Studies On Isolation And Purification Of An Osmotic Stress-induced Protein And Its Expression Regulation By Signal Substances

The maize specie of stress-resistance LuYu No.14 was cultivated by solution in the mode of simulated osmotic stress with NaCl and PEG. The characteristic and expression mechanism of stress protein in maize seedling roots under osmotic stress were studied. The experimental aim was to offer academic foundation for the studies of plant stress- resistant mechanism. The soluble proteins of maize seedling roots under different stress treatments were analyzed by SDS-PAGE gel electrophoresis,isoelectic focusing electrophoresis,ion-exchange chromatography and gel filtration. A novel induced-salt protein was isolated and purified. The main results are as follows:1.The induced-osmotic stress protein was isolated. Maize seedling root adapted to grow in medium containing 1.0％ NaCl and 10％PEG produced several new or enhanced polypeptide bands on SDS-PAGE gel electrophoresis. The 26.8-kilodalton polypeptide was present in both NaCl and PEG-induced osmotic stress roots but that was not detected in natural roots. The protein was a salt-adaptable protein because it was similar to osmotin. The 26.8-kilodalton polypeptide was intituled ZmR26.8 protein bacause it was synthesized in Zea Mays roots.2.The treating conditions of maize roots for purification were confirmed. It was necessary for the purified material to be cheap and got easily. And materials contained much target protein. The maize seedling roots after treated by different concentration NaCl ,PEG and by different time were examined by SDS-PAGE gel electrophoresis. The propriety treating condition was that : fistly, maize seedling roots were treated by 1.0％ NaCl for 72 hours; then they were treated by 1.5％ NaCl for 72 hours. 3. The sample was prepared. Fistly, the maize seedling roots which havebeen treated were ground into a fine powder. An equal volume of extraction buffer(100mmol/L Tris-HCl, 5mmol/L EDTA, 1mmol/L PMSF, 2％β-mercaptoethanol, pH 7.5 ) was added. Then the following steps were done at 4℃: After centrifugation at 5 000 xg for 20 min, proteins were precipitated from the supernatant by 60％ to 90％ saturation with ammonium sulfite. The precipitates were redissolved in chromatography buffer. Lastly, the fraction was dialyzed against chromatography buffer.4. ZmR26.8 protein was isolated by chromatography. The fraction was applied to separate columns of CM-Sepharose Fast Flow. The columns were washed with five bed volumes of washing buffer. Then fraction were eluted from the columns with a 0~ 0.5mol/L linear gradient of NaCl and amass liquor of every protein peak were monitored with SDS-PAGE gel electrophoresis. The appropriate protein peak was collected and concentrated to 2~3ml. The condense solution was applied to Sephacryl S-100 HR.Then the appropriate protein peak was collected and monitored with SDS-PAGE gel electrophoresis. We could get pure ZmR26.8 protein.5. Analysis of characteristic. The characteristic of ZmR26.8 protein were analyzed by SDS-PAGE gel electrophoresis,isoelectric focusing electrophoresis and heat-stable experimentation. The results were follows: its molecular weight is 26.8KD, its isoelectric point is 6.5, and it is a heat-stable protein.6.The expression mechanism of ZmR26.8 protein in response to stress. Maize seedling roots were treated by salt and fed with ABA, ancymidol (ABA inhibitor), neomycin sulfate and Butyl alcohol (PLD/PLC inhibitor), EGTA(Ca2+ inhibitor),TFP (CaM inhibitor). In addition, We fed maize seedling roots with K+, Salicylic acid and Tetraethylammonium chloride. The change content of ZmR26.8 protein were examined by SDS-PAGE gel electrophoresis. The result indicated: the signal substances ABA, PLD/PLC, Ca2+ /CaM had affect on the expression of salt-induced ZmR26.8 protein. But SA and K+ had no affect on the expression of induced-stress ZmR26.8 protein.