Background

Cloning and characterizing the drought-inducible promoters is essential for their use in crop resistance's genetic improvement. Previous studies have shown that the TaNRX1-D gene participates in regulating the response of wheat to drought stress. However, its promoter has not yet been identified.

Objective

In this study, we aimed to characterize the promoter of the TaNRX1-D gene.

Methods

The promoter of TaNRX1-D (named P0, 2081 bp) was isolated from common wheat with several cis-acting elements that regulate in response to abiotic stresses and some core cis-acting elements. Functional verification of the promoter, eight 5′-deletion fragments of TaNRX1-D promoter, was fused to the β-glucuronidase (GUS) gene P0::GUS ~ P7::GUS and transformed into Arabidopsis, respectively. Agrobacterium-mediated GUS transient assay the P6a and P6b promoter regions in tobacco leaves under normal, osmotic or ABA stress.

Results

Activity analysis of the full-length promoter (P0) showed that the intensity of stronger β-glucuronidase (GUS) staining in the roots and leaves was obtained during the growth of transgenic Arabidopsis. P0::GUS displayed the GUS activity was much higher in the roots and leaves than in other parts of the transgenic plant under normal conditions, which was similarly within wheat. Analysis of the 5′-deletion fragments revealed that P0::GUS ~ P6::GUS responded well upon exposure to osmotic (polyethylene glycol-6000, PEG6000) and abscisic acid (ABA) stress treatments and expressed significantly higher GUS activity than the CaMV35S promoter (35S::GUS), while P7::GUS did not. GUS transient assay in tobacco leaves showed that the GUS activities of P6a and P6b were lower than P6 in the PEG6000 and ABA stresses.

Conclusion

The 193 bp (P6) segment was considered the core region of TaNRX1-D responding to PEG6000 or ABA treatment. GUS activity assay in transgenic Arabidopsis showed that this segment was sufficient for the PEG6000 or ABA stress response. The identified 193 bp promoter of TaNRX1-D in this study will help breed osmotic or ABA tolerant crops. The 36 bp segment between P6 and P6b (−193 to −157 bp) was considered the critical sequence for the TaNRX1-D gene responding to PEG6000 or ABA treatment.

中文翻译：

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鉴定普通小麦TaNRX1-D基因的193 bp启动子区有助于转基因拟南芥的渗透或ABA胁迫诱导

背景

克隆和表征干旱诱导型启动子对于它们在作物抗性遗传改良中的应用至关重要。以往的研究表明，TaNRX1-D基因参与调控小麦对干旱胁迫的反应。然而，其启动子尚未确定。

客观的

在这项研究中，我们旨在表征TaNRX1-D基因的启动子。

方法

TaNRX1-D的启动子（命名为 P0，2081 bp）是从普通小麦中分离出来的，它具有几个响应非生物胁迫而调节的顺式作用元件和一些核心顺式作用元件。启动子的功能验证， TaNRX1-D启动子的 8 个 5'-缺失片段，与 β-葡萄糖醛酸酶 (GUS) 基因P0::GUS  ~  P7::GUS融合并分别转化到拟南芥中。农杆菌介导的GUS 瞬时测定在正常、渗透或 ABA 胁迫下烟叶中的 P6a 和 P6b 启动子区域。

结果

全长启动子（P0）的活性分析表明，在转基因拟南芥的生长过程中，获得了更强的根和叶中β-葡萄糖醛酸酶（GUS）染色的强度。P0::GUS在正常条件下，根和叶中的 GUS 活性比转基因植物的其他部分高得多，小麦中的情况也是如此。对 5'-缺失片段的分析表明，P0::GUS  ~  P6::GUS在暴露于渗透压（聚乙二醇-6000、PEG6000）和脱落酸 (ABA) 胁迫处理时反应良好，并且 GUS 活性显着高于 CaMV35S启动子（35S::GUS），而P7::GUS没有。烟叶中的 GUS 瞬时测定表明，在 PEG6000 和 ABA 胁迫下，P6a 和 P6b 的 GUS 活性低于 P6。

结论

193 bp (P6) 片段被认为是响应 PEG6000 或 ABA 处理的TaNRX1-D的核心区域。转基因拟南芥中的 GUS 活性测定表明，该片段足以应对 PEG6000 或 ABA 应激反应。本研究中鉴定的TaNRX1-D的 193 bp 启动子将有助于培育耐渗透或 ABA 作物。P6 和 P6b 之间的 36 bp 片段（-193 到 -157 bp）被认为是响应 PEG6000 或 ABA 处理的TaNRX1-D基因的关键序列。