分子生物学外文文献汇报1分析.ppt

Blue light-induced autophosphorylation of phototropin is a primary step for signaling蓝光诱导的向光素自我磷酸化是信号传导的重要步骤　　　　　　　  2012.10.20 Introduction Phototropin作为一种蓝光受体激酶能够调节植物的向光性弯曲、叶绿体运动、气孔开放和叶片伸展。人们已经在拟南芥、水稻、玉米等植物中发现了基因PHOT1、PHOT2分别编码两种向光素phot1和phot2蛋白，phot1和phot2都含有两个重要的多肽区域：　　1、 N-末端敏感区域:具有能与FMN结合的两个LOV(light, oxygen, voltage)－LOV1与LOV2；　　2、C-末端的Ser/Thr蛋白激酶区域。 作者首先测定了拟南芥保卫细胞中phot1在蓝光照射下发生磷酸化，进而对磷酸化位点进行定位，然后用Ala置换这些磷酸化位点的Aa残基，通过检测不同突变型phot1调节的气孔开放、叶绿体聚集、叶片伸展、向光性，分析这些位点磷酸化对蓝光诱导的信号传导所起的作用。 1.从32P标记的黄化幼苗中提取质膜微囊；2.phot1蛋白分离：immunoprecipitation,antibodies against phot13.磷酸化检测：Coomassie brilliant blue染色，放射自显影检测 蓝光照射下野生型phot1磷酸化水平比黑暗条件下高，D806N-2型磷酸化没有发生变化。说明体内实验蓝光诱导了野生型phot1自我磷酸化，黑暗条件下少量的磷酸化可能是其它激酶的作用。 Ⅲ、Mapping of in Vivo Phosphorylation Sites of Phot1. Salomon et al. determined eight phosphorylation sites in vitro in Avena phot1a. However, those sites did not include any of the sites in the kinase domain or in the C terminus shown in this study. The difference in results is probably due to the phosphorylation condition of the phot1 protein. We phosphorylated Arabidopsis phot1 in planta by an endogenous phot1 kinase, but Salomon et al. phosphorylated the recombinant Avena phot1a by PKA in vitro.Furthermore, the techniques used to identify phospho-rylation site differed between the two experiments. We may have missed phosphorylation sites that exist in too-long or -short peptides produced by tryptic digestion,because these peptides were beyond the detection range of LC-MS/MS. Fig5 Time courses of phosphorylation and dephos-phorylation of the Ser-851 in response to blue light Ⅳ、Functional Role of Phosphorylation in Phot1 in Stomatal Opening. The S849D S851D construct mimicked the phospho-rylation of the Ser residues in the activation loop. However, the construct did not induce any responses without blue light in planta . This is explained by a recent report that