高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

白桦BpSPL8启动子的克隆及异源过表达BpSPL8对拟南芥耐旱性的影响

张勇 胡晓晴 李豆 刘雪梅

张勇, 胡晓晴, 李豆, 刘雪梅. 白桦BpSPL8启动子的克隆及异源过表达BpSPL8对拟南芥耐旱性的影响[J]. 北京林业大学学报, 2019, 41(8): 67-75. doi: 10.13332/j.1000-1522.20190137
引用本文: 张勇, 胡晓晴, 李豆, 刘雪梅. 白桦BpSPL8启动子的克隆及异源过表达BpSPL8对拟南芥耐旱性的影响[J]. 北京林业大学学报, 2019, 41(8): 67-75. doi: 10.13332/j.1000-1522.20190137
Zhang Yong, Hu Xiaoqing, Li Dou, Liu Xuemei. Cloning the promoter of BpSPL8 from Betula platyphylla and overexpression of BpSPL8 gene affecting drought tolerance in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2019, 41(8): 67-75. doi: 10.13332/j.1000-1522.20190137
Citation: Zhang Yong, Hu Xiaoqing, Li Dou, Liu Xuemei. Cloning the promoter of BpSPL8 from Betula platyphylla and overexpression of BpSPL8 gene affecting drought tolerance in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2019, 41(8): 67-75. doi: 10.13332/j.1000-1522.20190137

白桦BpSPL8启动子的克隆及异源过表达BpSPL8对拟南芥耐旱性的影响

doi: 10.13332/j.1000-1522.20190137
基金项目: 中央高校基本科研业务费专项基金E类项目(2572015EA05)
详细信息
    作者简介:

    张勇。主要研究方向:植物基因工程。Email:202026713@qq.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126信箱

    责任作者:

    刘雪梅,教授,博士生导师。主要研究方向:植物发育与分子调控。Email:695898040@qq.com 地址:同上

  • 中图分类号: S 718.43; S792.153; Q943.2

Cloning the promoter of BpSPL8 from Betula platyphylla and overexpression of BpSPL8 gene affecting drought tolerance in Arabidopsis thaliana

  • 摘要: 目的目前对植物SPL8的基因功能研究主要集中在开花和育性方面,而其在干旱胁迫响应中的作用却鲜有报道。本文克隆、分析了白桦BpSPL8启动子,并研究了BpSPL8基因在拟南芥中响应干旱胁迫的功能。方法通过PCR克隆技术得到了白桦BpSPL8启动子;利用PLACE和PlantCARE软件对BpSPL8启动子顺式作用元件进行了预测。构建了BpSPL8启动子驱动GUS(β-葡萄糖苷酸酶编码基因)的植物表达载体,并采用浸花法将其转化至拟南芥中;继而利用GUS染色分析了BpSPL8启动子的组织表达模式;同时对BpSPL8在PEG处理下的表达水平进行了qRT-PCR分析。最后,以过表达BpSPL8拟南芥为材料来探究BpSPL8在干旱胁迫下的生物学功能。结果启动子元件分析显示,BpSPL8启动子中含有组织特异表达、光响应、激素响应及多个胁迫响应元件。GUS染色结果表明,BpSPL8启动子可在拟南芥的下胚轴、叶片、叶柄、根和花序中启动GUS基因表达。BpSPL8基因在PEG处理下的野生型白桦的根和叶片中均呈现先上调后下调的表达趋势。干旱胁迫下,过表达BpSPL8拟南芥的存活率和脯氨酸含量均显著低于野生型,丙二醛含量显著高于野生型;两个已知的抗逆基因DR29BP5CS1在干旱处理后的野生型和转基因拟南芥中均上调表达;但在转基因拟南芥中呈现出延迟上调的表达模式。结论异源过表达白桦BpSPL8能够降低拟南芥的耐旱性,并在干旱胁迫下影响抗性基因DR29BP5CS1的表达模式。

     

