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Screening of enviromental stress response elements and their application

环境应激反应元件筛选及其应用

基本信息

批准号：
11650813
负责人：
TAKEYAMA Haruko
金额：
$ 2.18万
依托单位：
Tokyo university of Agriculture & Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Various environmental stresses are present in the nature. The common stresses are physicochemical stresses such as temperature, osmotic pressure, radiation, pressure, gas composition, contamination of toxic chemicals/metals, etc. Microorganisms quickly respond to those environmental stresses at genetic level for their survival or homeostasis. The present study attempted to screen gene elements resulting gene expression caused by response to various stress conditions.At first, we attempted to construct a genome library of marine cyanobacterium, Synechococcus sp., using a marine cyanobacterium as a host organism. However, it was unexpectedly unsuitable to construct the library, since the transformation efficiency cyanobacteria was low. E.coli cells were, then, employed as the host organism for the construction of total genome library of the Synechococcus strain. Before the transformation of E.coli, a plasmid vector baring replication region for cyanobacteria was constructed. The lauxAB as a reporter gene was also inserted in the vector at the down stream of cloning site. Gene fragments between 0.5-2.0 kbp in length were used for cloning. Genome library with 2000 clones was constructed using E.coli as a host. Vectors were purified from 84 clones selected randomly. Size of inserted fragments was analyzed by the agarose gel eloctrophoresis. Only 25% of clones retained gene fragments between 0.5-2.0 kbp in length and the others retained smaller fragments. Luminescences due to the expression of LauxAB were observed from 20% of the clones, indicating that cloned fragments in those clones contain promoter region(s). These results indicated that the constructed vector system must be efficient for screening of stress response elements.

自然界中存在着各种环境压力。常见的应力是物理化学应力，如温度、渗透压、辐射、压力、气体成分、有毒化学物质/金属的污染等。微生物在遗传水平上对这些环境压力迅速作出反应，以维持其生存或体内平衡。本研究试图筛选各种应激条件下导致基因表达的基因元件。首先，我们尝试以一种海洋蓝藻作为宿主，构建一个海洋蓝藻的基因组文库。然而，由于蓝藻转化效率较低，因此不适合构建该文库。然后，以大肠杆菌细胞为宿主生物，构建聚球菌菌株的全基因组文库。在大肠杆菌转化前，构建了带蓝藻复制区的质粒载体。在克隆位点的下游将lauxAB作为报告基因插入载体。基因片段长度在0.5-2.0 kbp之间。以大肠杆菌为宿主，构建了包含2000个克隆的基因组文库。从随机选取的84个无性系中纯化载体。琼脂糖凝胶电泳分析插入片段的大小。只有25%的克隆保留了长度在0.5-2.0 kbp之间的基因片段，其余克隆保留了更小的片段。在20%的克隆中观察到由于表达LauxAB而产生的发光，表明这些克隆中的克隆片段含有启动子区。这些结果表明，所构建的向量系统对应力响应单元的筛选是有效的。