柽柳类锌指基因ThZFL酵母诱饵表达载体的构建及其表达验证

利用PCR技术从柽柳cDNA文库中克隆出ThZFL目的基因序列,连接到pMD18-T载体,转化大肠杆菌DH5α感受态,经检验后提取质粒,用BamH I和EcoR I限制性内切酶双酶切pMD18-T-ThZFL载体和pG-BKT7-BD空载体,并将酶切产物胶回收后,用T4 DNA连接酶连接过夜后转化大肠杆菌DH5α感受态...

Expression and Construction of Yeast Expression Plasmid Containing ThZFL Gene from Tamarix hispida in Yeast TwoHybrid System

The ThZFL gene was amplified by PCR from the Tamarix hispida cDNA library and inserted into pMD18-T vector,then transformed into E.coli DH5α to obtain the transformant.The pMD18-T-ThZFL and pGBKT7-BD were digested with BamH I and EcoR I,and resulting pGBKT7-ThZFL was transformed into E.coli DH5α.The positive clones were screened and identified by PCR,restriction endonuclease digestion and DNA sequence analysis.Then the positive recombinant plasmid pGBKT7-ThZFL was transformed into the competent yeast Y2Hgold strain and spread onto separate plates containing SD/-Trp,SD/-Trp/X,DDO and TDO.Results showed that no reporter genes were activated,and the bait vector was not toxic to Y2Hgold,suggesting the plasmid pGBKT7-ThZFL was constructed successfully,and could be used to screen target proteins interacting with the bait protein by the yeast two-hybrid system.