中华绒螯蟹细胞色素CYP2基因的克隆与表达分析

cDNA Cloning and Expression Analysis of CYP2 Gene in Chinese Mitten Crab Eriocheir sinensis

为研究细胞色素CYP2基因在中华绒螯蟹蜕皮及生长发育中的作用,本试验通过逆转录聚合酶链式RT-PCR反应以及cDNA末端快速扩增RACE技术获得了中华绒螯蟹CYP2基因cDNA全长。用实时荧光定量PCR技术,分析该基因在中华绒螯蟹蜕皮过程中各组织的相对表达量。试验结果显示,中华绒螯蟹CYP2的cDNA全长1772bp,编码491个氨基酸序列,包括一个84bp的5′端非编码区,一个212bp的3′端非编码区、一个1475bp的开放阅读框,经BLAST比对,该核苷酸序列与岸蟹、三疣梭子蟹的相似性分别为66%、64%。实时荧光定量PCR结果显示,中华绒螯蟹蜕皮前,CYP2基因在鳃中的表达量相对最高;在肝胰脏、肌肉中表达量略低于鳃;在Y器官、眼柄、胸神经节、脑神经节、肠中少量表达;在心和胃中表达量最低。中华绒螯蟹在不同的蜕皮时期中,通过荧光定量试验得出,肝胰脏中CYP2基因表达量在蜕皮前期和蜕皮期最高;眼柄中CYP2基因表达量从蜕皮期到蜕皮后期有上升趋势;鳃中CYP2基因表达量在蜕皮前期最高;Y器官、脑神经节和肠中CYP2基因表达量在蜕皮期最高;肌肉中CYP2基因表达量在蜕皮前期最高;胸神经节和胃中CYP2基因表达量在蜕皮后期最高;Y器官中CYP2基因在蜕皮前期表达量高于蜕皮后期和蜕皮期。以上试验结果表明,CYP2对中华绒螯蟹蜕皮生长中的调控可能起到重要作用。

The full-length cDNA of CYP2 gene was cloned using reverse transcriptase polymerase chain reaction（RT-PCR）and rapid-amplification of cDNA ends（RACE）to study the regulation of CYP2 gene in molting in Chinese mitten crab Eriocheir sinensis.Quantitative real-time PCR（qRT-PCR）was used to quantify the relative expression level of CYP2 in different tissues and molting process in Chinese mitten crab.The results showed that the full-length cDNA sequence of CYP2 was 1772bp,a 5′-untranslated region（UTR）of 84 bp,and a 3′-UTR of 212 bp and included a 1475 bp ORF encoding 491 amino acid residues.The alignment of CYP2 amino acid sequence of in Chinese mitten crab showed that it shared 66%identity with crab Carcinus maenas and shared 64%identity with swimming crab Portunus trituberculatus.The CYP2 mRNA was expressed in all tissues examined and highly in gills.CYP2 expression was slightly lower in hepatopancreas and muscle than in the gills,with small amount in Y organs,eye stalk,horaci ganglion,cerebral ganglion,and intestines and trace in heart and stomach.During the molting process,the expression levels of CYP2 mRNA of hepatopancreas was the maximum in D and E period.The amount of CYP2 expression in eye stalk was rising from E period to AB period,and CYP2 expression in gills was the maximum in D and AB period.CYP2 expression in Y organs,cerebral ganglion and intestines was the maximum in E period.CYP2 expression in muscle was the maximum in D period.CYP2 expression in thoracic ganglion and stomach was the maximum in AB period,and in Y organs higher in D period than in AB and E periods.The findings indicate that CYP2 might play an important role in regulation of the molting process.