Isolation And Polymorphic Analysis Of MHC Class ⅡBGenes In Rhscophorus Omeimontis

Major Histocompatibility Complex (MHC) are cell surface glycoproteins that trigger immune response in vertebrates. MHC genes belonging to a mulitigene family can encode these proteins. Many studies has proved that MHC is one of the most variable functional genes described in vertebrates, and the variability mainly appears on antigen binding sites. Because of their importance in immunity and fitness, many researches focus on the most variable regions. In terms of MHCⅡ genes, it is the exon2encoded antigen binding sites, so knowledge regarding the variable characteristics can provide many conveniences studying the problem such as adaptive evolution, sexual selection and so on. However, knowledge on the structure characteristics and evolution patterns associated with amphibian MHCⅡB genes remains limited. For the purpose of broadening our knowledge regarding the polymorphism of amphibian MHCⅡB gene system, the goal of the present study was to isolate MHCⅡB genes exon2from Rhscophorus omeimontis and examine the molecular mechanisms that maintain MHC diversity.We first successfully designed primers from introns to amplify MHCⅡB genes exon2in R. omeimontis. Two lines of evidence suggested that all of the newly isolated alleles belonged to MHCⅡB genes:(1) phylogenetic trees of all available anuran MHCⅡB genes indicated that the alleles isolated in this study clustered together with published MHCⅡB genes;(2) the conserved sites of MHCⅡB molecular also conserved at these new proteins.Then, we genotyped MHCⅡB genes in two populations (SCBX and HNBDGS) and detected126alleles in272individuals.104or62alleles were detected in Sichuan and Hunan population, respectively, with a maximum of7alleles per individual, indicating that at least four loci exist in R. omeimontis. The K-2P nucleotide distance (0.039±0.006) was lower than the poisson-corrected amino acid distance (0.079±0.016) computed in MEGA5, which indicated that these genes are under positive selection. There were13sites undergoing positive selection detected by PAML, FEL and MEME,9out of13sites were located at the putative ABS deduced from the crystal structure of human HLA-DR1. So we can concluded that pathogen drive the selection on MHCⅡB genes of R. omeimontis. However, we did not detect any recombination events in R. omeimontis.Therefore, pathgen-mediated selection and gene duplication played an important role in maintain the diversity of MHCⅡB genes in R. omeimontis. For the highly diversity of these genes, they can be used as a molecular marker to study the adaptive evolution in R. omeimontis.