乌龟MHCⅡ类B基因第二外显子的克隆及序列分析  

Molecular Cloning and Sequence Analysis at Exon 2 of MHC Class Ⅱ B Gene in Chinemys reevesii

作　　者：李恩[1] 吴孝兵[1] 晏鹏[1] 

机构地区：[1]安徽师范大学生命科学学院重要生物资源保护与利用省重点实验室,安徽芜湖241000

出　　处：《激光生物学报》2006年第1期58-64,共7页Acta Laser Biology Sinica

基　　金：国家自然科学基金(30270213);安徽省优秀青年基金(04043049);安徽师范大学青年基金(143-120059)

摘　　要：利用一对兼并性引物扩增了乌龟MHCⅡ类分子B基因第二外显子的部分片段,并对PCR产物进行了克隆和测序,结果得到8种长度为166 bp的不同序列。经分析,序列中有84个变异位点,核苷酸的非同义替换(dN)多于同义替换(dS),造成39个位点氨基酸的改变。氨基酸的替换趋于集中在假定的抗原结合位点附近。利用MEGA、PAUP软件分别构建NJ树和MP树,两种树极为相似,均分为两支。同一个体中出现有多种序列,提示乌龟MHCⅡ类分子B基因可能存在着座位重复。研究表明:乌龟MHCⅡ类分子B基因第二外显子有较高的多态性,有利于乌龟野生种群的遗传保护。The major histocompatibility complex （MHC） is a chromosomal region consisting of closely linked loci which are highly polymorphic, and plays a central role in the immune system. Here we investigated genetic variation at exon 2 （ including part of the putative antigen-binding site, pABS） of the class Ⅱ MHC B gene in Chinemys reevesii. A pair of degenerate primers was used to amplify 166 bp fragments of exon 2 of the class Ⅱ MHC B gene. PCR products were cloned into a pGEM -T Vector after purification. Eight different sequences were obtained from cloning and sequencing. All eight sequences did not show deletions, insertions, or stop codons, and therefore could be assumed functional in vivo. There were 84 variable sites in the nucleotide sequences, the ratio of nonsynonymous substitutions was higher than that of synonymous substitutions （ dN/ds = 28. 533/8. 324） , which caused 39 variable sites among amino acid sequences. Substitutions of amino acid tended to be clustered around the putative antigen-binding sites, suggesting that the variation at these sites would be responsible for recognition and presentation of different kinds of antigens. Phylogenetic relationships were constructed by neighbor-joining （NJ） method in MEGA 2.1 and maximum parsimony （MP） method in PAUP 4.0. The eight nucleotide sequences can be clustered into two clades in NJ and MP phylogenetic trees. More than twosequences were found in the same individual, suggesting there would be locus duplication in the class Ⅱ major histocompatibility complex B gene of C. reevesii. The high polymorphism of MHC class Ⅱ B gene in C. reevesii was of great benefit to genetic conservation in the wild population of C. reevesii.

关 键 词：乌龟 MHC Ⅱ类B基因第二外显子 序列分析 

分 类 号：Q78[生物学—分子生物学]