基本HTML版本
Bt Research 2015, Vol.6 No.1 1-8
ISSN 1925-1939
http://bt.biopublisher.ca
7
a total volume of 10
l, containing 4
l of the
amplified product from each isolate, 1
l of 10X
buffer, 1
l of enzyme (10 U), and 4
l of Milli-Q
sterilized water. The restriction reactions for the
vip3Aa
gene vip5/vip6 sequence were also performed
in a volume of 10
l, containing 2
l of the amplified
product from each isolate, 1
l of 10X buffer, 1
l of
enzyme (10 U), and 6
l of Milli-Q sterilized water.
Finally, for the
cry1Fa
gene, the total reaction volume
of 10
l contained 2
l of the amplified product from
each isolate, 1
l of 10X buffer, 1
l of enzyme (10 U),
and 6
l of Milli-Q sterilized water.
The reactions were incubated at 37ºC, after which 3
l
of each reaction was added to 3
l of sample buffer,
and this mixture was subjected to electrophoresis in a
1% agarose gel. Each gel also contained a sample of
the non-digested amplified product and the 1-kb DNA
ladder molecular marker to allow a comparison of the
results.
3.5 Bioassays
The bacterial isolates and standard strains used in this
work (Table 2) were cultivated in Petri dishes
containing Agar Nutrient medium and were incubated
for 7 days at 30ºC. To prepare spore/crystal solutions,
bacterial colonies at the surface of the medium were
collected and transferred to centrifuge tubes (Corning,
New York, USA) containing 9 ml of Milli-Q sterilized
water and 0.005% TWEEN-20®. After complete
homogenization via vortexing, the spores were diluted
and quantified using a Neubauer chamber (Barreto et
al., 1999), which allowed the solutions to be
standardized to a concentration of 3 ×10
8
spores/ml.
To purify the Vip3Aa and Chi proteins, aliquots of the
spore solutions from the
B. thuringiensis
samples
were inoculated into Petri dishes containing Terrific
Broth (TB) solid culture medium added to agar. The
Petri dishes were incubated at 30°C for approximately
20 h. Subsequently, a pre-culture of the sample was
prepared, for which one colony was collected,
incubated in a 125 ml Erlenmeyer flask containing 10
ml of TB medium and placed in an incubator shaker
(New Brunswick model G25) at 275 rpm and 30°C.
The OD
595 nm
was recorded every 20 min using a
spectrophotometer (Beckman DU 640B), with
expected values of 0.3 for each sample.
After the spectrophotometric measurements were
performed, 1 ml of the pre-culture of each sample was
inoculated into 40 ml of TB culture medium and
shaken at 275 rpm (30°C) for 12 h. The samples were
then centrifuged (Beckman J2-21) at 6368 x g for 20
min at 4°C, and the supernatants were stored until
later use in selective bioassays.
The selective bioassays were performed using
S.
frugiperda
neonatal larvae. The larval diet was poured
into a 16-cell insect-rearing tray. After the diet
solidified, 300
l of a bacterial suspension containing
one of the following solutions: Cry/VipAa/Chi,
Cry/Vip3Aa, Cry/Chi, or Vip3Aa/Chi was added,
according to the specified treatment. After this mixture
dried at room temperature (ca. 25°C), one larva was
transferred directly onto the surface of the diet in each
cell using a fine brush. Immediately after the larvae
were transferred, the trays were sealed with plastic
film and were stored in a room maintained at 26 ±
2ºC.
A randomized block design was adopted and the
bioassay was replicated four times (replicates). All
treatments were evaluated using 32 larvae per
treatment per replicate. Sterilized water was used in
the negative control. The larval mortality was assessed
1, 3, 5 and 7 days after the larvae were transferred to
the diet.
3.6 Statistical analysis
The data were subject to an analysis of variance, and
the means were compared using SAS PROC MIXED
(SAS Institute, 2004).
Authors' Contributions
ARNL, JAD and OAF conceived and designed the experiments.
ARNL, SCM, and JRVC performed the experiments. ECCA
and JRVC contributed reagents/material/analysis tools. ARNL,
JAD, MVFL, and OAF wrote the paper. All authors read and
approved the final manuscript.
Acknowledgments
The National Council for Scientific and Technological
Development (CNPq) for granting a scholarship.
References
Arantes Olivia et al., 2002,
Bacillus thuringiensis
: estratégias no controle