Agrobacterium tumefaciens precisely transfers the transferred DNA (T-DNA) between the right border (RB) and the left border (LB) and integrates it in the plant genome. However, transfer of the binary vector backbone (BVB) at the LB junction has been frequently reported. Here, we describe a complex T-DNA integration in the rice transgenic event CG27 with BVB at the RB junction. Amplification of an RB-plant DNA junction fragment by genome walking and its sequencing revealed that the T-DNA portion ended at the 1st bp of RB, but is followed by a binary vector backbone (BVB) along with an RB-flanking T-DNA portion, both in an inverse orientation. This is followed by a 5-bp filler sequence and the rice chromosome 1 sequence. The configuration of the BVB integration at the RB junction was independently confirmed by PCR. Disruption of native chromosome 1 sequence was confirmed by PCR with chromosome 1-specific primers designed from either side of the T-DNA integration site. BVB sequence close to the RB is transferred and integrated into the CG27 plant genome by a mechanism that does not involve LB-skipping and RB read-through. Therefore, a check for BVB sequences at both LB and RB junctions in a transgenic crop is important in biosafety evaluation.
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Binary vector backbone
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This work was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India [Project entitled “Development of sheath blight disease-resistant transgenic rice: Resistance tests in PR-protein-expressing transgenic rice and discovery of new RNA silencing strategies”, Project number: F.No: BT/PR6466/COE/34/16/2012]. Indian National Science Academy, New Delhi is acknowledged for the Senior Scientist Fellowship to K.V. Dr. P. Sundaresan, Aravind Medical Research Foundation, Madurai is thanked for DNA Sequencing Service.
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Rajapriya, V., Kannan, P., Sridevi, G. et al. A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction. J. Plant Biochem. Biotechnol. (2021). https://doi.org/10.1007/s13562-021-00647-3
- Complex T-DNA integration
- Event-specific PCR
- Genome walking
- Native site disruption
- Right border
- Vector backbone integration