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| 草鱼TGF-β1、Smad4基因的克隆及真核过表达和RNA干扰表达载体的构建 |
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张好放1,2, 郁二蒙1, 王广军1, 余德光1, 李志斐1, 谢骏1
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| (1.中国水产科学研究院 珠江水产研究所, 广州 510380;2.上海海洋大学 水产与生命学院, 上海 201306) |
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| 摘要: |
| 为研究TGF-β1/Smad4信号通路在草鱼（Ctenopharyngodon idellus）肌肉发育过程中的作用机制，首先克隆了草鱼TGF-β1、Smad4基因开放阅读框（ORF，Open reading frame）序列各1 134和1 644 bp。其次，在TGF-β1、Smad4序列两端分别加上相应的酶切位点，同时对pcDNA3.1（+）真核表达载体进行双酶切，将带酶切位点的片段正向插入到真核表达载体pcDNA3.1（+）中，构建TGF-β1、Smad4基因的真核过表达载体pcDNA3.1（+）-TGF-β1、pcDNA3.1（+）-Smad4。另一方面，根据TGF-β1、Smad4基因序列全长分别设计3对长度为48 bp的shRNA，然后将构建好的shRNA插入到载体pRNA-U6.1/Neo中，即可成功构建TGF-β1、Smad4基因的RNA干扰表达载体pRNA-U6.1/Neo-TGF-β1和pRNA-U6.1/Neo-Smad4。 |
| 关键词: 草鱼 克隆 真核表达载体 RNA干扰 |
| DOI：10.11841/j.issn.1007-4333.2017.04.13 |
| 投稿时间：2016-03-17 |
| 基金项目:国家自然科学基金资助项目（31402312）；国家现代农业产业技术体系建设专项（CARS-46-17） |
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| Cloning of TGF-β1, Smad4 and construction of eukaryon and RNAi expression vector from Ctenopharyngodon idellus |
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ZHANG Haofang1,2, YU Ermeng1, WANG Gangjun1, YU Deguang1, LI Zhifei1, XIE Jun1
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| (1.Pearl River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou 510380, China;2.College of Fisheries and Life, Shanghai Ocean University, Shanghai 201306, China) |
| Abstract: |
| In order to study the regulation of TGF-beta/Smad4 signaling pathways in regulating the growth of muscle in grass carp (Ctenopharyngodon idellus),the ORF(open reading frame) sequences of TGF-beta1,Smad4 gene were cloned.The results demonstrated that full-length ORF of TGF-beta1 was 1 134 bp and the full-length ORF of Smad4 was 1 644 bp.On one hand,Specific restriction sites were added to the both ends of ORF sequence and then the ORF sequences with enzyme sites were inserted into the eukaryotic expression vector pcDNA3.1(+) which was double digested by restriction enzyme.Two eukaryotic expression vector pcDNA3.1(+)-TGF-β1,pcDNA3.1(+)-Smad4 were constructed.On the other hand,according to TGF-β1、Smad4 gene sequence,three pairs siRNA sequences which were consisted of 48 bp have been designed.Then the completed shRNA was inserted into the carrier pRNA-U6.1/Neo.RNA interference expression vectors pRNA-U6.1/Neo-TGF beta1 and pRNA-U6.1/Neo-Smad4 were successfully constructed. |
| Key words: Ctenopharyngodon idellus clone eukaryotic expression vector RNA interference |
