Screening of enviromental stress response elements and their application

自然界中存在着各种环境压力。常见的应激包括温度、渗透压、辐射、压力、气体成分、有毒化学品/金属污染等物理化学应激。微生物在遗传水平上对这些环境应激做出快速反应，以求生存或保持自身的动态平衡。本研究以海洋蓝藻为寄主，构建了海洋蓝藻的基因组文库，旨在筛选各种胁迫条件下导致基因表达的基因元件。然而，由于蓝藻转化效率低，出人意料地不适合构建文库。然后，以大肠杆菌细胞为宿主构建聚球藻的全基因组文库。在大肠杆菌转化前，构建了蓝藻复制区质粒载体。在克隆位点的下游插入了报告基因laxAB。克隆的基因片段长度在0.5~2.0kbp之间。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。琼脂糖凝胶电泳法分析插入片段的大小。只有25%的克隆保留了长度在0.5-2.0kbp之间的基因片段，其余的克隆保留了较小的片段。有20%的克隆有Laux AB基因的表达，表明克隆片段中含有启动子区域(S)。这些结果表明，所构建的载体系统对于筛选胁迫响应元件是有效的。

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.