拟南芥AtCYS6基因酵母双杂交诱饵载体的构建及鉴定

为了筛选与拟南芥半胱氨酸蛋白酶抑制剂基因AtCYS6相互作用的蛋白,构建其酵母双杂交诱饵表达载体。本研究利用PCR方法扩增得到拟南芥AtCYS6的基因片段,与诱饵载体pGBKT7连接构建诱饵表达载体,经双酶切以及测序检测验证重组载体构建成功后,通过醋酸锂法将诱饵表达载体pGBKT7-AtCYS6与空载体pGBKT7分别转化到Y2HGold酵母菌株,检测其是否有自激活能力以及对宿主酵母菌株是否有毒性作用。结果表明:经PCR扩增后得到了AtCYS6基因,成功构建了无自激活和没有毒性的诱饵表达载体pGBKT7-AtCYS6。本研究结果可进一步为筛选与AtCYS6相互作用的蛋白质及功能研究提供科学依据。

Construction and Identification of Yeast Two-hybrid Bait Vector of AtCYS6 Gene in Arabidopsis thaliana

In order to screen the protein interacting with AtCYS6,a cysteine protease inhibitor gene of Arabidopsis thaliana,a yeast two hybrid bait expression vector was constructed.In the experiment,the coding sequences of AtCYS6 was obtained by using PCR amplification,then connected with bait vector pGBKT7 to construct bait recombinant vector.The recombinant vector was confirmed by double enzyme digestion and sequencing and named as pGBKT7-AtCYS6.The vector pGBKT7-AtCYS6 and empty vector pGBKT7 was transferred into yeast Y2HGold cells by using LiAc method to detect whether the recombinant vector has auto-activation and toxic effect to yeast cell.The results showed that the coding sequence of AtCYS6 was obtained by PCR amplification and the bait vector pGBKT7-AtCYS6 was constructed successfully.In addition,the results suggested that the bait vector had no auto-activation and toxic effect to yeast cell.The results of this study can provide scientific basis for further selecting the protein interacted with AtCYS6 using yeast two-hybrid technology.