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5
(1991). The molecular mass and integrity of the
proteins were determined by SDS-PAGE 10%
(Laemmli, 1970). The electrophoresis was realized in
Hoefer miniVE vertical electrophoresis system –
Amersham Pharmacia in voltage of 120 V three hours
and 30 minutes. The gel was stained and fixed in 40%
methanol, 10% acetic acid and Coomassie blue (0.1%)
for about 16 h, under slight shaking and it was
distained in 40% methanol and 10% acetic acid for 2 h,
with agitation. The HD-1 strain of
B. thuringiensis
subsp.
kurstaki
was used as control.
3.3.2 DNA sample preparation and PCR
The strains were grown on Embrapa agar medium
(Monnerat et al., 2007) for 14~16 h, at 30
℃
and
DNA was extracted as described by Bravo et al
.
(1998). Molecular characterization through PCR was
performed to identify the toxin-coding genes, by using
a variety of oligonucleotide pairs specific for the
following genes/gene families:
cry1, cry2, cry3, cry4,
cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry13,
cry14, cry17, cry19, cry21, cry24, cry25, cry27
,
cry29,
cry30, cry32, cry39, cry40, cyt1
and
cyt2
(Ceron et al.,
1994; Ceron et al
.,
1995; Ben-Dov et al., 1997; Bravo
et al., 1998; Ibarra et al., 2003). 15 µL of the
supernatant was used as DNA sample in the PCR
mixture containing 2.5 U
Taq
DNA polymerase
enzymes (5.0 U), 0.5 µM of each primer used in the
reactions and 0.2 mM of deoxynucleoside
triphosphates mix in a total volume of 50 µL. The
amplification reactions were carried out in PTC-100
(Programmable thermal Controller – MJ Research,
inc). The PCR products were visualized in 1% agarose
gel in tris-borate buffer at 150 V for 1 h and stained
with ethidium bromide (10 µg/mL).
3.3.3 Scanning and Transmission electron microscopy
In order to observe the spores and crystals produced
by the different strains in scanning electronic
microscopy, the strains were grown in sporulation
medium (Lereclus et al., 1995) at 28
℃
for 72 h.
Crystals were isolated by centrifugation in sucrose
gradients (Chang et al., 1993). These preparations
were washed and lyophilized before being deposited
on a metallic support. The samples were covered with
gold for 180 seconds, using a sputter coater
(EMITECH model K550) and observed in a ZEISS
model DSM 962 scanning electron microscope.
To observe the spores and crystals of
B. thuringiensis
by transmission electron microscopy
,
the culture was
centrifuged at 8000 rpm and fixed in 2.5%
glutaraldehyde and 0.1M sodium cacodylate buffer at
pH 7.0 for 4 h and maintained under agitation
overnight. Fixed bacteria were washed three times for
10 minutes with cacodylate buffer and twice with
water. The bacteria were post-fixed in 2% osmium
tetroxide and 0.2 M cacodylate buffer for 1h in dark
conditions. After washed with cacodylate buffer for
three times and twice with water for 10 minutes, the
pellets were dehydrated in a 10 to 100% ethanol series
for 20 minutes for each step and twice in pure ethanol
for completely dehydration. The bacteria were
embedded in Epon resin at 4
℃
under agitation and
incubated at 70
℃
. The ultrathin sections (60 µm)
were stained in 2% uranyl acetate at dark conditions
for 1 hour. The ultrathin sections were washed in
water and observed in a JEOL1011C Transmission
Electron Microscope. Between changes of solution,
the samples were centrifuged at 8000 rpm for few
seconds to form the pellets.
4 Conclusion
The strains S1905, S2122 and S2124 were toxic to
P.
xylostella
and present different genes. These strains
are important to the development of new
bioinsecticides that can be used in the control of
P.
xylostella
and the management of insect resistance in
the field.
Authors’ contribution
LP conducted all the research for this paper and prepared the
manuscript. CC participated in bioassays and in biochemistry
and molecular characterization. AG conducted the scanning and
transmission electron microscopy. RM reviewed the manuscript
and coordinated the project.
Acknowledgement
This work was financially supported by FAP-DF and Embrapa.
References
Ben-Dov E., Zaritsky A., Dahan E., Barak Z., Sinal R.,
Manasherob R., Khamraev A., Troitskaya E., Dubitsky A.,
Berezina N., and Margalith Y., 1997, Extended screening
by PCR for seven cry-group genes from field collected
strains of
Bacillus thuringiensis
, Applied and
Environmental Microbiology, 63(12): 4883-4890 PMid:
9406409 PMCid:168816
Bravo A., Sarabia S., Lopez L., Ontiveros H., Abarca C., Ortiz
A., Ortiz M., Lina L., Villa-lobos F.J., Guadalupe P.,