藏绵羊KLF7基因表达特征分析及其过表达对前脂肪细胞增殖分化的影响

doi:10.16431/j.cnki.1671-7236.2022.08.008

Abstract

Abstract: 【Objective】 The objective of this study was to analyze the expression profile of Krüppel-like factor 7 (KLF7) gene,and explore the effect of its overexpression on proliferation and differentiation of preadipocytes in Tibetan sheep.【Method】 Preadipocytes that isolated from adipose tissue in Tibetan sheep were cultured and induced differentiation.The mRNA relative expression of KLF7 gene in 7 tissues (brain,subcutaneous fat,kidney,longissimus dorsi muscle,rumen,testis and ileum) and preadipocytes at different differentiation stages (0,2nd,4th and 8th days) in Tibetan sheep were analyzed by Real-time quantitative PCR.The CDS sequence of KLF7 gene was amplified from adipose tissue by RT-PCR,and ligated into the pcDNA3.1(+) eukaryotic expression vector to conduct pcDNA3.1-KLF7 overexpression plasmid.The pcDNA3.1-KLF7 overexpression plasmid was transfected into preadipocytes,and the mRNA expression of adipocyte proliferation and differentiation marker genes were detected by Real-time quantitative PCR.The EdU-positive cell,cell viability and production of lipid droplets in KLF7 gene overexpressing preadipocytes were detected by EdU,CCK-8 and Oil red O staining methods,respectively.【Result】 KLF7 gene was expressed in 7 tissues of Tibetan sheep,and the highest expression was found in brain,followed by subcutaneous fat and kidney,which were significantly higher than other tissues (P<0.05).The mRNA expression of adipocytes which was induced at the 2nd,4th and 8th days were significantly higher than that of undifferentiated cell (P<0.05),and the highest expression was identified in adipocytes at the 2nd day after differentiation.In KLF7 gene overexpressing preadipocytes at the 2nd day,the expression of CDK4,CyclinB1 and CyclinD1 genes were significantly or extremely significantly inhibited (P<0.05 or P<0.01),and the cell viability and the number of EdU positive cell were also extremely significantly decreased (P<0.01).In KLF7 gene overexpressing preadipocytes at the 8th day,the expression of PPARγ,Glut4 and ELOVL6 genes were significantly or extremely significantly down-regulated (P<0.05 or P<0.01),and the lipid deposition was extremely significantly reduced (P<0.01). The results indicated that KLF7 gene overexpression inhibited the proliferation and differentiation of preadipocytes in Tibetan sheep.【Conclusion】 The widely expressed of KLF7 gene was found in tissues of Tibetan sheep,and there was a higher expression in brain,subcutaneous fat and kidney.The expression of differentiated adipocyte was significantly higher than that of undifferentiated,and the highest expression was observed at the 2nd day after differentiation.KLF7 gene overexpression could inhibit the proliferation and differentiation of preadipocytes in Tibetan sheep,which provided basic data for elucidating the molecular regulation mechanism of fat deposition in Tibetan sheep.

Key words: Tibetan sheep; KLF7 gene; expression; preadipocytes; proliferation; differentiation

CLC Number:

S826

ZHANG Xueping, SHI Bingang, JIN Xiayang, WANG Xiangyan, LAN Lijuan, SHI Yu, QI Youpeng, ZHAO Shijie, LI Shaobin, HU Jiang. Expression Characterization Analysis of KLF7 Gene and Effects of Its Overexpression on Proliferation and Differentiation of Preadipocytes in Tibetan Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2920-2930.

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Expression Characterization Analysis of KLF7 Gene and Effects of Its Overexpression on Proliferation and Differentiation of Preadipocytes in Tibetan Sheep

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