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T-DNA integration patterns in transgenic tobacco plants

한국식물학회지 2004년 47권 3호 p.179 ~ 186
 ( Lee Mi-Kyung ) - Kyung Hee University Graduate School of Biotechnology

김형석 ( Kim Hyoung-Seok ) - Kyung Hee University Graduate School of Biotechnology
김성훈 ( Kim Sung-Hoon ) - Kyung Hee University Graduate School of East-West Medical Science
박영두 ( Park Young-Doo ) - Kyung Hee University Graduate School of Biotechnology

Abstract


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.

키워드

Agrobacterium tumefaciens; flanking plant DNA; tagging; T-DNA integration
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