Analysis of transcriptional regulation in chloroplasts by DNA-associated transcrition factors by means of chloroplast transformation

叶绿体编码的光合作用基因由真细菌型RNA聚合酶（PEP）转录。因此，人们认为参与PEP调控的转录因子可能与细菌因子同源。然而，拟南芥基因组计划表明，叶绿体基因组和核基因组都没有编码任何原核转录因子。为了鉴定和表征参与PEP调控的叶绿体转录因子，应该采用新的叶绿体特异性生化和/或遗传方法。在这项研究中，我发现了新的核编码的叶绿体因子，它们特异性地与叶绿体sigma因子Sig1的4区相互作用。对叶绿体psbA启动子的体外转录分析表明，PEP启动子识别特性在小麦幼苗中受到发育调控。在年轻的叶绿体中，-35元件是psbA启动子转录起始所必需的，但在成熟的叶绿体中则不是。此外，叶绿体sigma因子Sig5可能参与了psbD光响应启动子蓝光依赖性转录激活。此外，我利用叶绿体转化技术研究了叶绿体中的转录调控，发现烟草中psbA启动子的转录起始需要-35元件。最后，我可视化了在转质体植物中表达GFP的基质，并描述了它们的生理功能。

Chloroplast-encoded photosynthesis genes are transcribed by eubacterial-type RNA polymerase (PEP). Thus it has been considered that transcription factors involved in the regulation of PEP would be homologous to the bacterial factors. Hewever, arabidopsis genome project revealed that neither chloroplast genome nor nuclear genome encoded any prokaryotic transcription factor. In order to identify and characterize the chloroplast transcription factors involved in the regulation of PEP, novel biochemical and/or genetic approaches specific for chloroplasts should be employed. In this study, I identified novel nuclear-encoded chloroplast factors that specifically interact with the region 4of chloroplast sigma factor, Sig1. In vitro transcription analysis of the chloroplast psbA promoter revealed that promoter recognition property of PEP was developmentally regulated in wheat seedlings. In young chloroplasts, the -35 element was essential for transcription initiation from the psbA promoter, but not in mature chloroplasts. It was also suggested that chloroplast sigma factor, Sig5 would be involved in the blue light-dependent activation of transcription at the psbD light-responsive promoter. Furthermore, I employed chloroplast transformation technique to study the trancriptional regulation in chloroplasts and revealed that the transcription initiation at the psbA promoter required the -35 element in tobacco. Finally, I visualized stromules in transplastomic plants expressing GFP in chloroplasts and characterized their physiological functions.