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Bt Research (Online) 2010, Vol.1 No.3
http://bt.sophiapublisher.com
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Figure 7 Localization of Cry1Ac22 protein in yeast
Note: A,B,C,D represent yeast localization in different views;
Cry1Ac22: pYES2 is ligated with Cry1Ac22-GFP fusion
protein; GFP: The control (pYES2 is ligated with GFP
protein); 1/4, 2/5, 3/6 indicates GFP, merged, and white
conditions, respectively; The shortest scale is 5.0 μm
Escherichia coli
multicopy shuttle plasmid. It is a
secretion type expression vector controlled by T7
promoter, and can replicate autonomously in
S.
cerevisiae
and
E. coli
. When multiclone was
inserted to recombinant vector, it can be screened
by the insertional inactivation of
LacZ
gene, which
also contains resistance gene.
PYES2 contains yeast URA gene, and can act as the
auxotroph screening tag. Taking the auxotroph
saccharomyces cerevisiae as model organism,
pYES2-Cry1Ac22-EGFP shuttle vector containing
reporter gene and enhanced green fluorescent
protein (EGFP) gene was constructed successfully
(Misteli and Spector, 1997). The vector can
replicate in the E. coli and express in the S.
cerevisiae. It forms fusion protein in the same open
reading fragment. EGFP proteins display green
fluorescence under the excitation of ultraviolet light.
And its ligation with target gene will not affect the
expression of target protein, Besides, it could fuse
with target gene and label the expression status of
target gene.
After the transformation of recombinant plasmid
into the auxotroph
S. cerevisiae
, the expression of
yeast in different times was observed under
fluorescence microscope. If the recombinant
plasmid can replicate in the cell, the EGFP will be
expressed displaying green fluorescence under the
excitation of ultraviolet light. The result indicated
that green fluorescence can be observed in the
transformed yeast under fluorescence microscope,
which tended to be most powerful after induction
for 12 h. No fluorescence was detected in the
control yeast.
In this study, we constructed the Cry1Ac22 fusion
protein marked with green fluorescent protein
(GFP), and investigate the expression and
localization of this fusion protein in
Saccharomyces
cerevisiae
. This study would provide the theoretical
guidance for studying the function of
Bt
insecticidal
proteins, and presents insights for the trail of tracing
transgenes protein.