RUI: Peroxide Metabolism in Legume Root Nodules

这项建议提出了一项实验计划，以研究大豆根瘤中防御活化形式氧的生物学机制。这种防御集中在清除过氧化氢的抗坏血酸-谷胱甘肽途径的4个酶上。生化研究将集中在谷胱甘肽还原酶上。该酶将被纯化，并从氨基酸序列、动力学性质、最适pH、抑制作用、辅因子需求、底物专一性、等电点和可能的同工酶等方面进行鉴定。多克隆抗体将被用来对酶进行免疫学表征，并用电子显微镜确定亚细胞位置。拟议工作的一个主要组成部分是分子研究，以1)确定调节谷胱甘肽还原酶和抗坏血酸过氧化物酶基因的生化机制，以及2)描述影响调节的环境和发育因素。谷胱甘肽还原酶和抗坏血酸过氧化物酶的基因将被克隆，以确定调控位点，并研究参与表达的顺式和反式作用因子。将研究各种环境因素(水分胁迫、热激和冷激、PO2)以及潜在的生化效应因子(GSH、H_2O_2、百草枯、根瘤酚信号)的调节作用。这些研究将包括分析整个根瘤和含有融合到启动子区域的电穿孔报告基因的大豆原生质体中的转录本和蛋白质水平。一个长期目标是通过使用携带反义基因的转基因植物来评估氧气防御的临界性。

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.