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Transgenes are integrated randomly into plant genomes by non-homologous recombination in plants, which gives them the potential to disrupt genes where they integrate. For example, the disruption of essential genes caused by transgene integration is likely to be lethal, particularly in the homozygous state. Such lethal transformants are therefore unlikely to persist in the breeding selection even if they could survive the plantlet regeneration process (Senior and Bavage, 2003). T-DNA integration can induce base substitutions, insertions and rearrangements at the insertion site (Afolabi et al., 2004; Zeng et al., 2010; Zhu et al., 2010), and unfavorable T-DNA rearrangements often abolish transgene expression (Kohli et al., 1998). Many reports have also shown that the transfer of non-T- NA region of vectors, which are referred to as ‘vector backbone’ sequences, is not uncommon in plants (De Buck et al., 2000; Kononov et al., 1997; Kuraya et al., 2004; Sallaud et al., 2003; Yin and Wang, 2000). These vector backbone sequences may influence transgene expression (Matzke and Matzke, 1998), leading to heightened public concern (Zhang et al., 2008). Zeng et al. (2010) suggested that regulatory authorities and consumers require commercial transgenic plants free of unnecessary genes such as selectable marker genes or vector backbone sequences. In practical transgenic breeding, transgenes may or may not follow Mendelian segregation; their expression can be significantly affected by the integration positions and structures of the transgenic DNA in host genomes (Zhong, 2001). Transgenes may become unstable over generations due to their genetic backgrounds and environmental conditions, leading them to have a significantly negative impact on the expression of endogenous genes. While deciphering a transgene into a desired phenotype is fundamentally challenging work, and a critical genetic issue in the success of transgenic breeding, it is often overlooked in breeding practice (Zhong et al., 1999). In this study, therefore, we analyzed the structure of the T-DNA and flanking region sequences of T-DNA insertion sites to understand the transgene behavior. In addition, we investigated T-DNA repeat formation and flanking region sequence of transgenic tomatoes, the transgene inheritance of which we have already characterized in our previous work (Ahn et al., 2011).

MATERIALS AND METHODS

Plant materials and transformation

 Ten-day-old cotyledons from seedlings of tomato (Solanum lycopersicum M.) cv. Momotaro-yoke were cut at both ends and incubated overnight on pre-culture medium. Cotyledons were infected with Agrobacterium tumefaciens for 5-8 min with gentle agitation, blotted on sterile paper towel, and co-cultivated in the dark for 72 h (Ahn, 2006). The transformation vector was pSB11, which contained cauliflower mosaic virus (CaMV) 35S promoter gene, the selectable marker phosphinotricin acetyltransferase II (patII, herbicide-resistant gene), and NOS terminator gene. Transgenic plants propagated in vitro were transferred to plastic plug trays for acclimatization. After acclimatization, the plantlets were transferred to soil and grown to maturity in a greenhouse.

Detection of repetitive T-DNA insertion repeats

 T-DNA repeats were identified using the reverse primer PCR (rpPCR) method (Kumar and Fladung, 2000a). Although this method utilizes a set of primer pairs oriented in opposite direction in the constructs, it is not possible to detect copy number of the transgenes were integrated into the host chromosome at various positions by PCR amplification. To increase the PCR detection efficiency, PCR was conducted using five different pairs of primers (1+2, 1+5, 1+3, 2+6, and 2+4) (Fig. 1). However, amplification products will always be obtained when transgenes are integrated in the form of multiple repeats. The results obtained with different primer pairs normally allow the orientation and state (complete or truncated) of the repeat to be deduced (Table 1).

Fig. 1. T-DNA of vector pSB11 (A), and pattern of T-DNA repeat (B, C, and D). A. Schematic diagram showing the T-DNA region with left (LB) and right (RB) borders of vector pSB11 including MARs (Matrix attachment regions, 1.3kb) containing patII gene used for tomato transformation in this study. The numbers indicate the locations of the various primers used to detect transgene repeats and arrowheads indicate their respective 5’ to 3’ orientations. B. A complete direct repeat of T-DNA in head-to-tail orientation. C and D. Inverted repeats in head-to-head (C) and tail-to-tail (D) orientation. 35S, cauliflower mosaic virus 35S promoter (0.74kb); patII, herbicide-resistant gene coding region (0.59kb); NOS, 3’ NOS terminator (0.28 kb).

Table 1. Determination of T-DNA repeats in transgenic plants with PCR using reverse primers (Kumar and Fladung, 2000a).

