OsRUS1酵母双杂交诱饵载体的构建及其自激活作用检测

Construction of OsRus1 Yeast Two-Hybrid Bait Vectors and Detection of Their Self-activated Activity

采用PCR技术扩增水稻(Oryza sativa L)根UV-B敏感基因1(ROOT UV-B SENSITIVE 1,RUS1)四个不同片段[OsRUS1(1-1782)、OsRUS1(1-504)、OsRUS1(510-1282)、OsRUS1(1188-1782)],连接到T载体pMD18-T-Simple上,测序无误后分别亚克隆到诱饵载体pGBKT7上,酶切和测序结果表明构建的4个OsRUS1基因片段的诱饵载体构建成功,读码框正确;转化重组载体于酵母感受态细胞Y187中,用LacZ、MEL1活性检测法和营养缺陷型培养基SD-Trp-DO培养法进行自激活检测和毒性检测,结果表明诱饵载体对酵母菌株Y187没有转录激活活性和毒害作用。说明构建的4个OsRUS1基因片段的诱饵载体可以用于酵母双杂交系统中,为下一步从水稻cDNA文库中筛选互作蛋白奠定了基础。

Four fragments of rice（Oryza sativa L）ROOT UV-B SENSITIVE 1（OsRUS1）,OsRUS1（1-1782）,OsRUS1（1-504）,OsRUS1（510-1282）,OsRUS1（1188-1782）,were amplified by PCR from cloned OsRUS1,and were ligated with pMD18-T-Simple,then transformed to E.coli TOP10 competent cell.The positive clones were selected and sequenced.The confirmed fragments were subcloned to bait vector pGBKT7.The four constructed pGBKT7 bait vectors were further confirmed by enzyme digestion and sequencing.The confirmed pGBKT7 bait vectors were transformed to Y187 yeast competent cell.The self-activation and toxicity of the plasmids to host yeast Y187 by LacZ and MEL1 activity assays and culturing in auxotroph medium SD-Trp-DO.Results showed that the four constructed plasmids had no self-transcriptional activity and not toxicity to yeast strain Y187.The four bait vectors constructed could be used in yeast two hybrid system,which laid the foundation for screening interactional proteins of OsRUS1 from rice cDNA library.