猪OLR1基因对肌内前脂肪细胞分化的影响

doi:10.3969/j.issn.1004-1524.2020.12.05

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (12): 2147-2153.DOI: 10.3969/j.issn.1004-1524.2020.12.05

猪OLR1基因对肌内前脂肪细胞分化的影响

乔木¹(), 黄藏宇¹, 吴俊静¹, 武华玉¹, 万绪玲², 周佳伟¹, 刘贵生¹, 梅书棋¹, 彭先文¹^,^*

1.湖北省农业科学院畜牧兽医研究所,动物胚胎工程及分子育种湖北省重点实验室,湖北武汉430064
2.武汉市江夏区动物卫生监督所,湖北武汉 430200

收稿日期:2020-09-27 出版日期:2020-12-25 发布日期:2020-12-25
通讯作者: 彭先文
作者简介:*彭先文,E-mail: pxwpal@163.com
乔木(1982—),女,辽宁建平人,博士,副研究员,主要从事猪遗传育种研究。E-mail: mqbetter@163.com
基金资助:
湖北省农业科学院人才项目(Q2018017);湖北省农业科技创新中心资助项目(2019-620-000-001-18);湖北省自然科学基金(2018CFA014);湖北省重大创新专项(2019ABA084);湖北省农业科技创新行动项目(2018skjcx05)

Effect of porcine OLR1 gene on intramuscular preadipocytes differentiation

QIAO Mu¹(), HUANG Cangyu¹, WU Junjing¹, WU Huayu¹, WAN Xuling², ZHOU Jiawei¹, LIU Guisheng¹, MEI Shuqi¹, PENG Xianwen1¹^,^*

1. Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal and Veterinary Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
2. Jiangxia District Animal Health Supervision Office, Wuhan 430020, China

Received:2020-09-27 Online:2020-12-25 Published:2020-12-25
Contact: PENG Xianwen1

摘要/Abstract

摘要：

研究氧化型低密度脂蛋白受体1(oxidized low-density lipoprotein receptor1, OLR1)基因在猪前脂肪细胞分化过程中的作用,为深入研究OLR1基因在脂肪沉积中的功能奠定基础。根据GenBank中猪OLR1基因序列(登录号:NM_213805),构建OLR1基因过表达载体OLR1-pcDNA3.1,同时合成干扰OLR1表达的siRNA。从恩施黑猪背最长肌中分离前脂肪细胞,分别将OLR1-pcDNA3.1和OLR1-siRNA转染前脂肪细胞,并对细胞进行诱导分化,至第6天,通过油红O染色、甘油三酯含量测定和荧光定量PCR检测OLR1基因对前脂肪细胞分化的影响。结果显示,OLR1过表达组脂滴明显多于对照组,甘油三酯含量显著(P<0.05)增加,成脂分化标志基因PPARγ的表达量极显著(P<0.01)升高、C/EBPα和FAS的表达量显著(P<0.05)升高;干扰OLR1后,细胞内脂滴减少,甘油三酯含量显著(P<0.05)下降,成脂分化标志基因PPARγ的表达量极显著(P<0.01)下降,C/EBPα和FAS的表达量显著(P<0.05)降低。综上表明,OLR1基因具有促进前脂肪细胞成脂分化的作用。

关键词: 猪, OLR1基因, 过表达, RNA干扰, 前脂肪细胞分化

Abstract:

The aim of this study was to investigate the role of oxidized low density lipoprotein receptor 1 (OLR1) gene in the differentiation of porcine preadipocytes, and to lay a foundation for further study of the function of OLR1 gene in fat deposition. According to the sequence of OLR1 gene in GenBank (GenBank No.: NM_213805), the over expression vector of OLR1-pcDNA3.1 was constructed and OLR1-siRNA was synthesized. Preadipocytes were isolated from the longissimus dorsi muscle of Enshi black pig. OLR1-pcDNA3.1 and OLR1-siRNA were transferred into the preadipocytes respectively. The cells were induced to differentiate to the 6th day. Then, oil red O staining, triglyceride content detection and fluorescence quantitative PCR were used to detect the effect of OLR1 gene on the preadipocyte differentiation. The results showed that the number of lipid droplets in the overexpression group was significantly(P<0.05) higher than that in the control group, the triglyceride content was significantly increased (P<0.05), and the expression levels of PPARγ, C/EBPα and FAS were significantly increased at P<0.01, P<0.05 and P<0.05 level, respectively. In contrast, after knock down OLR1 gene, the intracellular lipid droplets and triglyceride content were significantly decreased (P<0.05), and the expression of adipogenic differentiation marker genes PPARγ, C/EBPα and FAS were significantly decreased at P<0.01, P<0.05 and P<0.05 level, respectively. In conclusion, OLR1 gene could promote the adipogenic differentiation of preadipocyte.

