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GMO Biosafety Research 2012, Vol.3, No.1, 1-7
http://gmo.sophiapublisher.com
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developed based on the target genome walking PCR,
(Liu and Whitter, 1995) and the high-efficiency
TAIL-PCR (hiTAIL-PCR) (Liu and Chen, 2007).
In this study, Using hiTAIL-PCR and long-chain PCR
method, we attempted to obtain the flanking sequence
of exogenous DNA insertion sites in transgenic cotton
06N-119 in order to elucidate integration structure in
detail. While we try to design specific PCR primers
based on the flanking sequence of the 3' end of
transformants and to conduct qualitative PCR
amplification in order to establish the transforming
event-specific detection methods.
1 Results and Analysis
1.1Obtaining 3' flanking sequence of insertion fragment
The insertion fragment of the 3' flanking sequence and
the cotton genome sequence were obtained by
hiTAIL-PCR amplification experiments twice, and
then PCR validation was done. First, using 3-1sp1,
3-1sp2 and 3-1sp3 as specific primers, a 2776-bp
fragment was obtained by hiTAIL-PCR amplification
experiments using the sample DNA as template,
(Figure 1A), and then a long-range PCR validation
was carried out (primers 5'-F1/3'-R4) (Figure 1B).
Bioinformatics analysis showed that the 2200 bp of
the fragment was the
cry1Ac
gene sequence, while the
200 bp was the 7S-UTR, which were similar with the
sequences of Xinmian 33B.
The three specific primers, named 3-2sp1, 3-2sp2, and
3-2sp3 were designed based on the obtained fragments
of 2776 bp to conduct the second hiTAIL-PCR
amplification, a 1118 bp fragment was obtained.
BLASTn analysis showed that 200 bp of the fragment
was 7S-UTR sequence, and 800 bp of the fragment
was no any match of homologous sequences. The
forward primer, named MF-1 was designed according
to the 7S-UTR sequences, while reverse primer named
MR-5 was designed based on 800-bp sequence. PCR
validation was conducted to generate 926 bp fragment
of the sample 06N-119, while there is no amplified
band appearing in the positive and negative controls,
(Figure 1C). The primers (MF-3/MR-5,) were
designed based on the region of 800 bp sequence, the
PCR result showed that both of the cotton samples and
controls can be amplified the 506 bp band, which
indicated that the obtained 800 bp fragment are the
sequence of the cotton genomic sequence (Figure 1D).
1.2 Obtaining the 5' end flanking sequence of
insert fragment
The 5' flanking sequence of the insert fragment and
the cotton genome sequence were obtained by
hiTAIL-PCR and a long distance PCR amplification
experiments
four
times.
The
hiTAIL-PCR
amplification was performed four times toward the 5'
end sequence of the
cry1Ac
gene. A 1242 bp fragment
was amplified at the first amplification (Figure 2A),
which contained 200 bp of
cry1Ac
sequence, 521 bp
of the 35S promoter sequence, 500 bp of the vector
backbone; A 1678 bp fragment was amplified at
second amplification, which contained 276 bp of NOS
terminator, 600 bp of the
Npt II
gene sequence, and
the remaining of the vector backbone; A 690 bp
fragment was obtained at the third amplification,
which contained 195bp of
Npt II
gene sequences and
400 bp of the E35S promoter sequences.
Because the follow-up hiTAIL-PCR amplification
experiments failed to get the specific bands, we took
the following method to acquire the further 5' flanking
sequence. The direction of hiTAIL-PCR amplification
toward the direction of the 5' end sequence was
carried out by sing the negative non-genetically
modified cotton Ji 668 genomic DNA as a template,
and specific primers named c-1sp1, c-1sp2 and c-1sp3
designed based on the obtained 3' end cotton genomic
sequence, a 1400 bp cotton genome sequence was
obtained (Figure 2B), then the forward primer (5'-F5)
near the 1400 bp sequence and the reverse primer
(3'-R3) in the
Npt II
gene sequences were designed to
generate a 5 548 bp band (Figure 2C). The result of
sequencing and splicing showed that we obtained the
remaining unknown sequence of the 5' end of the
insert and 894 bp of cotton genome sequence.
1.3 The structural characteristics of the insert
By conducting the 3' and 5' end hiTAIL-PCR
amplification and long distance PCR amplification,
we obtained the entire sequence of the insert and the
cotton genome sequence of both flanking sides.
Bioinformatics analysis showed that exogenous
inserting DNA was 11578 bp in full length consisting