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.
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.
Lipman D.J. , 'Gapped BLAST and PSI-BLAST: a new generation of protein database search programs ' (1997 ) 25 Nucleic Acids Research : 3389 -3402 .
Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12 :8711–8721.
Bevan M. , 'Binary Agrobacterium vectors for plant transformation ' (1984 ) 12 Nucleic Acids Research : 8711 -8721 .
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.
Altosaar I. , 'Agrobacterium-transformed rice plants expressing synthetic crylA(b) and crylA(c) genes are highly toxic to striped stem borer and yellow stem borer ' (1998 ) 95 Proceedings of National Academy of Science, USA : 2767 -2772 .
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.
Offringa R. , 'A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR ' (1991 ) 17 Plant Molecular Biology : 151 -153 .
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.
Schmidt R. , 'A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome ' (2003 ) 52 Plant Molecular Biology : 161 -176 .
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.
Spiker S. , 'Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations ' (2005 ) 41 Plant Journal : 464 -477 .
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.
Gelvin S.B. , 'Agrobacterium and plant genes involved in T-DNA transfer and integration ' (2000 ) 51 Annual Review of Plant Physiology and Plant Molecular Biology : 223 -256 .
Gotoh, O. 1982. An improved algorithm for matching biological sequences. Journal of Molecular Biology 162 :705–708.
Gotoh O. , 'An improved algorithm for matching biological sequences ' (1982 ) 162 Journal of Molecular Biology : 705 -708 .
High, S.M., Cohen, M.B., Shu, Q.Y., Altosaar, I. 2004. Achieving successful deployment of Bt rice. Trends Plant Science 9 :286–292.
Altosaar I. , 'Achieving successful deployment of Bt rice ' (2004 ) 9 Trends Plant Science : 286 -292 .
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.
Yu S.M. , 'A rice gene activation/knockout mutant resource for high throughput functional genomics ' (2007 ) 63 Plant Molecular Biology : 351 -364 .
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.
Raina S.K. , 'Elite indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas) ' (2002 ) 11 Transgenic Research : 411 -423 .
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.
Back K. , 'Influence of a selectable marker gene hpt on agronomic performance in transgenic rice ' (2004 ) 32 Cereal Research Communications : 9 -16 .
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.
An G. , 'Transgene structures in T-DNA-inserted rice plants ' (2003 ) 52 Plant Molecular Biology : 761 -773 .
Kumar, S., Fladung, M. 2000. Determination of transgene repeat formation and promoter methylation in transgenic plants. Biotechniques 28 :1128–1137.
Fladung M. , 'Determination of transgene repeat formation and promoter methylation in transgenic plants ' (2000 ) 28 Biotechniques : 1128 -1137 .
Kumar, S., Fladung, M. 2002. Transgene integration in aspen: Structures of integration sites and mechanism of T-DNA integration. Plant Journal 31 :543–551.
Fladung M. , 'Transgene integration in aspen: Structures of integration sites and mechanism of T-DNA integration ' (2002 ) 31 Plant Journal : 543 -551 .
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.
Bouquet A. , 'Agrobacterium-mediated genetic-transformation of grapevine somatic embryos and regeneration of transgenic plants expressing the coat protein of grapevine chrome mosaic nepovirus (Gcmv) ' (1994 ) 102 Plant Science : 161 -170 .
Matsumoto, T. et al. (Group Author, International Rice Genome Sequencing Project) 2005. The map-based sequence of the rice genome. Nature 436 :793–800.
Matsumoto T. , 'The map-based sequence of the rice genome ' (2005 ) 436 Nature : 793 -800 .
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.
Mukai Y. , 'Rearrangements of large-insert T-DNAs in transgenic rice ' (2005 ) 273 Molecular Genetics and Genomics : 123 -129 .
Pearson, W.R., Lipman, D.J. 1988. Improved tools for biological sequence comparison. Proceedings of National Academy of Science, USA 85 :2444–2448.
Lipman D.J. , 'Improved tools for biological sequence comparison ' (1988 ) 85 Proceedings of National Academy of Science, USA : 2444 -2448 .
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.
Zhang J.L. , 'T-DNA integration category and mechanism in rice genome ' (2005 ) 47 Journal of Integrative Plant Biology : 350 -361 .
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.
Depicker A. , 'T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences ' (2003 ) 133 Plant Physiology : 2061 -2068 .
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.
Chong K. , 'Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana ' (2005 ) 57 Plant Molecular Biology : 773 -784 .
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.
Wang G.L. , 'Evidence of multiple complex patterns of T-DNA integration into the rice genome ' (2000 ) 100 Theoretical and Applied Genetics : 461 -470 .
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.
Altosaar I. , 'Ex situ application of foliar-produced lepidoptericides from transgenic rice to control Pieris rapae and Cry1Ab stability in vivo ' (2006 ) 25 Crop Protection : 748 -752 .