Key words: pig, OLR1 gene, overexpression, RNA interference, differentiation of preadipocytes

中图分类号:

S433.3

乔木, 黄藏宇, 吴俊静, 武华玉, 万绪玲, 周佳伟, 刘贵生, 梅书棋, 彭先文. 猪OLR1基因对肌内前脂肪细胞分化的影响[J]. 浙江农业学报, 2020, 32(12): 2147-2153.

QIAO Mu, HUANG Cangyu, WU Junjing, WU Huayu, WAN Xuling, ZHOU Jiawei, LIU Guisheng, MEI Shuqi, PENG Xianwen1. Effect of porcine OLR1 gene on intramuscular preadipocytes differentiation[J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2147-2153.

图/表 7

表1 PCR引物信息

Table 1 Information of PCR primers

引物名称 Primer name	引物序列 Primer sequences(5'-3')	退火温度 Annealing temperature/℃	产物长度 Product length/bp
F1	CCAAGCTTGCCACCATGACACTTGATGACCTCA	59	847
R1	CCCTCGAGTCACTGTGCTCTCAAGAG
pOLQF	AGTCTTTCCACTCGGCGGT	60	176
pOLQR	GCTAAAGCCTGTCCCTCCA
PPARγ-F	CTGACCAAAGCAAAGGCGAG	60	178
PPARγ-R	CGGAGCGAAACTGACACCC
CEBPα-F	GGCTACCTGGACGGCAGG	60	187
CEBPα-R	GCGGAGGGTGTGAATGCG
FAS-F	GTATCGCTGGACCACTGTCT	60	174
FAS-R	TCCAGAGCGAAGGAGAGATT
β-actinF	CCAGGTCATCACCATCGG	60	158
β-actinR	CCGTGTTGGCGTAGAGGT

表2 猪OLR1基因siRNA序列

Table 2 The siRNA sequence of porcine OLR1

siRNA	序列Sequences (5'-3')
siRNAF1	GGAUCAACCUGACGAGAAAUC
siRNAR1	UUUCUCGUCAGGUUGAUCCUU
siRNAF2	GCUAUACUUGGUCACUUAAGU
siRNAR2	UUAAGUGACCAAGUAUAGCUU
siRNAF3	GGAUUACUGGUGACCGUUAUA
siRNAR3	UAACGGUCACCAGUAAUCCCA

图1 OLR1基因扩增产物电泳结果 M, DL 2 000 marker; 1, PCR扩增片段。

Fig.1 Electrophoretic results of amplified products of OLR1 gene M, DL 2 000 marker; 1, PCR amplification fragment.

图2 OLR1基因过表达载体Hind Ⅲ和XhoΙ酶切鉴定结果 M,DL 10 000 marker;1,双酶切片段。

Fig.2 Digestion result of OLR1 overexpression vector by enzyme Hind Ⅲ and XhoⅠ M, DL 10 000 marker; 1, Double digestion fragment.

图3 转染siRNA后OLR1基因的相对表达量 *、**分别表示与对照组相比在P<0.05 和P<0.01 水平差异显著。下同。

Fig.3 Relative expression of OLR1 gene after transfection with siRNAs * and ** meant significant differences at the levels of P<0.05 and P<0.01 compared with the control group, respectively. The same as below.

图4 转染OLR1-pcDNA3.1或OLR1-siRNA后PPARγ,C/EBPα和FAS基因的相对表达量

Fig.4 Relative expression of PPARγ, C/EBPα and FAS genes after transfection with OLR1-pcDNA3.1 or OLR1-siRNA

图5 油红O染色和甘油三酯含量测定红色表示脂滴的沉积,*表示差异显著(P<0.05)。

Fig.5 Oil red O staining and determination of triglyceride content Red indicated the deposition of lipid droplets, * indicated significant difference at P<0.05.

