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铁皮石斛鲨烯单加氧酶基因的克隆与表达分析

林江波 王伟英 邹晖 戴艺民

林江波,王伟英,邹晖,等. 铁皮石斛鲨烯单加氧酶基因的克隆与表达分析 [J]. 福建农业学报,2020,35(5):495−502 doi: 10.19303/j.issn.1008-0384.2020.05.005
引用本文: 林江波,王伟英,邹晖,等. 铁皮石斛鲨烯单加氧酶基因的克隆与表达分析 [J]. 福建农业学报,2020,35(5):495−502 doi: 10.19303/j.issn.1008-0384.2020.05.005
LIN J B, WANG W Y, ZOU H, et al. Cloning and Expression of Squalene Monooxygenase Genes of Dendrobium officinale [J]. Fujian Journal of Agricultural Sciences,2020,35(5):495−502 doi: 10.19303/j.issn.1008-0384.2020.05.005
Citation: LIN J B, WANG W Y, ZOU H, et al. Cloning and Expression of Squalene Monooxygenase Genes of Dendrobium officinale [J]. Fujian Journal of Agricultural Sciences,2020,35(5):495−502 doi: 10.19303/j.issn.1008-0384.2020.05.005

铁皮石斛鲨烯单加氧酶基因的克隆与表达分析

doi: 10.19303/j.issn.1008-0384.2020.05.005
基金项目: 福建省科技计划公益类专项(2018R1024-3);福建省自然科学基金项目(2018J01119)
详细信息
    作者简介:

    林江波(1976−),男,硕士,副研究员,主要从事农业生物技术研究(E-mail:345953257@qq.com

    通讯作者:

    戴艺民(1969−),男,博士,研究员,主要从事农业生物技术研究(E-mail:dymttcn@163.com

  • 中图分类号: S 567.239

Cloning and Expression of Squalene Monooxygenase Genes of Dendrobium officinale

  • 摘要:   目的  克隆铁皮石斛(Dendrobium officinale)甾醇类化合物合成关键酶鲨烯单加氧酶(Squalene Monooxygenase,SQE)基因,并对其进行生物信息学分析和不同营养生长期茎和叶中的表达模式分析。  方法  根据铁皮石斛转录组测序获得带5’末端的SQE基因片段,设计DoSQE1DoSQE2基因的3’RACE引物,克隆全长cDNA,利用生物信息学分析软件对DoSQE1DoSQE2基因及其编码蛋白序列进行分析。运用实时荧光定量PCR检测DoSQE1DoSQE2基因在铁皮石斛营养生长期的8月、10月、12月茎和叶中的表达模式。  结果  DoSQE1基因cDNA序列全长1 796 bp(GenBank 登录号MT160182),含有1个1 554 bp的ORF,编码517个氨基酸;DoSQE2基因cDNA序列全长1 963 bp(GenBank 登录号MT160183),含有1个1 578 bp的ORF,编码525个氨基酸。DoSQE1具有2个跨膜区,分别在4~22 aa和55~72 aa;DoSQE2只有在5~23 aa的1个跨膜区。DoSQE1蛋白在204~476 aa、DoSQE2蛋白在211~484 aa处含有鲨烯环氧酶结构域。系统进化分析表明,DoSQE1与姬蝴蝶兰SQE(XP_020599860.1)的亲缘关系最近,DoSQE2与姬蝴蝶兰SQE(XP_020579136.1)的亲缘关系最近。qRT-PCR检测结果表明,茎和叶中都能检测到2个基因的表达,叶的表达量显著高于茎。DoSQE1基因在8月份表达量最高,DoSQE2基因在10月份表达量最高。  结论  本研究克隆得到DoSQE1DoSQE2基因,发现DoSQE1DoSQE2基因的表达模式存在差异,该结果为进一步研究铁皮石斛甾醇类化合物生物合成机理及代谢调控奠定基础。
  • 图  1  PCR产物电泳结果

    注:M. 分子量标记;A. DoSQE1基因3′RACE;B. DoSQE1基因开放阅读框;C. DoSQE2基因3′RACE;D. DoSQE2基因开放阅读框。

    Figure  1.  PCR products by electrophoresis

    Note: M. DNA marker; A. 3′RACE of DoSQE1; B. ORF of DoSQE1; C. 3′RACE of DoSQE2; D. ORF of DoSQE2.

