Genetic and Functional Dissection of the <i>NFKB2</i> Gene: Implications for Milk Fatty Acid Biosynthesis in Dairy Cattle

Milk fat, an essential component of bovine milk, significantly impacts human health and dairy industry profitability. We previously identified that <i>NFKB2</i> gene was a key candidate for milk fatty acids (FAs) in dairy cattle. This study aimed to investigate the role of <i>NFKB2</i> for regulating milk FAs in Holstein cattle through post-GWAS analysis. Knockdown of <i>NFKB2</i> using siRNA in bovine mammary epithelial cells (BMECs) significantly reduced triglyceride concentrations, lipid droplet secretion, and the expressions of 17 key genes involved in lipid metabolism pathways, including PI3K-AKT, NF-κB, MAPK, adipocytokine, PPAR, and AMPK signaling pathways. These findings highlight <i>NFKB2</i> as a critical regulator of milk FA composition through its interactions with key lipid biosynthesis pathways. Further, we constructed a new Chinese Holstein population including 1,065 cows, and performed the genotyping and milk FAs measurements in these individuals. By SNP identification and association analysis, SNP g.22889812C/T in the 5’ flanking region of <i>NFKB2</i> was significantly associated with multiple milk FAs, including higher C14:1, C17:0, C18:0, and C14 index levels in allele C group, and higher C6:0, C8:0, C14:0, C16:1, and C16 index levels in allele T group. Using computational predictions, luciferase assays, EMSA and super-shift EMSA, we revealed that SNP g.22889812C/T downregulated <i>NFKB2</i> transcription activity with allele T selectively binding with transcription factor (TF) ZNF282. This study not only advanced the understanding of genetic mechanisms underlying milk fat production but also identifies <i>NFKB2</i> and its causal mutation g.22889812C/T as the promising molecular markers for selective breeding to optimize milk fat traits.<br>