参考文献 25

[1]	KOCIUCKA B, JACKOWIAK H, KAMYCZEK M, et al. The relationship between adipocyte size and the transcript levels of SNAP23, BSCL2 and COPA genes in pigs[J]. Meat Science, 2016,121:12-18. URL PMID
[2]	HAUSMAN G J, DODSON M V, AJUWON K, et al. The biology and regulation of preadipocytes and adipocytes in meat animals[J]. Journal of Animal Science, 2009,87(4):1218-1246. URL PMID
[3]	候晔. 猪骨骼肌和脂肪组织microRNA调控元件鉴定及成肌细胞诱导成脂机理研究[D]. 武汉: 华中农业大学, 2019.
	HOU Y. Identification of microRNA regulatory elements in porcine skeletal muscle and adipose tissue and mechanism of myoblasts adipogenesis[D]. Wuhan: Huazhong Agricultural University, 2019.(in Chinese with English abstract)
[4]	SAWAMURA T, KUME N, AOYAMA T, et al. An endothelial receptor for oxidized low-density lipoprotein[J]. Nature, 1997,386(6620):73-77. URL PMID
[5]	KUME N, MURASE T, MORIWAKI H, et al. Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells[J]. Circulation Research, 1998,83(3):322-327. URL PMID
[6]	NAGASE M, HIROSE S, FUJITA T. Unique repetitive sequence and unexpected regulation of expression of rat endothelial receptor for oxidized low-density lipoprotein (LOX-1)[J]. The Biochemical Journal, 1998,330(Pt 3):1417-1422.
[7]	CHUI P C, GUAN H P, LEHRKE M, et al. PPARγ regulates adipocyte cholesterol metabolism via oxidized LDL receptor 1[J]. Journal of Clinical Investigation, 2005,115(8):2244-2256.
[8]	AJUWON K M, FRANKLIN D M, BANZ W J, et al. Linoleic acid regulates the expression of oxidized low density lipoprotein receptor (OLR1) in 3T3-L1 adipocytes via ERK and JNK pathways[J]. The FASEB Journal, 2007,21(5):455-459.
[9]	孙超, 刘春伟. 脂肪酸对小鼠前体脂肪细胞增殖分化及OLR1基因转录表达的作用[J]. 西北农林科技大学学报(自然科学版), 2009,37(3):1-6.
	SUN C, LIU C W. Effect of fatty acids on the proliferation and differentiation of mouse adipocytes as well as transcriptional expression of OLR1[J]. Journal of Northwest A & F University (Natural Science Edition), 2009,37(3):1-6.(in Chinese with English abstract)
[10]	KHATIB H, ROSA G J M, WEIGEL K, et al. Additional support for an association between OLR1 and milk fat traits in cattle[J]. Animal Genetics, 2007,38(3):308-310. DOI URL PMID
[11]	KOMISAREK J, DORYNEK Z. Effect of ABCG2, PPARGC1A, OLR1 and SCD1 gene polymorphism on estimated breeding values for functional and production traits in Polish Holstein-Friesian bulls[J]. Journal of Applied Genetics, 2009,50(2):125-132. DOI URL PMID
[12]	WANG X, PEÑAGARICANO F, TAL-STEIN R, et al. Short communication: association of an OLR1 polymorphism with milk production traits in the Israeli Holstein population[J]. Journal of Dairy Science, 2012,95(3):1565-1567. URL PMID
[13]	李托, 李陇平. 氧化型低密度脂蛋白受体1(OLR1)基因在家畜经济性状中的研究进展[J]. 榆林学院学报, 2020,30(2):56-60.
	LI T, LI L P. Research progress on animal breeding of OLR1 gene[J]. Journal of Yulin University, 2020,30(2):56-60.(in Chinese with English abstract)
[14]	郭咪咪, 乔木, 吴俊静, 等. 猪OLR1基因克隆及其与生产性状的关联、表达分析[J]. 华中农业大学学报, 2015,34(3):82-88.
