The Influences Of Melatonin On The Tolerance Formation Of The Dark Septate Endophytes Exophiala Pisciphila To Heavy Metals

Dark septate endophytes(DSE),one type of the mycorrhiza fungi,widely distribute in plant roots.Exophiala pisciphila(H93),one of DSE,was isolated with high heavy metal tolerance.H93 transcriptome data indicated that Cd2+ stress upregulated the expression of melatonin synthase genes(TDC,SNAT,ASMT).Melatonin is an ancient antioxidant that plays an important role in improving heavy metals tolerance.To explore the crosstalk between melatonin and H93 heavy metals tolerance,exogenous melatonin was utilized to investigate the behind mechanism.Then the bioinformatics analysis was implemented to explore H93 melatonin synthase genes.Furthermore,the response pattern of H93 melatonin and its synthetase genes to heavy metals(Cd2+,Cu2+,Zn2+,Pb2+)was studied.Two H93 melatonin synthase genes,EpTDC and EpASMT,were over expressed in Escherichia coli to attain the enzyme characteristics.At last,EpTDC,EpASMT were transformed in E.coli and Arabidopsis thaliana to investigate the relationship between melatonin synthase genes and heavy metal tolerance,and the mechanism behind it.The study achieved the following results:1.Exogenous melatonin reduced the H93 Cd2+,Zn2+ and Pb2+ accumulation mainly by regulating heavy metal absorption and transport system,or directly combining heavy metal.It also reduced the content of malondialdehyde and superoxide anion,and further lowering H93 oxidative stress caused by Cd2+,Zn2+ or Pb2+.In addition,exogenous melatonin increased the activity of superoxide dismutase to relieve H93 Pb2+ or Zn2+oxidative stress.2.Conservative domain analysis of EpTDC,EpSNAT,and EpASMT verified that all three enzymes contain conserved domains that have been identified in melatonin biosynthetic enzymes.This result indicating that the enzymes they encode may catalyze melatonin biosynthesis.Hydrophilic analysis,transmembrane structure analysis,and N-terminal sequence signal peptide analysis proved that the three encoded proteins are hydrophilic rather than transmembrane proteins.Subcellular localization prediction showed that EpTDC localized in cytoplasm,EpSANT in cytoplasm,while EpASMT in cytoplasm.Evolutionary phylogenetic tree demonstrated that H93 melatonin synthase belonged to the fungus separated from the animals and plants,indicating H93 melatonin synthase may evolve independently.3.Melatonin and its synthetase genes were proved to respond to heavy metal(Cd2+,Cu2+,Zn2+,Pb2+)stresses,the expression levels of H93 EpTDC and EpSNAT were consistent with melatonin content.And melatonin content increased significantly in the early stage of Cd2+,Cu2+,or Zn2+(2d).These results revealed that the heavy metal tolerance of H93 was closely related to melatonin and its expression of synthase genes.4.The enzyme activity analysis of EpTDC and EpASMT crude enzyme,obtained by E.coli,showed that its function of catalyzing the formation of tryptamine or 5-methoxytryptamine.5.Heterologous overexpression research further confirmed that melatonin synthase genes increased host's heavy metal tolerance and possibly through reducing the accumulation of heavy metals in A.thaliana.The introduction of EpTDC and EpASMT increased the OD600 of E.coli and the root long and fresh weight of A.thaliana under heavy metal stress,proving that EpTDC and EpASMT played an important role on improving H93 heavy metal tolerance.Moreover,the introduction of EpTDC and EpASMTreduced the root and plant of A.thaliana Cd2+ content under 40 mg/kg Cd2+stress,indicating that the two genes may increase the heavy metal tolerance of transgenic A.thaliana via reducing heavy metal content.