太平洋鳕ATG5基因的克隆及表达分析

Cloning and expression of ATG5 gene in Pacific cod Gadus macrocephalus

ATG5(Autophagy related gene 5)基因是一种自噬相关基因,是形成自噬体的重要基因,ATG5基因的表达对自噬调控的成熟有重要作用。本研究中通过克隆,首次得到太平洋鳕Gadus macrocephalus ATG5c DNA部分序列,该序列长为895 bp,含有完整的开放阅读框(Open reading frame,ORF),编码275个氨基酸,其编码的蛋白质相对分子量约为32 200;经核苷酸序列比对发现,太平洋鳕ATG5核苷酸序列与半滑舌鳎Cynoglossus semilaevis、尼罗罗非鱼Oreochromis niloticus ATG5核苷酸序列的相似性均高达87.27%;利用太平洋鳕与其他物种ATG5核苷酸序列构建系统进化树,结果显示,ATG5核苷酸序列系统进化树反映的亲缘关系基本符合传统分类学观点;氨基酸序列分析结果显示,ATG5蛋白氨基酸序列具有较高的保守性;利用绝对荧光定量PCR方法对ATG5基因在太平洋鳕各组织中的转录水平进行检测,结果显示,肝、脾和肾中的表达量高于其他组织,鳃、肾、脾、脑、肝和肌肉各组织中的ATG5 mRNA拷贝数分别为11.617 08、20.449 25、19.706 87、4.839 84、27.434 97、2.954 75 copies/ng。本研究结果将为进一步研究太平洋鳕细胞吞噬机制奠定基础,也为太平洋鳕的免疫机理和相关研究提供基础数据。

ATG5 as an abbreviation of autophagy related gene 5 is an important gene for the formation of autophago-some, and plays a vital role in the maturation of autophagy regulation. In this study, a cDNA sequence of ATG5 was first cloned in Pacific cod Gadus macrocephalus using gene cloning, and was found to be 895 bp encoding 275 amino acid residues with molecular weight of about 32 200 in length with a whole open reading frame（ ORF） . The nucleotide sequence alignment showed that there was as high as similarity of 87. 27% in ATG5 among Pacific cod, Nile tilapia Oreochromis niloticus, and half-smooth tongue-sole Cynoglossus semilaev. The phylogenetic tree con-structed by ATG5 nucleotide sequences of Pacific cod and some other animals reflected a genetic relationship which conformed to the viewpoint of traditional taxonomy. The amino acid sequence analysis revealed that ATG5 had high conservation. The expression of ATG5 in various tissues of Pacific cod using relative quantitative PCR and the abso-lute quantitative PCR showed that the higher expression levels of ATG5 mRNA were observed in liver, spleen and kidney than in other tissues, with 11. 617 08 copies/ng in gill, 20. 449 25 copies/ng in kidney, 19. 706 87 copies/ng in spleen, 4. 839 84 copies/ng in brain, 27. 434 97 copies/ng in liver, and 2. 954 75 copies/ng in muscle. The findings lay the foundation for further study of the phagocytosis mechanism and provide basic data with understand-ing of immune mechanisms in Pacific cod.