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Genomics and Applied Biology, 2011, Vol.2 No.1
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Figure 3 Open reading frame and amino acid sequence
1.2.3.2Analysis of
PutA Px
transmembrane domain
Analysis the
PutA Px
encoded transmembrane domain
by using ProtParam on line (http://www.cbs.dtu. dk/
cgi-bin/nph), it demonstrated that this protein had one
transmembrane domain, peroxisome, which was
consistent with barley HvAPx. PutA Px protein was a
secreted protein encoded by cytoplasmic peroxisome
gene, which was synthesized in cytoplasm and located
on
PutA Px
gene of peroxisome by protein transport
system (Figure 6).
1.3 Expression analysis of
PutA Px
under oxidative
stress
(H
2
O
2
)
in yeast
In this work, we designed to confirm the antioxidative
function of
PutA Px
by detecting the growth status of
PutAPx-overexpressed yeast under H
2
O
2
oxidative
stress.
1.3.1 Construction of yeast expression vector
The plasmid pT-PutA Px and vector pYES2 which
both have restriction enzyme sites of
Bam
H
Ⅰ
and
Xba
Ⅰ
were digested by these two restriction enzymes,
linked and transformed the products into
E. coli
JM109. selected the positive clones, extracted the
plasmid DNA and detectedby
Bam
H
Ⅰ
and
Xba
Ⅰ
digestion. It showed that a target fragment of 882 bp
was gained (Figure 7A), named it pYES2
-
PutA Px,
which indicated that we have constructed the yeast
expression vector successfully. The pYES2
-
PutA Px
was transformed into yeast strain INYSc1 by lithium
acetate, and the clones were detected by PCR with
primers F3 and R3. It showed that four positive clones
with a target band of 882 bp were selected (Figure 7B),
which proved that the target gene had been transformed.
Antioxidative stress detection of transformed yeast
strain INYSc1 showed that the growth status of
different oxidative stress (0, 2 mmol/L and 4 mmol/L
H
2
O
2
) has no obvious difference on SD medium.
Overexpression
PutA Px
of yeasts colonies
represented antioxidation differentia under 8 mmol/L
H
2
O
2
(Figure 8). Furthermore, with the increase of
dilution times, the growth status of overexpression
PutA Px
of yeasts colonies were better than that of the
control. When the dilution was 10
-4
, the control yeasts
could hardly grow, while the overexpression
PutA Px
clones grew well. The result indicted that
overexpression of
PutA Px
in yeast could improve the
growth status of yeast under oxidative stress,