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栾树查尔酮合酶基因克隆与表达分析

郭婷 黄赛 吴茹茜 安新民

郭婷, 黄赛, 吴茹茜, 安新民. 栾树查尔酮合酶基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(3): 27-35. doi: 10.12171/j.1000-1522.20200377
引用本文: 郭婷, 黄赛, 吴茹茜, 安新民. 栾树查尔酮合酶基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(3): 27-35. doi: 10.12171/j.1000-1522.20200377
Guo Ting, Huang Sai, Wu Ruqian, An Xinmin. Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata[J]. Journal of Beijing Forestry University, 2021, 43(3): 27-35. doi: 10.12171/j.1000-1522.20200377
Citation: Guo Ting, Huang Sai, Wu Ruqian, An Xinmin. Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata[J]. Journal of Beijing Forestry University, 2021, 43(3): 27-35. doi: 10.12171/j.1000-1522.20200377

栾树查尔酮合酶基因克隆与表达分析

doi: 10.12171/j.1000-1522.20200377
基金项目: 国家自然科学基金项目(31870652),2018年农业部华北都市农业重点实验室开放课题(kf2018012)
详细信息
    作者简介:

    郭婷。主要研究方向:林木基因组与分子育种。Email:guoting2020@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学院

    责任作者:

    安新民,教授。主要研究方向:林木基因组与分子育种。Email:anxinmin@bjfu.edu.cn 地址:同上

Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata

  • 摘要:   目的  查尔酮合酶(CHS)是苯丙烷途径的限速酶之一,在植物次生代谢物的合成中起着重要的作用。本研究通过对栾树CHS 基因进行克隆与生物信息学分析,以及分析栾树 CHS 基因表达与类黄酮合成的关系,期望为后续深入研究栾树类黄酮代谢途径其他相关基因、 CHS 基因家族以及锦叶栾呈色机制提供参考。  方法  以栾树叶片为材料,采用RT-PCR技术进行查尔酮合酶基因的克隆并进行生物信息学分析;通过实时定量PCR(qRT-PCR)技术分析 CHS 基因在栾树不同组织以及在5月、7月、9月的栾树和锦叶栾叶片中的表达模式;通过代谢组测定筛选出栾树与锦叶栾的类黄酮差异代谢物。  结果  克隆获得两个 CHS 基因的全长DNA,命名为 KpCHS1 和 KpCHS2 。其中 KpCHS1 序列全长为2 492 bp,ORF为1 173 bp,编码含有390个氨基酸的蛋白质; KpCHS2 序列全长为1 321 bp,ORF为1 182 bp,编码含有393个氨基酸的蛋白质;进一步的序列比对和系统发育分析表明,KpCHS1和KpCHS2蛋白高度同源,具有四个CHS特异性保守基序和一个查尔酮合成酶活性位点; KpCHS1 和 KpCHS2 在栾树根、茎、叶、种子中都普遍表达,其中, KpCHS2 在种子中的表达量最高, KpCHS1 在叶片中表达量高,而在根和茎中,两个基因的表达量相似且较低;表达模式分析显示,在栾树和锦叶栾叶片中,随着月份增加, KpCHS1 的表达量呈现出下降的趋势,而 KpCHS2 表达量未表现出明显的规律。在7月份的叶片样本中, KpCHS1 基因在锦叶栾中表达量显著高于栾树;代谢组结果显示,山奈酚-7-O-葡萄糖苷、7-羟基香豆素、槲皮素-3β-D-葡萄糖苷、以及类黄酮生物合成途径重要的中间产物山萘酚、柚皮苷等黄酮类物质在锦叶栾叶中含量显著升高。  结论   KpCHS1和KpCHS2属于栾树查尔酮合酶家族并且高度同源,但在系统进化树上分布在很远分支上,推测这两个蛋白在氨基酸活性催化功能上可能存在较大差异;KpCHS1和KpCHS2在根、茎、叶、种子中均有表达,且在叶和种子中较高。研究结果初步显示,KpCHS1基因与栾树类黄酮的生物合成高度相关。

     

  • 图  1  KpCHS1KpCHS2基因组全长及CDS序列

    M. DL3000 Marker;1、2、3、4分别代表KpCHS1全长基因、KpCHS1 CDS序列、KpCHS2全长基因、KpCHS2 CDS序列。M, DL3000 Marker; 1, 2, 3, 4 represent KpCHS1 full-length gene, KpCHS1 CDS sequence, KpCHS2 full-length gene and KpCHS2 CDS sequence, respectively.

    Figure  1.  Full length and CDS sequence of KpCHS1 and KpCHS2 genes

    图  2  KpCHS1KpCHS2基因结构

    Figure  2.  Gene structure of KpCHS1 and KpCHS2

    图  3  KpCHS1和KpCHS2蛋白二级结构分析

    a为KpCHS1蛋白,b为KpCHS2蛋白。a, KpCHS1 protein;b, KpCHS2 protein.

