| Salicylic acid(SA),also known as phthalic acid,as an immune signal molecule,it has been widely studied in plant stress.In recent years,it has been found that exogenous addition of SA affects plant growth and metabolism.As a large medicinal fungus,Ganoderma lucidum has high nutritional value and health care value.It has a long history in China.Ganoderic Acid as one of the secondary metabolites of G.lucidum,with antioxidant,antiviral and so on.It has been reported that SA can induce secondary metabolites in plants,however,there is no report in fungal.In this study,G lucidum was used as the research material,exogenous SA significantly induced GA content,and we also found that in this process ROS was involved.G lucidum was treated with NADPH oxidase inhibitors of diphenyleneiodonium(DPI)and used NOX silence transformants,and then subjected to SA treatment.The content of H2O2 was determined,the source of ROS was preliminary analysed,and then determination of mitochondrial respiration rate by Clark oxygen electrode,and also the activity of mitochondrial complex ? enzymes was measured,further analysis found that another source of ROS induced by SA was from mitochondrial.Achieved the following main conclusions:1.Exogenous SA could significantly induce the biosynthesis of GA,it was found that SA induced GA biosynthesis depended on dose time course.By choosing the optimum point based on response surface methodology,the GA content could be increased up to 229.03?g/100 mg,which was improved 66.38%compared with the control group.We also found that the transcription levels of key genes in the GA biosynthesis pathway—squalene(SQ)synthase(sqs),lanosterol(osc),and hydroxy-3-methylglutaryl-coenzyme A reductase(hmgr)—were induced and the content of lano and SQ were induced.2.Exogenous SA induced intracellular ROS accumulation,at the same time,we found that SA could induce partial enzymes related of ROS and increase the expression of related genes,the content of GA and ROS can be reduced by ROS scavenger,effectively.At the same time,the transcription levels of key genes sqs,osc,hmgr and the content of lano and SQ were induced by ROS,that is to say,the synthesis of GA regulated by ROS signal induced by SA.3.To explore the source of ROS,NADPH oxidase and NOX silence transformants were used,found that the content of ROS was decreased about 25-30%under SA induced,and the similar to GA content.The related of ROS enzyme activity and Key genes in ROS system induced by SA were relieved under NOX inhibitor.That is to say,Nox is involved in SA induced ROS production and plays a role in SA induced phenotypes.4.Further study on the source of ROS induced by SA,Mitochondrial ROS increased significantly,it is speculated that S A may have an effect on mitochondrial respiratory chain,study on the effects of mitochondrial inhibitors on GA content and effects of different treatment conditions on respiration rate of mitochondria which isolated from G lucidum by Clark oxygen electrode.The activity of mitochondrial complex ? was measured,found that the activity of mitochondrial complex ? was inhibited about 45-50%under SA treatment and the inhibition can not released by ROS scavenger.At the same time,the transcription of mitochondrial complex ? was measured,the similar trend as it activity.So,another source of ROS is from mitochondria,because the activity of mitochondrial complex ? was inhibited.In summary:SA induced ROS signaling is involved in the biosynthesis of GA,the NOX is one of the sources of ROS,SA through the inhibition of mitochondrial complex ?enzyme activity,causing mitochondrial ROS production,is also a source of ROS production.The results of this study provide a theoretical basis for the further study that is SA induced ROS signaling involved in the biosynthesis of GA. |
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