利用定点突变分析白念珠菌依赖铁基因FRP1启动子元件..docVIP

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?-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 ?- galactosidase assay. The results revealed that the FRP1 was up-regulated in alkaline pH and this was caused by iron starvation. The ?650 site, not the ?160 site, had an important role in FRP1 Rim101p-dependent expression. We conclude that Rim101p may interact with the ?650 binding site of the promoter to regulate the FRP1 expression. Keywords: site-directed mutation, Candida albicans, Rim101 protein, ferric reductase gene 铁是生物必需的重要营养元素, 铁离子是许多生化途径, 例如呼吸作用, 三羧酸循环, DNA和氨基酸的合成等过程中酶的辅助因子, 在自然界中, 生物体可直接吸收利用的铁非常有限, 因此微生物在长期的进化过程中形成高亲和的铁吸收系统来满足细胞铁离子需要。真菌中, 高亲和吸收系统在酿酒酵母(Saccharomyces cerevisiae)中研究最为详细[1,2], 这个系统主要有3个关键酶组成, 位于细胞膜上的高铁还原酶(Frep)首先将外界三价铁还原为二价铁离子后, 二价铁