Bt Research 2012, Vol.3, No.4, 20
-
28
21
performed with another
Bti
strain, have shown that
Bti
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).
So, in order to find out which protein was responsible
for the high level of toxicity we investigated the action
of Cry10Aa towards
A. grandis,
since
Bti
strains also
have a
cry10A
gene
.
In the present study, we have introduced the
cry10Aa
gene isolated from the Brazilian S1804
Bti
strain, into
the genome of a baculovirus and analyzed the
recombinant protein expression in cultured insect
cells and insect larvae. We have shown that the
recombinant Cry10A protein might be responsible for
the toxicity of this
Bti
strain to
A. grandis
larvae, since
it was shown to be toxic to this insect and bound to A
.
grandis
BBMVs p.
1
Results and Analysis
1.1
Amplification, cloning and sequencing of a
cry10Aa
gene from
B. thuringiensis
subsp.
israelensis
S1804 strain
The
cry10Aa
gene of
B. thuringiensis
S1804 strain,
was amplified by PCR using Cry10Aa-specific
oligonucleotides, which were designed from the
published
cry10Aa
gene sequence (GenBank
accession number: M12662) (Thorne et al., 1986),
and cloned into the pGEM
®
-
T Easy vector (data not
shown), resulting in the recombinant plasmid
pGemcry10Aa. The DNA fragment was sequenced
and the sequence analysis revealed that the entire
sequence of the
cry10A
gene was present but an error
in the design of the reverse oligo removed one
nucleotide (T) before the stop codon of the gene. Blast
analysis showed that, besides the missing base at the
3-
end, the sequence is practically identical to the
cry10Aa
gene described by (Thorne et al., 1986)
(
Genebank accession number: M12662), with only
two nucleotide differences at positions +1779 and
+1885, respectively (change of A to G and C to G).
These two nucleotide changes generate two amino acid
changes in the Cry10Aa protein (T589A and T624S).
1.2
Construction of recombinant baculoviruses
The DNA fragment containing the
cry10Aa
without
a stop codon was removed from the pGemcry10Aa
plasmid and cloned into the transfer vector
psSynXIV+X3 generating the plasmid pSyncry10Aa
(
Figure 1). When cloned into this plasmid, a new stop
codon was generated after 152 base pairs downstream
of the gene which resulted in a new ORF of 723
amino acids (not shown). The pSyncry10Aa was then
used in a co-transfection of insect cells with the DNA
of a linearized occluded negative virus (occ-)
vSynVI
-
gal and the recombinant vSyncry10Aa was
purified. The vSyncry10Aa virus, besides having a
cry
gene, has the
AcMNPV
polyhedrin gene, which makes
easy the purification of the recombinant by the
presence of viral occlusion bodies (OBs) inside the
nucleus of infected cells. In order to confirm the
insertion of the heterologous gene into the recombinant
virus genome, PCR reactions were carried out with
cry-specific oligonucleotides (data not shown).
Figure 1 Diagram showing the different plasmids and viruses
used in this work
Note: A: Plasmid pSynXIVVI+X3 containing the polyhedrin
promoter (Ppol), a mutated promoter derived from polyhedrin
promoter (PXIV), a synthetic promoter (pSyn) and the
polyhedrin gene (
Pol
);
B: Plasmid pSyncry10Aa, showing the
cry10Aa
gene inserted at the
Eco
R
Ⅰ
site of the
pSynXIVVI+X3 plasmid; C: region of the vSynVI-gal virus
genome where heterologous genes are inserted after
homologous recombination with transfer vectors based on the
pSynXIVVI+X3 plasmid; D: Region of the recombinant
vSyncry10Aa virus genome where the
cry10Aa
gene was
inserted. The
cry10Aa
was cloned into the plasmid
pSynXIVVI+X3 generating the pSyncry10Aa plasmid that was
co-transfected with vSynVI-gal DNA into insect cells,
generating the recombinant virus vSyncry10Aa by homologous
recombination
1.3
Structural and ultra-structural analysis of insect
cells and insects infected with the recombinant viruses
Six-well plates (TPP, Techno Plastic Products AG,
Switzerland) were seeded with BTI-TN5B1-4 cells
