The Analysis Of Structure, Polymorphism And Tissue Expression Of MHC Class Ⅰ Gene, And The Complete Mitochondrial Genome Of The Spotted Scat Scatophagus Argus

We used RACE-PCR and clone to get the complete sequence of MHC(Majorhistocompatibility complex) I α and β2m-microglobulin of Scatophagus argus. Thenwe analyzed its genome sructure, tissue and polymophysm of MHC I α. Also, thecomplete mitochondrial sequence of S. argus was obtained. These results will be usefulto MHC genome polymophysm and anti-disease, and enrich the mitochondrial genelibrary.1Analysis of genome structure and tissue expression of MHC I α of S.argusIn terms of RACE, it is the first time to get the complete sequence of MHC I αcDNA of S. argus with the length of2020bp. The Opening reading fram（ORF）is1068bp,which encoded a40.09kDa protein of365amino acids. A signal peptide wasdeposited in the1-21residues. The length of5’-Untranslated Region is87bp,3’-UTRis866bp. There was a polyadenylation tailing signal AATAAA and polyA tail. Aminoacid sequences have all the characteristics of the functional MHC I α molecular. Suchas, N-myristoylation site, N-glycosylation site, Protein kinase C phosphorylation site,Tyrosine kinase phosphorylation site, cAMP-and cGMP-dependent protein kinasephosphorylation site, Casein kinase II phosphorylation site, a IG-LIKE(Immunoglobulin-like region) at204~278site. The protein of MHC I α melocular of S.argus is mixed, helix is23.4%，strand is28.5%，loop is48.2%. It was revealed in theanalysis of amino aicd that S. argus had the most close relationship with giltheadseabream and sea bass, farther with salmon and trout, farther and farther with frog, birds and homan. MHC I α gene of S. argus contained6introns and7extrons. Thelength of the introns could be541bp、335bp、446bp、655bp/691bp、433bp、300bp.The intron4could be extended to two kinds of different lengths, which meanspolymophysm, while intron6can separete the3’-UTR. All introns donor/acceptor sitesof MHC I α gene of S. argus matched the consensus motifs (gt/ag). Furthermore,realtime PCR analysis demonstrated that the MHC I α gene were ubiquitouslyexpressed in ten tested tissues and their expression levels were different. In fact, highexpression levels of α gene were observed in immune organs such as kidney and spleen.While in organs such as gills, intestines and muscles that are likely to be frequentlyexposed to bacterial antigens, the expression levels was low.2the polymorphism of MHC I α gene of S. argusWe identified208different ecoding sequences of the MHC I α gene within210positive clones from42individuals for the spotted scat S. argus. The nucleotides were1065bp~1074bp in length and encoding345~357amino acid, which including signalpeptide, α1, α2, α3domains and TM/CY region.208cDNA sequencs ecoded163different amino acid sequences and could be identified into20alleles. The percentagesof polymorphic sites of MHC I α gene in nucleotide and amino acid were53.22%and70.22%. The ω（ω=dN/dS）of α1and α2domains for PBR were both above1, otherswere less than1, which seggesting that positive slection pressure conducted on theα1and α2domains. It was revealed in the analysis of amino aicd that S. argus had themost close relationship with Dicenrarchus labrax L., farther with Oncorhynchusmykiss, Sparas aurata, Cyprinus carpio and Brachydanio rerio.3Analysis of genome structure and tissue expression of β2m of S.argusIn terms of RACE-PCR, it is the first time to get the complete sequence of β2mfrom S. argus with the length of905bp. The Opening reading fram(ORF）is351bp,which encoded a13.01Kda protein of116amino acids. A signal peptide was depositedin the1-15residues. The length of5’-Untranslated Region is87bp,3’-UTR is467bp.There was a polyadenylation tailing aignal AATAAA and polyA tail. Amino acidsequences have all the characteristics of β2m functional molecular. Such as, oneN-myristoylation site, two Protein kinase C phosphorylation sites, one Casein kinase II phosphorylation site, one immunoglobulins and major histocompatibility complexproteins signature. The protein of β2m melocular of S. argus is mixed, helix is9.5%,strand is28.5%, loop is52.6%. It was revealed in the analysis of amino aicd that S.argus had the most close relationship with gilthead bream, farther with salmon andtrout, farther and farther with frog, birds and homan. S. argus β2m contains2intronsand3extrons. The length of the introns could be944bp and279bp. All intronsdonor/acceptor sites of MHC I genes of S. argus matched the consensus motifs (gt/ag).The expression levels of β2m gene in kidney, spleen and liver were higher than those instomach, eye and muscles. Finally, it was revealed in the NJ tree that S. argus had theclosest relationship with gilthead bream, far from sea bass, salmon, trout and Africanclawed frog, and still father from chicken, mouse and human.4The characteristics of complete mitochondrial genome of S. argusThe length of complete mitochondrial genome for S. argus was16783bp(GenBank: KC790398). It had a typical mitogenome structure including13protein-coding genes,22tRNA genes,2rRNA genes, and1control region. ND6andeight tRNA genes were encoded on the light strand, whereas the others were on heavystrand. Overall base composition was28.3%for A,15.8%for G,27.7%for T, and28.2%for C. The content A+T(56%) was higher than the G+C content. Allprotein-coding genes initiated with the orthodox ATG except for COI with GTG. Onecomplete stop codon (TAA) and two incomplete stop codons (TA-and T-) were used inthese protein-coding genes.