REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

植物氨基酸生物合成基因的调控

基本信息

批准号：
2668462
负责人：
Gloria CORUZZI
金额：
$ 24.99万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 2000-02-29
项目状态：
已结题

项目摘要

We are using Arabidopsis thaliana as a model to identify the structural and regulatory genes controlling nitrogen assimilation into glutamine and glutamate using molecular, biochemical and genetic approaches. Nitrogen assimilation into these amino acids affects plant growth and ultimately seeds quantity and quality. Thus, our basic studies on the genes that control this process in plants relate indirectly to human and animal nutrition. The structural genes under investigation are: glutamine synthetase (GS), glutamate synthase (Fd-GOGAT or NADH-GOGAT), and glutamate dehydrogenase (GDH). The gene families for each enzyme in Arabidopsis contain independently regulated members encoding distinct isoenzymes. Despite decades of in vitro studies conducted in many species, the in vivo roles of GS, GOGAT and GDH isoenzymes in plants can only be conjectured. We propose to conduct the first systematic isolation of plant mutants specifically defective in each isoenzyme of GS, Fd-GOGAT, NADH-GOGAT or GDH, in a single species. We have shown that it is possible to isolate Arabidopsis mutants defective in nitrogen assimilatory genes using isoenzyme screens unbiased for growth phenotype. We propose a detailed characterization of the mutant progeny, to quantify the effects of the loss of a single isoenzyme on processes such as primary nitrogen assimilation, photorespiration, and nitrogen mobilization during seed set. This analysis will define the key and rate-limiting enzymes that control the efficiency of nitrogen use in plants. In addition we may also uncover mutants in regulatory genes. We have begun to investigate the mechanisms controlling the regulation of nitrogen assimilatory genes. Based on gene regulation studies, we have developed a "metabolic control" model that proposes these nitrogen assimilatory genes are regulated in response to the ratio of carbon to nitrogen metabolites in a plant. We propose genetic screens to uncover the components of this machinery and have in hand two putative regulatory genes. Our specific aims are: 1) Isolate additional Arabidopsis gdh mutants and mutants in chloroplastic GS2 by isoenzyme screening, 2) Characterize existing Arabidopsis mutants defective in one of two genes for Fd-GOGAT (gls1) and isolate mutants in the second gene, 3) Create mutants in cytosolic GS1 or NADH-GOGAT by expressing dominant-negative subunits in transgenic plants, 4) Test the metabolic control model and define the metabolites senses, 5) Define components of the regulatory pathway using genetic screens and define the in vivo function of candidate genes in hand. Our basic studies on the mechanisms that regulate nitrogen assimilation in Arabidopsis may have implications for improving nitrogen use in crops not amenable to such molecular-genetic studies. Furthermore, as metabolic signaling also occurs in animals, insights into this process may be more readily obtained using a molecular-genetic strategy in Arabidopsis.

我们使用拟南芥作为模型来识别结构和控制氮同化为谷氨酰胺的调节基因使用分子、生物化学和遗传方法来检测谷氨酸。氮这些氨基酸的同化会影响植物生长，并最终种子数量和质量。因此，我们对基因的基础研究植物中控制这一过程与人类和动物间接相关营养。正在研究的结构基因是：谷氨酰胺合成酶 (GS)、谷氨酸合成酶（Fd-GOGAT 或 NADH-GOGAT），以及谷氨酸脱氢酶（GDH）。每种酶的基因家族拟南芥含有独立调控的成员，编码不同的同工酶。尽管在许多国家进行了数十年的体外研究植物中 GS、GOGAT 和 GDH 同工酶的体内作用可以只能推测。我们建议进行第一次系统隔离 GS、Fd-GOGAT 每种同工酶特异缺陷的植物突变体， NADH-GOGAT 或 GDH，在单一物种中。我们已经证明这是可能的分离氮同化基因缺陷的拟南芥突变体使用同工酶对生长表型进行公正的筛选。我们提出一个突变后代的详细表征，以量化影响诸如初级氮等过程中单一同工酶的损失结籽过程中的同化、光呼吸和氮动员。该分析将定义控制的关键酶和限速酶植物对氮的利用效率。此外我们还可能发现调节基因的突变体。我们已经开始研究机制控制氮同化基因的调节。基于基因调节研究，我们开发了一个“代谢控制”模型提出这些氮同化基因的调节是为了响应植物中碳与氮代谢物的比率。我们建议基因筛选以揭示该机器的组成部分并具有手上有两个假定的调控基因。我们的具体目标是：1）隔离其他拟南芥 gdh 突变体和叶绿体 GS2 突变体同工酶筛选，2) 表征现有拟南芥突变体 Fd-GOGAT (gls1) 的两个基因之一存在缺陷，并在其中分离出突变体第二个基因，3) 在胞质 GS1 或 NADH-GOGAT 中创建突变体在转基因植物中表达显性失活亚基，4) 测试代谢控制模型并定义代谢物感官，5）定义使用遗传筛选的调控途径的组成部分并定义手中候选基因的体内功能。我们的基础研究拟南芥氮同化调节机制可能有对改善不适合这种情况的作物氮利用的影响分子遗传学研究。此外，由于代谢信号也发生在动物身上，可能更容易获得对这个过程的了解在拟南芥中使用分子遗传策略。