Joint study on the search for phytochrome binding proteins

我们从菊苣基因组文库中鉴定出4个光敏色素基因（菊苣门1-4）。其中phy3是一个新的光感受器基因，其多肽具有光敏色素染色质结合结构域，该结构域与NPH1蛋白相关的大多肽相连，而NPH1蛋白是种子植物向光性的关键成分。用发色团前体藻蓝胆素重组酵母中表达的载脂蛋白。锌印迹分析和分光光度法实验表明，该植物PHY3是一种功能性光敏色素。PHY3的c端结构域具有两个LOV结构域，其次是Ser/Thr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/光和蓝光。Adaintum PHY3的Ser/Thr PK结构域具有所有必需的子结构域和Ser/Thr PK的保守氨基酸，表明该激酶活性受光调控。我们用酵母中表达的全长PHY3多肽和大肠杆菌中表达的激酶部分来检测蛋白激酶活性。但是，到目前为止我们还没有检测到这种活动。为了找到PHY3多肽的结合蛋白，我们尝试了双杂交系统。目前我们还没有找到合适的人选。我们研究了光敏色素依赖基因在铁蕨配子体和孢子体中的表达，发现铁蕨Lhcb基因的表达与高等植物一样受到光调控，但在铁蕨中没有检测到非常低的通量响应。还研究了光敏色素介导的原灌肠角龙的分支形成和卷曲生长。这些结果再次有力地证实了光敏色素在原角龙体内的二色性定位。

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.