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关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响
引用本文:孙金魁,许厚强,石鹏飞,阮涌.关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响[J].畜牧兽医学报,2023,54(2):584-595.
作者姓名:孙金魁  许厚强  石鹏飞  阮涌
作者单位:1. 贵州大学 高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室, 贵阳 550025;2. 贵州大学动物科学学院, 贵阳 550025
基金项目:国家自然科学基金(31571279);教育部促美大项目(【2015】2062号)
摘    要:旨在构建肌细胞增强因子MEF2A(myocyte enhancer factor 2A)基因的重组干扰载体,探究MEF2A基因对牛成肌细胞的影响。本研究选择3头健康的3日龄雌性关岭牛,体重约为21 kg,采集背最长肌组织成功培养成肌细胞。设计MEF2A基因的4对shRNA干扰序列和1对NC阴性对照序列,将其连接至pGPU6-GFP-Neo载体上,转染重组载体至关岭牛成肌细胞。采用qRT-PCR法筛选干扰效率最佳的载体,并检测干扰MEF2A基因对肌生成因子MEF2B、MEF2C、MEF2D,周期与凋亡因子CDK2、CCNA2、BCL2 mRNA表达水平的影响;随后利用流式细胞仪与酶标仪探究干扰载体对成肌细胞增殖生长的影响,各试验组别设置3个生物学重复。同时运用在线软件预测牛MEF2A蛋白理化性质与网络谱图。结果显示,本研究成功筛选出干扰效率最佳的shRNA-MEF2A-3载体(P<0.01)。MEF2A基因被抑制后,成肌细胞中MEF2B、MEF2C与MEF2D基因表达量均极显著上调(P<0.01);CDK2与BCL2表达量皆显著下调(P<0.05),CCNA2表达量极显...

关 键 词:关岭牛  MEF2A基因  干扰  成肌细胞  细胞增殖
收稿时间:2022-07-11

Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle
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.
Authors:SUN Jinkui  XU Houqiang  SHI Pengfei  RUAN Yong
Institution:1. Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountains Region of Ministry of Education, Guizhou University, Guiyang 550025 China;2. College of Animal Sciences, Guizhou University, Guiyang 550025, China
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.
Keywords:Guanling cattle  MEF2A gene  interference  myoblasts  cell proliferation  
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