CAJ | 학술논문

利用PeR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Nde Ⅰ和Sal Ⅰ酶切位点,双酶切后与同样经Nde Ⅰ和Sal Ⅰ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.
이용PeR방법확증향초홍소양환단백기인적보수결구역서렬CdRD화개방성열독광서렬FRD,병재기상하유분별인입Nde Ⅰ화Sal Ⅰ매절위점,쌍매절후여동양경Nde Ⅰ화Sal Ⅰ쌍매절적유이질립재체pGBKT7련접,구건중조유이질립pGBKT7-CdRD화pGBKT7-FRD,병장차량중조유이질립전입효모균주AH109중진행영양결함형분석검측독성급자격활.결과표명,성공구건료중조유이질립pGBKT7-CdRD화pGBKT7-FRD,병차기무자격활보고기인작용,대효모균주야무독성작용.저설명차량개중조유이질립가용우효모쌍잡교계통,위종향초협편cDNA문고중사선획득여향초홍소양환단백기인적보수결구역서렬CdRD화개방성열독광서렬FRD상호작용적수체단백기인전정료기출.
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.