Bt Research 2012, Vol.3, No.4, 20
-
28
23
1.6
Bioassays
The heterologous Cry10Aa proteins present in the
recombinant virus infected-insect extracts were shown
to be highly toxic to neonate
A. grandis
larvae with a
LC
50
of 7.12 µg/mL (Table 1). No toxic activity was
detected for second instar
A. gemmatalis
and
S.
frugiperda
larvae when incubated with the
heterologous Cry10Aa protein (data not shown).
Bioassays performed with IPS82 and S1804 strains
of
B. thuringiensis israelensiss
showed LC
50
of 740
and 300 µg/mL, respectively (Table 1).
Table 1 LC
50
values of recombinant Cry10Aa protein against
Anthonomus grandis
Sample
N
Slope (± SE) LC
50
(
FL) (μg/mL)
a
Cry10Aa protein 25 1.64 (± 0.26) 7.12 (5.27-9.8)
IPS82
Bt
strain
25 2.64 (
± 0.33) 740 (610-910)
S1804
Bt
stain
25 3.23 (
± 0.45) 300 (250-360)
Note: The results of three different bioassays with their
respective mean LC
50
values are shown. N: number of insects
used;
Bt
:
Bacillus thuringiensis
;
a
LC
50
(
LC: letal concentracion
and FL: Finducial Limites) calculated by Probit analysis
2
Discussion
The expression of
cry
genes in insect cells using
baculovirus expression vectors (BEVs) is an
alternative method for the study of single Cry proteins.
Some Cry proteins have already been expressed in
insect cells using BEVs , such as Cry1I Cry1C, Cry2A
(
Aguiar et al., 2006; Martens et al.. 1990; Martins et al.,
2008;
Lima et al., 2008), showing that the recombinant
proteins were biologically similar to their native
counterparts, with toxicity towards different insects.
Previous studies have shown that the
B. thuringiensis
subsp. israelensis
strain, was also toxic to
A. grandis
and this toxicity was not due to Cry4A
,
Cry4B
,
Cry11
and
Cyt
proteins
individually or their combination
(
Martins et al., 2007)
.
Since
Bti
strains also have a
cry10A
gene, we wanted to test the hyphotesis that the
toxicity of this strain towards
A. grandis
was in part
due to the Cry10A protein.
Currently various Coleopteran insects were shown to
be susceptible to the
B. thuringiens
Cry proteins, such
as:
Chrysomela scripta
(
Coleoptera: Chrysomelidae)
and
A. grandis
(
Martins et al., 2007; Martins et al.
2005;
Tailor et al., 1992)
,
Epilachna varivestis
(
Coleoptera: Chrysomelidae) (Tamez-Guerra et al.,
1999),
Hypothenemus
hampei
(
Coleoptera:
Scolytidae) (Arrieta et al., 2004; Bradley et al., 1995),
Xonthogaleruca luteola
(
Coleoptera: Chrysomellidae)
(
Arbab et al., 2001). Moreover,
A. grandis
have different
susceptibility to Cry proteins. For example, Cry1Ia
have shown a LC
50
of 21.5 µg/mL to
A. grandis
neonate
larvae (Martins et al., 2007), while, the Cry1Ba
a LC
50
of 305.32 µg/mL (Martins et al., 2010).
The recombinant Cry10Aa is equally or even more
toxic to
A. grandis
than the recombinant Cry1Ia and
Cry1Ba proteins also
expressed in insect cells, with a
LC
50
of 7.12 µg/mL (Martins et al., 2008).
Furthermore, the recombinant Cry10Aa protein had
the capacity of crystallization into cuboidal crystals
in insect cells. Since the
cry10Aa
gene is under
transcriptional control of two promoters in tandem,
pSyn and pXIV (Wang et al., 1991), the high
expression level of the Cry10Aa protein may have
contributed to the crystallization of the protein.
SDS-PAGE of purified crystals produced by
vSyncry10Aa recombinant viruses showed the
presence of a protein of molecular mass of
approximately 85 kD (Figure 3).
This difference in shape from the spherical form,
present in S1804 strain to a cuboidal form in insect
cells, probably could be associated with the presence
and association of others proteins during the formation
and assembly of the crystal or the presence of the
extra amino acids present in the C-terminal of the
recombinant protein. Cry proteins have been
expressed in high amounts in insect cells and some
formed larger crystals than when expressed in
Bt
,
suggesting that the size of the crystal produced in
Bt
is
limited by the size of the bacteria (Aguiar et al., 2006;
Ribeiro and Crook, 1993). The formation of crystals
by Cry proteins in insect cells infected with
recombinant baculoviruses has been reported for the
Cry1Ab, Cry1Ac, Cry11Aa, Cry1Ac, Cry1I, and
Cry1Ca proteins (Aguiar et al., 2006; Lima, 2008; Lu
and Miller, 1997; Martins et al., 2008; Ribeiro and
Crook, 1993).
The transcription analysis of the
cry10Aa
gene by
RT-PCR demonstrated that a
cry10Aa
gene transcript
was detected at 96 h.p.i. indicating that transcript was
present during the late phase of infection in
