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文冠果LEC1基因的克隆及表达分析

路蒙蒙 韩硕 杨琦 王俊秀 郭惠红

路蒙蒙, 韩硕, 杨琦, 王俊秀, 郭惠红. 文冠果LEC1基因的克隆及表达分析[J]. 北京林业大学学报, 2018, 40(1): 8-16. doi: 10.13332/j.1000-1522.20170314
引用本文: 路蒙蒙, 韩硕, 杨琦, 王俊秀, 郭惠红. 文冠果LEC1基因的克隆及表达分析[J]. 北京林业大学学报, 2018, 40(1): 8-16. doi: 10.13332/j.1000-1522.20170314
Lu Meng-meng, Han Shuo, Yang Qi, Wang Jun-xiu, Guo Hui-hong. Cloning and expression analysis of LEC1 gene from Xanthoceras sorbifolia embryos[J]. Journal of Beijing Forestry University, 2018, 40(1): 8-16. doi: 10.13332/j.1000-1522.20170314
Citation: Lu Meng-meng, Han Shuo, Yang Qi, Wang Jun-xiu, Guo Hui-hong. Cloning and expression analysis of LEC1 gene from Xanthoceras sorbifolia embryos[J]. Journal of Beijing Forestry University, 2018, 40(1): 8-16. doi: 10.13332/j.1000-1522.20170314

文冠果LEC1基因的克隆及表达分析

doi: 10.13332/j.1000-1522.20170314
基金项目: 

北京林业大学生物学理科基地人才培养支撑条件建设项目 J1310005

详细信息
    作者简介:

    路蒙蒙。主要研究方向:植物发育生物学。Email:lemon@bjfu.edu.cn 地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    郭惠红,副教授。主要研究方向:植物发育生物学。Email: guohh@bjfu.edu.cn 地址:同上

  • 中图分类号: Q78;S718.46

Cloning and expression analysis of LEC1 gene from Xanthoceras sorbifolia embryos

  • 摘要: 目的文冠果隶属无患子科, 是我国北方重要的油料树种, 种子含油量高, 是制备食用油、生物柴油等的优质原料。本研究旨在克隆文冠果Leafy Cotyledon 1(XsLEC1)基因, 进行序列及表达模式的分析。方法以文冠果种胚为试材, 利用RT-PCR和RACE技术克隆文冠果LEC1基因。采用Protparam及TMHMM2.0软件预测XsLEC1蛋白的理化性质和跨膜结构域, ProtComp9.0及Motif Scan软件预测XsLEC1蛋白的亚细胞定位及蛋白功能, BioEdit及MEGA7软件分析XsLEC1蛋白的多重序列比对和进化关系。运用RT-PCR和实时荧光定量PCR分析XsLEC1基因的表达模式。结果XsLEC1 cDNA全长1 035 bp, 包含一个长度为690 bp的开放阅读框(GenBank号为MF616360), 可编码229个氨基酸。推导的氨基酸序列包含一个HAP3亚基的保守功能域B。该保守功能域内含有组蛋白折叠中的3个α螺旋(α1、α2、α3)和两个短环结构(L1、L2)。在线预测该蛋白为稳定的, 亲水性蛋白, 无信号肽和跨膜区域, 定位于细胞核, 存在多个磷酸化位点。二级结构主要由α螺旋和无规则卷组成。进化分析表明, 该蛋白序列与同科龙眼亲缘关系最近, 其次为麻风树和毛果杨。RT-PCR实验得出, XsLEC1基因在文冠果的根、茎、叶及花中均无表达, 在种子中出现较高表达。实时荧光定量PCR结果进一步显示, XsLEC1基因在种胚中有明显的时序表达特性, 在种胚发育的前期(花后33、40、47 d)表达较高, 在种胚发育的后期(花后54、61、68 d)表达较低, 至花后75 d时仅有微量表达, 而在完全成熟的种胚(花后81 d)中未检测到XsLEC1的表达。结论文冠果LEC1基因的克隆及表达分析, 为以后深入研究XsLEC1的功能奠定了分子基础, 同时对生产实践中文冠果油脂品质的改良有一定的实践意义。

     

  • 图  1  基因的克隆

    A.中间序列PCR扩增;B.3′端PCR扩增;C.5′端PCR扩增;D.核苷酸与其推导的氨基酸序列。

    Figure  1.  Cloning of XsLEC1

    A, PCR amplification of central fragment; B, PCR amplification of 3′ end; C, PCR amplification of 5′ end; D, nucleotide and deduced amino acid sequences; M, marker.

