| Ganoderma lucidum is a precious medicinal fungus with pharmacological functions.Ganoderic acids(GAs)is an important active ingredient in G.lucidum.At present,the basic research of G.lucidum is very weak,which limits the development of its industry.Nicotinamide adenine dinucleotide(NAD+)is an important multifunctional molecule and nicotinamide mononucleotide adenylyl transferase(NMNAT)is a key enzyme in the NAD+biosynthesis pathways.Recently,it has been found that NMNAT-mediated production of NAD+could play an important role in regulating metabolisms and signals.However,the functions of NAD+ are rarely reported in basidiomycetes.Firstly,it was found that the NAD+content was increased about 90%,150%,74%and 42%respectively and the gene expression of NMNAT was increased about 283%,442%,133%and 92%respectively in(wild type)WT strains after heat stress(HS),ethylene,methyl jasmonate(MeJA)and salicylic acid(SA)treatment.Meanwhile,the GAs content WT strains was decreased about 30%after 150 μM gallotannin treatment and the GAs content in WT strains was increased about 33%and 42%respectively after 80 μM epigallocatechin gallate and 5 mM NAD+treatment.Then the NMNAT was identified and cloned in G.lucidum,and it was named GINMNAT.The length of the GINMNAT cDNA is 891 bp,and encodes a protein of 296 amino acids with a molecular mass of 32.89 kDa and an isoelectric point of 6.05.Evolutionary relationship analysis found GINMNAT was high identities with the other NMNAT of basidiomycetes,and domain analysis found that there were two highly conserved ATP-binding domains and a conservative tryptophan in GINMNAT.Next,NMNATi5 and NMNATi19 strains whose silencing efficiency of NMNAT were 91%and 86%respectively,and OE::NMNAT4 and OE::NMNAT19 strains whose relative expression of NMNAT approximately were 5.5-fold and 6.2-fold respectively were both constructed.When GINMNAT was silenced,the GAs content was decreased about 30%.When GINMNAT was overexpressed,the GAs content was increased about 40%.Subsequently,it was found that the activity of key enzymes in glycolysis(EMP)and tricarboxylic acid cycle(TCA)and the ATP content were significantly decreased,and the phosphorylation level of AMP-activated protein kinase(AMPK)were significantly increased in NMNAT-slienced strains,while there was a reverse effect in NMNAT-overexpression strains.Futher,the GAs content in NMNAT-slienced strains could recover to WT level after addition of 2 mM AMPK inhibitor treatment and the GAs content in NMNAT-overexpression strains could also recover to WT level after addition of 2 μM AMPK activator treatment.Finally,the NMNAT-AMPK co-silencing strains were constructed in this study and the GAs content in co-silencing strains did not change significantly treated by exogenous NAD+.These results suggest that NMNAT-mediated production of NAD+can regulate the biosynthesis of GAs through AMPK.Finally,it found that the reactive oxygen species(ROS)content and Ca2+concentration were decreased about 40%and 50%respectively in NMNAT-slienced strains,and the reactive oxygen species(ROS)content and Ca2+ concentration were increased about 50%and 50%respectively in NMNAT-overexpression strains.These results suggest NMNAT-mediated production of NAD+can affect intracellular ROS and Ca2+homeostasis.In addition,we found that the activity of carboxymethyl cellulase(CMCase)and pNPCase were decreased about 40%and 70%respectively in the NMNAT-silenced strains,and the activity of CMCase and pNPCase were increased about 60%and 100%respectively in NMNAT-overexpression strains.Futher,the activity of CMCase and pNPCase in NMNAT-slienced strains were significantly increased after addition of 5 mM calcium chloride(CaCl2)treatment,and the activity of CMCase and pNPCase in NMNAT-overexpression strains were significantly decreased after addition of 5 mM lanthanum chloride(LaCl3)treatment.These results suggest that NMNAT-mediated production of NAD+can regulate cellulase activity through Ca2+. |
PDF Full Text Request