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Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
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Figure 2 Phylogenetic tree of
ADH
in plants
Figure 3 Identification of recombinant plasmid pQE30-LjADH1
by restriction enzymes
Bam
H
Ⅰ
and
Sac
Ⅰ
M: λ DNA Marker; l: pQE30-LjADH1 digested by restriction
enzymes
Bam
H
Ⅰ
and
Sac
Ⅰ
Figure 4
SDS-PAGE analysis of fusion protein expressed with
time gradient under induction of 0.1 mmol/L IPTG
M: Protein molecular marker; 1: No induction; 2: 30 min
induction; 3: 60 min induction; 4: 120 min induction; 5: 180 min
induction; 6: 240 min induction; 7: 300 min induction; 8:
360 min induction
here E
340
is the absorbance increase within 5 minutes
at 340 nm (absorbency units 0.001), E
W
is the weight
(mg) of enzyme per mL enzyme solution. In this re-
search we measured that
absorbance change of Lj-
ADH1 in a minute was 0.108, so LjADH1 enzymatic
activity come out 48.2 U/mg (figure 5).
Figure 5 The absorbance of recombinant fusion protein at 340 nm
1.5
Resistant
analysis of the prokaryotic fusion
protein
We added H
2
O
2
into
E. coli
M15 recombinant strain
which harboring pQE30-LjADH1 and reference strain
with pQE30. The reference strain grew better than that
of recombinant strain in absence of H
2
O
2
. This phe-
nolmena might be due to the effect of overexpressed
heterologous proteins during bacteria growth stages.
Whereas when we added H
2
O
2
into recombinant strain
solution at 1 mmol/L H
2
O
2
final concentration, the
recombinant strain obviously grew better than that of
the reference strain (Figure 6). The results suggested
that alcohol dehydrogenase might have some fun-
ctions in the survival capability of prokaryotes under
oxidative stress.
1.6
Construction of eukaryotic expression vector
pYES2-LjADH1
In this research, we constructed a yeast expression
construct named pYES2-LjADH1, The construct was
validated by digestion of restriction enzymes
Bam
H
Ⅰ
and
Xba
Ⅰ
to produce 5.9 Kb and a 1.1 Kb fragment
by agarose gel electrophoresis(Liu et al., 2010, Wang
et al., 2010) (figure 7A), those fragments in size were
in accord with the size of pYES2 and
LjADH1
sequ-
ence. that was confirmed by digestion of restriction
enzymes
Bam
H and
Ⅰ
Xba
. The recombinant str
Ⅰ
-
ains were also identified by PCR to confirm that the
pYES2-LjADH1 were transferred into yeast (figure 7B).
1.7 Resistant analysis of LjADH1 protein ex-
pression in yeast
In order to understand how do the LjADH1 protein
response to different ion stresses, we inoculated the
9