School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia.
Plant Mol Biol. 2011 Jul;76(3-5):299-309. doi: 10.1007/s11103-011-9764-2. Epub 2011 Mar 15.
In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum (tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines (tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene (neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment.
在高等植物中，DNA 从质体（叶绿体）基因组转移到核是一个频繁的、持续的过程。然而，对于这种转移是通过直接的 DNA 机制发生，还是涉及 RNA 中间体，一直存在不确定性。以前利用转质体烟草（tp7 和 tp17）的实验使实时检测质体到核的转移成为可能。为了确定是否涉及 RNA 中间体，生成了含有核启动子驱动的卡那霉素抗性基因（neo）的转质体系（tpneoACG），该基因在其质体基因组中带有起始密码子，需要质体 RNA 编辑，但与 tp7 和 tp17 完全相同。因此，预计只有在 neo 通过 RNA 介导转移到核后，才会获得卡那霉素抗性。对 tpneoACG 后代的筛选揭示了几个卡那霉素抗性的植物，每个植物都含有位于核中的 neo 基因。令人惊讶的是，所有这些植物中的 neo 都没有经过编辑，这表明 neoACG 在没有编辑起始密码子的情况下是活跃的，并表明 RNA 中间体不参与转移。然而，对 tpneoACG 的分析表明，只有一小部分潜在能够介导 neo 转移的转录本经过编辑，因此不能明确得出 RNA 在质体到核转移中的作用。编辑转录本的低比例被发现是由于主要的反义 neo 转录本，而不是 sense 转录本的编辑效率低。本研究强调了在利用质体 RNA 编辑设计实验时需要考虑的一些重要因素。结果还表明，RNA 编辑位点减少但不能消除功能性质体到核基因转移。这在进化背景下以及将 RNA 编辑依赖的基因放置在质体基因组中作为转基因控制的手段都具有重要意义。
