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

烟草叶绿体 RNA 编辑因子的鉴定和表征

• 批准号: 250969564
• 负责人: Dr. Michael Philipp Tillich
• 金额: --
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/250969564?language=en
• 关键词: Identification; characterization; factors; chloroplast; RNA

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.

叶绿体RNA编辑对于叶绿体基因的正确表达至关重要，因此对于叶绿体生物发生至关重要。 RNA编辑迅速发展。这导致了特定物种的编辑位点模式。例如，Nicotiana tabacum（烟草）拥有40个叶绿体编辑地点，只有19个与拟南芥共享。总共发现了在种子植物的叶绿体中发现了大约100个不同的编辑位点。烟草已经很早就受雇于编辑研究。它是并且是定义编辑位点的顺式作用元素的映射和诱变的选择的模型生物，因为技术能够以有效和定向的方式操纵叶绿体基因组。然而，由于其基因组序列和遗传筛查的电阻性，因此在拟南芥中确定了执行叶绿体RNA编辑的核编码因子。该研究计划将使烟草作为研究较高植物中氯P的主要模型生物体复兴烟草。为了促进这一点，组装了假定的烟草编辑因子的迄今未知基因序列。此外，已经建立了用于预测22种被子植物物种的编辑因素/编辑位点关系的生物信息学框架。这些进步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素和编辑因子基因，从而系统地剖析叶绿体RNA编辑的机理。在该研究计划中，烟草中的反向遗传学将证实生物学预测的叶绿体RNA编辑因子，选定的因子将以最先进的分子生物学方法来表征。叶绿体编辑体的新成分的候选者已经过去并且将通过RNA亲和力色谱法隔离，并以其在RNA编辑中的作用而被特征。该设置将得到有效的体外编辑系统的强烈支持。获得的结果将使旨在通过在将来通过操纵两个相互作用伙伴来研究跨因素及其RNA目标之间的相互作用的高度精确的实验。这还包括来自其他物种的异源编辑因素/编辑站点夫妇。同样，基于获得的结果，可以采用叶绿体转化来阐明RNA编辑与拼接和翻译的相互作用。最后，将建立用于诱导或组织特异性生成转移起始密码子的方法，以通过采用异源RNA编辑位点及其同性编辑因素来控制叶绿体（trans-）基因的表达。