  • 图  1  ProSPL8::GUS拟南芥的组织化学GUS测定

    a. 野生型拟南芥未检测到GUS活性;b. GUS活性在转ProSPL8::GUS拟南芥的下胚轴、叶片、叶柄、根和花序检测到;c. 转ProSPL8::GUS拟南芥叶片;d. 转ProSPL8::GUS拟南芥叶柄;e. 转ProSPL8::GUS拟南芥根;f. 转ProSPL8::GUS拟南芥花序。a, wild type Arabidopsis thaliana without GUS activity; b, GUS activity was observed in hypocotyls, leaves, petioles, roots and inflorescences of ProSPL8::GUS transgenic Arabidopsis thaliana; c, leaves of ProSPL8::GUS; d, petiole of ProSPL8::GUS; e, root of ProSPL8::GUS; f, inflorescence of ProSPL8::GUS.

    Figure  1.  Histochemical GUS staining of ProSPL8::GUS transgenic Arabidopsis thaliana

    图  2  10%PEG处理下BpSPL8基因在野生型白桦叶和根中的表达模式

    Figure  2.  Expression patterns of BpSPL8 gene in Betula platyphylla leaves and roots under 10% PEG treatments

    图  3  干旱处理条件下野生型和35S::BpSPL8转基因拟南芥的生长状态及存活率

    WT为野生型拟南芥,35S::BpSPL8为BpSPL8过表达拟南芥。a. 处理前和干旱2周后野生型和35S::BpSPL8转基因拟南芥的表型;b. 干旱胁迫2周复水3 d后和正常生长条件下野生型和35S::BpSPL8转基因拟南芥的存活率(*P < 0.05)。WT is wild-type Arabidopsis thaliana, 35S::BpSPL8 is BpSPL8 overexpressing Arabidopsis thaliana. a, phenotype of wild-type and 35S::BpSPL8 transgenic Arabidopsis thaliana before treatment and 2 weeks after drought; b, survival rate of wild-type and 35S::BpSPL8 transgenic Arabidopsis thaliana under normal growth conditions and re-watering 3 days after 2 weeks of drought treatment (*P < 0.05).

    Figure  3.  Growth status and survival rate of wild-type and 35S::BpSPL8 transgenic Arabidopsis thaliana under drought stress

    图  4  正常生长和干旱7 d 35S::BpSPL8转基因和野生型拟南芥脯氨酸和丙二醛的含量测定

    WT为野生型拟南芥(**P < 0.01),35S::BpSPL8为BpSPL8过表达拟南芥。WT is wild-type Arabidopsis thaliana, 35S::BpSPL8 is BpSPL8 overexpressing Arabidopsis thaliana (**P < 0.01).

    Figure  4.  Contents of proline and malondialdehyde in wild type and 35S::BpSPL8 transgenic Arabidopsis thaliana under normal growth conditions and 7 days of drought treatment

    图  5  干旱处理下野生型和35S::BpSPL8转基因拟南芥DR29BP5CS1表达模式分析

    WT为野生型拟南芥;35S::BpSPL8为BpSPL8过表达拟南芥。WT is wild-type Arabidopsis thaliana, 35S::BpSPL8 is BpSPL8 overexpressing Arabidopsis thaliana.

    Figure  5.  Expression pattern analysis of DR29B and P5CS1 in wild type and 35S::BpSPL8 transgenic Arabidopsis thaliana under drought treatment

    表  1  BpSPL8启动子中的顺式作用元件及相关功能预测

    Table  1.   Cis-acting elements and predicted functions in the sequence of BpSPL8 promoter

