Construction of Eukaryotic Expression Vector of FGF10 Gene and Its Expression in Different Tissues of Congjiang Xiang Pig (Sus scrofa)
XU Min1, XU Hou-Qiang1,*, YANG Yang1, CHEN Wei1,2
1 College of Animal Science, Guizhou University/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/Guizhou Key Laboratory of Animal Genetics, Breeding and Reproduction, Guizhou 550025, China; 2 College of Life Science, Guizhou University, Guiyang 550025, China
Abstract The fibroblast growth factor 10 gene (FGF10) is a member of the fibroblast growth factor family, belonging to the FGF7 subfamily of six subfamilies of FGFs, it is the only FGF specifically expressed in white adipose tissue, and plays an important role in the development and metabolism of adipose tissue. The purpose of this study was to investigate the regulation of fat metabolism of FGF10 gene in the intramuscular preadipocytes of Congjiang Xiang pig (Sus scrofa), and the function of FGF10 in regulating fat deposition was revealed. The qRT-PCR was used to detect the relative expression level of FGF10 gene in different tissues of Congjiang Xiang pig. According to the CDS region of the cloned FGF10 gene, the expression vector pEGFP-C1-FGF10 was constructed. The intramuscular preadipocyte cells had been digested by type Ⅱ collagenase. The oil red O staining method was used identification after induction culture. qRT-PCR was used to detect the expression of FGF10 gene in precursors adipocytes in the intramuscular preadipocytes of Congjiang Xiang pig at different induction stages. The recombinant plasmid pEGFP-C1-FGF10 was transfected by liposome method in the intramuscular preadipocytes of Congjiang Xiang pig, and then detected the expression levels of peroxisome proliferator activated receptor γ gene (PPARγ), adiponectin gene (ADIPOQ), fatty acid binding protein 4 gene (FABP4), fatty acid synthase gene (FAS), adipose triglyceride lipase gene (ATGL), lipoprotein lipase gene (LPL), hormone-sensitive lipase gene (HSL)and acetyl-CoA carboxylase gene (ACC) after overexpression of FGF10. The results showed that the extremely higher expression levels of FGF10 gene in the stomach and significantly higher than other tissues (P<0.05), followed by high expression in the kidney and fat, which were the lowest in the dorsal longest muscle. At the early stage of induction, the expression level of FGF10 gene gradually increased, and the expression level of FGF10 gene was the highest at the induction and differentiation of 48 h. The pEGFP-C1-FGF10 expression vector was successfully constructed by double enzyme digestion and sequencing, and was successfully expressed in the intramuscular preadipocytes of Congjiang Xiang pig. Compared with blank controls, the expression levels of ACC, FABP4, FAS, LPL, and PPARγ gene had all increased, while the expression levels of ADIPOQ, ATGL, and HSL gene had all decreased. The results of this study showed that FGF10 gene had certain regulation effect on the fat deposition, this study provides basic data for revealing FGF10 gene's regulation of fat metabolism.
XU Min,XU Hou-Qiang,YANG Yang, et al. Construction of Eukaryotic Expression Vector of FGF10 Gene and Its Expression in Different Tissues of Congjiang Xiang Pig (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(3): 471-480.
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