Analysis Of The Tolerance Of Three Ganoderma Lucidum Strains To Metals And Oigopeptise Transport Gene Family In Ganoderma Lucidum

Compared with green plants,edible fungi can adsorb and accumulate high concentration of metals,and then have high potential for remediation of metal contaminated environments.The mechanisms of the tolerance and adsorption for metals are complex,including defense by extracellular polymeric substance(such as adsorption,precipitation),permeability of cell membrane,detoxification by intracellular proteins,and so on.Oligopeptide transport protein(OPT),which widely exists in prokaryotic and eukaryotic organisms,may be involved in various biological processes,including transport oligopeptides from the environment as nutrients,assist the transport of metal ions and detoxification of toxic metal ions.Plenty of researches about OPT have been carried out in plants,and their expression profiles were influenced by environmental conditions and developmental stages.However there is much less information about OPT protein in fungi,especially white rot fungi such as Ganoderma lucidum.G.lucidum is an edible-medicinal fungus,which has potential in immune regulation,anti-tumor,and anti-virus,and has achieved a wide range of artificial cultivation.Since covering soil is necessary for G.lucidum cultivation,so the growth of G.lucidum is extremely vulnerable to metals exist in the soil and cultivation matrix.Therefore,to analysze the mechanisms involved in metal tolerance and adsorption is of great inportance.In this research,metal-sensitive G.lucidum strain was selected through the analysis the tolerance and sensitivity to five kinds of metal of three G.lucidum strains(Meizhi,Chuanzhi and No.1 Rongbao).Using this metal-sensitive strain as material,the transcriptional expression patterns of OPT genes were analysed in different developmental stages,under different nutritional conditions and different metal stresses,and hope to reveal the potential roles of OPT in G.lucidum under different environmental stresses and developmental stages.The main contents and results are as follows:(1)Plate analysis was used to measure the mycelium growth under different metals and their different concentrations of the three G.lucidum strains.The results showed that the greater the concentration of four metals(Pb2+,Cd2+,Cu2+ and Se4+)were used,the stronger the inhibition were appeared,and decrease the radius and density of the colony;While under Fe2+ treatment,the radius and density of the colony is first inhibited and then promoted and then inhibited again.In general,the three strains were tolerant to Pb2+ and Fe2+,but sensitive to Cd2+,Cu2+ and Se4+.Among the three strains,Meizhi showed higher tolerance and No.1 Rongbao showed higher sensibility to the five metals.(2)Thirteen putative OPT genes were identified in G.lucidum genome database,which were named G0PT1-GIOPT13,The phylogenetic analysis of GlOPT and other OPT proteins with known function from other species showed that these OPTs can be divided into five groups(Group 1-5).Group 2 consists of OPT proteins from plants;Group 3 includes OPTs from C.albicans and S.cerevisiae,which belong to the basidiomycota;OPT proteins in the other three groups are those that originate from ascomycota and basidiomycota.The motif compositions were highly conserved in each group.Evolutionary relationships of GlOPT family were analyzed by gene structure,the results showed that GlOPT4,which possesses the most introns,may be more closely to the ancestral OPT gene.(3)Transcriptional expression profiles of GlOPT genes were analyzed by RT-qPCR in samples collected from different development stages,different nutritional conditions and different metal stresses.The results showed that no transcript was observed for three genes(GlOPT7,GlOPT8 and GIOPT9).While all other GlOPT genes were differentially and specificly expressed,indicating that these genes may be involved in the physiological metabolic processes of G.lucidum.Six genes(GIOPT,GIOPT2,GIOPT4,GIOPT5,GIOPT11 and GIOPT12)had relatively higher expression levels in the mycelial growth stage,and three genes(GlOPT3,GIOPTIO and GIOPT12)had relatively higher expression levels in the fruiting stage.Under carbon and nitrogen nutrient stresses,the transcriptional expression levels of five genes(GIOPT1,GIOPT5,GIOPT6,GlOPT11 and GIOPT13)were down-regulated and three genes(GlOPT2,GlOPT4 and GIOPT12)were up-regulated when cultured for 15 d;while the transcriptional expression levels of four genes(GlOPT4,GIOPT5,GIOPT10 and GIOPT11)were down-regulated and five genes(GlOPT1,GlOPT2,GlOPT3,GlOPT12 and GIOPT13)were up-regulated when cultured for 30 d.When treated with metals,transcriptional expression levels of three genes(GIOPT11,GIOPT12 and GIOPT13)were down-regulated under all five metal treatments,and only two genes(GIOPT6 and GIOPT10)were up-regulated under Cu2+ treatment from samples after 15 days of growth;while in the samples after 30 days of growth,Pb2+,Cd2+ and Cu2+ promoted the transcriptional expression levels of most OPT genes,Se4+ promoted the transcriptional expression of some OPT genes,and the transcriptional expression levels of all OPT genes were up-regulated under Fe2+ treatment.