• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

龙眼DlAGO4DlAGO6启动子的克隆及活性分析

Cloning and Activity of DlAGO4 and DlAGO6 Promoters from Dimocarpus longan

  • 摘要:
      目的  Argonaute(AGO)蛋白是RNA介导的沉默复合物RISC的核心成分,参与植物生长发育、组织形成、细胞增殖凋亡、病毒防御、逆境响应等多种生物过程。探究龙眼DlAGO4DlAGO6基因启动子区域的顺式作用元件,以及重组表达载体在不同激素处理下的表达模式,可为进一步研究DlAGO4DlAGO6基因的功能提供参考。
      方法  使用常规PCR技术克隆龙眼DlAGO4DlAGO6基因启动子序列,采用BPDG、PLACE和PlantCARE等生物信息学工具进行DlAGO4DlAGO6基因启动子序列的生物信息学分析,并构建全长启动子与GUS基因融合表达载体,转化烟草叶片,进行瞬时表达,通过GUS组织化学染色分析启动子的活性。
      结果  DlAGO4基因启动子片段长度1514 bp,DlAGO6基因启动子片段长度1784 bp。两个启动子序列均含有TATA-box和CAAT-box核心元件、茉莉酸甲酯、脱落酸和光响应元件;DlAGO4启动子序列还含有水杨酸、赤霉素响应元件和厌氧应答调控元件;DlAGO6启动子序列还含有生长素及昼夜节律调控元件。2个启动子片段均可驱动GUS基因表达,表达强度弱于CaMV35S。MeJA和ABA处理可显著提高转DlAGO6启动子烟草叶片中GUS基因的相对表达水平,SA和MeJA处理可提高转DlAGO4启动子烟草叶片GUS基因的相对表达水平。
      结论  成功克隆了龙眼DlAGO4DlAGO6基因启动子,二者为激素诱导型启动子,具有驱动下游GUS表达的活性,可能参与了植物体胚发育以及对激素的响应。

     

    Abstract:
      Objective   Argonaute (AGO) proteins play the important roles in a wide varieties of plant biological processes. To explore the cis --acting elements of DlAGO4 and DlAGO6 promoters, the expression patterns of recombinant vector of DlAGO4 and DlAGO6 promoters under different hormones treatments in longan, DlAGO4 and DlAGO6 promoters in longan were cloned and analyzed.
      Method  Promoters of DlAGO4 and DlAGO6 from Dimocarpus longan were cloned by PCR for a bioinformatic analysis. Fusion expression vectors of the full-length promoters and GUS gene were constructed. The Agrobacterium tumefaciens-mediated transformation was performed on protocorm of tobacco leaves to observe the transient expression of the genes, while promoter activity examined by GUS histochemical staining.
      Result   The sequence of DlAGO4 promoter was 1 514 bp and the sequence of DlAGO6 promoter was 1 784 bp. Both had the core elements of TATA- and CAAT-box as well as ababolic acid (ABA), methyl jasmonate (MeJA), and photoresponsive elements. The DlAGO4 promoter also contained salicylic acid (SA), gibberellin response, and anaerobic response regulatory elements, while the DlAGO6 promoter had auxin and circadian rhythm regulatory elements. The promoters exhibited a GUS-driving activity that was weaker than CaMV35S. The relative expression of GUS of PDlAGO6::GUS recombinant tobacco leaf was significantly elevated by the treatment of MeJA and ABA, whereas that of PDlAGO4::GUS recombinant tobacco leaf by SA and MeJA.
      Conclusion   The promoters of DlAGO4 and DlAGO6 from longan were successfully cloned. As hormone-induced promoters, they could activate the GUS expression and be involved in the somatic embryo development and hormone response of plants.

     

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