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起着核心作用。 GS催化氨与谷氨酸（GLU）的ATP依赖性凝结，以产生谷氨酰胺（GLN）。植物中的GS位于细胞质（GS 1）或叶绿体（GS 2）中。在几种作物植物中观察到了GS/1活性和产量之间的正相关关系，这表明GS/1可以代表氮的使用效率和产量的关键成分。因此，通过基因工程增加的GS/1活性是提高植物中氮利用效率的可行方法。但是，似乎对GS/1的过表达有广泛的反应，这似乎是特定于物种的，并且可能取决于该植物中氮同化的位点。 GS/1是一种非常高度调节的酶，该法规可能会说明 转基因表达的变异性。因此，了解GS/1基因表达的调节机制在我们增加转基因植物中GS/1水平的策略中至关重要。 由本构型CAMV 35S启动子驱动的GS/1基因的苜蓿转化子没有显示 增加GS/1多肽或GS活性。当转化物用硝酸盐处理时，转基因和内源性GS基因的转录水平也显示出显着下降。我们实验室的最新工作表明，从大豆GS/1转基因中除去3'UTR可取消苜蓿中转基因表达中的调节限制，转化剂显示转化剂的转录量增加，多肽和酶活性增加。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节中起着核心作用。这个项目的目标 是要了解植物中GS/1表达的转录后调节机制。该提案的目标是：i。鉴定在转录本周转和/或翻译起始中作用的GS/1 mRNA中的顺式作用元件； ii。确定该调节步骤的代谢信号； iii。分离和表征与这些顺式元素相互作用的反式因子。