    图  2  XsLEC1蛋白亲水性、信号肽以及跨膜结构的分析预测

    A.XsLEC1蛋白的亲水性分析,纵坐标正值表示疏水,负值表示亲水,负值越大表示越亲水,介于+0.5~-0.5之间的主要为两性氨基酸;B.XsLEC1蛋白信号肽预测;C.XsLEC1蛋白跨膜预测。图中红色线表示跨膜可能性。

    Figure  2.  Analysis and prediction of hydrophilicity, signal peptide and transmembrane domains of XsLEC1

    A, analysis of hydrophilicity of XsLEC1. The positive values on vertical axis indicate hydrophobicity and the negative values indicate hydrophilicity, the more negative the value is, the higher the hydrophilicity of the amino acids is, the values between +0.5 and -0.5 mainly denote amphoteric amino acids; B, prediction of signal peptide of XsLEC1; C, prediction of transmembrane domains of XsLEC1. The red line in the figure represents the possibility of transmembrane.

    图  3  LEC1蛋白序列多重比对和XsLEC1保守结构域的三级结构预测

    A.XsLEC1与其他植物来源的LEC1蛋白序列的多重比对。黑底和灰底部分分别代表几个物种相同和相似的氨基酸残基;黑色实线(α)和虚线(L)代表组蛋白折叠区的α螺旋和环结构;黑色方框代表DNA结合区和组蛋白亚基间的相互结合区;Xs.文冠果;Dl.龙眼;Jc.麻风树;Pt.毛果杨;At.拟南芥;Os.水稻;B.保守结构域的三级结构预测。α螺旋和环结构用蓝线画出。

    Figure  3.  Multiple sequence alignments of LEC1 and steric structure prediction of conserved domain in XsLEC1

    A, multiple sequence alignments of XsLEC1 with other plant LEC1s. Identical and similar amino acid residues are shaded in black and grey, respectively; the α-helices and loops in the histone fold motif were indicated as continuous and dashed black lines, respectively; the DNA-binding region and the histone subunit interaction region were shown in black boxes, respectively; Xs, Xanthoceras sorbifolia; Dl, Dimocarpus longan; Jc, Jatropha carcas; Pt, Populus trichocarpa; At, Arabidopsis thaliana; Os, Oryza sativa. B, steric structure prediction of conserved domain. The α-helices and loops were indicated as blue lines.

    图  4  XsLEC1蛋白的进化树分析

    文冠果.Xanthoceras sorbifolium;龙眼.Dimocarpus longan;麻风树.Jatropha curcas;毛果杨.Populus trichocarpa;大豆.Glycine max;花生.Arachis hypogaea;胭脂树.Bixa orellana;拟南芥.Arabidopsis thaliana;油菜.Brassica napus;水稻.Oryza sativa;玉米.Zea mays;江南卷柏.Selaginella moellendorffii;蔓出卷柏.Selaginella davidii;山胡椒.Lindera glauca;扭叶松.Pinus contorta;欧洲落叶松.Larix decidua;花旗松.Pseudotsuga menziesii;中华水韭.Isoetes sinensis。推导的文冠果LEC1序列位置用箭头标出。The position of deduced XsLEC1 was indicated by an arrow.

