三疣梭子蟹CYP302a1基因克隆及其表达分析  

Molecular cloning and expression analysis of CYP302a1 from Portunus trituberculatus

作　　者：柳志业 朱冬发[1] 谢熙[1] 邱锡尔[1] 周彦琦 沈锡权[1] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211

出　　处：《水产学报》2015年第5期628-637,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(41376152,40976098);浙江省自然科学基金(LY13C190006)

摘　　要：细胞色素P450(CYP)302a1是昆虫蜕皮激素合成通路中的关键酶,为了研究其在三疣梭子蟹蜕皮过程中的调控作用,实验采用反转录PCR(RT-PCR)和c DNA末端快速扩增(RACE)技术,克隆得到了三疣梭子蟹CYP302a1全长c DNA序列(Gen Bank登录号:KM596851)。该序列全长为3 171 bp,包含一个长度为1 626 bp的开放阅读框,编码541个氨基酸;序列分析显示该氨基酸含helix-C、helix-K、helix-I、PERF及heme-binding共5个P450特征保守区域;系统进化树分析发现三疣梭子蟹CYP302a1与日本剑水蚤CYP302a1聚为一小支,再与其他物种CYP302a1聚为一支。采用实时荧光定量PCR(qRT-PCR)技术分析了CYP302a1在不同组织和蜕皮过程中的表达水平变化。结果显示,CYP302a1的表达量在Y器中最高,而在其他组织中均极低(P<0.05);蜕皮过程中,CYP302a1在蜕皮后期(A、B期)表达量最低,从蜕皮间期(C期)开始上升,至蜕皮前期的D1亚期达到最大值,随后下降。结果表明CYP302a1在三疣梭子蟹蜕皮调控中可能起着重要作用。CYP302a1 is the key enzyme in biosynthesis of the ecdysteriods. The aim of this study is to analyze the function of CYP302a1 during the process of the molting in sw imming crab Portunus trituberculatus. The full-length cDNA of CYP302a1 is cloned from Y-organs of the P. tritubertulatus. The length of the CYP302a1（ Genbank accession NO. ： KM596851） is 3 171 bp and contains an ORF of 1 626 bp encoding 541 amino acid. The amino acid contains 5 conserved domains including helix-C,helix-K,helix-I,PERF and heme-binding. The phylogenetic tree show ed that CYP302a1 of P. trituberculatus was clustered with that of Tigriopus japonicas,w hich is separated from other CYPs. Tissue distribution show ed that transcript of CYP302a1 was mainly detected in the Y-organ（ YO） and far exceeded other tissues. The transcript level of CYP302a1 during molting cycle w as determined by qRT-PCR. During molting process,levels of CYP302a1 in YO were extremely low during postmolt（ stages A and B）,began to increase during intermolt（ stage C） and reached the peak at D1 stage,then declined. The combined results show ed that CYP302a1 might play an essential role in the molting of P. trituberculatus.

关 键 词：三疣梭子蟹 CYP302a1 蜕皮周期 基因克隆 表达水平 

分 类 号：Q785[生物学—分子生物学] S968.2[农业科学—水产养殖]