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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pGBKT7-Hcpro、pGBKT7-CP和pGBKT7-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。
Hcpro, CP and PIPO gene fragments that infect sugarcane sorghum mosaic virus (SrMV) were obtained by homologous cloning techniques. The gene fragment was ligated into pGBKT7 vector to construct yeast two-hybrid bait vector pGBKT7-Hcpro, pGBKT7-CP and pGBKT7-PIPO, respectively. After PCR, restriction enzyme digestion and sequencing, the recombinant bait vector was successfully constructed and introduced into Y2H Gold yeast strain to detect whether the expression product has toxicity to the yeast cell and whether the reporter gene is activated. The results showed that the yeast strains containing recombinant bait plasmids grew well on the SD / -Trp auxotrophic plates, indicating that the recombinant bait plasmid expression products are non-toxic to yeast cells. The yeast strain containing the recombinant bait plasmid could not grow on the SD / -Leu, SD / -Trp / -Ade and SD / -Trp / -His auxotrophic plates, indicating that the expressed recombinant bait plasmid had no self-activation on the ADE2 and HIS3 reporter genes effect. The results provided the basis for the next step to detect sugarcane protein interacting with Hcpro, CP and PIPO proteins of sorghum mosaic virus by yeast two-hybrid system.