Bt Research 2012, Vol.3, No.4, 20
-
28
24
vSyncry10Aa-infected insect cells (data not shown).
Consistent with the transcriptional analysis we have
detected the Cry10Aa protein of vSyncry10Aa by
SDS-PAGE in vSyncry10Aa-infected insect cells
extracts in the very late phase of the infection.
Several
Bt
toxins have already been identified and
their toxic activity described for several insects.
However, new toxins are still being discovered, as
current diversity in pesticidal activity of crystal
proteins does not yet approach the diversity reported
for naturally occurring
Bt
isolates (Frankenhuyzen,
2009).
The recombinant Cry10Aa was able to bind
specifically to BBMVs from
A. grandis
larvae and
previous research have shown that Cry proteins, after
activation by proteases localized in the midgut of the
target insect, bind to specific receptors localized in the
apical microvilli of the midgut cells of susceptible
insects from Lepidoptera (Hofmann et al., 1988),
Coleoptera (Bravo et al., 1992) and Diptera (Hofte and
Whiteley, 1989) orders. Recently, (Martins et al., 2010)
showed the binding of an activated C ry1B toxin to
BBMVs from
A grandis
.
Two GPI-anchored proteins
with alkaline phosphatase (ALP) activity and around
65
kD and 62 kD, respectively were shown to bind the
Cry1B toxin.
The study of the Cry toxins interactions to receptors
present on BBMVs of different insect is an important
tool for the understanding of molecular basis of
toxin-receptor interaction and will be useful for the
development of new recombinant Cry toxins with
novel specificities and improved toxic activities,
contributing to the management of insect resistance in
the field and development of new plant cultivars
resistant to
A. grandis
.
In recent studies, some Cry
toxin receptors have been characterized (Pigott and
Ellar, 2007). The best characterized among them are
the APN receptor (Pigott and Ellar, 2007) and the
cadherin-like receptor (Gahan et al., 2001;
Likitvivatanavong et al., 2011; Pacheco et al., 2009;
Valaitis et al., 2001) identified in lepidopterans. Other
putative receptors include ALP (Fernández et al., 2006;
Jurat-Fuentes and Adang, 2004; Jurat-Fuentes and
Adang, 2006), a 270 kD glycoconjugate (Valaitis et al.,
2001),
and a 252 kD protein (Hossain et al., 2004).
There is no transgenic plant resistant to boll weevil so
far, although this Coleopteran insect-pest is
economically important in cotton crop in different
producer countries, being the most devastating cotton
insect pest in Brazil. The commercially available
Bt
transgenic cotton expresses Cry1Ac/Cry2Ab toxins
that confers mild resistance against
Spodoptera
frugiperda
(
Lepidoptera: Noctuidae) (Hamilton et al.,
2004),
and no resistance towards
A. grandis
.
In this
work a recombinant version of the Cry10Aa from
B.
thuringiensis
was shown to be highly toxic to the
cotton boll weevil and its gene a promising candidate
to be inserted into cotton plants for the control of this
devastating cotton pest.
3
Materials and Methods
3.1
Cells, viruses and bacteria
Trichoplusia ni
(
BTI-Tn5B1-4) cells were maintained
at 27
℃
in TC-100 medium supplemented with 10%
fetal bovine serum (Gibco-BRL) and served as host
for the wild type virus AcMNPV, the recombinant
viruses vSynVI
–
gal (Wang et al., 1991), and
vSyncry10Aa (constructed in this work).
Bacillus
thuringiensis
subsp.
israelensis
S1804 and IPS82
strains were obtained from the
Bacillus
subsp. Bank
of the Embrapa Recursos Genéticos Biotecnologia
(
Brasília, Brazil).
3.2
Recombinant virus construction
The
cry10Aa
gene of
B. thuringiensis
S1804 strain,
was amplified by PCR using the oligonucleotides F1
(5’-
GGGATCC
GGGAGGAATAGATATGAATC-3’)
and R1 (5’-ATAGTGAATGATTTATTTGTAA
GGAT
CC
TTTCC-3’), which were designed from the
published
cry10Aa
gene sequence (GenBank
accession number: M12662) (Thorne et al., 1986).
The PCR reaction consisted of 100 pg of total DNA
from
B. thuringiensis
S1804, 0.4 µmol/L of each
oligonucleotide primer, 10 µmol/L of each dNTP, 2.5
μL of
Taq
DNA polymerase buffer (10×), 2 mmol/L
MgCl
2
and 5 U of
Taq
DNA polymerase (Invitrogen)
in a total volume of 50 μL. Amplification steps were:
94
℃
/30
s, followed by 35 cycles at 95
℃
/30
s, 52
℃
/30
s, 68
℃
/4
min and a final extension of 68
℃
/8
min.
Bam
H
Ⅰ
restriction sites were introduced into the
oligonucleotides F1 and R1 (letters in bold). The
amplified fragment was cloned into the plasmid
pGEM-T
®
easy
(
Promega
®
)
following
the
manufacturer’s instructions and sequenced (MEGA
