Effect Of Cys Mutation On Antioxidant Activity Of GcGAST Protein

Gibberellic acid-induced cysteine-rich proteins play key roles in several physiological processes in plants. In 2006, Eigoda et al reported that such a protein family member exhibited antioxidant activity in vivo. However, detailed mechanism was not available. Taking into consideration the fact that on one side these proteins share 12 conservative cysteine residues in their C-terminal, on the other side cysteine residues are found to not only stabilize protein, but also in some cases they could perform antioxidant capability. In this thesis we aimed at exploring the effect of these12 Cys on the antioxidant activity and the secondary structure using site-directed mutagenesis combined with some spectroscopic methods.Methods: Firstly we choose pET-28(a)-GcGAST as the expression vector to construct recombination plasmid and then make 12 Cysteine single mutants by PCR mutation method. We also use E.coli BL21(DE3) as the host cells and induce the expression with IPTG. On this basis, we tested the ability of degrade H2O2 in the format of whole-cell and inclusion bodies respectively. In addition, we use FTIR and absorption spectrum to study the secondary structure of GcGASA and its turbidity.Results: After we got the sequencing results, we found that gcgasa gene has successfully inserted into the pET-28(a) and 12 Cysteine mutations has been introduced.SDS-PAGE results showed that Inclusion bodys are found to be existing in target protein. The the antioxidant activity of E.coli experiments showed that the E.coli which expressed the wt GcGAST protein can still grow in the presence of H2O2, while the mutant protein,s antioxidant activity get lower. Assays of inclusion bodies suggest that wild-type proteins are capable to degrade H2O2, and this ability will become stronger with increasing the concentration. However, the mutants exhibit no significant H2O2-removing capability.After we use DTT, the results are exactly the opposite.FTIR analysis showed that ?-sheet dominated the secondary structure of IB. A slight decrease in the content of ?-sheet was found when Cys residue was mutated to Ser.This result is consistent with the turbidity results.