拟南芥At4g05060基因的酵母双杂交诱饵表达载体的构建、表达及自激活检测

Construction,Expression and Self-activation Detection of Bait Vector for Yeast Two-Hybrid of At4g05060 Gene in Arabidopsis thaliana

为研究At4g05060基因功能,采用PCR扩增拟南芥VAMP(vesicle-associated membrane protein)家族基因At4g05060的ORF序列,定向克隆至酵母双杂交诱饵表达载体pGBKT7,经酶切鉴定并测序后将其转化至酵母Y187细胞;然后采用Western blotting技术分析At4g05060基因在酵母中的表达水平,通过缺陷型培养基培养进行自激活检测。结果表明,成功构建酵母诱饵表达载体pGBKT7-At4g05060,并在酵母细胞正确表达,表达产物对报告基因无自激活作用。

In order to study of the function of At4g05060 gene,the opening read frame（ORF）sequence of Arabidopsis thaliana VAMP（vesicle-associated membrane protein）family At4g05060 gene was amplified by PCR and cloned into the bait vector pGBKT7 of yeast two-hybrid.After confirmed with restriction endonuclease digestion and sequence analysis,the recombinant bait vector pGBKT7_At4g05060 was transformed into the yeast strain Y187.Then the expression of the bait protein was analyzed by Western Blotting analysis.The self-activation detection was conducted by the culture on detective medium.The results showed that the bait expression vector pGBKT7_At4g05060 was constructed successfully.The recombinant fusion protein was expressed correctly and could not activate the transcription of reporter gene alone in yeast two-hybrid system.