马铃薯StSOD1基因酵母双杂交诱饵载体的构建及鉴定

为了筛选与马铃薯超氧化物歧化酶(superoxide dismutase,SOD)基因StSOD1作用的上游蛋白,构建其酵母双杂交诱饵重组载体。本研究利用PCR方法扩增得到马铃薯StSOD1的基因片段,通过与pMD-T18连接构建克隆载体,经验证构建成功后,与诱饵载体pGBKT7连接构建诱饵重组载体,经双酶切以及测序检测验证重组载体构建成功后,通过PEG/Li Ac法将诱饵载体pGBKT7-StSOD1与空载体pGBKT7分别转化到酵母AH109感受态细胞中,检测其是否有自激活作用和对酵母菌株的毒性作用。结果表明:经PCR扩增后得到了StSOD1基因,成功构建了诱饵重组载体pGBKT7-St SOD1,并且此诱饵载体无自激活活性和对酵母菌株的毒性作用。此研究结果可进一步为筛选与StSOD1互作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 StSOD1 Gene in Potato

In order to screen upstream protein interacted with potato superoxide dismutase(SOD)gene StSOD1,the yeast two-hybrid bait recombinant vector was constructed.In the experiment,the coding sequences of StSOD1 was obtained by using PCR amplification,then cloned into pMD-T18 to construct clone vector,after verified correctly,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-StSOD1.The vector pGBKT7-StSOD1 and empty vector pGBKT7 was transferred into yeast AH109 competent cell by using PEG/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 StSOD1 was obtained by PCR amplification and the bait vector pGBKT7-StSOD1 was constructed successfully.In addition,the results suggested that the bait vector had no auto-activation and toxic effect to yeast cell.The result of this study can provide scientific basis for further selecting the protein interacted with StSOD1 using yeast two-hybrid technology.