关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响

doi:10.11843/j.issn.0366-6964.2023.02.016

Abstract

Abstract: The aim of this study was to construct a recombinant interference vector of myocyte enhancer factor 2A (MEF2A) gene to explore the effect of MEF2A on bovine myoblasts. In this study, 3 healthy 3-day-old female Guanling cattle, with the body weight of about 21 kg, were selected to culture myoblastasts from longissimus dorsi muscle tissue. Four shRNA interference sequences of MEF2A gene and one NC negative control sequence were designed and connected to pGPU6-GFP-Neo vector, and the recombinant vector was transfected into Guanling bovine myoblasts. qRT-PCR was used to screen the vector with the best interference efficiency, and the effects of interfering MEF2A gene on the mRNA expression levels of myogenic factors MEF2B, MEF2C, MEF2D, cycle and apoptotic factors CDK2, CCNA2 and BCL2 were detected. Subsequently, flow cytometry and enzyme marker were used to investigate the effect of interference vector on myoblast proliferation and growth. Three biological replicates were set in each experimental group. Online software was used to predict the physicochemical properties and network spectra of bovine MEF2A protein. The results showed that the shRNA-MEF2A-3 vector with the best interference efficiency was selected successfully (P<0.01). After interfering MEF2A gene, MEF2B, MEF2C and MEF2D gene expressions in myoblasts were significantly up-regulated (P<0.01); The expressions of CDK2 and BCL2 were significantly down-regulated (P<0.05), the expression of CCNA2 was significantly down-regulated (P<0.01); Compared with the control group, the cell cycle in the interference group was significantly prolonged, and the proliferative activity of the interfered myoblasts was significantly lower than that in the control group (P<0.01). It can be preliminarily speculated that the MEF2A interference vector successfully transfected into bovine myoblasts can effectively inhibit the expression of MEF2A gene, change the gene expression pattern, and affect the division and proliferation of Guanling cattle myoblasts, which provides data support for further exploring the regulatory mechanism of MEF2A gene on the meat quality traits of Guanling cattle and mining local germplasm resources.

Key words: Guanling cattle, MEF2A gene, interference, myoblasts, cell proliferation

CLC Number:

S823.81

SUN Jinkui, XU Houqiang, SHI Pengfei, RUAN Yong. Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 584-595.

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Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle

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