    Figure  4.  Phylogenetic tree analysis of protein in XsLEC1

    图  5  XsLEC1基因表达模式分析

    A.XsLEC1在不同组织表达的RT-PCR分析。泳道1~5分别表示根、茎、叶、花和胚5个组织;B.XsLEC1在种胚发育不同阶段表达的RT-PCR分析。泳道1~8分别表示花后33、40、47、54、61、68、75和82 d;C.XsLEC1在种胚发育不同阶段表达的实时荧光定量PCR分析。不同字母表示差异性显著P<0.05。

    Figure  5.  Expression pattern analysis of XsLEC1

    A, RT-PCR analysis of XsLEC1 tissue-specific expression. Lanes 1-5 represent roots, stems, leaves, petals, and embryos; B, RT-PCR analysis of XsLEC1 expression at different embryo development stages. Lanes 1-8 represent 33, 40, 47, 54, 61, 68, 75 and 82 days after anthesis; C, quantitative real-time PCR analysis of XsLEC1 expression at different embryo development stages. Different letters indicate significant differences at P < 0.05 level.

    表  1  基因克隆所用的引物序列

    Table  1.   Primers used in gene cloning reactions

    引物名称
    Primer name
    引物序列
    Primer sequence
    LEC1-F1 5′-CATGCCWATHGCMAAYGT-3′
    LEC1-R1 5′-GTKATVGTCTTVCGTTGCTC-3′
    5′-GSP1 5′-CGGTAACGGTGGAGGTAGACTG-3′
    5′-GSP2 5′-TGTCAGGGGTTCAATGTAGTCG-3′
    3′-GSP1 5′-GATCATGCGTAAGATTCTCCCTC-3′
    3′-GSP2 5′-ATGCGTAAGATTCTCCCTCCG-3′
    5′-Outer-Primer 5′-CATGGCTACATGCTGACAGCCTA-3′
    5′-Inner- Primer 5′-CGCGGATCCACAGCCTACTGATGA
    TCAGTCGATG-3′
    3′-Outer- Primer 5′-TACCGTCGTTCCACTAGTGATTT-3′
    3′-Inner- Primer 5′-CGCGGATCCTCCACTAGTGATTTCA
    CTATAGG-3′
    下载: 导出CSV

    表  2  XsLEC1和AtLEC1蛋白亚细胞定位结果

    Table  2.   Results of subcellular localizations in XsLEC1 and AtLEC1

    亚细胞定位
    Subcellular localization
    XsLEC1蛋白的概率
    Probability of XsLEC1
    AtLEC1蛋白的概率
    Probability of the AtLEC1
    细胞核Nuclear 8.81 8.87
    质膜Plasma membrane 0.60 0.67
    细胞外Extracellular 0.04 0.02
    线粒体Mitochondrial 0.06 0.02
    过氧化物酶体Peroxisomal 0.08 0.05
    叶绿体Chloroplast 0.08 0.06
    液泡Vacuolar 0.33 0.31
    下载: 导出CSV

    表  3  XsLEC1和AtLEC1蛋白二级结构的比较

    Table  3.   Comparison of secondary structure in XsLEC1 and AtLEC1

    结构
    Structure
    XsLEC1蛋白概率
    Probability of XsLEC1
    AtLEC1蛋白概率
    Probability of AtLEC1
    α螺旋Alpha helix 98(42.79%) 59(24.79%)
    延伸链Extended strand 21(9.17%) 45(18.91%)
    β转角Beta turn 26(11.35%) 23(9.66%)
    无规则卷Random coil 84(36.68%) 111(46.64%)
    下载: 导出CSV

    表  4  XsLEC1蛋白motif预测

    Table  4.   Putative functional motif of XsLEC1

    功能位点
    Functional site
    氨基酸位置
    Amino acid
    position
    氨基酸序列
    Amino acid
    sequence
    N-糖基化位点
    N-glycosylation site
    15~18
    34~37
    41~44
    NSSS
    NQTN
    NNSD
    酪蛋白激酶Ⅱ磷酸化位点
    Phosphorylation site of casein kinaseⅡ
    49~52
    83~86
    112~115
    TVRE
    TIQE
    TAED
    豆蔻酰化位点
    N-myristoylation site
    5~10
    19~24
    147~152
    187~192
    201~206
    GGFHGY
    GLKLTE
    GSIRGE
    GGGGAT
    GTAGAT
    蛋白激酶C磷酸化位点
    Phosphorylation site of protein kinase C
    49~51
    148~150
    TVR
    SIR
    下载: 导出CSV
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  • 收稿日期:  2017-08-26
  • 修回日期:  2017-11-20
  • 刊出日期:  2018-01-01

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