Molecular Biotechnology

, Volume 53, Issue 2, pp 129–138 | Cite as

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

  • Ramu S. Vemanna
  • Babitha K. Chandrashekar
  • H. M. Hanumantha Rao
  • Shailesh K. Sathyanarayanagupta
  • K. S. Sarangi
  • Karaba N. Nataraja
  • M. Udayakumar


The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.


Gateway vectors Gene stacking β-Glucuronidase (GUS) reporter gene Cry genes Insect bioassay 

Supplementary material

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Supplementary material 1 (TIFF 1082 kb)
12033_2012_9499_MOESM2_ESM.docx (17 kb)
Supplementary material 2 (DOCX 17 kb)


  1. 1.
    Jacobsen, E., & Nataraja, K. N. (2008). Cisgenics—facilitating the second green revolution in India by improved traditional plant breeding. Current Science, 94, 1365–1366.Google Scholar
  2. 2.
    Capell, T., & Christou, P. (2004). Progress in plant metabolic engineering. Current Opinion in Biotechnology, 15, 148–154.CrossRefGoogle Scholar
  3. 3.
    Dafny-Yelin, M., & Tzfira, T. (2007). Delivery of multiple transgenes to plant cells. Plant Physiology, 145, 1118–1128.CrossRefGoogle Scholar
  4. 4.
    Halpin, C. (2005). Gene stacking in transgenic plants—the challenge for 21st century plant biotechnology. Plant Biotechnology Journal, 3, 141–155.CrossRefGoogle Scholar
  5. 5.
    Naqvi, S., Farre′, G., Sanahuja, G., Capell, T., Zhu, C., & Christou, P. (2010). When more is better: Multigene engineering in plants. Trends in Plant Science, 15, 48–56.CrossRefGoogle Scholar
  6. 6.
    Zhao, J. Z., Cao, J., Li, Y., Collins, H. L., Roush, R. T., Earle, E. D., et al. (2003). Transgenic plants expressing two Bacillus thuringiensis toxins delays insect resistance evolution. Nature Biotechnology, 21, 1493–1497.CrossRefGoogle Scholar
  7. 7.
    Wu, G., Truska, M., Datla, N., Vrinten, P., Bauer, J., Zank, T., et al. (2005). Step-wise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nature Biotechnology, 23, 1013–1017.CrossRefGoogle Scholar
  8. 8.
    Seitz, C., Vitten, M., Steinbach, P., Hartl, S., Hirsche, J., Rathje, W., et al. (2007). Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy. Phytochemistry, 68, 824–833.CrossRefGoogle Scholar
  9. 9.
    Singla-Pareek, S. L., Reddy, M. K., & Sopory, S. K. (2003). Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proceedings of the National Academy of Sciences of the United States of America, 100, 14672–14677.CrossRefGoogle Scholar
  10. 10.
    Lin, L., Liu, Y. G., Xu, X., & Li, B. (2003). Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proceedings of the National Academy of Sciences of the United States of America, 100, 5962–5967.CrossRefGoogle Scholar
  11. 11.
    Lucker, J., Schwab, W., van Hautum, B., Blaas, J., van der Plas, L. H., Bouwmeester, H. J., et al. (2004). Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiology, 134, 510–519.CrossRefGoogle Scholar
  12. 12.
    Hadi, M. Z., McMullen, M. D., & Finer, J. J. (1996). Transformation of 12 different plasmids into soybean via particle bombardment. Plant Cell Reports, 15, 500–505.CrossRefGoogle Scholar
  13. 13.
    Chen, L., Marmey, P., Taylor, N. J., Brizard, J. P., Espinoza, C., D’Cruz, P., et al. (1998). Expression and inheritance of multiple transgenes in rice plants. Nature Biotechnology, 16, 1060–1064.CrossRefGoogle Scholar
  14. 14.
    Ye, X., Al Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P., et al. (2000). Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287, 303–305.CrossRefGoogle Scholar
  15. 15.
    Nawrath, C., Poirier, Y., & Somerville, C. (1994). Targeting of the polyhydroxybutyrate biosynthetic-pathway to the plastids of Arabidopsis thaliana results in high-levels of polymer accumulation. Proceedings of the National Academy of Sciences of the United States of America, 91, 12760–12764.CrossRefGoogle Scholar
  16. 16.
