Abstract: The study aimed to explore the structural characteristics of specificity protein 1 (SP1) and its effect on milk fat synthesis of Chinese Holstein dairy cows. According to the SP1 gene sequence of Bos taurus published by NCBI (NM_001078027.1), the SP1 sequence conservation, physical and chemical properties, protein hydrophilicity, protein structure and interaction proteins of the encoded protein were analyzed by bioinformatics softwares. The CDS region of SP1 gene was amplified and cloned by PCR. Then, 6 healthy Holstein dairy cows were used to study the effect of SP1 on milk fat synthesis. The mammary tissues of lactating cows and dry cows were collected, and the expression of SP1 in mammary tissue at different stages was detected by real-time quantitative PCR and Western blot. Next, the mammary epithelial cells were isolated and purified from the lactating cows and the effect of SP1 on milk fat synthesis were investigated by SP1 overexpression and RNAi. Each experiment was preformed triplicate. The results showed that the sequence of SP1 gene was highly conserved among different species, and had the highest similarity with goat (98.94%). The CDS region of SP1 gene in dairy cows was 2 361 bp, encoding 786 amino acids. The molecular weight of the protein was 80 902.17 u and the theoretical isoelectric point was 6.942. The average hydrophobic index of SP1 was -0.438, which was an unstable hydrophilic protein. The SP1 contained 3 zinc finger structures, and the secondary structure of SP1 protein was mainly random coil (52.29%). The results of STRING protein interaction analysis showed that SP1 interacted with transcription factor AP-1 (JUN), estrogen receptor α (ERα), MYC proto oncogene protein (MYC), TATA box binding protein (TBP) and other proteins. Real-time quantitative PCR and Western blot results showed that the mRNA and protein levels of SP1 in mammary tissues of lactating cows were significantly higher than those in dry cows (P<0.01). Overexpression of SP1 in mammary epithelial cells of dairy cows significantly increased the triglyceride content (P<0.01), while knockdown of SP1 expression significantly decreased the intracellular triglyceride content (P<0.01). These results suggest that SP1 positively regulate the milk fat synthesis. The study on the structure and function of SP1 gene provides a theoretical basis for further study on the regulatory mechanism of SP1 on milk fat synthesis in lactating dairy cows.

Key words: Chinese Holstein cows, SP1 gene, gene cloning, bioinformatics, expression pattern