Screening for genes that respond to environmental stimulus by a real-time monitoring of gene expression by bioluminescence

为了扩大荧光素酶基因作为基因表达报告基因的潜力，以及通过使用冷却CCD相机的生物发光筛选生物体显示的各种现象的技术，我们计划了本研究项目。蓝细菌聚球藻7942基因组中启动子的荧光素酶报告基因文库含有太长而无法进一步分析的DNA片段。因此，我们用荧光素酶报告基因重新构建了一个基因组文库。新文库的平均DNA片段大小为600 bp，含有的独立大肠杆菌克隆可覆盖聚球藻基因组5倍。我们筛选了100，000个用这种DNA-lux构建体引入的聚球藻转化体，并获得了800个生物发光克隆。800个克隆均表现出生物发光的昼夜节律，证实了蓝藻的生物钟控制了大部分基因的表达 ...更多信息 通过高温、低温、光通量率改变、包括UV范围在内的单色光照射等刺激，选择具有独特反应的克隆。用生物发光监测的克隆的响应对于大多数克隆是相似的，但是我们发现其中几个克隆的响应是例外的。我们将详细分析这些选择的克隆的反应，并分析响应于每种刺激的启动子。我们对其中生物发光受到昼夜节律钟严格控制的10个克隆的DNA片段进行测序，并在集胞藻基因组中寻找插入片段的同源性（Kazusa DNA Research Institute）。六个基因被确定为时钟控制基因，并将分析这些基因的环境响应相关的控制的生理意义。同时，我们还将在高等植物中寻找同源基因，以研究这些基因在植物中的生物钟调控和环境响应，除了荧光素酶报告基因外，我们还发现将水母发光蛋白基因导入高等植物中可以报告植物细胞中的游离钙水平，并发现钙水平受生物钟和光照条件的调控。少

To expand a potential of luciferase gene as a reporter of gene expression and technology for screening by bioluminescence with a cooled-CCD camera for a various phenomena that living organisms show, we planed this research project. Luciferase reporter library of promoters in a genome of cyanobacteria Synechococcus sp.PCC 7942 contained DNA fragments that were too long for further analysis. Therefore, we re-constructed a genomic library with luciferase reporter gene. Average DNA fragment of new library was 600 bp and the library contained independent E.coil clone as much as to cover the genome of Synechococcus for five times. We screened for 100,000 Synechococcus transformants that were introduced with this DNA-lux constructs and obtained 800 bioluminescent clones. All of 800 clones displayd a circadian bioluminescence rhythms, that confirmed previous results that circadian clock of cyanobacteria controlled most of gene expressions.Then, we treated the 800 clones with various environmen … More tal stimulus, such as high temperature, low temperature, alteration in light fluence rate, irradiation of monochromatic light including UV range and selected for clones that displayd unique response. Response of clones as monitored with bioluminescence was similar for most of clones but we found several clones of which response was exceptional. We will analyze the response of these selected clones in detail and analyze the promoters that respond to each stimulus.We sequenced DNA fragments of 10 clones of which bioluminescence was tightly controlled by the circadian clock and search for homology of insert in the genome of Synechocystis (Kazusa DNA Research Institute). Six genes were identified as clock controlled genes and will analyze physiological significance of the control in relation to environmental responses of these genes. Homologous genes will also be search in higher plant to examined clock control and environmental response of these genes in plants.In addition to luciferase reporter gene, we demonstrated that aequorin genes which was introduced into higher plant can report free Ca level in plants cells and found that Ca level was under control of the circadian clock and light conditions. Less