Screening of enviromental stress response elements and their application

自然界中存在着各种各样的环境胁迫。常见的胁迫是物理化学胁迫，如温度、渗透压、辐射、压力、气体成分、有毒化学品/金属污染等。微生物在遗传水平上迅速响应这些环境胁迫，以维持生存或稳态。本研究试图筛选导致基因表达的基因元件，首先，我们试图构建海洋蓝细菌Synechococcus sp.的基因组文库，使用海洋蓝细菌作为宿主生物。然而，出乎意料的是，由于蓝藻的转化效率低，因此不适合构建文库。然后，采用大肠杆菌细胞作为构建聚球藻菌株全基因组文库的宿主生物。在转化大肠杆菌之前，先构建了带有蓝藻复制区的质粒载体。将lauxAB作为报告基因插入到载体的下游克隆位点。使用长度为0.5-2.0 kbp的基因片段进行克隆。以大肠杆菌为宿主构建了2000个克隆的基因组文库。从随机选择的84个克隆中纯化载体。通过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，其他克隆保留了较小的片段。从20%的克隆中观察到由于LauxAB表达而产生的发光，表明这些克隆中的克隆片段含有启动子区。这些结果表明，所构建的载体系统必须是有效的筛选胁迫反应元件。

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.