Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida - IFAS, Gainesville, FL, 32611, USA.
Department of Plant Resources, College of Industrial Science, Kongju National University, Yesan, 32439, Republic of Korea.
Plant Mol Biol. 2019 Jun;100(3):247-263. doi: 10.1007/s11103-019-00856-4. Epub 2019 Mar 27.
A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.
创建了一个无选择标记、高度表达的单拷贝基因座，其两侧为绝缘子，作为转基因堆叠的着陆垫在甘蔗中。与缺乏绝缘子的单拷贝转基因系相比，这些事件表现出更高的转基因表达。通过 FLPe/FRT 位点特异性重组支持从转基因甘蔗系中切除选择标记基因。甘蔗是禾本科（Poaceae）甘蔗属的热带 C4 草，占全球糖产量的近 80%，也是生物燃料生产的重要原料。生成具有可预测和稳定转基因表达的转基因甘蔗对于作物改良至关重要。在这项研究中，我们生成了一个高度表达的单拷贝基因座作为转基因堆叠的着陆垫。通过 NPTII ELISA 分析，与没有绝缘子的单拷贝事件相比，带有单个拷贝 nptII 表达盒和绝缘子侧翼的稳定整合的转基因甘蔗系表现出更高的转基因表达，同时降低了系间变异。随后，通过 FLPe/FRT 位点特异性重组系统有效地从甘蔗基因组中切除 nptII 选择标记基因，以创建无选择标记的植物。这项研究为未来使用位点特异性重组或基因组编辑工具进行基因堆叠提供了有价值的资源。
