| Glutathione (GSH) is the active tripeptide widely distributed in plants, animals and other organisms. GSH contains a sulfthydryl group which sequesters heavy metal ions to protect cells from stress damage. As an antioxidant, it can eliminate active oxygen free radical in cells that are under heavy metal stress conditions. So, increase of GSH content may increase the cells' tolerance to heavy metal stress. GSH is synthesized by the consecutive actions ofγ-glutamylcysteine synthetase and glutathione synthetase. Because the activity of the former is feedback regulated by GSH, GSH always maintains a certain level in the cell. A bifunctional fusion protein termed y-GCS-GS distributed in several gram-positive bacteria catalyzes both y-GCS and GS reaction. However, the activity of its y-GCS is not feedback regulated by GSH.In this study, the y-GCS-GS protein of S. thermophilus was analyzed by several bioinformatics web sites and softwares. The results showed that the y-GCS-GS was a 85.1kDa protein with an theoretical isoelectric point of 4.90, and average hydrophobicity of -0.268. This protein had an ATP binding site and contained a Glu_cys_ligase and a CPSase_L_D2 superfamily domain. Homology comparison showed that the protein sequence of y-GCS-GS shared 93%,73% and 63% similarity with S. salivarius, S sanguinis and S. agalactiae. Phylogenetic trees analysis showed that their genetic relationship was relatively approximate. The prediction of y-GCS-GS 3D structure showed that their y-GCS-GS protein had the similar space frame, so we speculated that they performed the similar function.To investigate the expression of StGCS-GS gene in prokaryotic system and its resistance function under heavy metal stress. His-γ-GCS-GS fusion protein was expressed after IPTG and the highly purified fusion protein was obtained after purification with His-Ni-NTP column. Western blot were then applied to identify target protein. The result showed that the fusion protein was His-y-GCS-GS. To investigate the role of StGCS-GS gene in heavy metal tolerance, the experiment was carried out on transformed E.coli under Cd, Ni and Cu stress. The results showed that transformed E.coli suffered less growth inhibition than the control one expecially under 1mM CdCl2 and 2mM NiSO4 stress. However, there were no much significant differences between transgenic and non-transgenic one under Cu2+ stress. These results suggested that the expression of StGCS-GS gene in E.coli could increase the tolerance of the transformed cells to Cd and Ni and play an important role in the growth under heavy metal stress.To further investigate whether StGCS-GS gene has conferred stress resistance on yeast under abiotic stress, and to study expression and subcellular location in yeast. StGCS-GS gene was cloned into yeast expression vector pYES2, and the pYES2-StGCS-GS was transformed into yeast (Saccharomyces cerevisiae) strain INVScl. The survival rate of cells under different stress was detected. The result showed that transformed yeast suffered less growth inhibition than the control one under CdCl2, NiSO4 and H2O2 stress. This difference was especially noticeable after CdCl2 stress. However, transformed yeast cells exhibited no tolerance under Cu and sorbitol stress. These results suggested that the expression of StGCS-GS gene increase the tolerance of the transformed cells to Cd, Ni and oxidative stress. Additionally, in order to study the subcellular location of StGCS-GS, theγ-GCS-GS-GFP fusion protein was expressed in yeast. The results showed that fusion protein located in the cytoplasm.To further investigate whether StGCS-GS gene could increase the tolerance of higher plant to heavy metal stress. The test of Heavy metal tolerance from 9 independent and stable expression T3 lines of transgenic tobacco was carried out. Statistic analysis on the root length, fresh weight and chlorophyll content showed that the transgenic plants suffered less growth inhibition than wild-type plants under Cd2+ and Ni2+ stress. However, transgenic plants exhibited no tolerance under Cu2+ stress. These results suggested that StGCS-GS gene conferred stress resistance on transgenic tobacco under Cd and Ni stress. In addition, the study on expression and subcellular location ofγ-GCS-GS-GFP in A. thaliana suspension cells showed that this protein was expressed in cells.
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