PnsICE1基因酵母双杂交诱饵载体构建及转录自激活检测

Construction of Yeast Two-Hybrid Bait Vector of PnsICE1 and Testing of Autonomous Transcriptional Activation

为了更好地研究与Pns ICE1基因互作的蛋白,利用PCR方法扩增含有p GBKT7载体同源序列的Pns ICE1基因片段,构建酵母双杂交载体。测序结果表明,构建的目标基因序列正确。将诱饵载体质粒转化酵母菌株Y2H感受态细胞,Pns ICE1转化菌在SD/-Trp营养缺陷型培养基中正常生长,在SD/-Trp/X-a-Gal筛选培养基上长出蓝色的酵母单菌落,说明Pns ICE1转录因子具有转录自激活活性。

In order to screen the PnsICE1 interacted proteins, the CDS sequence of PnsICE1 contains pGBKT7 vector homologous sequences was amplified using PCR and inserted into pGBKT7 vector to construct the bait vector for yeast two-hybrid （Y2H）system. Sequencing result showed that the PnsICE1 sequence was inserted into pGBKT7 vector in correct reading frame. Furthermore, the bait vector was transformed into Y2H yeast strain to test the autoaetivation. The transformed yeast grew very well on SD/-Trp plate,in the SD/-Trp/X-a-Gal screening yeast, grown on the medium blue single colony ,PasICE1 transcription factor has the transcriptional activation activity.