| In this paper,3 wild colony of Macaca mulatta from northwest Sichuan as our research subjects,with application of the PCR amplification, DGGE(Denaturing gradient gel electrophoresis) technic and gene cloning technic we analysing MHC-DRB(MHC class II gene DR sub-region, B chain) exon 2 polymorphism of 82 wild Macaca mulatta in Sichuan. Comparison of homology was done by NCBI for the partial DRB gene exon2 sequences of the test, the discovery of new alleles were named. Download the DRB exon2 sequences of other primates from the NCBI, together with the sequences got in our study, phylogenetic tree was constructed using N-J method and the evolutionary relationship between species was analyzed.Also we analyzed the allele frequency distribution, nucleotides composition, amino acid sequence variation, nucleotide diversity and genetic distance among populations. The following are the conclusions we obtained in this study:(1) 71 Mamu-DRB alleles were detected and of which 59 were new Mamu-DRB alleles obtained in this study. Taking 82 macaca mulutta individules as one big group, the highest Allele frequencie is Mamu-DRB4*0104(0.048). Analyses were underway among the three groups(Danba,Beichuan,Heishui) from Different geographical environment, The higher frequency distribution of alleles respectively were; View from the populations, some shared alleles existed(Mamu-DRB4*0104, Mamu-DRB*W2704, Mamu-DRB6*011201, Mamu-DRB6*0123) Otherwise, Many population-specific alleles in the three populations.Such as Mamu-DRB*W270302, Mamu-DRB*W270303, Mamu-DRB6*0114 etal, only belong to the the population of Danba; Mamu-DRB*W250903, Mamu-DRB1*1011, Mamu-DRB1*0328 etal, just appear in the Beichuan population; Mamu-DRB1*1012, Mamu-DRB*W108, Mamu-DRB1*041201 etal, have been detected only in the Heishui population.(2) Through the comparison of 80 amino acid sequence of 59 newly discovered Mamu-DRB alleles,62 amino acid residues mutations existed of which 9 (29,32,35, 39,43,45,52,62,70) for a single amino acid mutation, and the remaining 18 sites did not find mutations.80 amino acid residues contains 13 antigen binding sites, their analysis found that 9 of which mutated ABS sites, accounting for 69.23%; while in the remaining 66 Non-ABS the other sites in the 43 mutation, accounting for 64.17%, indicating that antigen binding regions in the amino acid substitution in the event higher than the non-antigen binding sites.(3) By analysing the phylogenetic tree (N-J)constructed by the MHC-DRB exon 2 sequences from different species, we can see that the 15 Mafa-DRB alleles didn't completely cluster in one branch but interspersed between the published and the new founed Mamu-DRB alleles. This shows that there exist cross-species allele polymorphism (Trans-species polymorphism, TSP) phenomenon, and we can deduced that the appearance of new alleles was caused by the convergent evolution due to The same or similar pressures of natural selection. Meanwhile,2 of the 9 HLA-DRB alleles we selected in the phylogenetic tree constructing were interspersed between the new founed Mamu-DRB alleles which indicated that some shared HLA-DRB sites and lineages existed between the new founed Mamu-DRB alleles and the HLA-DRB Alleles.(4)The nucleotide diversity (Pi) was obtained by the method of Fu and Li's test, in the three populations (Danba, Beichuan, Heishui), the Pi Were 0.13077,0.15278, 0.13513 respectively. So we can get a conclution that during the three populations, Beichuan population has the highest genetic diversity, and Danba population genetic diversity is lowest than the other two populations. Genetic recombination existed in the Beichuan group by RDP software analysis, this is one of the factors causing the result of the high genetic diversity of Beichuan group. (5) By analyzing the genetic distance during the populations, the genetic distance between Beichuan and Heishui groups was the highest (0.158), followed by between Beichuan and Danba (0.156), and the lowest is between Heishui and Danba groups(0.145). |
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