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紫红笛鲷Cyp1a基因克隆、表达及其对一溴联苯醚和十溴联苯醚胁迫的响应

张喆 巩秀玉 兰丽丽 王学锋 马胜伟 陈海刚 张林宝

张喆, 巩秀玉, 兰丽丽, 王学锋, 马胜伟, 陈海刚, 张林宝. 紫红笛鲷Cyp1a基因克隆、表达及其对一溴联苯醚和十溴联苯醚胁迫的响应[J]. 南方水产科学. doi: 10.12131/20210271
引用本文: 张喆, 巩秀玉, 兰丽丽, 王学锋, 马胜伟, 陈海刚, 张林宝. 紫红笛鲷Cyp1a基因克隆、表达及其对一溴联苯醚和十溴联苯醚胁迫的响应[J]. 南方水产科学. doi: 10.12131/20210271
ZHANG Zhe, GONG Xiuyu, LAN Lili, WANG Xuefeng, MA Shengwei, CHEN Haigang, ZHANG Linbao. Cloning and expression profiling of Cyp1a gene in Lutjanus argentimaculatus under 4-bromodiphenyl ether (BDE-3) and decabromodiphenyl ether (BDE-209) exposure[J]. South China Fisheries Science. doi: 10.12131/20210271
Citation: ZHANG Zhe, GONG Xiuyu, LAN Lili, WANG Xuefeng, MA Shengwei, CHEN Haigang, ZHANG Linbao. Cloning and expression profiling of Cyp1a gene in Lutjanus argentimaculatus under 4-bromodiphenyl ether (BDE-3) and decabromodiphenyl ether (BDE-209) exposure[J]. South China Fisheries Science. doi: 10.12131/20210271

紫红笛鲷Cyp1a基因克隆、表达及其对一溴联苯醚和十溴联苯醚胁迫的响应

doi: 10.12131/20210271
基金项目: 国家自然科学基金项目 (31702352);中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金项目 (2021SD17, 2019TS14);农业农村部财政专项项目 (NHYYSWZZZYKZX2020, NFZX2021)
详细信息
    作者简介:

    张喆:张 喆 (1981—),女,副研究员,博士,从事海洋生态学研究。E-mail: zhangzhe@scsfri.ac.cn

  • 中图分类号: X 171.5

Cloning and expression profiling of Cyp1a gene in Lutjanus argentimaculatus under 4-bromodiphenyl ether (BDE-3) and decabromodiphenyl ether (BDE-209) exposure

  • 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。
  • 图  1  紫红笛鲷Cyp1a核苷酸序列及其推导氨基酸序列

    粗体为起始密码子;粉色阴影为脯氨酸/甘氨酸丰富区;绿色阴影为底物识别位点;方框为硬骨鱼CYP1As保守序列;红色粗体体为CYPs不变序列;灰色阴影为血红素蛋白结合区;*为终止密码子;下划线为多腺苷酸信号区。

    Figure  1.  Nucleotide and deduced amino acid sequence of Cyp1a of L. argentimaculatus

    Bolds. Start condon; Pink shadows. Proline/glycine-rich region; Green shadows. Substrate recognition sites; Box. Highly conserved sequence among teleost CYP1As; Red bolds. Invariant sequence of CYPs; Gray shadows. Heme-binding corer; *. Stop condon; Underline. Polyadenylation signal.

    图  2  紫红笛鲷CYP1A二级和三级结构预测

    a. 二级结构, 粉色:α-螺旋,黄色:β-折叠,灰色:无规则卷曲:b. 三级结构。

    Figure  2.  Prediction of secondary structure and tertiary structure of L. argentimaculatus CYP1A

    a. Secondary structure. Pink. α-helix. Yellow. β-sheet. Gray. Coils. b. Tertiary structure.

    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  4  紫红笛鲷CYP1A进化树分析

    Figure  4.  Phylogenetic analysis of L. argentimaculatus CYP1A

    图  5  Cyp1a mRNA在紫红笛鲷的组织表达

    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

    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 (5'—3')
    Primer sequence (5'—3')
    用途
    Purpose
    cyp1a-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 cyp1a-R
    18S-qF GTCAAACCCTTTGTCTCCGA
    18S-qR CGATGATCAATGTGTCCTGC
    下载: 导出CSV
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  • 收稿日期:  2021-09-22
  • 修回日期:  2021-11-04
  • 录用日期:  2021-12-01
  • 网络出版日期:  2021-12-14

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