摘要
[目的]构建诱饵载体pGBKT7-Lescpth5,并检测其转录自激活活性及对酵母细胞的毒性。[方法]采用PCR技术扩增Lescpth5基因,连接到诱饵载体pGBKT7上,转化重组载体于酵母感受态细胞AH109中,在营养缺陷型培养基上进行自激活检测和毒性检测。[结果]酶切和测序鉴定结果显示诱饵载体pGBKT7-Lescpth5构建成功,读码框正确。自激活检测结果和毒性检测结果显示诱饵载体对酵母菌株AH109没有转录激活活性,对酵母菌也无毒害作用。[结论]成功构建的诱饵载体可以用于酵母双杂交系统中,为下一步的cD-NA文库筛选奠定基础。
[Objective]The paper was to construct bait vector pGBKT7-Lescpth5,and detect its self-transcriptional activity and the toxicity on yeast cells.[Method]Lescpth5 was amplified by PCR technique,and cloned into bait vector pGBKT7.The recombined vector was transformed into yeast competent cells and carried out self-transcriptional activity and toxicity detection in the auxotroph medium.[Result]Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was successfully constructed with correct reading frame.Self-activated activity and toxicity detection results showed that bait vector had no self-transcriptional activity on yeast strain AH109,which also had no toxicity on yeast.[Conclusion]Bait vector successfully constructed could be used in yeast two hybrid system,which laid the foundation for screening of cDNA library.
出处
《安徽农业科学》
CAS
北大核心
2011年第19期11512-11513,共2页
Journal of Anhui Agricultural Sciences
基金
北京市自然科学基金(6012017)
新疆生产建设兵团博士资金资助
关键词
番茄细菌性斑点病菌
诱饵载体
酵母双杂交
自激活
Pseudomonas syringae pv. Tomato
Bait vector
Yeast two hybrid
Self-activated transcriptional activity
