关岭牛IGF1基因干扰载体构建及其对成肌细胞的影响  

Construction of IGF1 Gene Interference Vector in Guanling Cattle(Bos taurus)and Its Effect on Myogenic Cells

作　　者：宋林锦 许厚强[1,2] 李永 孙金魁 SONG Lin-Jin;XU Hou-Qiang;LI Yong;SUN Jin-Kui(Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals,Ministry of Education,Guizhou University/Key Laboratory of Animal Genetic Breeding and Reproduction,Guizhou Province,Guiyang 550025,China;School of Animal Science,Guizhou University,Guiyang 550025,China;School of Life Sciences,Guizhou University,Guiyang 550025,China)

机构地区：[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 [3]贵州大学生命科学学院,贵阳550025

出　　处：《农业生物技术学报》2022年第5期896-907,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31571279)。

摘　　要：胰岛素样生长因子1(insulin like growth factor 1,IGF1)是通过调控骨骼肌蛋白的合成影响肌肉发育的单链多肽类激素,对细胞增殖等多种生理功能具有重要作用,本研究旨在探究IGF1基因对关岭牛(Bos taurus)成肌细胞的影响。选用健康的成年(2~3岁)关岭牛3头,采集心、肝、脾、肺、肾、前腿肌、后腿肌、背最长肌、斜方肌、臀肌、胃、小肠12个组织样提取RNA,采集背最长肌组织成功培养关岭牛成肌细胞。运用qPCR技术检测IGF1基因在不同组织中的相对表达量;运用在线软件对关岭牛IGF1蛋白质理化性质、二级结构和三级结构、亚细胞定位进行分析;同时通过在线软件设计IGF1基因的3对短发夹RNA(short hairpin RNA,shRNA)干扰序列和1对阴性对照序列,与pGPU6-GFP-Neo载体连接后进行测序,将构建成功的IGF1基因干扰载体转染至成肌细胞;运用qPCR技术检测并筛选出干扰效率最高的干扰载体,分析该干扰载体对IGF1基因表达的影响;运用qPCR技术检测干扰IGF1基因对细胞增殖相关基因细胞周期蛋白D1(cyclin D1)和细胞周期蛋白依赖性激酶2(cyclin-dependent kinases 2,CDK2)的相对表达量影响;运用CCK8(Cell Counting Kit-8)法检测成肌细胞增殖情况。结果表明,IGF1基因在关岭牛心、前腿肌、后腿肌、背最长肌、斜方肌、臀肌等12个组织样中均有表达,在肝中相对表达量最高,极显著高于其他组织(P<0.01);在培养关岭牛成肌细胞的基础上,成功构建关岭牛IGF1基因干扰载体,并筛选出shRNA-IGF1-1干扰效率最高(P<0.01);IGF1蛋白的分子式为C_(744)H_(1186)N_(214)O_(216)S_(15),相对分子质量为17.06581 kD,理论等电点为9.36,属碱性不稳定蛋白;IGF1基因被抑制后,细胞增殖相关基因cyclin D1和CDK2的表达量均极显著低于shRNA-NC(P<0.01)。CCK8法检测细胞增殖结果表明,IGF1基因被抑制后,在24和72 h成肌细胞增殖均显著低于对照组(P<0.05),48 h为极显著水平(P<0.01),表Insulin like growth factor 1(IGF1)is a single-chain peptide hormone that affects muscle development by regulating the synthesis of skeletal muscle proteins,and is important for cell proliferation and other physiological functions.Three healthy adult(2~3 years old)Guanling cattle(Bos taurus)were selected and 12 tissue samples were collected from heart,liver,spleen,lung,kidney,foreleg,hindleg,longest dorsal muscle,rhomboid,gluteus,stomach and small intestine for RNA extraction,respectively,and the longest dorsal muscle was successfully cultured in Guanling myogenic cells.The relative mRNA expression of IGF1 gene in different tissues was detected by qPCR;the physical and chemical properties,secondary and tertiary structures,and subcellular localization of IGF1 protein in Guanling bovine were analyzed by online software;meanwhile,three pairs of short hairpin RNA(shRNA)interference sequences and one pair of negative control sequences of IGF1 gene were designed by online software,and sequenced with pGPU6-GFP-Neo.IGF1 interference vector was sequenced and transfected into adult myoblasts.qPCR was used to detect and screen the most efficient interfering vector and analyze the effect of the interfering vector on IGF1 gene expression.The relative mRNA expression of cyclin-dependent kinases 2(CDK2)and cyclin-dependent kinases 1(Cyclin D1)was measured by qPCR,and the proliferation of myogenic cells was detected by CCK8.The results showed that IGF1 gene was expressed in 12 tissues including heart,foreleg,hindleg,longest back,rhomboid and gluteus muscles of Guanling cattle,and the relative mRNA expression of liver was the highest,which was significantly higher than that of other tissues(P<0.01).The IGF1 protein had a molecular formula of C_(744)H_(1186)N_(214)O_(216)S_(15),a relative molecular mass of 17.06581 kD and a theoretical isoelectric point of 9.36,and was a basic unstable protein;After the IGF1 gene was inhibited,the expression of cyclin D1 and CDK2,was significantly lower than that of shRNA-NC(P<0.01).The results of

关 键 词：关岭牛 胰岛素样生长因子1(IGF1) RNA干扰 成肌细胞 细胞增殖 

分 类 号：S823[农业科学—畜牧学]