Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 73-79.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1597

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

YU Jing1(), YANG Hui1, YU Shi-zhou1, ZHAO Hui-na1, ZHENG Qing-xia2, WANG Bing1, LEI Bo1()   

  1. 1. Guizhou Academy of Tobacco Science,Molecular Genetics Key Laboratory of China Tobacco,Guiyang 550081
    2. Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001
  • Received:2021-12-25 Online:2022-10-26 Published:2022-11-11
  • Contact: LEI Bo E-mail:yujingbio@126.com;leibo_1981@163.com

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