小麦h型硫氧还蛋白Trx-h酵母双杂交诱饵载体的构建和自激活检测  

Construction and Self-activation Detection of Yeast Two-hybrid Bait Vector for Trx-h of Wheat

作　　者：曹玲珑[1] 李冬兵[1] 熊大斌[1] 邓利[1] 牛洪斌[1] 姜玉梅[1] 尹钧[1] 

机构地区：[1]河南农业大学,河南粮食作物协同创新中心,小麦玉米作物学国家重点实验室,国家小麦工程技术研究中心,河南郑州450002

出　　处：《河南农业科学》2014年第7期19-22,共4页Journal of Henan Agricultural Sciences

基　　金：国家转基因新品种培育重大专项(2011ZX08002-003);“十二五”农村领域国家科技计划项目(2012AA101105)

摘　　要：为构建小麦h型硫氧还蛋白Trx-h酵母双杂交诱饵载体,并检测其对酵母细胞的自激活作用。利用RT-PCR扩增TaTrx-h基因的ORF区域,并与pGBKT7载体连接构建诱饵载体pGBKT7-Trx-h,利用PEG/LiAC法转化酵母AH109后,通过表型筛选检测诱饵蛋白对酵母有无自激活作用。结果表明,含pGBKT7-Trx-h质粒的酵母在SD/-Trp-Leu培养基上能正常生长,说明诱饵载体表达产物对酵母细胞无毒性作用;在SD/-Trp-Leu-His-Ade培养基上不能生长,说明诱饵质粒无自主激活报告基因的作用。因此,可以利用该诱饵载体通过酵母双杂交方法筛选与Trxh蛋白相互作用的蛋白。To construct the bait vector for Trx-h of wheat and to evaluate its self-activation in yeast two-hybrid system,the open reading frame（ORF）of Trx-h gene was amplified by RT-PCR and cloned into the vector pGBKT7 to construct the bait vector pGBKT7-Trx-h.The constructed bait vector pGBKT7-Trx-h was transformed into yeast strains AH109 by PEG/LiAC and its selfactivation was tested through the phenotype screening.The result showed that the yeast strain AH109 transformed with the bait plasmids grew well on SD/-Trp-Leu plate without the toxicity effect,whereas they could not grow on SD/-Trp-Leu-His-Ade plate,indicating that pGBKT7-Trxh could not activate the transcription of reporter gene alone in yeast two-hybrid system.So the pGBKT7-Trx-h could act as a bait to screen the interaction proteins with Trx-h by yeast twohybrid system.

关 键 词：小麦 硫氧还蛋白 酵母双杂 诱饵载体 自激活 

分 类 号：S512.1[农业科学—作物学]