Molecular Genetic Control of Nitrate Assimilation

植物、绿藻和一些真菌和细菌的硝酸盐同化是一个复杂的、消耗能量的过程，它提供了生物圈中大部分有机氮。硝酸盐吸收和减少所需的基因受到严格调控，并对各种环境和生理信号作出反应。该研究的长期目标是了解光合生物中参与硝酸盐同化的基因的表达是如何被调节的。本研究使用单细胞绿藻衣藻作为模型实验生物来鉴定和表征介导硝酸盐同化控制的基因。首先，我们将关注硝酸盐同化的产物铵如何抑制硝酸盐同化基因的表达。6个参与硝酸盐同化的基因聚集在染色体的一个区域内。这些基因的表达在铵存在时被协调地阻断，在除去铵时被抑制，在硝酸盐的作用下被诱导到高水平。这些基因的抑制和诱导都需要NIT2基因产物。NIT2基因似乎在介导该途径的正调控和负调控中起关键作用，因为NIT2基因本身被铵抑制。分离衣藻中破坏这种调节的突变体的能力为识别感知和转导铵信号为基因表达模式变化所需的细胞成分提供了有力的工具。在铵存在下组成表达硝酸盐同化基因的突变体迄今已鉴定出两个负调控基因，FARI和FAR2。这两个基因都是铵抑制N1T2基因所必需的。这一建议的第一个具体目标是使基因组充满定义硝酸盐同化负控制所需的反式作用因子的突变。破坏这种调节的突变体将为解决抑制是否发生在基因表达的一个或多个阶段以及通过一个或多个途径提供信息。第二个具体目标是对克隆的FARI基因进行表征，并利用突变体集合分离和表征FAR2基因和本研究中通过转座子标记鉴定的任何其他基因。

Nitrate assimilation by plants, green algae, and some fungi and bacteria, is a complex, energy-consuming process which provides most of the organic nitrogen in the biosphere. The genes required for nitrate uptake and its reduction are tightly regulated, and respond to a variety of environmental and physiological signals. The long-term goal of the proposed research is to understand how the expression of genes involved in nitrate assimilation is regulated in photosynthetic organism. This study uses the unicellular green alga, Chlamydomonas, as a model experimental organism to identify and characterize genes that mediate control of nitrate assimilation. Initially, attention will be focused on understanding how ammonium, the product of nitrate assimilation, represses the expression of nitrate assimilation genes. Six genes involved in nitrate assimilation are clustered within a single region of the chromosome. Expression of these genes is coordinately blocked when ammonium is present, derepressed upon removal of ammonium, and induced to a high level by nitrate. Both derepression and induction of these genes requires the NIT2 gene product. The NIT2 gene appears to play a key role in mediating both positive and negative control of the pathway, since the NIT2 gene itself is repressed by ammonium. The ability to isolate mutants in Chlamydomonas that disrupt this regulation provides a powerful tool for identifying the cellular components required for perceiving and transducing the ammonium signal into a change in the pattern of gene expression. Mutants that express nitrate assimilation genes constitutively in the presence of ammonium have so far identified two negative regulatory genes, FARI and FAR2. Both genes are required for repression of the N1T2 gene by ammonium. The first specific aim of this proposal is to saturate the genome with mutations that define trans-acting factors required for negative control of nitrate assimilation. Mutants that disrupt this regulation will be informative for resolving whether repression occurs at one or more stages of gene expression and by one or more pathways. The second specific aim is to characterize the cloned FARI gene, and to use the collection of mutants to isolate and characterize the FAR2 gene and any additional genes identified in this study by transposon tagging.