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Bt Research 2015, Vol.6 No.1 1-8
ISSN 1925-1939
http://bt.biopublisher.ca
2
genes for controlling the development of resistance to
Cry proteins.
Other proteins that have been investigated for their
potential use in the control of insects include
chitinases (Chi) (Arora et al., 2003; Ding et al., 2008),
which are also present in
B. thuringiensis
isolates.
Studies have demonstrated that chitinase hydrolyzes
the peritrophic membrane chitin in the insect gut,
which causes the formation of pores and facilitates the
binding of delta-endotoxins to their receptors located
in the gut epithelium, thus increasing the toxicity of
B.
thuringiensis
.
The objectives of this work were to select new
B.
thuringiensis
isolates and to verify the existence of
synergistic effects among the
cry1Fa
,
vip3Aa,
and
chi
genes in the control of
S. frugiperda
larvae. The gene
vip3Aa
appeared to act synergistically with
chi
and the
combination of all three genes increased mortality of
S.
frugiperda
. These results suggest potential
applications for biological control as well as
construction of pyramidized plants. Additionally, the
different modes of action of these genes could be used
to increase toxicity and to reduce the likelihood of the
development of resistant insect populations.
1 Results
1.1 Detection of the
cry1Fa
,
vip3Aa
, and
chi
genes
The presence of the
cry1Fa
,
vip3Aa
, and
chi
genes in
the isolates was confirmed via DNA amplification
using pairs of primers specific for the genes (Table 1).
It was verified that the isolates produced an
amplification product of the expected size (553 bp) for
the
cry1Fa
gene, as did the positive control,
B.
thuringiensis
var.
aizawai
HD137. For the
vip3Aa
gene, the expected 2439-bp product was obtained
from the isolates and from the positive control,
B.
thuringiensis
var. HD125. Finally, a
chi
gene product
with the expected size of 2031 bp was obtained from
the isolates and from the positive control,
B.
thuringiensis
var.
alesti
(Figure 1). Conversely, no
amplicons were obtained from
B. thuringiensis
var.
tenebrionis
(the negative control), a Coleoptera
-specific strain that does not carry the three studied
genes.
The PCR analysis revealed that among the 114 isolates,
Table 1. Specific oligonucleotide primers for the
cry1Fa, vip3A
,
and
chi
genes
Primers
Nucleotide sequence (5’-3’)
Fragment
size (bp)
vip3Aa
(a)
ATGACCAAGAATAATACTAAATTAAGC (f)
2370
GATCTTACTTAATAGAGACAT (r)
chi
(b)
ATGGTCATGAGGTCTC (f)
2031
CTATTTCGCTAATGACG (r)
cry1Fa
(c)
AATGTAGAGCCGTTTGTTAGTG (f)
553
CCCTCAAGTTATTTAGACCTG (r)
(f): forward; (r): reverse
(a)
LOGUERCIO et al., 2002;
(b)
LIN & XIONG, 2004;
(c)
LGBBA.
Figure 1. Agarose gel electrophoresis of the gene amplification
products: (A)
cry1Fa,
(B)
vip3Aa
and
(C)
chi
, from samples
isolated from
Bacillus thuringiensis;
M: 1 kb DNA ladder
molecular size marker; PC: positive control.
all three genes (
cry1Fa
+
vip3Aa
+
chi
) were
amplified in only 12 (10.53%); 79 isolates (69.30%)
amplified the
cry1Fa
gene only; 26 (22.81%)
amplified
vip3Aa
only; 54 (47.37%) amplified the
chi
gene only; 5 (4.38%) amplified the combination of