Lescpth5诱饵载体的构建及其自激活作用的检测  

Construction of Lescpth5 Bait Vector and Detection of its Self-activated Activity

作　　者：张月娟[1,2] 万一[1] 陈华民[2] 张琨[1] 赵廷昌[2] 

机构地区：[1]陕西省微生物研究所,陕西西安710043 [2]中国农业科学院植物保护研究所,北京100193

出　　处：《安徽农业科学》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

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.

关 键 词：番茄细菌性斑点病菌 诱饵载体 酵母双杂交 自激活 

分 类 号：S436.412.1[农业科学—农业昆虫与害虫防治]