Studies On Differential Proteome In Ovary And Testis Of Marsupenaeus Japonicus And Analysis Of Differential Expressed Gene Cathepsin B

Penaeid shrimp Marsupenaeus japonicus is an important economic crustacean aquaculturespecies in southeast China. With rapid development of the penaeid shrimp aquaculture andmolecular biology techniques, cloning and characterization of genes related gonad developmentin penaeid shrimp are focusd on aquaculture genetics and breeding. However, the studies aboutmolecular mechanisms of gonad differences are poor in penaeid shrimp. The researches ondifferentially expressed proteins in ovary and testis are scarcer. Therefore differential proteomicsin ovary and testis of M. japonicus will provide insights into the molecular mechanisms of thisimportant process.In the study, the gonad total protein of M. japonicus was prepared by a special reagent usedto extract DNA, RNA and protein simultaneously (RDP reagent). The extraction efficiency wascompared with traditional lysis solution method by two-dimensional electrophoresis (2-DE). The2-DE map of RDP was clear with less striations and more protein spots, while that of lysissolution method had horizontal and vertical striations with decrease of protein spots at thecathodic side. The results indicated that RDP method can remove salt and lipid from gonadsample effectively. The finding from the study provided not only technical support forproteomics research of M. japonicus, but also useful reference for gonad total protein extractionof other crustacean.2-DE approaches were utilized to study the differentially expressed proteins in the gonadtissue of M. japonicus. About45differentially expressed protein spots were found and15proteinspots were randomly chosen for further analysis by MALDI-TOF/TOF MS. As a result,3ovary-specific proteins (cathepsin B, cortical rod protein-2, crustacyanin-A),5ovaryup-regulated proteins (translationally controlled tumor protein, keratin, triosephosphateisomerase, eukaryotic translation initiation factor5A, NADPH-specific isocitratedehydrogenase),2ovary down-regulated proteins (Nesprin-1, proliferating cell nuclear antigen)and1testis-sepcific protein (peroxiredoxin-6) were indentified. Gene Ontology (GO) anlysisshowed cortical rod protein-2and cathepsin B involved in gonad development and oogoniacell-cycle control.Cathepsin B, one of differentially expressed proteins, was used for further research.According to conserved domains of closely related species of M. japonicus, degenerate primers were designed and partial sequence of cathepsin B was cloned. The full-length cDNA ofcathepsin B was obtained by SMART-RACE. It was of1525bp, including a5’-terminaluntranslated region (UTR) of24bp, a3’-terminal UTR of502bp and an open reading frame(ORF) of999bp. The translated protein was332amino acids in length, with a predictedmolecular weight (MW) of36.9kDa and a pI of6.64. The protein predicted by SignalP3.0ownssignal peptide of17amino acids. The Real-time PCR results show cathepsin B got the highestexpression level in ovary of M. japonicus, which was hardly detected in testis.pGEX-4T-2vector was used to constructe recombinant expression vector pGEX-CTSB.Plasmid of containing the right insertion was sequenced to confirm its identification andretransformed into E.coil BL21. Bacteria lysates prepared from IPTG induced cultures wereloaded directly onto SDS-PAGE, and a63KDa fusion protein was expressed.