    顺式元件 Cis-element数量 Number功能 Function功能分类 Function group
    MBS4MYB binding site involved in drought-inducibilityBinding site specific response element
    MRE1MYB binding site involved in light responsivenessBinding site specific response element
    circadian3Circadian controlCircadian
    Box-W11Fungal elicitor responsive elementElicitor specific responsive element
    ABRE1Abscisic acid responsivenessHormone responsive element
    TGA-element1Auxin-responsive elementHormone responsive element
    GARE-motif3Gibberellin-responsive elementHormone responsive element
    ERE1Ethylene-responsive elementHormone responsive element
    TCA-element1Salicylic acid responsivenessHormone responsive element
    AE-box2Light responseLight responsive element
    Box 43Light responseLight responsive element
    Box I3Light responsive elementLight responsive element
    CATT-motif4Light responsive elementLight responsive element
    GAG-motif1Light responsive elementLight responsive element
    GA-motif2Light responsive elementLight responsive element
    GT1-motif1Light responsive elementLight responsive element
    H-box1Light responsive elementLight responsive element
    Sp16Light responsive elementLight responsive element
    TCT-motif1Light responsive elementLight responsive element
    G-Box2Light responsivenessLight responsive element
    G-box3Light responsivenessLight responsive element
    ATGCAAAT motif1Associated to the TGAGTCA motifPlant tissue-specific element
    MSA-like1Cell cycle regulationPlant tissue-specific element
    GCN4_motif1Endosperm expressionPlant tissue-specific element
    Skn-1_motif3Endosperm expressionPlant tissue-specific element
    CAT-box1Meristem expressionPlant tissue-specific element
    AC-II1Negative regulation of phloem expressionPlant tissue-specific element
    as-2-box1Shoot-specific expression and light responsivenessPlant tissue-specific element
    ARE1Anaerobic response elementStress responsive element
    TC-rich repeats1Defense and stress responsivenessStress responsive element
    HSE2Heat stress responsivenessStress responsive element
    GCC box1Wounding and pathogen responsivenessStress responsive element
    W box1Wounding and pathogen responsivenessStress responsive element
    5UTR Py-rich stretch2Conferring high transcription levelsTranscription regulation element
    AAGAA-motif1Unknown
    Unnamed__12Unknown
    Unnamed__32Unknown
    Unnamed__47Unknown
    下载: 导出CSV
  • [1] Cardon G, Hohmann S, Klein J, et al. Molecular characterisation of the Arabidopsis SBP-box genes[J]. Gene, 1999, 237(1): 91−104. doi: 10.1016/S0378-1119(99)00308-X
    [2] Schwab R, Palatnik J F, Riester M, et al. Specific effects of microRNAs on the plant transcriptome[J]. Developmental Cell, 2005, 8(4): 517−527. doi: 10.1016/j.devcel.2005.01.018
    [3] Wu G, Poethig R S. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3[J]. Development, 2006, 133(18): 3539−3547. doi: 10.1242/dev.02521
    [4] Wang J W, Czech B, Weigel D. miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[J]. Cell, 2009, 138(4): 738−749. doi: 10.1016/j.cell.2009.06.014
    [5] Yu N, Cai W J, Wang S, et al. Temporal control of trichome distribution by microRNA156-targeted SPL genes in Arabidopsis thaliana[J]. Plant Cell, 2010, 22(7): 2322−2335. doi: 10.1105/tpc.109.072579
    [6] Jung J H, Seo P J, Kang S K, et al. miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions[J]. Plant Molecular Biology, 2011, 76(1−2): 35−45. doi: 10.1007/s11103-011-9759-z
    [7] Shikata M, Koyama T, Mitsuda N, et al. Arabidopsis SBP-box genes SPL10, SPL11 and SPL2 control morphological change in association with shoot maturation in the reproductive phase[J]. Plant and Cell Physiology, 2009, 50(12): 2133−2145. doi: 10.1093/pcp/pcp148
    [8] Schwarz S, Grande A V, Bujdoso N, et al. The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis[J]. Plant Molecular Biology, 2008, 67(1−2): 183−195. doi: 10.1007/s11103-008-9310-z
    [9] Yamasaki H, Hayashi M, Fukazawa M, et al. SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[J]. Plant Cell, 2009, 21(1): 347−361. doi: 10.1105/tpc.108.060137
    [10] Stone J M, Liang X, Nekl E R, et al. Arabidopsis AtSPL14, a plant-specific SBP-domain transcription factor, participates in plant development and sensitivity to fumonisin B1[J]. Plant Journal, 2005, 41(5): 744−754. doi: 10.1111/tpj.2005.41.issue-5
    [11] Chao L M, Liu Y Q, Chen D Y, et al. Arabidopsis transcription factors SPL1 and SPL12 confer plant thermotolerance at reproductive stage[J]. Molecular Plant, 2017, 10(5): 735−748. doi: 10.1016/j.molp.2017.03.010
    [12] Unte U S, Sorensen A M, Pesaresi P, et al. SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis[J]. Plant Cell, 2003, 15(4): 1009−1019. doi: 10.1105/tpc.010678
    [13] Zhang Y, Schwarz S, Saedler H, et al. SPL8, a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis[J]. Plant Molecular Biology, 2007, 63(3): 429−439. doi: 10.1007/s11103-006-9099-6
    [14] Xing S, Salinas M, Hohmann S, et al. miR156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis[J]. Plant Cell, 2010, 22(12): 3935−3950. doi: 10.1105/tpc.110.079343
    [15] Gou J, Debnath S, Sun L, et al. From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa[J]. Plant Biotechnology Journal, 2018, 16(4): 951−962. doi: 10.1111/pbi.2018.16.issue-4
    [16] 李双, 苏艳艳, 王厚领, 等. 胡杨miR1444b在拟南芥中正调控植物抗旱性[J]. 北京林业大学学报, 2018, 40(4):1−9.

