烟草尼古丁去甲基酶基因CYP82E4启动子结合蛋白的鉴定与分析

Identification and analysis of regulatory proteins binding to the promoter of nicotine demethylase gene CYP82E4 in tobacco

摘要: 以尼古丁去甲基酶编码基因(CYP82E4)上游的启动子功能元件作为诱饵,使用酵母单杂交方法从喷施乙烯利叶片的cDNA融合表达文库中钓取与之相互作用的结合蛋白.研究结果表明,诱饵载体pHIS2/pE4和cDNA融合表达载体pGADT7-Rec2共转化到酵母细胞中的效率为1×106.对文库的筛选结果表明,本研究共获得39个阳性克隆,且阳性克隆的插入片段在850～2 500 bp之间.将获得的39个阳性克隆进行测序,其中发现1个可能参与基因(CYP82E4)表达调控的锌指蛋白转录因子.下一步将对其功能进行分析,为通过分子育种手段培养低含量去甲基尼古丁的烟草提供实验基础.

Abstract: To identify proteins regulating the expression of the nicotine demethylase gene CYP82E4,a promoter fragment of the gene was used as the bait for screening of a cDNA library using yeast one-hybrid system.The results showed that the yeast cotransformation efficiency of bait vector pHIS2/pE4 and prey vector pGADT7-Rec2 reached 1×106.Thirty-nine positive clones were obtained and sequenced with the insertion size between 850 to 2 500 bp.Among these clones,a promising zinc finger transcription factor which could be involved in regulation of target gene was identified and its function will be charaterized to provide information for development of low-nornicotine tobacco variety via molecular breeding.