Analysis of T-DNA integration events in transgenic rice

Abstract

Agrobacterium-mediated plant transformation has been widely used for introducing transgene(s) into a plant genome and plant breeding. However, our understanding of T-DNA integration into rice genome remains limited relative to that in the model dicot Arabidopsis. To better elucidate the T-DNA integration into the rice genome, we investigated extensively the T-DNA ends and their flanking rice genomic sequences from two transgenic rice plants carrying Cowpea Trypsin Inhibitor (CpTI)-derived gene Signal-CpTI-KDEL (SCK) and Bacillus thuringiensis (BT) gene, respectively, by TAIL-PCR method. Analysis of the junction sequences between the T-DNA ends and rice genome DNA indicated that there were three joining patterns of microhomology, filler DNA sequences, and exact joining, and both the T-DNA ends tend to adopt identical manner to join the rice genome. After T-DNA integration, there were several variations of rice genomic sequences, including small deletions at the integration sites, superfluous DNA inserted between T-DNA and genome, and translocation of genomic DNA in the flanking regions. The translocation block could be from a noncontiguous region in the same chromosome or different chromosomes at the integration sites, and the originating position of the translocated block resulted in comparable deletion based on a cut/paste mechanism rather than a replication mechanism. Our study may lead to a better understand of T-DNA integration mechanism and facilitate functional genomic studies and further crop improvement.

Keywords: Cut/paste mechanism; Filler DNA; Microhomology; T-DNA integration; Transgenic rice; Translocation.