Cloning And Sequence Analysis Of The Major Histocompatibility Complex Gene In The Cyprinus Carpio Var Jian

Cyprinus carpio var.jian is a new breed developed by the use of the integrated breeding technology combining techniques of genealogical selection, hybridization of multi-line and gynogenesis development in Freshwater Fisheries Research Renter. Cyprinus carpio var.jian has a fastgrowth, strong adaptability and disease resistance. In recent years, the research focused on selection and breeding, about the immune-related genes are limited. Major histocompatibility complex is widely found in vertebrates, closely relation immune function and encode immunoglobulin-like receptors, a highly polymorphic genes. It plays a very important role in the immune system. This protein that gene encodes commonly known as MHC molecules or MHC antigens, according to the structure and function, it divided into Ⅰ, Ⅱ and Ⅲ categories. In this study, using homology cloning and RACE, we report the clonging and sequencing of three MHC genes from Cyprinus carpio var.jian, aquaculture species by Freshwater Fisheries Research Center.According to the mRNA sequence of MHC Ⅰ A gene registered in GeneBank, the Primer Premier5.0was adopted to design a pair of primers, amplified the target gene of Cyprinus carpio var.jian, cloning and sequencing, the full-length cDNA is1914bp. Analysis of Cyprinus carpio var.jian showed that MHC Ⅰ A comprises118bp5’terminal untranslated region,1044bp encoding region and752bp3’terminal untranslated region. The347amino acids encoded including the signal peptide, peptide binding region immunoglobulin-like region, transmembrane region, cytoplasmicregion.Bybiology software showed, there are three glycosylation sites, three protein kinase C phosphorylation sites, three casein kinase Ⅱ phosphorylation sites, four myristoylation sites, one Microbodies C-terminal targeting signal, one immunoglobulin and major histocompatibility complex protein signature. The alignment result showed that the identity between Cyprinus carpio var.jian MHC I A amino acid sequence and that of Cyprinus carpio was highly66.0%, and the identity with those of Oncorhynchus mykiss, Salmo salar, Oryzias latipes, Takifugu rubripes were54.5%,57.9%,44.3%,42.0%, the identities with those of Mus musculus, Rattus norvegicus, Homo sapiens were29.1%,28.7%,29.7%, respectively. According to the mRNA sequence of MHC ⅡA gene registered in GeneBank, the Primer Premier5.0was adopted to design a pair of primers, amplified the target gene of Cyprinus carpio var.jian, cloning and sequencing, The full-length cDNA is877bp. Analysis of Cyprinus carpio var.jian cDNA showed that MHC ⅡA comprises65bp5’terminal untranslated region,705bp encoding region and107bp3’terminal untranslated region. The235amino acids encoded including the signal peptide, peptide binding region immunoglobulin-like region, transmembrane region and cytoplasmic region. By biology software showed, there are one glycosylation site, one protein kinase C phosphorylation site, six casein kinase Ⅱ phosphorylation sites, five myristoylation sites, one immunoglobulin and major histocompatibility complex protein signature. The alignment result showed that the identity between the putative Cyprinus carpio var.jian MHC ⅡA amino acid sequence and that of European carp was highly89.7%, and the identity with Nile tilapia was47.6%.According to the mRNA sequence of MHC ⅡB gene registered in GeneBank, the Primer Premier5.0was adopted to design a pair of primers, amplified the target gene of Cyprinus carpio var.jian, cloning and sequencing, the full-length cDNA is1487bp. Analysis of Cyprinus carpio var.jian showed that MHC ⅡB comprises26bp5’terminal untranslated region,759bp encoding region and702bp3’terminal untranslated region. The252amino acids encoded including the signal peptide, peptide binding region immunoglobulin-like region, transmembrane region and cytoplasmic region. By biology software showed, there are one glycosylation site, one cyclic adenosine monophosphate and guanosine monophosphate-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, eight casein kinase Ⅱ phosphorylation sites, four myristoylation sites. The alignment result showed that the identity between the putative Cyprinus carpio var.jian MHC ⅡB amino acid sequence and that of European carp was highly88.3%, and the identity with danio rerio was71.6%, and the identities with those of salvelinus fontinalis, verasper variegates, oryzias latipes were only53.2%,52.2%,52.0%.As MHC is closely related with fish immunity, the paper which cloning and sequence analysis of Cyprinus carpio var.jian MHC, will provide a basis for further study about mechanism of MHC molecules and the relationship between MHC gene polymorphism and fish disease resistance.