4.5. Gene Cloning, Vector Construction and Transcriptional Activation Analysis in Yeast Cells

To further evaluation of transactivation activity of 12 BaAP2/ERF genes, we cloned these 12 genes into the pMD18-T clone vector. After sequence analysis, the PCR products of these genes were cloned separately into the pGBKT7 vector using the in-fusion PCR cloning system (Clontech, Mountain View, CA, USA). Positive plasmids containing different BaAP2/ERF genes were transformed into the Y2H yeast strain (Clontech, Mountain View, CA, USA). All primers used for cloning and vector construction are listed in Tables S3 and S4. The cell concentration of yeast positive transformants were adjusted to an OD600 of 2.0, the yeast cells were then diluted serially (1, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) and dropped with 2 μL on synthetic dropout (SD) medium without tryptophan (SD/−Trp), without tryptophan and histidine (SD/−Trp−His), and with SD/−Trp−His plates containing x-α-gal with the final concentration of 40 mg/L (SD/−Trp−His+x-α-gal). Yeast cells expressing the empty vector pGBKT7 was used as negative control. The plates were incubated at 30 °C for 2–4 days before photographing. Adobe Illustrator CS5 was used for image processing.