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Moleculaabiological analysis of phytochrome siggnal transduction

光敏色素信号转导的分子生物学分析

基本信息

批准号：
06454018
负责人：
NAGATANI Akira
金额：
$ 4.61万
依托单位：
UNIVERSITY of TOKYO (1995)The Institute of Physical and Chemical Research (1994)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Phytochrome is a chromoprotein which is common in green plants.Phytochrome is know to be act as a major photoreceptor mediating divergent plant responses to light.Although intensive work has been done on phytochrome, molecular mechanism in which phytochrome transduce the light signal to signal transduction factors.We addressed this problem with molecular as well as biochemical techniques in the present study.To examine biological activity of the C-terminal half, which has been speculated to be involved in the signal transduction process, we prepared transgenic Arabidopsis expressing a C-terminal fragment of phytochrome B(phyB).The analysis of those plants indicated that the C-terminal region alone could not interact with the endogenous signal transduction mechanism.Next, we investigated intracellular localization of phyB with the aid of a reporter protein.C-terminal fragments of phyB was fused to GUS reporter protein and expressed in transgenic Arabidopsis.Histochemical staining of GUS activity in those plants revealed that the fusion protein localized to the nucleus.Nuclear localization of endogenous phyB was confirmed by immunoblotting analysis of nuclei isolated from light-grown Arabidopsis.Furthermore, the level of phyB in the isolated nuclei was substantially decreased if plants was irradiated with far-red light and then kept in darkness.Taken together, is possible that nuclear localization of phyB is an important step of phytochrome signaling process.We have also examined biolochemical and biological activity of fern phytochrome expressed in higher plants.The results indicated that the fern phytochrome is photochemically active in higher plants.Nevertheless, it did show only very limited biological activity.So, we concluded that fern phytochrome is structurally too diverse from higher plant phytochrome to transduce the signal in higher plants.

光敏色素是绿色植物中常见的一种色素蛋白。光敏色素是一种主要的光感受器，介导植物对光的不同反应。虽然对光敏色素的研究已经非常深入，但光敏色素将光信号转导到信号转导因子的分子机制尚不清楚。在本研究中，我们用分子和生化技术解决了这个问题。为了检测c端部分的生物活性，我们制备了表达光敏色素B(phyB) c端片段的转基因拟南芥。对这些植物的分析表明，单独的c端区域不能与内源信号转导机制相互作用。接下来，我们利用一个报告蛋白研究了phyB在细胞内的定位，将phyB的c端片段与GUS报告蛋白融合并在转基因拟南芥中表达。GUS活性的组织化学染色表明，融合蛋白定位于细胞核。内源性phyB的核定位是通过免疫印迹分析从光生长的拟南芥分离的核证实。此外，植物在远红光照射后再置于黑暗环境中，离体细胞核中phyB的水平显著降低。综上所述，phyB的核定位可能是光敏色素信号传导过程的重要步骤。我们还研究了蕨类植物光敏色素在高等植物中表达的生物化学和生物活性。结果表明，蕨类植物光敏色素在高等植物中具有光化学活性。然而，它只显示出非常有限的生物活性。因此，我们认为蕨类植物的光敏色素与高等植物的光敏色素在结构上差异太大，无法在高等植物中转导信号。