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

高等植物的叶绿体基因由两种RNA聚合酶转录：一种是由叶绿体基因组编码的类似大肠杆菌的多亚单位酶(plettid-encoded plettid RNA聚合酶，简称PEP)，另一种是与线粒体RNA聚合酶相关的噬菌体RNA聚合酶(核编码的plettid RNA聚合酶，简称NEP)。由于NEP催化亚基与线粒体酶相关，因此假设NEP含有一个催化核心和一个特异性因子。需要资金来完成对两名新能源计划推动者的描述。第一个是I型启动子PatpB-289，它有一个新的、尚未鉴定的启动子元件(框II)。第二个是已知的唯一的II型启动子，PclpP-53。首先将使用一种独特的生物材料的提取物在体外对启动子进行表征，烟草植株由于rpoA基因的定向缺失而缺乏PEP。这些植物是非光合作用的，但可以通过将突变的枝条嫁接到野生型(具有光合作用的)砧木上而在温室中种植。体外研究的结论将通过转基因方法在体内得到验证。在体内，启动子活性之后将积累水母维多利亚绿色荧光蛋白(GFP)。该项目将确定高等植物中NEP启动子的结构，并确定这两个叶绿体启动子的组织特异性和发育表达。NEP的组成将通过从转基因植物中提纯复合体来确定，转基因植物在其核基因组中携带His标记的催化亚单位的基因。His亲和树脂将用于促进天然NEP的亲和纯化。含有NEP全酶的组份将在体外转录实验中通过特异性转录进行鉴定。利用SDS-PAGE对NEP蛋白进行分离、分离和测序。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数据库中表达的cDNA的搜索。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叶绿体基因表达的核控制的理解，并促进将叶绿体NEP启动子用于生物技术应用的工程。

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.