Abstract: To investigate the role of theid4gene in the early heart development in zebrafish, and to explore the potential regulatory relationship between theid4gene and key genes of heart development, this study firstly predicted and analyzed the promoter sequences of theid4gene, constructed theid4promoter vector and carried out the dual luciferase reporter assay (Luciferase). Subsequently, using the Tol2 transposase system, a transgenic zebrafish line was constructed to provide experimental material for further in-depth research. Promoter analysis results showed no methylated CpG islands in the promoter cloning region and multiple transcription factor binding sites were identified. The dual-luciferase reporter gene assay demonstrated that 293T cells transfected with theid4promoter exhibited a 7.3-fold increase in luciferase activity compared with the null control (P<0.0001), suggesting that the clonedid4promoter had a strong expression activity. Additionally, it was found that the key cardiac development genesnkx2.5andgata5significantly activated and inhibited theid4promoter, respectively. Observation of zebrafish embryos injected with Tol2-id4-EGFP and Tol2 transposase revealed fluorescent expression in the zebrafish heart region at 2 and 3 days. Results suggested thatid4gene played a role in early heart development, laying a foundation for further elucidation of its molecular regulatory network and providing a zebrafish model for studying the pathogenesis mechanisms of congenital heart disease.

Key words: id4, zebrafish, heart development, promoter, Tol2 transposon