Bt Research 2012, Vol.3, No.4, 20
-
28
20
Research Report Open Access
Cry10Aa Protein is Highly Toxic to
Anthonomus grandis
Boheman (Coleoptera:
Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields
Raimundo Wagner de Souza Aguiar
1
,
Érica Soares Martins
2,4
,
Bergmann Morais Ribeiro
3
,
Rose Gomes
Monnerat
4
1.
Fundação Universidade Federal do Tocantins, Campus Universitário de Gurupi, Gurupi, TO, Brazil;
2.
Faculdades Promove de Brasília, Campus Águas Claras, Brasília, DF;
3.
Universidade de Brasília, Departamento de Biologia Celular, Campus Universitário, Asa Norte, Brasília, DF, Brazil;
4.
Embrapa Recursos Genéticos e Biotecnologia, Controle biológico, Asa Norte, Brasília 70770-900, Brazil
Corresponding author email:
rose.monnerat@cenargen.embrapa.br;
Authors
Bt Research, 2012, Vol.3, No.4 doi: 10.5376/bt.2012.03.0004
Received: 22 May, 2012
Accepted: 19 Jul., 2012
Published: 20 Jul., 2012
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Preferred citation for this article:
de Souza Aguiar et al., 2012, Cry10Aa Protein is Highly Toxic to Anthonomus grandis Boheman (Coleoptera: Curculionidae), an Important Insect Pest in
Brazilian Cotton Crop Fields, Bt Research, Vol.3, No.4 20-28 (doi: 10.5376/bt.2012.03.0004)
Abstract
The
cry10Aa
gene from the Brazilian
Bacillus thuringiensis
subsp
israelensis
S1804 strain was introduced into the
genome of the baculovirus
Autographa californica multiple nucleopolyhedrovirus
(
AcMNPV) in order to evaluate its expression in
insect cells and its toxicity to
A. grandis.
The recombinant virus (vSyncry10Aa) was amplified in
Trichoplusia ni
(
BTI-Tn5B1-4)
cells and used to infect
Spodoptera frugiperda
larvae. Total extracts from
S. frugiperda
infected with the recombinant viruses were
analyzed by SDS-PAGE and showed the presence of a polypeptide around 85 kD. Cuboid-shaped protein crystals were observed in
insect extracts by light and scanning electron microscopy. Bioassays, using the recombinant virus infected-insect extracts, showed
high toxicity to
A. grandis
larvae, with a LC
50
of 7.12 µg/mL. Furthermore, a competition binding assay with the recombinant
biotin-labeled Cry10A protein and brush border membrane vesicles (BBMV) from
A. grandis
indicated that the toxin binds
specifically to BBMVs. Therefore, the Cry10A protein has a potential to be used in transgenic cotton plants for the control of this
important insect pest.
Keywords
Bacillus thuringiensis
;
Toxicity; Recombinant protein
Introduction
Bacillus thuringiensis
(
Bt
)
is a naturally occurring,
Gram positive, spore forming soil bacterium
.
The
entomocidal properties of
Bt
are mediated by Cry
proteins (δ endotoxins), which form parasporal crystal
inclusions during the bacterial stationary growth
phase (Monnerat and Bravo, 2000; Schnepf et al.,
1998).
These crystal inclusions are produced by one
or more insecticidal proteins, which can exhibit
toxicity and specificity toward a select group of
Lepidopteran, Coleopteran and Dipteran insect species
(
Schnepf et al., 1998). Different Cry
toxins showed
great potential for the control of several economically
devastating insect-pests when bioengineered into crop
plants (Betz et al., 2000; Chattopadhyay et al., 2004).
To date, many plant species have been genetically
modified with
cry
genes, resulting in transgenic plants
with high level of resistance to insect pests (Hilder
and Boulter, 1999; Christou et al., 2006; van Rie, 2000)
and a viable alternative to chemical control.
Cotton is one of the most important crops cultivated
worldwide. The cotton productive chain is one of the
most important in Brazil and in the world, as it
generates thousands of direct and indirect jobs and
annually only the Brazilian cotton industry generates
around US $ 1.5 billion (Martins et al., 2007). The
cotton boll weevil,
Anthonomus grandis
(
Coleoptera:
Curculionidae), is a devastating cotton pest responsible
for more than 50% of insecticide costs in Brazilian
cotton crop fields. Moreover,
A. grandis
larvae resides
inside floral buds and results in the destruction of fiber
quality and hampers chemical control, causing
considerable yield losses (Martins et al., 2007).
However, due to
A. grandis
economic importance,
the search for Cry toxins specific to this species is as
essential as the knowledge on the toxic processes involved.
Bacillus thuringiensis
subsp.
israelensis
(
Bti
)
S1804, is
a Brazilian soil strain serotype H-14, toxic to
Aedes
aegypti
(
Diptera: Culicidae). Previous work,
