Molecular Cloning, Expression Pattern, And3D Structural Prediction Of The Cold Inducible Rna-binding Protein (CIRP) In Japanese Flounder (paralichthys Olivaceus)

Cold-inducible RNA-binding protein(CIRP) is a kind of RNA binding proteinswhich is rich in Gly and it is an important molecular chaperone of mRNA. This is thefirst cold shock protein cloned from mammals. This protein plays an important roleswhen cells were treated with cold shock. Since the cold inducible RNA-bindingprotein was cloned from mammals, it has been researched widely in mammals andamphibians. With further research, it is found that CIRP plays important roles in manyphysiological processes. For example, CIRP is necessary for the hibernation ofamphibians, biological rhythm and developmental progress. There is little reportedabout the functions of CIRP in fish till now. Screening of EST-SSRs makers incommon carps shows that CIRP is related to growth traits and the CIRP is a sexrelated gene according to the comparative analysis of gene expression between femaleand male turbot.In this study, the cDNA of the CIRP gene of the Japanese flounder was clonedand sequenced through RACE and homology-based cloning. The cDNA of thepoCIRP is1596bp long and the length of the ORF is597bp which codes a proteincontains198amino acid. The poCIRP contains the RRM motif in the N terminal and aregion which conatins Arg-Gly-Gly(RGG) repeats and is of rich in Gly.The full length sequence of the poCIRP gene was got from PCR cloning. Thegenomic sequence of the poCIRP consists of seven exons and six introns. The5’flanking sequence was cloned by genome walking and many transcription factorbinding sites were predicted. Besides the TF binding sites, there was a CpGs regionwhich was predicted to be located in the exon I region. The methylation level wassequenced and the results show that the methylation level was low which indicating that the gene was a widely expressed gene.Quantitative real-time PCR analysis uncovered that the highest expression levelof the CIRP was in the ovary, and this gene was widely expressed in adult tissues ofJapanese flounder. The mRNA of the CIRP was maternally deposited and theexpression level of the gene was regulated up during the gastrula and neurula stages.In order to gain the information how the protein interacts with mRNA, weperformed the modeling of the3D structure of the Japanese flounder CIRP. Theresults showed there is a cleft existed on the surface of the molecular. In order to getthe protein, we constructed the expression vector which could produce the coldinducible RNA binding protein.Taken together, the results indicated that the CIRP was a multi-functionalmolecular in teleosts and the findings about the structure provided valuableinformation for understanding the mechanism of this protein’s function.