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

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.