The Study Of Different Rapeseed Materials Seedlings By2-dimensional Polyacrylamide Gel Electrophoresis

Rapeseed oil with high oleic acid have satisfied nutrition and health functions, so it attracted more and more attention in the world. But most of the studies of high oliec acid rapeseed fatty acid synthesis based on genes, and there has no such study based on protein has been reported. The15-day-seedlings of high oleic acid rapeseed and xiangyou15were used as raw materials in this study, and the results just as follows:(1) protein extractionLiquid nitrogen was used to grind rape seedlings, and the grinding powder according to1:10(W/V) with sample solution (2M thiourea,7M urea,2%CHAPS,0.7%DTT,0.01%Bio-Lyte). Then the sample was placed in a ultrasonic beaker which has the mixture of ice and water, the amplitude of ultrasonic beaker is set to21%(total order:ultrasound ultrasound for60seconds, super0.1second, stop one second). Treated with4fold volume of precool TCA acetone mixture, and then washed3times with the precool acetone, the required protein can be obtained.(2) Two-dimensional polyacrylamide gel electrophoresisThe first-dimensional electrophoresis was performed using an IPGphor II IEF system (Amersham Biosciences, Uppsala, Sweden) as described by the manufacturer. IPG strips of24cm were actively rehydrated with450μL of rehydration solution, which was contained1mg of protein. Immobilized pH gradients (pH3-10, Bio-Rad) were also used. After IEF separation, the strips were equilibrated twice for15min using equilibration buffer10mL (6M urea,50mM Tris-HCl,20%glycerol,2%SDS,0.1g DTT),10mL buffer II (6M urea,50mM Tris-HCl,20%glycerol,2%SDS,0.4g iodoacetamide). The separation in the second dimension was performed by12%SDS-PAGE with a constant electric current of10mA and then switched to20-30mA until the bromophenol blue frontier reached the bottom of the gel. The gel was stained in Coomassie brilliant blue buffer for1h, and then decolored until the background was clear.(3) electrophoresis and mass spectrometry analysis The IPG strips were analyzed by PDQest8.0image analysis software,277different point were obtained, and50points which has the fold change between3-5(forward, backward) were analyzed by mass spectrometry and bioinformatics, the names and types of the differential proteins were obtained. Most of the differential proteins involved in carbon metabolism and photosynthesis,such as ribulose-1bisphosphate carboxylase,5-two and ATP synthase.