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| 从江香猪PDK4基因干扰载体的构建与检测 | |
| 引用本文： | 杨洋,许厚强,陈伟,徐敏,谌颖莲.从江香猪PDK4基因干扰载体的构建与检测[J].中国畜牧兽医,2018,45(4):873-880. |
| 作者姓名： | 杨洋 许厚强 陈伟 徐敏 谌颖莲 |
| 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, 贵阳 550025; 2. 贵州省动物遗传育种与繁殖重点实验室, 贵阳 550025; 3. 贵州大学动物科学学院, 贵阳 550025; 4. 贵州大学生命科学学院, 贵阳 550025 |
| 基金项目： | 国家科技支撑计划（2015BAD03B02-3）；黔科合重大专项（黔科合NY字[2013]6008号） |
| 摘 要： | 为了进一步揭示PDK4基因在猪肌内前体脂肪细胞中的调控作用，本试验采集5日龄从江香猪背最长肌，采用Ⅱ型胶原酶进行消化，分离肌内前体脂肪细胞，进行原代和传代培养及形态学观察；根据克隆得到的PDK4基因序列，利用在线软件设计合成3对特异性单链siRNA序列和1对阴性对照序列，经退火后形成双链，连接pLVX-shRNA2-Puro载体，重组质粒采用双酶切及测序进行验证。针对干扰载体转染培养的从江香猪肌内前体脂肪细胞，采用实时荧光定量PCR方法筛选出最有效的干扰序列。结果显示，原代从江香猪肌内前体脂肪细胞约4 h开始贴壁，24 h后细胞形态均一，贴壁牢固，第5天汇合呈单层细胞，基本铺满培养板；重组质粒经双酶切及测序鉴定结果显示，设计合成的4对siRNA干扰序列与载体质粒均连接正确，表明成功构建了PDK4基因的干扰载体，并可成功转染猪肌内前体脂肪细胞；实时荧光定量PCR方法检测发现，干扰载体可有效减少从江香猪肌内脂肪前体脂肪细胞中PDK4基因mRNA的表达，最佳干扰效率可达81.90%。本试验成功培养了从江香猪肌内前体脂肪细胞，构建并得到了PDK4基因的最佳干扰载体，为进一步研究PDK4基因对脂肪代谢的调控机制奠定基础。 |
| 关 键 词： | 从江香猪 PDK4基因 肌内前体脂肪细胞 干扰载体 干扰效率 |
| 收稿时间： | 2017-10-16 |
Construction and Detection of the Interference Vector of PDK4 Gene in Congjiang Xiang Pig |
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| YANG Yang,XU Houqiang,CHEN Wei,XU Min,CHEN Yinglian.Construction and Detection of the Interference Vector of PDK4 Gene in Congjiang Xiang Pig[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):873-880. | |
| Authors: | YANG Yang XU Houqiang CHEN Wei XU Min CHEN Yinglian |
| Institution: | 1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Platean Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Guizhou Province, Guiyang 550025, China; 3. College of Animal Science, Guizhou University, Guiyang 550025, China; 4. College of Life Science, Guizhou University, Guiyang 550025, China |
| Abstract: | In order to further reveal the regulation function of PDK4 gene in intramuscular preadipocyte cells,this study collected the longissimus dorsi from the 5-day-old Congjiang Xiang pig.Type Ⅱ collagenase was adopted to digest the cells,and intramuscular preadipocyte cells were separated and cultured,and then the morphology were observed.According to the cloning PDK4 sequence,online software was used to design and synthesize three pairs of specific single siRNA and a pair of negative control sequences,annealing products were cloned into pLVX-shRNA2-Puro vector directly.The recombinant plasmids were verified by double enzyme digestion and sequencing.The constructed interference plasmids were transfected into the intramuscular preadipocyte cells of Congjiang Xiang pig.The most effective interference sequences were screened by Real-time PCR.The results showed that the primary culture of intramuscular preadipocyte cells started adherent in about 4 h,and the cell morphology was uniform after 24 h,the cells were firmly adherent.On the 5th day,the adherent cells were monolayer.The results of double enzyme digestion and sequencing showed that the four pairs of siRNA interference sequences designed and synthesized were correctly connected with the vector plasmid,indicating that the PDK4 gene interference vectors were constructed successfully,and the transfection of the intramuscular preadipocyte cells was successful.The results of Real-time PCR showed that the interfering vector could effectively reduce the mRNA expression of PDK4 gene in intramuscular preadipocyte cells with the best interference efficiency of 81.90%.In this study,we successfully cultured the intramuscular preadipocyte cells and constructed the interference vector of PDK4 gene,which laid the foundation for further research on the regulation mechanism of PDK4 gene on lipid metabolism. |
| Keywords: | Congjiang Xiang pig PDK4 gene intramuscular preadipocyte cell interference vector interference efficiency |
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