大菱鲆趋化因子受体CCR3和CCR9基因的克隆及组织表达  

Chemokine receptor genes CCR3 and CCR9 in turbot(Scophthalmus maximus): Cloning and tissue distribution

作　　者：孟艳青[1,2] 刘晓飞[1,2] 刘洋[1,2] 常亚青 王秀利[1,2] 姜志强[1,2] 

机构地区：[1]农业部北方海水增养殖重点实验室,大连海洋大学,辽宁大连116023 [2]大连海洋大学水产与生命学院,辽宁大连116023

出　　处：《中国水产科学》2013年第5期918-930,共13页Journal of Fishery Sciences of China

基　　金：辽宁省教育厅计划项目(2009A171)

摘　　要：本实验克隆了大菱鲆趋化因子受体基因CCR3和CCR9的全长cDNA,并进行了鉴定。其中,全长cDNA的克隆采用了5′和3′-RACE。CCR3 cDNA全长1 451 bp,包括92 bp的5′非编码区,276 bp的3′非编码区以及编码360个氨基酸的1 083 bp的开放阅读框。CCR9 cDNA全长1 441 bp,包括59 bp的5′非编码区,278 bp的3′非编码区,以及编码367个氨基酸的1 104 bp的开放阅读框。两种受体均具有7次跨膜结构,符合G蛋白偶联受体的序列特征。系统进化分析表明,大菱鲆CCR3与其他鱼类CCR3聚为一支,大菱鲆CCR9也与其他鱼类的CCR9聚为一支;系统进化树显示的亲缘关系符合各物种的进化地位。实时定量PCR(qRT-PCR)表明这两个基因在大菱鲆正常组织中均有一定量的表达,尤其是它们在头肾、脾及心脏中均有高水平的表达。在脂多糖(Lipopolysaccharide,LPS)诱导之后,CCR3在脾和肝中的表达水平与对照组有显著性差异(P<0.05),而CCR9的表达水平仅在肝中与对照组有显著性差异(P<0.05)。两个基因在免疫相关组织中表达水平较高,并且能够对LPS产生免疫应答,说明它们在大菱鲆免疫系统中发挥了重要功能。Turbot is an important aquaculture species in the eastern North Atlantic and Mediterranean regions, China, and Korea. Research on the molecular mechanisms of the turbot immune system could contribute to improving the economic performance of turbot aquaculture. In this study, we cloned and characterized the full-length cDNA of CCR3 and CCR9 in turbot （Scophthalmus maximus）. The full-length cDNAs were obtained by the 5′ and 3′-RACE method. The CCR9 cDNA was 1 441 bp in length, consisting of a 59 bp 5′ UTR, a 278 bp 3′ UTR, and a 1 104 bp ORF encoding a 367 amino acid polypeptide. The CCR3 cDNA was 1 451 bp in length and contained a 92 bp 5′ UTR, a 267 bp 3′ UTR, and a 1 083 bp ORF encoding 360 amino acids. The TMHMM （TransMembrane prediction using Hidden Markov Models） analysis confirmed that they were seven-transmembrane-spanning proteins. A phylogenetic tree based on the amino acid sequences was constructed by the neighbor-joining （NJ） method using Mega4. The results of phylogenetic analysis revealed that turbot CCR3 and CCR9 were more similar to the cDNAs of other teleosts than to each other. The relationships exhibited in the tree are consistent with their evolutionary relationships. The CCR9 and CCR3 mRNA expression levels in various tissues was measured by quantitative RT-PCR （qRT-PCR）. Both gene transcripts were expressed in all of the tissues analyzed, with the highest expression being in the spleen, head kidney, and heart. The lipopolysaccharide （LPS） challenge results suggest that CCR9 expression in liver was more sensitive than in the other three tissues, while CCR3 expression in spleen and liver was more sensitive than in head kidney or blood. CCR3 and CCR9 expression levels were both high in immune-related tissues and after induction by LPS. Our results indicate that both genes play a role in the turbot immune system.This work further elucidates the functions of these genes in immune responses, which will help to better understand the important relationship between fish

关 键 词：大菱鲆 趋化因子受体 CCR3 CCR9 克隆 表达 

分 类 号：S917[农业科学—水产科学]