Identification and characterization of factors for chloroplast RNA editing in Nicotiana tabacum

叶绿体RNA编辑对于叶绿体基因的正确表达以及因此对于叶绿体生物发生是必需的。RNA编辑发展迅速。这导致了物种特异性的编辑位点模式。例如，烟草（Nicotiana tabacum）拥有40个叶绿体编辑位点，而拟南芥（Arabidopsis thaliana）只有19个。在种子植物的叶绿体中总共发现了大约100个不同的编辑位点。烟草很早就被用于编辑研究。它过去和现在都是定义编辑位点的顺式作用元件的作图和诱变的选择模式生物，因为它具有以有效和定向的方式操纵叶绿体基因组的技术能力。然而，由于拟南芥基因组序列的可获得性和遗传筛选的可操作性，在拟南芥中鉴定了进行叶绿体RNA编辑的核编码因子，该研究计划将恢复烟草作为研究高等植物叶绿体RNA编辑的主要模式生物。为了促进这一点，迄今未知的基因序列的推定编辑因子的烟草组装。在此基础上，建立了一个预测22种被子植物编辑因子/编辑位点关系的生物信息学框架。这些进展与叶绿体转化相结合，使我们能够同时操纵同一物种中的顺式元件和编辑因子基因，从而系统地剖析叶绿体RNA编辑的机制。在这项研究计划中，生物信息学预测的叶绿体RNA编辑因子将通过烟草中的反向遗传学来证实，所选因子将通过最先进的分子生物学方法来表征。叶绿体编辑体的新组分的候选物已经并将通过RNA亲和色谱分离，并表征其在RNA编辑中的作用。这一设置将得到一个有效的体外编辑系统的有力支持。所获得的结果将使设计高度精确的实验，旨在研究反式因子和它们的RNA靶之间的相互作用，在未来的相互作用的合作伙伴的操纵。这还包括来自其他物种的异源编辑因子/编辑位点对。同样，基于所获得的结果，将有可能采用叶绿体转化来阐明RNA编辑与剪接和翻译的相互作用。最后，将建立用于翻译起始密码子的诱导型或组织特异性生成的方法，以便通过采用异源RNA编辑位点及其同源编辑因子来控制叶绿体（反式）基因的表达。

Chloroplast RNA editing is essential for the proper expression of chloroplast genes and, thus, for chloroplast biogenesis. RNA editing evolves rapidly. This results in species-specific patterns of editing sites. Nicotiana tabacum (tobacco), for instance, possesses 40 chloroplast editing sites and only 19 are shared with Arabidopsis thaliana. In total, about 100 different editing sites have been found in the chloroplasts of seed plants. Tobacco has been early on employed in editing research. It was and is the model organism of choice for the mapping and mutagenesis of the cis-acting elements that define editing sites, because of the technical ability to manipulate the chloroplast genome in an efficient and directed manner. However, the nuclear encoded factors that carry out chloroplast RNA editing were identified in Arabidopsis, because of the availability its genomic sequence and the conductibility of genetic screens.This research program will revive tobacco as prime model organism for studying chloroplast RNA editing in higher plants. To facilitate this, the hitherto unknown gene sequences of the putative editing factors of tobacco were assembled. Furthermore, a bioinformatic framework for the prediction of editing factor/editing site relationships for 22 angiosperm species has been established. These advancements in combination with chloroplast transformation allow us to simultaneously manipulate both cis-elements and editing factor genes in the same species and thus, to systematically dissect the mechanism of chloroplast RNA editing. Within this research program, bioinformatically predicted chloroplast RNA editing factors will be confirmed by reverse genetics in tobacco and selected factors will be characterized by state-of-the-art molecular biological methods. Candidates for novel components of the chloroplast editosomes have been and will be isolated by RNA-affinity chromatography and characterized for their role in RNA editing. This set-up will be strongly supported by an efficient in vitro editing system.The obtained results will enable to design highly precise experiments aimed to study the interaction between trans-factors and their RNA targets by the manipulation of both interaction partners in the future. This includes also heterologous editing factor/editing site couples from other species. Likewise, based on the obtained results, it will be possible to employ chloroplast transformation to elucidate the interplay of RNA editing with splicing and translation. Finally, approaches will be established for the inducible or tissue-specific generation of translational start codons in order to control the expression of chloroplast (trans-)genes by employing heterologous RNA editing sites and their cognate editing factors.