| The green mud crab(Scylla paramamosain) is the one of most prevalent crabs on the southeast coast of China and an important commercial crab in aquaculture. More attention is being paid to the molecular regulation mechanisms of the sex determination and gonadal differentiation. This is because of the huge different biological and economic characters between the male and female crabs. In this study, in order to compare specifically/differentially expressed genes between testis and ovary of S. paramamosain, Roche 454 GS FLX massively parallel DNA sequencing technology was used to develop a deep-coverage transcriptomic sequencing of testis and ovary from the crab. In addition, some important functional genes were chosen to analyze their expression. This study will help us better understand the molecular regulational mechanism of sexual differentiation and gonadal development in S. paramamosain and provide useful insight and information for further research.Through Roche 454 sequencing, a total of 365116 reads(testis 171962, ovary 193154) were produced with an average sequence length of 285.4 bp from two cDNA libraries. After trimming and assembling, the two libraries generated 21791 isotigs, leaving 22814 reads as singlets. Using the Blastx program, 3471 unique sequences(2275 contigs and 1196 singletons) were annotated with known protein sequences from the Nr protein database. Gene Ontology and KEGG analysis showed that 224 unique sequences annotated with 224 Enzyme Codes were mapped into 174 KEGG pathways. On the basis of bioinformatics analysis, 4199 differentially expressed genes between ovary and testis, 10522 ovary-specifically expressed genes and 19013 testis-specially expressed genes were identified. Moreover, 33 ovary-specifically transcripts, 13testis-specifically transcripts and 34 gonad-differentially transcripts were confirmed by semi-quantitative and real-time PCR. Meanwhile, 8610 putative SSRs and 23879 potential SNPs were also identified.The full-length cDNA of Sp-OTUB is 1261 bp, including a 804 bp open-reading frame which encodes a protein of 365 amino acids. The protein contains a typical OTU-like cysteine protease catalytic domain and a putative catalytic triad which was composed of conserved cysteine, histidine, and possibly the aspartate residues. Real-time quantitative PCR showed that Sp-OTUB was expressed in all detected tissues of mature crabs with the highest expression in haemolymph and the lowest expression in testis. In the process of ovarian development,Sp-OTUB showed the largest expression in proliferation stage and had significant difference with other development stages. In secondary spermatocytes stage of testis, the expression of Sp-OTUB reached the highest and was significantly higher than primary spermatocytes stage and spermatids stage. Phylogenetic analysis based on the OTUB protein homologue sequences indicated that Sp-OTUB was separated together with OTUB1 of most species, and Sp-OTUB had the closest genetic relationship with Ixodes scapularis and Strongylocentrotus purpuratus.The full-length cDNA of Sp-wds is 1433 bp, including a 975 bp open-reading frame which encodes a protein of 365 amino acids. The protein contains seven WD repeat motif, forming a highly symmetrical cyclic "propeller" structure. Real-time quantitative PCR showed that Sp-wds was expressed in all detected tissues of mature crabs. The expression of Sp-wds in ovary was significantly higher than other tissues(P < 0.5), and its expression level in testis also is higher. In ovarian development process, the expression of Sp-wds showed a trend of rising first and then falling. It reached the highest level in secondary virellogenesis stage of ovary. With the mature of testis, the expression level of Sp-wds reduces gradually. It reached the minimum in the spermatids stage, significantly lower than the other two stages(P < 0.5). |
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