Abstract:

The study aims to investigate the expression characteristics of growth factor independent 1B (GFI1B) and Tropomodulin (TMODs) family member TMOD4 gene in common carp (Cyprinus carpio) following acute and chronic infection with Aeromonas veronii (A. veronii), in order to reveal the molecular mechanisms by which these two genes participate in antibacterial infection through the regulation of hematopoietic cell generation and immune cell migration pathways. In this study, the full-length coding regions (CDS) of GFI1B and TMOD4 in common carp were cloned, and their sequences were subjected to bioinformatics analysis. The expression patterns of GFI1B and TMOD4 in response to A. veronii infection were analyzed using two models: chronic infection in carp for 15 days and acute infection in carp leukocyte cell lines for 24 hours. Protein-protein interaction networks of GFI1B and TMOD4 were predicted using STRING12.0 software based on zebrafish homologous proteins, followed by joint analysis incorporating expression data. The GFI1B CDS region spanned 762 bp and encoded a 253-amino acid protein, whereas the TMOD4 CDS comprised 1032 bp and translates into a 343-amino acid protein. The GFI1B protein features a conserved zinc finger domain at its C-terminus, while TMOD4 contains both Tropomodulin and ribonuclease inhibitor domains. The common carp GFI1B shared over 91% identity with Sinocyclocheilus anshuiensis but only 43.92% to 65.56% identity with other teleosts, in contrast to TMOD4, which exhibited 80.88% to 96.79% similarity across teleosts. Both genes were expressed in all tested tissues of healthy carp. GFI1B exhibited the highest expression level in the head kidney, while TMOD4 showed expression levels second only to muscles and skin in the head kidney. STRING prediction revealed that GFI1B interacts with multiple hematopoietic transcription factors, and TMOD4 interacts with myotonic structural proteins and cytoskeletal regulatory factors. After 15 days A. veronii infection, GFI1B and TMOD4 transcripts were significantly upregulated in the head kidney, reaching 6.75-fold and 5.66-fold higher levels compared to controls, respectively. In carp leukocyte cell line, acute infection with A. veronii, the expression levels of GFI1B and TMOD4 genes rapidly increased at 6 hours post-infection, significantly decreased at 12 hours, and rose again at 24 hours. These results demonstrated that after acute and chronic A. veronii infections, GFI1B and TMOD4 exhibited high expression in the head kidney tissue and carp leukocyte cells, highlighting these two genes may synergistically play a crucial role in anti-A. veronii defense. Overall, this work provides a foundational basis for further investigation into the antibacterial regulatory functions of GFI1B and TMOD4 in teleosts.

Key words: Cyprinus carpio, growth factor independent 1B transcriptional repressor (GFI1B), Tropomodulin 4 (TMOD4), Aeromonas veronii (A. veronii), carp leucocyte cell line (CLC)