RUI: Peroxide Metabolism in Legume Root Nodules

RUI：豆科植物根瘤中的过氧化物代谢

• 批准号: 9206453
• 负责人: David Dalton
• 金额: $ 20.36万
• 依托单位: Reed College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1996-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9206453&HistoricalAwards=false
• 关键词: RUI; Peroxide; Metabolism; Legume; Root

This proposal presents an experimental plan to investigate biological mechanisms to defend against activated forms of oxygen in soybean root nodules. This defense is centered on the 4 enzymes of the ascorbate-glutathione pathway in wich H2O2 is scavenged. Biochemical studies will concentrate on glutathione reductase. This enzyme will be purified and characterized with regard to amino acid sequence, kinetic properties, pH optimum, inhibitor effects, cofactor requirements, substrate specificity, pI values, and possible isozymes. Polyclonal antibodies will be used to immunologically characterize the enzyme and to determine the subcellular location with electron microscopy. A major component of the proposed work consists of molecular studies to 1) identify the biochemical mechanisms that regulate the genes encoding glutathione reductase and ascorbate peroxidase and 2) describe environomental and developmental factors that affect regulation. The genes for glutathione reductase and ascorbate peroxidase will be cloned in order to identify regulatory sites and study cis- and trans-acting factors involved in express. The regulatory role of various environmental factors (water stress, heat and chilling shock, pO2) and of potential biochemical effectors (GSH, H2O2, paraquat, rhizobiol signals) will be examined. These studies will involved analysis of transcript and protein levels in whole nodules and in soybean protoplasts bearing electroporated reporter genes fused to promoter regions. A long term goal is to assess the criticality of oxygen defense through use of transgenic plants bearing antisense genes.

本文提出了一项实验计划，以研究大豆根瘤中活性氧的生物防御机制。这种防御集中在抗坏血酸-谷胱甘肽途径的4种酶上，在这些酶中H2O2被清除。生化研究将集中于谷胱甘肽还原酶。将对该酶进行纯化，并对其氨基酸序列、动力学特性、最佳pH值、抑制剂作用、辅因子需求、底物特异性、pI值和可能的同工酶进行表征。多克隆抗体将用于酶的免疫学表征，并通过电子显微镜确定亚细胞位置。这项工作的主要组成部分包括分子研究：1)确定调节编码谷胱甘肽还原酶和抗坏血酸过氧化物酶基因的生化机制；2)描述影响调节的环境和发育因素。克隆谷胱甘肽还原酶和抗坏血酸过氧化物酶基因，以确定调控位点，研究参与表达的顺式和反式因子。各种环境因子（水分胁迫、冷热冲击、pO2）和潜在生化效应因子（谷胱甘肽、H2O2、百草枯、根瘤素信号）的调控作用将被研究。这些研究将包括分析整个根瘤和携带电穿孔报告基因融合到启动子区域的大豆原生质体的转录物和蛋白质水平。一个长期的目标是通过使用带有反义基因的转基因植物来评估氧气防御的重要性。