Establishment And Application Of A Two-Dimensional Electrophoresis Protocol On Proteins In Mesocarp And Sclerised Endocarp During Peach (Prunus Persica) Fruit Development

Most activities in living cells are performed by proteins. Proteomics is a subject to study the compositions and changes of all proteins expressed by the genome in an organism. As one of the three significant approaches, 2-DE(two-dimensional electrophoresis) is an exclusive technique to separate continuously and display thousands of standard protein in one gel, which has been widely used in every aspect of biology at the present time. Along with the EST(expressed sequence tags)were obtained from peach fruits at different developmental stages, functional genomics of peach fruit will be confronted with a new opportunity. Peach(Prunus persica L.cv.Okubao)mesocarp was adopted in this experiment. Comparing with different effects of 2-DE patterns that were involved with quantitative loading of sample, sample buffer, equilibration buffer, IPG (immobilized pH gradients) strips with two types of pH range and the style of staining respectively to explore influence factors of two-dimensional electrophoresis patterns and expect to establish an available protocol for peach fruit. Approximately 90μg of protein was brought to a total volume of 375μL sample buffer which included urea, thiourea CHAPS, sulfphobetaine 10 and ampholyte and loaded on 17 cm IPG strip, and the strip was treated in one step by equilibration buffer with tributyl phosphine. After isoelectric focusing completed, sodium dodecyl sulfatepolyacrylamide gel electrophoresis was processed. The gel stained by coomassie brilliant blue appeared favorable effects, furthermores the gel stained by silver contained more protein spots than it and had a clear background. The results demonstrated that this protocol was suited to proteomic analysis of peach mesocarp. Subsequently, using pH 5.0~8.0 gradients strip and applying above-mentioned method to analysis 2-DE patterns of proteins between mesocarp and sclerised endocarp during peach fruit development indicated that the 2-DE gel of mesocarp took on 1318±125 protein spots, in contrast to the pattern of endocarp which had 1415±133 protein spots, and there were at least 24.77% discripancies of two gels, whereas the identification of variant proteins would be accomplished ulteriorly by matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry.