Studies On MHC Class Ⅱ B Genes Of Chinese Alligator (Alligator Sinensis) And DNA Extraction From Its Tannage

MHC is an extended cluster of genes with extraordinary polymorphism, which consists of three classes of genes (Class I , Class II and ClassIII) .They are different from each other in their structure, expression, mode of evolution and so on. Class II genes are further divided into two subclasses and the MHC genes spread over nearly all vertebrates extensively. Besides the immunological functions, MHC genes also play important roles in many other respects. The polymorphism of MHC genes, especially of MHC Class II genes, is the most essential property. Moreover, MHC genes are usually used to analyze the genetic structure and genetic variation in conservation genetics. The polymorphic MHC genes will be very important to genetic conservation of endangered species.In order to study MHC genes of Chinese a\\igator(Alligator sinensis), a pair of degenerate primers were used to amplify the segments of MHC class II B genes from the genomic DNA of Chinese alligator. Ten 166 bp (one 160bp) -long different nucleotide sequences, which were divided into two groups (A, B), were obtained from cloning and sequencing. The segments showed high polymorphism in both nucleotide and amino acid sequences. There were 38 variable sites among ten nucleotide sequences, and 23 variable sites among amino acid sequences. The numbers of synonymous (ds) and nonsynonymous (dN) substitutions of putative antigen-binding sites(pABS) were analyzed by MEGA. Phylogenetic relationship of sequences was constructed by neighbor-joining method in MEGA and maximum parsimony algorithm in PAUP. The same two groups were showed in the results, and the group B sequences were more similar to two sequences of Caiman crocodilus. The high polymorphism of MHC genes in Chinese alligator would be of great benefit to genetic conservation in the captive population of Chinese alligator.DNA extraction from different specimens is the foundation of conservation genetics. It is difficult to extract total DNA from tannage of Chinese alligator with usual method. In this paper, total DNA from tannage, tail skin, scales and the skin treated with salt were successfully extracted with an improved methodfor DNA extraction. To verify the results, four pairs of primers, which were universal primers for 12S rRNA gene, diagnostic primers of Chinese alligator meat, microsatellite primers and RAPD primer, were used to do PCR amplifications. Some amplified fragments were sequenced, either. The results showed that DNA of tannage could be extracted by this method and used as samples in conservation genetics. It is a good idea for molecular identification of leather things and amplification or sequencing of genes. DNA of other specimens could also be extracted and amplified well.