The Study Of Cell-cycle And UPP Related Genes In The Gonad Development Of Mud Crab, Scylla Paramamosain

The mud crab Scylla paramamosain is one of important economic breeding crabs aroundthe southeast coast of China. To date, there are few reports about the molecular mechanism inthe regulation of gonad development of Scylla. The genes in the cell-cycle and UPP have beenreported in gonad development of other animal species. Therefore, cloning and study these geneswhich participate in the gonad development of Scylla paramamosain may shed lights onmolecular mechanism of gonad development of crab.This study selects the genes fragment about cell-cycle and UPP identified from the ESTlibrary of S. paramamosain constructed by our labratory, and clone their full length of cDNAsequence by employing SMART RACE technology. The expression level of these genes in thedifferent tissues of female crab and different gonad development stages are assessed by usingreal-time PCR and in situ hybridization technologies. The results are reported as follows:1) The full length cDNA of Sp-cdc2gene was of1593bp encoded an deduced proteincomposed by299amino acids. Real-time PCR analysis revealed that its expression level inovary was extreme higher than other tissues (P<0.01);and the gonad expression level of Sp-cdc2was no significant difference in different stages of ovary (P>0.05), and in T3stage was higherthan T1and T2stages (P<0.05).2) The full length cDNA of Sp-cycB was1492bp encoded391amino acids. The Real-timePCR results showed that its expression level in ovary was extreme higher than othertissues(P<0.01), and in the hepatopancreas was followed by the ovary; And the gonad expressionlevel in O6stage was significantly higher than front5stages and the testis(P<0.05),and in T2stage was also significantly higher than T1stage(P<0.05).3) The full length of Sp-cycH gene was1523bp which encoded a protein of332animo acids.The expression level of of Sp-cycH in ovary was extremely higher than other tissues (P<0.01),and muscle was followed. Meanwile,the expression level of Sp-cycH in O6atage was the highestamong other stages with significant disparation(P<0.05);Its level in the O5and T2stages weresignificantly higher than in T1stage.4) The full length cDNA of Sp-CKS1B was722bp that encoded a peptide of86amino acids.Its tissue expression level in the ovary was extremely higher than in other tissues (P<0.01), andfollwing in the blood, stomach and eye. And the highest gonad expression level of Sp-CKS1Bwas in T1stage, and was higher than previous5stages of ovary and T3stage (P<0.05), and theO6and T2stage were following the T1stage. 5) The full length of Sp-Ub was555bp which encoded a protein of154amino acids. Thereal-time PCR results showed that there were no significant different among the different tissues,whereas the highest was in the stomach, and the following was ovary. And its gonad expressionlevel in O6stage was the highest, and T2followed. The expression level in both of them weresignificantly higher than O2, O4and T3stages (P<0.05）, and no significant difference amongthe other stages.6) The full length cDNA of Sp-uce2was971bp which encoded a protein of160amino acids.The expression level of this gene in ovary was significantly higher than in othertissues(P<0.05),and the following was in stomach and heart. Its expression level at T2stage wassignificantly higher than other stages(P<0.05), and the O5stage was followed which also higherthan at other stages(P<0.05).7) The full length cDNA of Sp-UCHL3was of1804bp that encoded a protein of228aminoacids. Its expression level in the ovary was extremely higher than other tissues(P<0.01);Thegonad expression of Sp-UCHL3gene in O6stage was the highest, then in the O1and T3stages,and the lowest expression level was detected in T2stage. The expression level of Sp-UCHL3inboth the O1and O6were significantly higher than O3,O4,O5,T1and T2stages (P<0.05), and inT3was significantly higher than in O4,T1and T2stage (P<0.05).8) The full length cDNA of Sp-UCHL5was1217bp which encoded a protein of337aminoacids. The Sp-UCHL5expression level was highest in ovary which was extremely higher than inother tissues(P<0.01). The tissue with following level was eye. The gonad expression level in O6stage was the highest, and then following in the O3stage. Its expression level in both of thesetwo stages were extremely higher than in O4,T1,T2and T3stages. Its expression level in the3stages of testes was no significant difference.9）In situ hybridization analysis showed that the distribution patterns of Sp-cdc2andSp-cycB mRNA were identical in the crab ovaries and testes at different stages of the crab.TheSp-cdc2and Sp-cycB mRNA were presented in the spermatocyte abundantly but nohybridization signals of these two genes were detected in the spermatogonium duringspermatogenesis. Similarly, they were presented in the follicle cells and no hybridization signalsof these two genes were detected in the oocytes during oogenesis. Thus, it appears that Sp-cdc2and Sp-cycB transcripts have similar expression patterns during oogenesis and spermatogenesis.Conclusively, results of this project indicated that genes in the cell-cycle and UPP may playessential roles in the oogenesis and spermatogenesis.