Molecular Characterization, Expression Pattern, DNA Methylation and Gene Disruption of Figla in Blotched Snakehead (Channa maculata)

Abstract

<i>Figla</i> is one of the earliest expressed genes in the oocyte during ovarian development. In this study, <i>Figla</i> was characterized in <i>C</i>. <i>maculata</i>, one of the main aquaculture species in China, and designated as <i>CmFigla</i>. The length of <i>CmFigla</i> cDNA was 1303 bp, encoding 197 amino acids that contained a conserved bHLH domain. <i>CmFigla</i> revealed a female-biased expression patterns in the gonads of adult fish, and <i>CmFigla</i> expression was far higher in ovaries than that in testes at all gonadal development stages, especially at 60~180 days post-fertilization (dpf). Furthermore, a noteworthy inverse relationship was observed between <i>CmFigla</i> expression and the methylation of its promoter in the adult gonads. Gonads at 90 dpf were used for in situ hybridization (ISH), and <i>CmFigla</i> transcripts were mainly concentrated in oogonia and the primary oocytes in ovaries, but undetectable in the testes. These results indicated that Figla would play vital roles in the ovarian development in <i>C. maculata</i>. Additionally, the frame-shift mutations of <i>CmFigla</i> were successfully constructed through the CRISPR/Cas9 system, which established a positive foundation for further investigation on the role of <i>Figla</i> in the ovarian development of <i>C. maculata</i>. Our study provides valuable clues for exploring the regulatory mechanism of <i>Figla</i> in the fish ovarian development and maintenance, which would be useful for the sex control and reproduction of fish in aquaculture.