The PsiYAB11, PtoYAB11 PSC , and PtoNGAL1 genes were cloned into the pGADT7-rec vector (Clontech, Mountain View, CA, USA) with the GAL4 activation domain. The promoters of PtoNGAL1, PtoCUC2, and PtoRBCL were inserted into a pAbAi-BR yeast-integrating vector (Clontech), which was used as a bait-reporter construct. pGADT7-PsiYAB11, pGADT7-PtoYAB11 PSC , pGADT7-PtoNGAL1, and pAbAi-Pro (PtoNGAL1, PtoCUC2, and PtoRBCL) were co-transformed into the Y1HGold yeast strain using a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). The full-length CDS of PsiYAB11 was inserted into the pCzn1 vector (Zoonbio, Nanjing, China) to produce recombinant PsiYAB11 in Escherichia coli BL21. The His-PsiYAB11 fusion protein was purified using PureCube Ni-NTA Agarose (Cube Biotech, Monheim, Germany). Reactions were incubated at 25°C for 30 min after adding 2 mg of protein from the His purification eluate. The primers and probes used for these analyses are listed in Supplemental Table 7 (Supplemental Methods 11).
Pabai br yeast integrating vector
Manufactured by Takara Bio
The PAbAi-BR yeast-integrating vector is a tool designed for gene expression studies in Saccharomyces cerevisiae. It enables the integration of a target gene into the yeast genome, allowing for stable and consistent expression of the gene of interest.
Automatically generated - may contain errors
2 protocols using pabai br yeast integrating vector
1
Yeast One-Hybrid Assay for Transcriptional Interactions
2
Yeast One-Hybrid Assay for Plant Transcription Factor Interactions
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The PsiYAB11 and PtoYAB11 PSC genes were cloned into the pGADT7-rec vector (Clontech, Mountain View, CA, USA) with the GAL4 activation domain. The promoters of PtoNGAL1, PtoNGAL1, PtoCUC2, PtoRBCL, PtoATPA, PtoATPE and PtoPSBB were inserted into a pAbAi-BR yeast-integrating vector (Clontech), which was used as a bait-reporter construct. pGADT7-PsiYAB11, pGADT7-PtoYAB11 PSC , and pAbAi-Pro (PtoNGAL1, PtoNGAL1, PtoCUC2, PtoRBCL, PtoATPA, PtoATPE and PtoPSBB) were co-transformed into the Y1HGold yeast strain using the Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech), respectively. Co-transformed yeast cells were selected on synthetic dextrose minimal medium (SD) lacking uracil (Ura) and leucine (Leu) (SD/-Ura-Leu), with or without 0.6 mg/L Aureobasidin A (AbA, Sigma-Aldrich, St. Loui, MO, USA), and were then incubated for 5 days at 30°C. pGAD-p53+p53-pAbAi and pGADT7-AD+ pAbAi-Pro were carried out in the same manner as for the positive and negative controls, respectively. The primers used for these analyses are listed in Supplemental Table S1 (Supplemental Methods S11).
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