Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants
Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants
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DOI：
10.1007/s11248-009-9246-3
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发表时间：
2009-08-01
影响因子：
3
通讯作者：
Hanson, Maureen R.
中科院分区：
文献类型：
--
作者：
Gray, Benjamin N.;Ahner, Beth A.;Hanson, Maureen R.
质体内同源重组指导质体基因组转化，用于外源基因表达和质体基因功能的研究。虽然转基因通常被有效地靶向其所需的插入位点，但在质体转化期间已经观察到非预期的同源重组事件。为了了解这些重组事件的性质和丰度，我们分析了来自三种不同质体转化载体的转质体烟草品系，这些质体转化载体利用两个不同的外源基因插入位点。从转化载体中包含的每个调节质体DNA元件形成两个非预期的重组质体DNA种类。这些重组DNA种类中的一些在T0植物中积累了多达所需整合的转基因序列量的10 - 60%。一些重组DNA种类经历进一步的"次级"重组事件，导致甚至更多数量的重组质体DNA种类。新的重组DNA种类的丰度在T0植物中比在T1后代中更高，这表明这里描述的辅助重组事件可能在转化体的选择和再生过程中具有最大的影响。鉴定了一个转质体烟草系，其含有由于非预期重组事件而与预期转基因插入不连锁的抗生素抗性基因，表明本文所述的同源重组事件可能阻碍含有所需转基因的质体转化体的有效回收。
Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10-60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, "secondary" recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene.