Isolation And Polymorphic Analysis Of MHC Class Ⅰ And Class Ⅱ Genes In Tiger Frog (Hoplobatrachus Chinensis)

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 MHC genes, it are the exon2 and exon3 encoded antigen sites, so knowledge regarding the variable characteristics can provide many conveniences studying the problem such as adaptive evolution patterns associated with amphibian MHC genes remains limited. For the purpose of broadening our knowledge regarding the polymorphism of amphibian MHC gene system, the goal of the present study was to isolate MHC genes exon3 and exon2 from tiger frog(Hoplobatrachus chinensis) and examine the molecular mech-anisms that maintain MHC diversity.Firstly, We designed universal primers and successfully gained MHC gene class Ⅰ exon3 126bp, MHC Ⅱ class exon2 132bp.Then we received a total of tiger frog MHC Ⅰ genes 1020bp,class Ⅱ genes 2020bp by conventional methods and Genomic Walking PCR method, which contains all of the exons.We design specific primers,based on the obtained sequence tiger frog,so as to amplified tiger frog MHC Ⅰ class and class Ⅱ.we genotyped the MHC in three populations(Bilian Daruoyan and Xialiao) and detected the MHC diversity of tiger frog. Our major results were: 1、We detected ten alleles of the class Ⅰ, with a maximum of 7 alleles per individual,indicating that at least four loci exist in tiger frog. We detected seven alleles of the class Ⅱ,with a maximum of 3 alleles per individual, indicating that at least two loci exist in tiger frog.2、Nucleotide sequence variation sites by DNAsp software calculates its class Ⅰ genes in 10 alleles was 27.3%; nucleotide sequence variation sites Ⅱ genes of 7 allele is 13%.3、The K-2P nucleotide distance was lower than the poisson-corrected amino acid distance computed in MEGA6.0,which indicated that these genes are under positive selection.4、About the class I gene,There were 6 sites undergoing positive selection detected by MEGA, FEL and MEME,3 out of 6 sites were located at the putative ABS deduced from the crystal structure of human HLA-DRl.About the class Ⅱ gene,There were 5 sites undergoing positive selection detected by MEGA, FEL and MEME,2 out of 5 sites were located at the putative ABS deduced from the crystal structure of human HLA-DRl.So we can concluded that pathogen drive the selection on MHC genes of tiger frog.5、we did not detect any recombination events and trans-species polymorphism in tiger frog.The result can be used as a molecular maricer to study the adaptive evolution in tiger frog.First, tiger frog MHC polymorphism gene sequence is higher, and there is significant positive selection signal can be used as a function-related molecular markers to study the geographical variation in different geographic populations of tiger frog, also tiger frogs and other amphibians may be linked to the evolutionary model system Interpretation amphibian genes. Second,the gene can resist the ability to pathogens,which can reveal the molecular mechanism of tiger frog disease. Third,this gene which is link to spouses choices, can reveal mate selection mechanism.