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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- 英文论文题名：Cloning of TGF-β1、Smad4 and construction of eukaryon and RNAi expression vector from Ctenopharyngodon idellus
- 论文作者：张好放 ; 郁二蒙 ; 王广军 ; 余德光 ; 李志斐 ; 谢骏
- 英文论文作者：ZHANG Hao-fang ; YU Er-meng ; WANG Guang-jun ; YU De-guang ; LI Zhi-fei ; XIE Jun ; Pearl River Fishery Research Institute ; Chinese Academy of Fisheries Sciences ; College of Fisheries and Life ; Shanghai Ocean University
- 年：2016
- 作者机构：中国水产科学研究院珠江水产研究所;上海海洋大学水产与生命学院;
- 论文关键词：草鱼 ; 克隆 ; 真核表达载体 ; RNA干扰
- 英文论文关键词：Ctenopharyngodon idellus ; clone ; eukaryotic expression vector ; RNA interference
- 会议召开时间：2016-11-02
- 会议录名称：2016年中国水产学会学术年会论文摘要集
- 语种：中文
- 分类号：S917.4
- 学会代码：OGSB
- 会议名称：2016年中国水产学会学术年会
- 会议地点：中国四川成都
- 主办单位：中国水产学会、四川省水产学会
- 学会名称：中国水产学会
- 页数：1
- 文件大小：625k
- 原文格式：D
- 会议级别：全国
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。
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 1134 bp and the full-length ORF of Smad4 was 1644 bp. Secondly, the specific restriction sites were added to the both ends of ORF sequenceand then 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 si RNA sequences which were consisted of 48 bp have been designed. Then the completed shRNA was inserted into the carrier pRNA-U6.1/Neo. We successfully constructed the RNA interference expression vectors pRNA-U6.1/Neo-TGF beta1 and pRNA-U6.1/Neo-Smad4. The construction of RNA interference expression vectors and eukaryotic expression vector based on TGF-β1, Smad4 gene will lay the foundation for the next study of TGF-β1/Smad4 signaling pathways in the muscle tissue of grass carp.
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 1134 bp and the full-length ORF of Smad4 was 1644 bp. Secondly, the specific restriction sites were added to the both ends of ORF sequenceand then 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 si RNA sequences which were consisted of 48 bp have been designed. Then the completed shRNA was inserted into the carrier pRNA-U6.1/Neo. We successfully constructed the RNA interference expression vectors pRNA-U6.1/Neo-TGF beta1 and pRNA-U6.1/Neo-Smad4. The construction of RNA interference expression vectors and eukaryotic expression vector based on TGF-β1, Smad4 gene will lay the foundation for the next study of TGF-β1/Smad4 signaling pathways in the muscle tissue of grass carp.
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