牙鲆MHC-DAA结构及其等位基因多态性

Genomic structure of DAA gene and polymorphism within MHC-DAA alleles in Japanese flounder (Paralichthys olivaceus)

为了探讨牙鲆MHC-DAA等位基因的多态性,根据牙鲆(Paralichthys olivaceus)MHC-DAA的cDNA序列设计特异引物,扩增内含子序列。牙鲆DAA基因由4个外显子和3个内含子组成,与大西洋鲑(Salmo salar)DAA基因结构相同,在内含子2中存在一个高度多态的微卫星位点(GT)n。根据获得的MHC-DAA基因组序列设计特异性引物,在45尾牙鲆中扩增了包括完整外显子2和内含子2的长度约670bp的DNA片段,克隆测序后共发现30个MHC-DAA等位基因,各等位基因的频次及其各主型中亚型的数目都不均衡。在249bp的外显子2序列中共有55个位点发生变异,核苷酸多样性指数为0.0887,编码的氨基酸序列中多态变异位点31个,其中简约信息位点30个,单变异位点1个。非同义替代与同义替代的比率在PBR(Peptide binding region)和非PBR结合区分别为3.30和2.43,分析证明平衡选择是牙鲆存在众多DAA等位基因的发生机制。

To investigate the allele polymorphism of MHC-DAA in Japanese flounder （Paralichthys olivaceus）, specific primers were designed based on the published DAA-cDNA sequence of P. olivaceus to amplify the entire introns from flounder. Gene DAA consisted of four exons and three introns. The exon-intron structure was similar as that of Atlantic salmon. A polymorphic microsatellite was detected in intron 2. Subsequently, specific primer was designed based on the DAA genomic sequences. The genetic polymorphism within exon 2 of MHC-DAA was investigated by means of sequence analysis. Forty-five individuals were selected to amplify exon 2 and intron 2. One hundred and twenty-nine fragments revealed 30 novel alleles belonging to 18 allele major type. The frequency of alleles and the numbers of subtypes were not distributed equally. There were 55 variable sites in exon 2. The diversity of nucleotide within the sequence of exon 2 was 0.0887. The nucleotide sequence was corresponded with a putative peptide. Among the 83 amino acids in this peptide, 37.80% were variable, and 30 parsimony informative sites were observed. The rates of non-synonymous substitution （dN） were 3.30 and 2.43 times higher than the rate of synonymous substitution （dS） in the peptide-binding regions （PBR） and non-PBR, respectively. In this study, the high ratio of dN/dS value and high allelic diversity observed in flounder suggested a balancing selection in the exon2 of the MHC class HA gene, which explained the high polymorphism of MHC-DAA gene in Paralichthys olivaceus.