Objective: To identify and functionally characterize the pig septin12 gene, including its expression pattern, subcellular localization, and interacting proteins, to explore its potential roles in male reproduction.Methods: Full-length septin12 cDNA was obtained from the Banna mini-pig inbred line (BMI) testis using RACE.qPCR and western blot were employed to assess tissue-specific expression in BMI pigs.Subcellular localization was determined by fluorescence microscopy following transfection of EGFP-tagged septin12.A yeast two-hybrid (Y2H) screen using an adult BMI testis cDNA library identified candidate interacting proteins, which were validated by Co-IP and IF.GO analysis was performed to determine the functional categories of the interactors. Results:The cloned septin12 cDNA from the BMI testis was 1289 bp in length.Septin12 mRNA and protein expression was predominant in the BMI testis, suggesting its critical role in male reproductive function.Septin12 localized to both fibrous filamentous and punctate cytoplasmic structures.Thirteen interacting proteins were identified by Y2H, including ATAD5, ATP1B3, CENPL, ENO1, and septin5, with GO analysis indicating enrichment in mitotic nuclear division, cell growth, and G2/M phase transition.Further, Co-IP and IF confirmed interactions between septin12 and ETFA as well as LDHB, supporting the results of the Y2H A c c e p t e d A r t i c l e 4 screen. Conclusion:This study identified and characterized the septin12 gene in BMI pig, highlighting its predominant expression in the testis and implicating it in male reproductive function.Protein interaction analysis uncovered partners involved in cell division and metabolism, suggesting potential mechanisms by which septin12 may influence spermatogenesis.Co-IP and IF further validated interactions with ETFA and LDHB.These findings lay the groundwork for future investigations into the role of septin12 in male fertility.
Wang et al. (Thu,) studied this question.