## 3 Results
### Search for a Possible Reason for the Biological Activity Difference between 311 and B6 Lines
#### 3.2.4 The Structure of the Junctions of T-DNA and Genomic DNA  
Rearrangements occurred in the terminal part of the napaline synthase terminator of the 311 line (Figure 6). For B6 plants, the full-length NOS terminator is present, separated from the plant genome by a region of plasmid DNA corresponding to the shortened left border. For the 311 line, any LB fragments are absent at both junction points of T-DNA and plant genomic DNA, and the NOST was shortened by 160-200 bp. The most interesting structure is the site of two inserts joining in the head-to-tail orientation: the 95 bp T-DNA region, including the NOST and LB fragments, was turned 180 degrees (Figure 6). These rearrangements did not affect the functionality of the NOST, judging by the fact that 311 line plants successfully passed the stage of kanamycin selection. The exact structure of the \"tail-to-tail\" junction of the inserts remains unknown--this region could not be fully amplified either during FPNI-PCR or standard PCR with 35 Sm-SP primers. This indicates a large size of the intermediate section. According to the literature, there are cases of insertion into the genome of a transgenic plant of additional sequences formed by plant and/or plasmid DNA [20]. Figure 5: PCR results with primers to the tobacco genomic DNA flanking transgenic inserts. Sample WT—DNA of non-transgenic tobacco plant, samples 311 and B6—DNA of transgenic plants of lines Inter311 and InterB6. (**A**): primers 311_L1 + 311_R1. The corresponding fragment (1093 bp) is absent in sample 311, in which the T-DNA was inserted between the binding sites of these primers. (**B**): primers B6_L2 + B6_R1. High level of non-specific binding was shown for all samples, which confirms the insertion of the T-DNA into the region of repeats. (**C**): primers B6_R1 (tobacco genomic DNA) + Nost_SP2 (T-DNA region). The amplified fragment appeared only in sample B6. decided to sequence the transgenic inserts of both lines. As a result, no differences were found in the T-DNA sequence between plants of 311 and B6 lines, which could explain the difference in the biological activity of their extracts. It should be noted that in the case of line 311 with multiple insertions, the mutation in one of them may be hidden by the suppressive level of the PCR product from the others. The next hypothesis explains the differences in the productivity of transgenic plants by the integration of transgenes into regions of the genome with different activities. In order to test this hypothesis, localization of the T-DNA insertion sites in both studied lines was required, which was performed by the \"genome walking\" method (FPNI-PCR). This made it possible to obtain sequences of genomic DNA regions flanking transgenic inserts. Analysis of the obtained sequences showed that, in the case of the 311 line, the insertion actually occurred in the transcriptionally active region of the genome. For the B6 line, an insert was found in the region of centromeric repeats. The results of the genome walking were confirmed by PCR with specific primers (Figure 5). Thus, the data obtained confirmed the hypothesis of T-DNA insertion into genome regions with different transcriptional activity. Integration into an inactive region of the genome can explain the lower biological activity of the extract from B6 line plants. Although early studies showed that T-DNA is predominantly incorporated into the active regions of the genome, more recent studies have confirmed that T-DNA is incorporated equally likely into all regions of the genome [22; 23]. The sequences obtained during FPNI-PCR showed that the insert in the 311 line has a complex trimeric structure and consists of three identical copies of T-DNA in a tail-to-tail-head-to-tail orientation. This fact could not be detected by genetic analysis, which demonstrated monogenic inheritance of the transgene. Multi-copy insertion often causes silencing (decrease in transgene expression) [20].