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| 三疣梭子蟹CYP4C基因的cDNA克隆及表达分析 |
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张晓燕,李健,刘萍,陈萍,孙铭
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黄海水产研究所,黄海水产研究所,黄海水产研究所,黄海水产研究所,黄海水产研究所
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| 摘要: |
| 采用RT-PCR及Smart-TM Race技术，首次获得三疣梭子蟹CYP4C基因cDNA全长序列。该基因全长1888bp，编码一个由514个氨基酸组成的多肽，预测分子量大小为59.54kDa，理论等电点为8.08。氨基酸序列中含有CYP基因家族特有的K螺旋保守序列（ExxR）和血红素结合区（FxxGxxxCxG）。同源性及系统进化分析表明，三疣梭子蟹CYP4C基因与岸蟹、凡纳滨对虾、日本沼虾、中国对虾、沟虾、红螯螯虾的同源性分别为88%、72%、66%、59%、60%、59%。荧光定量RT-PCR结果表明，CYP4C在肝胰腺、肌肉、眼柄、鳃和心脏中均有分布，在肝胰腺中表达量最高。高盐（45）胁迫后，三疣梭子蟹CYP4C的表达量显著高于对照组，随着胁迫时间的延长出现逐渐降低的趋势，在胁迫48h后表达量达到对照组水平；低盐（11）胁迫条件下，CYP4C的表达水平随着胁迫时间的延长呈现逐渐下降的趋势，且从胁迫6h开始显著低于对照组。研究推测，CYP4C基因参与三疣梭子蟹盐度适应调节，是蜕皮机制的调控因子之一。 |
| 关键词: 三疣梭子蟹，CYP4C，基因克隆，表达，盐度胁迫 |
| DOI：10.11693/hyhz20121227001 |
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| 基金项目:国家863计划课题（2012AA10A409），中央级公益性科研院所基本科研业务费项目资助（20603022012021） |
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| Cloning and expression analysis of CYP4C gene in Portunus trituberculatus |
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zhangxiaoyan,lijian,liuping,chenping and sunming
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Yellow Sea Fisheries Research Institute,Yellow Sea Fisheries Research Institute,Yellow Sea Fisheries Research Institute,Yellow Sea Fisheries Research Institute,Yellow Sea Fisheries Research Institute
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| Abstract: |
| The complete cDNA sequence of CYP4C gene in Portunus trituberculatus was first cloned through RT-PCR and Smart-TM Race technology. The length of the CYP4C gene is 1888bp and encodes the protein of 514 amino acids. Bioinformatics analysis deduced the CYP4C gene protein molecular weight of 59.54kDa with a theoretical pI 8.08. The amino acid sequence has CYP conserved domains of helix K （ExxR）and heme-binding motif（FxxGxxxCxG）. Blast analysis revealed that the similarities of CYP4C with C. maenas、L. vannamei、Macrobrachium nipponense、Fenneropenaeus chinensis、O. limosus、C. quadricarinatus were 88%、72%、66%、59%、60%、59%, respectively. Real-time fluorescent quantitative PCR was used to assess the mRNA expression of CYP4C in different tissues and its expression level of CYP4C after the salinity stress. The results showed that CYP4C expression existed in all tested tissues of Portunus trituberculatus, including hepatopancreas, muscle, eyes, gills, and heart. The highest expression level was observed in hepatopancreas, while the lowest in heart. The expression level of CYP4C was up-regulated distinctly in the hepatopancreas of Portunus trituberculatus at salinity of 45 and gradually reduced with the time extended that achieved the level of control group after 48 h; CYP4C expression gradually reduced with the time extended at salinity of 11 and significantly lower than those of the control group at the 6 h. The results implied that CYP4C might participate in the salinity accommodation and play an important role in the molting mechanism. |
| Key words: Portunus trituberculatus, CYP4C, Gene cloning, Expression, Salinity stress |