	GUO M M, QIAO M, WU J J, et al. Cloning, association with production traits and expression analysis of the porcine OLR1 gene[J]. Journal of Huazhong Agricultural University, 2015,34(3):82-88.(in Chinese with English abstract)
[15]	张罕星, 朱晓彤, 束刚, 等. 猪肌内脂肪前体细胞与皮下脂肪前体细胞分化过程中基因差异表达分析[J]. 中国农业科学, 2008,41(11):3760-3768.
	ZHANG H X, ZHU X T, SHU G, et al. Differential mRNA expression profiles of porcine intramuscular preadipocytes compared with subcutaneous preadipocytes during differentiation[J]. Scientia Agricultura Sinica, 2008,41(11):3760-3768.(in Chinese with English abstract)
[16]	QIAO M, HUANG J S, WU H Y, et al. Molecular characterization, transcriptional regulation and association analysis with carcass traits of porcine TCAP gene[J]. Gene, 2014,538(2):273-279. URL PMID
[17]	胡濒月, 胡杨, 成文敏, 等. Leptin过表达对猪前脂肪细胞脂滴形成的研究[J]. 生物技术通报, 2020,36(8):111-119.
	HU B Y, HU Y, CHENG W M, et al. Lipid droplet formation in the pre-adipocytes of leptin-overexpressed pig[J]. Biotechnology Bulletin, 2020,36(8):111-119.(in Chinese with English abstract)
[18]	孙瑞萍, 王峰, 晁哲, 等. 屯昌猪PDK4基因克隆及其组织表达分析[J]. 浙江农业学报, 2020,32(6):978-985.
	SUN R P, WANG F, CHAO Z, et al. Cloning and tissue expression analysis of PDK4 gene in Tunchang pig[J]. Acta Agriculturae Zhejiangensis, 2020,32(6):978-985.
[19]	张凤, 曾倩晖, 陶虎, 等. 牛ACOX1基因过表达载体构建及对脂肪沉积的初步研究[J]. 中国畜牧杂志, 2017,53(11):42-47.
	ZHANG F, ZENG Q H, TAO H, et al. Construction of over-expression vector of bovine ACOX1 gene and preliminary study on fat deposition[J]. Chinese Journal of Animal Science, 2017,53(11):42-47.(in Chinese with English abstract)
[20]	瞿秋红, 黄叶, 蒋钦杨, 等. 影响猪脂肪沉积主要候选基因的研究进展[J]. 动物营养学报, 2019,31(7):2981-2987.
	QU Q H, HUANG Y, JIANG Q Y, et al. Research progress of major candidate genes affecting pig fat deposition[J]. Chinese Journal of Animal Nutrition, 2019,31(7):2981-2987.(in Chinese with English abstract)
[21]	SUN C, LIU C W, ZHANG Z P. Cloning of OLR1 gene in pig adipose tissue and preliminary study on its lipid-accumulating effect[J]. Asian-Australasian Journal of Animal Sciences, 2009,22(10):1420-1428.
[22]	OTTO T C, LANE M D. Adipose development: from stem cell to adipocyte[J]. Critical Reviews in Biochemistry and Molecular Biology, 2005,40(4):229-242. URL PMID
[23]	LUO X, LI H X, LIU R X, et al. Beta-catenin protein utilized by tumour necrosis factor-alpha in porcine preadipocytes to suppress differentiation[J]. BMB Reports, 2009,42(6):338-343. URL PMID
[24]	姜俊芳. 脂肪细胞分化相关因子基因表达在猪生长过程中的变化规律研究[D]. 杭州: 浙江大学, 2006.
	JIANG J F. Study on gene expression rules of adipocyte determination factors in pig development[D]. Hangzhou: Zhejiang University, 2006.(in Chinese with English abstract)
[25]	郭红芳, 宁越, 成功, 等. Krüppel-Like Factor 3(KLF₃)基因对牛脂肪沉积的作用[J]. 中国农业科学, 2019,52(7):1272-1281.
	GUO H F, NING Y, CHENG G, et al. The effect of krüppel-like factor 3(KLF₃) gene on bovine fat deposition[J]. Scientia Agricultura Sinica, 2019,52(7):1272-1281.(in Chinese with English abstract)

猪OLR1基因对肌内前脂肪细胞分化的影响

Effect of porcine OLR1 gene on intramuscular preadipocytes differentiation

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