Thermal asymmetric interlaced (TAIL)-PCR procedure

 TAIL-PCR was performed essentially as described by Liu et al. (1995) using arbitrary degenerate (AD) primers with minor modifications (Table 2). Nucleotide sequences were designed from the left border. The following three nested and target-specific primers were designed: SP1 (5’-CTCTCCTGTCACTGAGCTGCCACCAC-3’), SP2 (5’-TGTCTCCCAAGACAAAGGACACA-CAGC-3’), and SP3 (5’-CAACTGCC-GTTTTCTGAGCACGCATAG-3’). Each of primers was used in combination with three AD primers (15- or 16-mers): AD1, 5’-NTCGA(G/C)T(A/T)T(G/C)-G(A/T)GTT-3’; AD2, 5’-NGTCGA(G/C)(A/T)GAN-A(A/T)GAA-3’; and AD3, 5’-(A/T)GTGNAG-(A/T)ANCANA-3’. The TAIL-PCR products were analyzed by nucleotide sequencing.

Table 2. Cycle settings used for TAIL-PCR.

 TAIL-PCR was conducted as summarized in Table 2 using thermal cyclers (Biometra, Germany). A difference in product size consistent with primer position was used as the criterion for judging a product to be insertion-specific. TAIL-PCR products were separated on 1% agarose gel and evaluated by the bands generated on the products of expected size to judge whether they had been inserted in specific locations in the host genome.

Database search

 With 300-400 bp nucleotides of T-DNA flanking regions, NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and Sol genomics network (http://www.sgn.comell.edu/tools/blast/simple.pl) databases were searched for nucleotide similarities by using the BLASTN algorithm.

RESULTS AND DISCUSSION

Identification of T-DNA repeats

 Of the sixty independent transformants obtained by transformation of tomato cv. Momotaro-yoke with patII gene in our previous work, 42 were analyzed for the transgene copy numbers by Southern blotting and for the segregation ratio by herbicide bioassay (Ahn et al., 2011). Several independent transgenic lines showed the inconsistent pattern of segregation in phenotype with inserted copy number. Among them, transgenic lines with 1, 2, 3, and 4 copies were screened for possible T-DNA repeats using an rpPCR (Table 3). As the primers in each pair faced away from each other, amplified bands should be produced only from transgenic lines with direct or inverted repeats of transgene.

Table 3. Segregation ratio of bialaphos resistance in the T₁ generation.

 No amplification was observed in transgenic line T₀-19 (Table 4 and Fig. 2), which was attributed to the reverse facing orientation of the primer pairs. Two transgenic lines (T₀-2 and T₀-32) showed different band patterns from common. The copy number of patII was one. Both results showed that T-DNA was single-copy integration. In contrast, the transgenic lines (T₀-21, -26, and -34) showed positive results for rpPCR. PCR amplification products were obtained with some primer pairs but not with others, indicating that TDNA repeats were present in truncated forms in these lines. In T₀-21, -26, and -34, while PCR products of the expected size were obtained from rpPCR with primer pairs 1+2 and 2+6. Expected size of product for primer pair 1+2 was about 700bp (including RB and LB, but without Matrix attachment regions, MARs), and primer pair 2+6 was about 2.7kb (Fig. 1 and Fig. 2). No amplifications were obtained with primer combinations 1+5, 1+3, and 2+4 (Table 4 and Fig. 2). This suggests that the binding sites for primers 3, 4 and 5 were missed in this transgenic line and that T-DNA copies were truncated. The transgenic lines of tomatoes, in which there was not the match between segregation ratio and copy number, showed similar phenotype as compared with the non-transgenic tomato cv. Momotaro-yoke.

Table 4. T-DNA repeats in transgenic tomato cv. Momotaro-yoke.

Fig. 2. Molecular analysis for T-DNA repeats in transgenic tomato. Amplification products obtained from different transgenic tomato lines (A and B). Arrow head indicates expected band size. M, marker; No 19, T₀-19 (singlecopy T-DNA integration); No 21, 26, and 34, T₀-21, -26, and -34, respectively (both T-DNA copies are truncated). Results are summarized in Table 3.

 While we have previously reported a segregation ratio of 3:1 (Ahn et al., 2011), the 3 copies of patII gene were detected by Southern blot analysis in T₀-26 plant. Vain et al. (2003) reported a similar result in which one line containing three fragments in hybridization patterns was segregated with a ratio of 3:1 as one Mendelian locus. This indicated that the 3 T-DNAs appeared to be linked as tandem repeats(head-to-tail). The Southern blot analysis results revealed that T₀-21 and -34 contained 4 T-DNA copies. However, in a previous study (Ahn et al., 2011) segregating of the transgene phenotype did not show 256:1 but 15:1 and 63:1, suggesting that they have 2 and 3 loci. This indicated that 2 or 3 T-DNAs were apparently arranged as direct repeats in one site and the other T-DNAs could be integrated in other sites. This result is consistent with Kumar and Fladung (2000b)’s finding that mostly more than one copy of the transgene integrated in the analyzed lines was present in linked form at one site in transgenic aspen. T-DNA repeat formation under routine transformation conditions has also been reported in rice (Kohli et al., 1999), petunia (Cluster et al., 1996), and birch (Zeng et al., 2010). The transgenic lines that were found to be positive for T-DNA repeats using this method were previously reported to contain more than one transgene copy on the basis of Southern hybridization analysis (Fladung, 1999; Fladung et al., 1997).

