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Bt Research (Online) 2010, Vol.1 No.2
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three-dimensional structure was identified by
X-Ray analysis, is highly similar to
cry1Aa
and
cry3Aa
(
http://swissmodel.expasy.org/workspace/
index.php?func=modelling_simple1
).
1.6 Expression of
cry1Ac22
gene and bioassay analysis
The
cry1Ac22
was ligated into pQE30 to construct
recombinant plasmid pQE30
-
cry1Ac22
. The express-
ion of
cry1Ac22
was induced by IPTG in the strain
E. coli
M15. SDS-PAGE analysis indicated that the
band of 133 kD inclusion protein was present in the
whole cell lysate with IPTG induction and no band
was present without IPTG induction (Figure 8). The
inclusion of
cry1Ac22
can be hydrolysed to a
trypsin-activated form with a molecular weight of
about 80 kD with the 1 µmol/L trypsin treatment
(Figure 8). Bioassay of a trypsin-activated form of
cry1Ac22
was carried out and the results showed that
expressed
cry1Ac22
protein exhibited high toxicity to
second instar larvae of
Plutella xylostella
(LC
50
:
4.135×10
8
cfu/mL; 95%FL: 3.368~5.122×10
8
cfu/mL).
Figure 7 Predicted 3
-
D crystal structure of Cry1Ac22 protein
Figure 8 SDS-PAGE analysis of
cry1Ac22
gene expressed in
Escherichia coli
M15 cells
Note: M: Protein molecular weight marker; Lane 1:
E. coli
M15 cells harboring pQE30
-
cry1Ac22
plasmids incubated
without IPTG induction; Lane 2:
E. coli
M15 cells harboring
pQE30
-
cry1Ac22
plasmids induced by IPTG; Lane3: 80 kD
trypin-active peptides hydrolysed from expressed inclusions
of Cry1Ac22 by 1 µmol/L trypsin treatment
2 Methods and Materials
2.1 Collection and anatomy of Silkworm
One hundred diseased and dead silkworm larvae
with brown or black diapause symptoms were
collected from farms in the Hangjiahu region of
Zhejiang Province and placed in individual 15 mL
tubes. The larvae were dissected to obtain the
midgut tissue and slime for isolating bacteria.
2.2 Isolation of
Bacillus
and
Bacillus thuringiensis
Bacillus
isolates were obtained with the use of high
temperature sodium acetate according to Xie et al.
(2009) and Hossain et al. (1997). The midgut tissue
and slime were completely dissolved in 20 mL BPA
medium incubated for 4~5 hours at 30
℃
with
shaking at 220 r/min and then moved to a water
bath at 75
℃
for 15 min, 1 mL of dissolved solution
and was proportionally diluted in 9 mL sterilized
water to a final dilution ratio of 10
-
2
and 10
-
3
for
plating. The isolates were grown in NB solid
medium plate for three days (beef extract 5 g,
peptone 10 g, NaCl 34 g, distilled water 1,000 mL,
pH value 7.0~7.4, 1.5% (w/v) agar, at 121
℃
for 20
min autoclave sterilization). Single colonies were
re-plated and then used to observe the morphology
and parasporal crystal through oil lens optical
microscopey and scanning electronic microscopey.
The isolates were stored in 50% glycerin solution at
low temperature refrigeration.
2.3 Strains, plasmids and growth conditions
The strains and plasmids used in this study are
listed in Table 2.
B. thuringiensis
W015
-
1 was
isolated from the diapausing silkworm larvae.
B.
thuringiensis
subsp.
kurstaki
and
B. thuringiensis
subsp.
israelensis
were used as reference strains.
pQE30 and pMD 18T were the vectors for cloning
and expression of cry gene.
Bt
strains were grown in
BP medium (Lecadet et al., 1980) and G-Tris
medium (Aronson and Thompson, 1971) at 30 .
℃
All
E. coli
strains were grown at 37 in
℃
Luria-Bertani
(LB) medium and Terrific Broth (TB) medium (12 g
Bacto-tryptone, 24 g yeast extract, 4 mL glycerol
ddH
2
O to 900 mL). All culture medium was
autoclaved at 121 for 20
℃
min before using. For
solid media 1% agar was added. Ampicillin
(100 μg/mL) or Kanamycin (12.5 μg/mL) were
added to culture media as required.