Cloning and expression profiling of Cyp1a gene in Lutjanus argentimaculatus under 4-bromodiphenyl ether (BDE-3) and decabromodiphenyl ether (BDE-209) exposure
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摘要: 细胞色素P450酶 (Cytochrome P450, CYPs) 由P450基因编码，其中Cyp1a基因参与不同类型外源物质的生物转化和代谢。克隆了紫红笛鲷 (Lutjanus argentimaculatus) Cyp1a基因，对其组织表达模式进行分析，探讨了不同质量浓度 (10、50和250 μg·L−1) 一溴联苯醚 (4-bromodiphenyl ether, BDE-3) 和十溴联苯醚 (decabromodiphenyl ether, BDE-209) 胁迫对紫红笛鲷肝脏Cyp1a表达及7-乙氧基香豆素-O-脱乙基酶 (7-ethoxyresorufin O-deethylase, EROD) 活性的影响。结果表明，紫红笛鲷Cyp1a cDNA全长2540 bp，开放阅读框长1 566 bp，编码521个氨基酸。同源分析结果表明紫红笛鲷CYP1A与花鲈 (Lateolabrax maculatus) CYP1A蛋白相似性最高 (92.69%)，进化树分析与白梭吻鲈 (Sander lucioperca) 聚为一支，进化地位最近。Cyp1a基因在紫红笛鲷肝脏表达量最高，其次是脑和鳃，肌肉最低。10 μg·L−1 BDE-3和BDE-209未对Cyp1a基因表达和EROD活性产生影响，而50和250 μg·L−1BDE-3胁迫7~15 d则对两者产生显著抑制，且呈现剂量效应。与BDE-3相反，50和250 μg·L−1 BDE-209 处理组Cyp1a基因表达和EROD活性显著增加，且Cyp1a基因表达与EROD活性呈显著正相关。高浓度BDE-3和BDE-209可对紫红笛鲷肝脏Cyp1a基因的表达产生影响，但两者的影响模式不同。Abstract: Cytochrome P450 enzymes (CYPs) are encoded by P450 genes, in which cytochrome P450 1A (Cyp1a) genes mainly involve in biotransformation and metabolism of numerous xenobiotics. In this study, we cloned the Cyp1a gene from Lutjanus argentimaculatus, and investigated its tissue expression pattern. In addition, we evaluated different concentrations (10, 50 and 250 μg·L−1) of BDE-3 and BDE-209 on Cyp1a gene profile and 7-ethoxyresorufin-O-deethylase (EROD) activity in liver of L. argentimaculatus. The results show that the full length of Cyp1a cDNA was 2 540 bp with 1 566 open reading frame encoding 521 amino acids. The sequence homology of L. argentimaculatus CYP1A was the highest (92.69%) with that of Lateolabrax maculatus. Phylogenetic analysis results indicate that CYP1A was closely aligned with Sander lucioperca protein. Cyp1a transcripts were most abundant in liver, followed by brain and gill, but lowest in muscle. 10 μg·L−1 of BDE-3 and BDE-209 showed no effects on both Cyp1a expression and EROD activity, while high concentrations (50 and 250 μg·L−1) of BDE-3 down-regulated both of them significantly in a concentration-dependent manner on 7th-15th day. In contrast, exposure to 50 and 250 μg·L−1 of BDE-209 resulted in increasing of hepatic Cyp1a level and EROD activity. Moreover, Cyp1a genes levels and EROD activities showed a good correlation. High concentrations of BDE-3 and BDE-209 can affect Cyp1a gene expression in liver of L. argentimaculatus in different manners.
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图 1 紫红笛鲷Cyp1a核苷酸序列及其推导氨基酸序列
Figure 1. Nucleotide and deduced amino acid sequence of Cyp1a of L. argentimaculatus
图 2 紫红笛鲷CYP1A二级 (a) 和三级结构 (b) 预测
Figure 2. Prediction of secondary structure (a) and tertiary structure (b) of L. argentimaculatus CYP1A
图 3 紫红笛鲷CYP1A推导氨基酸多重序列比较
Figure 3. Multiple sequence alignment of deduced amino acid sequence of CYP1A of L. argentimaculatus
图 5 Cyp1a mRNA在紫红笛鲷的组织表达
注：不同字母代表存在显著性差异(P<0.05)， 后图同此。
Figure 5. Tissue expression of Cyp1a mRNA in L. argentimaculatus
Note: Different ltters represent significant dfference (P<0.05). The same case in fllowing figures.
图 6 BDE-3 (a) 和BDE-209 (b) 胁迫对紫红笛鲷肝脏Cyp1a基因表达的影响
Figure 6. Effects of BDE-3 (a) and BDE-209 (b) on Cyp1a expression in liver of L. argentimaculatus
图 7 BDE-3 (a) 和BDE-209(b) 胁迫对紫红笛鲷肝脏EROD活性的影响
Figure 7. Effects of BDE-3 (a) and BDE-209 (b) on EROD activities in liver of L. argentimaculatus
图 8 Cyp1a基因表达量与7-乙氧基香豆素-O-脱乙基酶活性相关性分析
Figure 8. Correlation between Cyp1a relative expression and EROD activity
表 1 实验所用引物
Table 1. Primers used in experiment
引物名称
Primer name引物序列
Primer sequence (5'–3')用途
Purposecyp1a-F GTCTCCGTTGCTAATGTGATCTGTGG Cyp1a中间片段克隆 cyp1a-R GTGATGTCCCGAATGTTGTCCTTGTC cyp1a-5P1 GACCATGACAGGGCAGTGGATATG 5' RACE PCR cyp1a-5P2 GAGTCAGTGATGTCACGAATGTTG cyp1a-3P1 AATGTGCTTTGGCCGACGCTACAA 3' RACE PCR cyp1a-3P2 CTGCTCAGCTTGGTGAACCTCAGT cyp1a-YZF CTCGGGCAAGAACTTTACTA 序列全长验证 cyp1a-YZR GTATCTCCTTATACTTCACT cyp1a-qF GTCTCTGTTGCTAACGTGATCTGTGG RT-PCR[22] cyp1a-qR 同cyp1a-R 18S-qF GTCAAACCCTTTGTCTCCGA 18S-qR CGATGATCAATGTGTCCTGC 
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