    Halpin, C., Barakate, A., Askari, B. M., Abbott, J. C., & Ryan, M. D. (2001). Enabling technologies for manipulating multiple genes on complex pathways. Plant Molecular Biology, 47, 295–310.CrossRefGoogle Scholar
  17. 17.
    Daniell, H., & Dhingra, A. (2002). Multigene engineering: Dawn of an exciting new era in biotechnology. Current Opinion in Biotechnology, 13, 136–141.CrossRefGoogle Scholar
  18. 18.
    Ma, C. L., & Mitra, A. (2002). Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker. Molecular Breeding, 9, 191–199.CrossRefGoogle Scholar
  19. 19.
    Karimi, M., Bleys, A., Vanderhaeghen, R., & Hilson, P. (2007). Building blocks for plant gene assembly. Plant Physiology, 145, 1183–1191.CrossRefGoogle Scholar
  20. 20.
    Walhout, A., Temple, G., Brasch, M., Hartley, J., Lorson, M., van den Heuvel, S., et al. (2000). GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods in Enzymology, 328, 575–592.CrossRefGoogle Scholar
  21. 21.
    Chen, Q. J., Zhou, H. M., Chen, J., & Wang, X. C. (2006). A Gateway-based platform for multigene plant transformation. Plant Molecular Biology, 62, 927–936.CrossRefGoogle Scholar
  22. 22.
    Mahmood. T., Zar. T., & Saqlan Naqvi. S. M. (2008). Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens. Electronic J Biotechnology. ISSN: 0717-3458 11:1–4.Google Scholar
  23. 23.
    Clough, S. J., & Bent, A. F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735–743.CrossRefGoogle Scholar
  24. 24.
    Datta, K., Schmidt, A., & Marcus, A. (1989). Characterization of two soybean repetitive proline-rich proteins and a cognate cdna from geminated axes. Plant Cell, 1, 945–952.Google Scholar
  25. 25.
    Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6, 3901–3907.Google Scholar
  26. 26.
    Sundaram, K. M. S., & Sundaram, A. (1992). An insect bioassay method to determine persistence of Bacillus thuringiensis var. Kurstaki (B.t.k.) protein in oak foliage, following application of a commercial formulation under field and laboratory conditions. Journal of Environmental Science and Health. Part B, 27, 73–112.CrossRefGoogle Scholar
  27. 27.
    Halpin, C., & Boerjan, W. (2003). Stacking transgenes in forest trees. Trends in Plant Science, 8, 363–365.CrossRefGoogle Scholar
  28. 28.
    Curtis, M. D., & Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology, 133, 462–469.CrossRefGoogle Scholar
  29. 29.
    Earley, K. W., Haag, J. R., Pontes, O., Opper, K., Juehne, T., Song, K., et al. (2006). Gateway-compatible vectors for plant functional genomics and proteomics. The Plant Journal, 45, 616–629.CrossRefGoogle Scholar
  30. 30.
    Karimi, M., Inze, D., & Depicker, A. (2002). GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science, 7, 193–195.CrossRefGoogle Scholar
  31. 31.
    Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., et al. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. The Plant Journal, 40, 428–438.CrossRefGoogle Scholar
  32. 32.
    Rozwoadowski, K., Yang, W., & Kagale, S. (2008). Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems. BMC Biotechnology, 8, 88.CrossRefGoogle Scholar
  33. 33.
    Chakrabarthi, S. K., Mandaokar, A. D., Ananda kumar, P., & Sharma, R. P. (1998). Synergistic effect of cry1Ac and cry1F δ-endotoxins of Bacillus thuringiensis on cotton bollworm, Helicoverpa armigera. Current Science, 75, 663–664.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ramu S. Vemanna
    • 1
  • Babitha K. Chandrashekar
    • 1
  • H. M. Hanumantha Rao
    • 1
  • Shailesh K. Sathyanarayanagupta
    • 1
  • K. S. Sarangi
    • 2
  • Karaba N. Nataraja
    • 1
  • M. Udayakumar
    • 1
  1. 1.Department of Crop PhysiologyUniversity of Agricultural SciencesBangaloreIndia
  2. 2.Department of Microbiology & BiotechnologyBangalore UniversityBangaloreIndia

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