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金线莲甘露糖-1-磷酸尿苷转移酶基因的克隆与表达分析

李和平 林江波 黄惠明 邹晖 戴艺民

李和平,林江波,黄惠明,等. 金线莲甘露糖-1-磷酸尿苷转移酶基因的克隆与表达分析 [J]. 福建农业学报,2023,38(5):537−544 doi: 10.19303/j.issn.1008-0384.2023.05.004
引用本文: 李和平,林江波,黄惠明,等. 金线莲甘露糖-1-磷酸尿苷转移酶基因的克隆与表达分析 [J]. 福建农业学报,2023,38(5):537−544 doi: 10.19303/j.issn.1008-0384.2023.05.004
LI H P, LIN J B, HUANG H M, et al. Cloning and Expression of Mannose-1-phosphate Guanyltransferase Gene in Anoectochilus roxburghii [J]. Fujian Journal of Agricultural Sciences,2023,38(5):537−544 doi: 10.19303/j.issn.1008-0384.2023.05.004
Citation: LI H P, LIN J B, HUANG H M, et al. Cloning and Expression of Mannose-1-phosphate Guanyltransferase Gene in Anoectochilus roxburghii [J]. Fujian Journal of Agricultural Sciences,2023,38(5):537−544 doi: 10.19303/j.issn.1008-0384.2023.05.004

金线莲甘露糖-1-磷酸尿苷转移酶基因的克隆与表达分析

doi: 10.19303/j.issn.1008-0384.2023.05.004
基金项目: 福建省科技计划公益类专项(2020R1030003);福建省农业科学院科技创新团队建设项目( CXTD2021001-2)
详细信息
    作者简介:

    李和平(1982-),男,硕士,助理研究员,研究方向:经济作物育种与分子生物学(E-mail:hepingli_1982@126.com

    通讯作者:

    戴艺民(1969-),男,博士,研究员,研究方向:药用植物创新利用(E-mail:dymttcn@163.com

  • 中图分类号: Q786

Cloning and Expression of Mannose-1-phosphate Guanyltransferase Gene in Anoectochilus roxburghii

  • 摘要:   目的  甘露糖是金线莲多糖重要组成成分,对甘露糖-1-磷酸尿苷转移酶(GMP)基因进行克隆和基因表达调控分析,为进一步研究金线莲多糖的生物合成奠定基础。  方法  以梅花山金线莲植株为材料,克隆ArGMP基因的cDNA序列和基因组序列,利用在线软件进行生物信息学分析,并对其基因表达调控模式进行qRT-PCR分析。  结果  金线莲ArGMP基因ORF区序列长1086 bp,共编码361个氨基酸;基因组序列长度为1760 bp,含有3个内含子,GenBank登录号OQ030271。生物信息学分析表明:ArGMP蛋白是一种较稳定的、无跨膜结构的亲水蛋白,该蛋白与铁皮石斛、深圳拟兰、蝴蝶兰等兰科植物的亲缘关系较近。qRT-PCR结果显示:ArGMP基因在金线莲不同组织中的表达量差异显著,在花中的表达量最高;在不同种植温度处理条件下,25 ℃时表达量最高,高温严重抑制其表达;35 ℃高温处理不同时间显示,处理3 h后ArGMP基因表达量显著下降;而不同浓度NaCl胁迫处理对ArGMP基因表达基本无影响。  结论  克隆了甘露糖-1-磷酸尿苷转移酶基因的cDNA序列和基因组序列,发现该基因具有组织特异性表达的特点,且该基因受温度调控,而不受盐胁迫调控,这为进一步研究金线莲多糖生物合成调控机制奠定理论基础。
  • 图  1  ArGMP基因cDNA序列(A)和基因组序列扩增(B)

    M1:DL2000 DNA Marker;1:cDNA扩增产物;M2:DL2000 plus DNA Marker;2:基因组扩增产物。

    Figure  1.  Amplification results of ArGMP cDNA(A) and genome sequence(B)

    M1: DL2000 DNA marker; 1: cDNA amplification product; M2: DL2000 plus DNA marker; 2: amplified genome products.

    图  2  ArGMP基因序列结构和蛋白序列预测

    Figure  2.  Predicted ArGMP structure and sequence

    图  3  ArGMP蛋白的亲水性、二级和三级结构预测

    A:ArGMP蛋白亲水性预测;B:ArGMP蛋白二级结构;C:ArGMP蛋白三级结构。

    Figure  3.  Predicted hydrophilicity and secondary and tertiary structures of ArGMP protein

    A: Predicted hydrophilicity of ArGMP protein; B: secondary structure of ArGMP protein; C: tertiary structure of ArGMP protein.

    图  4  基于GMP蛋白氨基酸序列同源性构建的系统发育进化树

    Figure  4.  Phylogenetic tree based on amino acid sequence of GMP protein from different species

    图  5  ArGMP基因在不同处理的表达情况

    A:不同组织样品;B:不同种植温度处理;C:高温处理不同时间;D:盐胁迫处理。不同大、小写字母表示处理间差异极显著(P<0.01)或显著(P<0.05)。

    Figure  5.  Expressions of ArGMP under different treatments

    A: Different tissues; B: different temperatures; C: high temperature treatment; D: salt stress. Data with different capital letters indicate extremely significant differences atP<0.01; those with different lowercase letters, significant differences at P<0.05.

    表  1  基因克隆和RT-qPCR引物序列

    Table  1.   Sequences of cloned gene and RT-qPCR primer

    引物名称
    Primer
    正向引物(5′-3′)
    Forward primer(5′-3′)
    反向引物(5′-3′)
    Reverse primer(5′-3′)
    作用
    Function
    gGMPACCATGAAAGCCCTAATTCTTGTCACATAACAATCTCAGGCT基因克隆 Gene clone
    GMP-RTCCTTCTAAGCTGGCTTTCGGCAACTCTTGCCCACTGTCCCqRT-PCR
    ActinAGATGAGGCACAGTCCAAGAGCTGGAACATTGAAGGTCTC内参基因 Reference gene
    下载: 导出CSV

    表  2  ArGMP蛋白的基元和结构域分析

    Table  2.   Motifs and domains of ArGMP

    结构域
    Motifs or Domains
    结构域功能中文描述
    Function of domain
    结构域在ArGMP中的位置
    Domain position in ArGMP
    ASN_GLYCOSYLATIONN-糖基化aa323~326
    CAMP_PHOSPHO_SITE依赖cAMP和cGMP蛋白激酶磷酸化aa238~242
    CK2_PHOSPHO_SITE酪蛋白激酶II磷酸化aa137~140,aa182~185,aa192~195,aa262~265
    MYRISTYLN-蛋白质豆蔻酰化aa8~13,aa72~77,aa129~134,aa169~174,aa278~283,aa284~289
    PKC_PHOSPHO_SITE蛋白激酶 C 磷酸化aa154~156,aa236~238
    TYR_PHOSPHO_SITE酪氨酸激酶磷酸化aa138~145,aa201~209
    下载: 导出CSV
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  • 收稿日期:  2022-12-22
  • 修回日期:  2023-04-06
  • 网络出版日期:  2023-05-24
  • 刊出日期:  2023-05-28

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