Moleculaabiological analysis of phytochrome siggnal transduction

光敏色素是绿色植物中普遍存在的一种色素蛋白。光敏色素被认为是介导不同植物对光反应的主要光感受器。尽管人们对光敏色素进行了大量的研究，但光敏色素将光信号转导到信号转导因子的分子机制已经做了大量的工作。本文结合分子和生化技术对这一问题进行了研究。为了检测C末端的生物活性，它被认为参与了信号转导过程。我们制备了表达植物光敏色素B 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。其次，我们利用报告蛋白研究了光敏色素B的C端片段在细胞内的定位。将光敏B的C端片段与GUS报告蛋白融合并在转基因拟南芥中表达。对这些植物的GUS活性进行了组织化学染色，结果表明融合蛋白定位于细胞核。通过免疫印迹分析，证实了内源PhyB的核定位。此外，还对从轻度生长的拟南芥中分离的细胞核进行了免疫印迹分析，证实了该融合蛋白的核定位如果植物受到远红光照射并保持黑暗状态，细胞核中光敏B的水平显著降低。综上所述，光敏B的核定位可能是光敏色素信号传递过程的重要步骤。我们还检测了高等植物中表达的蕨类光敏色素的生物化学和生物活性。结果表明，蕨类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，我们得出结论，蕨类光敏色素在结构上与高等植物光敏色素的结构差异太大，无法在高等植物中传递信号。

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.