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Fig. 1 Circadian expression of the roraa gene in zebrafish is regulated by the circadian clock. A Under LD (light/dark) conditions, qRT‒PCR was used to measure the mRNA expression levels of the roraa gene in wild-type (WT) zebrafish. JTK cycle analysis revealed that roraa expression exhibited significant circadian oscillation (p < 0.05). B In situ hybridization was performed to detect roraa gene expression under LD conditions, revealing that roraa expression during the day was significantly greater than that at night, demonstrating circadian rhythmic differences. C Statistical results from in situ hybridization under LD conditions revealed that roraa expression was significantly higher during the day than at night. D Potential cis-regulatory elements on the roraa promoter fragment were predicted via the JASPAR database; two D-box elements, two E-box elements, and one RORE element, which may play key regulatory roles in the circadian expression of roraa, were identified. E Dual-luciferase reporter assays revealed that the heterodimers formed by the Bmal1b and Clocka proteins, as well as the Tefa, Roraa, Rorb, and Rorc proteins, significantly activated the expression of the roraa gene. F Diagram showing the deletion of E-box elements in the roraa-luc plasmid. G The results from dual-luciferase reporter assays demonstrated that the activation of the roraa gene by Bmal1b and Clocka was significantly reduced when E-box1 and E-box2 elements were deleted from the roraa-luc plasmid, indicating the important role of E-box elements in the regulation of roraa expression. All the data are presented as the means ± standard errors of the means (SEMs) (n = 3); ** indicates p < 0.01, **** indicates p < 0.0001 (Student's t test)