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:
- 金额:$ 4.03万
- 依托单位:
- 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
- 来源:
- 关键词:
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者的潜力,并利用冷却ccd相机进行生物发光筛选,以显示生物体的各种现象,我们计划了这个研究项目。聚藻球菌pcc 7942基因组启动子荧光素酶报告文库中含有过长的DNA片段,无法进行进一步分析。因此,我们重新构建了含有荧光素酶报告基因的基因组文库。新文库的平均DNA片段长度为600 bp,文库中含有独立的E.coil克隆,覆盖了聚球菌基因组的5倍。我们筛选了10万个引入DNA-lux构建体的聚珠球菌转化体,获得了800个生物发光克隆。所有800个克隆都显示出昼夜生物发光节律,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的表达。然后,对800个无性系进行高温、低温、光通量变化、单色光(包括紫外)照射等多种环境刺激,筛选出具有独特反应的无性系。在生物发光监测下,大多数克隆的反应是相似的,但我们发现有几个克隆的反应是例外的。我们将详细分析这些选择的克隆的反应,并分析对每种刺激作出反应的启动子。我们对10个生物发光受生物钟严格控制的克隆进行了DNA片段测序,并在聚囊藻(Kazusa DNA研究所)基因组中寻找插入的同源性。6个基因被确定为生物钟控制基因,并将分析这些基因的环境反应与生物钟控制的生理意义。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控制和环境反应。除了荧光素酶报告基因外,我们还证实了导入高等植物的aequorin基因可以报告植物细胞内的游离钙水平,并且发现钙水平受生物钟和光照条件的控制。少
项目成果
期刊论文数量(25)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Liu, Y., N.F.Tsinoremas, S.S.Golden, T.Kondo, and C.H.Johnson: "Circadian expression of genes involved in the purine biosynthetic pathway of cyanobacterium Synechococcus sp.strain PCC 7942." Mol.Microbiol. 20. 1071-1081 (1996)
Liu, Y.、N.F.Tsinoremas、S.S.Golden、T.Kondo 和 C.H.Johnson:“参与蓝藻聚球藻菌株 PCC 7942 嘌呤生物合成途径的基因的昼夜表达。”
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Liu,Y.,et al.: "Circadian expression of genes involved in the purine biosynthetic pathway of cyanobacterium Synechococcus sp.strain PCC7942." Mol.Icrobiol.20. 1071-1081 (1996)
Liu,Y.,et al.:“蓝藻聚球藻菌株 PCC7942 嘌呤生物合成途径相关基因的昼夜表达。”
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Kondo, T., T.Mori, N.V.Lebedeva, S.Aoki, M.Ishiura and S.S.Golden: "Circadian rhythms in rapidly dividing cyanobacteria." Science. 275. 224-227 (1997)
Kondo, T.、T.Mori、N.V.Lebedeva、S.Aoki、M.Ishiura 和 S.S.Golden:“快速分裂的蓝细菌的昼夜节律。”
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
S.S.Golden, M.Ishiura, C.H.Johnson and T.Kondo: "Cyanobacterial Circadian Rhythms." Ann Rev.Plant Physiol. 48. 327-354 (1997)
S.S.Golden、M.Ishiura、C.H.Johnson 和 T.Kondo:“蓝藻昼夜节律”。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Aoki, S., T.Kondo and M.Ishiura: "Circadian rhythm of dnaK gene expression in cyanobacteria Synechocystis sp.PCC 6803." J.Bacteriol.177. 5606-5611 (1995)
Aoki, S.、T.Kondo 和 M.Ishiura:“蓝藻集胞藻属 sp.PCC 6803 中 dnaK 基因表达的昼夜节律。”
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
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KONDO Takao其他文献
KONDO Takao的其他文献
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{{ truncateString('KONDO Takao', 18)}}的其他基金
Circadian pacemaker of cyanobacteria by clock protein KaiC
通过时钟蛋白 KaiC 实现蓝藻的昼夜节律起搏器
- 批准号:
24000016 - 财政年份:2012
- 资助金额:
$ 4.03万 - 项目类别:
Grant-in-Aid for Specially Promoted Research
Temporal integration of cellular system by circadian clock in cyanobacteria
蓝藻生物钟对细胞系统的时间整合
- 批准号:
15GS0308 - 财政年份:2003
- 资助金额:
$ 4.03万 - 项目类别:
Grant-in-Aid for Creative Scientific Research
Molecular genetic studies on kai gene : mechanism of circadian oscillator
kai基因的分子遗传学研究:昼夜节律振荡器的机制
- 批准号:
11440234 - 财政年份:1999
- 资助金额:
$ 4.03万 - 项目类别:
Grant-in-Aid for Scientific Research (B).
Real-time monitoring of gene expression by bioluminescence
通过生物发光实时监测基因表达
- 批准号:
11558089 - 财政年份:1999
- 资助金额:
$ 4.03万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Molecular basis of circadian rhythms : function of clock genes
昼夜节律的分子基础:时钟基因的功能
- 批准号:
11694199 - 财政年份:1999
- 资助金额:
$ 4.03万 - 项目类别:
Grant-in-Aid for Scientific Research (A).
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