Molecular cloning and expression analysis of cytochrome P450 3A gene in the turbot Scophthalmus maximus
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Molecular cloning and expression analysis of cytochrome P450 3A gene in the turbot Scophthalmus maximus Airong Sun • Jian Li • Jingzhou Huang Zhiqiang Chang • Jitao Li • Qi Wang
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Received: 14 August 2012 / Accepted: 25 February 2013 / Published online: 23 March 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract In this study, the cytochrome P450 3A (CYP3A) gene was cloned from the turbot Scophthalmus maximus for the first time using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches. The amino acid sequences were analyzed with corresponding software programs. The cDNA of CYP3A was 1,969 bp in length, which contained a 50 -untranslated region (UTR) of 34 bp, a 30 -UTR of 404 bp and an open reading frame of 1,530 bp encoding a predicted protein of 509 amino acids (GenBank accession No.
A. Sun J. Li (&) Z. Chang J. Li Q. Wang Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, People’s Republic of China e-mail: [email protected]
JN216889). The deduced protein had a molecular weight of 58.09 kDa and an isoelectric point of 5.75. Amino acid sequence alignment indicated that turbot CYP3A shared 60–67 % homology with other fish species. It consists of a signal peptide, six conservative substrate recognition sites (SRS 1–6) and the conserved heme-binding motif FXXGXXXCXG in all CYP3As. Quantitative real-time RT-PCR analysis indicated that turbot CYP3A mRNA was widely expressed in liver, kidney, gill, muscle, stomach, intestine, gallbladder and spleen, with the highest level in liver and the lowest in muscle. After oral administration of sulfamethazine, CYP3A expression in all experimental groups enhanced compared with control, and the expression varied with administration time. It suggested that CYP3A expression could be induced by sulfamethazine. Our findings provided molecular characterization and expression profile of turbot CYP3A, and revealed the important role that turbot CYP3A played in drug metabolisms.
A. Sun College of Fisheries and Life Science, Shanghai Ocean University, 999 Shanghai City Loop Road, Shanghai 201306, People’s Republic of China
Keywords Scophthalmus maximus Cytochrome P450 3A Cloning Sulfamethazine Expression
J. Huang Department of Experimental Oncology, University of Alberta, Edmonton, AB T6G 2H7, Canada
Introduction
Q. Wang Fisheries College, Ocean University of China, 238 Songling Road, Qingdao 266003, People’s Republic of China
The cytochrome P450 (abbreviated as CYP) is the terminal oxidase of cytochrome P450 monooxygenases, which plays an important role in binding substrates and transferring electrons from NADPH to
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NADPH cytochrome P450 reductase (Nelson et al. 1993; Feyerisen 1999). CYP has been identified as the major enzyme involved in drug metabolism (Anzenbacher and Anzenbacherova 2001), and it is widely distributed in different organis
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