Molecular Cloning, Expression And Polymorphism Analysis Of MHC Gene Of Nile Tilapia (Oreochromis Niloticus)

Major histocompatibility complex (MHC) is a highly polymorphic genomic region withan important role in the immune system of vertebrates. This research studied the cloning,polymorphism, and the expression of major histocompatibility complex (MHC) by PCR,homology cloning, RACE and RT-PCR from an economically important fish in our country,Nile tilapia, Oreochromis niloticus. These results are shown as follows:(1) In this study, the partial sequence of MHCⅠA gene and the full-length of MHCⅡAand ⅡB cDNA were obtained from Nile tilapia. A length of957bp MHCⅠA cDNAsequence was obtained, which included666bp encoding region, as well as291bp3’-UTR witha canonical polyadenylation signal (AATAAA) and a29bp poly (A) tail. The putative aminoacid sequence contains221amino acid residues, with partial peptide binding region, completeimmunoglobulin constant region, a transmembrane region and a cytoplasmic domain. Thefull-length cDNA sequence of MHC Ⅱ A (Orni-DBA*0101, GenBank No: JF719813)comprises1205bp with a720bp open reading frame (ORF). The predicted protein contains aleader peptide, two extracellular domains (α-1and α-2domains), a transmembrane region anda cytoplasmic domain. In addition, there are four conserved cysteine residues and an abundantof phosphorylation sites. The deduced amino acid sequence of MHCⅡA shares23%-65%identity with those of other vertebrates. A full length of MHCⅡB cDNA fragment of1143bp(Orni-DAB*0101, GenBank No: JF713026) was obtained, which included35bp5’-UTR,756bp encoding region, as well as352bp3’-UTR. The coded protein was251amino acidresidues, with a leader peptide, two extracellular domains (β-1and β-2domains), a connectingpeptide, a transmembrane region, and a cytoplasmic domain. Sequence comparison showedthat the deduced amino acid sequence of MHCⅡB had27%-70%identity with those of otherspecies.(2) The genomic sequence of MHCⅡA and MHCⅡB were obtained from healthy Niletilapia, respectively. The genomic sequence of MHCⅡA was identified to be1388bp, whichcontains four exons and three introns. Exon1encodes the single peptide. Exon2encodes theα-1domain. Exon3encodes the α-2domain. Exon4encodes the transmembrane region and the cytoplasmic domain. In addition, a2428bp genomic sequence of Nile tilapia MHCⅡBwas obtained. Six exons and five introns were identified. Exon1encodes the single peptide.Exon2encodes the β-1domain. Exon3and4encode the β-2domain. Exon5and6encodethe connecting peptide, the transmembrane region, and the cytoplasmic region.(3) Both MHCⅡA and ⅡB Moleculars were coded by multiple gene loci. And there area wealth of polymorphism between alleles. Eight classⅡA alleles were identified from fourhealthy Nile tilapia individuals. The variability existed in the α-1region was higher than thatin other regions. Four different alleles originating from a single individual suggested theexistence of at least two classⅡA loci in the genome. Ten alleles of MHCⅡB were obtainedfrom five health individuals. The sequence variation among alleles was abundant, and anextensive variability was detected in the β-1domain. Five different alleles observed in asingle individual may imply the existence of three MHCⅡB loci at least.(4) RT-PCR demonstrated that the Nile tilapia MHCⅡA and ⅡB mRNA wereubiquitously expressed in all tested tissues, but the expression level was distinctly different.High levels of transcripts of MHCⅡA was detected in spleen, kidney, intestine, gill, gonad,liver and heart, and low expression in bladder and muscle. High expression of MHCⅡB wasdetected in spleen, kidney, gill and intestine, and low expression in bladder and muscle. Thispattern demonstrates the adaptability of MHC molecules in both expression and function.(5) Challenge of Nile tilapia with Aeromonas hydrophila, results in different degreechanges of the expression of MHCⅡA and ⅡB in the liver, spleen, kidney, gills andintestine. The result implied that MHCⅡA and ⅡB might play an important role in theimmune system.(6) Phylogenetic trees were constructed based on the IGC region of these three genes.The topology structures of all three genes are similarly. These results showed that the IGCsequences of MHCⅠA、ⅡA and ⅡB are informative for phylogenetic studies in fishes.