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De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology.

ABSTRACT: The soft-shelled turtle Pelodiscus sinensis is a high-profile turtle species because of its nutritional and medicinal value in Asian countries. However, little is known about the genes that are involved in formation of their nutritional quality traits, especially the molecular mechanisms responsible for unsaturated fatty acid and collagen biosynthesis. In the present study, the transcriptomes from six tissues from Pelodiscus sinensis were sequenced using an Illumina paired-end sequencing platform. We obtained more than 47 million sequencing reads and 73954 unigenes with an average size of 754 bp by de-novo assembly. In total, 55.19% of the unigenes (40814) had significant similarity with proteins in the National Center of Biotechnology Information (NCBI) non-redundant protein database and Swiss-Prot database (E-value <10(-5)). Of these annotated unigenes, 9156 and 11947 unigenes were assigned to 52 gene ontology categories (GO) and 25 clusters of orthologous groups (COG), respectively. In total, 26496 (35.83%) unigenes were assigned to 242 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, we found a number of highly expressed genes involved in the regulation of P. sinensis unsaturated fatty acid biosynthesis and collagen formation, including desaturases, growth factors, transcription factors, and extracellular matrix components. Our data represent the most comprehensive sequence resource available for the Chinese soft-shelled turtle and could provide a basis for new research on this turtle as well as the molecular genetics and functional genomics of other terrapins. To our knowledge, we report for the first time, the large-scale RNA sequencing (RNA-Seq) of terrapin animals and would enrich the knowledge of turtles for future research.

SUBMITTER: Wang W

PROVIDER: S-EPMC3542959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Chinese soft-shelled (Pelodiscus sinensis) turtle exhibits obvious sex dimorphism, which leads to the higher economic and nutritional value of male individuals. Exogenous hormones can cause the transformation from male to female phenotype during gonadal differentiation. However, the molecular mechanism related to the sexual reversal process is unclear. In this study, we compared the difference between the small RNAs of male, female, and pseudo-female turtles by small RNA-seq to understand the sexual reversal process of Chinese soft-shelled turtles. A certain dose of estrogen can cause the transformation of Chinese soft-shelled turtles from male to female, which are called pseudo-female individuals. The result of small RNA-seq has revealed that the characteristics of pseudo-females are very similar to females, but are strikingly different from males. The number of the microRNAs (miRNAs) of male individuals was significantly less than the number of female individuals or pseudo-female individuals, while the expression level of miRNAs of male individuals were significantly higher than the other two types. Furthermore, we found 533 differentially expressed miRNAs, including 173 up-regulated miRNAs and 360 down-regulated miRNAs, in the process of transformation from male to female phenotype. Cluster analysis of the total 602 differential miRNAs among females, males, and pseudo-females showed that miRNAs played a crucial role during the sexual differentiation. Among these differential miRNAs, we found 12 miRNAs related to gonadal development and verified their expression by qPCR. The TR-qPCR results confirmed the differential expression of 6 of the 12 miRNAs: miR-26a-5p, miR-212-5p, miR-202-5p, miR-301a, miR-181b-3p and miR-96-5p were involved in sexual reversal process, which was consistent with the results of omics. Using these six miRNAs and some of their target genes, we constructed a network diagram related to gonadal development. We suggest that these miRNAs may play an important role in the process of effective sex reversal, which would contribute to the breeding of all male strains of Chinese soft-shelled turtles.

Project description:BackgroundAquatic animals show diverse body coloration, and the formation of animal body colour is a complicated process. Increasing evidence has shown that microRNAs (miRNAs) play important regulatory roles in many life processes. The role of miRNAs in pigmentation has been investigated in some species. However, the regulatory patterns of miRNAs in reptile pigmentation remain to be elucidated. In this study, we performed an integrated analysis of miRNA and mRNA expression profiles to explore corresponding regulatory patterns in embryonic body colour formation in the soft-shelled turtle Pelodiscus sinensis.ResultsWe identified 8 866 novel genes and 9 061 mature miRNAs in the skin of Chinese soft-shelled turtles in three embryonic stages (initial period: IP, middle period: MP, final period: FP). A total of 16 563 target genes of the miRNAs were identified. Furthermore, we identified 2 867, 1 840 and 4 290 different expression genes (DEGs) and 227, 158 and 678 different expression miRNAs (DEMs) in IP vs. MP, MP vs. FP, and IP vs. FP, respectively. Among which 72 genes and 25 miRNAs may be related to turtle pigmentation in embryonic development. Further analysis of the novel miRNA families revealed that some novel miRNAs related to pigmentation belong to the miR-7386, miR-138, miR-19 and miR-129 families. Novel_miR_2622 and novel_miR_2173 belong to the miR-19 family and target Kit and Gpnmb, respectively. The quantification of novel_miR_2622 and Kit revealed negative regulation, indicating that novel_miR_2622 may participate in embryonic pigmentation in P. sinensis by negatively regulating the expression of Kit.ConclusionsmiRNA act as master regulators of biological processes by controlling the expression of mRNAs. Considering their importance, the identified miRNAs and their target genes in Chinese soft-shelled turtle might be useful for investigating the molecular processes involved in pigmentation. All the results of this study may aid in the improvement of P. sinensis breeding traits for aquaculture.

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De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology.

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