Screening and Identification of Hnf1ba-slc12a1 Signal Pathway in Response to Low-Salinity Stress in Marine Medaka (Oryzias melastigma)

Euryhaline fishes provide excellent material for the theoretical study of the broad-spectrum adaptability of organisms and the use of low-salinity and even freshwater environments, or high-salinity and seawater environments, for the domestication of fishes. Here, we studied the molecular mechanisms of osmotic pressure regulation in a euryhaline fish, marine medaka (<i>Oryzias melastigma</i>). As the fish progressed from seawater to freshwater, the changes in stress indicators (cortisol-COR; malondialdehyde-MDA; reactive oxygen species-ROS; superoxide dismutase-SOD) indicated that they gradually adapted to the freshwater environment. The transcriptome analysis also showed that there were 6850 DEGs (differentially expressed genes) involved in the process. By analyzing these DEGs deeply, we screened and identified the Hnf1ba-<i>slc12a1</i> signal pathway involved in osmotic pressure regulation. The results of a dual-luciferase reporter assay in HEK293T cells, as well as an overexpression experiment by in vitro cultured gill cells of <i>O. melastigma</i>, confirmed that Hnf1ba transcriptionally regulates the <i>slc12a1</i> gene. Fragment deletion and site-directed mutagenesis assays revealed a Hnf1ba-binding sequence (GATTAATCATTTACT, located at -1877 to -1863) in the <i>slc12a1</i> promoter. Based on this result, we conducted a targeted regulation experiment on the <i>slc12a1</i> gene using the CRISPR-dCas9 & Sun-Tag system. The most effective activation of <i>slc12a1</i> gene expression was observed in the sgRNA2 group. These results enhance our understanding of adaptation mechanisms in salt-tolerant fish and provide a reference for efficiently promoting the domestication of fish adaptive to salinity changes.