The mechanism of T-DNA integration in plant genome is still largely unknown
although the Agrobacterium-mediated transformation has become one
of the most widely used techniques for creating genetic modification of
crops and researches on plant genes. The difficulty in producing transgenic
monocots by this technique has limited its application in many important
food crops such as maize, wheat and rice until recently.
Early in a series of experiments, we obtained transgenic rice plants of
five Chinese varieties including maintainer and restorer lines of hybrid
rice via Agrobacterium-mediated transformation (Zhai et al.
2000). The wide spectrum resistance gene for resistance to bacterial blight,
Xa21, was integrated and stably inherited through several generations.
Most of the T0 plants and their T1 progenies showed
high level of resistance to the disease. These transgenic rice plants
are not only useful in breeding excellent rice varieties with bacterial
blight resistance, but are also valuable for the study of the mechanism
of T -DNA integration in a monocot.
In this study, TAIL-PCR (Thermal asymmetric interlaced PCR) technique
was used to amplify T-DNA flanking sequences in 20 resistance positive
transgenic rice plants, including 6 T0 and 14 T1
plants. Twenty-four fragments were amplified and sequenced, of which 14
were rice DNA, 9 contained vector backbone sequences of three types, and
the remaining one was a segment of the exogenous gene Xa21.
Of the 14 flanking sequences whose nature was rice DNA, 11 were at the
left border (LB) of T -DNA and 3 at the right border (RB). No homology
was found between these sequences and the sequence of transfer vector
pCXK 1301. Moreover, strong hybridization signals could be detected when
they were hybridized with rice genomic DNA. The sequences of these T-DNA
end-joining regions are compiled in Fig. 1 in a linear form and the schematic
map of this type of junction is shown in Fig. 2A.
None of these sequences had an AT-content over 70% (Data not shown), of
which six had even lower AT-content than the average of rice genomic DNA
(61.5%). The length of these fragments did not have significant influence
on amount of AT-content. All but two of them bore just a few motifs such
as A-box, T-box, AT-tract, ATC-tract and the recognition sites of DNA
topoisomerase which were characteristics of scaffold/matrixattachment
region (S/MAR) (Takano et al. 1997, Sawasaki et al. 1998).
The copy numbers of these sequences in rice genome was determined when
hybridized with rice genome DNA, and most of them were single copy (Data
not shown). However, exogenous genes introduced by direct gene transfer
methods such as particle bombardment and calcium phosphate have been found
to have a preference to integrate in the repeated sequences and AT -rich
regions bearing motifs of S/MAR in plant genomes (Takano et al.
1997, Sawasaki et al. 1998).
Studies on T-DNA processing have shown that T-strand theoretically contains
border sequences of 22nt (Nucleotide) and 3nt in its left and
right ends, respectively, when it is produced from T-DNA. No 22nt LB remnants
remained intact in the 14 fragments we obtained. In L608, L612, L593 and
L590, the whole LB sequence and several nucleotides adjacent to LB were
deleted. However, deletion of the whole RB was not found, and at least
one (in R612 and R003) or three (in R010) nucleotides of the RB sequence
were retained, correlating with the position of major and minor nick sites
identified during T-DNA processing in Agrobacterium (Wang et
al. 1987). This suggests that the right T-DNA end remained intact,
to some extent, during transfer and integration while the left border
junctions showed more variations. This is in agreement with that found
in many dicots indicating that the T-DNA integration in monocots possibly
involves the same mechanism as dicots (Guus et al. 1989, Reinhold
et al. 1991, Maria et al. 1997). It has been known that
VirD2 protein remains to covalently join the right end of the T-strand
after nicking the T-DNA borders. It pilots and protects T-DNA during the
transfer and integration while LB are often partially or all deleted for
lacking the protection.
Transgenic plant No.585 was the progeny of No.45 and their flanking sequences
were almost the same, indicating that the integrated T-DNA could inherit
with no changes in the insertion site. A BLAST X search against GeneBank
was performed for these rice DNA bearing sequences but no homology to
any protein-coding genes could be found.
Of the 24 sequenced fragments, 9 contained vector backbone sequences of
three types. The backbone sequence is referred to the bacterial replicon
region of binary vector (Maria et al. 1997). Type I is the backbone
sequence directly linked to the T-DNA across either LB or RB (Fig. 2B).
In three amplified fragments at LB (L301, L591 and L592-4) and three at
RB (R301, R591 and R597), sequences linked to either border were found
to read through the LB or RB. In addition, Southern hybridization in transgenic
plants No.301 and 591 showed - that both contained only one copy of the
transgene Xa21, indicating that the whole transfer vector possibly
is integrated into rice genome in both cases.
