| Pleurotus ostreatus is the most widely cultivated edible fungi in China,the yield and quality of which are significantly affected by the nitrogen supply levels.However,the molecular mechanisms of nitrogen absorption,transportation and assimilation of P.ostreatus are still unclear untile now.The glutamine synthetase(GS)palys a key role in nitrogen assimilation in organisms,and a hypothetical gene,encoding glutamine synthetases(designated as GS99),was obtained by genomic analysis of P.ostreatus PC 15.In order to identification of GS99 gene,P.ostreatus New 831 strain,the most widely cultivated specie in Henan province,was used as the experimental material.Firstly,the gDNA fragment and CDS fragment of the GS99 gene were amplified by separately using the genomic DNA and cDNA as templates,and the obtained PCR products were sequenced and analyzed through bioinformatics.Secondly,the prokaryotic expression vector pET22b-GS99 was constructed for heterologous expression,and the recombinant GS99 protein was inducibly expressed,purified and determined by enzymatic analysis.Besides,the molecular weight and configuration of GS99 protein were analyzed by Native-PGAE and gel permeation chromatography(GPC).Thirdly,the temporal and spatial expression of GS99 gene was analyzed by RT-qPCR,and the nutritional composition and amino acid composition of different parts of fruiting body were also analyzed.Lastly,the overexpression vector pTHG1300-ovGS99 and antisense silencing vector pTHG1300-siGS99 of GS99 gene were constructed.The main conclusions of this study are as follows1.The CDS fragment(1,390 bp)and the gDNA fragment(1,062 bp)of GS99 gene were obtained by using P.ostreatus New 831 genomic DNA and cDNA as templates,and the GenBank accession number of GS99 gene was MG680443.1.The results of bioinformatic analysis showed that GS99 gene,encoding 353 aa,was composed of a 5’ UTR,four introns and 5 exons.The native GS99 protein was decamer,and the molecular wigth(Mw)and pI of GS99 monomer of which were 39.05 kDa and 5.95,respectively.The typical N terminal domain(Gln-synt N),C terminal catabolic domain(Gln-synt C),ATP binding sites and the Glycine enrichment site GLNA 1 of type II glutamine synthetase were all found in GS99 protein,which indicated that the GS99 protein may have the glutamine synthetase activity2.The recombinant plasmid pET22b-GS99 was constructed,and then GS99 protein was inducibly expressed and analyzed by SDS-PAGE.The results showed that the molecular weight of GS99 protein monomer is 39 kDa.The highly purified(>90%)GS99 protein was obtained by Ni column affinity chromatography,and the configuration of native GS99 protein was detected by Native-PAGE and GPC system,the results showed that the Mw of the native GS99 protein was nearly 400 kDa,which indicated that the native GS99 protein was a decamer as expected3.The enzymatic properties of GS99 were dtermined,the results showed that the optimum reaction temperature and the optimum pH value were 40℃ and 7.5,respectively.Addition of Mg2+could significantly improve its activity,the acitivity of GS99 reached the highest level at 40 mM Mg2+,and there was no obvious incrase above 40 mM.The effect of Mn(2+)concentration on GS99 activity showed a phenomenon of low-promotion and high-inhibition,and the highest activity was obtained at 5 mM Mn(2+).Low temperature pretreatment could increase GS99 activity,but high temperature lead to its inactivation4.GS99 gene were all expressed in different parts and at different periods.The temporal expression profile of GS99 gene was as follows:the primordium stage>the mycelial stage>the fruiting period,and the spatial expression profile of GS99 gene was as follows:gill>cap>stipe,which indicated that GS99 gene was not only involved in the nitrogen assimilation in P.ostreatus,but also may be closely related to fruiting body development.Besides,the nutrition composition of different parts of the fruiting body were significantly,the total amino acids content and the total nitrogen content were all highest in gill,then in stipe,while lowest in stipe.The total sugar was higest in stipe,then in cap,and lowest in gill.The contents of 17 amino acis were all highest in gill,and glutamate was the richest amnio acid specie,asparate was the second richest one,which indicated that glutamate and asparate may function as the nitrogen donors for other nitrogen content compounds5.Theβ-tubulin and gpd gene promoters of the P.ostreatus New 831 strain were amplified,and then the overexpression and antisense silencing plmasid pTHG1300 was construncted.The GS99 gene overexpression plasmid pTHG1300-ovGS99 and antisense silencing plasmid pTHG1300-siGS99 were separately constructed,which lays a foundation for further charactierzie GS99 gene in the nitrogen metabolism and fruitbody development of P.ostreatus. |
