Proteomic Research Of Rice Root Tip Plasma Membrane Under Salt Stress

A highly purified plasma membrane fraction from japonica rice Xudao 3(oryza sativa) root tip was isolated using two phase partition system,then we studied plasma membrane proteome under salt stress with IEF/SDS-PAGE two dimensional gel electrophoresis.The main contents are as follow:1.Optimization of two dimensional gel eleetrophoresis of plasma membrane proteome from rice rootA highly purified(90%) plasma membrane fraction was isolated by using an aqueous Dextran-Polyethylene Glycol two phase partition system,and solubilized by four different kinds of rehydration.Solubilized plasma membrane fraction was separated with IEF/SDS-PAGE two dimensional gel electrophoresis and then analyzed by MALDI-TOF/TOF MS. The results showed that the most efficient rehydration buffer in this case consisted of 7 mol/L urea,2 mol/L thiourea,20 mmol/L DTE,1%amidosulfobetaine-14(ASB-14),and 4 %3-[(3-Cholamidopropyl) dimethyl- ammonio]-1-propanesulfonate(CHAPS).9 of 16 spots analyzed by MS were plasma membrane-associated proteins,5 spots were unknown proteins,and only 2 spots represented the contaminated proteins from other organdies.The study indicated that low concentration of ASB-14 is more suitable for the proteomic analysis of plant plasma membrane.IEF/SDS-PAGE two dimensional gel electrophoresis in such optimized condition is suitable for separation of hydrophilic peripheral proteins from plasma membrane of rice root tips.2.Separation of plasma membrane proteome from rice root tips by SDS-PAGE and analysis of transmembrane proteomeHigh-salt solutions(0.6 mol/L KCl) and gentle detergents(15 mmol/L CHAPS) had been used to wash plasma membrane proteome in order to reduce the complexity of the fraction.According to the results of the SDS-PAGE and ESI-MS/MS,8 integral proteins that contained several transmembrane domains had been identified.The result suggested that the complexity of the purified membrane was reduced.Each strip of the SDS-PAGE contained one or two protein,which are mostly hydrophobic transmembrane protein.3.New changes of plasma membrane proteome in rice root tips under salt stressIn order to gain a better understanding of salt stress response in plants,a proteomic approach was applied to investigate the changes of rice(Oryza sativa) root-tip plasma membrane proteins following the treatment of 150 mmol/L NaCl.With or without 48h salt treatment,plasma membrane fractions from root tip cells of a rice salt sensitive cultivar, were separated by IEF/SDS-PAGE.Comparative analysis between three independent biological replicates revealed that the expressions of 34 proteins were changed,16were upregulated, and 18 were down-regulated,respectively.Mass spectrometry analysis indicated that most of these membrane associated proteins are involved in important physiological processes such as membrane stabilization,ion homeostasis,and signal transduction.Three changed proteins were selected to perform real time PCR analysis.Compared to the translational level,two proteins showed the similar changes at transcriptional level except for the third one,which indicated the different regulation between transcription and translation of eukaryotic genes under salt stress.