PRSV NIa-pro、NIa-vpg、NIb酵母双杂交诱饵表达载体的构建及自激活检测  

Construction and Identification of Bait Vector Containing PRSV NIa-pro/NIa-vpg/NIb Gene in Yeast Two-hybrid System

作　　者：王明钦[1] 沈文涛[1] 周鹏[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,海口571101

出　　处：《热带作物学报》2008年第3期369-373,共5页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(编号:30760134;30560082);中央级科研院所基本科研业务费项目资助

摘　　要：用PCR技术获得NIa-pro、NIa-vpg、NIb基因片段,将这些基因分别克隆到pBD-GAL4载体中,构建酵母双杂交系统的诱饵载体pBD-GAL4-NIa-pro、pBD-GAL4-NIa-vpg和pBD-GAL4-NIb。测序正确后,将重组质粒导入YRG-2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,获得了正确的NIa-pro、NIa-vpg、NIb基因片段,并成功克隆到pBD-GAL4诱饵载体中,且转化有诱饵载体的YRG-2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,对报告基因也无自激活作用,为下一步利用酵母双杂交系统检测与NIa-pro、NIa-vpg、NIb蛋白相互作用的蛋白奠定基础。NIa-pro, NIa-vpg and NIb genes were amplified by PCR and then fused with pBD-GAIA. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell YRG-2, and its toxicity and transcriptional activation was tested by color assay. As a result, the NIa-pro, NIa-vpg and NIb were successfully amplified and cloned into pBD-GAIA. The YRG-2 transformed with bait plasmids grew well on SD/-trp plate, without toxicity and autonomous activation effect. The bait vectors, pBD-GAIA-NIa-pro, pBD-GAIA-NIa-vpg and pBD-GAL4-Nib, constructed were expressed correctly, and could not activate the transcription of reporter gene alone in yeast two-hybrid system.

关 键 词：PRSV 酵母双杂交 诱饵载体 构建 检测 

分 类 号：S432.4[农业科学—植物病理学] Q78[农业科学—农业昆虫与害虫防治]