香蕉ROP1基因酵母双杂交体系的诱饵载体构建及毒性与自激活效应检测

Construction of Bait Vector Containing ROP1 Gene from Banana in Yeast Two-hybrid System and Identification of Its Toxicity and Autonomous Activation Effects

用PCR技术获得香蕉ROP1基因片段,构建酵母双杂交系统表达载体pGBKT7-ROP1,测序正确后将重组质粒导入AH109酵母菌株,检测其表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。结果表明,用PCR技术获得了正确的ROP1基因片段,并正确构建了ROP1基因的酵母双杂交表达载体,并转化到AH109酵母菌株中,含诱饵载体的AH109在SD/-Trp营养缺陷平板上生长良好,说明表达产物对酵母细胞无毒性,含诱饵载体的AH109在His、Trp二重营养缺陷平板上不能生长,说明对报告基因无自激活作用,为下一步利用酵母双杂交系统筛选与ROP1蛋白相互作用的蛋白奠定基础。

ROP1 gene was amplified by the PCR and then fused with pGBKT7. After confirmed by using sequence analysis, the plasmid was transformed into the cell of yeast strain AH109, and tested in terms of toxicity and transcriptional activation by nutrition disfigurement assay. As a result, the ROP1 was successfully amplified and cloned into the pGBKT7. The yeast strain AH109 cells harboring bait plasmids grew well on SD/-Trp plate, without toxicity and autonomous activation effect, which lays the foundation for using yeast two-hybrid system to screen the protein interacting with ROP1 protein.