牛FoxO1基因CDS区克隆及其在脂肪细胞分化过程中的表达分析

doi:10.3969/j.issn.1004-1524.2023.05.05

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1016-1027.DOI: 10.3969/j.issn.1004-1524.2023.05.05

牛FoxO1基因CDS区克隆及其在脂肪细胞分化过程中的表达分析

宋雅萍¹(), 雷召雄¹, 赵毅昂¹, 姜超¹, 王兴平¹^,², 罗仍卓么¹^,², 马云¹^,², 魏大为¹^,²^,^*()

1.宁夏大学农学院,宁夏银川 750021
2.宁夏大学宁夏反刍动物分子细胞育种重点实验室,宁夏银川 750021

收稿日期:2022-01-06 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:宋雅萍(1999—),女,甘肃靖远人,硕士研究生,研究方向为动物遗传育种。E-mail:songyaping0905@163.com
通讯作者: *魏大为,E-mail: weidaweiwdw@163.com
基金资助:
国家自然科学基金(32060744);国家自然科学基金(32202641);宁夏自然科学基金(2021AAC05007);宁夏青年托举工程(TJGC2019076)

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

摘要/Abstract

摘要：

本试验旨在克隆牛叉头转录因子O1(Forkhead box O1,FoxO1)基因编码区序列并探究其在牛脂肪细胞分化过程中的表达规律。利用PCR扩增牛FoxO1基因CDS区序列,通过生物信息学方法预测牛FoxO1的理化特性和功能,并运用实时荧光定量PCR技术检测牛脂肪细胞不同分化阶段FoxO1和脂肪标志基因PPARγ、C/EBPα、C/EBPβ、FABP4和LPL的表达规律。结果显示,牛FoxO1基因的CDS区全长1 998 bp,编码665个氨基酸,其蛋白分子式为C₃₀₁₆H₄₇₁₁N₈₇₁O₉₇₉S₃₅,理论等电点为6.38。预测发现,FoxO1与脂肪生长发育调控蛋白AKT1、AKT2、AKT3、SIRT1、PRKACA、CDK2和MAPK8等之间存在紧密互作关系。系统进化树结果显示,牛FoxO1进化保守,与亲缘关系较近的绵羊和山羊物种同源性最高。检测牛脂肪细胞不同分化时期基因的时序表达,结果显示,与分化第0天相比,牛FoxO1基因在第6天表达量达到峰值(P<0.01),随后下调;PPARγ和C/EBPβ均在第0天就有较高的表达量,且二者在诱导分化第4、6天表达量都极显著高于第0天(P<0.01);C/EBPα的表达量在分化第2、4、6和10天都极显著高于第0天(P<0.01);FABP4在分化第0、2天表达量较低,于分化第4天表达量上升达峰值(P<0.01),随后逐渐下调(P<0.05);LPL随分化进程推进其表达量不断上升,于分化第10天达到峰值(P<0.01)。以上研究结果可为进一步探究FoxO1调节牛脂肪生成的分子机制提供参考信息。

关键词: 牛, FoxO1, 生物信息学分析, 脂肪细胞分化

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

中图分类号:

S823

宋雅萍, 雷召雄, 赵毅昂, 姜超, 王兴平, 罗仍卓么, 马云, 魏大为. 牛FoxO1基因CDS区克隆及其在脂肪细胞分化过程中的表达分析[J]. 浙江农业学报, 2023, 35(5): 1016-1027.

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.

图/表 10

参考文献 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

牛FoxO1基因CDS区克隆及其在脂肪细胞分化过程中的表达分析

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

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