    图  2  DoSQE1与DoSQE2蛋白跨膜区预测

    Figure  2.  Predicted transmembrane regions in DoSQE1 and DoSQE2

    图  3  铁皮石斛DoSQE1和DoSQE2蛋白的二级结构预测

    注:大写字母:氨基酸序列;小写字母:二级结构;c:无规则卷曲;h:α螺旋;e:β折叠。

    Figure  3.  Predicted secondary structures of DoSQE1 and DoSQE2 from D. officinale

    Note:capital letters show amino acid sequence; lowercase letters show amino acid chain structure, as c for random coil, h for α-helix, and e for β-extended form.

    图  4  预测的铁皮石斛DoSQE1和DoSQE2蛋白三维结构

    Figure  4.  Predicted 3D structures of DoSQE1 and DoSQE2 from D. officinale

    图  5  铁皮石斛DoSQE蛋白系统进化树分析

    注:图中各节点处数字代表可信度。

    Figure  5.  Phylogenetic tree analysis on DoSQE from D. officinale

    Note: the numbers at the node of the figure is credibility.

    图  6  DoSQE1DoSQE2基因在铁皮石斛茎和叶中的相对表达量

    Figure  6.  Expressions of DoSQE1 and DoSQE2 in stems and leaves of D. officinale

    表  1  PCR引物序列信息

    Table  1.   Sequence of PCR primers

    引物名称
    Primer name
    引物序列(5′-3′)   
    Primer sequence(5′-3′)   
    引物用途   
    Primer usage   
    3DoSQE1F1 CCTTCCATTGCTAACGGAGAGA DoSQE1基因 3′RACE引物3′RACE primers of DoSQE1
    3DoSQE1F2 CCTGGAGCACTTCTAATGGGAG
    3DoSQE2F1 CTCTTCCTACACCTGGAGCACT DoSQE2基因3′RACE引物 3′RACE primers of DoSQE2
    3DoSQE2F2 TATACCTTGCGTAAGCCCGTTG
    dT-adaptor CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT 3′RACE接头引物 Adaptor primers of 3′RACE
    adaptor CTGATCTAGAGGTACCGGATCC
    DoSQE1F CGCGGTACCATGATGCTTCTCCAGTACA DoSQE1基因ORF克隆引物 primers of DoSQE1 ORF clone
    DoSQE1R CGCGTCGACGGAGTTCTCATTGTGTAGG
    DoSQE2F CGCGGTACCATGGTGATGCCACTTTCGTA DoSQE2基因ORF克隆引物 primers of DoSQE2 ORF clone
    DoSQE2R CGCGTCGACGTTGGAAGTTCACCCACGAG
    DoSQE1-F AGGACGCAAACAGCGAGAG DoSQE1基因表达分析引物 Expression analysis primers of DoSQE1
    DoSQE1-R CACCAACGATTCTATGAGGC
    DoSQE2-F CCCCAGATACCGAGTCAG DoSQE2基因表达分析引物 Expression analysis primers of DoSQE2
    DoSQE2-R CCCATCAGAAGTGCTCCAG
    DoACT-F AGGAAGGCGGCTTTGAATC 内参基因 Reference gene
    DoACT-R CCATGCCAACCATGACACC
    注:下划线碱基为酶切位点。
    Note:The ud bases were the enzyme site.
    下载: 导出CSV
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  • 收稿日期:  2020-03-14
  • 修回日期:  2020-04-30
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