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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1597

摘要/Abstract

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。

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

Abstract:

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.

Key words: yeast one-hybrid, transcription factors, Nicotiana tabacum L., promoter, NtCBT gene, cembranoids

余婧, 杨慧, 余世洲, 赵会纳, 郑庆霞, 王兵, 雷波. 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[J]. 生物技术通报, 2022, 38(10): 73-79.

YU Jing, YANG Hui, YU Shi-zhou, ZHAO Hui-na, ZHENG Qing-xia, WANG Bing, LEI Bo. Construction of Yeast One-hybrid Bait Vector of Tobacco NtCBT Gene Promoter and Screening of Interacted Proteins[J]. Biotechnology Bulletin, 2022, 38(10): 73-79.

图/表 8

图1 重组诱饵载体pAbAi-Px线性化 M：Marker；泳道1，3，5，7，9，11：分别为未酶切质粒pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5，pAbAi-P6；泳道2，4，6，8，10，12：分别为BstB I酶切线性化质粒：pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5，pAbAi-P6

Fig.1 Linearization of recombinant bait vector pAbAi-Px M：Marker. Lane 1，3，5，7，9，and 11：Undigested plasmid pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5 and pAbAi-P6，respectively. Lane 2，4，6，8，10，and 12：Plasmid pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5 and pAbAi-P6，digested with BstB I restricted enzyme，respectively

图2 pAbAi-Px诱饵载体自激活 A-E：分别为诱饵菌株Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3]，Y1H[pAbAi-P4]，Y1H[pAbAi-P5]在SD/-Ura培养基上的生长情况；F-J：分别为诱饵菌株Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3]，Y1H[pAbAi-P4]，Y1H[pAbAi-P5]在SD/-Ura/AbA（200ng/mL）培养基上的生长情况

Fig.2 Self-activation of pAbAi-Px bait vector A-E：Yeast transformants of Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3]，Y1H[pAbAi-P4] and Y1H[pAbAi-P5]in SD/-Ura medium，respectively. F-J：Yeast transformants of Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3] ，Y1H[pAbAi-P4] and Y1H[pAbAi-P5]in SD/-Ura/AbA（200ng/mL）medium，respectively

图3 pAbAi-P6诱饵载体自激活诱饵菌株Y1H[pAbAi-P6]分别在A：SD/-Ura，B：SD/-Ura/AbA（200 ng/mL），C：SD/-Ura/AbA（500 ng/mL），D：SD/-Ura/AbA（800 ng/mL），E：SD/-Ura/AbA（1 000 ng/mL）培养基上的生长情况

Fig.3 Self-activation of pAbAi-P6 bait vector Yeast transformants of Y1H[pAbAi-P6]in A：SD/-Ura，B：SD/-Ura/AbA（200 ng/mL），C：SD/-Ura/AbA（500 ng/mL），D：SD/-Ura/AbA（800 ng/mL）and E：SD/-Ura/AbA（1 000 ng/mL）medium，respectively

图4 NtCBT基因启动子P1-P6区域示意图及P5区域转录因子结合motif预测 P1：-929 - -750 bp；P2：-769 - -590 bp；P3：-609 - -430 bp；P4：-449 - -270 bp；P5：-279 - -119 bp；P6：-129 - +10 bp。向下箭头表示转录起始位点

Fig.4 Schematic diagram of P1-P6 regions of NtCBT promoter and prediction of transcription factor binding motif in the P5 region Downward arrow indicates the transcription start site

图5 PCR鉴定Y1H[pAbAi-P5]酵母菌落 M：Marker；泳道1，2：菌落PCR扩增产物

Fig.5 Identification of Y1H[pAbAi -P5]yeast colonies by PCR M：Marker. Lane 1 and 2：PCR-amplified products

图6 NtCBT基因启动子P5区域结合蛋白的初筛 A，B：初筛中获得的部分菌落

Fig.6 Preliminary screening of binding protein interacting with P5 regions of NtCBT promoter A and B：Partial colonies during the preliminary screening

图7 NtCBT基因启动子P5区域结合蛋白的二次筛选 1-50：1#-50#酵母菌落；+：阳性对照；-：阴性对照

Fig.7 Secondary screening of binding protein interacting with P5 regions of NtCBT promoter 1#：ANL2；41#：NF-YA3；44#：ML1 1-50：Colonies of 1#-50# yeasts. +：Positive control. -：Negative control

图8 部分阳性菌落的PCR检测 M：DL2000 DNA marker；泳道1-24：PCR扩增产物

Fig.8 Identification of partial positive colonies by PCR M：DL2000 DNA marker. Lane 1-24：PCR-amplified products

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