Impairment of Root GS Activity in Alfalfa by Antesense RNA Technology and Its Effect on NO3-Assimilation, Nodulation and N2-Fixation

反义RNA技术对苜蓿根部GS活性的损害及其对NO3同化、结瘤和N2固定的影响

• 批准号: 9220142
• 负责人: Champa Sengupta-Gopalan
• 金额: $ 24.89万
• 依托单位: New Mexico State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9220142&HistoricalAwards=false
• 关键词: Impairment; Root; GS; Activity; Alfalfa

This project is aimed at testing the hypotheses that impairing glutamine synthetase (GS) activity in roots of nitrate fed and N2- fixing alfalfa plants perform better than normal plants. The working hypothesis for the NO3-fed, root GS impaired plants is that the NO3-taken up by the root instead of being distributed between the root and leaves for assimilation is now exclusively assimilated in the leaves. Being energetically more efficient to assimilate NO3-in the photosynthetic tissue, the result is more efficient usage of nitrogen. In the case of the nodulated alfalfa plants, it is postulated that all the photosynthate normally distributed between the roots and the nodules is now directed primarily to the nodules. The increased flow of photosynthate to the nodules provides more energy substrate for the bacteroides to fix N2 and more C-skeleton for assimilating the fixed N2, resulting in increased N2-fixation rates and ammonia assimilation. The experimental approach to impair root GS activity is to use the antisense RNA technology. To specifically target the root GS, antisense gene constructs consisting of either the constitutive 35S promoter and a root GS specific sequence or a root specific/enhanced promoter and a GS coding sequence in antisense orientation have been introduced into alfalfa via Agrobacterium tumefaciens mediated transformation of leaf discs. To ensure that GS activity in nodules is not impaired, root GS impaired alfalfa transformants will be transformed with GS gene sense contructs behind the leghemoglobin gene promoter or the nodule-specific GS gene promoter. Since the effects of increased GS activity is not known, alfalfa plants have also been transformed with GS sense constructs under the control of constitutive and root specific/enhanced promoters. Both molecular and biochemical analysis of the different tissues will be performed to test for changes in expression of genes encoding key enzymes in N- and C- metabolism. Besides allowing us to understand why root GS impaired plants perform better, this analyses will allow us to determine if changes in N-metabolism has any effect on C-metabolism. Availability of root GS impaired plants will provide us with the necessary tools to test our hypothesis with and eventually allow us to manipulate plants for better usage of N and C.

本项目旨在验证硝酸盐饲喂和固氮苜蓿根系受损谷氨酰胺合成酶（GS）活性优于正常植株的假设。对供给no3的根系GS受损植物的工作假设是，no3被根系吸收，而不是在根系和叶片之间分配同化，现在完全被叶片同化了。在光合组织中吸收no3的能量效率更高，其结果是氮的利用效率更高。在结瘤的紫花苜蓿植物的情况下，假定所有正态分布在根和根瘤之间的光合作用现在主要指向根瘤。光合产物流向根瘤的增加为拟杆菌固定N2提供了更多的能量底物，并为吸收固定的N2提供了更多的c -骨架，从而提高了N2固定率和氨同化。利用反义RNA技术破坏根GS活性的实验方法。为了特异性靶向根GS，通过农杆菌介导的叶盘转化，将由35S启动子和根GS特异性序列或根特异性/增强启动子和根GS编码序列组成的反义基因构建体引入苜蓿。为了确保GS在根瘤中的活性不受损，将在根瘤GS受损的苜蓿转化体中加入豆血红蛋白基因启动子或根瘤特异性GS基因启动子后面的GS基因感构合体。由于GS活性增加的影响尚不清楚，在组成型启动子和根特异性/增强启动子的控制下，苜蓿植株也被转化为GS感结构。将对不同组织进行分子和生化分析，以检测编码N-和C-代谢关键酶的基因表达的变化。除了让我们了解为什么根系GS受损的植物表现更好，这个分析将使我们能够确定n代谢的变化是否对c代谢有任何影响。根系GS受损植物的可用性将为我们提供必要的工具来检验我们的假设，并最终使我们能够操纵植物更好地利用N和C。