过表达山羊APOC3基因促进肌内脂肪细胞分化  

Effect of Overexpression of Goat APOC3 Gene on Differentiation of Intramuscular Adipocytes

作　　者：冉黎 吕锦诗 张浩[1,2] 王永 朱江江[1,2] 李艳艳[1,2] 孟庆勇[3] 林亚秋[1,2] RAN Li;Lü Jinshi;ZHANG Hao;WANG Yong;ZHU Jiangjiang;LI Yanyan;MENG Qingyong;LIN Yaqiu(Key Laboratory of Ministry of Education, Sichuan Province for Qinghai-Tibetan Plateau Animal Geneyic Resource Reservation and Exploitation, Chengdu 610041,China;College of Animal &Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;State Key Laboratories for Agrobiotechnology,College of Biological Sciences,China Agricultural University,Beijing 100193,China)

机构地区：[1]青藏高原动物遗传资源保护与利用教育部,四川省重点实验室,四川成都610041 [2]西南民族大学畜牧兽医学院,四川成都610041 [3]中国农业大学生物学院,农业生物技术国家重点实验室,北京100193

出　　处：《华北农学报》2022年第3期223-230,共8页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(32072723);四川省重点研发项目(2021YFYZ0003);中央高校基本科研业务费专项基金项目(3300221815);农业生物技术国家重点实验室2021年度开放课题(2021SKLAB6-1)。

摘　　要：为了明确APOC3基因在山羊肌内脂肪细胞分化中的作用,采用RT-PCR技术克隆山羊APOC3基因序列,并通过在线软件进行生物信息学分析;利用实时荧光定量PCR(qPCR)技术检测山羊APOC3基因在各组织和不同分化阶段的肌内脂肪细胞中的表达规律;在利用双酶切法构建山羊APOC3过表达载体的基础上,利用油红O染色确定山羊APOC3基因过表达对肌内脂肪细胞脂滴聚集的影响,同时利用qPCR方法检测成脂分化标志基因mRNA的相对表达水平,进而明确其可能发挥作用的途径。结果表明,获得山羊APOC3的ORF区长294 bp,编码97个氨基酸,功能结构域区在第23—88个氨基酸处。山羊APOC3在心脏、肝脏、脾脏等14个组织中均有表达,且在肝脏中的表达量最高;山羊APOC3在诱导分化48 h表达量最高,极显著高于分化前;过表达山羊APOC3后肌内脂肪细胞中脂滴积聚增多,成脂分化标志基因SREBP1和CEBPβ的相对表达水平极显著上调,PPARγ的相对表达水平显著上调,而Pref-1相对表达水平显著下调。结果表明,山羊APOC3可能通过上调SREBP1、CEBPβ、PPARγ及下调Pref-1来发挥作用促进肌内脂肪细胞的分化。In order to clarify the role of APOC3 gene in the differentiation of intramuscular adipocytes of goats,the APOC3 gene sequence was cloned by RT-PCR,and the biological information was analyzed by online software.Quantitative Real-time PCR(qPCR)was used to detect the expression of APOC3 gene in intramuscular adipocytes of goats at different tissues and differentiation stages.APOC3 overexpression vector was constructed by double-enzyme digestion method,and the effects of APOC3 gene overexpression on lipid droplet accumulation were determined by oil red O staining.At the same time,the mRNA relative expression level of marker gene of adipogenic differentiation was detected by qPCR.The results showed that the ORF region of APOC3 was 294 bp in length and encoded 97 amino acids with a functional domain of 23—88 aa.APOC3 was expressed in 14 tissues including heart,liver and spleen,and so on,which the highest level of APOC3 was found in liver.APOC3 expression was the highest at 48 h and extremely significantly higher than that before differentiation.After APOC3 was overexpressed,lipid drop accumulation increased,and the relative expression levels of marker genes SREBP1 and CEBPβwere extremely significantly up-regulated,PPARγwas significantly up-regulated,and Pref-1 was significantly down-regulated.APOC3 might promote the differentiation of intramuscular adipocytes by up-regulating SREBP1,CEBPβ,PPARγand down-regulating Pref-1.

关 键 词：山羊 APOC3基因 肌内脂肪细胞 过表达 细胞分化 

分 类 号：Q78[生物学—分子生物学] S826[农业科学—畜牧学]