Joint study on the search for phytochrome binding proteins

我们从铁线蕨基因组文库中鉴定了4个光敏色素基因（铁线蕨phyl 1 -4）。其中一个（phy 3）是一种新的感光基因，其推定的多肽具有光敏色素发色团结合结构域，该结构域与NPH 1蛋白相关的大多肽相连，NPH 1蛋白是种子植物向光性的关键组分。酵母中表达的载脂蛋白与发色团前体藻蓝胆素重组。锌印迹分析和分光光度实验表明铁线蕨PHY 3是一种功能性光敏色素。PHY 3的C-末端结构域具有两个LOV结构域，随后是Ser/Thr蛋白激酶（PK）结构域。已经提出LOV结构域具有黄素结合位点，这意味着PHY 3可以吸收红光/远红光和蓝光。Adaintum PHY 3的Ser/Thr PK结构域具有Ser/Thr PK的所有必需亚结构域和保守氨基酸，表明该激酶活性是光调节的。我们使用在酵母中表达的全长PHY 3多肽以及在大肠杆菌中表达的激酶部分测试蛋白激酶活性。为了寻找与该多肽结合的蛋白质，我们正在尝试双杂交系统。我们研究了植物光敏色素依赖基因在铁线蕨配子体和孢子体中的表达，发现铁线蕨Lhcb基因的表达与高等植物一样受光调控，但在这种蕨类植物中没有检测到极低的光通量响应。这些结果再次有力地证实了角齿藻光敏色素的二色性定位。

We identified 4 phytochrome genes (Adiantum phyl1-4) from Adiatum genomic library. One of them (phy3) was a novel photoreceptor gene whose putative polypeptide has a phytochrome chromophore-binding domain, that is tethered to a large polypeptide related to the NPH1 protein which is a key component in seed plant phototropism. Apoprotein expressed in yeasts was reconstituted with a chromophore precursor phycocyanobilin. Zinc blot analysis and spectrophotometric experiments suggested that the Adiantum PHY3 was a functional phytochrome. C-terminal domain of PHY3 possesses two LOV domains, followed by Ser/Thr protein kinase (PK) domain. The LOV domain has been proposed to have flavin binding site, meaning tht PHY3 may absorb red/farred and blue light. The Ser/Thr PK domain of Adaintum PHY3 has all of the essential subdomains and conserved amino acids of Ser/Thr PKs, suggesting that this kinase activity is light-regulated. We tested protein kinase activity using full length PHY3 polypeptides expressed in yeasts and also using kinase part expressed in E.coli. However, we do not detect the activity so far.To find a binding protein to this PHY3 polypeptide, we are trying two-hybrid system. We do not find any candidate at the moment.We studied phytochrome dependent gene expression in Adiantum gametophtyes as well as in sporophytes, and found taht expression of Lhcb gene (s) of Adiantum is light regulated as in higher plants, however very low fluence rate response could not be detected in this fern.Phytochrome-mediated branch formation and curling growth of Ceratodon protoenemata were also studied. These results strongly reconfirmed dichroic localization of phytochrome in Ceratodon protonemata.