The Mechanism Research Of Nadph Dehydrogenase Regulates The Energy And Fatty Acid Metabolism And Influences The Formation Of Fruiting Body In Filamentous Fungus Podospora Anserina

NADPH dehydrogenase is a flavin protein involved in electron transport of mitochondrial respiratory chain.It can regulate the concentration of NADP(H),a key component in redox metabolism,to affect the thermodynamic driving force of many reactions in vivo,and participates in many important physiological,biochemical and energy metabolism processes.The formation of fruiting body involves a variety of complex mechanisms such as external signal induction and intracellular signal transduction,and is closely related to the transformation of intracellular material energy metabolism.However,the current research between NADPH dehydrogenase and fruiting body formation is relatively rare.In order to better understand the function of NADPH dehydrogenase and its mechanism during the growth and development of filamentous fungi,we used homologous recombination to knock out three NADPH dehydrogenase genes Pa?1?9760,Pa?4?60,and Pa?6?6330 in the Ascomycota fungus Podospora anserina,and obtained three NADPH dehydrogenase single mutants nph1^?,nph2^?,and nph3^?.The double mutants nph1^?nph2^?,nph1^?nph3^?,nph2^?nph3^?and the triple mutant nph1^?nph2^?nph3^?(nph^???)were constructed by genetic hybridization of heteronuclear hyphae.Subsequently,wild-type strain and the mutant strains were compared and analyzed in terms of enzyme activity measurement,growth observation,fruit body development,antioxidant capacity,mycelial reduction capacity,central metabolic pathway and fatty acid metabolism.The following results were obtained from the experiment:(1)Evolutionary analysis showed that the protein sequences encoded by the three putative NADPH dehydrogenase genes were closely related to NADPH dehydrogenase in fungi.In addition,the activity of NADPH dehydrogenase decreased in all mutant strains,and the most obvious was nph^???strain,which decreased by 28%compared with the wild type.The results showed that these three genes do encode NADPH dehydrogenase.(2)There was no significant difference in nutrition growth,colony size,pigmentation and cellulose degradation among NADPH dehydrogenase gene-deficient strains and wild type strain.However,the number of fruit body of NADPH dehydrogenase gene-deficient strains increased.The fruit body number of strain nph^???increased by nearly 35%,and it was more sensitive to oxidative stress,indicating that NADPH dehydrogenase has important physiological functions in P.anserina.(3)The results of fluorescent quantitative PCR(q PCR)showed that the expression of complex CIV in respiratory chain of nph^???decreased,while the expression of key genes in PPP and TCA cycle(such as glucose 6-phosphate dehydrogenase and malic enzyme)were significantly up-regulated,indicating that the increase of substrate-level phosphorylation could make up for the decrease of oxidative phosphorylation caused by NADPH dehydrogenase gene deletion.(4)NADPH/NADP⁺ratio which is the driving force of the biosynthesis was increased in NADPH dehydrogenase gene-deficient strains compared with that of wild type,especially nph^???increased by nearly twice,indicating that the reducing power in NADPH dehydrogenase gene-deficient strain was increased.(5)The up-regulation of fatty acid synthesis-related genes such as fatty acid synthase(FAS)and the down-regulation of fatty acid degradation-related genes such as acyl-Co A dehydrogenase indicated that the fatty acid metabolism pathway in mutant nph^???was more inclined to fatty acid synthesis and inhibited fatty acid degradation.And the growth of nph^???strain on oleic acid medium was inhibited,which further proved the metabolic reprogramming in NADPH dehydrogenase gene-deficient strains.The above results showed that the NADPH dehydrogenase gene-deficient strains through increased substrate level phosphorylation to compensated for the damage caused by oxidative phosphorylation,and used more energy and substances for the formation of fruiting body,which is a protective mechanism in P.anserina.Our research not only proved that NADPH dehydrogenase has a significant role in regulating related gene expression,energy metabolism balance and fruiting body formation in P.anserina,but also provided genetic evidence for the potential regulatory mechanisms of NADPH dehydrogenase to participate in intracellular oxidative defense.