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卵形鲳鲹脂肪酸延长酶(Elovl4-like)基因特征与功能研究

宋岭 朱克诚 郭华阳 江世贵 张殿昌

引用本文:
Citation:

卵形鲳鲹脂肪酸延长酶(Elovl4-like)基因特征与功能研究

    作者简介: 宋 岭(1991—),女,硕士研究生,从事水产动物遗传育种和分子生物学研究。E-mail: 1126319294@qq.com;
    通讯作者: 张殿昌, zhangdch@scsfri.ac.cn
  • 中图分类号: S917.4

Characterization and function analysis of Elovl4-like elongase gene in golden pompano (Trachinotus ovatus)

    Corresponding author: Dianchang ZHANG, zhangdch@scsfri.ac.cn
  • CLC number: S917.4

  • 摘要: 长链脂肪酸延长酶(elongases of very long chain fatty acids, Elovls)是长链多不饱和脂肪酸(long chain polyunsaturated fatty acid, LC-PUFA)生物合成途径中的关键限速酶,能够延长PUFA的碳链。为探究Elovl4在卵形鲳鲹(Trachinotus ovatus)PUFA生物合成中的功能,该研究克隆了卵形鲳鲹的Elovl4基因的cDNA序列,命名为ToElovl4-likeToElovl4-like开放阅读框(open reading frame,ORF)为792 bp,编码263个氨基酸。生物信息学分析结果表明,编码的氨基酸包含Elovl家族的显著结构特征如组氨酸盒子、多个跨膜区和ER滞留信号等。序列比对结果显示,卵形鲳鲹Elovl4-like基因高度保守,且该基因编码的蛋白序列与其他鱼类的相似度为73%~86%,其中与大西洋鳕(Gadus morhua)的相似度最高为86%。系统进化分析表明,Elovl4主要聚为Elovl4a、Elovl4b和Elovl4-like三类。其中ToElovl4-like与其他鱼类的Elovl4-like亲缘关系最近。实时荧光定量PCR表明ToElovl4-like基因在各组织中均有表达,其中在性腺和脾中mRNA表达量最高,在脑中mRNA表达量最低。酵母异源表达分析表明,ToElovl4-like可以分别将亚油酸(18:2n-6)、二十碳五烯酸(20:5n-3)延长为二十碳二烯酸(20:2n-6)、二十二碳五烯酸(22:5n-3)。研究结果为了解鱼类长链多不饱和脂肪酸(LC-PUFA)的生物合成提供了理论基础。
  • 图 1  Elovl4-like cDNA序列及推测的氨基酸序列

    Figure 1.  Full cDNA and deduced amino acid sequence of Elovl4-like

    图 2  卵形鲳鲹Elovl4-like与其他物种的氨基酸序列比对

    Figure 2.  Multiple alignment of deduced amino acid sequence of Elovl4-like from T. ovatus and other species

    图 3  卵形鲳鲹与其他物种Elovl4的ML系统进化树

    Figure 3.  ML phylogenrtic tree based on amino acids of Elovl4 of T. ovatus and other species

    图 4  卵形鲳鲹Elovl4-like mRNA在不同组织中的表达分布

    Figure 4.  Expression level of T. ovatus Elovl4-like gene in different tissuesDifferent letters indicate significant difference at 0.05 level among different tissues

    图 5  Elovl4-like基因的功能鉴定

    Figure 5.  Functional identification of Elovl4-like gene

    表 2  用于序列比对和构建系统进化树所用物种的基因及序列登录号

    Table 2.  Genes and sequence accession numbers used for alignment and construction of phylogenetic tree

    物种
    species
    基因
    gene
    登录号
    accession No.
    原鸡 Gallus gallus Elovl4 ENSGALG00000015876
    红鳍东方鲀 Takifugu rubripes Elovl4a ENSTRUG00000004612
    红鳍东方鲀 Takifugu rubripes Elovl4b ENSTRUG00000014717
    红鳍东方鲀 Takifugu rubripes Elovl4-like XP_003974148.1
    Homo sapiens Elovl4 ENSG00000118402
    斑马鱼 Danio rerio Elovl4a ENSDARG00000006773
    斑马鱼 Danio rerio Elovl4-like1 ENSDARG00000068551
    斑马鱼 Danio rerio Elovl4-like2 ENSDARG00000057365
    大西洋鳕 Gadus morhua Elovl4a ENSGMOT00000002680.1
    大西洋鳕 Gadus morhua Elovl4-like ENSGMOT00000020430.1
    三刺鱼 Gasterosteus aculeatus Elovl4a ENSGACT00000008132.1
    三刺鱼 Gasterosteus aculeatus Elovl4b ENSGACT00000017607.1
    三刺鱼 Gasterosteus aculeatus Elovl4-like1 ENSGACT00000016161.1
    三刺鱼 Gasterosteus aculeatus Elovl4-like2 ENSGACG00000012189
    三刺鱼 Gasterosteus aculeatus Elovl4-like3 ENSGACG00000012118
    河鲀 Tetraodon nigroviridis Elovl4a ENSTNIG00000002416
    河鲀 Tetraodon nigroviridis Elovl4b ENSTNIG00000010936
    河鲀 Tetraodon nigroviridis Elovl4-like ENSTNIG00000010693
    卵形鲳鲹 Trachinotus ovatus Elovl4a MG674424
    卵形鲳鲹 Trachinotus ovatus Elovl4b MG674425
    卵形鲳鲹 Trachinotus ovatus Elovl4-like MK215859
    斑剑尾鱼 Xiphophorus maculatus Elovl4a ENSXMAP00000006115.1
    斑剑尾鱼 Xiphophorus maculatus Elovl4b ENSXMAP00000007217.1
    斑剑尾鱼 Xiphophorus maculatus Elovl4-like1 ENSXMAP00000011156.1
    斑剑尾鱼 Xiphophorus maculatus Elovl4-like2 ENSXMAP00000013098.1
    眼斑雀鳝 Lepisosteus oculatus Elovl4-like ENSLOCP00000021081.1
    墨西哥脂鲤 Astyanax mexicanus Elovl4a ENSAMXP00000017270.1
    墨西哥脂鲤 Astyanax mexicanus Elovl4b ENSAMXP00000006663.1
    墨西哥脂鲤 Astyanax mexicanus Elovl4-like ENSAMXP00000005396.1
    秀美花鳉 Poecilia formosa Elovl4a ENSPFOP00000018605.2
    青鳉 Oryzias latipes Elovl4b ENSORLG00000017019
    尼罗罗非鱼 Oreochromis niloticus Elovl4a ENSONIP00000009094.1
    尼罗罗非鱼 Oreochromis niloticus Elovl4b ENSONIP00000000842.1
    尼罗罗非鱼 Oreochromis niloticus Elovl4-like1 ENSONIP00000011380.1
    尼罗罗非鱼 Oreochromis niloticus Elovl4-like2 ENSONIP00000002524.1
    下载: 导出CSV