    Li S, Su Y Y, Wang H L, et al. Populus euphratica miR1444b positively regulates plants response to drought stress in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2018, 40(4): 1−9.
    [17] 姚琨, 练从龙, 王菁菁, 等. 胡杨PePEX11基因参与调节盐胁迫下拟南芥的抗氧化能力[J]. 北京林业大学学报, 2018, 40(5):19−28.

    Yao K, Lian C L, Wang J J, et al. PePEX11 functions in regulating antioxidant capacity of Arabidopsis thaliana under salt stress[J]. Journal of Beijing Forestry University, 2018, 40(5): 19−28.
    [18] Sakuma Y, Maruyama K, Osakabe Y, et al. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression[J]. Plant Cell, 2006, 18(5): 1292−1309. doi: 10.1105/tpc.105.035881
    [19] Strizhov N, Abraham E, Okresz L, et al. Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis[J]. Plant Journal, 1997, 12(3): 557−569.
    [20] 李蕾蕾, 孙丰坤, 李天宇, 等. 白桦BpGT14基因启动子克隆及表达活性分析[J]. 北京林业大学学报, 2016, 38(7):16−24.

    Li L L, Sun F K, Li T Y, et al. Cloning and activity analysis of BpGT14 gene promoter in Betula platyphylla[J]. Journal of Beijing Forestry University, 2016, 38(7): 16−24.
    [21] 张一南, 王洋, 张会龙, 等. 过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性[J]. 北京林业大学学报, 2017, 39(11):1−8.

    Zhang Y N, Wang Y, Zhang H L, et al. Overexpression of PeRIN4 enhanced salinity tolerance through up regulation of PM H+-ATPase in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2017, 39(11): 1−8.
    [22] Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J]. EMBO Journal, 1987, 6(13): 3901−3907. doi: 10.1002/embj.1987.6.issue-13
    [23] Liu C, Guan M X, Hu X Q, et al. Complex regulatory network of Betula BplSPL8 in planta[J]. Journal of Forestry Research, 2017, 28(5): 881−889. doi: 10.1007/s11676-017-0372-0
    [24] Yu X, Liu Y, Wang S, et al. CarNAC4, a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis[J]. Plant Cell Reports, 2016, 35(3): 613−627. doi: 10.1007/s00299-015-1907-5
    [25] 牛素贞, 宋勤飞, 樊卫国, 等. 干旱胁迫对喀斯特地区野生茶树幼苗生理特性及根系生长的影响[J]. 生态学报, 2017, 37(21):7333−7341.

