青石斑鱼MHCclassI基因的克隆与分析  

Cloning and Sequence Analysis of the Major Histocompatibility Class I Gene in the Teleost Garrupa(Epinephelus awoara)

作　　者：王海燕[1] 刘勇[1] 张甲[1] 张冰[1] 

机构地区：[1]中国农业大学动物医学院,北京100094

出　　处：《中国比较医学杂志》2008年第11期14-20,共7页Chinese Journal of Comparative Medicine

摘　　要：目的克隆青石斑鱼MHCclassI基因,为研究石斑鱼的免疫系统和MHCclassI基因库提供材料。方法首先利用MHCclassI基因的特征,根据已知MHCclassI基因及其特点设计简并引物得到MHCclassI部分序列,然后分别利用5′RACE、3′RACE延伸至全基因的两端。将得到的序列拼接,经NCBI上BLASTx分析青石斑鱼MHCclassI基因核心区域,设计引物得到青石斑鱼MHCclassI基因。最后,将所得MHCclassI基因所对应氨基酸与几种鱼和人类MHCclass分子利用DNAMAN和MEGA软件分别进行比对和绘制分子进化树。结果成功得到青石斑鱼5个MHCclassI基因和绘制了MHCclassI分子进化树。同源性分析发现5个MHCclassI基因氨基酸同源性85.69%即高度的多变性。经氨基酸序列分析,青石斑鱼与已知鱼类和人MHCclassI基因的氨基酸序列存在30.27%～73.46%同源性其中不同鱼类同源性比对的比值表现为不同种类的鱼亲缘关系越近同源性比值越高。结论青石斑鱼MHCclassI基因具有已知物种MHCclassI基因特征。MHCclassI分子可以为生物进化分析提供依据。Objective To do further research in Garrupa immounity system and provide materials for MHCclassl gene pool, we firstly obtained the MHCclassl of Garrupa. Methods Firstly, according to high conservative amino acids of MHCclassl, a pair of degenerated primers were designed and a special DNA fragment was gained from Garrupa kidney by RT-PCR. Then based on the gained sequence, some new primers were designed, and two terminals of MHCclassl gene were cloned by 3′RACE and 5′ RACE respectively. Splicing the fragments of sequence, and by BLASTx software in the NCBI, we designed primers and gained the Open reading frame region of MHCclassl gene of Garrupa. Finally, analyze the sequence by using DNAMAN and MEGA. Results We obtained five MHCclassl genes of Garrupa successfully and drew a MHCclassl evolutionary tree. By the analysis of the five different MHCclassI genes amino acids sequences, they showed high polymorphism identity of 85.69% in the Garrupa. Compared the amino acid of Garrupa MHCclassl to humans and some fish showed that the key amino acids of the peptide binding sites all are conserved, had identity of 30.27% - 73.46% , moreover the closer relationship among fish were in favor to higher ratio. Conclusion The MHCclassl of Garrupa have genetic characteristics, which shared in all species. MHCclassl molecule can provide the basis for biological evolution.

关 键 词：青石斑鱼 MHCclassI 进化指征 

分 类 号：Q341[生物学—遗传学]