CAJ | 학술논문

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。
목적：재전통방안적기출상건립일충경제유효차역우조작적류전록격활효응자전록인자（TALE‐TFs）적구건화공능검측방안。방법채용기우PCR적Golden gate극륭법분별상시구건류전록격활효응물（TALEs）적륙취체、오취체、사취체화삼취체，비교구건결과，선택최우효과방안진행TALE‐TFs적구건。채용편단치환반응（FSR）구건료함유 TALE‐TFs결합편단적RFP질립pminCMV ，병여TALE‐TFs진행공전염，관찰홍색형광험증TALE‐TFs적전록활성。결과 TALEs중소함적천련중복모괴월소，획득적구건산물월다。공전염시，TALE‐TFs사득pminCMV성공계동표체。결론해연구위불동조건하실험방안적선택제공료의거，병이용함유TALE‐TF결합편단화홍색형광적질립건립료쾌첩직관적전록활성험증방법。
Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,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 .