一氧化氮通过酪氨酸硝基化修饰调控灵芝三萜合成的分子机制

Ganoderic acid is one of the important medicinal ingredients of Ganoderma lucidum, the contents of which determine the medicinal value of G. lucidum. Previous studies have found that different nitrogen sources could affect intracellular NO content, while NO is the key to inhibit the biosynthesis of ganoderic acid. However, the detailed mechanism is not clear. Further, the intracellular NO content was positively correlated with the level of tyrosine nitration, and multiple tyrosine nitration sites were found in the functional proteins in G. lucidum. Therefore, this project intends to further explore the downstream proteins that can affect the biosynthesis of ganoderic acid by nitration modification. Firstly, it is important to confirm the regulatory proteins affected by nitration, which have been confirmed to have functions in ganoderic acid biosynthesis. Secondly, screen the unknown functional proteins that can be nitrated by NO through nitroproteome analysis. Then the effect of NO on the activity and nitration of the selected proteins will be confirmed. Finally, 3-5 nitrated modified target proteins were identified and the mutant strains of the related proteins will be constructed to confirm their functions on regulating the biosynthesis of ganoderic acid. These results will not only clarify the important roles of proteins regulated by tyrosine nitration modification in biosynthesis of ganoderic acid, but also provide target genes for improving the content of ganoderic acid by genetic engineering techniques. Meanwhile, it will also provide the new understanding and studying methods for the NO signal transduction involved in the secondary metabolism in G. lucidum.

灵芝三萜是灵芝的重要药用成分之一，其含量的多少决定了灵芝品质的高低。申请者前期研究发现氮源通过影响胞内NO含量调控灵芝三萜的生物合成，但是NO对灵芝三萜合成的调控机制并不清晰；进一步研究发现灵芝胞内NO含量与酪氨酸硝基化水平正相关，同时在灵芝三萜合成相关的关键调控蛋白中预测出硝基化位点。因此，本项目拟在此基础上进一步深入筛选NO通过酪氨酸硝基化修饰影响灵芝三萜生物合成的靶蛋白。首先明确NO通过硝基化修饰调控的、已知与灵芝三萜生物合成相关的调控蛋白；其次通过硝基化蛋白质组学筛选能被NO硝基化修饰调控的未知的功能蛋白；最终确定3-5个目标蛋白，构建相关蛋白的基因突变菌株，验证目标蛋白在灵芝三萜合成中的作用。研究结果不仅明确了NO通过酪氨酸硝基化修饰调控灵芝三萜生物合成的分子机制，为通过基因工程技术手段提高灵芝三萜含量提供靶基因，同时也为NO信号转导途径参与灵芝次级代谢调控提供新的研究方向。

灵芝三萜是灵芝的重要药用成分之一，其含量的多少决定了灵芝品质的高低。氮源能够显著影响灵芝三萜的生物合成，但是其调控机制不清晰。申请者前期研究发现氮源能够通过调控NO含量调控灵芝三萜的生物合成。因此，本项目在此基础上，（1）首先发现灵芝谷氨酰胺合成酶不仅能够被氮代谢关键转录因子AreA激活，而且也能被NO硝基化修饰。通过构谷氨酰胺合成酶蛋白的基因沉默菌株发现，谷氨酰胺合成酶活性的抑制能够显著影响灵芝三萜的生物合成。此外，NO能够通过硝基化修饰抑制谷氨酰胺合成酶活性，进而负调控灵芝三萜的生物合成。（2）其次发现了参与氮代谢的重要代谢酶——谷草转氨酶，能够通过影响胞内NADH含量以及抗氧化酶相关基因的酶活，影响线粒体ROS含量，进而调控灵芝三萜的生物合成。研究结果不仅明确了NO通过酪氨酸硝基化修饰调控灵芝三萜生物合成的分子机制，也明确了参与氮代谢的关键酶基因在氮源调控中的重要作用。该研究为通过基因工程技术手段提高灵芝三萜含量提供靶基因，也为氮源调控灵芝次级代谢调控提供新的研究方向。