Pgadt7 rec p53

The yeast one-hybrid test was performed as previously described (2 (link)). The primer pair (Table S2) corresponding to different motifs was mixed, heated, and annealed by PCR to form double strands, which were then inserted into the pAbAi vector (Clontech) digested by HindIII and XhoI to generate the pBait-AbAi construct. The resulting construct was confirmed by AflII and XmaI digestion. Full-length BbWor1 was amplified from cDNA with the p9/p10 primers (Table S2) and then cloned into the NdeI and EcoRI sites to construct the pGADT7-Rec-BbWor1 vector. The pGADT7-Rec-BbWor1 plasmid was transformed to a bait-specific reporter strain and then selected on appropriate selection plates (leucine-free SD medium with 250 ng/ml aureobasidin A; AbA). Transformation of yeast cells with the pGADT7-Rec-BbWor1 vector and one blank vector (pAbAi) was used as the negative control, while transformation of yeast cells with the p53-AbAi vector and pGADT7-Rec-p53 (Clontech) was used as the positive control. The positive colonies displayed an interaction of BbWor1 and the tested gene motifs.

The yeast one-hybrid test was performed as previously described (2 (link)). The primer pair (Table S2) corresponding to different motifs was mixed, heated, and annealed by PCR to form double strands, which were then inserted into the pAbAi vector (Clontech) digested by HindIII and XhoI to generate the pBait-AbAi construct. The resulting construct was confirmed by AflII and XmaI digestion. Full-length BbWor1 was amplified from cDNA with the p9/p10 primers (Table S2) and then cloned into the NdeI and EcoRI sites to construct the pGADT7-Rec-BbWor1 vector. The pGADT7-Rec-BbWor1 plasmid was transformed to a bait-specific reporter strain and then selected on appropriate selection plates (leucine-free SD medium with 250 ng/ml aureobasidin A; AbA). Transformation of yeast cells with the pGADT7-Rec-BbWor1 vector and one blank vector (pAbAi) was used as the negative control, while transformation of yeast cells with the p53-AbAi vector and pGADT7-Rec-p53 (Clontech) was used as the positive control. The positive colonies displayed an interaction of BbWor1 and the tested gene motifs.