RUI: Peroxide Metabolism in Legume Root Nodules

该提案提出了一个实验计划， 生物机制来抵御活性形式的氧气 在大豆根瘤中。 这种防御主要集中在4种酶上， 的抗坏血酸-谷胱甘肽途径，其中H2 O2被清除。 生化研究将集中在谷胱甘肽还原酶。 将对该酶进行纯化并对其氨基进行表征。 酸序，动力学性质，最适pH，缓蚀剂作用， 辅因子要求、底物特异性、pI值和 可能的同工酶 多克隆抗体将用于 对酶进行免疫学表征并确定 亚细胞定位。 拟议工作的一个主要组成部分包括分子 研究，以1）确定调节的生化机制， 编码谷胱甘肽还原酶和抗坏血酸过氧化物酶的基因， 2)描述影响的心理和发育因素 调控 谷胱甘肽还原酶和抗坏血酸的基因 将克隆过氧化物酶以鉴定调控位点， 研究参与表达的顺式和反式作用因子。 的 各种环境因素的调节作用（水分胁迫， 热激和冷激，pO 2）和潜在的生化 效应物（GSH、H2 O2、百草枯、根瘤菌信号）将被 考察 这些研究将涉及对转录本的分析， 在整个根瘤和大豆原生质体中的蛋白质水平 电穿孔的报告基因与启动子区融合。 很长 本学期的目标是评估氧气防御的重要性， 携带反义基因的转基因植物的用途。

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.