TALE-TFs构建方法的建立及优化

Establishment and optimization for TALE-TFs construction

目的在传统方案的基础上建立一种经济有效且易于操作的类转录激活效应子转录因子(TALE-TFs)的构建和功能检测方案。方法采用基于PCR的Golden gate克隆法分别尝试构建类转录激活效应物(TALEs)的六聚体、五聚体、四聚体和三聚体,比较构建结果,选择最优效果方案进行TALE-TFs的构建。采用片段置换反应(FSR)构建了含有TALE-TFs结合片段的RFP质粒pminCMV,并与TALE-TFs进行共转染,观察红色荧光验证TALE-TFs的转录活性。结果 TALEs中所含的串联重复模块越少,获得的构建产物越多。共转染时,TALE-TFs使得pminCMV成功启动表达。结论该研究为不同条件下实验方案的选择提供了依据,并利用含有TALE-TF结合片段和红色荧光的质粒建立了快捷直观的转录活性验证方法。

Objective To optimize the method of transcription activator‐like effector transcription factors （TALE‐TFs） construction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction （FSR） .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .