Identification And Characterization Of A Glutamate Dehydrogenase Coding Gene(PoGDH)from Pleurotus Ostreatus

Pleurotus ostreatus is the most widely cultivated and consumed edible fungi in China,the yield and quality of which are significantly affected by many nutrients including nitrogen source.Even through nitrogen is an essential macronutrient required for the growth and development of P.ostreatus,the molecular mechanism of nitrogen metabolism still remains to be elucidated.As reported,the glutamate dehydrogenase(GDH),catalyzing reversible transformation of glutamate to ammonium and α-ketoglutarate,plays a crucial role in coupling carbon and nitrogen metabolism in organism and overexpression of GDH gene can improve the abilities of nitrogen assimilation and stress tolerance of organism.PLEOSDRAFT 40761 gene,the GO functional annotation of which is "glutamate dehydrogenase 2"in P.ostreatus PC 15 genome,was obtained through genome-wide analysis and designated as PoGDH in this study.In order to further elucidate the function and the role in nitrogen assimilation of PoGDH gene,the P.ostreatus New 831 strain,widely cultivated in Henan province,was chosen as the experimental material.Firstly,the full-length genomic DNA(gDNA)and coding sequence(CDS)were amplified,sequenced,and analyzed by bioinformatics.Based on that,the PoGDH protein was inducibly expressed in Escherichia coli by using pCold-TFV as expression vector and purified,and then the purified PoGDH was subjected to analysis of enzymatic properties and native molecule determination.Meanwhile,the PoGDH protein was also heterologously expressed by using the Saccharomyces cerevisiae INVScl(pYES2/NT A)system,and then the subcellular localization and enzyme activity were analyzed.Lastly,the spatiotemporal expression patterns of PoGDH gene,and the effects of different nitrogen forms and treatment period on PoGDH gene expression were measured.The main results of this study were as follows.1.The full-length of gDNA(4,209 bp)and CDS(3,135 bp)fragment of PoGDH geac from P.ostreatus New 831 were successfully obtained by separately using the genome DNA and cDNA as templates.The bioinformatics analysis showed that PoGDH gene was composed of 20 exons,19 introns and 1 3’ UTR.And the deduced PoGDH protein sequence contained 1,044 amino acids,which shared high similarity with NAD[H]-dependent glutamate dehydrogenase(NAD[H]-GDH)from other organisms.The molecular weight(Mw)and pI of PoGDH protein were 117 kDa and 6.11,respectively.Similar to NAD[H]-GDH protein from other organisms,the spatial structure of PoGDH protein was homohexamer as well.2.The soluble TFV-PGDH fusion protein was successfully expressed by using the cold-shock vector pCold-TFV.After purified by nickel affinity chromatography,the TFV-PoGDH protein was applied to enzymatic analysis.The optimal conditions for inducing TFV-PoGDH protein were induced with 0.3 mM IPTG at 16℃ for 12 h.And SDS-PAGE analysis revealed that the Mw of TFV-PoGDH protein monomer is about 170 kDa as expected.After cleaved by TEV protease,the obtained PoGDH protein was subjected to SDS-PAFE and Native-PAGE analysis,and the results showed that the Mw of monomer and polymer of PoGDH were separately 117 kDa and 700 kDa,which indicates that the native PoGDH is homohexamer as expected.The optimal coenzyme of TFV-PoGDH was determined by separately using NAD+,NADH,NADP+,and NADPH as coenzymes,the results revealed that NADH was the optimal coenzyme of PoGDH.And the optimal reaction temperature and pH of TFV-PoGDH were 30℃ and 7.5,respectively.The thermal stability experiments exhibited that TFV-PoGDH protein was stable at temperatures lower than 40℃.The Michaelis constants(Km)of TFV-PoGDH for ammonium,α-ketoglutarate and glutamate were separately 2.953 mg/mL,0.184 mg/mL and 0.318 mg/mL,which indicates that PoGDH has higher affinity to α-ketoglutarate and mainly catalyzes the ammoniation reaction of α-ketoglutarate to synthesize glutamate.3.In order to determine the subcellular localization of PoGDH protein,the PoGDH-gfp fusion gene was constructed.Then the PoGDH-GFP fusion protein was inducibly expressed in S.cerevisiae,and the glutamate dehydrogenase activity measurement and fluorescence microscopic observation were conducted.SDS-PAGE analysis showed that a target band of about 144 kDa was obtained in the recombinant yeast strain,which indicated that the PoGDH-GFP fusion protein was successfully expressed.And the recombinant yeast strain had 15-fold higher glutamate dehydrogenase activity than the control ones.The result of fluorescence microscopic observation showed that green fluorescence was mainly visualized in the cytoplasm,which indicated that PoGDH protein was localized in the cytoplasm.4.The results of the differential spatiotemporal expression of PoGDH gene in P.ostreatus showed that PoGDH gene was expressed at all developmental stages and in all parts of fruiting body.The expression level of PoGDH gene was gradually increased along with the growth and development of P.ostreatus,and the highest expression level was observed at the stage of the mature fruiting body.The expression level of PoGDH gene was significantly different among different fruiting body parts,which was highest in stipe,then was in pileus,and lowest in gill.Thus,PoGDH gene may play different physiological roles at different developmental stage and in different parts of fruiting body.5.The differential expression patterns of PoGDH gene responding to different nitrogen forms and treatment periods were determined in this study.PoGDH gene was differentially expressed when the mycelia were treated with different forms of nitrogen for different periods.Under nitrogen starvation,the expression levels of PoGDH gene showed the trends of first increasing and then falling with the prolongation of nitrogen starvation,and the highest expression lever was obtained at 30 min.The effects of different forms of nitrogen source on the expression pattern of PoGDH gene were different.No obvious differential expression patterns were observed when treated with 10 mM glutamine or 10 mM NH4+.And the expression levels of PoGDH gene were gradually decreased along with the prolongation of 10 mM glutamate or 10 mM arginine treatment.