摘要
研究旨在对鸡APOC3蛋白进行生物信息学分析及构建APOC3基因组织表达谱,并探究其对肌内前体脂肪细胞分化的影响。试验从NCBI(https://www.ncbi.nlm.nih.gov/)数据库下载APOC3蛋白氨基酸序列并进行生物信息学分析;利用PCR技术扩增APOC3基因CDS序列,采用同源重组方法构建APOC3基因过表达载体;利用qPCR检测APOC3基因在鸡不同组织中的表达量,分析APOC3基因表达规律;利用差速贴壁法分离14日龄AA肉鸡胸肌原代肌内前脂肪细胞;在肌内前脂肪细胞中过表达APOC3基因,利用qPCR、检测甘油三酯(Triglyceride,TG)含量、油红O染色评估APOC3基因对肌内前体脂肪细胞分化成脂的影响。生物信息学分析结果显示,同源基因APOC3在鸡与火鸡之间序列保守度较高,与小鼠保守度最低;APOC3蛋白是定位于细胞外、无跨膜结构域的分泌型蛋白。组织表达分析结果显示,APOC3基因在肝脏中特异性表达。体外试验结果分析显示,与对照组相比,试验组APOC3 mRNA表达水平升高了60倍,甘油三酯含量显著增加(P<0.05),细胞内脂滴含量极显著增多(P<0.01),脂肪细胞分化相关标志基因过氧化物增殖物激活受体γ(Peroxisome proliferator-activated receptor gamma,PPARγ)、脂肪酸结合蛋白4(Fatty acid binding protein 4,FABP4)、脂肪酸结合蛋白5(Fatty acid binding protein 5,FABP5)、CCAAT/增强子结合蛋白α(CCAAT/enhancer binding protein alpha,C/EBPα)、脂蛋白脂肪酶(Lipoprotein lipase,LPL)mRNA的表达水平显著上升(P<0.05);APOC3蛋白与载脂蛋白A1(APOA1)、异质核核糖核蛋白A/B(HNRNPAB)、高温需求因子A3(HTRA3)、肿瘤坏死因子受体超家族成员6B(TNFRSF6B)、载脂蛋白D(APOD)存在相互作用。研究表明,鸡APOC3蛋白属于分泌型蛋白,其编码基因在肝脏中特异性表达且与火鸡之间保守序列相似度较高;鸡APOC3基因过表达能够促进鸡肌内前体脂肪细胞分化与脂质积累。
The aim of the study was to perform bioinformatics analysis and construct tissue expression profiles of APOC3 gene in chicken APOC3 protein,and to investigate its effect on the differentiation of intramuscular precursor adipocytes.The amino acid sequence of APOC3 protein was downloaded from NCBI(https://www.ncbi.nlm.nih.gov/)database and analyzed by bioinformatics;The CDS sequence of APOC3 gene was amplified by PCR,and the APOC3 gene overexpression vector was constructed by homologous recombination;The expression level of APOC3 gene in different tissues of chickens was detected by qPCR,and the expression level of APOC3 gene was analyzed by qPCR.The expression pattern of APOC3 gene was analyzed;Primary intramuscular preadipocytes was isolated from the breast muscle of 14-day-age AA broilers using differential wall attachment;APOC3 gene was overexpressed in intramuscular preadipocytes,and the effect of APOC3 gene on the differentiation of intramuscular preadipocytes into adipocytes was evaluated by qPCR,detection of triglyceride(TG)content and oil red O staining.Bioinformatics analysis showed that the homologous gene APOC3 was more conserved between chickens and turkeys,and the least conserved between chickens and mice,and APOC3 protein was a secreted protein localized in the extracellular region without a transmembrane structural domain.Tissue expression analysis showed that the APOC3 gene was specifically expressed in the liver.Analysis of the results of overexpression of APOC3 in intramuscular preadipocyte cells showed that the APOC3 mRNA of the overexpression group,as compared with that of the control group,was significantly higher than the APOC3 mRNA of the control group.APOC3 mRNA expression level in the overexpression group increased 60-fold compared with that in the control group,triglyceride content was significantly increased(P<0.05),the content of intracellular lipid droplets was significantly increased(P<0.01),and the mRNA expression levels of adipocyte differentiation-related marker genes peroxisome proliferator-activated receptor gamma(PPARγ),fatty acid binding protein 4(FABP4),fatty acid binding protein 5(FABP5),CCAAT/enhancer binding protein alpha(C/EBPα)and lipoprotein lipase(LPL)were significantly increased(P<0.05).APOC3 protein interacted with apolipoprotein A1(APOA1),heterogeneous nuclear ribonucleoprotein A/B(HNRNPAB),high temperature demand factor A3(HTRA3),tumor necrosis factor receptor superfamily member 6B(TNFRSF6B)and apolipoprotein D(APOD).The study showed that chicken APOC3 protein was a secreted protein,and its coding gene was specifically expressed in the liver and had a high degree of sequence similarity with that of turkey;The overexpression of chicken APOC3 gene promoted the differentiation of intramuscular precursor adipocyte cells and the accumulation of lipids in chicken muscle.
作者
邢雨欣
管宏波
马乘霖
申颖
申家宁
卢镕镕
田亚东
李转见
田卫华
刘小军
李红
XING Yuxin;GUAN Hongbo;MA Chenglin;SHEN Ying;SHEN Jianing;LU Rongrong;TIAN Yadong;LI Zhuanjian;TIAN Weihua;LIU Xiaojun;LI Hong(College of Animal Science and Technology,Henan Agricultural University,Zhengzhou,Henan 450046;Key Laboratory of Livestock and Poultry Resources(Poultry)Evaluation and Utilization of Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou,Henan 450046;Henan Innovative Engineering Research Center of Poultry Germplasm Resource,Henan Agricultural University,Zhengzhou,Henan 450046)
出处
《中国家禽》
北大核心
2025年第6期19-28,共10页
China Poultry
基金
河南省高等学校重点科研项目(No.24A230012)。
