Molecular Control of Ammonia Assimilation in Alfalfa Root Nodules

这项计划中的研究将集中在寄主植物调节磷酸烯醇式丙酮酸羧基酶和NADH-谷氨酸合成酶的生化和分子机制，这两种酶是紫花苜蓿(Medicago sativa L.)同化共生固定氮的关键酶。根瘤。这项提议基于两个假设。第一种是根瘤PEPC和NADH-GOGAT基因的表达受顺式作用元件(S)的调控，这些顺式作用元件与一个或多个反式作用因子相互作用，这些因子的活性是以下一个或多个因素的函数：根瘤器官发生、有效细菌、铵离子有效性和/或氧浓度。二是根瘤PEPC和/或NADH-GOGAT酶活性的直接遗传变化对根瘤固氮和氨同化的影响。具体目标包括：1)PEPC和NADH-GOGAT基因组克隆的分离和序列分析，并通过缺失分析和蛋白质-DNA结合研究初步鉴定这些基因的启动子区域：2)通过免疫胶体金染色和原位杂交定位PEPC和NADH-GOGAT蛋白和mRNAs；(3)通过表达转化基因直接修饰酶的合成，改变PEPC和NADH-GOGAT的活性。从这些研究中获得的知识将加强我们对参与氨同化的植物基因的基本了解，并可能通过制定改变氮代谢的策略来促进作物改良。

The research in this proposal will focus on host plant biochemical and molecular mechanisms that regulate phosphoenolpyruvate carboxylase and NADH-glutamate synthase, key enzymes involved in assimilation of symbiotically fixed nitrogen in alfalfa (Medicago sativa L.) root nodules. This proposal is based on two hypotheses. The first is that expression of the nodule PEPC and NADH-GOGAT genes is regulated by cis-acting element(s) that interact with one or more trans-acting factors and the activity of these factors is a function of one or more of the following: nodule organogenesis, effective bacteroids, ammonium ion availability and/or oxygen concentration. The second is that direct genetic alteration of nodule PEPC and/or NADH-GOGAT enzyme activities impacts upon nodule nitrogen fixation and ammonia assimilation. The specific objectives include: 1) isolation and sequence analysis of PEPC and NADH- GOGAT genomic clones and initial characterization of the promoter regions of these gene by deletion analysis and by protein-DNA binding studies: 2) localization in planta of PEPC and NADH- GOGAT proteins and mRNAs by immunogold staining and by in situ hybridization; and 3) alteration in planta of PEPC and NADH- GOGAT activities through direct modification of enzyme synthesis by the expression of transformed genes. Knowledge gained from these studies will enhance our fundamental understanding of plant genes involved in ammonia assimilation and may facilitate crop improvement through the development of strategies to modify nitrogen metabolism.