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: 3282081
• 负责人: Gloria CORUZZI
• 金额: $ 20.01万
• 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 1996-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282081
• 关键词: DNA binding protein; Leguminoseae; aminoacid biosynthesis; asparagine; autoradiography; cell type; chloroplasts; developmental genetics; gene deletion mutation; gene expression; genetic library; genetic manipulation; genetic promoter element; genetically modified plants; glutamate ammonia ligase; lighting; molecular cloning; nitrogen fixation; nonvisual photosensitivity; nucleic acid probes; nucleic acid sequence; photosynthesis; plant genetics; plant growth /development; plant proteins; radiotracer; reporter genes; transcription factor

The long-term objectives of this proposal ar to understand global control of gene expression in plants. In particular we are investigating the mechanisms which coordinate the expression of genes belonging to integrated amino acid biosynthetic pathways. Insights into this process are beginning to emerge from recent work in our laboratory on the gene families for glutamine synthetase (GS) and asparagine synthetase (AS). The integrated expression of these key enzymes serves to regulate nitrogen assimilation and transport in plants. Our studies have revealed that individual members of the GS and AS gene families encode distinct products which are differentially expressed in particular cell-types, at particular times in development, and in response to environmental signals. Light induces the expression of the nuclear gene for chloroplast GS2 and concomitantly represses the expression of the AS1 gene. The switch from GS2 expression in the light to AS1 expression in the dark is dramatic and has biological significance to nitrogen economy in plants. We propose to elucidate the novel mechanism of light- repressed expression of AS1 with particular attention to its potential as an inducible promoter system. Several models will be tested to determine whether light-activation of GS2 and light- repression of AS1 are mediated by a "repressor/activator" protein or by distinct factors. GS and AS gene expression are also coordinated when nitrogen must be mobilized from nitrogen-rich sources to metabolic sinks. Accordingly, genes for cytosolic GS3A and AS1 are coordinately induced during germination and in nitrogen-fixing root nodules. We propose to identify factors involved in the phloem- specific and developmentally-regulated expression of these genes. Preliminary results suggest that a number of factors interact with the promoters of the GS and AS genes and suggest that the mechanisms underlying the integrated regulation of these genes are complex and multifaceted. The proposed studies on factors which coordinate the regulation of GS and AS genes during development will provide some of the first insights into global control of gene expression in plants. The specific aims of this proposal are to: 1) Define Light Regulatory Elements (LREs) involved in activation of GS2 expression and/or repression (nLRE) of AS1 expression by light using a reporter gene system in transgenic plants. We will elucidate the novel mechanism of light-repressed gene expression and develop AS1 for use as a "dark" inducible promoter system in plants. 2) Determine the nature of unique binding activities in "light" and "dark" extracts detected with cis-elements of the GS2 and AS1 promoters. 3) Identify DNA sequences involved in phloem-specific and developmentally regulated expression of GS3A and AS1 in cotyledons and nitrogen- fixing nodules. 4) Detect phloem-specific DNA binding activities utilizing a novel purification procedure. 5) Characterize cDNA clones encoding DNA binding protein factors and determine the nature of factor:factor interactions. 6) Test ability of DNA-binding factor to activate or repress transcription in vitro. 7) Determine cell- specific and in vivo regulation of cloned factor. 8) Test models for integrated GS and AS regulation by ectopic expression of factor(s) in transgenic plants harboring GS-GUS and AS-GUS transgenes. 9) Determine whether ectopic expression of factor modulates endogenous GS and AS gene expression and/or affects nitrogen assimilation.

该提议的长期目标是了解全球 控制植物中基因表达。 特别是我们是 研究协调基因表达的机制 属于综合氨基酸生物合成途径。 见解 进入这一过程的开始是从我们的最新工作中出现的 谷氨酰胺合成酶（GS）基因家族的实验室和 天冬酰胺合成酶（AS）。 这些键的综合表达 酶用于调节氮同化和转运 植物。 我们的研究表明，GS的个别成员 并且随着基因家族的编码不同的产品 在特定细胞类型中差异表达 开发时期以及对环境信号的响应。 光诱导核成形体GS2的核基因的表达 并同时抑制AS1基因的表达。 这 从光中的GS2表达转换为黑暗中的AS1表达 具有戏剧性，对氮经济具有生物学意义 植物。 我们建议阐明光的新机理 抑制AS1的表达，特别注意其 作为诱导启动子系统的潜力。 几种型号将是 经过测试以确定GS2和光的光激活是否是否 AS1的抑制是由“抑制剂/激活剂”蛋白介导的 通过不同的因素。 GS和基因表达也是协调的 当必须从富含氮的来源动员氮时 代谢水槽。 因此，胞质GS3A和AS1的基因是 在发芽期间和固氮根中协调诱导 结节。 我们建议确定涉及韧皮部的因素 - 这些基因的特定和发育调节的表达。 初步结果表明，许多因素与 GS的启动子和作为基因，并建议该机制 这些基因的综合调控是复杂的，并且 多方面。 提出的关于协调因素的研究 GS的调节和开发过程中的基因将提供一些 对植物中基因表达的全球控制的首次见解。 该提议的具体目的是：1）定义光 参与GS2表达激活的调节元件（LRE） 和/或抑制（NLRE）AS1表达的光线表达使用者 转基因植物中的基因系统。 我们将阐明小说 浅色抑制基因表达和开发AS1的机理 作为植物中的“黑暗”诱导启动子系统。 2）确定 “光”和“黑暗”提取物中独特的结合活动的性质 用GS2和AS1启动子的顺式元素检测。 3）识别 参与韧皮部特异性和发育的DNA序列 在子叶和氮中，GS3A和AS1的调节表达 固定结节。 4）检测韧皮部特异性DNA结合活性 利用一种新颖的纯化程序。 5）表征cDNA 编码DNA结合蛋白因子并确定性质的克隆 因素：因子相互作用。 6）DNA结合因子的测试能力 在体外激活或抑制转录。 7）确定细胞 - 克隆因子的特异性和体内调节。 8）测试模型 综合GS并通过异位表达因子的调节 具有GS-GUS和AS-GUS转基因的转基因植物。 9） 确定因子的异位表达是否调节内源性 GS和基因表达和/或影响氮同化。