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

Liu, Binghua; Lin, Lei; Wang, Meng; Zhang, Jingjing; Yang, Yu; Wang, Hong-Yan; Shao, Changwei

doi:10.3390/ijms262311402

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

by

Binghua Liu

^1,2

,

Lei Lin

^1,2,

Meng Wang

^1,2,

Jingjing Zhang

^1,2,

Yu Yang

^1,2,

Hong-Yan Wang

^1,2

and

Changwei Shao

^1,2,*

¹

State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

²

Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266237, China

^*

Author to whom correspondence should be addressed.

Int. J. Mol. Sci. 2025, 26(23), 11402; https://doi.org/10.3390/ijms262311402

Submission received: 10 September 2025 / Revised: 18 October 2025 / Accepted: 23 October 2025 / Published: 25 November 2025

(This article belongs to the Section Molecular Biology)

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Abstract

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 (Oryzias melastigma). 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-slc12a1 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 O. melastigma, confirmed that Hnf1ba transcriptionally regulates the slc12a1 gene. Fragment deletion and site-directed mutagenesis assays revealed a Hnf1ba-binding sequence (GATTAATCATTTACT, located at −1877 to −1863) in the slc12a1 promoter. Based on this result, we conducted a targeted regulation experiment on the slc12a1 gene using the CRISPR-dCas9 & Sun-Tag system. The most effective activation of slc12a1 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.

Keywords: osmotic pressure regulation; Oryzias melastigma; Hnf1ba-slc12a1 signal pathway; transcription binding site; CRISPR-dCas9 & Sun-Tag system; targeted regulation experiment

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MDPI and ACS Style

Liu, B.; Lin, L.; Wang, M.; Zhang, J.; Yang, Y.; Wang, H.-Y.; Shao, C. Screening and Identification of Hnf1ba-slc12a1 Signal Pathway in Response to Low-Salinity Stress in Marine Medaka (Oryzias melastigma). Int. J. Mol. Sci. 2025, 26, 11402. https://doi.org/10.3390/ijms262311402

AMA Style

Liu B, Lin L, Wang M, Zhang J, Yang Y, Wang H-Y, Shao C. Screening and Identification of Hnf1ba-slc12a1 Signal Pathway in Response to Low-Salinity Stress in Marine Medaka (Oryzias melastigma). International Journal of Molecular Sciences. 2025; 26(23):11402. https://doi.org/10.3390/ijms262311402

Chicago/Turabian Style

Liu, Binghua, Lei Lin, Meng Wang, Jingjing Zhang, Yu Yang, Hong-Yan Wang, and Changwei Shao. 2025. "Screening and Identification of Hnf1ba-slc12a1 Signal Pathway in Response to Low-Salinity Stress in Marine Medaka (Oryzias melastigma)" International Journal of Molecular Sciences 26, no. 23: 11402. https://doi.org/10.3390/ijms262311402

APA Style

Liu, B., Lin, L., Wang, M., Zhang, J., Yang, Y., Wang, H.-Y., & Shao, C. (2025). Screening and Identification of Hnf1ba-slc12a1 Signal Pathway in Response to Low-Salinity Stress in Marine Medaka (Oryzias melastigma). International Journal of Molecular Sciences, 26(23), 11402. https://doi.org/10.3390/ijms262311402

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

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