Effect of porcine OLR1 gene on intramuscular preadipocytes differentiation

doi:10.3969/j.issn.1004-1524.2020.12.05

Acta Agriculturae Zhejiangensis ›› 2020, Vol. 32 ›› Issue (12): 2147-2153.DOI: 10.3969/j.issn.1004-1524.2020.12.05

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S433.3

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.

Figures/Tables 7

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

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

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

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

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.

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

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.

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Effect of porcine OLR1 gene on intramuscular preadipocytes differentiation

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