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Bt Research (Online) 2010, Vol.1 No.2
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most devastating pests of forests, crops, and stored
products as well as some insects important to
humans, such as the silkworm,
Bombyx mori
. The
silkworm is a lepidopteran of economic value
highly domesticated by man (Kristensen et al., 2007;
Khajuria et al., 2009).
Sericulture has several thousand years of history in
China. Asericulturist always faces the challenge of
rearing silkworms free of bacterial infection which
results in growth retardation and death. The infected
larvae cause a severe symptom of diapause. The
diapausing larvae of silkworm mostly are typically
infected by the common soil bacterial called
Bacillus thuringiensis
(Ohba, 1996). The annual
economic losses caused by
Bacillus thuringiensis
infection in the Hangjiahu regions of Zhejiang
Province, known as the home of silk in China are
incalculable.
The spore-forming bacterium
Bacillus thuringiensis
synthesizes parasporal crystal toxin during sporulation.
Bt
toxin usually works in the insect midgut, where
Bt
protoxins are activated by gut protease to produce
activated toxins, which bind to specific receptors to
confer toxicity. Large numbers of studies show that
the reactions between
Bt
toxins and insect gut are
determined by many gene products expressed in the
insect gut. These include many proteins and
enzymes involved in
Bt
protoxin activities, toxins
bound to receptors and toxin degradation products.
These results imply that there are interacting systems
for
Bt
toxin functions existing in the insect midgut.
Changes in these systems might cause particular
Bt
toxin specificity and efficacy, and could affect
Bt
toxic lethal action to a variety of insects (Crickmore
et al., 1998; Knowles, 1994).
Silkworm was the first lepidopteran to have its
complete genome sequenced and has become the
model insect species for Lepidoptera research
(Mita1 et al., 2004). Mining highly toxic Bt strains
from the midgut of diseased silkworm larvae and
identifying the
Bt
toxin functional gene has
significant importance for studying insect resistance
to
Bt
toxins.
In this study, we collected 100 diapausing silkworm
larvaefrom different farms in the Hangjiahu region in
Zhejiang Province. Sodium acetate & temperature
separation was used to isolate
Bacillus
isolates from
the midgut tissue and slime of collected samples.
The
Bacillus thuringiensis
strains were further
characterized by using staining, crystal shape
observation, SDS-PAGE, plasmid profile and
bioassay to make clear the genotype and
Bt
toxin
genes.
1 Results
1.1 Isolation of
Bacillus
strains and
Bacillus
thuringiensis
isolates
We dissected 100 diapausing silkworm larvae
collected from Hangjiahu of Zhejiang Province and
used midgut tissue and slime to isolate the
Bacillus
strains using sodium acetate & temperature separation.
From 218 bacillus strains were harvested, six
isolates were further identified to be
Bacillus
thuringiensis
based on parasporal crystal formation
observed under the oil lens optical microscope and
scanning electron microscope. Larvicidal assays
were carried out using crude proteins and the results
indicated that
Bt
strains W015
-
1 was highly toxic to
the lepidopteran
Plutella xylostella
of lepidopteran
insects (data not shown). It was deposited in the
HITAR
Bacillus
Collections with Accession No.
20050509W015.
1.2 Parasporal inclusion morphology of W015
-
1
isolate
Strain W015
-
1 was grown in BP solid medium at 30
℃
for 3 days until the parasporal crystal were observaed
through oil lens light microscopy, and then the
crystals were examined by scanning electron
microscopy (SEM). It was clear that the parasporal
crystals from
Bt
W015
-
1 were typically bipyramidal in
shape (Figure 1). SDS-PAGE analysis indicated that
the intact parasporal crystal of W015
-
1 has a
dominant polypeptide of about 130 kD after grown
for 20 hours during the sporulation stage (Figure 2).
The crystal proteins of W015
-
1 were processed into
about 80 kD fragments with 1 µmol/L trypsin
treatment (Figure not shown).
1.3 Plasmid profiles of W015
-
1
Bacillus thuringiensis
commonly harbors a varied
number of large plasmids with different molecular
mass. Most of the
cry
genes are located on these