    表 1  Elovl4-like ORF扩增、组织表达和功能验证所需引物

    Table 1.  Sequences of primers used for Elovl4-like cDNA cloning, functional characterization and real-time quantitative PCR

    引物名称
    primer name
    引物序列(5’-3’)
    primer sequence
    用途
    application
    Elovl4-like F1 ATGGCTTCTGCATGG ORF扩增
    Elovl4-like R2 TTATTTCTGCTTCTTC
    Elovl4-like F2 CGG GGTACCATGGCTTCTGCATGGCAAAGT
    Elovl4-like R2 CCG CTCGAGTTTCTGCTTCTTCTTGCTGA
    Elovl4-like qF GTCAGCCTGTCAACTACT qRT-PCR
    Elovl4-like qR CCACCAGAGGATGAACAT
    EF-1αF CCCCTTGGTCGTTTTGCC 内参
    EF-1αR GCCTTGGTTGTCTTTCCGCT
    下载: 导出CSV

    表 3  卵形鲳鲹Elovl4-like基因在酿酒酵母中的功能鉴定

    Table 3.  Functional characterization of Elovl4-like gene in T. ovatus

    底物脂肪酸
    FA substrate
    产物
    product
    转化率/%
    conversion
    活性
    activity
    亚油酸18:2n-6 20:2n-6 0.83 C18 → C20
    γ-亚麻酸18:3n-6 20:3n-6 0 C18 → C20
    二十碳五烯酸20:5n-3 22:5n-3 0.84 C20 → C22
    α-亚麻酸18:3n-3 20:3n-3 0 C18 → C20
    十八碳四烯酸18:4n-3 20:4n-3 0 C18 → C20
    花生四烯酸20:4n-6 22:4n-6 0 C20 → C22
    二十二碳四烯酸22:4n-6 24:4n-6 0 C22 → C24
    二十二碳五烯酸22:5n-3 24:5n-3 0 C22 → C24
    二十二碳六烯酸22:6n-3 24:6n-3 0 C22 → C24
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-12-04
  • 录用日期:  2019-01-06
  • 网络出版日期:  2019-02-18

卵形鲳鲹脂肪酸延长酶(Elovl4-like)基因特征与功能研究

    作者简介:宋 岭(1991—),女,硕士研究生,从事水产动物遗传育种和分子生物学研究。E-mail: 1126319294@qq.com
    通讯作者: 张殿昌, zhangdch@scsfri.ac.cn
  • 1. 中国水产科学研究院南海水产研究所,农业农村部南海渔业资源开发利用重点实验室,广东 广州 510300
  • 2. 上海海洋大学水产与生命学院,上海 201306
  • 3. 广东省海洋生物种业工程技术研究中心,广东 广州 510300

摘要: 长链脂肪酸延长酶(elongases of very long chain fatty acids, Elovls)是长链多不饱和脂肪酸(long chain polyunsaturated fatty acid, LC-PUFA)生物合成途径中的关键限速酶,能够延长PUFA的碳链。为探究Elovl4在卵形鲳鲹(Trachinotus ovatus)PUFA生物合成中的功能,该研究克隆了卵形鲳鲹的Elovl4基因的cDNA序列,命名为ToElovl4-likeToElovl4-like开放阅读框(open reading frame,ORF)为792 bp,编码263个氨基酸。生物信息学分析结果表明,编码的氨基酸包含Elovl家族的显著结构特征如组氨酸盒子、多个跨膜区和ER滞留信号等。序列比对结果显示,卵形鲳鲹Elovl4-like基因高度保守,且该基因编码的蛋白序列与其他鱼类的相似度为73%~86%,其中与大西洋鳕(Gadus morhua)的相似度最高为86%。系统进化分析表明,Elovl4主要聚为Elovl4a、Elovl4b和Elovl4-like三类。其中ToElovl4-like与其他鱼类的Elovl4-like亲缘关系最近。实时荧光定量PCR表明ToElovl4-like基因在各组织中均有表达,其中在性腺和脾中mRNA表达量最高,在脑中mRNA表达量最低。酵母异源表达分析表明,ToElovl4-like可以分别将亚油酸(18:2n-6)、二十碳五烯酸(20:5n-3)延长为二十碳二烯酸(20:2n-6)、二十二碳五烯酸(22:5n-3)。研究结果为了解鱼类长链多不饱和脂肪酸(LC-PUFA)的生物合成提供了理论基础。

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