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| Construction of Overexpression Vector and Functional Verification of Atg5 Gene in Chicken (Gallus gallus) |
| HAN Qi, WANG Qian-Qian, XU Yu-Fang, YU Jian-Feng, XU Lu*, GU Zhi-Liang* |
| School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China |
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Abstract Chicken occupies the first place in meat consumption, and excessive lipid deposition will reduce meat quality. Studies have shown that autophagy participates in liver lipid metabolism, improves lipid deposition and regulates lipid metabolism in poultry. This study aims to investigate the effect of autophagy related gene 5 (Atg5) on lipid metabolism related genes in chicken fibroblast cell line (DF-1). According to the amino acid sequence of chicken ATG5 protein in NCBI database, the physical and chemical properties of chicken ATG5 protein were analyzed by ExPASy database. The expression of Atg5 in chicken tissues was detected by qPCR. The overexpressed recombinant plasmid pcDNA3.1-Atg5 was constructed, and the expression of Atg5 was interfered by RNAi technology. The expressions of lipid metabolism-related genes: Acetyl CoA carboxylase (ApoB), fatty acid synthase (FASN), acetyl CoA carboxylase (ACACA), peroxisome proliferator-activated receptor-α (PPARα), forkhead box protein-1 (Foxo1), fatty acid-binding protein 7 (FABP7) and sortilin 1 (Sort1) were detected after interference and overexpression of Atg5. The results showed that chicken ATG5 protein was an unstable protein with relative molecular weight of 32.478 kD. Then Atg5 was expressed in all tissues, and the highest mRNA expression was found in breast muscle tissue. In addition, the overexpression vector pcDNA3.1-Atg5 was successfully constructed, and optimal interfering siRNA was siRNA-330. Overexpression or interference of Atg5 gene affected the expression of other lipid metabolism-related genes. Among them, after overexpression of Atg5, the expression level of ApoB decreased significantly, and the expression level of Sort1 increased significantly, the change of FABP7 was not significant. After interference with Atg5, the expression levels of FABP7 and ApoB were significantly increased, but the change of Sort1 was not significant, and it was speculated that the expression levels of Sort1, FABP7 and ApoB might be potentially related to Atg5. This study provides a reference basis for further study on the function of Atg5 gene in chicken.
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Received: 27 May 2021
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Corresponding Authors:
*sunnyxl2017@163.com; zhilianggu88@hotmail.com
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