T-DNA integration patterns in transgenic tobacco plants |
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Authors: | Mi?-Kyung?Lee Hyoung?-Seok?Kim Sung?-Hoon?Kim Email author" target="_blank">Young?-Doo?ParkEmail author |
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Institution: | (1) Graduate School of Biotechnology, Kyung Hee University, 447-501 Kyunggi, Korea;(2) Craduate school of East-West Medical Science, Kyung Hee University, 447-501 Kyunggi, Korea |
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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. |
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Keywords: | Agrobacterium tumefaciens flanking plant DNA tagging T-DNA integration |
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