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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。
In order to study the role of cytochrome CYP2 in the molting and growth of Eriocheir sinensis, cDNA of CYP2 gene of Eriocheir sinensis was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) long. Real-time quantitative PCR was used to analyze the relative expression level of each gene in the process of molting of Eriocheir sinensis. The results showed that CYP2 cDNA of Chinese mitten crab (Eriocheir sinensis) was 1772bp in length and encoded 491 amino acid sequences, including a 84bp 5 ’untranslated region, a 212bp 3’ noncoding region and a 1475bp open reading frame. BLAST. The similarity of this nucleotide sequence to Portland crab and Portunus trituberculatus was 66% and 64%, respectively. The results of real-time PCR showed that CYP2 gene expression in gill was the highest before ecdysis, slightly lower than in gill in liver, pancreas and muscle. In Y organs, eyelids, thoracic ganglia, Cerebellum, small intestine expression; the lowest expression in the heart and stomach. In different molting periods, the Chinese mitten crab (Eriocheir sinensis) showed that the expression level of CYP2 gene in hepatopancreas was the highest during the early molting and molting stages, and the expression level of CYP2 in the eyelid increased from the molting stage to the late molting stage ; CYP2 gene expression in the gills in the pre-molting phase the highest; Y organs, brain ganglia and intestine CYP2 gene expression in the molting phase the highest; muscle CYP2 gene expression in the pre-molting phase; thoracic ganglia and stomach CYP2 gene expression The amount of CYP2 in Y organs was higher in the early ecdysis than in the late ecdysis and ecdysis. The above experimental results show that CYP2 may play an important role in the regulation of molting growth of Eriocheir sinensis.