    Figure  3.  Secondary structure analysis of KpCHS1 and KpCHS2 protein

    图  4  KpCHS1和KpCHS2蛋白预测的三级结构图

    Figure  4.  Three-dimensional structures of thededuced KpCHS1 and KpCHS2

    图  5  KpCHS1和KpCHS2编码的氨基酸同源比对

    在所有序列中高度保守的残基以黑色背景表示,在序列中仅部分保守的残基以白色和灰色背景显示。蛋白活性位点、催化残基、CoA结合位点、其他高度保守的残基和不同的残基分别表示为◆、△、●、□和♡。红框为查尔酮合成酶活性位点(RLMMYQQGCFAGGTVLR)。Highly conserved residues in all sequences are indicated in black background and only partially conserved residues in the CHS sequences are showed in black with gray background. The protein sequences of KpCHS have four CHS-specific conserved motifs (marked Motif Ⅰ, Ⅱ, Ⅲ and Ⅳ). Active site, catalytic residues, CoA-binding site, other highly conserved residues and different residues are indicated as ◆, △, ●, □ and ♡, respectively. The red box is the active site of chalcone synthase (RLMMYQQGCFAGGTVLR).

    Figure  5.  Amino acid sequence alignment encoded by KpCHS1 and KpCHS2

    图  6  KpCHS1、KpCHS2与其他物种CHS蛋白系统进化树分析

    Figure  6.  Phylogenetic trees of KpCHS1, KpCHS2 and CHSs protein in other plants

    图  7  栾树不同器官以及不同月份栾树和锦叶栾叶片中KpCHS1KpCHS2基因表达分析

    a. KpCHS1和KpCHS2的组织特异性表达;b. KpCHS1在WT和GL的5月、7月、9月叶片中的表达量分析;c. KpCHS2在WT和GL的5月、7月、9月叶片中的表达量分析;WT为栾树;GL为锦叶栾。a, tissue specific expression of KpCHS1 and KpCHS2; b, expression of KpCHS1 in the leaves of WT and GL in May, July and September; c, expression of KpCHS2 in the leaves of WT and GL in May, July and September; WT, Koelreuteria paniculata; GL, K. paniculata ‘Jinye’.

    Figure  7.  Analysis of KpCHS1 and KpCHS2 expression in different organs and leaves of Koelreuteria paniculata and K. paniculata ‘Jinye’ in different months

    图  8  差异的类黄酮物质热图

              WT为栾树,GL为锦叶栾。WT, Koelreuteria paniculata; GL, K. paniculata ‘Jinye’

    Figure  8.  Heatmap of differential flavonoids

    表  1  基因克隆中使用引物列表

    Table  1.   List of primers in gene cloning

    引物名称 Primer name序列(5′—3′) Sequence (5′−3′)Tm值 Tm value用途 Usage
    CHS1-F ATGGTGACTGTCGAGGAAGTC 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification
    CHS1-R CTAAGCAGAGGCAACAGAGTGG 55.00 KpCHS1 基因扩增 KpCHS1 gene amplification
    CHS2-F ATCTCACTCCTAAACCCCCTTC 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification
    CHS2-R TTTAATAAAAGGAACAGTATCCAGA 56.00 KpCHS2 基因扩增 KpCHS2 gene amplification
    Actin-F AAATTAACGAGGACACCAATGC 58.00 qRT-PCR
    Actin-R GGGTATGGATATGGCGATCTTA 58.00 qRT-PCR
    Y-CHS1-F GTGTCGAAAAGCCCATGTATGA 58.99 qRT-PCR
    Y-CHS1-R TTGAAATCAGCCCAGGAACATC 58.91 qRT-PCR
    Y-CHS2-F AAGAACATCGAGAAAAGCTTGG 59.89 qRT-PCR
    Y-CHS2-R GCCCTGAGTTTCTCTTCTTTGA 60.00 qRT-PCR
    下载: 导出CSV

    表  2  CHS蛋白理化性质

    Table  2.   Physicochemical properties of CHS protein

    蛋白名称
    Protein name
    氨基酸数量
    Quantity of
    amino acids
    分子量
    Molecular mass/kD
    等电点
    Isoelectric point (pI)
    亲/疏水性
    Hydrophilicity/
    hydrophobicity
    跨膜区域
    Transmembrane
    region
    亚细胞定位预测
    Prediction of protein
    subcellular localization
    KpCHS1 390 42.59 6.12 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm
    KpCHS2 393 42.91 6.57 疏水性 Hydrophobicity 不具有 No 细胞质 Cytoplasm
    下载: 导出CSV
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  • 收稿日期:  2020-11-28
  • 修回日期:  2020-12-18
  • 网络出版日期:  2021-01-26
  • 刊出日期:  2021-04-16

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