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水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析

刘春浩 梁楠松 于磊 赵兴堂 刘颖 孙爽 王紫晴 詹亚光

刘春浩, 梁楠松, 于磊, 赵兴堂, 刘颖, 孙爽, 王紫晴, 詹亚光. 水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析[J]. 北京林业大学学报, 2017, 39(6): 22-31. doi: 10.13332/j.1000-1522.20160359
引用本文: 刘春浩, 梁楠松, 于磊, 赵兴堂, 刘颖, 孙爽, 王紫晴, 詹亚光. 水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析[J]. 北京林业大学学报, 2017, 39(6): 22-31. doi: 10.13332/j.1000-1522.20160359
LIU Chun-hao, LIANG Nan-song, YU Lei, ZHAO Xing-tang, LIU Ying, SUN Shuang, WANG Zi-qing, ZHAN Ya-guang. Cloning, analysing and homologous expression of TCP4 transcription factor under abiotic stress and hormone signal in Fraxinus mandschurica[J]. Journal of Beijing Forestry University, 2017, 39(6): 22-31. doi: 10.13332/j.1000-1522.20160359
Citation: LIU Chun-hao, LIANG Nan-song, YU Lei, ZHAO Xing-tang, LIU Ying, SUN Shuang, WANG Zi-qing, ZHAN Ya-guang. Cloning, analysing and homologous expression of TCP4 transcription factor under abiotic stress and hormone signal in Fraxinus mandschurica[J]. Journal of Beijing Forestry University, 2017, 39(6): 22-31. doi: 10.13332/j.1000-1522.20160359

水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析

doi: 10.13332/j.1000-1522.20160359
基金项目: 

东北林业大学大学生创新创业训练计划项目 201610225233

“十二五”国家科技支撑计划项目 2012BAD21B020106

国家自然科学基金项目 31270697

详细信息
    作者简介:

    刘春浩。主要研究方向:植物基因工程。Email: DLliuchunhao@163.com  地址:150040  黑龙江省哈尔滨市香坊区和兴路26号东北林业大学生命科学学院

    责任作者:

    詹亚光,博士,教授。主要研究方向:林木遗传育种。Emailyaguangzhan@126.com  地址:同上

  • 中图分类号: S718.46;S792.41;Q943.2

Cloning, analysing and homologous expression of TCP4 transcription factor under abiotic stress and hormone signal in Fraxinus mandschurica

  • 摘要: TCP转录因子家族是植物特异的一类调控生长发育和逆境胁迫的重要转录因子。本文揭示了水曲柳FmTCP4在非生物胁迫及激素信号诱导调控的表达特征,为该基因在水曲柳中调控生长发育以及逆境胁迫响应功能的研究奠定基础。通过前期研究克隆获得了水曲柳TCP4基因序列,并命名为FmTCP4,应用生物信息学软件对水曲柳FmTCP4基因的分子结构特征进行了分析,利用4 ℃低温、盐(NaCl)以及干旱(PEG6000)进行非生物胁迫处理,利用脱落酸(ABA)和赤霉素(GA3)进行激素信号诱导,分析FmTCP4基因的表达特征。生物信息学分析表明该基因全长1 251 bp,具有完整的开放阅读框,编码416个氨基酸。FmTCP4具有螺旋-环-螺旋结构,为亲水性的不稳定蛋白,不存在信号肽,具有跨膜能力,亚细胞定位预测存在于细胞核中,并在细胞质到细胞核的调控中起作用。同源序列比对结果表明,FmTCP4与芝麻、烟草等物种的同源蛋白序列相比具有很高的同源性。非生物胁迫处理后,FmTCP4的表达量随处理时间而改变,但其变化趋势不同,表明FmTCP4明显响应了这3种非生物胁迫。激素信号诱导结果表明,外源植物激素调控了FmTCP4基因的表达,基因表达量相对于对照组具有显著性差异。非生物胁迫和激素信号诱导下的基因表达分析表明,FmTCP4响应了寒冷、盐、干旱等非生物胁迫以及激素信号,说明其参与了植物的生长发育和逆境胁迫的平衡。

     

  • 图  1  FmTCP4基因PCR扩增片段(M:DL2 000)

    Figure  1.  PCR amplified fragments of FmTCP4 (M:DL2 000)

    图  2  FmTCP4基因编码区序列及推测的氨基酸序列

    Figure  2.  Gene coding region sequence and amino acid sequence of FmTCP4

    图  3  FmTCP4蛋白信号肽的预测和分析

    C.原始剪切位点的分值; S.信号肽的分值; Y.综合剪切位点的分值。

    Figure  3.  Prediction and analysis of signal peptides in FmTCP4 protein using the neural network algorithm

    C, the score of the original cut site; S, the score of signal peptide; Y, the score of the integrated cut site.

    图  4  FmTCP4蛋白跨膜结构域预测和分析

    箭头所示为预测的FmTCP4的跨膜结构域。

    Figure  4.  Prediction and analysis of trans-membrane domain in FmTCP4 protein

    Arrow means trans-membrane domain of predicted FmTCP4.

    图  5  FmTCP4蛋白三级结构预测(三视图)

    Figure  5.  Three-dimensional structure prediction of FmTCP4 protein(three views)

    图  6  FmTCP4蛋白质残基二面角预测

    Figure  6.  Prediction of residue dihedral alangles in FmTCP4 protein

    图  7  FmTCP4基因编码的氨基酸序列比对

    Figure  7.  Multiple alignment of amino acid sequence of FmTCP4

    图  8  FmTCP4蛋白质结构域预测

    Figure  8.  FmTCP4 protein domain prediction

    图  9  根据FmTCP4蛋白NCBI Blastp比对结果构建的系统进化树

    Figure  9.  Phylogenetic tree of FmTCP4 protein based on the Blastp results from NCBI

    图  10  FmTCP4基因在非生物胁迫下的相对表达量

    a, b, c, d表示处理下不同时间点的基因表达差异显著性(P<0.05), 下同。

    Figure  10.  Relative expression level of FmTCP4 under abiotic stress treatment

    a, b, c and d mean significant difference in gene expression at different time points under processing (P<0.05), same as below.

    图  11  FmTCP4在植物激素信号下的相对表达量

    Figure  11.  Relative expression level of FmTCP4 under hormone signal

    表  1  克隆FmTCP4基因编码区对应引物

    Table  1.   Primers used for cloning the coding regions in FmTCP4 gene

    引物名称
    Primer name
    引物序列
    Primer sequence
    FmTCP4-F 5ˊ-CCGCTCTACTCCACAACTCCC-3ˊ
    FmTCP4-R5ˊ-GCTACTAGCTCACCCAGACAATAAAC-3ˊ
    下载: 导出CSV

    表  2  实时荧光定量PCR采用的引物序列

    Table  2.   Primers for quantitative real-time PCR

    引物名称
    Primer name
    引物序列
    Primer sequence
    Tu-F 5′-AGGACGCTGCCAACAACTTT-3′
    Tu-R 5′-TTGAGGGGAAGGGTAAATAGTG-3′
    qFmTCP4-F 5′-CTTCGGCTGAGTCCAGTTCC-3′
    qFmTCP4-R 5′-TGCTGTGGCTGTTGTTGGTTAT-3′
    下载: 导出CSV
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  • 收稿日期:  2016-11-24
  • 修回日期:  2016-03-20
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