Type II has the backbone sequence not directly linked to a T-DNA border
(Fig. 2C). The fragment L010 has a total length of 273nt with 15nt remnants
at LB. The sequence between its 57thnt and 162thnt
is a backbone fragment. The two end segments (i.e. the segment between
lstnt and 56thnt and the segment between 163thnt
and 273thnt) are homologous to vector pCXK1301. Moreover, the
whole fragment showed strong hybridization signals in Southern analysis
probed with rice genomic DNA, indicating that both of its end segments
possibly originated from rice DNA.
Type III is rather complicated. As in transgenic plants R615 and R592-1
the flanking sequences outside RB were from the backbone sequence linked
to LB in the vector (Fig. 2D), which might be attributed to integration
of tandem T-DNAs into the rice genome. The other unusual flanking sequence
was found in transgenic plant No.52, in which the flanking sequence outside
LB is a segment of the exogenous gene Xa21 (Fig. 2E). Until recently,
it has been assumed that during Agrobacterium-mediated plant transformation,
only the sequence between the two T -DNA borders is transferred to the
plant. Indeed, T -DNA is traditionally defined as the sequences that lie
between these borders. However, evidences in the literature suggest that
DNA sequences residing outside the T -DNA border may be occasionally transferred
to the plant. Martineau et al.(1994) reported that binary vector
sequence outside T -DNA left border could integrate into the genomes of
transgenic plants by a rate of 20-30%. In addition, Maria et al.(1997)
found backbone sequences in over 75% transgenic tobaccos. In this study,
37.5% (9/24) sequenced flanking fragments had backbone sequences, slightly
higher than the statistic of Martineau et al. In agreement with
that found by Maria et al., the integrated backbone sequences were
either linked to the T -DNA across the border sequence or independent
of the T -DNA.
The results from the present study provided information about the characteristics
of flanking sequences of integrated T-DNA in rice genome. They will contribute
to utilization of the Ti-plasmid transformation system in a more comprehensive
way and get full use of the system in monocot transformation. The flanking
sequences can be used to map the T -DNA insertion sites in rice genome
and to facilitate the research into the positional effect and stability
of exogenous gene in transgenic plants. Finally, we think that the evaluation
of transgenic plants should include the possible effect caused by vector
Guus, B., K. Zdena, G. Nigel and H. Barbara, 1989. Recovery of Agrobacterium
tumefaciens T-DNA molecules from whole plants early after transfer.
Cell 57: 847-857.
Enoki, H., T. Izawa, M. Kawahara, M. Komatsu, S. Koh, J. Kyozuka and K.
Shimamoto, 1999. Ac as a tool for the functional genomics of rice. Plant
J. 19(5): 605-613.
Takano, M., H. Egawa, J.-E. Ikeda and K. Wakasa, 1997. The structures
of integration sites in transgenic rice. Plant J. 11(3): 353-361.
Maria, E., B. Burgund and B. Stanton, 1997. Integration of T-DNA binary
vector 'backbone' sequences into the tobacco genome: evidence for multiple
complex patterns of integration. Plant J. 11(5): 945-957.
Martineau, B., T. Voelker and R. Sanders, 1994. On defining T-DNA. Plant
Cell 6: 1032-1033.
Reinhold, M., K. Zsuzsanna, N. Christiane, B. Guus, C. Andreas, A. Karel,
P. George, S. Jeff, H. Barbara and K. Csaba, 1991. T-DNA integration:
a mode of illegitimate recombination in plants. EMBO J. 10(3):
Sawasaki, T., M. Takahashi, N. Goshima and H. Morikawa, 1998. Structures
of transgene loci in transgenic Arabidopsis plants obtained by
particle bombardment: Junction regions can bind to nuclear matrices. Gene
Wang, K., S.E. Stachel, B. Yimmerman, M. Van Montagu and P.C. Zambryski,
1987. Site-specific nick in the T -DNA border sequence as a result of
Agrobacterium vir gene expression. Science 235: 587-591.
Zhai, W., X. Li, W. Tian, Y. Zhou, S. Pian, S. Cao, S. Zhao, B. Zhao,
Q. Zhang and L. Zhu, 2000. Introduction of a blight resistance gene, Xa21,
into Chinese rice varieties through an Agrobacterium-mediated system.
Science in China (Series C) 43(4): 361-368.