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Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
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Figure 6 Effects of H
2
O
2
on the growth of recombinant
E. coli
M15 strain
Figure 7
identification of eukaryotic expression vector pYES2-
LjADH1 by
Bam
H
Ⅰ
and
Xba
double digestion
Ⅰ
Note: A: Identification of pYES2-LjADH1 digested by
Bam
H
Ⅰ
and
Xba
; B: Identification of recombinant yeast pYES2
Ⅰ
-
LjADH1 by PCR
recombinant yeast strain with pYES2-LjADH1 and
wild type strain pYES2 in the YPG media with differ-
rent ion concentrations, respectively (Figure 8). The
responses showed that the growth status between the
recombinant and its wild type yeast had very similar
in the YPG medium without any treatment, but the
recombinant yeast grew better than the wild type in
the YPG media with final concentrations of 10 mmol/L
CuCl
2
, 100 μmol/L CdCl
2
, 150 μmol/L CdCl
2
and
3.5 mmol/L H
2
O
2
, respectively. There is no any res-
ponse between the recombinant and wild type in con-
Figure 8 Over expression of pYES2-LjADH1 under different
abiotic stresses
Note: A: The controls, Yeast cells harboring pYES2-LjADH1
and pYES2 vector, respectively, were inoculated in YPD
medium, growth status was observed after incubated 2~3 days
at 30 ; B~F: Yeast cells harboring pYES2
℃
-LjADH1 and
pYES2 vector were respectively inoculated in YPD media with
different concentrations of 10 mmol/L CuCl
2
, 1.8 mmol/L
NiCl
2
, 100 μmol/L CdCl
2
, 150 μmol/L CdCl
2
, and 3.5 mmol/L
H
2
O
2
, growth status observation after 2~3 days incubation at 30
℃
centration of 1.8 mmol/L NiCl
2
. The results indicated
that the LjADH1 protein has some resistant functions
to oxidative stress in yeast. With reference to evidence
of the recombinant yeast growth in the concentration
of 3.5 mmol/L H
2
O
2
we considered that LjADH1
might have certain resistant functions to abiotic stre-
sses. There was a report that the plants will lack of re-
sistance to waterlogging while the ADH gene in plant
is going to silence, whereas overexpressing ADH alone
might not yet improve the plant resistance to water-
logging (Ismond et al., 2003). Therefore the functions
of LjADH1 in plants need to be further studied in future.
2 Conlusions
In this research, we cloned
alcohol dehydrogenase
gene from
Lotus japonicus
MG20, and considered that
the gene encodes a typical zinc-binding alcohol dehy-
drogenase, named as
LjADH1.
In this research, the gene was ligated to pQE30 expre-
ssion vector with a 6×His tag, and a batch of non-
denaturalized proteins was expressed in
E. coli
and
purified by Ni
+
-NTA gel column under the optimum
induced expression conditions. The enzymatic activity
of the LjADH1 was determined by method proposed
by Vallee and Hoch, which demonstrated strong alco-
10