T-DNA integration patterns in transgenic tobacco plants
To investigate the various integration patterns of T-DNA generated by infection withAgrobacterium, we developed a vector (pRCV2) for the effective T-DNA tagging and applied it to tobacco (Nicotiana tabacum cv. Havana SR1). pRCV2 was constructed for isolating not only intact T-DNA inserts containing both side borders of T-DNA, but also for partial T-DNA inserts that comprise only the right or left side. We also designed PCR confirmation primer sets that can amplify in several important regions within pRCV2 to detect various unpredictable integration patterns. These can also be used for the direct inverse PCR. Leaf disks of tobacco were transformed withAgrobacterium tumefaciens LBA4404 harboring pRCV2. PCR and Southern analysis revealed the expected 584 bp product for thehpt gene as well as one of 600 bp for thegus gene in all transformants; one or two copies were identified for these integrated genes. Flanking plant genomic DNA sequences from the transgenic tobacco were obtained via plasmid rescue and then sequenced. Abnormal integration patterns in the tobacco genome were found in many transgenic lines. Of the 17 lines examined, 11 contained intact vector backbone; a somewhat larger deletion of the left T-DNA portion was encountered in 4 lines. Because nicking sites at the right border showed irregular patterns when the T-DNA was integrated, it was difficult to predict the junction regions between the vector and the flanking plant DNA.
KeywordsAgrobacterium tumefaciens flanking plant DNA tagging T-DNA integration
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- Cho YG, Eun MY, McCouch SR, Chae YA (1994) The semi-dwarf gene, sd-1, of rice (Oryza sativa L). II. Molecular mapping and marker-assisted selection. Theor Appl Genet 89: 54–59Google Scholar
- Kim HS, Kim SH, Park YD (2003) Development of rescue cloning vector with phophinothricin resistant gene for effective T-DNA tagging. J Kor Soc Hort Sci 44: 407–411Google Scholar
- Koes R, Souer E, van Houwelingen A, Mur L, Spelt C, Quattrocchio F, Wing J, Oppedijk B, Ahmed S, Maes T, Gerats T, Hoogeveen P, Meesters M, Kloos D (1995) Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants. Proc Nat Acad Sci USA 92: 8149–8153PubMedCrossRefGoogle Scholar
- Park YD, Kim HS (2000) Expression and inheritance patterns of gus gene driven by an endosperm-specific promoter in transgenic tobacco. Kor J Hort Sci Tech 18: 594–598Google Scholar
- Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura Y, Cheng Z, Nagamura Y, Antonio BA, Kanamori H, Hosokawa S, Masukawa M, Arikawa K, Chiden Y, Hayashi M, Okamoto M, Ando T, Aoki H, Arita K, Hamada M, Harada C, Hijishita S, Honda M, Ichikawa Y, Idonuma A, lijima M, Ikeda M, Ikeno M, Ito S, Ito T, Ito Y, Ito Y, Iwabuchi A, Kamiya K, Karasawa W, Katagiri S, Kikuta A, Kobayashi N, Kono I, Machita K, Maehara T, Mizuno M, Mizubayashi T, Mukai Y, Nagasaki H, Nakashima M, Nakama Y, Nakamichi Y, Nakamura M, Namiki N, Negishi M, Ohta I, Ono N, Saji S, Sakai K, Shibata M, Shimokawa T, Shomura A, Song J, Takazaki Y, Terasawa K, Tsuji K, Waki K, Yamagata H, Yamane H, Yoshiki S, Yoshihara R, Yukawa K, Zhong H, Iwama H, Endo T, Ito H, Hahn JH, Kim HI, Eun MY, Yano M, Jiang J, Gojobori T (2002) The genome sequence and structure of rice chromosome 1. Nature 420: 312–316PubMedCrossRefGoogle Scholar
- Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2003) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 3: 1423–1429Google Scholar
- Tinland B, Hohn B (1995) Recombination between prokaryotic and eukaryotic DNA: integration ofAgro-bacterium tumefaciens T-DNA into the plant genome.In JK Setlow, ed, Genetic Engineering, Vol 17. Plenum Press, New York, pp 209–229Google Scholar