Research On The Mechanisms Of Ornithine Decarboxylase Antizyme Regulating Intracellular Polyamines Content And Secondary Metabolism In Ganoderma Lucidum

Polyamines(PAs)mainly include putrescine(Put),spermidine(Spd)and spermine(Spm).The primary PAs—Put in fungi is formed by the decarboxylation of ornithine through ornithine decarboxylase(ODC).In organisms,PAs have a variety of biological functions and play an important role in the growth and development.Therefore,PAs homeostasis are involved in physiological metabolism.Triterpenoids of Ganoderma lucidum(also known as ganoderic acids,GAs),as a secondary metabolite of G.lucidum,are also the important medicinal components and have values of scientific research or medical treatment.Our previous studies showed that PAs could affect the synthesis of GAs in G.lucidum.Therefore,we further explored the function of ornithine decarboxylase antienzyme(AZ),a major regulator of PAs system,in intracellular PAs homeostasis and secondary metabolism of G.lucidum.Firstly,we identified and cloned homologous genes of AZ in the whole genome of G.lucidum and named it GIAZ.The gene is 1000 bp in length,which matches the number of nucleic acid sequences of AZ-specific 3n+1.Further,evolutionary tree and domain alignment with other species revealed that GlAZ was closer to the AZ protein of basidiomycetes.And GlAZ also had conserved dipeptides,nascent signaling peptides and AZ functional domains.Meanwhile,to better detecting the protein levels of intracellular functional AZ,we constructed a GlAZ protein heterologous expression vector.Then GlAZ protein was expressed and purified.The Anti-GIAZ rabbit polyclonal antibody was prepared and used for subsequent experiments.Secondly,we uesd three systems to verify protein interaction,including yeast bihybrid,co-IP and bimolecular fluorescence complementation(BIFC).These results show that GlAZ could interact with GlODC.Then,we constructed the silencing mutant strains and overexpression strains of GlAZ.GlODC protein level in the GlAZi strains were significantly higher than that in the wild type(WT)strains.While the GlODC was reduced in the GlAZ overexpression strains than that in the WT strains.The results show that GlAZ could make GlODC degradation by interacting with GlODC.Then,we performed the assay with Put by HPLC.In the assay,compared with the WT strains,the Put content in the GlAZi strains increased by about 40%,while the Put content in the GlAZ overexpressed strains decreased by about 63%.Moreover,after adding the ODC inhibitor DFMO in the GlAZi strains,the intracellular Put content was lower than that in the untreated GlAZi strains.These results indicate that GlAZi could affect the intracellular Put content by interacting with GlODC.In addition,we also found that GAs levels in GlAZi strains respectively decreased by 33%and 38%compared with the GAs levels of the WT strains.After exogenous addition of DFMO in GlAZii strains,intracellular GAs returned to the levels of the WT strains almost.In GlAZ overexpression strains,GAs content increased by 70%and 41%compared with the WT strains’,while GAs content in the overexpression strains with exogenous Put were lower than untreated overexpression strains.Except the effect on secondary metabolism,the expression level of GlAZ also affects the mycelia growth and the accumulation of biomass in G.luciduum.These results support the idea that that GlAZ might affect the secondary metabolism of G.lucidum by regulating GlODC/Put levels.Finally,we preliminarily explored AZ upstream and focused on target of rapamycin(TOR).GlAZ1 levels are determined by western blot.Compared with untreated WT strains,GlAZ increased in G.lucidum TORi strains and the WT strains treated with TOR inhibitor.Further,inhibiting ribosomal S6 protein(S6K)of the TOR downstream,which regulates ribosome translation,could also increase the GlAZ significantly.These results indicate that G.lucidum TOR(GlTOR)might inhibit translational regulation of GlAZ through S6K.By detecting the Put content,it was found that the Put content in the strains with GlTOR inhibition was 55%lower than that in the WT strains.After that,we constructed the GlTORGlAZ co-silencing strains and found that the Put content in the co-silencing strains were same as the WT strains’.In comparison,Put content reduced in the single-silent GITOR strains.Moreover,the content of GAs in the GlTOR-GlAZ co-silencing strains were similar as that in the WT strains,while the content of GAs in the single-silent GITOR strain were on rise.These results indicate that GlAZ is affected by the upstream regulator GITOR,which changes the intracellular Put level and the anabolism of GAs.In summary,GlAZ not only could play a role in regulating the intracellular Put content of G.lucidum through interaction with GlODC,but also could affect the synthesis of intracellular secondary metabolites.Moreover,the preliminary study on the upstream regulatory factors of GlAZ showed that GlAZ was regulated by the upstream regulator GlTOR.Then GlAZ affects the intracellular Put level and the anabolism of GAs.This study maybe helpful to broaden the cognition of the PAs regulation systems,which also being beneficial for the study on the relationship between PAs and secondary metabolism in fungi.