Construction of MEF2A Gene Overexpression Vector in Guanling Cattle (Bos taurus) and Its Effect on Myoblasts
SUN Jin-Kui, XU Hou-Qiang*, RUAN Yong, SHI Peng-Fei, XIONG Xun
College of Animal Sciences/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountains Region of Ministry of Education, Guizhou University, Guiyang 550025, China
Abstract Myocyte enhancer factor 2A (MEF2A) is an important member of myocyte enhancer factor family, which plays an important role in the process of muscle genesis and regeneration. In this study, the CDS region of the MEF2A gene was amplified and the overexpression vector pEGFP-C1-MEF2A was constructed. The gene sequence and protein structure of MEF2A were analyzed. The expression characteristics of MEF2A in various tissues of Guanling cattle (Bos taurus) at different stages were detected by qRT-PCR. Meanwhile, the overexpressed vector was transfected into Guanling bovine myoblasts, and the effect of the overexpressed vector on the proliferation and growth of the myoblasts was investigated by flow cytometry and cell growth analyzer. The results showed that MEF2A gene could be expressed in multiple tissues of 3-days-old cattle and 30-months-old cattle, and the difference of expression level was extremely significant (P<0.01). MEF2A gene expression was extremely significantly up regulated after transfection of overexpressed vector into cells (P<0.01), MEF2B, MEF2C and MEF2D were extremely significantly up-regulated (P<0.01); The expression of cyclin-dependent kinase 2 (CDK2) was not significantly different, the expression of cell cycle protein A2 (CCNA2) was extremely significantly up-regulated (P<0.01). Compared with pEGFP-C1 group, the cell cycle of pEGFP-C1-MEF2A group was extremely significantly shortened, and the proliferative activity of myoblasts in pEGFP-C1 group was extremely significantly higher than that in pEGFP-C1 group at 6 h (P<0.01), indicating that MEF2A overexpression vector transfected into Guanling bovine myoblasts can effectively up-regulate MEF2A gene expression and change gene expression pattern. In this study, pEGFP-C1-MEF2A overexpression vector was successfully constructed to detect the effect of MEF2A gene on the expression of quality-related genes in Guanling cattle. This study provides basic data support for further exploring the regulatory mechanism of MEF2A gene on quality traits in Guanling cattle.
SUN Jin-Kui,XU Hou-Qiang,RUAN Yong, et al. Construction of MEF2A Gene Overexpression Vector in Guanling Cattle (Bos taurus) and Its Effect on Myoblasts[J]. 农业生物技术学报, 2023, 31(11): 2294-2303.
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