OsRUS2.1酵母双杂交猎物载体的构建及其细胞毒性和自激活作用检测

Construction of OsRUS 2.1 yeast two hybrid prey vectors and detection of their toxicity and self activated activity

关键词:  水稻根对UVB敏感基因2.1  猎物载体  酵母双杂交  自激活  毒性检测

Key words:  rice ROOT UV B SENSITIVE 2.1(OsRUS2.1)  prey vector  yeast two hybrid  self activated transcriptional activity  toxicity detection

摘要:

采用PCR技术扩增水稻根UV B敏感基因2.1（ROOT UV B SENSITIVE 2.1，RUS2.1）四个不同片段[OsRUS2.1(1 1317)，OsRUS2.1(1 138)，OsRUS2.1(139 879)，OsRUS2.1(880 1317)]，连接到T载体pMD18 T Simple上，测序无误后分别亚克隆到猎物表达载体pGADT7上。结果表明：四个OsRUS2.1基因片段的表达载体构建成功，读码框正确；分别转化这四个猎物表达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 Leu DO培养法进行自激活检测和毒性检测，结果表明构建的四个OsRUS2.1不同片段的猎物表达载体对酵母菌株Y187均没有转录激活活性和毒害作用，可用于后续研究。

Abstract:

Four fragments of rice(Oryza sativa)ROOT UV B SENSITIVE 2.1(OsRUS2.1)，OsRUS2.1(1 1317)，OsRUS2.1(1 138)，OsRUS2.1(139 879)，OsRUS2.1(880 1317)，were amplified by PCR from cloned OsRUS2.1 plasmid，and were ligated with pMD18 T simple vector，then transformed to E.coli TOP10 competent cell. The positive clones were selected and sequenced. The confirmed fragments were subcloned to prey vector pGADT7. The four constructed pGADT7 prey vectors were further confirmed by enzyme digestion and sequencing. The confirmed 4 types of pGADT7 prey vectors were transformed to Y187 yeast competent cells. The self activation and toxicity of the plasmids to host yeast Y187 were detected by LacZ and MEL1 activity assays and culturing in auxotroph medium SD Leu DO． Results showed that the four constructed plasmids had no self transcriptional activity and not toxicity to yeast strain Y187，and they could be used in the following yeast two hybrid experiments．