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云南逸散紫花苜蓿MsVDE基因的克隆与表达分析

许超 何承刚 姜华

引用本文: 许超,何承刚,姜华. 云南逸散紫花苜蓿基因的克隆与表达分析. 草业科学, 2022, 39(6): 1-11 doi: shu
Citation:  XU C, HE C G, JIANG H. Cloning and expression analysis of in escaped alfalfa in Yunnan, China. Pratacultural Science, 2022, 39(6): 1-11 doi: shu

云南逸散紫花苜蓿MsVDE基因的克隆与表达分析

    作者简介: 许超(1985-),女,云南东川人,博士,研究方向为草地植物资源与遗传育种。E-mail: xuchao@ynlky.org.cn
    通讯作者: 何承刚(1972-),男,甘肃民勤人,副教授,博士,研究方向为作物耕作与栽培。E-mail: chengganghe63@ 163.com
  • 基金项目:   国家自然科学基金(31660682);云南省技术创新人才(2018HB075)

摘要: 叶黄素循环是植物应对环境胁迫进行热耗散的光保护途径之一,紫黄质脱环氧化酶基因(VDE)是叶黄素循环脱环氧化过程的关键基因。本研究采用RACE技术首次克隆了云南逸散紫花苜蓿(Medicago sativa)的MsVDE基因,同时采用实时荧光定量分析干热胁迫下的MsVDE基因表达特征。结果表明,MsVDE全长1 678 bp,CDS编码区1 608 bp,编码535个氨基酸。氨基酸序列保守区域具有3个VDE基因的特征区,即N-端半胱氨酸富集区、脂质运载蛋白特征区和C-端谷氨酸富集区。进化树分析表明该基因与蒺藜苜蓿(Medicago truncatula) VDE基因的氨基酸序列亲缘关系最近。干热胁迫第4天和第8天时的MsVDE基因表达量分别比正常条件上调了2.13倍和1.97倍;喷洒二硫苏糖醇(1, 4-Dithiothreitol, DTT)溶液后MsVDE基因表达量平均下调了55%。本研究初步得出,云南逸散紫花苜蓿MsVDE基因在干热胁迫下可通过调控叶黄素循环,从而进行其光保护作用。

English

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  • 凯时66

    图 1  MsVDE基因扩增结果

    Figure 1.  Amplification results of MsVDE gene

    左为3′片段扩增结果,中间1、2、3分别为引物MsVDE-1F/1R、MsVDE-2F/2R、MsVDE-3F/3R扩增结果,右为5′片段扩增结果。

    Left panel presents the 3′ RACE product. The panel with lanes 1, 2, and 3 show the amplification results of primers MsVDE-1F/1R, MsVDE-2F/2R, and MsVDE-3F/3R, respectively. The right panel presents the product of 5′ RACE.

    图 2  紫花苜蓿 MsVDE 基因编码蛋白质的保守域

    Figure 2.  Conserved domain of protein encoded by MsVDE gene in alfalfa

    图 3  逸散紫花苜蓿与不同植物的VDE基因氨基酸序列比对分析

    Figure 3.  Schematic description of amino acid sequence alignment of MsVDE and homologous proteins

    黑色表示完全保守域;红色为75%以上的物种保守域,绿色为50%以上的保守区域;蓝色细实线表示脂运蛋白保守域(PLN02372),黑色粗实线表示GDNF/GAS1域(GDNF),红色实线方框为N-端半胱氨酸(C)富集区,红色虚线方框为C-端谷氨酸(E)富集区。

    Multiple alignment with conserved residues shaded in black. Red and green shading indicate similar residues in more than 75% and 50%, respectively, of all sequences. The blue thin solid underline indicates the conserved lipoprotein domain (PLN02372). The black thick solid underline indicates the GDNF/GAS1 domain (GDNF). The red solid line box denotes the N-terminal cysteine (C) rich region. The red dotted line box denotes the C-terminal glutamate (E) rich region.

    图 4  紫花苜蓿与其他物种VDE基因推导氨基酸进化树

    Figure 4.  Phylogenetic tree of deduced amino acid sequences of VDE in escaped alfalfa and other species

    图 5  干热胁迫对MsVDE基因表达的影响

    Figure 5.  Effects of drought and heat stresses on MsVDE gene expression

    *、**和***分别表示在同一时间不同处理条件之间在0.05、0.01和0.001水平存在显著性差异;不同小写字母表示同一处理条件不同时间段之间差异显著(P < 0.05);下图同。

    *, **, and *** indicate significant differences at the same treatment period between different treatment conditions at the 0.05, 0.01, and 0.001 levels, respectively; different lowercase letters indicate significant differences at the same treatment conditions between different treatment period at the 0.05 level; this is applicable for the following figures as well.

    图 6  喷洒DTT溶液对MsVDE基因表达的影响

    Figure 6.  Effects of spraying dithiothreitol solution on MsVDE gene expression

    图 7  干热胁迫下叶黄素循环抑制剂对MsVDE基因相对表达量的影响

    Figure 7.  Effects of xanthophyll cycle inhibitors on relative expression of MsVDE gene under drought and heat stresses

    表 1  MsVDE基因克隆及荧光定量的引物序列

    Table 1.  Gene cloning and fluorescence quantification primer sequences of MsVDE gene

    引物名称
    Primer name
    引物序列(5′ -3′)
    Primer sequence (5′ -3′)
    备注
    Remark
    5′adaptorGCTGTCAACGATACGCTACGTAACGGCATGACAGTGCCCCCCCCCCCCCCC接头引物
    Splice primers
    3′adaptorGCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT
    5.3′outerGCTGTCAACGATACGCTACGTAAC
    5.3′innerGCTACGTAACGGCATGACAGTG
    MsVDE-1FATGTCCTTCAAACTGCAACTTCA中间序列扩增引物
    Intermediate sequence amplification primers
    MsVDE-1RAGATTGAATGCACTCGTCGGTA
    MsVDE-2FTCCTGTGAGGATGGTGGCTATA
    MsVDE-2RCCATATACCTTACCTCTCTTAACCCTA
    MsVDE-3FTGTTTGTTGGTTGGTTGGGG
    MsVDE-3RGTCCTCTCTCTTTCATTTGG
    MsVDE-F1GAACCGGTGTTTCAGGTAAAAACATTTGAA3′ RACE 特异性引物
    3′RACE specific primers
    MsVDE-F2TAGGGTTAAGAGAGGTAAGGTATATGGCACATT
    MsVDE-R6AGAATGAGCATGGAGAAGAATAGTAAAAGGGAA5′ RACE 特异性引物
    5′RACE specific primers
    MsVDE-R5GAGGAAAGCAATAAGGAGTAGTGGTGTGTGAA
    MsVDE-RT2GCTTTAGCAGCTCCATGATA
    MsVDE-RT1ATATGCTCCATCTGGACTAA
    MsVDE upstreamCCAACTGCAACTTCATCTTCC产物为213 bp
    The product is 213 bp
    MsVDE downstreamGGCTCATCCATCGTCACATC
    18SrRNA upstreamGAGAAACGGCTACCACATCCA
    18SrRNA upstreamCCCAACCCAAGGTCCAACTAC
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  • 通讯作者:  何承刚, jianghua15@163.com
  • 收稿日期:  2021-11-02
  • 接受日期:  2022-01-13
  • 网络出版日期:  2022-03-29
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