Functions And The Vital Roles Of ABC Transporter Protein-encoding Genes In The Tolerance Of Exophiala Pisciphila To Heavy Metals

Dark septate endophytes(DSE)commonly colonize the roots of plants grown in heavy metals(HMs)polluted environments,and enhance heavy metal tolerance of host plants.Meanwhile,DSE from the HMs contaminated environments also show strong tolerance to HMs in general.ATP-binding cassette transporters(ABC transporter proteins),ubiquitously existed in the cells of almost all organisms,are multifunctional proteins classified as a super family of transporters,which play an important role in improving the host tolerance to various biotic and abiotic stresses.In this thesis,the DSE strain(Exophiala pisciphila)with a relatively strong tolerance to Pb,Zn and Cd,isolated from a HM mine tailing in Yunnan,was targeted as the fungal strain.Based on transcriptome data of E.pisciphila cultured under Cdz+ stress and no-Cd supplements as negative control,we selected the up-regulated ABC transporter protein-encoding genes by real-time fluorescence quantification PCR(qPCR)assay under the stress of Pb,Zn and Cd EC50 concentrations,respectively.Then,bioinformatics analysis of the targeted genes was performed and three ABC transporter protein-encoding genes were selected for the further study.Accordingly,the expressions of the three target genes were evaluated under the stress of a series of Pb,Zn and Cd concentrations.The subcellular localization of the three genes was carried out using the immunofluorescence technique and the heterogeneous expression of three recombinant genes in Saccharomyces cerevisiae and Arabidopsis thaliana in the function verification for HM tolerance was tested.And at last,the mechanism and the vital roles of ABC transporter protein-encoding genes in the tolerance of E.pisciphila to heavy metals was discussed.The main results of this thesis are as follows:1.Based on transcriptome data of E.pisciphila,in total 42 ABC transporter genes were identified from the unigene libraries,which belonged to 4 subfamilies,i.e.ABCB,ABCC,ABCD and ABCG.Then,EpABCl,EpABC2 and EpABC3 were targeted for next research according to the qPCR results under the stress of Pb,Zn and Cd EC50 concentrations,respectively.Bioinformatics analysis indicated that the ORF length of EpABC1,EpABC2 and EpABC3 were 2127 bp,2601 bp and 4659 bp,respectively.Isoelectric point and protein molecular weight were 8.90,80.91 KDa,5.65,96.23 KDa and 8.07,172.791 KDa,respectively.EpABC1,EpABC2 and EpABC3 belonged to subfamilies of ABCD,ABCB and ABCC.Both EpABC1 and EpABC2 were half-size transporters,and EpABC3 was a full-size transporter.Moreover,EpABC2 and EpABC3 had the characteristic transmembrane domain structure,while EpABC1 had no corresponding domain.None of them had.signal peptide.Subcellular localization of proteins encoded by EpABCl,EpABC2 and EpABC3 were predicted to be localized in vacuole/peroxidase,vacuole/nucleus and vacuoles/plasma membrane,respectively.2.EpABC1 EpABC2 and EpABC3 were up-regulation expression responding to the elevated stresses of Pb,Zn and Cd concentrations in E.pisciphila.However,the expression characteristics were slightly different among various HM species.Under the stress of Pb and Zn,the expression level of the three genes were gradually increased with the elevated metal concentrations,and in comparison to the no metal stress,the maximium values were 13.3,30.7 and 26.8 for Pb and 3.7,21.7 and 5.6 folds for Zn,respectively.The expression of the three genes showed the same tendency under the stress of different Cd2+ concentrations,and increased to the maximium at the EC50 Cd stress,then decreased gradually with the increased stess,and the highest expression levels of EpABC1,EpABC2 and EpABC3 were 8.6,9.6 and 29.3 folds to the no metal stress,respectively.Among the expression patterns of three genes,the response sensitivity of EpABC2 and EpABC3 to heavy metals was obviously higher than EpABC1 which suggested that EpABC2 and EpABC3 might play more important roles on enhancing the tolerance ofE.pisciphila to Pb,Zn and Cd than EpABC1.3.The subcellular location of the three genes by the technique of immunofluorescent revealed that all proteins encoded by the three genes were localized in cytoplasm with strong signal and high expression levels.4.The heterogeneous expression of three recombinant genes in S.cerevisiae,showed that both the transformants of Wild type and sensitive mutant with target genes grew better than the transformants with vector under the stress of different Pb2+,Zn2+ and Cd2+ concentrations,which suggested that EpABCl,EpABC2 and EpABC3 improved the tolerance of 3 transformants to Pb,Zn and Cd.Besides,the content of Pb,Zn or Cd in the transformants with WT-EpABC2 or WT-EpABC3 was significantly higher than the transformant of WT-pYES2.However,the content of HMs in the transformant WT-EpABC1 was lower than WT-pYES2,which suggested that different detoxification mechanisms may exist in EpABCl,EpABC2 and EpABC3.Comprehensively,EpABC2 and EpABC3 play more important role on improving the tolerance of yeast transformants to Pb,Zn and Cd.5.The three genes were successfully transformed into A.thaliana.EpABC3 has been stably inherited and overexpressed in A.thaliana(stably expressed in generation T2),while EpABCl and EpABC2 were only determined in T1 transgenic lines.Both EpABC2 and EpABC3 transformed in A.thaliana could response to the stress of Cd2+,and enhanced the tolerance;especially it was true forEpABC3.Besides,the expression of EpABC2 and EpABC3 transformed in A.thaliana accumulated more Cd2+ than the wild type.However,the EpABCl transformed A.thaliana was not sensitive to the Cd2+ stress and didn't enhance the tolerance.Interestingly,EpABCl,EpABC2 and EpABCS transformed A.thaliana could significantly improve the biomass than the wild type.The function of three ABC transporter protein-encoding genes(EpABCl,EpABC2 and EpABC3)on the improved DSE(E.pisciphila)tolerance to HMs was documented for first time in this thesis.Molecular structure and their functional mechanisms of EpABCl,EpABC2 and EpABCS in terms of the enhanced HM tolerance for their hosts were also explained.It would not only provide theoretical basis and reference of research methods for studying DSE HM tolerance mechanism at the molecular level,but also provide theoretical and technical supports for exploring the HM tolerant genes from DSE for transgenic breeding of HM tolerance crops.The results also give new insights into the phytoremediation of heavy metal polluted soil theoretical by the use of transgenic plants both practically and theoretically.