山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证  

Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene

作　　者：陆健[1,2] 张世芳[2] 张小宁[2] 刘佳森[2,3] 李碧春[1] 赵福平[2] 张莉[2] 魏彩虹[2] 杜立新[2] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009 [2]中国农业科学院北京畜牧兽医研究所国家畜禽分子遗传育种中心,北京100193 [3]内蒙古农牧科学院畜牧研究所,内蒙古呼和浩特010031

出　　处：《中国畜牧兽医》2012年第9期1-6,共6页China Animal Husbandry & Veterinary Medicine

基　　金：转基因生物新品种培育重大专项--转基因肉羊育种新材料创制课题(2009ZX08008-003B)

摘　　要：myostatin基因是肌肉生长和发育的关键负调控因子之一,该基因突变或失活的物种都呈现肌肉增大的双肌表型,暗示在家畜中使该基因失活可以增加其产肉量。为了进一步揭示myostatin在山羊胎儿成纤维细胞中的生物学功能以及制备可有效失活myostatin基因的工具,将筛选的myostatin基因潜在RNA干扰靶位点连接到慢病毒干扰载体的转移质粒中,构建慢病毒介导的RNA干扰载体,验证其干扰效果。结果表明,干扰序列与转移质粒连接正确,成功构建myostatin基因慢病毒干扰载体,慢病毒三质粒共转293T细胞,获得的慢病毒颗粒可高效转染山羊胎儿成纤维细胞,Real-time PCR检测发现慢病毒干扰载体Lv322有效减少山羊胎儿成纤维细胞中myostatin基因mRNA 75%的表达(P<0.01),Western blotting检测显示myostatin蛋白则有效降低了94%(P<0.01)。本试验为研究myostatin基因的功能及制备失活转基因动物提供有效工具以及理论基础。Myostatin gene was an important regulator of muscle growth and development,and the gene mutation or inactivation of the species also displayed the double muscle phenotype of the skeletal hypermuscular,suggesting that modulating myostatin gene expression may allow for increased meat production of livestock.To explore the biological function of myostatin in goat fetal fibroblast and generate the inactivation tool of myostatin,we had cloned the potent targeted site of myostatin into the transfer vector of the lentiviral RNA interference system,constructed the lentiviral mediated RNA interference vector to knockdown myostatin and detected the knockdown efficiency.The results demonstrated that the sequences of RNA interference had been inserted into the transfer vector rightly,and we had constructed the RNA interference of myostatin gene mediated by the lentiviral vector successfully.In addition,we obtained the lentiviral particles by co-transfected of the three plasmids into 293T cells could be transfected into goat fetal fibroblasts with high effective.The results of Real-time PCR demonstrated that the lentiviral vector Lv322 had reduced the myostatin mRNA by 75%（P〈0.01）,and Western blotting also demonstrated that the protein level of myostatin had been decreased by 94%（P〈0.01）.The results would provide the important foundation and effective tool to detect the function of myostatin and to generate the inactivation of transgenic animal in the future.

关 键 词：MYOSTATIN基因 山羊胎儿成纤维细胞 慢病毒载体 RNA干扰 

分 类 号：Q78[生物学—分子生物学]