Cloning of CDS region of bovine FoxO1 gene and analysing expression pattern during adipocyte differentiation

doi:10.3969/j.issn.1004-1524.2023.05.05

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (5): 1016-1027.DOI: 10.3969/j.issn.1004-1524.2023.05.05

• Animal Science • Previous Articles Next Articles

Cloning of CDS region of bovine FoxO1 gene and analysing expression pattern during adipocyte differentiation

SONG Yaping¹(), LEI Zhaoxiong¹, ZHAO Yi’ang¹, JIANG Chao¹, WANG Xingping¹^,², LUORENG Zhuoma¹^,², MA Yun¹^,², WEI Dawei¹^,²^,^*()

1. School of Agriculture, Ningxia University, Yinchuan 750021, China
2. Key Laboratory of Ruminant Molecular Cell Breeding, Ningxia University, Yinchuan 750021, China

Received:2022-01-06 Online:2023-05-25 Published:2023-06-01

Abstract

Abstract:

The aim of this study was to clone the coding region of bovine Forkhead box O1 (FoxO1) gene and explore its expression pattern during bovine adipocyte differentiation stages. Polymerase chain reaction (PCR) and bioinformatics analysis were used to amplify the CDS sequence of bovine FoxO1 gene and predict the physicochemical properties and functions of bovine FoxO1 respectively. Additionally, the expression patterns of FoxO1 and adipocyte-specific genes (PPARγ, C/EBPα, C/EBPβ, FABP4 and LPL) were measured during bovine adipocyte differentiation stages by qPCR. The results showed that the CDS region of FoxO1 was 1 998 bp in length and encoded 665 amino acids. Its molecular formula was C₃₀₁₆H₄₇₁₁N₈₇₁O₉₇₉S₃₅, theoretical isoelectric point was 6.38. It was predicted that FoxO1 interacted closely with adipose growth and development regulatory proteins AKT1, AKT2, AKT3, SIRT1, PRKACA, CDK2 and MAPK8. Phylogenetic tree results showed that bovine FoxO1 was evolutionarily conserved and had the highest homology with closely related sheep and goat species. Temporal expression of FoxO1 in bovine adipocyte differentiation stages showed that the relative expression level reached a peak on day 6 compared with day 0 of differentiation (P<0.01) and was subsequently down-regulated. Both PPARγ and C/EBPβ had higher expression on day 0, and the levels of them on day 4 and 6 were significantly higher than those on day 0(P<0.01). The expression level of C/EBPα on day 2, 4, 6, 10 of differentiation was significantly higher than that on day 0 (P<0.01). However, FABP4 it was lower at on day 0 and 2 of differentiation, and increased to the peak on day 4 of differentiation (P<0.01), then decreased gradually (P<0.05). It increased with the progress of differentiation on LPL and peaked on day 10 (P<0.01). These data will provide the reference information for further exploration of the molecular mechanism of bovine FoxO1.

Key words: bovine, FoxO1, bioinformatics analysis, adipocyte differentiation

CLC Number:

S823

SONG Yaping, LEI Zhaoxiong, ZHAO Yi’ang, JIANG Chao, WANG Xingping, LUORENG Zhuoma, MA Yun, WEI Dawei. Cloning of CDS region of bovine FoxO1 gene and analysing expression pattern during adipocyte differentiation[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1016-1027.

Figures/Tables 10

References 43

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基因 Gene	引物序列 Primer sequence(5'→3')	退火温度 Tm/℃	扩增片段长度 Product length/bp	NCBI登录号 GenBank No.	用途 Purpose
FoxO1	F: ATGGCCGAAGCGCCCCAGGTGGT	61.5	1 998	XM_025000053.1	克隆Clone
	R: TCAGCCTGACACCCAGCTGTGTGTT
FoxO1	F: CAGATTTACGAGTGGATG	60.0	96	XM_025000053.1	实时荧光定量PCR
	R: CAGATTATGACGAATTGAAT				(qRT-PCR)
PPARγ	F: AGGATGGGGTCCTCATATCC	60.0	121	NM_181024.2	qRT-PCR
	R: GCGTTGAACTTCACAGCAAA
C/EBPα	F: TGGACAAGAACAGCAACGAG	60.0	130	NM_176784	qRT-PCR
	R: TTGTCACTGGTCAGCTCCAG
C/EBPβ	F: TTCCTCTCCGACCTCTTCTC	60.0	79	NM_176788	qRT-PCR
	R: CCAGACTCACGTAGCCGTACT
FABP4	F: AAGTCAAGAGCATCGTAA	60.0	111	NM_174314.2	qRT-PCR
	R: CCAGCACCATCTTATCAT
LPL	F: ACGATTATTGCTCAGCATGG	60.0	130	NM_001075120.1	qRT-PCR
	R: ACTTTGTACAGGCACAACCG
GAPDH	F: CCAACGTGTCTGTTGTGGAT	60.0	80	NM_001034034.2	qRT-PCR
	R: CTGCTTCACCACCTTCTTGA

