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

这个项目的目的是验证这样的假说，即损害硝酸盐供应和固氮的紫花苜蓿植株根部谷氨酰胺合成酶(GS)活性的表现优于正常植株。对于硝酸营养、根GS受损植物的工作假说是，被根吸收的硝酸根现在只在叶中被同化，而不是分配在根和叶之间进行同化。在能量上更有效地吸收光合作用组织中的NO3-，结果是更有效地利用氮。就结瘤紫花苜蓿而言，据推测，所有正常分布在根部和根瘤之间的光合作用现在主要指向根瘤。光合作用产物流向根瘤的增加为类杆菌固定氮气提供了更多的能量底物，并为吸收固定的氮气提供了更多的C骨架，从而提高了氮气固定速率和氨的同化。利用反义RNA技术是降低根系GS活性的实验方法。通过根癌农杆菌介导的叶盘转化，将含有35S启动子和根GS特异序列或根特异/增强型启动子和反义GS编码序列的反义基因载体导入紫花苜蓿，获得了针对根GS的反义基因。为了确保根瘤中GS活性不受损害，将GS基因正义结构置于豆红蛋白基因启动子或根瘤特异性GS基因启动子后面，转化根GS受损的紫花苜蓿。由于GS活性增加的影响尚不清楚，紫花苜蓿也已在结构性启动子和根特异/增强启动子的控制下，将GS正义结构转化到紫花苜蓿植株中。将对不同组织进行分子和生化分析，以测试编码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，这一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GS受损植物的可用性将为我们提供必要的工具来验证我们的假设，并最终允许我们操纵植物以更好地利用N和C。

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.