利用定点突变分析白念珠菌依赖铁基因FRP1启动子元件.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, August 25; 24(8): 1348-1353 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 利用定点突变分析白念珠菌依赖铁基因FRP1 启动子元件 桂磊, 梁勇, 魏东盛, 郑雯, 邢来君, 李明春 南开大学微生物学系 分子微生物学与技术教育部重点实验室, 天津 300071 摘 要: 通过对白念珠菌高铁还原酶基因FRP1 启动子进行突变分析, 确认启动子中特殊调控元件。我们通过分析FRP1 起始密码子上游 1000 bp 序列发现在−160 和−650 处有2 个推测的Rim101p 结合位点, 对其分别进行定点突变, 然后构 建启动子与报告基因LacZ 融合质粒, 转化整合到白念珠菌 rim101-/-株和野生株中, 检测不同缺铁条件下β-半乳糖苷酶 活性。结果发现碱性条件, Rim101p 能够正向调控FRP1 的表达; 启动子−160 处突变对启动子功能影响较弱, 而−650 突 变使启动子活性大大降低, 此结果和双突变的结果相同, 表明 Rim101p 主要通过与启动子−650 处结合位点相互作用来 调控FRP1 的表达。 关键词: 定点突变, 白念珠菌, Rim101 蛋白, 高铁还原酶基因 Regulating Promoter Element of Iron-dependent Gene FRP1 in Candida albicans by Site-directed Mutation Lei Gui, Yong Liang, Dongsheng Wei, Wen Zheng, Laijun Xing, and Mingchun Li Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin 300071, China Abstract: Microarray analysis revealed that the expression of ferric reductase (FRP1) can be regulated by the Rim101 protein. In order to find new transcriptional regulatory element in the promoter of FRP1, we analyzed the 1000 bp sequence upstream of ATG to find 2 potential Rim101p binding sites. We generated site-specific mutations in each of the two sites and fused these mutated promoters to LacZ . Then the promoter-LacZ fusion construct was recombinant into wild type and rim101-/- strains for β- galactosi