The Study Of Genes Involved In Sumoylation In The Gonad Development Of Green Mud Crab(Scylla Paramamosain)

Sumoylation is an important post-translational modification of proteins which regulates thetranscriptional regulation, intracelluar transport, signaling, cell cycle, mitochondrial fission,chromosome segregation, DNA repair, genomic stability and other biological processes.Recently, sumoylation involved in reproductive regulation have been reported in some animal,especially mammals and fruit fly. To date, little is known about the distinct roles of sumoylationin crustacean. In the present study, we report the identification and characterization of genesinvolved in sumoylation from green mud crab Scylla paramamosain using an approach whichcombines expressed sequence tag (EST) and rapid amplification cDNA end (RACE).Meanwhile, the tissue distribution and the expression profiles of these genes in the differentgonad developing stages of mud crab were determined by real-time PCR. Then, the transcriptfrom SpSUMO-1during gametogenesis and the transcripts from Sp-Uba2、Sp-Uce2andSp-SENP1during oogenesis were detected by in situ hybridization. Furthermore, the expressionof SpSUMO-1protein during gametogenesis was examined using immunocytochemical method.The results were reported as follows:1) The full length cDNA of SpSUMO-1gene (GenBank: HM581660) is of732bp, including a282bp open reading frame which encodes a protein of93amino acids. The full lengthcDNA of Sp-Uba2(GenBank: JX081268) is of2519bp, including a1944bp open readingframe which encodes a protein of647amino acids. The full length cDNA of Sp-NSE2(GenBank: JX081269) is of1144bp, including a633bp open reading frame which encodesa protein of210amino acids. The full length cDNA of Sp-SENP1(GenBank: JX081270) isof2077bp, including a1455bp open reading frame which encodes a protein of484aminoacids.2) Tissue distribution analysis showed that SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1were expressed more abundantly in the ovary than in other tissues (P<0.05). Moreover, theexpression profiles of SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1in the different gonaddeveloping stages revealed that the highest expression level of SpSUMO-1was detected atproliferation stage, gradually reduced as the ovarian development progressed, and in testis,the expression level of SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1were relativelystable at different stages of testis development. Interestingly, the expression level of SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1in early stages (Stage I-III) of ovary issignificantly higher than in all development stages of testis (P<0.05).3) The distribution of SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1mRNA were observed inthe crab gametogenesis by in situ hybridization. In oogenesis, SpSUMO-1、Sp-Uba2、Sp-NSE2and Sp-SENP1transcripts presented at cytoplasm and nucleus of oocytes fromproliferation stage to primary vitellogenesis stage, but only appeared in the nucleus ofoocytes in secondary and tertiary vitellogenesis stage. In spermatogenesis, the strongpositive signals of SpSUMO-1presented at nuclei of primary and secondary spermatocytes,spermatids and spermatozoa. Interestingly, acrosomal tubules of spermatozoa were alsodetected the positive signal.4) The distribution of SpSUMO-1protein was observed in the crab gametogenesis byimmunocytochemical method. SpSUMO-1protein was localized in cytoplasm of oogoniaand different developing oocytes during oogenesis. On the other hand, SpSUMO-1proteinwas localized in spermatogonium, primary spermatocyte, secondary spermatocyte andspermatid, but the positive signal was only detected in the nucleus of spermatozoa inspermatogenesis.All these results suggested that sumoylation may play essential roles in the gametogenesisof the crustacea.