过表达山羊<i>APOC3</i>基因促进肌内脂肪细胞分化

doi:10.7668/hbnxb.20192779

摘要/Abstract

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。

关键词: 山羊, APOC3基因, 肌内脂肪细胞, 过表达, 细胞分化

Abstract:

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 CEBPβ were extremely significantly up-regulated,PPARγ was significantly up-regulated,and Pref-1 was significantly down-regulated.APOC3 might promote the differentiation of intramuscular adipocytes by up-regulating SREBP1,CEBPβ,PPARγ and down-regulating Pref-1.

Key words: Goat, APOC3 gene, Intramuscular adipocytes, Overexpression, Cell differentiation

冉黎, 吕锦诗, 张浩, 王永, 朱江江, 李艳艳, 孟庆勇, 林亚秋. 过表达山羊APOC3基因促进肌内脂肪细胞分化[J]. 华北农学报, 2022, 37(3): 223-230. doi: 10.7668/hbnxb.20192779.

RAN Li, LÜ Jinshi, ZHANG Hao, WANG Yong, ZHU Jiangjiang, LI Yanyan, MENG Qingyong, LIN Yaqiu. Effect of Overexpression of Goat APOC3 Gene on Differentiation of Intramuscular Adipocytes[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 223-230. doi: 10.7668/hbnxb.20192779.

http://www.hbnxb.net/CN/Y2022/V37/I3/223

图/表 7

表1 PCR和实时荧光定量PCR(qPCR)引物信息

Tab.1 Primers information for PCR and quantitative real-time PCR(qPCR)

基因 Gene name	序列(5'-3') Sequence(5'-3')	退火温度/℃ TM	产物长度/bp Products length	用途 Purpose
APOC3	S:GCAGTTCATCCCAAGAGGC	58	370	克隆
	A:AGGATGGACAGGGGGACTT	58	370	克隆
APOC3	S:ACTCCTGGTCTTGCCCGAAACT	64	207	qPCR
	A:GTGAACTTGCCCTTGAACGAGC	64	207	qPCR
TBP	S: AACAGCCTCCCACCTTATGC	60	155	内参
	A: TGCTGCTCCTCCAAAATAGAC	60	155	内参
UXT	S: GCAAGTGGATTTGGGCTGTAAC	60	173	内参
	A: ATGGAGTCCTTGGTGAGGTTGT	60	173	内参

表2 山羊APOC3生物信息的方法

Tab.2 Method of goat APOC3 biological information

分析内容 Analytical contents	运用软件或在线工具 Applied software or online tools
开放阅读框预测、氨基酸序列翻译 Open reading frame prediction, amino acid sequence translation	ORF Finder
蛋白质理化性质分析 Analysis of physical and chemical properties of protein	ExPASy ProtParam
蛋白质二级结构预测 Protein secondary structure prediction	ExPASy
信号肽分析 Signal peptide analysis	Signal P4.1 Server
蛋白质磷酸化、糖基化位点预测 Prediction of protein phosphorylation and glycosylation sites	NetPhos 3.1、NetOGlyc 4.0、NetNGlyc 1.0
跨膜结构域预测 Transmembrane domain prediction	TMHMM
亚细胞定位 Subcellular localization	PSORT Ⅱ Prediction
核苷酸序列对比 Nucleotide sequence comparison	DNAMAN
进化树构建 Evolutionary tree construction	MEGA 5.0
功能结构域 Functional domain	Conserved Domain

图1 山羊APOC3基因生物学分析 A:M.DL2000 Marker;1.APOC3基因。B:横线.克隆的引物;橙色字体.蛋白质磷酸化位点;方框.功能结构域;椭圆形.信号肽切割位点;ATG.起始密码子;^*.终止密码子。

Fig.1 Bioinformatics analysis of APOC3 gene of goats A:M.DL2000 Marker;1.APOC3 gene.B:The horizontal line.The cloned primer;The orange font.Protein phosphorylation sites; Boxes.Simple domains;The ellipse.The signal peptide cleavage site;ATG.The starting codon;^*.Stop codon.

图2 山羊APOC3基因氨基酸序列分析 A.山羊APOC3蛋白二级结构预测;B.山羊APOC3的氨基酸序列与其他物种间的同源性;C.APOC3氨基酸序列物种进化树。

Fig.2 Amino acid sequence analysis of APOC3 gene in goat A. Secondary structure prediction of APOC3 protein in goat;B.The amino acid sequence of goat APOC3 was homologous with other species;C.APOC3 amino acid sequence species evolutionary tree.

图3 山羊APOC3基因组织及肌内脂肪细胞不同分化阶段的相对表达水平 A.以肺脏表达水平作为对照,TBP作为内参基因;B.以0 d表达水平作为对照,UXT作为内参基因。不同大写字母表示差异极显著(P<0.01)。

Fig.3 Relative expression levels of APOC3 gene in goat tissues and intramuscular adipocytes at different differentiation stages A.The lung expression level was used as the control,TBP as the internal reference gene;B.The expression level of 0 d was used as control, UXT as an internal reference gene.Different letters have extremely significant differences(P < 0.01).

图4 山羊APOC3过表达效率及油红O染色结果 A.pcDNA3.1-APOC3载体在山羊肌内脂肪细胞中的转染效率;B、C.肌内脂肪油红O染色形态学观察(200×和400×);不同大写字母表示差异极显著(P<0.01)。

Fig.4 Goat APOC3 overexpression efficiency and oil red O staining A.Transfection efficiency of pcDNA3.1-APOC3 vector in goat intramuscular adipocytes;B,C.Morphological observation of intramuscular fat oil red O staining(200×and 400×);Different uppercase indicate extremely significant difference(P<0.01).

图5 过表达山羊APOC3对成脂分化标志基因的影响不同大写字母表示对照组与试验组差异极显著(P<0.01);不同小写字母表示对照组与试验组差异显著( P<0.05)。

Fig.5 Effect of overexpression of goat APOC3 on adipogenic differentiation marker gene

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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

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