高效PiggyBac转座酶的构建与筛选  

Construction and screening of hyperactive PiggyBac transposases

作　　者：张龙[1] 梁明福 张智英[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2015年第8期1-9,18,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：农业部"948"计划项目"高效PiggyBac转座酶引进及改造"(2013-Z17)

摘　　要：【目的】构建能够定点转座的高效PiggyBac转座酶系统。【方法】以野生型PiggyBac转座酶为模板,通过易错PCR方法随机突变转座酶基因,构建转座酶突变库,并将其与能够靶向识别并结合DNA序列的锌指蛋白(ZFP)融合表达,构建ZFP-PiggyBac转座酶突变库系统。将转座酶载体突变库、转座子载体和报告载体共转到酿酒酵母JMY1中,通过Ura-5FOA酵母筛选系统筛选高效定点作用的转座酶。【结果】成功构建ZFP-PiggyBac转座酶突变库系统,经过3轮系统筛选获得5个高效作用的突变转座酶。【结论】新构建的ZFP-PiggyBac转座酶突变库系统具有在锌指蛋白ZFP靶向识别域Rosa26BS位点处进行高效转座的能力。[Objective] This study aimed to construct highly efficient and site-directed PiggyBac trans- posase system. [Method] Wild-type PiggyBac transposase was used as template to construct mutant trans- posase library with random mutation transposase gene by error-prone PCR. Fusion expression with the ZFP protein that can recognize and bind the target DNA sequence was conducted to get ZFP-PiggyBac mutant transposase library system. Then the mutant transposase library, transposon and report vector were co- transformed into Saccharomyces cerevisiae JMY1. Highly efficient and site-specific transposases were screened by Ura-5FOA yeast screening system. [Result] Highly efficient ZFP-PiggyBac mutant trans- posase library was successfully constructed. After three times of screening, five hyperactively mutant trans- posases were obtained. [Conclusion] Newly constructed ZFP-PiggyBac mutant transposase library had the site-specific and efficient transposable ability at the ZFP protein recognition domain Rosa 26BS.

关 键 词：5FOA 转座酶 锌指蛋白 

分 类 号：Q78[生物学—分子生物学]