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Bt Research (Online) 2010, Vol.1 No.3
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management of agricultural pests.
Figuring out the expression and localization of
Cry1Ac22 in Eukaryotic cell is of great significance
to the study regarding the expression of
cry1Ac22
in
higher plant. In this research, we constructed
GFP-labeled Cry1Ac22 fusion protein by applying
the GFP of
Aequrea victoria
as reporter molecules,
and investigated preliminarily the expression and
localization distribution of the fusion protein in
yeast, expecting to provide theoretical guidance for
the research of
Bt
transgenic plants utilizing
cry1Ac22
gene.
1 Results and Analysis
1.1 Construction and identification of pGFP-
Cry1Ac22 vector
The
GFP
gene used in this study is from mammalian
expression vector pGFP.
Bam
H
Ⅰ
and
Kpn
Ⅰ
were
inserted into the amphi of the linear vector through
the enzyme digestion and ligation. pMD18
-
T-Cry1Ac22 was constructed with the primers
designed according to the
cry1Ac22
gene sequence,
digested by
Bam
H
Ⅰ
and
Kpn
Ⅰ
, and obtained a 2
178 bp fragment (Figure 1B); pGFP vector is
digested by the
Bam
H
Ⅰ
and
Kpn
Ⅰ
and obtained a
3 400 bp pGFP linear fragment (Figure 1A). The
targeted fragments were recycled, ligated at 16
℃
,
and then transformed into JM109 competent cells.
Identification and screening of the positive clones
were performed by
Bam
H
Ⅰ
and
Kpn
Ⅰ
digestion
(Figure 2). From figure 2, we can see that lane No.
1,2,3,4,5,6,7,9,10 are positive recombinants
showing a 2 178 bp
cry1Ac22
target fragment and a
3 400 bp pGFP vector fragment. The results
indicated that the pGFP-Cry1Ac22 is constructed
successfully.
1.2 Construction and identification of pYES2
-
Cry1Ac22-GFP yeast expression vector
Positive clones was digested by
Bam
H
Ⅰ
and
Not
Ⅰ
and obtained a 3 000 bp gene fragment, which is a
fusion gene of
cry1Ac22
gene and GFP gene
(Figure 3). pYES2 vector was digested by BamHІ
and NotІ and obtained a 5.9 kb pYES2 vector
fragment (Figure 3). The targeted fragments were
Figure 1 Enzyme digestion of pGFP (A) vector and pMD18
-
T-Cry1Ac22 (B) with
Bam
H
Ⅰ
and
Kpn
Ⅰ
Note: M:
λ
DNA/
Hin
d
Ⅲ
marker; 1~2: Enzyme digestion
of pGFP with
Bam
H
Ⅰ
and
Kpn
Ⅰ
; 3~4: Enzyme digestion
of pMD18
-
T-Cry1Ac22 with
Bam
H
Ⅰ
and
Kpn
Ⅰ
Figure 2 Identification of transformants using
Bam
H
Ⅰ
and
Kpn
Ⅰ
Note: M:
λ
DNA/
Hin
d
Ⅲ
marker; 1~10: Transformants
picked
Figure 3 Enzyme digestion of pGFP-Cry1Ac22 and pYES2
with
Bam
H
Ⅰ
and
Not
Ⅰ
Note: M: λDNA/
Hin
d
Ⅲ
marker; 1: Enzyme digestion of
pGFP-Cry1Ac22; 2: Enzyme digestion of pYES2
recycled, ligated at 16
℃
, and then transformed into
JM109 competent cells. The recombinant was
digested with
Bam
H
Ⅰ
and
Not
Ⅰ
, and obtained a
3000bp target gene fragment and a 5.9 kb pYES2
vector fragment (Figure 4). These results indicated
that No. 1,2,3,4 were positive recombinants and the
yeast expression vector pYES2
-
Cry1Ac22
-
EGFP
was constructed successfully.