Page 10 - ME-436-v3-3

Bt Research (Online) 2010, Vol.1 No.2
http://bt.sophiapublisher.com
- 8 -
EDTA (pH 8)]. Lysozyme was added to the
suspension at a final concentration of 10 mg/mL
and incubated 1 hour at 37℃ for enzymatic lysis of
cell walls. Lysozyme (1.0% SDS; 0.8 mol/L NaOH)
was added at 2 × volume and mixed gently by
inverting the suspension 6 to 8 times. A half volume
of 3 mol/L sodium acetate was added and incubated
on ice bath for more than 4 h. After centrifugation at
4℃ at 12,000 g for 15 min, the supern-atant was
extracted with an equal volume of phenol-
chloroform-isoamyl alcohol (25:24:1) and then the
plasmid DNA was precipitated from the aqueous
phase with 2 volumes of cold 96% ethanol.
CHEF Mapper® XA Pulsed Field Electrophoresis
System (Bio-Rad, USA)was employed to detect the
plasmid profiles of Bt W015-1 in accordance with
the manufacturer’s instructions. Parameters were set
for automatic separation of a plasmid DNA with
size range of 15 kb to 500 kb (voltage: 6 V/cm,
running time: 20 hours, included angle: 120°, initial
switch interval time: 1.19 s, final switch interval time:
44.69 s). The plasmid DNA was subjected to
electrophoresis in 1% low-melting-point agarose
(Amresco, USA) at 14℃ with 0.5× TBE buffer
(45 mmol/L Tris•Cl, 1 mmol/L EDTA). The plasmids
DNA was then stained in ethidium bromide (5
µg/mL) after electrophoresis for 30 min, followed
by destaining in double-distilled water (at 4℃) for 2
hours. Water was changed three times in the course
of decoloring and plasmids were then observed
under ultraviolet light.
2.6 SDS-PAGE analysis of Bt strains
Bt W015- 1 was inoculated in 5 mL of liquid LB
medium and grown overnight at 30℃ with shaking
at 200 r/min, at 12 hours post inoculation, the
mixture was transferred into Eppendorf tubes with
200 mL BP medium by 1% volume ratio to continue
culturing at 30℃ while shaking at 200 r/min. A
1 mL spore-crystal mixture was harvested every two
hours SDS-PAGE analysis after centrifugating at 4
℃ at 12,000 g for 5 min and re-suspended the
pellet was in 100 µL sterilized water after removing
the supernatant. After 25 µL NaOH (0.5 mol/L) was
added, samples were placed at room temperature for
10 min and 63 µL of a 3×loading sample buffer
(3.63 g Tris, 0.3 g bromophenol blue, 6 g SDS, 30 mL
glycerol, 15 mL b-mercaptoethanol were dissolved
in 100 mL double-distilled water and pH 6.8) was
added to the suspension. The suspension was boiled
at 100 ℃ for 5 min and then centrifugated at
12,000 g for 5 min. The supernatant was loaded
onto 7.5% gel immediately for SDS-PAGE analysis.
Laemmli’s electrophoresis procedures were followed in
this study (1970).
2.7 Identification of cry genotype by PCR-RFLP
DNA templates for PCR were prepared following
Yu’s (2006) method. Thirty four primer pairs were
used to identify the cry gene (Xie et al., 2009),
including the primer pairs of K5un2/K3un2 and
K5un3/K3un3 for screening the Bt strain cry-type
genes (Table 2) (Kuo and Chak, 1996). 50 μL of
PCR reaction volume sample contains 5 μL 10×
PCR buffer (promega, USA), 0.5 μL (4 U/μL) Taq
polymerase (promega, USA), 1 μL dNTP mixture
(to 250 μmmol/L final concentration), 1 μL each
primer, 1 μL (50~100 ng) template DNA, after
adding 40.5 μL ddH 2O. The PCR was performed in
a PTC -200 Thermo Cycler (MJ Research, USA)
with the procedures as follows: pre-denaturation at
94℃ for 5 min, followed by 30 cycles (94℃ 1 min,
53℃ 1min and 72℃ 3 min), and finished at 72℃
for 10 min. PCR products were examined by 1%
agarose gel electrophoresis and purified using the
TIANgen Midi Purification Kit (Tiangen, Beijing,
China). PCR-RFLP analysis followed the procedures
of Kuo and Chak (1996a). Table 3 shows the
PCR-RFLP banding patterns of cry1Ac1revealed by
using 2% agarose gel electrophoresis (Kuo and
Chak, 1996).
Table 3 Typical banding pattern of cry1Ac1 by PCR-RFLP
analysis
Primer pairs Sizes of PCR
productions (bp)
The size of restri-
cted fragments
K5un2/K3un2 1,600 322,801,518a
K5un3/K3un3 1,450 726,434,244,59b
Note: PCR amplicon was digested with restricted enzymes;
a: PstI/XbaI; b: PstI/EcoRI
2.8 Cloning of cry type gene
The PCR products amplified by primer pairs
K5un2/K3un2 and K5un3/K3un3 were ligated into
the Easy Vector pMD18T to construt recombinant