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Bt Research (Online) 2010, Vol.1 No.3
http://bt.sophiapublisher.com
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Figure 4 Identification of YES2-Cry1Ac22-GFP transformants
using BamHⅠ and NotⅠ
Note: M: λ DNA/Hind Ⅲ marker; 1~4: Transformants picked
1.3 Transformation of pYES2-Cry1Ac22-GFPvector
In order to study the expression and localization of
Cry1Ac22- GFP in Eukaryotic cell, transformation
into yeast was carried out. Theoretically, GFP-
Cry1Ac22 would expressed in yeast after
transformation. pYES2-Cry1Ac22-GFP vector was
transformed into the Saccharomyces cerevisiae
INVScl strains. Observation by fluorescence micro-
scope shows that flourescence was distributed in the
form of puncta, which indicated that Cry1Ac22-
GFP protein can be expressed in yeast cells (Figure 5).
Figure 5 Fluorescent fusion protein expressed in yeast under
ordinary fluorescent microscope
1.4 Expression of Cry1Ac22-EGFP fusion gene in
yeast
The recombinant yeast induced by galactose was
collected at different times, and detected by SDS-
PAGE (Figure 6). A new protein was detected in the
yeast crude protein induced by the galactose, with a
molecular weight of about 130 kD. However, no
such protein was detected in the crude protein that
is not induced by the galactose (lane one) .The
results primarily indicate that the recombinant
pYES2-Cry1Ac22-GFP expressed Cry1Ac22-GFP
fusion protein under the induction of galactose, and
Figure 6 The expression of Cry1Ac22-GFP fusion protein in
yeast
Note: M: Protein Marker; 1: The control (the recombinant
yeast that is not induced by galactose); 2~4: The
recombinant yeasts after galactose induction for 6, 12, 24
hours, respectively; Arrow indicates the expressed
Cry1Ac22-GFP fusion proteins
the expression was of high performance. The
expression level tend to be stable as the elongation
of induction.
1.5 Localization of cry1Ac22 gene in yeast
PYES2-Cry1Ac22- GFP vector was transformed
into the yeast, induced by the galactose, and directly
observed under confocal laser scanning microscopy.
After induction for 12 h, 60% yeast cells are
observed with green fluorescent under inversion
fluorescence microscope. Fluorescent proteins were
distributed in the form of dispersion in the whole
yeast cell transformed with pYES2-GFP vector
(Figure 7), while in the yeast transformed with
pYES2 -Cry1Ac22-GFP, fluorescent proteins were
distribute in plasma membrane and cytoplasm, but
not in nucleus. Domain Ⅰ in the N-terminal of
cry1Ac22 gene is constituted by a group of α helical
bundle formed by six or seven amphiphilic α
helixes surrounding a hydrophobic α helix. The
hydrophobic part acts as the function domain of
specific virulence and participates in the perforation
of cell membrane. The result is in accordance with
the localization prediction.
2 Discussion
PYES2 (5 900 bp) used in this study is a yeast-