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

doi:10.7668/hbnxb.20192779

Abstract

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

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.

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

Figures/Tables 7

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

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

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.

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.

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).

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).

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

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