The Roles Of Glutamine And Nitric Oxide Signaling In Triterpenoids Synthesis Of Ganoderma Lucidum Under Different Nitrogen Sources

Nitrogen is an important element of living organisms.It is involved in the synthesis of proteins,nucleic acids and other nitrogen-containing compounds,and can affect cell growth,metabolism and other life activities.More and more studies in fungi have shown that different nitrogen sources can influence the synthesis of secondary metabolites.As the secondary metabolites of Ganoderma lucidum,triterpenoids have multiple effects,such as anti-tumor,immune regulation and lipid lowering.Researches show that nitrogen source changes affect the synthesis of triterpenoids in G.lucidum,but the mechanism is not clear.The completion of the whole genome sequencing of G.lucidum and the establishment of the genetic transformation system provide new means to study the regulation mechanism of triterpenoids synthesis.The effects of glutamine and nitric oxide(NO)signal on the synthesis of triterpenoids by different nitrogen sources were studied.In this study,the content of triterpenoids in G.lucidum was determined,and the accumulation of triterpenoids showed significant difference under different nitrogen sources.Subsequently,the contents of intracellular free amino acids were detected.The content of glutamine showed a great change,and it was significantly reduced with nitrate cultured.In addition,high concentration of glutamine promoted the synthesis of triterpenoids in G.lucidum cultured with different concentrations of glutamine as a single nitrogen source.By measuring the activities of intracellular glutamine synthesis and degradation enzymes,the activity of glutamine synthetase was significantly increased cultured with ammonium salt,proline and nitrate as a single nitrogen source respectively compared with glutamine.The addition of glutamine synthetase inhibitor MSX could reduce the content of intracellular glutamine,and the accumulation of triterpenoids was also significantly reduced under the condition of ammonium salt,which further indicated that glutamine might be an important regulator,affecting the synthesis of triterpenoids under different nitrogen sources.By constructing gs silenced and overexpressed transformants,it was found that the growth of gs silenced transformants was inhibited,and the content of triterpenoids was also significantly reduced with the ammonium salt cultured;overexpressed transformants showed the opposite effects.The results indicated that glutamine was involved in the regulation of triterpenoids synthesis in G.lucidum under different nitrogen sources.NO is widely involved in a variety of life activities in organisms,and its production is closely related to nitrogen metabolism.The results of NO staining showed that the content of NO in hyphaes increased in different degrees cultured with proline and nitrate as a single nitrogen source respectively compared with ammonium salt.The addition of NO donor significantly increased the intracellular content of NO and inhibited the accumulation of triterpenoids.Further experiments showed that the increase of NO content inhibited the enzyme activity of intracellular GS,and reduced the content of glutamine.The results showed that NO was involved in the regulation of triterpenoids synthesis in G.lucidum by nitrogen sources.At the same time,NO might inhibit the accumulation of triterpenoids by inhibiting GS activity and reducing the intracellular content of glutamine.This paper showed that glutamine and nitric oxide were involved in the regulation of triterpenoids synthesis of G.lucidum under different nitrogen sources cultured.It preliminarily proved the way of which nitrogen sources regulating the triterpenoids synthesis of G.lucidum,providing a basis for further research.