Comparative Analysis of the Male and Female Gonadal Transcriptome of Thamnaconus septentrionali

Abstract

Thamnaconus septentrionali has excellent breeding characteristics, is omnivorous, easy to domesticate, is especially suitable for netting, cleans the netting, and effectively reduces labor-costs. T. septentrionali is available in various sizes and is raised in the market at 100 g. Under water temperature of 18–25 ℃, after 5–6 months of breeding, it reaches commercial size. As the Yellow Sea and Bohai Sea are cold in winter, a suitable breeding cycle for green fins is from May to November annually. Under natural conditions, the spawning period of T. septentrionali in the Yellow Sea and Bohai Sea is from early May to early June, making good use of the suitable breeding cycle of net box culture. Therefore, reproductive regulation is required to advance the reproductive period of T. septentrionali. Previous studies have primarily focused on the expression and functional analysis of individual genes. In contrast, no studies exist on the overall expression analysis of gonad-related genes based on histology. Herein, we conducted the first transcriptome sequencing analysis of the spermatophores and ovaries of T. septentrionali using an Illumina high-throughput sequencing platform. Overall, 165, 981, 523 raw reads were sequenced from the cDNA, and 161, 234, 846 clean reads were obtained after quality control. The Q20 of each sample was > 98.43%, Q30 was > 95.25%, and GC content of the sample bases was greater than 52.31%. Our findings indicate that the sequencing data are accurate, of good quality, and were used for subsequent analyses. The obtained unigenes were annotated in the NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), PFAM (protein family), KOG (euKaryotic Ortholog Groups), SwissProt (a manually annotated and reviewed protein sequence database), GO (Gene Ontology), and KO (KEGG Orthology) databases, and 24, 009, 35, 057, 18, 453, 26, 971, 30, 294, 11, 420, and 21, 613 unigenes were annotated, respectively. The KEGG annotation results were divided into five major categories: organic systems, with 2, 115 genes in nine pathways; metabolism, with 1, 444 genes in 12 pathways; environmental information processing, with 1, 200 genes in three pathways; and genetic information processing, with 1, 128 genes in four pathways. Environmental information processing (1, 200 genes in three pathways), genetic information processing (1, 128 genes in four pathways), and cellular processes (1, 128 genes in four pathways). The number of genes enriched in cellular process-related pathways was 927 distributed in four related pathways. The signal transduction pathway was the most annotated KEGG pathway with 804 genes. The experiment used 24, 546 unigenes annotated in the KOG database, which were then classified into 26 categories based on their function. The largest number of unigenes were annotated in the signal transduction mechanism category (5, 411), followed by the general function prediction-only category (4, 252 unigenes), and posttranslational modification, protein folding and chaperone category (1, 713 unigenes). Unigene posttranslational modification, protein turnover, chaperones; transcription, 1, 613; and other related functions. There were 18, 954 differentially expressed genes (DEGs) in the spermatophore and ovary transcriptomes of the T. septentrionali, with 11, 265 genes up-regulated and 7, 689 down-regulated in the ovary relative to the spermatophore. The GO functional enrichment analysis revealed that DEGs were most enriched in the cellular component of biological processes, intracellular component of the cellular component subclass, and nucleic acid binding of the molecular functional subclass. GO analysis of the DEGs in males and females provided a partial list of genes associated with the reproductive process (GO: 0022414), sexual reproduction (GO: 0019953), gamete formation (GO: 0007276,), sex differentiation (GO: 0007548), and gonad development (GO: 00084060). To further characterize the specific functions of the DEGs in the spermatophores and ovaries of T. septentrionali, the enriched signaling pathways were further analyzed using the KEGG database. In total, 154 KEGG pathways were included, of which the top 30 were selected. The functional pathways with the highest number of expressed genes were closely associated with gonadal sex differentiation and development, including the insulin signaling pathway, steroid hormone biosynthesis, FoxO signaling pathway, M-TOR signaling pathway, and progesterone-mediated oocyte maturation. Among these, the FoxO signaling pathway regulatory genes are expressed in multiple processes, such as cell cycle control, apoptosis, and gluconeogenesis. The insulin signaling pathway plays an important role in regulating developmental, metabolic, and lifespan physiological processes, and the insulin signaling pathway is involved in gonadal development and maturation. Nine DEGs, bmp2, sox3, figla, hsd17b1, cyp19a, cyp17, foxl2, star, and amh, were selected for real-time fluorescence quantitative PCR (qRT-PCR) validation. The qPCR results were consistent with those of RNA-seq analysis. GO and KEGG enrichment results were analyzed and revealed that amh, cyp17, and star are imperative in male spermatogenesis of T. septentrionali; bmp2, foxl2, cyp19a, figla, and hsd17b1 are essential in female oogenesis and ovarian steroidogenesis. By comparing the transcriptome expression differences between the spermatophore and ovary of T. septentrionali, we elucidated the gene expression characteristics of the spermatophore and ovary, laying the foundation for future research on the mechanism of reproductive development of T. septentrionali and providing theoretical support for optimizing reproductive regulation techniques.

Keywords

• thamnaconus septentrionalis
• transcriptome
• gonads
• differentially expressed gene