The Polymorphism And Evolution Of MHC Class I Genes In The Anuran Amphibians

The major histocompatibility complex (MHC) is one of the most polymorphism gene family in most vertebrate animals, which encode the glycoproteins on the cell surface, present the shelf or non-shelf peptide to the T lymphocytes, confirm that the genes are critical to the vertebrate immune system. The extremely high polymorphism of MHC, which is an result of the long term evolution of the gene, is considered to be related to the resistance to the parasites. Thus the research on the evolution of MHC can reveal the molecular mechanisms of shaping the polymorphism of MHC, understanding the evolution of immunity-related proteins. Most knowledge of the evolution of MHC gene comes from the mammals, the fishes, the birds, limited knowledge from the amphibians, so the polymorphism and evolutionary mechanism of the amphibious MHC gene are still unclear. The amphibians possess a significant status in the evolution, because of the first terrestrial tetrapods, and the characteristic of life resulting in a more complex microorganisms environment. To having an comprehensive and systematic understanding of the evolution of amphibian MHC systems, broaden the knowledge about evolution of MHC gene, we chosen nine anuran amphibious species(Megophrys sangzhiensis, Vibrissaphora boringii, Vibrissaphora leishanensis, Bombina orientalis, Bufo gargarizans, Fejervarya Limnocharis, Pelophylax nigromaculata, Rhacophorus dennysi, Rhscophorus Chenfui) from five family(Megophryidae, Bombinidae, Bufonidae, Ranidae, Rhacophoridae) as the research objects, and isolated MHC class I gene sequences by molecular clone and DNA sequence, region from the exon 2 to 4, then to analyse the polymorphism and the evolutionary mechanism.In this study, we successfully design 2 pairs of special primers based on the partial obtained sequences of the M.sangzhiensis, V. leishanensis and R.dennysi by the RNA-Seq technology. We isolated 1800 sequeces of MHC class I gene of the 9 species, including about 730bp fragment, containing the entire exon 3 and partial exon 2 and 4, with 2 pairs of newly designed special primers and a pair of published degenerate primers. We totally identify 78 MHC class I alleles from 90 individuals, R.Chenfui possess 12 alleles, R.dennysi possess 9 alleles, P.nigromaculata possess 6 alleles, F.Limnocharis possess 11 alleles, B.gargarizans possess 8 alleles, V.leishanensis possess 10 alleles, V.boringii possess 9 alleles, M.sangzhiensis possess 5 alleles, B.orientalis possess 8 alleles. The numbers of alleles differ from individuals, and the maximum is 4 in an individual in this study. We then reasonably deduced that most of the focal species—R. Chenfui, F. limnocharis, B. gargarizans, V. leishanensis, B. orientalis possess at least more than 2 loci; V.boringii, Msangzhiensis, P. nigromaculata and R.dennysi show only 1 loci. The multiple gene loci suggested the gene duplication events were occurred in the evolution, according with the Birth-and-death model.The 78 MHC class I alleles from the 9 species all display a high levels of polymorphism in both nucleotide and amino acid. In 3 areas, the polymorphism of exon2 and exon3 are higher than that of exon4. The exon2 is the highest of all, and the amino acid sequence of the exon4 are much more conserved. This suggests that the balancing selection and gene recombination are the important evolutionary mechanism of amphibious MHC class I gene in the evolution of maintaining polymorphism, besides gene complication.In addition, we compared the non-synonymous to synonymous substitutions and used 5 codon-based models(SLAC,REL,FEL,MEME,Codeml) to detect the signals of natural selection in different exons. First, the distance of amino acid is higher than that of nucleotide, suggested the existence of natural selection. And, the value of the non-synonymous compared to synonymous substitutions is higher than 1 in most area, especially in ABS, illustrated that the MHC gene had been underwent positive selection. The 5 models had detected 52 amino acid sites, which were underwent selection, most in the exon2 and 3,containing 10 putative antigen-binding sites, suggested that the balancing selection plays an important role in the evolution. The phylogenetic tree was constructed, and we found the trans-polymorphism exist between the Ranidae and Rhacophoridae, and in the difference Megophryidae species, which is a further evidence of balancing selection.Recombination events involving the entire exons were examined, and the breakpoints most located near the boundary of exon2 and exon3,exon3 and exon4, which is similar with the pattern of the bony fish. We also found that 37 alleles were underwent gene recombination in the 78 alleles, confirmed that the gene recombination is an critical evolutionary mechanism for the amphibious MHC class I.In condition, we constructed phylogenetic tree to have a better understanding of the evolutionary relationship of the MHC class I gene. To compared with the other vertebrates, the amphibians own an old MHC I gene lineage.Our results suggested the MHC gene owns a high level of polymorphism. We concluded that gene recombination, balancing selection and recombination are play extreme important roles in the evolution of MHC class I evolution.