Differential Proteomic Analysis In Ovary And Testis And Their Different Developing Stages Of Scylla Paramamosain

In order to develop an efficient protein preparation method suitable for green mud crabScylla paramamosain gonad proteomic analysis, four methods, i.e. urea-thiourea extraction,trichloroacetic acid/acetone precipitation, Triton X100-urea extraction and Tris-urea extractionwere compared. The results showed that the Tris-urea extraction method was the mostappropriate for proteomic analysis on account of the highest resolution, the most protein spots.The method can be an efficient and simple protocol for proteomics in mud crab gonads. Asuitable two-dimensional gel electrophoresis (2-DE) system for total proteins in mud crabgonads was established. The isoelectric focusing process, the concentrations of SDS-PAGE geland silver staining method compatible with mass spectra (MS) were optimized.Using2-DE and ImageMaster2D Platinum6.0, changes of the proteins from differentstages of ovary of mud crab (early-developing stage, developing stage and ripe stage) wereinvestigated and69differentially expressed protein spots were found.15spots were identified byMALDI-TOF/TOF MS and bioinformatics analysis. And5proteins (vitellogenin, ubiquitincarboxyl-terminal esterase L3, elongation factor, glutathione S-transferase, cryptocyanin) relatedto the ovary development were identified. These proteins involved in nutrient accumulation,protein synthesis and breakdown, cellular immunity in the development of mud crab ovary.The differential proteomics at spermatocyte stage, spermatid stage and sperm stage of testiswas also studied by2-DE and ImageMaster2D Platinum6.0, and49differentially expressedprotein spots were found.9differentially expressed proteins related to testis development, suchas aldehyde dehydrogenase1family-L2, glyceraldehyde-3-phosphate dehydrogenase, argininekinase, proteasome subunit alpha type, microtubule protein, cryptocyanin, failed axonconnections protein and keratin were identified by MALDI-TOF/TOF MS and bioinformaticsanalysis. These proteins involved in metabolism, protein synthesis, cytoskeleton and cellularimmunity of mud crab ovary development.The ovary at developing stage and testis at spermatid stage were compared by2-DE andImageMaster2D Platinum6.0, and71differentially expressed protein spots were found.7differentially expressed proteins in ovary and testis, i.e. vitellogenin, alpha-tubulin, fatty acidbinding protein, heat shock protein70, inositol-phosphate phosphatase and superoxide dismutasemicrotubule protein, were identified by MALDI-TOF/TOF MS and bioinformatics analysis. These proteins involved in nutrient accumulation, metabolism, antioxidant and cytoskeleton inthe process of gonadal differentiation of mud crab.