烟草NtCBT基因启动子酵母单杂诱饵载体构建及互作蛋白筛选  

Construction of Yeast One-hybrid Bait Vector of Tobacco NtCBT Gene Promoter and Screening of Interacted Proteins

作　　者：余婧 杨慧 余世洲 赵会纳 郑庆霞[2] 王兵 雷波 YU Jing;YANG Hui;YU Shi-zhou;ZHAO Hui-na;ZHENG Qing-xia;WANG Bing;LEI Bo(Guizhou Academy of Tobacco Science,Molecular Genetics Key Laboratory of China Tobacco,Guiyang 550081;Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001)

机构地区：[1]贵州省烟草科学研究院烟草行业烟草分子遗传重点实验室,贵阳550081 [2]中国烟草总公司郑州烟草研究院,郑州450001

出　　处：《生物技术通报》2022年第10期73-79,共7页Biotechnology Bulletin

基　　金：中国烟草总公司贵州省公司科技项目(2021XM02);中国烟草总公司科技项目[110202101005(JY-05),110202001021(JY-04)]。

摘　　要：为了筛选烟草类西柏烷生物合成途径中西柏三烯一醇合酶基因(NtCBT)的上游调控转录因子,将NtCBT基因启动子截为6段(P1-P6区域),分别构建酵母单杂诱饵载体pAbAi-Px,将pAbAi-Px转化Y1H酵母感受态细胞构建诱饵菌株并进行自激活检测,从烟草腺毛酵母cDNA文库中筛选与P5区域(-279--119 bp)互作的转录因子。结果表明,P1-P5区域所构建的诱饵菌株,在AbA浓度为200 ng/mL时转录自激活得到有效抑制;在以P5区域为诱饵菌株的筛库实验中,共获得49个阳性克隆,去除冗余序列后35个克隆为非重复序列,其中3个克隆注释为ANL2、ML1及NF-Y转录因子。以上结果为进一步研究NtCBT基因的表达调控机制奠定了基础。To screen the upstream regulatory transcription factors of cembratrienol synthase(NtCBT)in the cembranoids synthesis pathway of tobacco(Nicotiana tabacum L.),the promoter of NtCBT gene was cut into P1 to P6 regions,six yeast one-hybrid bait vectors pAbAi-Px were constructed and transformed into Y1H competent yeast cells,bait strains were constructed,and a self-activation experiment was completed.Furthermore,a screening was finished from yeast cDNA library of tobacco trichomes in order to obtain the transcription factors that interacted with P5 regions(-279--119 bp).The results showed that,the growths of the strains containing P1 to P5 regions bait strain were inhibited on the medium added with 200 ng/mL AbA.A total of 49 positive colonies were obtained through Y1H screening when P5 regions as bait strain,among them,35 colonies were non-repeating sequences,and 3 of colonies were annotated as transcription factors of ANL2,ML1 and NF-Y.Above results lay a foundation for further study of gene expression and regulation mechanism of NtCBT.

关 键 词：酵母单杂交 转录因子 栽培烟草 启动子 NtCBT基因 类西柏烷 

分 类 号：Q943.2[生物学—植物学] S572[农业科学—烟草工业]