Post-transcriptional Regulation of Glutamine Synthetase

谷氨酰胺合成酶的转录后调控

• 批准号: 6766186
• 负责人: CHAMPA SENGUPTA-GOPALAN
• 金额: $ 17.4万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766186
• 关键词: alfalfa; ammonia; biological signal transduction; chemical stability; electron microscopy; genetic regulation; genetically modified plants; glutamate ammonia ligase; glutamates; glutamine; intermolecular interaction; messenger RNA; nitrogen metabolism; nucleic acid structure; plant genetics; plant growth /development; plant physiology; plant proteins; posttranscriptional RNA processing; protein structure function

Glutamine synthetase (GS) plays a central role in nitrogen metabolism in all plants. GS catalyzes the ATP dependent condensation of ammonia with glutamate (Glu), to yield glutamine (Gln). GS in plants is located either in the cytosol (GS 1) or in the chloroplast (GS 2). A positive correlation between GS/1 activity and yield has been observed in several crop plants, suggesting that GS/1 could represent a key component in nitrogen use efficiency and yield. Thus increasing GS/1 activity by genetic engineering is a viable approach to improve the nitrogen use efficiency in plants. However, there appears to be a wide range of responses to overexpression of GS/1 which appears to be species specific and may depend on the site of nitrogen assimilation in that plant. GS/1 is a very highly regulated enzyme and this regulation may account for the variability in the expression of the transgene. Thus, understanding the regulatory mechanism underlying the expression of GS/1 genes is critical in our strategies to increase GS/1 levels in transgenic plants. Alfalfa transformants with a GS/1 gene driven by a constitutive CaMV 35S promoter showed no increase in GS/1 polypeptide or GS activity. The transcript level for the transgene and the endogenous GS genes also showed a significant drop when the transformants were treated with nitrate. More recent work from our laboratory has shown that the removal of the 3'UTR from the soybean GS/1 transgene abolishes the regulatory constraints in the expression of the transgene in alfalfa and the transformants show increased transgene transcript, polypeptide and enzyme activity. Our hypothesis is that sequences in the 3'UTR of GS genes play a central role in the posttranscriptional regulation of GS/1 gene expression. The goal of this project is to understand the posttranscriptional regulatory mechanism underlying GS/1 expression in plants. The objectives of this proposal are: i. to identify cis-acting elements in GS/1 mRNA with a role in transcript turnover and/or translation initiation; ii. to identify the metabolic signal for this regulatory step; iii. to isolate and characterize the trans-factors that interact with these cis-elements.

谷氨酰胺合成酶（GS）在所有植物的氮代谢中发挥着核心作用。 GS 催化氨与谷氨酸 (Glu) 的 ATP 依赖性缩合，产生谷氨酰胺 (Gln)。植物中的 GS 位于细胞质 (GS 1) 或叶绿体 (GS 2) 中。在几种作物中观察到 GS/1 活性与产量之间呈正相关，这表明 GS/1 可能代表氮利用效率和产量的关键组成部分。因此，通过基因工程提高GS/1活性是提高植物氮素利用效率的可行方法。然而，似乎对 GS/1 的过度表达有多种反应，这似乎是物种特异性的，并且可能取决于该植物中氮同化的位点。 GS/1 是一种受到高度调控的酶，这种调控可能是 转基因表达的变异性。因此，了解 GS/1 基因表达的调控机制对于我们提高转基因植物中 GS/1 水平的策略至关重要。 具有由组成型 CaMV 35S 启动子驱动的 GS/1 基因的苜蓿转化体没有表现出 GS/1 多肽或 GS 活性增加。当用硝酸盐处理转化体时，转基因和内源 GS 基因的转录水平也显示出显着下降。我们实验室最近的工作表明，从大豆 GS/1 转基因中去除 3'UTR 消除了苜蓿中转基因表达的调控限制，并且转化体显示出增加的转基因转录物、多肽和酶活性。我们的假设是 GS 基因 3'UTR 中的序列在 GS/1 基因表达的转录后调控中发挥核心作用。该项目的目标 目的是了解植物中 GS/1 表达的转录后调控机制。该提案的目标是：鉴定 GS/1 mRNA 中在转录本周转和/或翻译起始中发挥作用的顺式作用元件；二.识别该调节步骤的代谢信号；三.分离并表征与这些顺式元件相互作用的反式因子。