Research On The Functions Of Ganoderma Lucidum AMPK Protein Kinase ? Subunit (Glsnf1) In Degradation Of Cellulose And Heat Stress Adaption

Ganoderma lucidum is one of Chinese traditional medicine treasures and belongs to medicinal macro basidiomycete.G.lucidum contains a variety of biological activities,such as anti-tumor,improve immunity and so on.During cultivation,environmental factors such as changes in nutritional conditions and heat stress will affect its normal growth.However,how G.lucidum deals with environmental factors stimulus is still unclear.AMP-activated protein kinase(AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1(SNF1),as a key factor for sensing energy signals in vivo,participates in a variety of biological physiological processes.The functions of G.lucidum AMPKa subunit(Glsnfl)in degradation of cellulose and heat stress adaption were studied.The mainly results are as follows:1.Analysis of the functions of Glsnfl in environmental stress adaption.Firstly,AMPK protein kinase ? subunit was identified in G.lucidum by homology comparison and was named Glsnf1.Then,RNA-mediated gene silencing technology was used to construct Glsnf1-silenced transformants,and two transformants with a silencing efficiency of about 70%were screened for subsequent experiments.Compared with the wild type strains,the growth of hyphae was inhibited and the hyphae spacing increased in the Glsnf1-silenced strains.Chemical stress sensitivity test showed that Glsnf1-silenced strains were more sensitive to the cell wall stress substances calcofluor white(200 ?g/mL),sodium dodecyl sulfate(0.05%)and concomitant red(1 mg/mL).In terms of nutritional stress tolerance,carbon starvation and non-preferred carbon sources significantly increased the phosphorylation level of Glsnf1.Glsnf1 silencing reduced the biomass of G.lucidum and ATP content under carbon starvation.In addition,Glsnf1 silencing weakened the utilization ability of non-preferred carbon sources.Heat stress significantly increased the phosphorylation level of Glsnfl,and Glsnf1 silencing hindered the growth of G.lucidum hyphae under heat stress.2.The regulation of Glsnfl in the utilization of cellulose.The phosphorylation level of Glsnfl increased by about 2.7-fold under the induction of cellulose.Then the AMPK specific activator 5-aminoimidaze-4-carboxamide-1-?-D-ribofuranoside(AICAR)and inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]-pyrimidine(Com C)were used.Results showed that AICAR(2 mM)significantly increased cellulase activity(about 4.0-fold of that in the control strain)and the transcription levels of most cellulase genes.However,Com C(20 ?M)significantly reduced the cellulase activity(with a decrease rate of about 61.8%)and the transcription levels of most cellulase genes.Similarly,silencing of Glsnfl significantly reduced cellulase activity(about 48.3%-59.1%of the WT strain)and transcriptional level of most cellulase genes,while overexpression of Glsnf1 got the opposite results.The transcriptional levels of cellulase key transcription factors were detected.Results showed that the transcriptional levels of carbon catabolite repressor CreA and the corepressor Acel significantly increased in the Glsnf1-silenced strains(increased about 5.0 and 0.8-fold,respectively)and significantly decreased in the Glsnf1-overexpressed strains(decreased by about 69.0%and 19.8%,respectively).Amino acid sequence analysis showed that GlCreA contained AMPKa conserved binding sites.GlCreA-silenced strains and Glsnf1-GlCreA co-silencing strains were further constructed,and it was found that GlcreA had a negative regulation effect on the cellulase activity,and co-silencing Glsnf1 and GlCreA could partially inhibit the decrease of cellulase activity caused by Glsnf1 silencing(cellulase activity decreased by about 59%in the Glsnf1-silenced strains,and decreased by about 25%in the co-silencing strains).Finally,protein level analysis showed that Glsnfl could interact with GlCreA and inhibit the localization of GlCreA in the nucleus.3.The regulation of Glsnfl in heat stress adaption.Firstly,the gene expression and phosphorylation level of Glsnfl increased significantly under heat stress;In addition,the content of hydrogen peroxide(H2O2)increased significantly and the biomass reduced significantly under heat stress.Adding reactive oxygen species(ROS)scavengers N-acetyl-L-cysteine(0.5 mM)and vitamin C(1 mM)under heat stress could partially restore the biomass of G.lucidum.These results indicated that ROS produced under heat stress could inhibit the biomass accumulation of G.lucidum.The content of H2O2 increased significantly and the biomass decreased significantly under Com C(20 ?M)treatment.Similar results were obtained by silencing Glsnf1.Oxygen consumption rate and ATP content were used to measure mitochondrial function,and glycolysis key genes expression and lactic acid content were used to measure glycolysis function.Results showed that the oxygen consumption rate and ATP content firstly increased and then decreased significantly during the extension of heat stress time.The glycolysis key genes expression and the lactic acid content increased significantly during the extension of heat stress time.It suggested that the metabolic rate of anaerobic glycolysis was enhanced under heat stress.Glycolysis was then inhibited by adding 10 mM glycolysis inhibitor 2-deoxidation-D-glucose(2-DG)and silencing glycolytic key genes(fructokinase,hexokinase and pyruvate kinase).Results showed that H2O2 content significantly increased and the biomass decreased significantly after inhibiting glycolysis.In addition,the content of antioxidant substances NADPH and GSH and the expression levels of antioxidant genes decreased significantly after inhibiting glycolysis.It indicated that the increase of anaerobic glycolysis was helpful for G.lucidum to cope with ROS damage.Addition of Com C(20?M)and silencing of Glsnf1 did not change the mitochondrial oxygen consumption rate,but inhibited the accumulation of lactic acid under heat stress and reduced activities of glycolytic key enzymes and the expression of glycolytic key enzyme genes,which indicated that Glsnfl was involved in the enhancement of glycolysis under heat stress.In addition,Glsnfl inhibited the accumulation of triterpenoids in G.lucidum.In summary,Glsnfl responded to environmental stress such as chemical stress,nutritional stress and physical stress,and is a universal stress response protein.Glsnfl enhanced cellulase gene transcription and cellulase activity by inhibiting the transcription of GlCreA gene and its protein distribution in the nucleus.Glsnfl-mediated changes in metabolic flow were involved in the adaptation to heat stress and the accumulation of secondary metabolites.This study is helpful to further broaden and improve the mechanism research of AMPK/SNF1 pathway in fungi,and provides reference for other basidiomycetes to study the functions of AMPK/SNF1 pathway.