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Bioscience Methods
BM 2011, Vol.2, No.3 http://bm.sophiapublisher.com
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3.6 Identification of Agrobacterium recombinant
constructs by PCR
These two constructed plant expression vectors were
transformed into the Agrobacterium competent cells.
The Agrobacterium plasmid DNA was extracted using
the alkali split kits, the positive clones were identified
by using the primers that were used to constructs of
pBI121-Cry1Ac22F and pBI121- Cry1Ac22T in the
table 1. PCR procedure is followed as: 94℃ , 5 min for
pre-denaturation, 30 cycles of 94℃, 1 min, 54℃, 1 min
and 72℃ , 30 s, finally 72℃ , 10 min for extension and
then 4℃ for ever. The PCR products were electrophor
etically separated in the agarose gel.
3.7 Transformation of Arabidopsis
Arabidopsis ( Arabidopsis thaliana) was transformed
by the approach of inflorescence infiltration, followed
as the procedures of Clough with slight modifications
(Clough et al., 1998). Agrobacterium baterials with
constructs of pBI121- Cry1Ac22F and pBI121- Cry1A
c22T respectively were inoculated in 10 mL LB liquid
medium, and shaked incubation at 28℃ over a night,
and then added the incubating liquid into 500 mL LB
liquid medium on the ratio of 1% bacteria. While the
density of Agrobacterium reached OD600=1.0~1.2 at
28℃ , the strains were collected by centrifuged with
3 000 r/min at 4℃ for 15 min , After removing the
supernatant liquid and the precipitate was dissolved
into medium with 1/2 MS with 5% (w/v) Sucrose and
0.044 umol/L Benzylamine purine to suspend
Agrobacterium and then make its final density reach
5 μL/L by adding surfactant SilwetL-77.
The inflorescence of Arabidopsis thaliana was immersed
into the infiltration utensils filled with 500 mL
suspensions by soaking in Agrobacterium liquid
medium for 10~15 min and then continuing to culture
in darkness for 2 days. Transformed plants were
cultivated in the normal growth conditions.
3.8 Validation of positive transgenic Arabidopsis by
kanamycin selection and PCR detection
Kanamycin resistant selection and PCR detection were
carried out according to the experimental methods of
Clough et al (1998). Putative transgenic Arabidopsis
seeds were soaked by Agrobacterium, treated by the
95% ethanol and 3.0% Sodium hypochlorite containing
0.05% Tween and then rinsed by 4~5 times in the
sterile water. Steriled seeds were suspended in 2 mL
0.1% steriled agarose and seeded into 9 mm dish in
diameter, 4 mg seeds per dish which contained
1/2×MS with 50 μg/mL kanamycin (KM) and 0.8%
agorose. Culture conditions were as follows:
Firstly, these dishes were treated at 4℃ 2 days, then
treated at 24 ℃ for 23 h light and 1 h darkness
cultured 7~10 days. Finally, the status of plantlet
colour and root developmental condition was easily
judged whether the plantlets harbored resistant gene.
The resistant plantlets were transplanted into culture
pan. The transgenic Arabidopsis genomic DNA were
extracted from the Arabidopsis young leaves carrying
the kanamycin resistant gene by using modified
CTAB method. Then employing non-transgenic
Arabidopsis genomic DNA as negative control,
pBI121-Cry1Ac22F and pBI121 -Cry1Ac22T plasmid
as positive control, we used transgenomic DNA as
template to identify the target genes by using specific
primers for cry1Ac22F and cry1Ac22F, the expected
amplified products were 3 534 bp and 1 959 bp
respectively. PCR procedure was as follows: 94℃ 5 min
pre-denaturation, 30 cycles of 94℃ 1 min, 54 ℃ 1 min
and 72℃ 30 s, final cycle , 72℃ 10 min for extension
and then 4 ℃ for ever. The amplified products were
separated by agarose gel electrophoresis.
Author Contributions
ZL is the person who designed and conducted this experiment;
ZL and YZ finished the data analysis and paper preparation. SL
conducted experimental design and result analysis; XF is the PI
of this project, involving in project design, data analysis, paper
modification. All authors had read and consented the final text.
Acknowledgements
This research is partly sponsored by the National 863 Project
( No. 2004AA2111112). Authors would like to appreciate Dr X
Zhang who provided solid technological supports and helpful
advice during the experiment in ASNESC of Northeast Forestry
University. Thanks for two anonymous reviewers with their
critical comments. In this paper we mentioned some reagent
suppliers and sequencing service providers, that doesn’t mean
we would like to recommend or endorse the production of
theirs.
References
Clough S.J., and Bent A.F., 1998, Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant
J., 16(6): 735-743 doi:10.1046/j.1365-313x.1998.00343.x PMid: 10069079
Lenin K., Mariam M.A. and Udayasuriyan V., 2001, Expression of a cry2Aa
gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of