Basic HTML Version
Bt Research (Online) 2010, Vol.1 No.2
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- 4 -
Figure 1 Scanning electron micrograph of the spores and
crystal proteins from
Bt
W015
-
1 (6.6 mm×15 k)
Note: A,B,C are spore, paraspore and bacterial cell,
respectively
Figure 2 SDS-PAGE profiles of parasporal inclusion proteins
of
Bt
W015
-
1
Note: M: Protein molecular weight marker; Lane 1~9:
Bt
W015 parasporal inclusion protein grown after 4 h, 16 h, 18 h,
20 h, 22 h, 26 h, 30 h, 34 h and 48 h
large plasmids (Carlson et al., 1996). Therefore,
plasmid size and number are usually considered as a
tools to identify the strain characteristics (Procar et
al., 1999; Vilas-Boas et al., 2004). In this study, the
Pulsed-Field Gel Electrophoresis (PFGE) was emp-
loyed to compare plasmid profiles of
Bt
W015
-
1
and the reference strains
Bti
AND508, HD1 and
HD73. The plasmid profiles of
Bt
W015
-
1, in size
and number are the same as HD71, but significantly
different from
Bti
AND508 and HD1 (Figure 3).
1.4 Identification of cry-type toxin genes
The polymerase chain reaction-fragment length
polymorphism (PCR-RFLP) was used to identify
the cry genotype of W015
-
1 (All tested universal
primers not listed in this paper). The primer pairs of
K5un2/K3un2 and K5un3/K3un3 produced PCR
fragments in size of 1.6 kb and 1.4 kb, respectively
(Figure 4A). Both PCR amplicons were digested
with
Pst
Ⅰ
and
Xba
I,
Pst
Ⅰ
and
Eco
R
Ⅰ
, respectively
(Figure 4B). The 1.6 kb fragments digested by
Pst
Ⅰ
and
Xba
I are 820 bp, 550 bp and 320 bp in size
which are obviously larger than those of expecting
sizes (801 bp, 518 bp and 322 bp). Similarly, The 1.4 kb
fragments digested by
Pst
Ⅰ
and
Eco
R
Ⅰ
are 800 bp,
470 bp and 280 bp in size which are a little larger
than those of expecting sizes (726 bp, 434 bp, 244 bp
and 59 bp), it is clear that 59 bp digested fragment
Figure 3 PFGE banding patterns of large plasmids from
Bt
strain W015
-
1
Note: Lane1:
Bt
subsp
israelensis
AND508, haboring the
large plasmids of pXO16 (350 kb) and pBtoxis (128 kb),
used as a size marker; Lane 2:
Bt
subsp
kurstaki
HD
-
1; Lane
3:
B. thuringiensis
subsp
kurstaki
HD
-
73; Lane 4:
Bt
W015
-
1
Figure 4 Identification of cry1A genotype of
Bt
strain
W015
-
1 and HD73 based on PCR-RFLP approach
Note: A: Agrose gel electrophoreisis of PCR amplication;
1,2 are PCR products amplified from HD73 by primer
pairs K5un2/K3un2 and K5un3/K3un3, respectively; 3,4 are
PCR products from W015
-
1 by primer pairs K5un2/K3un2
and K5un3/K3un3, respectively; B: RFLP anlysis of the
PCR fragment, 1,2 are PCR fragments by primer pairs
K5un3/K3un3 and K5un2/K3un2digested with
Pst
I/
Eco
RI
and
Pst
I/
Xba
I restricted enzymes