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| Effects of AGPAT4 Gene Interference and Overexpression on Lipid Metabolism in Mammary Epithelial Cells of Xinong Saanen Dairy Goats (Capra hircus) |
| YAO Wei-Wei, LUO Jun*, WANG Xin-Pei, QU Xiao-Peng, TIAN Hui-Bin, NIU Hui-Min |
| College of Animal Science and Technology, Northwest A&F University/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, China |
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Abstract AGPAT4 (1-acylglycerol-3-phosphate O-acyltransferase 4) is a member of the 1-acylglycerol-3-phosphate O acyltransferase family of enzymes that catalyze synthesis of the triacylglycerol (TAG). In order to clarify the gene sequence and protein structure of AGPAT4 and to preliminarily analyze the effect of AGPAT4 on lipid metabolism in goat mammary epithelial cells (GMECs), In this study, the mammary epithelial cells of Xinong Saanen dairy goat (Capra hircus) were used as experimental materials, the AGPAT4 gene was cloned by reverse transcription-PCR (RT-PCR). Based on the cloned sequence, the bioinformatics analysis was conducted using online website and tools. qPCR was applied to detect the expression of GMECs lipid metabolism-related genes after AGPAT4 interference and overexpression. Then, the content of triacylglycerol in GMECs was detected after interference or overexpression of AGPAT4. The results showed that the CDS region of AGPAT4 gene was successfully cloned, with 1 137 bp (sequence ID: XM_005684959.3), encoding 378 amino acids, protein molecular weight of 43 939.35 u, theoretical pI value of 8.92, transmembrane structure and no signal peptide, which was an unstable protein with positive charge. The AGPAT4 protein interacted with AGPAT1, AGPAT2, lipin3 (LPIN3), diacylglycerol kinases (DGK), phospholipase D (PLD), cytidine diphosphate-diacylglycerol synthase (CDS) and other proteins. The downregulation of AGPAT4 via mRNA interference caused a significant reduction in acetyl coa carboxylase (ACC) gene expression (P<0.05). Overexpression of AGPAT4 significantly up-regulated the expression of stearoyl-coenzyme A desaturase (SCD1) and ACC genes (P<0.05) and significantly promoted the accumulation of intracellular TAG (P<0.05). These results indicated that AGPAT4 gene had positive regulation on lipid synthesis in GMECs. This study provides basic data for further research on the regulatory mechanism of AGPAT4 gene on milk fat metabolism of goat milk.
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Received: 25 July 2022
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Corresponding Authors:
* luojun@nwafu.edu.cn
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