Post-transcriptional Regulation of Glutamine Synthetase

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催化依赖于ATP的氨与谷氨酸(Glu)缩合生成谷氨酰胺(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活性与产量呈正相关，这表明GS/1可能是氮素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途径。然而，对于GS/1的过度表达似乎有广泛的反应，这似乎是物种特有的，可能取决于植物中氮素同化的位置。GS/1是一种高度调控的酶，这种调控可能解释了 转基因表达的可变性。因此，了解GS/1基因表达的调控机制对于我们在转基因植物中提高GS/1水平的策略至关重要。 含有CaMV 35S启动子驱动的GS/1基因的紫花苜蓿转化植株 GS/1多肽或GS活性增加。在硝酸盐处理下，转基因和内源GS基因的转录水平也显著下降。我们实验室的最新工作表明，从大豆GS/1转基因中去除3‘非编码区，取消了对转基因在紫花苜蓿中表达的调控限制，转化体显示出更高的转基因转录物、多肽和酶活性。我们的假设是GS基因3‘端非编码区的序列在GS/1基因表达的转录后调控中起核心作用。这个项目的目标是 目的是了解植物GS/1表达的转录后调控机制。这项建议的目标是：1.确定GS/1mRNA中在转录翻转和/或翻译启动中起作用的顺式作用元件；2.确定这一调节步骤的代谢信号；分离和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.