Expression Of A Wheat Methyl-binding Protein TaMBD1 In Vitro And Identification Of Its Interacting Proteins

In order to investigate biology function of TaMBD1, the expression vector was constructed. Expression conditions of fusion protein were optimized. Separating interacted protein of TaMBD1 utilized affinity chromatography and 2D electrophoresis. The main results showed as follows:1. Base on the complete ORF of TaMBD1, pulling in restriction site by PCR, prokaryotic expression vector pET28a-TaMBD1 was constructed. And vectors were transformed in Escherichia coli BL21 (DE3). Expression of BL21 induced by IPTG was successful. The expression of fusion protein TaMBD1 was tested and verified by SDS-PAGE and western blot.2. We tested different temperature and different induced hours, in order to optimize expression condition of fusion protein of TaMBD1. Results showed that fusion protein TaMBD1 can express on 28℃and 37℃, 1.0 mmol·L^-1 IPTG, induced 3, 6, 9 hours. But target protein didn't increase obviously after inducing 6 h, non-target protein increased much more. In general, the most suitable induction condition for the fusion protein was 6 h induction under 1.0 mmol·L^-1 IPTG, 28℃.3. In order to obtain high purify proteins, we tested different condition of Binding Buffer, Elution Buffer and speed. Results showed that binding buffer including 40mM/L imidazole had more effective in getting rid of non target protein; Elution Buffer including 500mM/L imidazole can elute all protein that bind on chromatography. Speed of binding was 0.5mL/min, and speed of elution was 1.2mL/min.4. Utilized purification and 2D-electrophoresis, we found 6 slots in 2D gel. 2 of 6 slots analysis of MALDI-TOF-MS, the 2 protein slots were ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and Isocitrate dehydrogenase [NADP]. Large subunit was important part in ribulose-1,5-bisphosphate carboxylase/oxygenase, and active domain in large subunit. Ribulose-1,5-bisphosphate carboxylase/oxygenase were important enzyme in land plants. This enzyme played key role in photosynthesis and photorespiration. Isocitrate dehydrogenase [NADP] existed several domains in cells, such as cytoplasm, mitochondria, chloroplast and peroxisome. It correlated metabolism about synthesizing and break down. Under the PEG mediated water-stress, the expression of TaMBD1 was increased after 1 hour, 12hours and 48hours in the leaves of LuohanNo. 2. It implied that TaMBD1 could response to external stimulus by adjusting metabolism.