Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane
Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane
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DOI：
10.1007/s11103-019-00856-4
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发表时间：
2019-03
影响因子：
5.1
通讯作者：
Yang Zhao;J. Y. Kim;R. Karan;J. Jung;B. Pathak;Bruce Williamson;Baskaran Kannan;Duoduo Wang;Chunyang Fan;Wenjin Yu;Shujie Dong;V. Srivastava;F. Altpeter
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文献类型：
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作者：
Yang Zhao;J. Y. Kim;R. Karan;J. Jung;B. Pathak;Bruce Williamson;Baskaran Kannan;Duoduo Wang;Chunyang Fan;Wenjin Yu;Shujie Dong;V. Srivastava;F. Altpeter
在甘蔗中建立了一个可选择的无标记、高表达的单拷贝基因座，该基因座两侧为绝缘子，可作为转基因堆积的着陆点。与缺乏绝缘子的单拷贝转基因系相比，这些事件表现出优越的转基因表达。通过FLPe/ frtsite特异性重组，可以从转基因甘蔗株系中切除选择标记基因。【摘要】甘蔗是一种热带C4禾本科植物，占全球食糖产量的近80%，也是生物燃料生产的重要原料。培育可预测、稳定表达的转基因甘蔗是作物改良的关键。在这项研究中，我们产生了一个高表达的单拷贝位点作为转基因堆叠的着陆点。通过NPTII酶联免疫吸附分析，与没有绝缘子的单拷贝事件相比，稳定整合带有绝缘子的单拷贝表达盒的转基因甘蔗株系支持更高的转基因表达，同时减少了株系间的变异。随后，通过FLPe/ frtsite特异性重组系统从甘蔗基因组中高效地切除了ptii选择标记基因，获得了无选择标记植株。该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宝贵的资源。
Key messageA 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/FRTsite-specific recombination.AbstractSugarcane, a tropical C4 grass in the genusSaccharum(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 copynptII 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, thenptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRTsite-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.