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

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 1081 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 (DE3).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.