Cloning of Nile tilapia IL-34 Gene and its Function in Antibacterial Immune Response

Objective To clone the interleukin-34 (IL-34) gene in Nile tilapia, conducting bioinformatics analysis, and investigating its expression pattern in different tissues of tilapia. Further more, the expression differences of IL-34 in the head kidney, spleen and peripheral blood under different stimuli were explored to provide a reference for the immune regulatory role of IL-34 in bacterial infection of tilapia.

Method Primers were designed based on the OnIL-34 gene sequence of Nile tilapia, homologous genes were identified, and the coding region (CDS) of OnIL-34 was cloned. The amino acid sequence, protein domain composition, and phylogenetic analysis were conducted, followed by real-time quantitative PCR (qRT-PCR) to detect gene distribution in healthy tissues and expression changes induced by Streptococcus agalactiae, Aeromonas hydrophila, and lipopolysaccharide (LPS).

Result The open reading frame (ORF) of OnIL-34 is 648 bp long, encoding 215 amino acids with a predicted molecular weight of 25.26 kDa and theoretical isoelectric point of 8.52. The protein sequence contains a 20-amino-acid transmembrane domain and an 189-amino-acid extracellular domain at the N-terminus. Phylogenetic analysis showed that the Nile tilapia IL-34 amino acid sequence exhibited the highest similarity (98.6%) to Simochromis diagramma, with phylogenetic tree results indicating coalescence with the S. diagramma IL-34 lineage. qRT-PCR results demonstrated that OnIL-34 is expressed in all tissues of healthy Nile tilapia, with the highest expression detected in the stomach (26.26-fold higher than intestinal tissue). The liver, heart, and gill tissues showed significantly lower levels at 26.09, 17.14, and 15.85 times the intestinal level, respectively. After infection by S. agalactiae and A. hydrophila, IL-34 expression in the head kidney was significantly elevated (P < 0.05). Following LPS stimulation for 6 hours, peripheral blood IL-34 levels increased markedly, with a significant rise in the head kidney at 24 hours. All tissues including head kidney, spleen, and peripheral blood leukocytes exhibited elevated IL-34 expression following pathogen exposure or LPS stimulation.

Conclusion The upregulated IL-34 in immune tissues following pathogen infection and LPS stimulation preliminarily confirms its critical role in host immunity and inflammatory responses. However, the specific immunomodulatory mechanisms require further investigation.