Cloning and expression of NQO1 gene and its regulatory relationship with the Nrf2 signaling pathway in Hyriopsis cumingii

This study cloned and functionally characterize the NAD(P)H quinone oxidoreductase 1 gene from Hyriopsis cumingii (HcNQO1), and to elucidated its potential interaction with the H. cumingii transcription factor Nrf2 (HcNrf2). The full-length cDNA HcNQO1 was cloned using the rapid amplification of cDNA ends technique. The tissue-specific expression pattern of the HcNQO1 mRNA was analyzed by real-time quantitative PCR (RT-qPCR). The promoter sequence of the HcNQO1 was amplified via high-efficiency thermal asymmetric interleaved PCR, and a dual-luciferase reporter assay was used to identify the HcNrf2 binding site. Additionally, protein expression levels of the HcNQO1 in different tissues were assessed using Western blot analysis. The HcNQO1 gene was found to contain an open reading frame of 1 005 bp, encoding a protein of 264 amino acids. Sequence alignment analysis revealed high conservation of HcNQO1 with homologous proteins from other bivalves. RT-qPCR results demonstrated that the HcNQO1 mRNA was expressed in multiple tissues, with significantly higher expression levels observed in the gonads and gills compared to other tissues. A 1 109 bp upstream promoter sequence of HcNQO1 was successfully obtained, and two antioxidant response elements were identified, confirming their essential roles in HcNrf2 binding. Furthermore, polyclonal antibodies against the HcNQO1 protein were generated, and Western blot analysis revealed that the protein expression pattern across tissues differed from that of its mRNA expression. This study successfully cloned and characterized the HcNQO1 gene of H. cumingii and elucidated its interaction with HcNrf2. These findings provide a fundamental theoretical basis for a deeper understanding of the biological function of the NQO1 gene in bivalves and its regulatory role in oxidative stress responses.