Identification Of MHC Class Ⅱ Genes And Their Relation To Purulent Disease In The Forest Musk Deer

Forest musk deer are one of the most valuable game animals in China. The secretion from the preputial gland of the male deer, the musk, plays an important role in the medicine and the perfume industry. Over the past decades, the great demand in the world market, the extensive hunting and poaching and the lost of the habitats push this animal to the edge of extinction. The captive program has been executed for more than fifty years, but the captive population is still small for many reasons, especially the high susceptibility to many diseases. So, it is very necessary to study this animal and protect this species from extinction.The major histocompatibility complex (MHC) is the most polymorphic super-gene family in the vertebrates. The glycoproteins on the cell surface encoded by MHC genes play an important role in immune responses through recognizing and presenting the foreign peptides to T cells. Functional MHC class II molecules are heterodimers of alpha and beta chains encoded by corresponding A and B genes respectively. Exon2fragments of A and B genes, coding for the first domain of a functional MHC class II molecule and sites associated with the peptide binding are highly polymorphic, science the peptide binding region is considered to be related to the resistance to the parasites and the adaptability to the complex environments. A total of115individuals sampled from2005to2007in Sichuan Musk Deer Breeding Institution were studied here. After the identification of classical DR and DQ genes, the analysis of genetic variation and the correlation analysis with purulent disease, we found the mechanisms that maintain the MHC class II genes polymorphism and the relationship between MHC class II genes and purulent disease of forest musk deer. The main results are listed below:1. We obtained part of the exon2sequences using the universal primers, and then we used the genome-walking method and the long range PCR to obtain the flanking regions. After that, we designed the locus specific primers based on the obtained sequences. At last, we isolated one DRA and one DKB (DRB3) loci.2. Regarding the Mobe-DQ genes, we first used the universal primers to amplify the exon2sequences and then adopted the long range PCR to obtain the entire intron2sequences. The intron1fragments were isolated by the primers that were designed based on the homologous sequences. Finally, we obtained two DQA (DQA1&DQA2) and two DOB (DOB1&DQB2) loci.3. We obtained six classical MHC Ⅱ loci in this study. Thirty-four alleles were confirmed by the locus specific PCR-SSCP genotyping and sequencing methods (DRA,4; DRB3,10; DQA1,4; DOA2,7; DOB1,4; DQB2,5). The six loci of forest musk deer are all polymorphic and showed high numbers of alleles per locus comparing with the other endangered animals, implying that the captive forest musk deer still contain an abundant of genetic diversity.4. The different loci of forest musk deer experienced different selection pressures. All MHC Ⅱ A genes experienced purifying selection, while all the B genes experienced positive selection, especially the peptide binding region. The phylogenetic analysis showed the trans-species evolution was an important mechanism to maintain the polymorphism of the forest musk deer MHC Ⅱ genes.5. The relationship between the forest musk deer MHC class Ⅱ genetic variation and the purulent disease was analyzed by the statistical analysis. We found10alleles.13genotypes and6super haplotypes were related to the susceptibility/resistance to the purulent disease. Four alleles (Mobe-DQA1*01, Mobe-DQA2*03, Mobe-DOA2*04and Mobe-DQB2*03) were found to be resistant to the purulent disease significantly, and six alleles (Mobe-DRB3*07, Mobe-DQA1*03(or Mobe-DQA1*04), Mobe-DOA2*05(or Mobe-DOA2*06) and Mobe-DQB2*02) were found to be susceptible to the purulent disease significantly. Six genotypes were related to the resistance to the purulent disease [DRB3*01/DRB3*03, DOA1*01/DQA1*01, DOA1*01/DOA1*02, DQA2*01/DQA2*03, DQA2*02/DOA2*04and DQB2*03/DQB2*04(P<0.05)], and7genotypes related to the susceptibility to the purulent disease [DRB3*07/DRB3*07, DQA1*03/DOA1*03, DQA2*01/DQA2*01, DQA2*05/DOA2*05, DQA2*05/DQA2*06, DOB1*01/DOB1*03and DQB2*01/DOB2*02(P<0.05)]. The super haplotypes related to the resistance to the purulent disease were H1(DRA*hl/DRB3*hl/DOA1*h1/ DQA2*h2/DQB1*h1/DQB2*h2) and H2(DRA*h1/DRB3*h4/DQA1*h1/DQA2*h2/DQB1*h1/DQB2*hl). The haplotypes related to the susceptibility to the purulent disease were H3(DRA*h1/DRB3*h1/DQA1*h3/DQA2*h3/DQB1*h3/DQB2*h2), H4(DRA*h1/DRB3*h5/DQA1*h3/DQA2*h3/DQB1*h1/DQB2*h1), H5(DRA*h1/DRB3*h7/DQA1*h3/DQA2*h1/DQB1*h3/DQB2*h2) and H6(DRA*h1/DRB3*h7/DQA1*h3/DQA2*h3/DQB1*h1/DQB2*h1). The frequency of DRA*02and related genotypes were relatively high in the resistant group for which was linked to the most resistant super haplotyes. Meanwhile, the frequency of DRA*03(or DRA*04) and related genotypes were relatively high in the susceptible group for which was linked to the most susceptible super haplotyes. The peptide binding sites of Mobe-DQB2*02and Mobe-DQB2*03were completely same, but Mobe-DQB2*02had a higher frequency in the susceptible group and Mobe-DQB2*03had a higher frequency in the resistant group for they were linked to different super haplotypes.The number of the susceptible alleles, genotypes and super haplotypes exceeded that of the resistant ones, implying a correlation with the high susceptibility to the purulent disease of forest musk deer. So, it is necessary to introduce new individuals to timprove current genetic structure of captive populations and enhance ability to resist pathogen infection.