The Role of the Nuclear-Encoded Plastid RNA Polymerase in Plastid Function and Development

摘要高等植物质体基因由两种RNA聚合酶转录：一种是多亚基RNA聚合酶，另一种是E.由质体基因组编码的大肠杆菌样酶（质体编码的质体RNA聚合酶或PEP）和与线粒体RNA聚合酶相关的噬菌体型RNA聚合酶（核编码的质体RNA聚合酶或NEP）。由于NEP催化亚基与线粒体酶相关，因此推测NEP包含催化核心和特异性因子。要求提供资金，以完成对两名新经济政策倡导者的定性。第一个是I型启动子PatpB-289，它具有一个新的、尚未表征的启动子元件（框II）。第二个是唯一已知的II型启动子，PclpP-53。 启动子首先将使用来自独特生物材料的提取物在体外表征，烟草植物由于rpoA基因的靶向缺失而缺乏PEP。这些植物是非光合作用的，但可以通过将突变体芽嫁接到野生型（光合能力强）砧木上而在温室中生长。将通过转基因方法在体内验证体外研究的结论。体内启动子活性之后是水母维多利亚水母绿色荧光蛋白（GFP）的积累。 该项目将定义高等植物中的NEP启动子结构，并确定两种质体启动子的组织特异性和发育表达。NEP组成将通过从转基因植物中纯化复合物来确定，所述转基因植物在其核基因组中携带His标记的催化亚基的基因。His亲和树脂将用于促进天然NEP的亲和纯化。含有NEP全酶的级分将通过体外转录测定中的特异性转录来鉴定。将通过SDS-PAGE分离NEP蛋白，分离并测序。微序列数据将有助于设计合适的引物进行克隆，并将用于EST数据库搜索表达的cDNA。克隆的直接目标将是NEP特异性因子。该项目将进一步加深我们对质体基因表达的核控制的理解，并促进质体NEP启动子的生物技术应用。

MCB-9905043PI: Pal MaligaABSTRACTThe plastid genes of higher plants are transcribed by two RNA polymerases: a multisubunit, E. coli-like enzyme encoded by the plastid genome (plastid-encoded plastid RNA polymerase or PEP) and a phage-type RNA polymerase related to the mitochondrial RNA polymerase (the nuclear-encoded plastid RNA polymerase or NEP). Since the NEP catalytic subunit is related to the mitochondrial enzyme, it is assumed that NEP contains a catalytic core and a specificity factor. Funds are requested to complete characterization of two NEP promoters. The first is a Type I promoter, PatpB-289, which has a novel, as yet uncharacterized, promoter element (Box II). The second is the only known Type II promoter, PclpP-53. Promoters first will be characterized in vitro using extracts from a unique biomaterial, tobacco plants lacking PEP due to targeted deletion of the rpoA gene. These plants are non-photosynthetic, but can be grown in the greenhouse by grafting mutant shoots onto wild-type (photosynthetically competent) rootstocks. Conclusions from the in vitro studies will be verified in vivo via transgenic approaches. In vivo promoter activity will be followed by accumulation of the jellyfish Aequorea victoria green fluorescence protein (GFP). The project will define NEP promoter architecture in higher plants, and determine tissue-specific and developmental expression of the two plastid promoters. NEP composition will be determined by purifying the complex from transgenic plants which carry a gene for a His-tagged catalytic subunit in their nuclear genome. His-affinity resins will be used to facilitate affinity purification of the native NEP. Fractions containing NEP holo-enzyme will be identified by specific transcription in in vitro transcription assays. NEP proteins will be separated by SDS-PAGE, isolated and sequenced. The microsequence data will facilitate the design of suitable primers for cloning and will serve in EST database searches for expressed cDNAs. The immediate target for cloning will be the NEP specificity factor.This project will further our understanding of the nuclear control of plastid gene expression and facilitate the engineering of plastid NEP promoters for biotechnological applications.