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

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

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。

The conserved domain and open reading frame of Musa acuminata rubredoxin （ MaRD ） was amplified by PCR with the NdeⅠand SalⅠrestriction 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 auxotrophic plate assay.The results suggested that the recombinant plasmids pGBKT7-CdRD and pGBKT7-FRD were constructed,which neither had the ability 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.