Post-transcriptional Regulation of Glutamine Synthetase

谷氨酰胺合成酶（GS）在植物氮代谢中起着重要作用。GS催化氨与谷氨酸（Glu）的ATP依赖性缩合，以产生谷氨酰胺（Gln）。GS在植物中位于细胞质（GS 1）或叶绿体（GS 2）中。GS/1活性与产量之间的正相关性已在几种作物中观察到，这表明GS/1可以代表氮利用效率和产量的关键组分。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一条可行途径。然而，似乎有一个广泛的响应过度GS/1，这似乎是物种特异性，并可能取决于该植物中的氮同化的网站。GS/1是一种高度调节的酶，这种调节可以解释 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CaMV 35 S组成型启动子驱动的GS/1基因的Alcohol转化体显示， GS/1多肽或GS活性增加。当转化体用硝酸盐处理时，转基因和内源GS基因的转录水平也显示出显著下降。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节限制，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基因的3 'UTR序列在GS/1基因表达的转录后调控中起着重要作用。这个项目的目标 目的是了解GS/1在植物中表达的转录后调控机制。本建议的目标是：鉴定GS/1 mRNA中在转录物周转和/或翻译起始中起作用的顺式作用元件; ii.鉴定用于该调节步骤的代谢信号; iii.分离并鉴定与这些顺式元件相互作用的反式因子。

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.