Screening Of Differentially Expressed Proteins Of Different Grape Seed Developmental Stages

In this study, we take the seed of Pinot Noir grapes and seedless grapes after 20 d,30 d and 40 d of their flowering. Using differential proteomics research methods and technology for protein seperation, identification and bioinformatic analysis of different materials, in order to find abortion-related key factors in protein level to provide a theoretical reference for the seedless grape breeding. The main findings are as follows.1. After using TCA/acetone precipitation method to extract protein in grape seed,using vortex, ultrasonic and phenol protein extraction method to solute protein.Through comparative analysis, we found that improved TCA/acetone and benzene extraction method is relatively suitable for grape seed extracting protein. Solubility of the extracted protein increased and have a clear background with less stripes in the subsequent two-dimensional electrophoresis analysis, which is suitable for differential protein research.2. By comparing the effect of IPG with different pH, different sample volume,different isoelectric focusing parameters and different concentration of SDS-PAGE separation gel on two-dimensional electrophoresis effect, we established a twodimensional electrophoresis system suitable for grape seed protein. That is pH5-8 IPG strips, 600μg of sample volume, focusing 80000 Vh, a concentration of 11%separating gel.3. Analysis of proteins at different developmental periods by two-dimensional electrophoresis showed that pinot noir has 162 protein spots, while seedless white grapes has 105. Through mass spectrometry, we identified 161 and 97 differentially expressed proteins respectively with a 92% success rate. However, due to protein modification process, the protein gels in different locations may be the same protein.As a result, the actural protein spots is 135 and 87 respectively. These differential proteins are primarily involved in cell processes, energy metabolism, stimulate response, regulatory and other related processes.4. Functional analysis of differential expressed proteins. In the Identified differential expressed proteins, The biological function of identified differential expressed proteins in seedless white grape and Pinot Noir grape are distributed as follows: cellular processes(44% and 38%), metabolic processes(each 15%),stimulation response(14% and 11%), biological regulation(9% and 10%), positioning(5% and 8%), development process(2% and 4%), interact with cells and organs(1%and 4%), immune system processes(each 1%), protein of unknown function(each9%). Analysis on proteins associated with cellular processes and metabolic processes showed that differential expressed proteins in seedless grapes is more relevant with seed development. This differential expressed proteins may associated with embryo abortion. While differential expressed proteins at different developmental periods in Pinot Noir grape seed is mainly related with metabolic regulation.5. After screening we found these proteins have a direct correlation with fail fertility of seed: Calreticulin, formic acid glutathione hydrolase(SFGH), cell division cycle protein 48(Cdc48p), glycyl-tRNA synthetase(GlyRS), hydroxymethyl-coenzyme A synthetase(HMGS), aspartic acid synthase(Ast), inositol phosphate synthase(MIPS), ribosomal protein L3(RPL3), catalase, phosphatase glycerol dehydrogenase(PGDH), amidino mannose phosphate transferase(MPG) and phosphatase(PLD).