绵羊Musclin基因真核表达载体的构建及其对肌细胞糖代谢和胰岛素作用的影响

doi:10.11843/j.issn.0366-6964.2021.03.008

Abstract

Abstract: To investigate the effects of sheep Musclin on glucose metabolism and insulin function, the structure properties of sheep Musclin protein were predicted by using various bioinformatics softwares and the eukaryotic expression vector of Musclin gene was constructed. The sheep fetal myoblasts were divided into 4 groups and treated as follows:mock-vector group (pcDNA3.1), Musclin-overexpressed group (pcDNA3.1-Musclin), mock-vector + insulin group (pcDNA3.1 + Insulin) and Musclin-overexpressed + insulin group (pcDNA3.1-Musclin + Insulin). Then the cells were collected at 48 and 72 h after differentiation induction, and the effects of Musclin on glucose metabolism in normal or insulin-treated differentiating myoblasts were analyzed and the changes of the related gene expression were detected by real-time PCR. The restriction endonuclease digestion and sequencing results indicated that the expression vector (pcDNA3.1-Musclin) was successfully constructed. The results of bioinformatics analysis showed that the sheep Musclin protein contained a signal peptide and had a restriction site at the 28th amino acid. The subcellular localization showed that it was in the extracellular space, and belonged to the secretory protein. Its secondary and tertiary structure mainly included alpha helixes and random coils. The cellular assays showed that the overexpression of sheep Musclin gene significantly reduced the glycogen content in the normal or insulin-treated sheep differentiating myoblasts and increased the glucose content in medium. The overexpression of Musclin inhibited the expressions of IRS1, AKT1, AKT2, GLUT1, GLUT4 mRNA, and promoted the expression of GSK3β mRNA in the Musclin-overexpressed group and Musclin-overexpressed + insulin group sheep myoblasts induced differentiation for 72 h, while only the expression of GLUT4 mRNA was inhibited after cells were induced differentiation for 48 h. In conclusion, the overexpression of the sheep Musclin gene affected the glucose metabolism and attenuated the functions of insulin in sheep differentiating myoblasts, and they coordinated with each other to maintain the homeostasis of glucose metabolism. The functional mechanisms of sheep Musclin were involved in the expression changes of the above genes and were related to the differentiation state of sheep myoblasts.

Key words: sheep, Musclin gene, bioinformatics, eukaryotic expression, glucose metabolism, insulin

CLC Number:

S826.2

ZHENG Tengfei, XIN Xiang, HAN Gaolian, LI Mengxin, LI Junling, QIN Jian, DU Rong. Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 641-652.

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Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes

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