CAJ | 학술논문

AtCRPI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AtCRP7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／-Tip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。
AtCRPI기인시의남개적일개종동진탕기기인，목전기생리공능지지심소。위료경호지연구여AtCRP7기인적호작단백，필자이용PCR기술종휴대AtGRP7기인서렬적질립중확증해기인적편마서렬，연후장목표서렬삽입pGBKT7재체적NcoⅠ화PstⅠ2개매절위점지간，구성효모쌍잡체계적유이재체。매절화측서결과표명，구건적재체목표기인서렬화열독광시정학적。지후，장해재체전입감수태효모Y2HGold균주중，병검측기표체물대보고기인적격활정황。AtGRP7효모전화균재SD／-Tip평판상장세량호；재SD／-Trp-Ade평판상장세교약；재SD／-Trp／-His화SD／-Trp／-Ade／-His평판상칙몰유생장。저설명His합성상관기인몰유피전록화번역，AtGRP7기인재해효모쌍잡체계중몰유자전록격활。필자재효모쌍잡적초보사선중，환획득궤개가능여AtGRP7호작적기인。
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.