REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

植物氨基酸生物合成基因的调控

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/2668462
• 关键词: Arabidopsis; aminoacid biosynthesis; autoradiography; chloroplasts; gene mutation; genetic regulation; genetically modified plants; glutamate ammonia ligase; glutamate dehydrogenase; isozymes; nitrogen fixation; nitrogen metabolism; nucleic acid probes; photosynthesis; plant genetics; plant proteins; radiotracer; regulatory gene; structural genes; western blottings

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识别结构的模型 以及控制氮同化谷氨酰胺和的调节基因 使用分子，生化和遗传方法的谷氨酸。 氮 这些氨基酸的同化会影响植物的生长，最终会影响 种子数量和质量。 因此，我们对基因的基础研究 在植物中控制这一过程与人类和动物间接相关 营养。 所研究的结构基因是：谷氨酰胺 合成酶（GS），谷氨酸合酶（FD-GOGAT或NADH-GOGAT）和 谷氨酸脱氢酶（GDH）。 每个酶的基因家族 拟南芥包含独立监管的成员编码不同的成员 同工酶。 尽管在许多人中进行了数十年的体外研究 物种，GS，GoGat和GDH同工酶在植物中的体内作用 仅是猜想。 我们建议进行第一个系统的隔离 植物突变体在GS的每个同工酶，Fd-Gogat， nadh-gogat或gdh，在一个物种中。 我们已经证明了这是可能的 分离拟南芥中有缺陷的拟南芥突变基因 使用无偏生长表型的同工酶筛选。 我们提出了一个 突变后代的详细表征，以量化影响 在诸如原发性氮等过程中失去单个同工酶的损失 种子集中的同化，光呼吸和氮动员。 该分析将定义控制的密钥和限制酶 在植物中使用氮的效率。 另外，我们也可能会发现 调节基因中的突变体。 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受到调节 植物中碳与氮代谢物的比率。 我们建议 遗传筛选以发现该机械的组件，并具有 手两个推定的调节基因。 我们的具体目的是：1）隔离 其他拟南芥GDH突变体和叶绿体GS2中的突变体 同工酶筛选，2）表征现有的拟南芥突变体 FD-GOGAT（GLS1）和分离物突变体的两个基因之一中有缺陷 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阴性亚基，4）测试 代谢控制模型并定义代谢物感官，5）定义 使用遗传筛选的调节途径的组成部分，并定义 候选基因的体内功能。 我们关于 调节拟南芥中氮同化的机制可能具有 对改善农作物中氮的使用的影响不适合这种 分子遗传学研究。 此外，作为代谢信号传导 发生在动物中，对此过程的见解可能更容易获得 在拟南芥中使用分子遗传学策略。