| Pelodiscus sinensis is highly nutritious,and is of high economical value.Domestic researches on the carapace of Pelodiscus sinensis mainly focus on its medicinal value,yet the mechanism of carapace formation has rarely been investigated.The carapacial ridge of Pelodiscus sinensis is a longitudinal ridge consisting of epithelial cells and mesenchymal cells that emerges at the 14th stage(TK14)of embryonic development on the ventral side of the body,which directs the arrangement of the ribs and plays a key role in the formation of carapase.However,it has not been clarified how carapacial ridge development is regulated.Therefore,this study identified the key candidate genes involved in the regulation of carapacial ridge formation through transcriptomic and translatomic analysis,then the candidate genes were further verified using molecular biotechnology such as fluorescence quantification and in situ hybridization,which provided the basis for elucidating the molecular mechanism of carapace development of Pelodiscus sinensis.The main contents and results of this study are as follows:(1)Using the Illumina HiSeq4000 sequencing platform,we performed high-throughput sequencing of the carapace tissue of Pelodiscus sinensis at TK14,TK15 and TK16 stages.The results demonstrated that a total of 20077 genes were transcribed and 18669 genes were translated at TK14;20045 transcribed and 17383 translated at TK15;19984 transcribed and 15028 translated at TK16.(2)Comparison of the transcriptomes between TK14 and TK15 revealed 349 differentially expressed genes,including 263 upregulated and 86 downregulated;comparison between TK15 and TK16 revealed 342 differentially expressed genes,including 206 upregulated and 136 downregulated;and comparison between TK14 and TK16 revealed 1203 differentially expressed genes,including 713 upregulated and 490 downregulated.Comparison of the translatomes between TK14 and TK15 revealed 3439 differentially expressed genes,including 1674 upregulated and 1764 downregulated;comparison of the translatomes between TK15 and TK16 revealed 3430 differentially expressed genes,including 1461 upregulated and 1969 downregulated;and comparison of the transcriptomes between TK14 and TK16 revealed 2594 differentially expressed genes,including 1969 upregulated and 900 downregulated.(3)GO functional enrichment analysis was performed for the differentially expressed genes in the transcriptomic and translatomic analyses.It was found that the differentially transcribed genes enriched in 38 GO functional classifications,and differentially translated genes enriched in 46 GO functional classifications.Many unigenes were involved in cellular process,metabolic process,cell,cell part,binding,or catalytic activity.KEGG enrichment analysis of the differentially expressed genes in transcriptome and translatome revealed enrichment in the Wnt signaling pathway,AMPK signaling pathway,TGF-beta signaling pathway and FoxO signaling pathway.It has been reported that Wnt signaling pathway may play an important regulatory role in carapace development of Pelodiscus sinensis,so this study further analyzed transcription and translation of genes involved in the Wnt signaling pathway during carapacial ridge development at TK14,TK15 and TK16.The results showed that many members of the Wnt signaling pathway showed differences in transcription and translation during carapacial ridge development at the 3 stages.Yet,Wnt5a was the only Wnt ligand that showed significant changes in both transcriptional and translational levels,so Wnt5a was considered a key candidate gene in the regulation of carapacial ridge development.(4)Temporal expression of Wnt5a in carapacial ridge at TK14,TK15,and TK16 was further verified by Real-time fluorescent quantitative PCR.The results showed that,consistent with transcriptome sequencing results,expression of Wnt5a was consistently up-regulated at TK14,TK15 and TK16,indicating that the sequencing data was reliable.(5)Whole-mount in situ hybridization(WISH)of the embryo further confirmed expression of Wnt5a in the carapacial ridge of Pelodiscus sinensis at TK14,TK15,and TK16.In this study,we detected significant changes in transcription and translation of components of the Wnt signaling pathway by transcriptomic and translatomic analysis,and identified Wnt5a as the only Wnt ligand that showed significant differences in both transcription and translation.Then we further verified spatial and temporal expression of Wnt5a in carapacial ridge of Pelodiscus sinensis by Real-time fluorescent quantitative PCR and WISH,which provided reference for studies on the mechanism of carapace formation of Pelodiscus sinensis. |
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