Analysis Of Catalytic Sites Of Cytosolic TaGAPC And Enzyme Activity Assays Of Leaf GAPDH Under Abiotic Stresses In Wheat(triticum Aestivum L.)

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was a key and ubiquitous enzyme involved in glycolysis. As a “housekeeping” gene/protein, GAPDH has been widely used as a control in Western bolt experiments and quantitative real-time polymerase chain reaction(qRT-PCR) assays.However, recent studies demonstrated that GAPDH played crucial roles in many cellular non-metabolic processes including plant resistanting to stress. In this study, cysteine Cys154、Cys158 residues of changwu 134 cytosolic TaGAPC protein and total enzyme activity of leaf GAPDH of Changwu 134 in period of second leaves was analyzed. The results were mainly as follows:(1) The Cys154、Cys158 of Triticum aestivum L. cytoplasmic TaGAPC were site-directed mutated into serine by overlap-extension PCR, and recombinant vectors pGEMT-easy-TaCys154S/TaCys158 S were constructed to confirm the success of site-directed mutagenesis. Then three prokaryotic expression vectors pET-28a(+)-TaGAPC/TaCys154S/ TaCys158 S were further constructed. The optimized conditions for prokaryotic expression were as follows: 25°C, 0.25mmol/L IPTG for 12h;(2) Purified fusion proteins of TaGAPC and TaCys154S/TaCys158 S were obtained by Ni-NTA after sonication of the collected pellets obtained according to the optimized conditions. Then enzyme activity assays revealed that these two mutant proteins(TaCys154S and TaCys158S) were virtually inactive except TaGAPC wild type with specific activity of 48.84U/mg. So both cysteines Cys154 and Cys158 had significant influences on activity of TaGAPC protein, and were catalytic active residues of TaGAPC;(3) The seedlings of Changwu 134 were subjected to abiotic stresses in the period of second leaves. Treatment groups were as follows: drought stress of 20%PEG8000, different concentrations of H2O2(including 10μmol/L, 100μmol/L, 1000μmol/L) and 20%PEG8000 with 100μmol/L H2O2, respectively, and the samples were taken at 0h, 2h, 6h, 12 h, 24 h for enzyme activity assays of leaf GAPDH. The results showed that both 100μmol/L H2O2 and 20%PEG8000 had obvious promoting effects on leaf GAPDH enzyme activities, however, 20%PEG8000 with 100μmol/L H2O2 inhibited the increase of its total enzyme activities. Overall, the obtained results indicated that total activity of leaf GAPDH was related to catalytic cysteine or mutanted sites of GAPDH, this provided certain theoretical basis for further study of mechanism of TaGAPC in responding to drought-resistance.