    Niu S Z, Song Q F, Fan W G, et al. Effects of drought stress on leaf physiological characteristics and root growth of the clone seedlings of wild tea plants[J]. Acta Ecologica Sinica, 2017, 37(21): 7333−7341.
    [26] Yu N, Niu Q W, Ng K H, et al. The role of miR156/SPLs modules in Arabidopsis lateral root development[J]. Plant Journal, 2015, 83(4): 673−685. doi: 10.1111/tpj.12919
    [27] Gao R, Wang Y, Gruber M Y, et al. miR156/SPL10 modulates lateral root development, branching and leaf morphology in Arabidopsis by silencing AGAMOUS-LIKE 79[J/OL]. Frontiers in Plant Science, 2017, 8 (2017−01−04) [2018−06−20]. https://doi.org/10.3389/fpls.2017.02226.
    [28] 刘闯. 18个白桦SPLs基因的鉴定及BpSPL8基因的功能分析[D]. 哈尔滨: 东北林业大学, 2017.

    Liu C. Identification of 18 SPL gene family members and functional analysis of BpSPL8 in Betula platyphylla[D]. Harbin: Northeast Forestry University, 2017.
    [29] Saini S, Sharma I, Kaur N, et al. Auxin: a master regulator in plant root development[J]. Plant Cell Reports, 2013, 32(6): 741−757. doi: 10.1007/s00299-013-1430-5
    [30] Hao Y J, Wei W, Song Q X, et al. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants[J]. Plant Journal, 2011, 68(2): 302−313. doi: 10.1111/j.1365-313X.2011.04687.x
    [31] Chen D, Richardson T, Chai S, et al. Drought-up-regulated TaNAC69-1 is a transcriptional repressor of TaSHY2 and TaIAA7, and enhances root length and biomass in wheat[J]. Plant and Cell Physiology, 2016, 57(10): 2076−2090. doi: 10.1093/pcp/pcw126
    [32] Laplaze L, Benkova E, Casimiro I, et al. Cytokinins act directly on lateral root founder cells to inhibit root initiation[J]. Plant Cell, 2007, 19(12): 3889−3900. doi: 10.1105/tpc.107.055863
    [33] Loutfy N, El-Tayeb M A, Hassanen A M, et al. Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat (Triticum aestivum)[J]. Journal of Plant Research, 2012, 125(1): 173−184. doi: 10.1007/s10265-011-0419-9
    [34] Ivanchenko M G, Muday G K, Dubrovsky J G. Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana[J]. Plant Journal, 2008, 55(2): 335−347. doi: 10.1111/tpj.2008.55.issue-2
    [35] Ruiz-Lozano J M, Aroca R, Zamarreno A M, et al. Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and tomato[J]. Plant Cell and Environment, 2016, 39(2): 441−452. doi: 10.1111/pce.v39.2
    [36] Sanchez-Romera B, Ruiz-Lozano J M, Zamarreno A M, et al. Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought[J]. Mycorrhiza, 2016, 26(2): 111−122. doi: 10.1007/s00572-015-0650-7
    [37] Rowe J H, Topping J F, Liu J, et al. Abscisic acid regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin[J]. New Phytologist, 2016, 211(1): 225−239. doi: 10.1111/nph.13882
    [38] 赵婉莹, 于太飞, 杨军峰, 等. 大豆GmbZIP16的抗旱功能验证及分析[J]. 中国农业科学, 2018, 51(15):6−18.

    Zhao W Y, Yu T F, Yang J F, et al. Verification and analyses of soybean GmbZIP16 gene resistance to drought[J]. Scientia Agricultura Sinica, 2018, 51(15): 6−18.
  • 加载中
图(5) / 表(1)
计量
  • 文章访问数:  1254
  • HTML全文浏览量:  1108
  • PDF下载量:  58
  • 被引次数: 0
出版历程
  • 收稿日期:  2019-03-13
  • 修回日期:  2019-04-18
  • 网络出版日期:  2019-06-19
  • 刊出日期:  2019-08-01

目录

    /

    返回文章
    返回