鸡<em>Atg5</em>基因过表达载体的构建与功能验证

doi:10.3969/j.issn.1674-7968.2022.04.010

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摘要鸡肉占肉质消费品首位,过多的脂质沉积会降低肉质。研究表明自噬参与肝脏脂质代谢,改善脂质沉积,从而调控禽类脂质代谢。本研究旨在探究自噬相关基因5 (autophagy related gene 5, Atg5)对鸡(Gallus gallus)成纤维细胞系DF-1细胞内的脂质代谢相关基因的影响。根据NCBI数据库中鸡ATG5蛋白(NP_001006409.1)氨基酸序列,利用ExPASy数据库对鸡ATG5蛋白进行理化特性分析;利用qPCR技术检测鸡各组织中Atg5基因的表达情况;构建过表达重组质粒pcDNA3.1-Atg5,并且利用RNAi技术干扰Atg5表达;最后,检测干扰和过表达Atg5后相关脂代谢基因：载脂蛋白B (apolipoprotein, ApoB)、脂肪酸合成酶(fatty acid synthase, FASN)、乙酰辅酶A羧化酶(acetyl CoA carboxylase, ACACA)、过氧化物酶体增殖物激活受体-α (peroxisome proliferator-activated receptor-α, PPARα)、叉头转录因子-1 (forkhead box protein-1, Foxo1)、脂肪酸结合蛋白7 (fatty acid-binding protein 7, FABP7)、分拣蛋白1 (sortilin 1, Sort1)的表达情况;结果表明：鸡ATG5蛋白是相对分子质量为32.478 kD的不稳定蛋白质,Atg5在各组织中均有表达,其中以胸肌组织中表达量最高;此外,成功构建过表达载体pcDNA3.1-Atg5,筛选出最优干扰siRNA—siRNA-330,过表达或干扰Atg5基因对其他脂代谢相关基因的表达情况均有影响;其中,过表达Atg5后,ApoB的表达量显著降低,Sort1的表达量显著增加,FABP7变化不显著;干扰Atg5后,FABP7和ApoB的表达量显著提高,Sort1变化不显著,推测三者与Atg5可能存在潜在联系。本研究为进一步研究鸡Atg5基因的功能提供参考依据。

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	韩琪
	王茜倩
	徐钰芳
	郁建锋
	徐璐
	顾志良

关键词 ：自噬相关基因5 (Atg5), 自噬, 脂质代谢, 鸡

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.

Key words： Autophagy related gene 5 (Atg5) Autophagy Lipid metabolism Chicken

收稿日期: 2021-05-27

ZTFLH:

S831.2

基金资助:国家自然科学基金(31802050); 江苏省自然科学基金(BK20191476)

通讯作者: *sunnyxl2017@163.com;zhilianggu88@hotmail.com

引用本文:

韩琪, 王茜倩, 徐钰芳, 郁建锋, 徐璐, 顾志良. 鸡Atg5基因过表达载体的构建与功能验证[J]. 农业生物技术学报, 2022, 30(4): 716-725.
HAN Qi, WANG Qian-Qian, XU Yu-Fang, YU Jian-Feng, XU Lu, GU Zhi-Liang. Construction of Overexpression Vector and Functional Verification of Atg5 Gene in Chicken (Gallus gallus). 农业生物技术学报, 2022, 30(4): 716-725.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.04.010 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I4/716

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