Orel Nadiya, Puchta Holger
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, 06466 Gatersleben, Germany.
Plant Mol Biol. 2003 Mar;51(4):523-31. doi: 10.1023/a:1022324205661.
Surprising species-specific differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks (DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum. In Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of the nature of the ends (blunt, 5' or 3' overhangs), linearized full-length DNA molecules were, on average, more stable in tobacco than in Arabidopsis. The relative expression of a beta-glucuronidase gene encoded by the plasmid was similar in both plant species when the break was distant from the marker gene. However, if a DSB was introduced between the promoter and the open reading frame of the marker, transient expression was halved in Arabidopsis as compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the Arabidopsis genome is more than twenty times smaller than the tobacco genome.
对于两种双子叶植物拟南芥和烟草，已有报道称基因组双链断裂（DSB）的非同源末端连接（NHEJ）存在惊人的物种特异性差异。在拟南芥中，缺失平均比烟草中的更大，且与插入无关。为了确定该现象的分子基础，我们通过用线性化质粒分子对叶片组织进行基因枪转化，分析了这两种植物中游离DNA末端的命运。Southern杂交表明，无论末端的性质如何（平端、5'或3'突出端），线性化的全长DNA分子在烟草中平均比在拟南芥中更稳定。当断裂远离标记基因时，质粒编码的β-葡萄糖醛酸酶基因在两种植物中的相对表达相似。然而，如果在标记基因的启动子和开放阅读框之间引入DSB，与烟草相比，拟南芥中的瞬时表达减半。这些结果表明，游离DNA末端在烟草中比在拟南芥中更稳定，这可能是由于较低的DNA核酸外切酶活性，或者是由于对DNA断裂末端的更好保护，或者两者兼而有之。由于拟南芥基因组比烟草基因组小二十多倍，DNA末端的核酸外切酶降解可能是基因组大小进化的驱动力。
