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

通过生物发光实时监测基因表达来筛选对环境刺激有反应的基因

基本信息

批准号：
07554045
负责人：
KONDO Takao
金额：
$ 4.03万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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

为了扩大荧光素酶基因作为基因表达报告基因的潜力，以及通过冷却 CCD 相机进行生物发光筛选生物体显示的各种现象的技术，我们计划了这个蓝藻基因组启动子荧光素酶报告基因库的研究项目。聚球藻 PCC 7942 含有太长的 DNA 片段，无法进行进一步分析，因此，我们用新文库的荧光素酶报告基因的平均 DNA 片段重建了基因组文库。为 600 bp，文库包含足以覆盖聚球藻基因组的独立大肠杆菌克隆，我们筛选了 100,000 个引入该 DNA-lux 构建体的聚球藻转化体，并获得了 800 个生物发光克隆。显示出昼夜节律生物发光节律，证实了先前的结果，即蓝藻的昼夜节律时钟控制了大部分基因然后，我们用各种环境刺激（例如高温、低温、光通量率的变化、包括紫外线范围的单色光照射）处理这 800 个克隆，并选择表现出独特反应的克隆。大多数克隆的生物发光监测结果相似，但我们发现了几个反应异常的克隆，我们将详细分析这些选定克隆的反应，并分析对每个克隆做出反应的启动子。我们对10个生物发光受到生物钟严格控制的克隆的DNA片段进行了测序，并在集胞藻基因组中寻找插入片段的同源性（Kazusa DNA研究所），其中六个基因被鉴定为时钟控制基因，并将分析其生理意义。还将在高等植物中寻找与这些基因的环境反应相关的控制的同源基因，以检查这些基因在植物中的时钟控制和环境反应。除了荧光素酶报告基因之外，我们还证明了这些基因。引入高等植物的水母发光蛋白基因可以报告植物细胞中的游离Ca水平，并发现Ca水平受到生物钟和光照条件的控制。