筛选dbat启动子顺式元件结合蛋白的酵母单杂交文库的构建  

Construction of Yeast One-hybrid Library for Screening the Binding Proteins of dbat Promoter Cis-element

作　　者：姚瑞枫[1] 张蒙[1] 张鹏[1] 李书涛[1] 陈丽[1] 付春华[1] 余龙江[1] 

机构地区：[1]华中科技大学生命科学与技术学院,武汉430074

出　　处：《生物技术通报》2009年第9期117-120,共4页Biotechnology Bulletin

基　　金：国家自然科学基金面上项目(NSFC20776058);2006年教育部新世纪优秀人才支持计划(NCET-06-0646);国家大学生创新性实验计划项目(2008055)

摘　　要：旨在构建酵母单杂交文库用于筛选与dbat基因启动子顺式元件结合的转录因子。首先,分离dbat启动子上的顺式作用元件,并进行3个拷贝的重复后,和报告质粒pHis 2.1连接构建诱饵载体pHis 2.1-dp3。然后,从茉莉酸甲酯诱导的红豆杉细胞中提取总RNA,以纯化出的mRNA为模板,依次完成ss cDNA和ds cDNA的合成,并将纯化的ds cDNA、线性化载体pGADT7-Rec2和诱饵载体pHis 2.1-dp3共转化酵母细胞Y187,各取100μl分别涂布于SD/-leu和SD/-leu/-trp平板,用于计算重组效率和转化效率,剩余转化液涂布于SD/-leu/-trp/-his/20 mM3-AT平板,用于筛选阳性克隆。结果表明,重组效率为1.49×106CFU/μg,共转化效率为1.93×105CFU/μg;对阳性克隆质粒进行酶切、测序分析,得到6个可编码结合蛋白的基因。以上工作为筛选调控紫杉醇合成关键酶基因dbat表达的转录因子奠定了基础。It was to screen the binding proteins of dbat promoter cis-element, a yeast one-hybrid library was constructed. Firstly, the dbat promoter cis-element was cloned and repeated three copies, then ligated with reporter vector pHis 2.1 to construct the bait vector pHis 2. 1-dp3. Secondly, total RNA was extracted from Taxus media cells induced by methyl jasmonate, and the mRNA purified from total RNA was used as template to synthesize ss cDNA, then the ss cDNA was used as the template to synthesize ds cDNA. The purified ds cDNA, lined pGADTT-Rec2 and pHis 2.1-dp3 were transformed into yeast strain Y187 together by LiAc-mediated method. 100 μl of the transformation liquid was respectively coated on SD/-leu agar plate and SD/-leu/-trp agar plate to calculate the recombi- nation efficiency and the transformation efficiency, and the residual transformation liquid was coated on SD/-leu/-trp/-his/20 mM 3-AT agar plates to screen positive clones. The results showed that the recombination efficiency of the library was 1.49 × 10^6 CFU/μg, and the co-transformation efficiency was 1.93 × 10^5 CFU/μg, and the restriction analysis and sequence analysis results showed that six genes encoding binding proteins of dbat promoter cis-element were obtained. These results have laid a foundation for screening the transcription factors that regulate the expression of dbat gene, which is a key gene involved in the biosynthesis of Taxol.

关 键 词：红豆杉细胞 dbat基因 酵母单杂交文库 结合蛋白 转录调控 

分 类 号：Q78[生物学—分子生物学]