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Functional analysis of chloroplast genes by chloroplast transformation

通过叶绿体转化进行叶绿体基因的功能分析

基本信息

批准号：
10440238
负责人：
SUGITA Mamoru
金额：
$ 8.83万
依托单位：
Nagova University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10440238/
关键词：
tobacco cyanobacteria chloroplast transformation ycf small stable RNA knockout ribonucleoprotein phage-type RNA polymerase 葉緑体 ycf5 光合成 DNAマイクロアレイ原色素体 DNA

项目摘要

This research purpose is to define the function of hypothetical chloroplast reading frames (ycf) and to understand the mechanism of regulation of chloroplast gene expression. The novel findings obtained are as follows.1. Two targeted ycf5-disruptants were obtained. These disruptants were uncapable of electron-fow from photosystem II to photosystem I.This indicates that ycf5 is involved in electron transport in photosynthesis.2. The gene encoding a small stable RNA (tmRNA) was isolated and characterized in the unicellular cyanobacterium, Synechococcus PCC6301. A tmRNA gene is not present in higher plant chloroplast DNA, suggesting that a ancestral chloroplast tmRNA gene lost during evolution.3. By immunoprecipitation, gel filtration, and western blot analysis, we demonstrated that tobacco chloroplast ribonucleoproteins (cpRNPs) are abundant stromal proteins that exist as complexes with ribosome-free mRNAs. In addition, an in vitro mRNA degradation assay revealed that the cpRNPs act as stabilizing factors for nonribosome-bound mRNAs in the stroma.4. We isolated and characterized the three genes, NsRpoT-A, NsRpoT-B and NsRpoT-C, encoding phage-type single subunit RNA polymerase from Nicotiana sylvestris. The gene product of NsRpoT-B was demonstrated to be imported into mitochondria and plastids. This provides a new insight of evolution of plant organelles.

本研究的目的是明确叶绿体阅读框架（ycf）的功能，并了解叶绿体基因表达的调控机制。取得的新发现如下：1.获得了两种靶向ycf 5-破坏物。这些干扰物不能使电子从光系统II流向光系统I，这表明ycf 5参与了光合作用中的电子传递.在单细胞蓝细菌聚球藻6301中分离并鉴定了编码小稳定RNA（tmRNA）的基因。高等植物叶绿体DNA中不存在tmRNA基因，表明在进化过程中有一个祖先叶绿体tmRNA基因丢失.通过免疫沉淀、凝胶过滤和蛋白质印迹分析，我们证明了烟草叶绿体核糖核蛋白（cpRNP）是丰富的基质蛋白，与无核糖体的mRNA形成复合物。此外，体外mRNA降解试验表明，cpRNP在基质中非核糖体结合mRNA的稳定因子.我们从欧洲烟草（Nicotiana sylvestris）中分离并鉴定了编码噬菌体型单亚基RNA聚合酶的三个基因NsRpoT-A、NsRpoT-B和NsRpoT-C。NsRpoT-B的基因产物被证明被导入线粒体和质体。这为植物细胞器的进化提供了新的视角。