秦川肉牛脂肪细胞PLIN2基因干扰siRNA筛选及过表达载体效率检测

doi:10.16431/j.cnki.1671-7236.2021.08.005

Abstract

Abstract: In order to obtain the high interference efficiency siRNA of adipocyte differentiation associated protein 2 (PLIN2) gene and the high overexpression efficiency gene overexpression vector of Qinchuan new beef line (hereinafter referred to as "Qinchuan beef cattle"), and provide experimental basis for the follow-up study of PLIN2 gene function in the process of adipocyte differentiation of Qinchuan beef cattle, in this study, si-PLIN2 targeting bovine PLIN2 mRNA sequence was synthesized by siRNA mediated target gene silencing and eukaryotic pcDNA3.1(+) -PLIN2 overexpression vector was constructed.FuGENE^® 6 low toxic transfection reagent was used to prepare transfection complex, transfect Qinchuan beef cattle adipocytes and induce differentiation.Real-time quantitative PCR was used to detect the relative expression of PLIN2 gene in each experimental group and control group, and the interference and overexpression efficiency of the target gene were calculated.Western blotting was used to detect the protein expression and the phenotype was observed by BODIPY staining.The results showed that after transfection of preadipocytes in Qinchuan beef cattle, three groups of si-PLIN2 (si-PLIN2_01, si-PLIN2_02 and si-PLIN2_03) compared with control group (si-PLIN2-NC), the expression of PLIN2 gene decreased (P<0.01), and the interference efficiency was 71%, 54% and 50%, respectively.The protein level of the si-PLIN2_01 and si-PLIN2_03 groups showed a downward trend compared with control group.Compared with control group, the number of lipid droplets in the interference group decreased significantly.Compared with control group, the expression of PLIN2 gene in overexpression group pcDNA3.1(+)-PLIN2 was significantly increased by 35 times (P<0.01), and the number of lipid droplets was significantly increased.In conclusion, the silencing of PLIN2 gene mediated by siRNA or the expression vector mediated by pcDNA3.1(+) in vitro could successfully interfere with or overexpress PLIN2 gene in the process of adipocyte differentiation in Qinchuan beef cattle, and affect the number of lipid droplets in the process of adipocyte differentiation.This study laid the foundation for further exploring the function of PLIN2 in the process of adipocyte differentiation in Qinchuan beef cattle.

Key words: Qinchuan beef cattle; PLIN2; gene interference; vector construction

CLC Number:

S813.3

LI Peiwei, ZHANG Weiyi, CHENG Gong, ZAN Linsen. The Selection of siRNA Efficiency and Detection of Overexpression Vector for PLIN2 Gene of Adipocytes in Qinchuan Beef Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2727-2735.

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The Selection of siRNA Efficiency and Detection of Overexpression Vector for PLIN2 Gene of Adipocytes in Qinchuan Beef Cattle

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