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Bt Research 2015, Vol.6 No.1 1-8
ISSN 1925-1939
http://bt.biopublisher.ca
6
in the production of bioinsecticides for the control of
this pest.
3 Materials and Methods
3.1 Insects
S. frugiperda
larvae from a laboratory colony
maintained for four years at the Applied Ecology
Laboratory of UNESP, Jaboticabal campus, Brazil,
were used. The insects were reared using an artificial
diet (larva) or 5% honey (adults) and were maintained
under controlled conditions (25 ±1ºC, 70 ±10% RH
and a 14 h photophase) (Barreto et al., 1999).
3.2 Bacterial isolates and cultivation conditions
The
B. thuringiensis
isolates are part of the collection
maintained at the Laboratory of Bacterial Genetics
and Applied Biotechnology (LGBBA) of UNESP,
Jaboticabal campus. The isolates were originally
collected in soils from different states in the
southeastern region of Brazil (São Paulo, Minas
Gerais, and Paranástates).
We analyzed 114 isolates and the following four
standard strains:
B. thuringiensis
var.
alesti
,
B.
thuringiensis
var. HD125 and
B. thuringiensis
var.
aizawai
HD137, which were used as positive controls
and
B. thuringiensis
var.
tenebrionis
, which was used
as a negative control in the bioassays. All the isolates,
including the standard strains, were multiplied in
nutrient agar medium (meat extract 3 g/l,
bacteriological peptone 5 g/l and agar 15 g/l) and
incubated in a BOD chamber for 12 h at 30°C,
followed by the extraction of total DNA.
3.3 PCR amplification of the
vip3Aa
,
chi
, and
cry1Fa
gene regions
Total DNA was extracted using the InstaGene Matrix
kit (BioRad, Richmond, CA, USA) according to the
manufacturer's recommendations. To detect the
cry1Fa
,
vip3Aa
, and
chi
genes from the
B.
thuringiensis
isolates, we employed oligonucleotide
primers developed by the LGBBA research group at
FCAV/UNESP, Loguercio et al. (2002), and Lin and
Xiong (2004), respectively.
The amplification reactions for the
cry1Fa
,
vip3Aa
,
and
chi
genes were optimized according to the
sequences of the primers and contained 1X buffer
solution, 1.5-2 mM MgCl
2
, 200-250 µM dNTPs, 0.3-1
mM of each primer, 1 U of Taq DNA polymerase, 2-3
µl of the DNA sample and sterilized, distilled water
(q.s. 20 µl). The PCR cycling conditions were
optimized according to the annealing temperatures of
the individual oligonucleotide primers. The following
conditions were used for the
cry1Fa
gene: an initial
denaturation step for 5 min at 95ºC followed by 30
cycles of 1 min at 95ºC for denaturation, 1 min at
50ºC for annealing and 1 min at 72ºC for extension,
with a final step for 5 min at 72ºC to complete the
extension. For the
vip3Aa
gene, the conditions
consisted of an initial denaturation step for 2 min at
94ºC followed by 30 cycles of 30 s at 94ºC for
denaturation, 45 s at 53ºC for primer annealing and 1
min at 72ºC for extension, with a final step for 5 min
at 72ºC to complete the extension. Finally, the
conditions used for the
chi
gene involved an initial
denaturation step for 5 min at 94ºC followed by 30
cycles of 1 min at 94ºC for denaturation, 1 min at
48ºC for primer annealing and 1.5 min at 72ºC for
extension, with an additional step of 10 min at 72ºC to
complete the extension. At the end of the cycling
program, the samples were maintained at 10ºC.
The amplified fragments were analyzed in agarose
gels that contained ethidium bromide (0.5 µg/ml):
1.5% gels for the
cry1Fa
and
vip3Aa
genes and 1%
gels for the
chi
gene. The samples were compared to a
1-kb DNA ladder molecular marker (Fermentas,
Vilnius, Lithuania), then visualized under UV light
and photographed using GEL DOC 2000 (BioRad)
and Quantity-one software (Bio-Rad, New York,
USA).
3.4 Detection of polymorphisms
The restriction enzymes used for the PCR-RFLP
analyses were selected using the pDRAW32 program
(http://www.acaclone.com) based on the sequences of
the studied genes. Bromophenol blue (0.5%) in
glycerol (50%; 4 µl) was added to the digested
products. After electrophoresis using 1X TBE buffer
(89 mM Tris, 89 mM boric acid and 2.5 mM EDTA,
pH 8.2), the agarose gels (1%) were analyzed using
the GEL DOC 2000 documentation system (Bio-Rad).
The reactions using the
chi
primers were performed in