Unité Mixte de Recherche 1095-Institut National de la Recherche Agronomique-Génétique, Diversité et Écophysiologie des Céréales, Domaine de Crouël, 63039, Clermont-Ferrand Cedex 2, France.
Plant Cell Rep. 2017 Oct;36(10):1547-1559. doi: 10.1007/s00299-017-2173-5. Epub 2017 Jun 30.
A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.
一个 53kbp 的大片段完整 DNA 片段被插入小麦（Triticum aestivum L.）基因组中。对单个转基因事件的 FISH 分析显示存在多个完整片段的插入。将包含多个抗性基因簇或完整代谢途径的大片段完整 DNA 片段转移到小麦基因组中仍然是一个挑战。在之前的工作中，我们表明，使用去磷酸化的盒进行小麦转化可以产生简单的整合模式。在这里，我们使用相同的技术生产了一个包含 44kb 的拟南芥 BAC 的盒，两端分别为一个选择基因和一个报告基因。这个 53kb 的线性盒通过生物弹道转化整合到面包小麦（Triticum aestivum L.）基因组中。我们的结果表明，生成了携带整个盒的转基因植物。在 T1 和 T2 代中证明了盒的可遗传性。令人惊讶的是，对来自独立事件的 T1 后代进行的 FISH 分析表明，在非同源位置存在完整片段的双基因组插入。通过对 T1 代进行 FISH 分析证明了这些双插入的可遗传性。讨论了可以从分子或 FISH 分析中得出的相关结论，以及未来用于小麦转化或基因组编辑的大片段工程的前景。
