高粱花叶病毒Hcpro、CP和PIPO基因酵母双杂交载体构建及自激活验证

摘要

采用同源克隆技术获得侵染甘蔗高粱花叶病毒(Sorghum mosaic virus,SrMV)的Hcpro、CP、PIPO基因片段.将该基因片段连接至pGBKT7载体,分别构建酵母双杂交诱饵载体pGBKTT-Hcpro、pGBKT7-CP和pGBKTT-PIPO,经PCR、酶切及测序鉴定,证明重组诱饵载体构建成功后,将重组质粒导入Y2H Gold酵母菌株,检测其表达产物对酵母细胞有无毒性及对报告基因有无激活作用.结果表明,含重组诱饵质粒的酵母菌株在SD/-Trp营养缺陷平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性.含重组诱饵质粒的酵母菌株在SD/-Leu、SD/-Trp/-Ade和SD/-Trp/-His营养缺陷平板上不能生长,表明重组诱饵质粒表达产物对ADE2、HIS3报告基因无自激活作用.研究结果为下一步利用酵母双杂交系统检测与高粱花叶病毒Hcpro、CP、PIPO蛋白相互作用的甘蔗蛋白提供基础.%The HcPro, CP and PIPO genes encoding fragments from sorghum mosaic virus (SrMV) were obtained by homologous cloning and then fused with the pGBKT7 vector to construct bait vectors pGBKT7 -Hcpro, pGBKT7-CP and pGBKT7-PIPO. After confirmation with PCR, enzyme restriction and sequencing analysis, the plasmid was transformed into the yeast cell Y2H Gold, and its toxicity and transcriptional activation was tested by color assay. The yeast strain Y2H Gold transformed with bait plasmids grew well on SD/-trp plate, without toxicity effect, whereas, they could not grow on SD/-Leu, SD/-Trp/-Ade and SD/-Trp/-His plates, which indicating there were no autonomous activities between the bait proteins and ADE2, HIS3 report genes. These results lay the foundation for using yeast two-hybrid system to screen the sugarcane proteins interacting with HcPro, CP and PIPO genes.