基因 Gene	引物序列 Primer sequence(5'→3')	退火温度 Tm/℃	扩增片段长度 Product length/bp	NCBI登录号 GenBank No.	用途 Purpose
FoxO1	F: ATGGCCGAAGCGCCCCAGGTGGT	61.5	1 998	XM_025000053.1	克隆Clone
	R: TCAGCCTGACACCCAGCTGTGTGTT
FoxO1	F: CAGATTTACGAGTGGATG	60.0	96	XM_025000053.1	实时荧光定量PCR
	R: CAGATTATGACGAATTGAAT				(qRT-PCR)
PPARγ	F: AGGATGGGGTCCTCATATCC	60.0	121	NM_181024.2	qRT-PCR
	R: GCGTTGAACTTCACAGCAAA
C/EBPα	F: TGGACAAGAACAGCAACGAG	60.0	130	NM_176784	qRT-PCR
	R: TTGTCACTGGTCAGCTCCAG
C/EBPβ	F: TTCCTCTCCGACCTCTTCTC	60.0	79	NM_176788	qRT-PCR
	R: CCAGACTCACGTAGCCGTACT
FABP4	F: AAGTCAAGAGCATCGTAA	60.0	111	NM_174314.2	qRT-PCR
	R: CCAGCACCATCTTATCAT
LPL	F: ACGATTATTGCTCAGCATGG	60.0	130	NM_001075120.1	qRT-PCR
	R: ACTTTGTACAGGCACAACCG
GAPDH	F: CCAACGTGTCTGTTGTGGAT	60.0	80	NM_001034034.2	qRT-PCR
	R: CTGCTTCACCACCTTCTTGA

分析内容 Analysis of the content	分析软件或者网址 Analysis software and online tool
理化性质分析Analysis of physical and chemical properties	http://www.expasy.org/tools/protparam.html
亚细胞定位Subcellular localization	https://psort.hgc.jp/form2.html
信号肽剪切位点预测Prediction of signal peptide splicing sites	http://www.cbs.dtu.dk/services/SignalP-3.0/
跨膜螺旋结构预测Prediction of transmembrane helical structure	http://www.cbs.dtu.dk/services/TMHMM
保守结构域分析Conservative domain analysis	http://smart.embl-heidelberg.de/
亲疏水性分析Hydrophilic and hydrophobic analysis	http://ca.expasy.org/tools/protscale.html
磷酸化位点Phosphorylation site	http://www.cbs.dtu.dk/services/net-Phos/
二级结构预测Secondary structure prediction	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
三级结构预测Prediction of tertiary structure	https://www.swissmodel.expasy.org/
互作蛋白预测Interaction protein prediction	https://string-db.org/
系统进化树Phylogenetic tree	MEGA software

分析内容 Analysis of the content	分析软件或者网址 Analysis software and online tool
理化性质分析Analysis of physical and chemical properties	http://www.expasy.org/tools/protparam.html
亚细胞定位Subcellular localization	https://psort.hgc.jp/form2.html
信号肽剪切位点预测Prediction of signal peptide splicing sites	http://www.cbs.dtu.dk/services/SignalP-3.0/
跨膜螺旋结构预测Prediction of transmembrane helical structure	http://www.cbs.dtu.dk/services/TMHMM
保守结构域分析Conservative domain analysis	http://smart.embl-heidelberg.de/
亲疏水性分析Hydrophilic and hydrophobic analysis	http://ca.expasy.org/tools/protscale.html
磷酸化位点Phosphorylation site	http://www.cbs.dtu.dk/services/net-Phos/
二级结构预测Secondary structure prediction	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
三级结构预测Prediction of tertiary structure	https://www.swissmodel.expasy.org/
互作蛋白预测Interaction protein prediction	https://string-db.org/
系统进化树Phylogenetic tree	MEGA software

物种名称 The name of the species	登录号 GenBank No.
牛Bos taurus	XP_024855821.1
猪Sus scrofa	NP_999179.3
绵羊Ovis aries	XP_027829397.1
山羊Capra hircus	QYC62012.1
人Homo sapiens	NP_002006.2
小鼠Mus musculus	NP_062713.2
瘤牛Bos indicus	XP_019827292
长臂猿Hylobates moloch	XP_032006629.1

Cloning of CDS region of bovine FoxO1 gene and analysing expression pattern during adipocyte differentiation

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