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梭鲈ho1基因的克隆及其低氧胁迫下表达分析

吉宇丹 孙志鹏 吕伟华 鲁翠云 曹顶臣 刘天奇 周佳 郑先虎

吉宇丹, 孙志鹏, 吕伟华, 鲁翠云, 曹顶臣, 刘天奇, 周佳, 郑先虎. 梭鲈ho1基因的克隆及其低氧胁迫下表达分析[J]. 南方水产科学. doi: 10.12131/20220187
引用本文: 吉宇丹, 孙志鹏, 吕伟华, 鲁翠云, 曹顶臣, 刘天奇, 周佳, 郑先虎. 梭鲈ho1基因的克隆及其低氧胁迫下表达分析[J]. 南方水产科学. doi: 10.12131/20220187
JI Yudan, SUN Zhipeng, LYU Weihua, LU Cuiyun, CAO Dingchen, LIU Tianqi, ZHOU Jia, ZHENG Xianhu. Characterization and expression analysis of ho1 from Sander lucioperca under acute hypoxia stress[J]. South China Fisheries Science. doi: 10.12131/20220187
Citation: JI Yudan, SUN Zhipeng, LYU Weihua, LU Cuiyun, CAO Dingchen, LIU Tianqi, ZHOU Jia, ZHENG Xianhu. Characterization and expression analysis of ho1 from Sander lucioperca under acute hypoxia stress[J]. South China Fisheries Science. doi: 10.12131/20220187

梭鲈ho1基因的克隆及其低氧胁迫下表达分析

doi: 10.12131/20220187
基金项目: 中央级公益性科研院所基本科研业务费专项 (HSY202009Q);国家重点研发计划项目 (2019YFD0900405);中央引导地方科技发展专项 (ZY21C03)
详细信息
    作者简介:

    吉宇丹 (1997—),女,硕士研究生,研究方向为水产生物技术与遗传育种。E-mail: jiyudan1127@163.com

    通讯作者:

    郑先虎 (1982—),男,研究员,博士,从事水产生物技术与遗传育种研究。E-mail: zhengxianhu@hrfri.ac.cn

  • 中图分类号: (S965.1)

Characterization and expression analysis of ho1 from Sander lucioperca under acute hypoxia stress

  • 摘要: 梭鲈 (Sander lucioperca) 对低氧极敏感,在集约化养殖和苗种运输过程中易发生低氧应激和死亡现象。为探究血红素加氧酶1 (Heme oxygenase 1, HO1) 在梭鲈响应低氧过程中的调节作用,通过RACE (Rapid amplification of cDNA ends) 技术克隆了梭鲈ho1基因,其cDNA全长为1 256 bp,包含840 bp的开放阅读框 (Open reading frame, ORF)、162 bp的5'非编码区(Untranslated region, 5'-UTR)和254 bp的3'非编码区(3'-UTR),编码279个氨基酸。多重序列比对显示,梭鲈Ho1与翘嘴鳜 (Siniperca chuatsi)、舌齿鲈 (Dicentrarchus labrax) 和大口黑鲈 (Micropterus salmoides) 的氨基酸序列相似性分别为91.84%、88.69%和88.11%。实时荧光定量结果显示,ho1基因在所有检测组织中均有表达,其中脑组织中高表达,其次是肾、肝、鳃等组织。低氧刺激前3 h,梭鲈ho1主要在皮肤、鳃中做出响应;低氧胁迫3 h之后,ho1主要在梭鲈心、肝、肾中发挥转录调控作用。复氧12 h,除肝脏外,梭鲈其他组织ho1的相对表达量均可恢复正常,低氧刺激对肝组织ho1的表达产生了较大影响。研究表明,ho1基因参与梭鲈响应低氧的分子调节机制并在其中发挥着重要的生物学功能,可为深入了解梭鲈低氧胁迫遗传机制提供理论参考。
  • 图  1  梭鲈各组织总RNA电泳图

    Figure  1.  Electrophoresis of total RNA in tissues of S. lucioperca

    图  2  梭鲈ho1基因PCR扩增产物电泳图

    M: DL 2000 Marker; 1. 鳃组织中ORF扩增结果; 2. 鳃组织5'末端扩增结果; 3. 鳃组织3'末端扩增结果

    Figure  2.  Electrophoresis of PCR products of ho1 in S. lucioperca

    M: DL 2000 Marker; 1. ORF of gill; 2. 5' RACE of gill; 3. 3' RACE of gill.

    图  3  梭鲈ho1基因cDNA全长序列及推导的氨基酸序列

    Figure  3.  Nucleotide and predicted protein sequences of ho1 of S. lucioperca

    图  4  梭鲈与其他物种Ho1氨基酸同源性比对

    深蓝色区域表示完全相似,浅蓝色区域表示部分相似;下划虚线表示跨膜结构域;下划实线表示Pfam功能结构域;矩形框表示细胞膜外氨基酸残基。

    Figure  4.  Multiple alignment of Ho1 amino acid sequences between S. lucioperca and other species

    Dark blue areas indicate complete similarity and light blue areas indicate partial similarity; transmembrane domain (256−278) is highlighted by dotted line; Pfam functional domain (17−221) is highlighted by underlines; extracellular amino acid residues are marked with rectangular frames.

    图  5  基于不同物种Ho1氨基酸序列构建的系统发育进化树 (NJ法)

    Figure  5.  Phylogenetic tree based on Ho1 amino acid sequences of different species (Neighbor-Joining method)

    图  6  梭鲈ho1 在各组织的相对表达量

    注:图中数值为“平均值±标准差”(N=3),不同字母表示组间差异极显著(P<0.01),后图同此。

    Figure  6.  Relative expression of ho1 in different tissues of S. lucioperca

    Note: The values are $ { \minibfont{\bar X} \pm {\minifont \rm{SD}}}$ (N=3). Different letters indicate extremely significant difference between the tissues (P<0.01). The same as below.

    图  7  急性低氧胁迫与常氧恢复下梭鲈ho1在组织中表达变化

    Figure  7.  Expression of ho1 in S. lucioperca under acute hypoxia stress and normoxia recovery

    表  1  引物序列

    Table  1.   Primers used in this study

    基因
    Gene
    目的
    Purpose
    引物
    Primer
    引物序列
    Sequence (5'–3')
    退火温度
    Annealing temperature/℃
    ho1ORF扩增ho1-F1GGAGCCAGAGAAGAAGACTCAG59.8
    ho1-R1TGCAGCTCGTTTTCAGTGAC60.2
    RACE扩增5'GSPACACCGGGGAAGGCGAAGAATGAC72.0
    5'nGSPCACTGGGGTGGTTGGAGTTCCTGTC70.9
    3'GSPGAGGGCAGGTCCTGGGTCGAATC71.2
    3'nGSPATGGGGCTAAAGGGCAGCGAAGGTC73.2
    RT-PCRho1-F1CTGTGCTCGCTGTATGAGGT59.1
    ho1-R1CCAGTCCTGGCCATAGAAGT59.2
    gapdhRT-PCRgapdh1-FATGTTCGTCATGGGCGTCAA60
    gapdh1-RCAGGCCCTCAATGATGACGA60
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-07-05
  • 修回日期:  2022-08-16
  • 录用日期:  2022-08-30
  • 网络出版日期:  2022-10-10

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