## 3 Results
### Search for a Possible Reason for the Biological Activity Difference between 311 and B6 Lines
#### 3.2.3 Search for T-DNA Insertion Sites  
The next hypothesis explains the differences between the InterB and Inter311 lines in the production of the recombinant protein by the transgene integration into regions of the genome with different transcriptional activity.  
For sequencing of the tobacco genomic DNA regions flanking the T-DNA insert, the FPNI-PCR \"genome walking\" method was used. Primers were picked for the T-DNA regions close to the right and left borders of the previously sequenced inserts (Table 1). As a result, fragments up to 968 bp in size were obtained, including sequences of tobacco genomic DNA up to 750 bp.  
By using BLAST, it was found that T-DNA in the case of the B6 line was integrated into the centromeric region of the indeterminate chromosome, among repeats and retrotransposons (Figure 4A). The obtained sequences of genomic DNA fragments correspond to regions of the sequence NW_015800096.1 (NCBI Reference Sequence).  
For 311 line, insertion also occurred in an undefined region of the genome, but in a more active one, next to it the genes \"_Nicotiana tabacum_ 60S ribosomal protein L39-like\" (LOC107799127), \"eukaryotic translation initiation factor 3 subunit M-like\" (LOC107799143), receptor-like protein kinase FERONIA (LOC104086005), and chloroplastic methylesterase 11 (LOC107799161) are located. Insertion did not destroy any of the open reading frames, i.e., no T-DNA insertional mutagenesis event occurred [19].  
Moreover, as a result of genome walking, it was found that 311 line carries a cluster of at least three insertions in a tail-to-tail-head-to-tail orientation (Figure 4B). PCR with specific primers (including a number of PCR reactions using Phusion polymerase to obtain large fragments) confirms that only NOS terminator sequences bind to genomic DNA, and there is a head-to-tail connection in the genome. A difference in the sizes of the insertion sites was found--in the 311 line, T-DNA displaced only nine nucleotides of genomic DNA, while 344 nucleotides of the original sequence disappeared in the B6 line.  
In order to confirm the correctness of the determination of the insertion sites, primers were designed for the genomic DNA sequences flanking the insertion sites of both plant lines (Table 1). PCR with primers for line 311 showed that the corresponding fragment (1093 bp, Figure 4B) appeared in wild-type and B6 samples but was absent in sample 311,  
Figure 4: T-DNA insert structures. The figure shows the location of the sequences obtained as a result of genome walking, the length of the overlapping regions, and the percentage of similarity are indicated. The binding sites of the primers used to confirm the genome walking data, and the length of the resulting PCR fragment are also indicated. (**A**): Insert structure from line B6 (into sequence NW_015800096.1). (**B**): Insert structure from line 311 (into sequence NW_015801991.1). Numbers I, II, III denote copies of T-DNA.  
in which a large region (T-DNA) was inserted between the binding sites of these primers (Figure 5A). It confirms the correctness of the localization of the insertion site, as well as the fact that no unmodified copies of this region remained in the genome of plants of 311 line. This means that the 311 line is homozygous for the recombinant gene.  
A similar reaction with primers for the insertion site of the InterB6 line (Figure 4A) showed a high level of non-specific binding for all samples (Figure 5B). This confirms the incorporation of T-DNA into the repeat region, which makes it impossible to accurately localize the insertion site, as in the case of plants of 311 line. PCR with primers to the B6 genomic DNA region (B6_R1) and the T-DNA region (Nost_SP2) gives the best result--an amplified fragment appeared only in a sample with B6 line DNA, which confirms the correctness of the localization (Figure 5C).