Analysis of T-DNA junction sequence

 To assess the distribution of inserts, 52 flanking sequences were amplified by TAIL-PCR (Fig. 3). Analysis of the nucleotide sequences of 22 flanking regions by direct sequencing of PCR products revealed that two showed higher than 90% homology in nucleotide sequences to sequenced tomato centromeric region of chromosome 12, and that five showed 97% homology to tobacco chloroplast genome DNA. In addition, the results of nucleotide homology search indicated that 12 of the 22 flanking sequences showed various vector sequences and unknown DNA sequences, that one flanking sequence showed 98% homology to tomato clone 135006R, mRNA sequence of tomato trichom, and that the others showed homology to zebrafish and human DNAs. Thirty flanking sequences showed no significant similarity to any released tomato genomic regions.

Fig. 3. Agarose gel analysis of TAIL-PCR products amplified from T-DNA insertion lines. AD1 was used for all the lines. Lane designations II and III indicate products of the secondary and tertiary reactions, respectively. Arrows indicates the bands analyzed by direct nucleotide sequencing. M, molecular marker; C, non-transgenic plant; No 1-8, 10, 22, 32, 33, 37, 39, 41, 42, 44, 45, 31, and 34, transgenic plants.

 In Agrobacterium-mediated gene transfer, it was initially considered that only regions between the T-DNA borders were introduced into the plant genome. However, Ramanathan and Veluthambi (1995) reported that many transformants contained vector sequences linked to the TDNA insert. Further investigations have shown that vector sequence transfer is probably a common event (Cluster et al., 1996). Wenck et al. (1997) also found that traditional root transformation resulted in vector transfer in 33% of all transformants, whereas vacuum infiltration resulted in 62% vector transfer in Arabidopsis thaliana. Vain et al. (2003) reported that backbone transfer was frequent in plant lines transformed with either pGreen-based, pRT18 (37%) or with pSoup-based, pRT47 (72%) vectors. These results are consistent with work using other binary vectors in transgenic dicotyledonous (De Buck et al., 2000; Martineau et al., 1994) and monocotyledonous (Upadhyaya et al., 2000; Yin and Wang, 2000) plant species.

 The transfer of the vector backbone may have resulted from the read-through integrated at the left border of the TDNA sequences. Read-through could proceed along the entire vector sequence up to the right or left border, resulting in the transfer of one or two T-DNAs separated by the entire vector backbone (De Buck et al., 2000). In addition, inner border sequences strongly influence the frequency of vector backbone transfer in Nicotiana and Arabidopsis. McCormac et al. (2001) proposed that co-transformation of different TDNAs in Nicotiana could be influenced by their relative sizes. This suggests that the sequences surrounding the border repeats or the size of the T-DNA (by itself or relative to the backbone size) could influence vector backbone transfer.

 In the present study, we analyzed the molecular structure of T-DNAs. Rearrangement or partial deletion occurred during the T-DNA integration process. In 3 transgenic lines, TDNA repeats were present in truncated forms and appeared to be linked as tandem repeats. Based on TAIL-PCR sequence analysis, 54.5% of the flanking sequences (12 of 22) showed various vector sequences. The presence of vector sequences may influence transgene expression and raise public biosafety concern (Zeng et al., 2010). In transgenic plant breeding, stable expression of transgene(s) is one of the most important factors for both commercialization and ecological risk-assessment studies. Therefore, molecular analysis to understand the transgene behavior should be performed in transgenic plants on the verge of commercial use.

적 요

 제초제 저항성 유전자가 도입된 형질전환 토마토에서 T DNA의 특성을 분석하였다. 형질전환 토마토의 T-DNA 도입 형태를 분석하기 위한 primer쌍을 제작하여 rpPCR을 실시하였다. T0-21(15:1, 4 copy), 26(3:1, 3 copy), 34(63:1, 4 copy)의 경우에는 5쌍의 primer를 이용하여 수행한 rpPCR 결과에서 양쪽이 절단된 불완전한 T-DNA 반복이 확인되었다. 도입된 T-DNA의 주변 서열 및 분포를 알아보기 위하여 총 52개체의 형질전환체에 대하여 TAIL-PCR을 실시하였다. 이 중 22개 형질전환체의 TAIL-PCR 산물의 염기서열을 분석하였는데, 2개의 형질전환체에서 나온 염기서열 결과는 토마토의 12번 염색체와 90%, 1개체는 토마토 clone 135006R과 98%, 5개체는 담배의 엽록체와 97% 상동성을 보였으며, 12개체는 다양한 vector의 염기서열을 나타내었고, 그 외 나머지 개체들은 토마토 유전체 정보와 는 유사성을 찾을 수 없었다.