拟南芥AtGRP7基因诱饵载体的构建及酵母双杂的初筛

Construction and Autoactivation Test of a Bait Vector of AtGRP7 for Two-hybrid System

AtGRP7基因是拟南芥的一个从动振荡器基因,目前其生理功能知之甚少。为了更好地研究与AtGRP7基因的互作蛋白,笔者利用PCR技术从携带AtGRP7基因序列的质粒中扩增该基因的编码序列,然后将目标序列插入pGBKT7载体的NcoⅠ和PstⅠ2个酶切位点之间,构成酵母双杂体系的诱饵载体。酶切和测序结果表明,构建的载体目标基因序列和阅读框是正确的。之后,将该载体转入感受态酵母Y2HGold菌株中,并检测其表达物对报告基因的激活情况。AtGRP7酵母转化菌在SD/-Trp平板上长势良好;在SD/-Trp/-Ade平板上长势较弱;在SD/-Trp/-His和SD/-Trp/-Ade/-His平板上则没有生长。这说明His合成相关基因没有被转录和翻译,AtGRP7基因在该酵母双杂体系中没有自转录激活。笔者在酵母双杂的初步筛选中,还获得几个可能与AtGRP7互作的基因。

AtGRP7 is one of slave oscillator of Arabidopsis thaliana, but its physiological role is so far less known. In order to screen the AtGRP7 interacted proteins, the CDS sequence of AtGRP7 was first amplified using PCR and inserted into pGBKT7 vector between the clone site Nco I and Pst I to construct the bait vector for yeast two-hybrid （Y2H） system. Restricted digestion and sequencing result showed that the AtGRP7 sequence was inserted into pGBKT7 vector in correct reading frame. Furthermore, the bait vector was transformed into Y2H Gold yeast strain to test the autoactivation and toxicity. The transformed yeast grew very well on SD/-Trp plate, but weakly on SD/-Trp/-Ade plate. No yeasts grew on SD/-Trp/-His and SD/-Trp/-Ade/-His plates,suggesting that histidine synthase gene was not transcribed and translated. This indicated that AtGRP7 was not autoactivated in the Y2H system.