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调控的转录因子与细菌因子同源。Hewever，拟南芥基因组计划揭示，叶绿体基因组和核基因组都不编码任何原核转录因子。为了鉴定和表征参与PEP调节的叶绿体转录因子，应采用对叶绿体特异的新的生物化学和/或遗传学方法。在这项研究中，我确定了新的核编码的叶绿体因子，特别是与叶绿体sigma因子，Sig 1的区域4相互作用。对叶绿体psbA启动子的体外转录分析表明，PEP的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，-35元件是必不可少的转录起始psbA启动子，但不是在成熟的叶绿体。这也表明，叶绿体sigma因子，Sig 5将参与在psbD光响应启动子的蓝光依赖性转录激活。此外，我还利用叶绿体转化技术研究了烟草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，我在叶绿体中表达绿色荧光蛋白的转质体植物中观察到了基质，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.