| 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。 |
| 关键词: 5FOA 转座酶 锌指蛋白 |
| DOI： |
| 分类号: |
| 基金项目:农业部“948”计划项目“高效PiggyBac转座酶引进及改造”（2013-Z17） |
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| Construction and screening of hyperactive PiggyBac transposases |
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ZHANG Long,LIANG Ming-fu,ZHANG Zhi-ying
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| Abstract: |
| 【Objective】This study aimed to construct highly efficient and site-directed PiggyBac transposase system.【Method】Wild-type PiggyBac transposase was used as template to construct mutant transposase 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 transposase library was successfully constructed.After three times of screening,five hyperactively mutant transposases 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. |
| Key words: 5FOA transposase zinc finger protein |
