两个参与水稻（Oryza sativa L ssp.）绿色组织特异性启动子活性的新型正调控元件。
Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.).
机构信息
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
出版信息
Plant Cell Rep. 2012 Jul;31(7):1159-72. doi: 10.1007/s00299-012-1238-8. Epub 2012 Mar 3.
UNLABELLED
In plant genetic engineering, using tissue-specific promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there are few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application. In this study, based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120). The expression pattern of DX1 gene promoter was examined by using the β-glucuronidase (GUS) reporter gene and analyzed in transgenic rice plants in different tissues. Histochemical assays and quantitative analyses of GUS activity confirmed that P (DX1):GUS was highly expressed in green tissues. To identify the regulatory elements controlling the expression of the DX1 gene, a series of 5' and 3' deletions of DX1 promoter were fused to GUS gene and stably introduced into rice plants. In addition, gel mobility shift assays and site-directed mutagenesis studies were used, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P(DX1). GSE1 acted as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acted as a positive regulator only in sheath and stem tissue, and had a weaker effect on gene expression. In addition, P(DX1):GUS was not expressed in anther and seed, this characteristic reduced the potential ecological risk and potential food safety issues. Taken together, our results strongly suggest that the identified promoter, P(DX1), and its cis regulatory elements, GSE1 and GSE2, are potentially useful in the field of rice transgenic breeding.
KEY MESSAGE
We have isolated and characterized the rice green tissue-specific promoter P(DX1), and identified two novel positive cis-acting elements in P(DX1).
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在植物基因工程中，使用组织特异性启动子来控制靶基因的表达是避免使用组成型启动子可能带来的潜在负面影响（如代谢负担等）的有效方法。然而，到目前为止，能够用于作物生物技术应用的具有强而可靠表达的组织特异性启动子仍然很少。在本研究中，我们基于微阵列和 RT-PCR 数据，鉴定了一个水稻绿色组织特异性表达基因 DX1（LOC_Os12g33120）。通过使用β-葡萄糖醛酸酶（GUS）报告基因检测 DX1 基因启动子的表达模式，并在不同组织的转基因水稻植株中进行分析。组织化学分析和 GUS 活性的定量分析证实，P（DX1）：GUS 在绿色组织中高度表达。为了鉴定控制 DX1 基因表达的调控元件，我们将 DX1 启动子的一系列 5'和 3'缺失片段与 GUS 基因融合，并稳定导入水稻植株中。此外，我们还使用凝胶迁移率变动分析和定点突变研究，鉴定了 P（DX1）内的两个新的组织特异性顺式作用元件（GSE1 和 GSE2）。GSE1 在所有绿色组织（叶、鞘、茎和穗）中作为正调控因子发挥作用。与 GSE1 相比，GSE2 仅在鞘和茎组织中作为正调控因子发挥作用，对基因表达的影响较弱。此外，P（DX1）：GUS 在花药和种子中不表达，这一特性降低了潜在的生态风险和潜在的食品安全问题。综上所述，我们的研究结果强烈表明，所鉴定的启动子 P（DX1）及其顺式调控元件 GSE1 和 GSE2，在水稻转基因育种领域具有潜在的应用价值。
关键信息
我们分离并鉴定了水稻绿色组织特异性启动子 P（DX1），并在 P（DX1）中鉴定了两个新的正顺式作用元件。
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