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| Construction of Eukaryotic Expression Vector for Chicken (Gallus gallus) PGC-1α Gene and Its Effect on Lipid Metabolism |
| ZHOU Bo, WEI Zhi-Heng, LIU Shi-Hao, LIU Shu-Ming, XIA Lei, XU Lu, YU Jian-Feng, GU Zhi-Liang* |
| School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China |
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Abstract Peroxisome proliferator activated receptor γ coactivator-1 alpha (PGC-1α) is a transcriptional coactivator that influences various energy metabolism processes and plays a crucial role in fat deposition. To investigate the function and underlying mechanisms of the PGC-1α gene in lipid metabolism, a series of experiments were performed. The expression of PGC-1α in 12 different tissues was examined in chickens (Gallus gallus). Using RT-PCR, the CDS of the chicken PGC-1α gene was amplified and obtained, and a eukaryotic expression vector was constructed. The vector was then transfected into the leghorn male hepatoma (LMH) cell line, and Western blot analysis was used to confirm the expression of the PGC-1α protein. After overexpressing and knocking down PGC-1α gene in LMH cells, lipid droplet deposition was assessed using oil red O staining, total triglyceride (TG) content was measured by colorimetric assay, and the expression of lipid metabolism-related genes was analyzed through qPCR. The results showed that PGC-1α had the highest expression in cardiac tissue, with relatively high levels in leg muscle, kidney, and brain tissues. Western blot analysis confirmed that the overexpression vector successfully expressed the fusion protein in LMH cells. Overexpression of PGC-1α led to a extremely significant reduction in lipid deposition and TG content (P<0.01), while knockdown of PGC-1α resulted in a trend of increased lipid deposition and a significant rise in TG content (P<0.05). Moreover, overexpression of PGC-1α significantly downregulated the expression of fatty acid synthetase (FASN), peroxisome proliferator activated receptor γ (PPARγ) and acetyl-CoA carboxylase alpha (ACACA) genes (P<0.05 or P<0.01), while extremely significantly upregulated phosphoenolpyruvate carboxykinase 1 (PCK1) (P<0.01). Knockdown of PGC-1α extremely significantly decreased PCK1 expression (P<0.01). These findings suggested that the chicken PGC-1α gene likely exerts its effects by regulating the expression of these functional genes. By constructing overexpression and knockdown models of the PGC-1α gene, this study reveals its key regulatory role in chicken lipid metabolism, particularly through modulating the expression of FASN, PPARγ, ACACA, and PCK1, which affect lipid deposition and triglyceride levels. This study provides a valuable reference for further exploration of the functional mechanisms of PGC-1α in chicken lipid metabolism.
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Received: 15 July 2024
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Corresponding Authors:
* Zhilianggu88@hotmail.com
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