香蕉红素氧还蛋白基因在酵母双杂交系统中的自激活检测

利用PCR方法扩增香蕉红素氧还蛋白基因的保守结构域序列CdRD和开放性阅读框序列FRD,并在其上下游分别引入NdeⅠ和SalⅠ酶切位点,双酶切后与同样经NdeⅠ和SalⅠ双酶切的诱饵质粒载体pGBKT7连接,构建重组诱饵质粒pGBKT7-CdRD和pGBKT7-FRD,并将此两重组诱饵质粒转入酵母菌株AH109中进行营养缺陷型分析检测毒性及自激活。结果表明,成功构建了重组诱饵质粒pGBKT7-CdRD和pGBKT7-FRD,并且其无自激活报告基因作用,对酵母菌株也无毒性作用。这说明此两个重组诱饵质粒可用于酵母双杂交系统,为从香蕉叶片cDNA文库中筛选获得与香蕉红素氧还蛋白基因的保守结构域序列CdRD和开放性阅读框序列FRD相互作用的受体蛋白基因奠定了基础。

Testing for self-activation of Musa acuminata rubredoxin (MaRD) in yeast two-hybrid system

The conserved domain and open reading frame of Musa acuminata rubredoxin (MaRD) was amplified by PCR with the Nde Ⅰ and Sal Ⅰ restriction sites incorporated into the primers,digested by restriction enzymes and ligated with the vector pGBKT7 which also digested by the same restriction enzymes to construct recombinant bait plasmid pGBKT7-CdRD and pGBKT7-FRD.After the transform of recombinant plasmid into yeast strain AH109,the autonomous reporter gene activation was then observed with attxotrophic plate assay.The results suggested that the recombinant plasmids pGBKT7-CdRD and pGBKT7-FRD were constructed,which neither had the abihty of self-activation,nor yeast cell toxicity.Therefore,the recombinant plasmid can be used in yeast two-hybrid system to screen a banana cDNA library for proteins interacted with the bait protein pGBKT7-CdRD and pGBKT7-FRD.