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龙眼MYB14的生物信息学分析及其基因表达载体构建

李浩然 丁峰 张树伟 彭宏祥 潘介春 何新华 王颖 李琳 王金英 黄幸 谭春露 徐炯志

李浩然,丁峰,张树伟,等. 龙眼 MYB14的生物信息学分析及其基因表达载体构建 [J]. 福建农业学报,2020,35(9):929−936 doi: 10.19303/j.issn.1008-0384.2020.09.002
引用本文: 李浩然,丁峰,张树伟,等. 龙眼 MYB14 的生物信息学分析及其基因表达载体构建 [J]. 福建农业学报,2020,35(9):929−936 doi: 10.19303/j.issn.1008-0384.2020.09.002
LI H R, DING F, ZHANG S W, et al. Bioinformatics and Construction of Expression Vector for Longan MYB14 [J]. Fujian Journal of Agricultural Sciences,2020,35(9):929−936 doi: 10.19303/j.issn.1008-0384.2020.09.002
Citation: LI H R, DING F, ZHANG S W, et al. Bioinformatics and Construction of Expression Vector for Longan MYB14 [J]. Fujian Journal of Agricultural Sciences,2020,35(9):929−936 doi: 10.19303/j.issn.1008-0384.2020.09.002

龙眼MYB14的生物信息学分析及其基因表达载体构建

doi: 10.19303/j.issn.1008-0384.2020.09.002
基金项目: 国家荔枝龙眼产业技术体系建设专项(CARS-33-10);国家现代农业产业技术体系广西荔枝龙眼创新团队项目(nycytxgxcxtd-02);国家荔枝龙眼产业技术体系良种繁育与生产配套技术岗位(CARS-32-04)
详细信息
    作者简介:

    李浩然(1993−),男,硕士,主要从事果树分子生物研究(E-mail:513371452@qq.com

    通讯作者:

    丁峰(1984−),男,副研究员,主要从事果树分子生物学研究(E-mail:dingfeng418@163.com

    徐炯志(1964−),男,研究员,主要从事果树栽培与生理研究(E-mail:xujiongzhi-2@163.com

  • 中图分类号: S 667.2

Bioinformatics and Construction of Expression Vector for Longan MYB14

  • 摘要:   目的  克隆龙眼MYB-related基因家族中MYB14基因,并对其进行生物信息学分析和基因表达载体的构建,为进一步研究该基因的生物学功能提供基础数据。  方法  通过转录组测序及生物信息学分析得到龙眼MYB14基因全长序列,进一步设计特异引物对其开放阅读框(ORF)全长序列进行克隆和生物信息学分析,同时构建超表达载体。  结果  MYB14基因属于MYB-related家族的TBP-like亚家族,ORF长度831 bp,编码276个氨基酸,有一个MYB结合位点。该蛋白属于非跨膜亲水蛋白,亚细胞定位预测定位于细胞质,存在57个氨基酸磷酸化位点。其二级结构主要由无规则卷曲以及α-螺旋构成,二级结构与三级结构预测结果高度一致,同源基因进化树分析表明该基因与大麻亲缘关系最近,同时成功构建了植物超表达载体pBI121-MYB14。  结论  本研究克隆得到了龙眼MYB14基因,成功构建表达载体pBI121-MYB14,为该基因功能的研究奠定了坚实的基础。
  • 图  1  基因克隆结果检测

    注:M:Maker DL2000;1:目的条带。a:cDNA为模板的MYB14 PCR扩增产物。b:pMD18-T MYB14质粒为模板的菌液PCR电泳结果。

    Figure  1.  Detection of cloned gene

    Note: M: Maker DL2000; 1: target band. a: PCR amplification products of MYB14 using cDNA as template. b: electrophoresis of microbial PCR using pMD18-T MYB14 plasmid as template.

    图  2  MYB14基因ORF序列及编码蛋白质

    注:*为终止子

    Figure  2.  ORF sequence and encoding protein of MYB14

    Note: * was terminator.

    图  3  NCBI中龙眼MYB14氨基酸序列保守结构域

    Figure  3.  Conserved domain in longan MYB14 amino acid sequence by NCBI

    图  4  龙眼MYB14蛋白磷酸化位点预测

    Figure  4.  Predicted phosphorylation sites on MYB14 protein

    图  5  不同物种MYB蛋白三级结构预测

    注:A:龙眼MYB14三级结构;B:柑橘MYB组蛋白三级结构;C:大麻类MYB组蛋白三级结构;D:山茶端粒重复结合因子三级结构。

    Figure  5.  Predicted tertiary structure of MYB proteins from different species

    Note: A: predicted tertiary structures of MYB14 in longan; B: that of Citrus sinensis single MYB histone 4-like; C: that of Cannabis sativa single MYB histone 3-like; and, D: that of Camellia sinensis telomere repeat-binding factor 4-like.

    图  6  龙眼MYB14蛋白与其他物种MYB蛋白的系统进化树比较

    Figure  6.  Phylogenetic trees of longan MYB14 and MYB proteins of other species

    图  7  不同物种同源基因的氨基酸序列比对结果

    Figure  7.  Homologous amino acid sequence alignment on genes of different species

    图  8  载体构建结果检测

    注:M:Maker DL2000;1:目的条带。a:MYB14 PCR扩增条带。b:Npt II PCR 扩增条带。

    Figure  8.  Detection of constructed vector

    Note: M: Maker DL2000; 1: Target band. a: MYB14 PCR amplification band. b: Npt II PCR amplification band.

    表  1  引物序列

    Table  1.   Sequence of primers

    引物名称 Primer name序列(5′-3′)Sequences(5′-3′)引物用途 Application
    MYB14 FGCTTATTTTCGTAGCAGAGGCA基因克隆 Gene Cloning
    MYB14 R1CTCTACTTGATGCGAACCATAT
    MYB14 R2ACTGTAGTCCGTTACTGTGAGA
    CBD MYB14-FCACGGGGGACTCTAGAATGGGAAATCCGAAGCAGAAAT(下划线为Xba I酶切位点)载体构建 Construct Expression Vector
    CBD MYB14-RTCCTTTACCCATCCCGGGCTATGCCATGATCACAATTTCA(下划线为Sma I酶切位点)
    MF47CGCCAGGGTTTTCCCAGTCACGAC克隆检测 Clone detection
    MR48AGCGGATAACAATTTCACACAGGA
    下载: 导出CSV
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  • 收稿日期:  2020-03-26
  • 修回日期:  2020-08-01
  • 刊出日期:  2020-09-28

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