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

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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