Rogalski Marcelo, Ruf Stephanie, Bock Ralph
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1 D-14476, Golm, Germany.
Nucleic Acids Res. 2006;34(16):4537-45. doi: 10.1093/nar/gkl634. Epub 2006 Aug 31.
Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Deltarps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.
质体基因组包含一组保守的基因，其中大多数基因参与光合作用或基因表达。在高等植物质体基因组中存在的核糖体蛋白基因中，rps18很特别，因为在几种非绿色单细胞生物的质体基因组中不存在该基因，包括长眼虫和刚地弓形虫。在这里，我们通过从烟草质体基因组中删除rps18基因来测试核糖体蛋白S18是否是翻译所必需的。我们报告说，虽然很容易获得rps18基因的缺失，但无法分离出同质性的Δrps18植物或叶片区域。相反，向同质性的分离导致叶片发育出现严重缺陷，这表明rps18的敲除是致命的，S18蛋白是细胞存活所必需的。我们的数据表明，S18对于烟草中的质体核糖体功能是不可或缺的，并支持质体翻译在植物发育中的重要作用。此外，我们证明了在短反向重复序列上发生了翻转重组，这产生了转化质体基因组的不同异构体，这些异构体在大单拷贝区域中一个70 kb片段的方向上有所不同。然而，翻转重组的发生率较低以及质体基因组的随机分离导致在许多组织样本中仅一种异构体占主导地位。讨论了对叶绿体转化实验解释和载体设计的影响。
