Abstract
The SCD is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids (MUFAs) in dairy cows; however, its role in the mammary gland of buffalo is not well understood. In this study, we isolated and characterized the complete coding sequence (CDS) of the buffalo SCD gene from mammary gland tissue and investigated its effects on milk fat synthesis using bioinformatics analyses, tissue differential expression detection, and cellular functional experiments. The cloned SCD gene has a CDS length of 1080 bp, encoding a protein of 359 amino acids. This protein is hydrophilic, lacks a signal peptide, and contains four transmembrane domains, including 10 conserved motifs and a Delta9-FADS domain, characteristic of the fatty acid desaturase family involved in unsaturated fatty acid biosynthesis within the endoplasmic reticulum. Molecular characterization revealed that the physicochemical properties, conserved domains, structures, and functions of buffalo SCD are highly similar to those in other Bovidae species. Among the tissues analyzed, SCD expression was highest in the mammary gland during lactation and in the cerebellum during dry-off period. Notably, SCD expression in the mammary gland was significantly higher during lactation compared to the dry-off period. Subcellular localization experiments confirmed that SCD functions in the endoplasmic reticulum of buffalo mammary epithelial cells (BuMECs). Functional overexpression and interference experiments in BuMECs demonstrated that SCD promotes milk fat synthesis by affecting the expression of lipid synthesis-related genes such as ACACA, FASN, and DGAT1, as well as milk fat regulatory genes like SREBFs and PPARG, thereby influencing intracellular triglyceride (TAG) content. Additionally, 18 single-nucleotide polymorphisms (SNPs) were identified in the buffalo SCD gene, with a specific SNP at c.-605, showing potential as molecular markers for improving milk production traits. These findings highlight that the SCD gene is a key gene in buffalo milk fat synthesis, involved in the de novo synthesis of milk fatty acids.