Proteomics In Ovary Developmentof Penaeus Monodom

Black tiger shrimp,Penaeus monodom is one of the most economically important penaeid species of aquaculture industry in the world. Especially,the research of gonadal development regulation of P.monodon is paid more attention by many researchers. While following the Genomics, Proteomics has become one of the hotspots of molecular biology and been widely applied to the study of the molecular biology of cultured organisms. Currently, proteomics studies on ovarian development of P.monodon is still less, and the molecular mechanisms of ovarian development of P.monodon is not clear. Therefore, proteomics and genomics methods are applied for researching the ovary at different developmental stage of P.monodon, which will provide more basic data for further sutdy on the molecular mechanism of ovary development of Penaeus mondon.In this study,Penaeus monodon ovary at stage Ⅱ、Ⅲ、Ⅳ is used. protein extraction by grounding in iquid N2, protein separation by 2-Dimemional gel Electeophoresis,taking photos and analysising the different protein spots. Protein data obtained by mass spectrometry.Selecting the differentially expressed reproduction-related proteins are further characterized by RACE-PCR and RT-PCR.Ubiquitin-ribosomal fusion proteins S27 and L40 screened out from the transcriptome database of Penaeus monodon are denote as Pm Ub S27 and Pm Ub L40,which are further characterized by RACE-PCR and RT-PCR.Some important progresses are as followes:(1)Proteomic expression profiles with high resolution and good reproduction for Penaeus monodon ovary at stage Ⅱ、Ⅲ、Ⅳ was obtained using 2-DE.(2)Based on the expression profiles,the expression protein changes of Penaeus monodon ovary at stage Ⅱ、Ⅲ、Ⅳ were investigated using proteomic approach.In ovary of Penaeus monodon at stage Ⅱ、Ⅲ、Ⅳ, 4147、4125、3972 expressed protein spot were identified respectively.In the stage Ⅱ and Ⅲ ovary, 418 differentially expressed protein spots were identified(159 up-regulated,259 down-regulated). In the stage Ⅲ and Ⅳovary, 460 differentially expressed protein spots were identified(181 up-regulated,279 down-regulated).The differentially expressed proteins in three stage with the same trend are 165(19 up-regulated,146 down-regulated).Choosing 18 differentially expressed proteins with more than 2-fold expression difference from 165 spots for mass spectrometry.12 protein were successfully identified. up-regulated protein are vitellogenin and beta-actin. down-regulated are Hemocyanin 、delta-1-pyrroline-5-carboxylate dehydrogenase、tropomyosin、prostaglandin reductase 1、enhancer-binding factor 3 homolog isoform X4、actin 1、endoplasmic reticulum protein 57、glutathione S-transferases、elongation factor 1 alpha and beta tubulin.(3)For further research, the mitochondria pyrroline 5-carboxylate reductase and glutathione S-transferase enzyme was selected from 12 kinds identificated proteins,which expression level was detected in various tissues and ovary of different stage of P. monodon by real time PCR.The result showed that Pm P5 CDh expresses highest in muscle,followed by the brain,there was no significant difference in the other tissues.Pm GST expresses highest in testis and hemolymph, followed by abdominal nerve, liver, pancreas and ovaries.In stage 3 ovary, Pm P5 CDh expressed highest and Pm GST expressed highest in stage 5 ovary of P. monodon.(4)Pm Ub S27 and Pm Ub L40 which full length c DNA were 506bp、571bp contained a 465-nucleotide open reading frame(OFR) and a 390-nucleotide OFR respectively. Pm Ub S27 encoded a protein of 154 amino acids. Pm Ub L40 encoded a protein of 129 amino acids. Pm Ub S27 contained a Ubiquitin(Ub) domain and the ribosomal protein S27 domain. Pm Ub L40 contained a Ubiquitin(Ub) domain and the ribosomal protein S27 domain. By real-time PCR, the expression level of Pm Ub S27 was shown to change significantly in ovary, expressing highest in stage 2 ovary. Pm Ub L40 was shown to change less significantly in ovary, expressing highest in stage 2 ovary.Based on these results, the mechanism of ovary development of P. monodon and find the important protein for ovary development.This study set a basis for further research on other shrimps.