ran Gene in Color Crucian Carp

根据Ran的保守区序列设计简并引物 ,结合SMARTcDNA合成及RACE PCR技术 ,克隆到彩鲫(Carassiusauratuscolorvariety)的Ran基因的cDNA全长 ,其编码区全长为 6 4 8bp ,编码 2 15个氨基酸。采用BLAST程序在NCBI数据库中对其进行同源基因搜索 ,结果表明 ,其推测的编码氨基酸序列与斑马鱼和鲑鱼的Ran基因编码的氨基酸序列的同源性分别高达 98%和 97%。还对其编码区全长序列进行了原核表达 ,以经纯化的表达蛋白免疫家兔 ,制备出了具有较高特异性的多克隆抗体。采用Westernblotting进行的组织特异性分析表明 ,Ran蛋白在卵巢、精巢和肾中表达 ,在心、脑、肝、脾和肌肉中不表达。本工作为采用免疫耗竭法、免疫共沉淀、体外系统模拟等方法进一步研究鱼类Ran基因的生理功能及分离鉴定其结合蛋白提供了良好基础。

A full-length cDNA of Ran gene was cloned from color crucian carp (Carassius auratus color variety) by using SMART cDNA synthesis and RACE-PCR with a pair of degenerate primers designed according to the conserved region sequence of Ran gene.The Ran cDNA was found to be 1?081 bp in length with a 67 bp 5UTR and a 366 bp 3 UTR.The coding region included 648 nucleotide acids and encoded 215 amino acids.Searching homologous genes by using this nucleotide sequence in NCBI database showed that the deduced amino acids sequence of Ran gene of color crucian carp shared high identity with Ran genes of Dano rerio (98%) and Salmo salar (97%).Moreover,the full-length sequence of its coding region was expressed in E.coli (DE 3).To acquire multiclonal antibody,the expressed protein was purified and applied to immunize rabbits.Western blotting results indicated that the multiclonal antibody prepared by us was highly specific to recognize Ran proteins expressed in E.coli or that from eggs of color crucian carp.In addition,analysis on tissue expression specificity indicated that Ran protein was expressed in ovary,testis and kidney,whereas was not detected in heart,brain,liver,spleen and muscle.The present studies will facilitate our future studies on the physiological functions of Ran gene,the isolation and identification of its binding proteins in fish by using immunodepletion,coimmunoprecipitation,and simulative systems in vitro.