Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane
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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.
KeywordsSugarcane Selection marker removal Site-specific recombination Insulator FLP FLPe
The authors are thankful to Syngenta Crop Protection, LLC. and CPBR for financial support. This work was also co-funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the U.S. Department of Energy. The authors would like to thank Dr. Hardev Sandhu (Everglades Research and Educational Center, UF-IFAS, Belle Glade, FL) for providing tops of sugarcane cultivar CP 88-1762 and Sun Gro Horticulture, Apopka, FL, for donation of the Fafard #2 potting mix.
F.A. and V.S. conceived and designed the experiments; Y.Z., J.Y.K. and J.H.J. constructed the plasmids; Y.Z., B.P. and B.W. generated the transgenic plants; Y.Z., B.K. and D.W. analyzed the transgenic plants; C.F., W.Y. and S.D. performed the TaqMan® qPCR assay; R.K. and Y.Z. carried out Southern Blot hybridization; Y.Z. and F.A. wrote the manuscript. All authors read and approved the final manuscript.
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