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33
2025年8月11日 星期一
农业生物技术学报  2025, Vol. 33 Issue (2): 344-354    DOI: 10.3969/j.issn.1674-7968.2025.02.009
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
PGC-基因真核表达载体的构建及其对脂类代谢的作用
周博, 魏志恒, 刘世昊, 刘书鸣, 夏磊, 徐璐, 郁建锋, 顾志良*
常熟理工学院 生物与食品工程学院,常熟 215500
Construction of Eukaryotic Expression Vector for Chicken (Gallus gallus) PGC- 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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摘要 过氧化物酶体增殖物激活受体γ共激活因子-1α (peroxisome proliferator activated receptor γ coactivator-1 alpha, PGC-1α)是一种转录共激活因子,参与调控体内各类能量代谢过程,对脂肪沉积起着关键作用。为探讨PGC-基因在鸡(Gallus gallus)脂质代谢中的作用和机制,本研究检测了PGC-基因在鸡12种组织中的表达情况,通过RT-PCR的方法,扩增并获得鸡PGC-基因CDS片段,构建真核表达载体,Western blot检测表达载体转染鸡肝癌细胞系(leghorn male hepatoma, LMH)后能否表达PGC-1α蛋白;过表达和敲低LMH细胞中PGC-基因后,油红O染色检测脂滴沉积变化,比色法测定总甘油三酯(total triglyceride, TG)含量的变化,qPCR检测对脂质代谢相关基因表达的影响。结果显示,PGC-基因在心脏组织中表达量最高,在腿肌、肾和脑组织中表达量较高;Western blot检测发现构建的过表达载体转染LMH细胞能成功表达融合蛋白;LMH细胞中过表达PGC-基因会使脂质沉积和TG含量极显著下降(P<0.01),而PGC-基因敲低后脂质沉积有增加趋势,且TG含量显著增加(P<0.05)。LMH细胞中PGC-基因过表达使得脂肪酸合成酶(fatty acid synthetase, FASN)基因和过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor γ, PPARγ)基因的表达极显著下调(P<0.01),乙酰辅酶A羧化酶α(acetyl-CoA carboxylase alpha, ACACA)基因的表达显著下调(P<0.05),磷酸烯醇式丙酮酸羧激酶1 (phosphoenolpyruvate carboxykinase 1, PCK1)基因表达极显著上调(P<0.01);PGC-基因敲低使PCK1基因表达极显著下降(P<0.01)。上述结果表明,鸡PGC-基因可能是通过调节上述功能基因表达而发挥作用。本研究通过构建PGC-基因的过表达和敲低模型,揭示了该基因在鸡脂质代谢中的关键调控作用,尤其是通过调节FASNPPARγACACAPCK1等基因的表达,影响脂质沉积和甘油三酯含量,表明PGC-基因可能在调控鸡体内脂肪代谢中扮演重要角色。本研究为深入理解PGC-在鸡脂质代谢中的功能机制提供了参考依据。
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周博
魏志恒
刘世昊
刘书鸣
夏磊
徐璐
郁建锋
顾志良
关键词 过氧化物酶体增殖物激活受体λ共激活因子-1α (PGC-)基因真核表达鸡肝癌细胞系(LMH)细胞脂代谢脂肪沉积    
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- gene in lipid metabolism, a series of experiments were performed. The expression of PGC- in 12 different tissues was examined in chickens (Gallus gallus). Using RT-PCR, the CDS of the chicken PGC- 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- 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- 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- led to a extremely significant reduction in lipid deposition and TG content (P<0.01), while knockdown of PGC- resulted in a trend of increased lipid deposition and a significant rise in TG content (P<0.05). Moreover, overexpression of PGC- 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- extremely significantly decreased PCK1 expression (P<0.01). These findings suggested that the chicken PGC- gene likely exerts its effects by regulating the expression of these functional genes. By constructing overexpression and knockdown models of the PGC- 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- in chicken lipid metabolism.
Key wordsChicken    Peroxisome proliferator-activated recepto λ coactivator-1α (PGC-) gene    Eukaryotic expression    Leghorn male hepatoma (LMH) cells    Lipid metabolism    Fat deposition
收稿日期: 2024-07-15     
中图分类号: S831.2
基金资助:江苏省自然科学基金(BK20191476); 常熟理工学院2023大学生创业创新计划资助
通讯作者: * Zhilianggu88@hotmail.com   
引用本文:   
周博, 魏志恒, 刘世昊, 刘书鸣, 夏磊, 徐璐, 郁建锋, 顾志良. 鸡PGC-基因真核表达载体的构建及其对脂类代谢的作用[J]. 农业生物技术学报, 2025, 33(2): 344-354.
ZHOU Bo, WEI Zhi-Heng, LIU Shi-Hao, LIU Shu-Ming, XIA Lei, XU Lu, YU Jian-Feng, GU Zhi-Liang. Construction of Eukaryotic Expression Vector for Chicken (Gallus gallus) PGC- Gene and Its Effect on Lipid Metabolism. 农业生物技术学报, 2025, 33(2): 344-354.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2025.02.009     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2025/V33/I2/344
 
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