甘蔗条纹花叶病毒 HC－Pro、P3N－PIPO、CP 和 VPg基因酵母双杂交诱饵表达载体的构建及自激活检测

摘要

为探索甘蔗条纹花叶病毒与甘蔗的互作机制，利用酵母双杂交技术筛选与 SCSMV 互作的甘蔗因子基因，利用 PCR 技术克隆了 SCSMV 的 HC－Pro、P3N－PIPO、CP 和 VPg 的编码区，构建到酵母双杂交诱饵载体 pGBKT7上，获得了 pGBKT7－HC－Pro、pGBKT7－P3N－PIPO、pGBKT7－CP 和 pGBKT7－VPg 4个诱饵载体。结果显示，将重组质粒转化酵母 Y2HGold 酵母菌株后，酵母菌株在 SD／－Trp 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；在SD／－Trp／X－α－gal 平板上长出白色菌落未变蓝，在 SD／－Leu、SD／－Trp／－Ade、SD／－Trp／－His 和 SD／－Trp／X－α－gal／AbA 平板上不能生长，表明重组诱饵质粒表达产物对 MEL1、ADE2、HIS3和 AUR1－C 报告基因无自激活作用。构建的诱饵表达载体可以作为诱饵用于文库筛选，为探索 SCSMV 侵染机制和发病机理奠定了基础。%Sugarcane streak mosaic virus (SCSMV) is one of the main pathogens causing mosaic syndrome on sugarcane.Yeast two hybrid system (Y2HS) is the main technique to investigate the mechanism between pathogen with host plant.In order to elucidate the interaction between the SCSMV and sugarcane by Y2HS,the coding se-quences of HC-Pro,P3N-PIPO,CP and VPg from SCSMV were cloned into the pGBKT7,respectively,to generate 4 bait vectors,pGBKT7-HC-Pro,pGBKT7-P3N-PIPO,pGBKT7-CP and pGBKT7-VPg.Then the 4 bait vectors were transformed into the yeast strain Y2HGold,respectively.All the Y2HGold strains containing the bait vectors grew well on the SD/-Trp media as the control.It suggested that the proteins encoded by the baits were not toxic to yeast. All these 4 transformed yeast strains developed white but not blue plaques on the SD/-Trp/X-α-gal media.And they could not survive on the SD/-Leu,SD/-Trp/-Ade,SD/-Trp/-His or SD/-Trp/X-α-gal/AbA media,which indicating the bait proteins could not activate the report genes,such as MEL1,ADE2,HIS3 or AUR1-C.These 4 bait vectors constructed in this study could be used to screen the host proteins interacting with them to investigate the molecular mechanism and pathogenesis of SCSMV infection on sugarcane.