| Yunnan province, known as "the kingdom of nonferrous metals", have to face the environmental problems with strong metal-contamination due to over-exploitation of lead-zinc in mine areas. Our previous studies found that natural revegatation plants were usually colonized by dark septate endophytes (DSE) in the lead-zinc areas which is mainly polluted by Cd, Zn and Pb. It was suggested that DSE may play an important role in promotion to plant adaptation to the heavy metals polluted environments. It is of significance to understand the Cd tolerant mechanism of DSE for clarifying the ecological function of DSE in the heavy metal polluted mining ecosystem, and to assess the relationship between DSE and heavy metal tolerance of plants in lead-zinc district.Zip transport protein (Zinc regulating transport protein and iron regulating transport protein) family is ubiquitious, which consisit of numerious members from various bacteria, fungi, animals and plants with function as broad specificity transition metal transporters. Previous studies showed that Zip transporter family functions as the transport of divalent metal cations, but studies on Zip transporter genes and their functions have been cloned and assessed in only several fungi. In this thesis, we annotated41Zip genes from the transcriptome data base constructed from Exophiala pisciphila (a tipical DSE strain) by RNA-seq. Then, the gene EpZipl (GenBank accession:KR270804), which was the most significant different expressed was chosen for the purpose of clone, bioinformatic analysis, yeast heterologous complementary functional verification, subcellular localization and differential expression analysis under different heavy metals stress, and major results were as he followings.1. Cloning and bioinformatic analysis of EpZipl geneThe full length of open reading frame (ORF) in comp6001was1578bp, encoding525amino acids with Mw about55.4kDa and pI about5.2and named EpZipl. Bioinformatics analysis showed that most of amino acids in EpZIP were hydrophobic, and EpZIP contained a signal peptide and eight typical transmembrane domains of Zip family. Bioinformatic analysis indicated that the EpZipl would be expressed only on the cell membrane, and a transmembrane transporter protein. Phylogenetic analysis showed that EpZipl gene has a high homology to the other family members of Zip genes.2.The functional complementary of EpZipl geneWe used the methods of yeast functional complementary validation to construct the recombinant plasmid which contains comp6001gene ORF (EpZipl) to inducibly fused in the specific expressed plasmid pFL61. By using the method of lithium acetate transformation, through observing the growth status of the wild type yeast DY1457and iron deficient yeast DHY1453and zinc deficient yeast ZHY3in specific culture medium to verify the function of the protein EpZIPl encoded by EpZipl whether can transport the Zn2+or Fe2+.The functional complementary of EpZipl gene in Yeast showed that EpZIP can partly or entirely transport the Zn2+in Saccharomyces cerevisiae, but not for divalent Fe2+.3. Sub-cellular localization and expression analysis of EpZipl geneRebuilding the plasmid pDDGFP-2which contains comp6001gene ORF (EpZipl) to inducibly expressed in Saccharomyces cerevisiae ZHY3, the microscope observation showed EpZipl gene encodes a protein located on the cell membrane. The results of qRT-PCR show that on the conditions of Cd2+or Zn2+stress, the expression of EpZipl gene in H93respectively showed a downward trend in varying degrees with elevated concentrations of heavy metals. The expression of EpZipl gene is regulated by the extracellular concentration of divalent cation Cd2+and Zn2+. It can be inferred that EpZipl gene may play the role of absorbing or transporting the extracellular divalent cations to intracellular in H93. |
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