条件启动子pCTR4在隐球菌基因表达调控中的应用  

Application of conditional promoter pCTR4 in Cryptococcal gene expression regulation

作　　者：赵静宇[1,2] 法振宗 方伟[1,2] 孟云芳[1,2] 张超[1,2] 周兆婧 潘炜华[1,2] 廖万清[1,2] 

机构地区：[1]上海长征医院皮肤病与真菌病研究所 全军真菌病重点实验室 [2]第二军医大学附属长征医院皮肤科,上海200003

出　　处：《中国真菌学杂志》2014年第1期1-6,共6页Chinese Journal of Mycology

基　　金：国家973项目(2013CB531601);国家自然基金(31170139);国家星火传染病专项(2013CB531606);上海市自然科学基金(12ZR1454400;12JC1411000)

摘　　要：目的通过条件启动子pCTR4的质粒构建以及其在新生隐球菌中的同源置换,研究其在隐球菌基因表达调控中的应用。方法应用套叠PCR,构建含报告基因NEO的铜离子抑制性启动子质粒pNEO/CTR4和启动子同源重组框,并利用基因枪将其转化入新生隐球菌感受态细胞,常规及实时定量PCR检测条件启动子对目的基因的转录调控效应。结果成功构建了质粒pNEO/CTR和隐球菌条件启动子重建菌株,条件启动子pCTR4对目的基因具有预期的转录诱导和抑制效果。结论新建铜离子抑制性启动子质粒pNEO/CTR4可以应用于对隐球菌目的基因表达水平的调控;隐球菌泛素编码基因UBI 1并非致死性关键基因。我们的研究为今后新生隐球菌泛素系统的分子致病机制研究奠定了基础。Objective To explore the application of conditional promoter pCTR4 in Cryptococcal gene expression regulation via construction of new vector pNEO/CTR4 and promoter replacement.Methods Constructed the new plasmid pNEO/CTR4 fusing report gene NEO and copper-repressive promoter pCTR4 via overlap PCR.On the basis,constructed its promoter replacement cassette,and then transformed them into Cryptococcal competent cells via gene gun to get its reconstitution strains,which were tested via regular and real-time PCR.Results Correct new vector pNEO/CTR4 and Cryptococcal reconstitution strains were obtained and confirmed.Conditional promoter pCTR4 played induced or repressed effect in UBI 1 transcription under different media as expected.Conclusion The new vector of copper-repressed promoter was applicable in regulating Cryptococcal gene expression.And the ubiquitin encoding gene UBI 1 was not essential for viability in Cryptococcus neoformans.Our study laid the groundwork for following functional studies of ubiquitin system in Cryptococcus neoformans.

关 键 词：新生隐球菌 条件启动子 泛素 致病机制 

分 类 号：R379.5[医药卫生—病原生物学]