Advertisement

Cereal Research Communications

, Volume 35, Issue 3, pp 1375–1383 | Cite as

Characterization of Left-border Flanking Sequences of T-DNA Integration in Transgenic Rice (Oryza sativa L.) Expressing cry1Ab

  • M. A. Zaidi
  • X. Y. Cheng
  • I. AltosaarEmail author
Open Access
Article

Abstract

To understand the molecular details of T-DNA integration, the left border (LB) sequences and flanking plant DNA of 16 independent T-DNA insertions in transgenic cry1Ab rice were analyzed by an inverse PCR approach. DNA sequencing indicated that five of the 16 fragments (31%) were found to have simple or rearranged tandem repeats of right border sequences in a head to tail fashion. Mirror truncations of LB of the T-DNA, as well as mirror rearrangements, such as point mutations, small deletions and inversions were found in the region close to the LB breakpoints in some inserts. Host plant DNA flanking the T-DNA endpoints were also sequenced. The A+T contents in the plant DNA within 50 bp adjacent to the T-DNA endpoints were between 30–76% (average 52.5%), not different from the average genome value. Despite minor mutations and some rearrangements, it appears that T-DNA, harbouring a synthetic cry1Ab coding sequence of 49% GC (as well as uidA and hph), still carries such a foreign gene into ‘transcriptionally active regions’ of the rice genome, which are 55.8% GC on average as predicted from the rice genome sequence.

Keywords

rice cry1Ab Agrobacterium T-DNA left-border right-border repeat DNA sequence promoter 

References

  1. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:3389–3402.CrossRefGoogle Scholar
  2. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12:8711–8721.CrossRefGoogle Scholar
  3. Cheng, X., Sardana, R., Kaplan, H., Altosaar, I. 1998. Agrobacterium-transformed rice plants expressing synthetic crylA(b) and crylA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proceedings of National Academy of Science, USA 95:2767–2772.CrossRefGoogle Scholar
  4. Does, M.P., Dekker, B.M., de Groot, M.J., Offringa, R. 1991. A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR. Plant Molecular Biology 17:151–153.CrossRefGoogle Scholar
  5. Forsbach, A., Schubert, D., Lechtenberg, B., Gils, M., Schmidt, R. 2003. A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant Molecular Biology 52:161–176.CrossRefGoogle Scholar
  6. Francis, K.E., Spiker, S. 2005. Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant Journal 41:464–477.CrossRefGoogle Scholar
  7. Gelvin, S.B. 2000. Agrobacterium and plant genes involved in T-DNA transfer and integration. Annual Review of Plant Physiology and Plant Molecular Biology 51:223–256.CrossRefGoogle Scholar
  8. Gotoh, O. 1982. An improved algorithm for matching biological sequences. Journal of Molecular Biology 162:705–708.CrossRefGoogle Scholar
  9. High, S.M., Cohen, M.B., Shu, Q.Y., Altosaar, I. 2004. Achieving successful deployment of Bt rice. Trends Plant Science 9:286–292.CrossRefGoogle Scholar
  10. Hsing, Y.I., Chern, C.G., Fan, M.J., Lu, P.C., Chen, K.T., Lo, S.F., Sun, P.K., Ho, S.L., Lee, K.W., Wang, Y.C., Huang, W.L., Ko, S.S., Chen, S., Chen, J.L., Chung, C.I., Lin, Y.C., Hour, A.L., Wang, Y.W., Chang, Y.C., Tsai, M.W., Lin, Y.S., Chen, Y.C., Yen, H.M., Li, C.P., Wey, C.K., Tseng, C.S, Lai, M.H., Huang, S.C., Chen, L.J., Yu, S.M. 2007. A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Molecular Biology 63: 351–364.CrossRefGoogle Scholar
  11. Khanna, H.K., Raina, S.K. 2002. Elite indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas). Transgenic Research 11:411–423.CrossRefGoogle Scholar
  12. Kim, K.M., Lee, D.E., Song, H., Kuk, Y.I., Jung, S., Guh, J.O., Baenziger, P.S., Back, K. 2004. Influence of a selectable marker gene hpt on agronomic performance in transgenic rice. Cereal Research Communications 32:9–16.Google Scholar
  13. Kim, S.R., Lee, J., Jun, S.H., Park, S., Kang, H.G., Kwon, S., An, G. 2003. Transgene structures in T-DNA-inserted rice plants. Plant Molecular Biology 52:761–773.CrossRefGoogle Scholar
  14. Kumar, S., Fladung, M. 2000. Determination of transgene repeat formation and promoter methylation in transgenic plants. Biotechniques 28:1128–1137.CrossRefGoogle Scholar
  15. Kumar, S., Fladung, M. 2002. Transgene integration in aspen: Structures of integration sites and mechanism of T-DNA integration. Plant Journal 31:543–551.CrossRefGoogle Scholar
  16. Le Gall, O., Torregrosa, L., Danglot, Y., Candresse, T., Bouquet, A. 1994. Agrobacterium-mediated genetic-transformation of grapevine somatic embryos and regeneration of transgenic plants expressing the coat protein of grapevine chrome mosaic nepovirus (Gcmv). Plant Science 102:161–170.CrossRefGoogle Scholar
  17. Matsumoto, T. et al. (Group Author, International Rice Genome Sequencing Project) 2005. The map-based sequence of the rice genome. Nature 436:793–800.CrossRefGoogle Scholar
  18. Nakano, A., Suzuki, G., Yamamoto, M., Turnbull, K., Rahman, S., Mukai, Y. 2005. Rearrangements of large-insert T-DNAs in transgenic rice. Molecular Genetics and Genomics 273:123–129.CrossRefGoogle Scholar
  19. Pearson, W.R., Lipman, D.J. 1988. Improved tools for biological sequence comparison. Proceedings of National Academy of Science, USA 85:2444–2448.CrossRefGoogle Scholar
  20. Wang, J., Li, L., Shi, Z.Y., Wan, X.S., An, L.S., Zhang, J.L. 2005. T-DNA integration category and mechanism in rice genome. Journal of Integrative Plant Biology 47:350–361.CrossRefGoogle Scholar
  21. Windels, P., De Buck, S., Van Bockstaele, E., De Loose, M., Depicker, A. 2003. T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences. Plant Physiology 133:2061–2068.CrossRefGoogle Scholar
  22. Xu, Y.Y., Wang, X.M., Li, J., Li, J.H., Wu, J.S., Walker, J., Xu, Z.H., Chong, K. 2005. Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana. Plant Molecular Biology 57:773–784.CrossRefGoogle Scholar
  23. Yin, Z., Wang, G.L. 2000. Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theoretical and Applied Genetics 100:461–470.CrossRefGoogle Scholar
  24. Zaidi, M.A., Cheng, X., Xu, H., Altosaar, I. 2006. Ex situ application of foliar-produced lepidoptericides from transgenic rice to control Pieris rapae and Cry1Ab stability in vivo. Crop Protection 25:748–752.CrossRefGoogle Scholar

Copyright information

© Akadémiai Kiadó, Budapest 2007

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Agricultural Biotechnology Laboratories, Department of Biochemistry, Microbiology and ImmunologyUniversity of OttawaOttawaCanada
  2. 2.Syngenta Biotechnology, Inc.Research Triangle ParkUSA
  3. 3.Proteins Easy Corp.OttawaCanada

Personalised recommendations