灵芝酸（GAs）是灵芝的主要药用成分，但因其含量较低，限制了灵芝药用价值的开发。申请者前期发现灵芝硝酸还原酶（NR）能够通过产生NO，调控GAs的生物合成，而这一调控作用的分子机制还不清楚。本项目拟采用基因沉默和过表达的手段，构建NR基因突变型菌株，并结合细胞生物学、分子生物学等手段，明确NR产生的NO对GAs生物合成的调控作用。进而结合前期研究所发现的ROS对GAs生物合成的调控作用，考察在GAs生物合成调控过程中，NO信号途径与ROS信号途径之间的交互机制。并通过转录组测序、荧光定量PCR等方法，探查在调控GAs生物合成过程中，响应NO信号转导途径调控的下游信号途径，进一步分析NO信号途径对GAs生物合成调控作用的分子机制。本研究有助于加深对GAs生物合成调控机制的认识，为生产上提高GAs含量提供理论基础，同时为更深入研究其它重要萜烯类化合物和担子菌其它活性成分的代谢调控提供借鉴。

Ganoderma lucidum is an important medicinal fungi. As the major pharmacological activity compounds from G. lucidum, Ganoderic acids (GAs) content has been taken as an important measurement of the quality of G. lucidum. Our recent results showed that nitrate reductase (NR) might regulate the biosynthesis of GAs by producing nitric oxide (NO). However, the regulatory mechanism of this phenomenon is not clear. For further analysis of this regulatory mechanism, in this project, we construct the mutant strains of NR gene by RNA silence and gene over expression. Combined with the previous results that the regulatory function of ROS signaling pathway in GAs biosynthesis, we will analyze the interaction of NO signaling pathway and ROS signaling pathway in the regulation of GAs biosynthesis. By using laser scanning confocal microscope (LSCM), combined with the application of the NO donors, scavenger and synthetic inhibitors, the temporal of NO content, the transcripts of genes involved in GAs biosynthesis, as well as the changes of GAs contents, will be compared. At the same time, through the transcriptome sequencing and qRT-PCR, we will analyze the relationship among NO signaling, its downstream components and other signal transduction pathway (especially related to ROS). Based on this research, the molecular mechanism of the NO signaling pathway of GAs biosynthesis will be described clearly. These results will not only provide the knowledge of biological roles of NO in G. lucidum, but also provide theoretical basis for the development of other important terpenoid compounds and basidiomycete of other active ingredients to provide reference.