Study On The Regulation Of Exogenous Elicitors On Ganoderma Lucidum Sterol Biosynthesis Pathway And Related Genes

Ganoderma lucidum is a traditional and valuable large medicinal fungus,which has very high medicinal and health care value.Ganoderma lucidum sterols are important active components in Ganoderma lucidum,but the studies on the active components of Ganoderma lucidum are mostly focused on Ganoderma lucidum polysaccharides and triterpenoids.Pharmacological studies have shown that Ganoderma lucidum sterols have outstanding anti-tumor,anti-inflammatory and antiviral activities,which is an important material basis for the efficacy of Ganoderma lucidum.Although many sterols have been isolated and identified from Ganoderma lucidum,the content of sterols is very low,which greatly limits the efficacy of Ganoderma lucidum and the development of new products,and hinders the high-quality development of Ganoderma lucidum industry.increasing the content of sterols in Ganoderma lucidum is of great significance to improve the quality of Ganoderma lucidum and the development of Ganoderma lucidum industry.Therefore,in view of the low content of sterols in Ganoderma lucidum and the low cost of obtaining sterols from cultivated Ganoderma lucidum fruiting bodies and destroying the ecological environment,three exogenous elicitors,salicylate（SA）,sodium nitroprusside（SNP）and hydrogen peroxide（H₂O₂）,were used to improve the sterols in Ganoderma lucidum,and the optimal induction conditions of the three exogenous elicitors were optimized.At the same time,the regulation modes of three exogenous elicitors on genes related to Ganoderma lucidum sterol biosynthesis pathway were investigated to provide theoretical guidance for the preparation of Ganoderma lucidum sterols by industrial large-scale fermentation.The main results of this paper are as follows:The main results are as follows:（1）Different fermentation methods have great influence on the biosynthesis of Ganoderma lucidum sterols.Among the three culture methods of static,oscillatory and oscillatory-static two-stage fermentation,the content of Ganoderma lucidum sterols in oscillatory culture is the highest,which is significantly higher than that in static culture and oscillatory-static two-stage culture.the possible reason is that the dissolved oxygen is different in different culture methods,and the oxygen demand of Ganoderma lucidum sterol fermentation is higher.（2）Exogenous elicitor SA could significantly induce the biosynthesis of Ganoderma lucidum sterols,and the regulation of Ganoderma lucidum sterols induced by SA showed concentration effect and time effect.In the concentration range of 10-200μM,the sterol content of Ganoderma lucidum increased at first and then decreased with the increase of SA concentration.The sterol content of Ganoderma lucidum reached the highest when the concentration of SA was 150μM.The optimization of SA addition time showed that the sterol content of Ganoderma lucidum was the highest when SA was added on the first day of fermentation culture.The effect of SA on gene transcription of key enzymes in Ganoderma lucidum sterol biosynthesis pathway was determined by fluorescence quantitative PCR.The results showed that the response of each gene to SA induction was different under different concentrations and different induction time of SA.EGR11,ERG2 and ERG4 were most significantly up-regulated by SA at the optimal induction concentration of 150um.All genes responded to SA induction at different induction time,but the responses of different genes were different.ERG3 and ERG5 were significantly up-regulated at 12 h after SA induction.ERG2 was significantly up-regulated at 24 h,SQS and LS were significantly up-regulated at 60 h,and ERG4,ERG6 and ERG11 were significantly up-regulated at 72 h.（3）As a signal molecule,NO can induce many physiological and metabolic responses.In this paper,the effect of NO donor sodium nitroprusside（SNP）on the synthesis of Ganoderma lucidum sterols was investigated.The results showed that SNP could significantly increase the content of Ganoderma lucidum sterols.The optimum induction conditions of SNP were obtained as follows:1.5μM SNP,was added on the first day of Ganoderma lucidum fermentation.The induction and regulation modes of key enzyme genes of Ganoderma lucidum sterol synthesis pathway were different in different concentrations and different induction time of SNP.LS and EGR11 were most significantly up-regulated by SNP at the optimal concentration of 1.5μM.The genes of key enzymes in Ganoderma lucidum sterol synthesis pathway were all up-regulated by SA at different induction time.ERG3 was significantly up-regulated at 2 h,ERG5 and ERG6 were significantly up-regulated at 36 h,and ERG4 and ERG11were significantly up-regulated at 60 h.SQS and LS were significantly up-regulated at 72 h after induction.（4）Oxidative stress is an important way to mediate microbial secondary metabolism.The effect of oxidative stress activator H₂O₂on Ganoderma lucidum sterol synthesis was investigated.It was found that H₂O₂could significantly increase Ganoderma lucidum sterol biosynthesis.When the concentration of H₂O₂was 50μM,the content of Ganoderma lucidum sterol reached the highest on the 3rd day of fermentation.The results of fluorescence quantitative PCR showed that the responses of key enzyme genes of Ganoderma lucidum sterol biosynthesis pathway were different under different concentrations and different induction time of H₂O₂.Under the optimal induction concentration of 50μM,EGR11,ERG2 and ERG4 were most significantly up-regulated by H₂O₂,and the responses of key enzyme genes of Ganoderma lucidum sterol synthesis pathway induced by H₂O₂were different at different time.HMGR,ERG2,ERG3,ERG4 and ERG11 were significantly up-regulated at 2h and ERG5 was significantly up-regulated at 24 h.To sum up,three exogenous elicitors SA,SNP（NO）and H₂O₂could significantly induce Ganoderma lucidum sterol biosynthesis,and all showed concentration effect and time effect.At the same time,three kinds of exogenous elicitors can induce and regulate the key enzyme genes of Ganoderma lucidum sterol biosynthesis pathway,but the induction and regulation modes of different elicitors are different and have potential synergistic regulation.