Studies On Differential Proteomics During Embryo Development Of Ginkgo Biloba L.

In this study, to explore the dormancy breaking mechanism of seeds with after-ripening characteristics, the seeds of Ginkgo biloba L.as a representative,which embryos are the research object.The 2-DE gels are involved in different protein extraction methods (the direct extraction method, the phenol extraction method, the Tris-HCl method, the Trizol extraction method and the TCA-acetone-phenol method), different concentration of urea in lysis buffer(6 mol/L,7 mol/L,8 mol/L,9 mol/L),different protein loading dosages(1500 μg,1650 μg,1800 μg) and different separation gel concentrations (10%,12.5%,15%), which are tested.And select the suitable methed from above conditions.According to the shape and size,the embryo of Ginkgo biloba is classified, and determined the ability of different developments during the embryo germination;On this basis, according to the differences in shape and size of Ginkgo biloba embryo development,select differentially expressed proteins, which are related to embryo development. And identify these expressed proteins to perform mass spectral analysis.To explore Ginkgo biloba embryo development mechanism of post-maturation and lay the foundation for revealing the molecular mechanisms of seed germination. Main results of this study are as follows:l.The optimized system included the following steps:extracting total protein from Ginkgo biloba embryo by TCA-acetone-phenol method, dissolving proteins with lysis buffer containing 8 mol/Lurea,1650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration. Reproducible profiles with high resolution and clean background are obtained by this optimized two dimensional electrophoresis of embryonic total protein.2.According to the length of Ginkgo biloba embryos,the embryo development is divided into five phases:0-0.3 cm as stage Ⅰ,0.3-0.5 cm as stage Ⅱ,0.5-0.8 cm as stage Ⅲ,0.8-1.0 cm as stage Ⅳ,> 1.0 cm as stage Ⅴ.Five-stage embryo were cultured in vitro respectively.The days of five-stage embryo which could reach the germination length 1.5 cm respectively are> 20 days,14 days,8 days,5 days and 3 days.Therefore,there are different germination abilities among five-stage Ginkgo biloba embryo.3.According to two-dimensional electrophoresis results,there are 310 protein spots detected among five-stage sample.60 protein points with expression changes more than 1.5 times are selected and analyzed by MS+MS/MS technology.Using Viridiplantae NCBI and the EST database to search,55 differentially expressed proteins are identified,and the success rate is 92%.In the result,20 proteins are up-regulated,21 proteins are down-regulated, and 14 proteins are fluctuated.4.Bioinformatics analysis shows that these proteins belong to nine functional groups,including:synthesis and folding (13%); DNA and RNA synthesis (9%); stress and defense (22%); metabolism (18%); storage proteins (3%); transportation (3%); embryo growth(3%); unique proteins (7%);unknown proteins (22%).