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Bt Research (Online) 2010, Vol.1 No.2
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- 9 -
plasimds pMDK2 and pMDK3, then sequenced by
Automatic DNA Sequencer (ABI
-
3730XL, Huda,
Beijing). According to the sequences, primer pairs
of cry1I5/cry1I3 were designed for the inverted
PCR amplification. Plasmid DNA of
Bt
W015
-
1
(1.0 to 2.0 μg) was digested completely by
Ned
I,
Sal
Ⅰ
,
Bgl
Ⅱ
and
Bam
H
Ⅰ
in a 37
℃
water bath for 3
hours, respectively, prior to inactivating with
restrictive endonuclease in a 65
℃
water bath for 15
min, T4 ligase was then added to connect the
digested fragments at 4
℃
for overnight (over 12
hours). The inverted PCR was performed using
connected products as the template and the inverted
PCR products were inserted into the pMD18T
vector to construct a recombinant plasmid named
pMDIS for sequencing. Finally, three sequences were
assembled from the recombineants of pMDK2,
pMDK3 and pMDIS to make the full length gene
sequence (Triglia et al., 1988).
2.9 Expression of
cry1Ac22
and bioassay
The primer pair E1A5/E1A3, was designed based
on the full sequence of
cry1Ac22
, inwhich the
Bam
H
I and
Sal
I restrictive endonuclease sites were
introduced into the 5’ end of the forward and reverse
primers, respectively. The sequence confirmed gene
was ligated into the prokaryotic expressing vector
pEQ30 to make the recombinant vector pEQ30
-
cry1Ac22. The recombinant plasmid was further
transformed into
Escherichia coli
M15 and then
incubated in TB medium adding 12.5 µg/mL
Kanamycin at 37
℃
for about 1.5 h until the value
of OD
600
reached about 0.5 IPTG was added to the
mixture at the final concentration of 1 µmol/L for
inducing Cry1A22 for 4~10 hours at 30
℃
. The
expression cells were collected by centrifugation at
12,000 g at 4
℃
, for 10 min and completely
suspended in an equal volume of TE. Lysozyme
was then added to digest at the final concentration
of 20 mg/mL and incubated at 37
℃
while shaking
at 200 r/min for 30 min. The cells and proteins were
re-collected by centrifugation, the pellet was
washed with an equal volume of 1 mol/L NaCl
three times and then broken up by ultrasonic
treatment (Model VC
-
130, Sonics and Materials Inc,
USA) for 20 min. The concentration of expressed
Cry1Ac22 protein was measured by the Lowry
assay with a standard marker protein of bovine
serum albumin (BSA) (Lowry et al., 1951). The
expressed proteins were disolved in 50 mmol/L
Na
2
CO
3
(pH 10.0) with 1mol/L typsin added to digest
during incubation at 37°C for 1 hour. SDS-PAGE
procedures were the same as that mentioned above.
To bioassay, 8 cm diameter disks of Chinese
cabbage leaves were immersed in different
concentrations of typsin treated proteins, containing
0.02% Triton X
-
100, for 10 sec and air dried
naturally at room temperature for 2 hours. Each leaf
disks was placed in an individual Petri dish (10 cm
diameter) lined with moistened filter paper and 10
second-instar larvae of
Plutella xylostella
were
introduced into each Petri dish. The experiment was
replicated six times and sterilized water were used
for reference and blank. Larvae assays were
performed at 26
℃
and 65% relative humidity for
96 hrs, with a photoperiod of 14 hrs light/10 hrs
dark. The LC
50
with the 95% confidence intervals
were estimated by SPSS software for windows
(SPSS Inc., Chicago, USA) (Sayyed et al., 2001).
3 Conclusions
In this paper, we isolated and characterized
Bacillus thuringiensis
W015
-
1, from the diapausing
silkworm larvae. It showed high insecticidal activity
against the lepidopteran,
P. xylostella
.
Bt
W015
-
1
synthesizes a bipyramidal crystal with a molecular
weight of 130 kD during sporulation. The plasmid
profiles of
Bt
W015
-
1 are similarity to to the
reference HD73 whereas there were dissimilarrities
in size and number to HD1 and
Bt
i. The cry genotype of
W015
-
1 has obvious differences in the restricted
enzyme sites with that of model strain HD73.
We cloned the
cry1Ac22
gene that has a length
of 3,537 bps encoding 1,178 amino acid residues.
Cry1Ac22 has high amino acid sequence identity to
Cry1Ac1 with the differences existing in the sites of
233 (T/R), 448 (M/I) and 1158 (K/E). The Cry1Ac22
inclusion protein, with a molecular weight of 133 kD,
was expressed in
E. coli
induced by ITPG. The
trypsin-activated form of the recombinant protein
was found to have high insecticidal activity against
larvae of
Plutella xylostella
compared to that of
model strain HD 73.
With respect to the reference strains W015
-
1 has