Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 73-79.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1597
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YU Jing1(), YANG Hui1, YU Shi-zhou1, ZHAO Hui-na1, ZHENG Qing-xia2, WANG Bing1, LEI Bo1()
Received:
2021-12-25
Online:
2022-10-26
Published:
2022-11-11
Contact:
LEI Bo
E-mail:yujingbio@126.com;leibo_1981@163.com
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.
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
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
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
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
Fig.6 Preliminary screening of binding protein interacting with P5 regions of NtCBT promoter A and B:Partial colonies during the preliminary screening
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
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