三疣梭子蟹(Portunus trituberculatus)CYP2基因的cDNA克隆及表达分析

通过RT-PCR及Smart?TM Race技术,首次克隆了三疣梭子蟹(Portunus trituberculatus)CYP2基因cDNA全长序列。该基因cDNA全长1662bp,编码一个由492个氨基酸组成的多肽,预测理论等电点为6.348,分子量大小为56.68kD。氨基酸序列中含有CYP基因家族所特有的K螺旋保守序列(ExxR)和血红素结合区(FxxGxxxCxG)。经氨基酸序列比对及系统进化树分析发现,与岸蟹(Carcinus maenas)的同源性最高,达到75%。实时荧光定量PCR结果表明,CYP2基因在肝胰腺、鳃、肌肉、血淋巴、心脏和眼柄中均有分布,在肝胰腺中表达量最高。肌肉注射磺胺嘧啶后,三疣梭子蟹高、中、低三剂量组CYP2基因表达较对照组都有上调,并具有时间差异性,低剂量组表达量逐渐降低,趋于对照组,中剂量组和高剂量组表达量先升高后降低,6h后同一时间点,均是高剂量组表达最高,低剂量组最低。表明磺胺嘧啶可诱导三疣梭子蟹CYP2基因,CYP2基因可能参与三疣梭子蟹的药物代谢反应。

CLONING AND EXPRESSION OF CYP2 GENE IN Portunus trituberculatus

The complete cDNA sequence of CYP2 gene in swimming crab Portunus trituberculatus was cloned first time in RT-PCR and Smart^TM Race technology. The full-length cDNA of CYP2 was 1662bp encoding a 492 amino-acid polypeptide with the predicted molecular weight of 56.68kD and estimated isoelectric point of 6.38. The conserved K helik sequence (ExxR) and heme-binding motif (FxxGxxxCxG) of cytochrome CYP450 monooxygenases identified in CYP2 suggested that the CYP2 belonged to the cytochrome CYP450 subgroup. Comparison of amino acid sequences showed that the amino acid homology of CYP2 between P. trituberculatus and Carcinus maenas was >75%. Real time RT-PCR was used to assess the mRNA expression of CYP2 in organs or tissues and its expression level of CYP2 under different concentrations of sulfadiazine. The results show that CYP2 was widely expressed in all detected tissues, including hepatopancreas, gills, muscle, hemolymph, and eyestalk. The expression level of CYP2 was up-regulated distinctly in the hepatopancreas after intramuscular administration of sulfadiazine, gradually reduced with the time, and achieved the level of the control in 72h (P<0.05). Therefore, CYP2 could be induced by sulfadiazine and might be involved in the drug-metabolic response of P. trituberculatus.