Arnaiz Ana, Martinez Manuel, Gonzalez-Melendi Pablo, Grbic Vojislava, Diaz Isabel, Santamaria M Estrella
Centro de Biotecnología y Genómica de Plantas, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Universidad Politécnica de Madrid, Madrid, Spain.
Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain.
Front Plant Sci. 2019 Jul 17;10:930. doi: 10.3389/fpls.2019.00930. eCollection 2019.
The plant defense responses to pests results in the synchronized change of a complex network of interconnected genes and signaling pathways. An essential part of this process is mediated by the binding of transcription factors to the specific responsive -elements within in the promoters of phytophagous-responsive genes. In this work, it is reported the identification and characterization of a bidirectional promoter that simultaneously co-regulate two divergent genes, and , upon arthropod feeding. Computational analysis identified the presence of -elements within the intergenic region between two loci, mainly from the DOF but also from the AP2/ERF, Golden 2-like and bHLH families. The function of the bidirectional promoter was analyzed using two enhanced variants of the and reporter genes, in both orientations, in transient tobacco and stably transformed Arabidopsis plants. Promoter activity was tested in response to feeding of and , as well as wounding, flagellin and chitin treatments. Using RT-qPCR assays and confocal microscopy, it was shown that all treatments resulted in the induction of both reporter genes. Furthermore, our findings revealed the asymmetric character of the promoter with stronger activity in the forward than in the reverse orientation. This study provides an example of a bidirectional promoter with a strong potential to be used in plant biotechnology in pest control that requires stacking of the defense genes.
植物对害虫的防御反应导致相互关联的基因和信号通路组成的复杂网络发生同步变化。这一过程的一个重要部分是由转录因子与植食性反应基因启动子内的特定反应元件结合介导的。在这项工作中，报告了一个双向启动子的鉴定和表征，该启动子在节肢动物取食时同时共同调控两个不同的基因 和 。计算分析确定了两个基因座之间基因间区域内存在 元件，主要来自DOF家族，但也来自AP2/ERF、Golden 2-like和bHLH家族。使用 和 报告基因的两个增强变体，在两个方向上，在瞬时烟草和稳定转化的拟南芥植物中分析了双向启动子的功能。测试了启动子活性对 和 的取食以及伤口、鞭毛蛋白和几丁质处理的响应。使用RT-qPCR分析和共聚焦显微镜，结果表明所有处理均导致两个报告基因的诱导。此外，我们的研究结果揭示了启动子的不对称特性，正向活性强于反向活性。这项研究提供了一个双向启动子的例子，该启动子在需要堆叠防御基因的害虫控制植物生物技术中具有很强的应用潜力。
