Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa
Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa
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DOI：
10.1071/fp18237
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发表时间：
2019-01-01
影响因子：
3
通讯作者：
Sudarsono, Sudarsono
中科院分区：
文献类型：
--
作者：
Apriana, Aniversari;Sisharmini, Atmitri;Sudarsono, Sudarsono
根特异性启动子可用于植物基因工程，主要是为了改善水和养分的吸收。本研究的目的是克隆并鉴定编码 2-烯醛还原酶（一种 NADPH 依赖性氧化还原酶）的稻烯醛还原酶 (OsAER1) 基因的启动子。使用定量实时 PCR 的表达分析证实了 OsAER1 基因的根特异性表达。随后，从印度尼西亚当地水稻品种 Awan Kuning 中分离出 OsAER1 启动子的 3082 bp 片段。对 3082 bp 启动子片段 (PA-5) 进行测序和进一步的核苷酸序列分析揭示了至少 10 个根特异性顺式调控元件的存在，这些元件可能负责 OsAER1 根特异性表达。使用 3082 bp 启动子片段驱动 GUS 报告基因转基因的表达证实了 OsAER1 启动子是根特异性的。此外，分析表明，在水稻发育的发芽、营养、孕穗和生殖阶段，叶子、叶柄和芽中不存在 OsAER1 启动子活性。相反，在开花后7-20天，启动子活性存在于未成熟种子的花药和糊粉层中。此外，在成熟种子的糊粉层中没有观察到启动子活性。 OsAER1 启动子活性由铝毒性、氯化钠和淹没胁迫诱导，表明 OsAER1 启动子活性是由这些胁迫诱导的。用脱落酸和吲哚乙酸对携带 PA-5 启动子构建体的转基因植物进行外源处理也诱导了 GUS 报告转基因的表达，表明植物生长调节剂在控制 OsAER1 启动子活性中的作用。进行启动子缺失分析以鉴定负责控制根特异性表达的启动子的顺式作用元件。 GUS报告基因与OsAER1启动子的各种缺失片段融合，并将所得构建体转化到水稻植物中以产生转基因植物。该分析的结果表明，控制根特异性表达的顺式作用元件位于OsAER1 CDS的-1562至-1026bp之间。在这里，我们讨论了分析的结果、OsAER1 在水稻生长和发育中的可能作用、可能的贡献以及这些发现在未来植物研究中的潜在用途。
Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7-20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between -1562 to -1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.