Study Of ERK Singal Transduction Pathway Relative Genes On The Ovarian Development In Scylla Paramamosain

Mud crab Scylla paramamosain is one of the commercially important crustacean speciesaround the coast of southeast China. With the development of crab artificial culture technologyand the molecular biotechnology, researchers pay more attention to egg quality and themolecular mechanisms of ovarian development. Extracellular signal-regulated protein kinase(ERK) signal transduction pathway is an old pathway which exists in all eukaryotic cells and isinvolved widely in cell growth, proliferation, differentiation, reproduction, apoptosis, anti-stressand other physiological processes. It was reported that ERK pathway takes a significant role inoocyte meiosis.In this study, ESTs containing partial sequences of ERK signal transduction pathway relatedgenes, derived from a normalized gonadal cDNA library of mud crab constructed in ourlaboratory, were selected for further investigation. SMART-RACE and other moleculartechniques were used to clone the full-length cDNA of ERK2, Ras-related protein Rap-1bprecursor (Rap-1b precursor), PAK1interacting protein1(PAK1-IP1) and G protein-coupledreceptor89(GPR89). The expression of these genes in crab tissues, organs and different stagesof ovaries were analyzed by quantitative real-time PCR. In situ hybridization histochemistry wasemployed to clarify the expression of Sp-erk2and Sp-gpr89mRNA. The consequences arereported as follows:(1) The full-length cDNA of Sp-erk2is1516bp, including a5’-terminal untranslated region(UTR) of19bp, a3’-terminal UTR (including a poly(A)+tail of18bp) of399bp and an openreading frame (ORF) of1098bp. The translated protein was365amino acids in length, with apredicted molecular weight (MW) of42kDa; and a pI of5.98. The real-time quantitative PCRresults show that Sp-erk2expressed in ovary (O) with a much higher significant expression level(P <0.05) when compared to other tissues and organs. The expression level of Sp-erk2raisedgradually as the ovarian development reached the highest expression peak at stage6(O6).Expression level of stage6ovary is significantly different from the first four stages (P <0.05),while there is no significant difference between O6and O5(P>0.05). In situ hybridizationshows that positive signals in early developing ovary were detected in follicular cells. Inrelatively mature and fully mature ovarian tissues, Sp-erk2was distributed in both follicular cellsand oocytes.(2) The full-length cDNA of Sp-rap-1b precursor is1078bp, including a5’-terminaluntranslated region (UTR) of142bp, a3’-terminal UTR (including a poly(A)+tail of17bp) of 381bp and an open reading frame (ORF) of555bp. The translated protein was184amino acidsin length, with a predicted MW of20.76kDa and a pI of5.25. The real-time quantitative PCRresults show that Sp-rap-1b precursor has the highest expression level in blood and ovary in thesecond place. The expression level of Sp-rap-1b precursor gradually rose as the ovariandevelopment, reached the highest expression peak at O6. The expression level of O6ovary issignificantly different with respect to the first five stages (P <0.01).(3) The full-length cDNA of Sp-pak1-ip1is1252bp, consisting of a5’-terminal UTR of31bp, a3’-terminal UTR of57bp (including a poly(A)+tail of31bp) and an ORF of1164bp.The translated protein is composed of387amino acids, with an estimated MW of43.12kDa; anda pI of6.23. The real-time quantitative PCR results reveals that Sp-pak1-ip1is expressed inovary with a significantly high expression level (P <0.05) compared with other tissues andorgans. The expression level of Sp-pak1-ip1raised gradually as the ovarian development reachedthe highest expression peak at O6. The expression level of O6ovary is significant different fromthe first five stages (P <0.05).(4) The full-length cDNA of Sp-gpr89is2053bp, consisting of a5’-terminal UTR of114bp, a3’-terminal UTR of541bp (including a poly(A)+tail of18bp) and an ORF of1398bp.The translated protein is composed of465amino acids, with an estimated MW of54kDa; and apI of8.95. The real-time quantitative PCR results reveals that the expression level of Sp-gpr89inovary is significantly higher (P <0.01) compared with other tissues and organs. In general,Sp-gpr89expression rose gradually as the ovary development, reaching the highest expressionlevel at O6. The expression level of O6ovary is significantly different with the other five stages(P <0.05). The expression levels were lower in the first five stages. In situ hybridization showsthat positive signals in early developing ovary were detected in cytoplasm. In relatively matureovarian tissues, Sp-gpr89was distributed not only in cytoplasm but also in the nucleus andaround cell membrane, expecially in the nucleolus.