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GMO Biosafety Research 2012, Vol.3, No.1, 1-7
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4
consistent with the promoter sequences of the
cry1Ac
gene. The
cry1Ac
gene cassette structure between
cotton 06N-119 and Xinmian 33B was complete
identical, but 8 base of the
cry1Ac
gene in 06N-119
was different with the XInmian 33B. The specific
primer for Mon531 line (Yang et al., 2005) was
employed to detect on the 06N-119, the results failed
to detect the specific bands, indicating that Mon531
line-specific detection method should not be suitable
for the detection of 06N-119.
Comparison between the cotton genomic sequences of
the sides of the insert fragments in 06N-119 and the
same regional of cotton genomic sequences in
non-transgenic cotton Ji 668, we found that a 75 bp
cotton genomic fragment was lost during the insertion
event occurred, but did not found gene recombinant
phenomena in the insertion site region (Figure 3).
Figure 3 The sketch of inserting site and inserting fragment structure
Note: The underline stands for the missing base sequence;
Ori322: Replion; OriV: Replion; E35S: double CaMV 35S
Promoter;
Npt
Ⅱ
: Neomycin phosphotransferase ; TnNOS:
Ⅱ
NOS terminator; add: spc/str coding region;
cry1A
c:
Bacillus
thuringiensis
gene; 7S-3': 7S-UTR terminator
1.4 Establishment of specific PCR detection
method for 3' end of the transformation event
In order to establish the specific PCR detection
method for 3' end of the transformation event, we used
genomic DNA as template to screen the primes and their
combinations based on the amplification efficiency and
specificity, total of 10 combinations among the five
forward primers and six reverse primers were screened to
determine the MF-1/MR-2 as the 3' end specific
detection primers of genetically modified cotton. Then
using this screened primers to detect the specificity on
different crops, the results showed that only 06N-119
contained the expected 445 bp band, while the other 32
samples including cotton, rice, corn, soybean, rapeseed,
sugar beet, and wheat were unable to amplify target
band, which indicated that the establishment of
transformant specific qualitative PCR detection method
for cotton 06N-119 should be high specificity.
The genomic DNA of GM 06N-119 were gradient
diluted by using the non-transgenic gene cotton
genomic DNA (100 ng of/μL), the diluted
concentration were respectively 10.00%, 1.00%,
0.50%, 0.10%, 0.05% and 0%, which used as
templates for PCR amplification with MF-1/MR-2
primer pair. The sensitivity testing results showed that
the primer pair MF-1/MR-2 could still amplify the
specific target fragment in genomic DNA
concentration of 06N-119 that was diluted to 0.05%
(Figure 4), which indicated that the MF-1/MR-2
detection sensitivity should be at least 0.05% level,
namely about 11 copies.
Figure 4 Sensitivity detection for event-specific primer pair
MF-1/MR-2
M: 100bp DNA ladder; 1: 10.00%; 2: 1.00%; 3: 0.50%; 4:
0.10%; 5: 0.05%; 6: 0%
2 Discussions
Wang et al (2011) found that the transgenic cotton
EZaMian 1 Hao share the same construct with
Monsanto's Xin Mian 33B, but the transformation
event is not the same as the Monsanto’s. By detecting
the 5' end flanking sequence of EZAmian 1 Hao, the 5'
end of strain-specific detection method was
established, but it has not yet to report the 3' end
flanking sequence. In this study, we obtained the
complete structure of the insertion of transgenic cotton
06N-119 and 5' end and 3' flanking sequence, in which
the 5' flanking sequence of EZAmian 1 Hao was
complete identical to that reported by Wang et al (2011).
At present, the cotton genome sequencing is in
progress. In this study, although insert fragment
flanking sequence of 5' end 894 bp and 3' end 800 bp
in transgenic cotton 06N-119 did not yet search for
homologous sequences in GenBank, it was proved to