Joint study on the search for phytochrome binding proteins

我们从铁线蕨光敏色素基因文库中鉴定出4个光敏色素基因(Adiantum Phyl1-4)。其中Phy3是一个新的光感受器基因，其多肽具有光敏色素生色团结合区，与种子植物向光性的关键成分NPH1蛋白相关的一个大的多肽相连。在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，Adiantum PHY3是一种功能性光敏色素。PHY3的C末端结构域有两个LOV结构域，其次是丝氨酸/苏氨酸蛋白激酶(PK)结构域。LOV结构域被认为具有黄素结合位点，这意味着PHY3可能吸收红/远红光和蓝光。Adaintom PHY3的Ser/Thr PK结构域具有Ser/Thr PKs的所有必需亚区和保守氨基酸，表明该蛋白的活性受光调控。我们用酵母中表达的全长PHY3多肽和在大肠杆菌中表达的蛋白激酶部分来检测蛋白激酶活性。然而，到目前为止，我们还没有检测到这种活性。为了找到与这种PHY3多肽结合的蛋白，我们正在尝试双杂交系统。我们目前没有找到任何候选基因。我们研究了光敏色素依赖基因在铁线莲配子体和孢子体中的表达，发现铁线藻的LHCb基因(S)的表达与高等植物一样是光调控的，但在这种蕨类植物中没有检测到极低的光敏率响应。这些结果有力地证实了光敏色素在原驼鹿体内的二色性定位。

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.