Genetics of Rhizobium-Legume Symbiotic N2 Fixation

本研究的目标是对根瘤菌-豆科植物共生进行分子遗传分析，根瘤菌-豆科共生在世界粮食生产中具有重要的农艺意义。在分子水平上对共生的研究向我们提出了挑战，要求我们理解两种不同的生物如何将它们的基本生化生命过程相互交织在一起，并相互调节负责这些过程的基因。为了实现这一点，作为遗传探针，将通过完美的基因替换，在根瘤菌固定氮基因与E.coliLacZ(编码B-半乳糖苷酶)、uidA(编码B-葡萄糖醛酸酶)或V.facherii lux(编码细菌荧光素酶)基因之间构建翻译的框内基因融合。为了了解在共生和自由生长的细菌生长过程中固氮是如何进行的，使用这些基因工程根瘤菌菌株，将在培养和植物中比较固氮过程的调节。为了了解这些过程是如何受到基因控制的，将分离负责这一调控的基因，并构建这些基因已被改变的根瘤菌突变体，并对这些基因改变所产生的表型进行测试。因为它提供了一种以特定方式操纵基因的强大方法，所以通过噬菌体向任何细菌传递定义的DNA序列的能力将被开发出来。根瘤菌是一种革兰氏阴性细菌，能侵袭豆科植物的根部，促进根瘤的发育。在这种共生关系中，细菌和植物能够通过被称为固氮的过程将大气中的氮(氮素)转化为生物有用的形式。这个项目的目标是了解导致这种共生的基因调控事件。重点将放在分离参与固氮的根瘤菌基因，并确定调节这些基因表达的机制。这是一个经济上重要的研究领域，因为共生固氮是氮肥的有效替代品。

The objectives of this research program are to conduct molecular genetic analyses of the Rhizobium-Legume symbiosis, which, in world food production, is of great agronomic importance. The study of symbioses at the molecular level challenges us to understand how two different organisms intertwine their fundamental biochemical life processes and interregulate the genes responsible for these processes. To accomplish this, as genetic probes, translational, in-frame gene fusions will be constructed between Rhizobium N 2 fixation genes and either E. coli lacZ (encoding B-galactosidase), uidA (encoding B- glucuronidase), or V. fischerii lux (encoding bacterial luciferase) genes by perfect gene replacement. To understand how N 2 fixation is orchestrated during both symbiosis and free- living bacterial growth, using these gene-engineered Rhizobium strains, the regulation of N 2 fixation processes will be compared in culture and in planta. To understand how these processes are genetically controlled, the genes responsible for this regulation, will be isolated defined Rhizobium mutants in which these genes have been altered, will be constructed and the phenotypes resulting from such alterations will be tested. Because it offers a powerful method to manipulate genes in defined ways, the capacity to deliver to any bacterium defined DNA sequences via phage will be exploited. Rhizobium is a genus of gram-negative bacteria which can invade the roots of legumes and stimulate the development of root nodules. ln this symbiotic relationship, the bacteria and plant are able to convert atmospheric nitrogen (dinitrogen) to biologically useful forms by the process known as nitrogen fixation. The goal of this project is to understand the genetic regulatory events that bring about this symbiosis. Emphasis will be placed on isolating the Rhizobium genes that participate in nitrogen fixation and on identifying the mechanisms that regulate the expression of these genes. This is an economically important area of research as symbiotic nitrogen fixation represents an efficient alternative to nitrogen fertilizers.