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    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

    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。

       

      Abstract:
      ObjectiveCurrently, researches on the gene function of plant SPL8 are mainly focused on flowering and fertility, but there are fewer reports about its SPL8 function in drought stress response. In this paper, BpSPL8 promoter was cloned and analyzed from Betula platyphylla, and the function of BpSPL8 gene in response to drought stress was studied in Arabidopsis thaliana.
      MethodPromoter sequence of BpSPL8 gene was isolated from Betula platyphylla by PCR technology, and the cis-element prediction of BpSPL8 promoter was performed using PLACE and PlantCARE software. The plant expression vectors with GUS (β-glucuronidase coding gene) expression driven by the promoters of BpSPL8 were constructed and transformed into Arabidopsis thaliana by the floral dip method. Through the detection of GUS activity, the tissue expression pattern of the BpSPL8 promoter in Arabidopsis thaliana was analyzed. QRT-PCR analysis was performed on the expression level of BpSPL8 under PEG treatment. Finally, the overexpression of BpSPL8 Arabidopsis thaliana was used to explore the function of BpSPL8 in the drought process.
      ResultPromoter element analysis revealed that BpSPL8 promoter contained elements for tissue-specific expression, light-responsive, hormone-responsive and stress-responsive. GUS histochemical staining results showed that GUS activity was observed in hypocotyls, leaves, petioles, roots and inflorescences of transgenic Arabidopsis thaliana carrying the BpSPL8 promoter. The expression patterns of BpSPL8 gene in roots and leaves of birch were up-regulated and then down-regulated under PEG treatment. Drought stress tolerance pointed out that the transgenic plants showed significantly lower survival rate and proline content than wild type, while malondialdehyde content was higher than wild type. Two known stress-resistant genes, DR29B and P5CS1, were up-regulated in wild-type and transgenic Arabidopsis thaliana under drought stress. However, compared with wild-type, they showed delayed up-regulation in transgenic Arabidopsis thaliana.
      ConclusionEctopic overexpression of BpSPL8 can reduce the drought tolerance of Arabidopsis thaliana and affect the expression patterns of resistance genes DR29B and P5CS1 under drought stress.

       

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