| 摘要: |
| 通过RT-PCR及SmartTM 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。 |
| 关键词: 三疣梭子蟹 CYP2 基因克隆 实时荧光定量PCR 磺胺嘧啶 |
| DOI：10.11693/hyhz20131100186 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863 计划), 2012AA10A409 号; 山东省自主创新专项, 2013CX80202 号; 农业科技成果转化资金项目, 2013GB23260589 号。 |
相关附件： 图1.三疣梭子蟹CYP2基因cDNA的核甘酸序列及其推导的氨基酸序列.pdf 图2 三疣梭子蟹CYP2氨基酸序列与其他动物CYP2的氨基酸序列比对.emf 图3.不同物种的CYP2基因系统进化树.emf 图4 三疣梭子蟹CYP2基因在组织中表达量.emf 图5 不同浓度SD对三疣梭子蟹肝胰腺CYP2基因表达的影响.emf |
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| CLONING AND EXPRESSION OF CYP2 GENE IN Portunus trituberculatus |
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feng yan yan,li jian,liu ping and zhang de ning
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Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;2.Ocean University of Shanghai,,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;2.Ocean University of Shanghai,
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| Abstract: |
| The complete cDNA sequence of CYP2 gene in swimming crab Portunus trituberculatus was cloned first time in RT-PCR and SmartTM 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. |
| Key words: Portunus trituberculatus CYP2 gene cloning expression sulfadiazine |
