T-DNA Integration Patterns In Transgenic Maize Lines Mediated By Agrobacterium Tumefaciens

Agrobacterium tumefaciens-mediated transformation is the most widely utilized technique to generate transgenic events of plants. The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. It was complex to elucidation of the integration mechanism of T-DNA in the host genome. Illegitimate integration by non-homologous recombination was suggested for T-DNA integration in plant chromosomes, whereas site-specific integration and homologous recombination were identified in many other transformed events. Sometimes, the integration of T-DNA can induce chromosomal rearrangement including translocation, inversion, deletion and insertion. Furthermore, various lengths of the bacterial plasmid backbone DNA sequence were found contained in the host genome of Agrobacterium-mediated transformats. Based on the sequence analysis of T-DNA transgenic, believed that multiple mechanisms are involved in T-DNA integration in plants.In Arabidopsis and tobacco, the T-DNA integration pattern was found to be highly determined. The complex repetition and diversity of the maize genome make it a bigger challenge to explore the distribution and characterization of T-DNA tags integration than other plants. Up to now, few documents have been available on T-DNA integration patterns in maize. In the present study, we improved the protocol of thermal asymmetric interlaced PCR (TAIL-PCR), amplified the flanking sequences around T-DNA integration sites from70independent transgenic maize lines mediated by Agrobacterium tumefaciens, and analyse the molecular characteristics of T-DNA integration by the Blast, to explore the T-DNA integration patterns in the maize genome.The results were summarized as follows:1. Amplification of the flanking sequences around T-DNA integration sitesIn70transgenic maize plants of "18-599" by Agrobacterium tumefaciens, the protocol of thermal asymmetric interlaced PCR which we improved was performed to rescue the flanking sequences of T-DNA insertions in the maize genome.75flanking sequences were successfully rescued and the ratio was approximately up to53.6%.By the repeat PCR amplification,64out of the75recovered products were confirmed to be specific fragments amplified from transgenic maize lines. Another11flanking sequences were non-specific franments amplified. The ratio was approximately up to85.3%.With the six arbitrary degenerate primers, the amplification efficiency of AD4and AD6was higher than the other arbitrary degenerate primers. The ratio was31.0%and26.2%, respectively.2. The datum arounds T-DNA integrations sits analysisBy the sequence analysis with the maize "B73",41out of the64specific fragments were demonstrated to be the flanking sequences outside the left border (26fragments) or right border (15fragments) of the integrated T-DNA sequences. Thirty-two and nine of them are homologous to the sequences of the maize genome and the expression plasmid, respectively.21out of the32flanking sequences were found homologous to the sequences at a single physical site on one chromosome, and another9sequences at more than one physical site on three to ten chromosomes.3. Discussion of the molecular characteristics of T-DNA integration21precisely indentified integration sites distributed on all the10chromosomes. But T-DNA integration has some hot spots on maize chromosomes, and preference to the distal chromosomal ends and non-repetitive sequences. And the hot spots had nothing to do with the A/T.Analysed the cleavage, the deleted sequences and the backbone sequences (21.4%) were in the T-DNA integration sites. The cleavage occurs not only during the T-DNA borders but also inside or outside the borders.For26of them, a filler sequence of3to63bp long was found flanking the maize genomic sequence. These filler sequences were homologous neither among themselves nor to the sequences of the maize genome or the expression plasmids. The pre-integrated sites were not found in the T-DNA integration. To sum up, the amplification of non-specific franments reduced besides increasing efficiency by improved TAIL-PCR. The characteristics, filler sequences and hot spots, were same between transgenic Arabidosis thaliana or rice and transgenic maize plants in the T-DNA integration, while, T-borders were not the cleavages and the pre-integrated sites were inexistent in the transgenic maize lines.