Studies Of The Major Histocompatibility Complex (MHC) Class Ⅰ Genes From The Giant Panda (Ailuropoda Melanoleuca)

Giant Panda (Ailuropoda melanoleuca) is an endangered species that is endemic to China and has received great attention for its conservation and survivability. Historically, it is widely distributed across the major land, but due to glacial effect and human activities, currently the range of giant panda is shrunk to six isolated mountain areas: qinling, Minshan, Qionglai, liangshan, Daxiangling, Xiaoxiangling mountains. Because of the historical isolation from other populations, the qinling species has differentiated to a new Qinling subspecies {Ailuropoda m. qinlingensis).As part of the conservation strategies to conserve gene resources of the giant panda, we successfully constructed a high quality genomic BAC library, which is also applicable and important for subsequent study of comparative genomics and functional genes from the giant panda. In view of its great importance of the role of major histocompatibility complex (MHC) genes in peptide presentation to T cells, MHC genes were widely studied in various fields. Here we conducted a first characterization of MHC class I genes in the giant panda. All the results are listed as follows:·Fresh blood was used to construct the genomic BAC library, which is consisted with totally 205,800 clones and stored in 2,100 96-plates. According to the studies of 174 clones randomly selected, the average size of the BAC library is estimated to be 97kb, with empty clones of 5.74 percent. Using the genomic size of black bear and Himalaya bear as reference, the whole library is estimate to be 6.8-fold of the giant panda genome, and the probability to get the positive clone is 99.93 percent. The results of screening the library with 16 giant panda primers showed that the number of positive clones ranged from 2 to 16, with an average of 6.2.·A 4D PCR method was applied to screen positive clones from the BAC library. We divided the whole library into 43 suppercool each having 34 sub-supercool, including seven 1D, seven 2D, eight 3D and twelve 4D. To obtain a positive clone, First we screen the positive supercool in 43 PCR reactions, then it only needs 7+7+8+12=77 PCR reactions to determine the place of the positive clone in the sub-supercool.·We successfully characterized three genes from mRNA and genomic BAC library. Analyzing the gene structure and characteristics, protein structure modeling, motifs of regulatory elements, phylogenetic trees all demonstrated that the three genes are typical of MHC class I genes. We designated them as Aime-128, Aime-152 and Aime-1906.·Aime-1906 is a very special nonclassical class I gene divergent from all other giant panda MHC class I genes. And the reason is as follows: First, Aime-1906 has a shortened Cytoplasmid tail owing to the pre-stop codon at the beginning of exon 7. Only five residues are conserved concerning the ten amino acids which are conserved across nearly all mammalian classical class I genes; Second, the GC content in the third base of the codon in peptide binding region and non-peptide binding region are both less than another two genes, suggesting it is more inclined to be a Ib gene; third, investigation of amino acid substitution in exon 2 and 3 of its alleles is found that only one-third of the changes are located at the peptide binding region, which differed from classical class I genes; Fourth, in the phylogenetic trees, Aime-1906 always clustered with DLA-79 and separated from the other class I genes of carnivore species, indicating a different ancestral source of the Aime-1906 gene.·Aime-128 and Aime-152 were believed to be derived from two loci in the giant panda. First, sequence composition is very different in the two genes. Pairwise comparisons of the two sequences from 5'UTR, 3'UTR and exon 1 are 84%,87% and 88% respectively; two regular substitutions were found from Aime-152 in the ten amino acids which are conserved in nearly all mammalian classical class I genes while in Aime-128 all of them are well conserved; Nucleotide variations are found in the core regulatory elements between Aime-128 and Aime-152; Second, the GC content in the third base of the codon in peptide binding region and non-peptide binding region is also different in these two genes, which is higher in Aime-128; Third, in the phylogenetic trees, the Aime-128 cluster with its alleles and is sperated from Aime-152, indicating there is at least another class I gene more related with Aime-128 than Aime-152 does.·Calculation of synonymous and nonsynonymous substitution for these class I genes demonstrated that strong positive selection existed in the peptide-binding region of giant panda class I genes.·MHC class I genes of giant panda is also a complex multigene family, similar to other mammalian MHC class I regions.