Basic HTML Version
Molecular Soil Biology (online), 2011, Vol. 2 No.2, 9
-
14
ISSN1925-2005
http://msb.sophiapublisher.com
- 13 -
Figure 5 Growth assay of yeast expressing
PutSTE24
and
AtSTE24
in the stresses of various of metal ions
Note: Yeast cells were incubated as described in materials and
methods; Serial dilutions were spotted onto solid yeast YPD
medium supplemented with or without metal cations, including
K
+
1 mol/L, Mg
2+
0.8 mol/L, Cu
2+
8 mmol/L, Fe
3+
10 mmol/L,
Cd
2+
180 µmol/L, Ca
2+
100 mmol/L, Mn
2+
1.2 mmol/L, Ba
2+
6 mmol/L, Co
2+
0.5 mmol/L, Ni
2+
1.5 mmol/L and Zn
2+
4 mmol/L,
growth were monitored for 3~7 d at 30
℃
growth of the two
STE24
transformants seemed
hyper-sensitive in the presence of Co
2+
, Ni
2+
and Zn
2+
.
These results indicate that
STE24
is a gene related to
some metal ions stresses besides Al
3+
, which have not
been reported previously. This responsive relationship
is deduced to caused by the post-translation modification
of some cations transporters under the action of
STE24 protease.
2 Discussions
In this study, the growth of yeast transformed with
PutSTE24
and
AtSTE24
was assayed in the presence
of various of abiotic stresses. We have got the
conclusions that
STE24
is a gene related to some
metal ion stresses, but the molecular mechanism
involved in have not been clear.
3 Materials and methods
3.1 Materials
Yeast full-length cDNA library of Puccinellia
tenuiflora (1 865 000 clones), cDNAs of
Arabidopsis
thaliana
,
Escherichia coli
strain JM109, Yeast strain
(
Saccharomyces cerevisiae
)
InVSCI
.
3.2 Cloning
PutSTE24
and
AtSTE24
from plant
and sequence analysis
The ORF portion of Put
STE24
was amplified from
the yeast expression library of
P. tenuiflora
with the
primers F-F: 5’
-
GCAGCTGTAATACGACTCAC
-
3’
and F-R: 5’
-
TTACATGATGCGGCCCTCTA
-
3’. The
ORF portion of
AtSTE24
was amplified from the yeast
expression library of
Arabidopsis thaliana
with the
primers F-F: 5’
-
GGTCACTCTTTTCTCAGCCATG
-
3’
and F-R: 5’
-
ACAAGAGACGAGTTAAGCGGAC
-
3’.
Homologous comparison was obtained with other
plants according to the amino acid sequence of the
two genes.
3.3 Plasmids construction
of
pAUR123
-
PutSTE24
and pAUR123
-
AtSTE24 and yeast transformation
The modified form of
PutSTE24
was constructed:
SgsI-PutSTE24
-
SfaAI. The forward primer (F-P:
5’
-
GCGATCGCGCACTGTAATACGACTCAC
-
3’)
was designed to add
SgsI
site and the reverse primer
(R-P: 5’
-
CTCGAGTTACACAAAAAAGCTTG
-
3’)
was designed to add
SfaAI
.
The modified form of
AtSTE24
was constructed:
SgsI
-
AtSTE24
-
SfaAI. The forward primer (F-P: 5’
-
GGTACCTTTTCTCAGCCATG
-
3’) was designed to
add SgsI site and the reverse primer (R-P: 5’
-
GGCG
CGCCTCTAGATGCATGCTCGAG
-
3’) was designed
to add
SfaAI
.
All amplified fragments were cloned into the
pAUR123 vector (Invitrogen) and the constructed
vectors were introduced into yeast mutant
InVSCI
using the LiAc/PEG method. The yeast transformants
were selected on medium supplied with Aureobasidin A.
3.4 Tolerance of
PutSTE24/AtSTE24
overexpressing
cells to various stress
For growth response assay, the yeast transformants of
pAUR123, pAUR123
-
PutSTE24 and pAUR123
-
AtSTE24, were cultured in liquid YPD medium until
OD
600
≈0.6 respectively, and diluted 10
-1
, 10
-2
, 10
-3
,
10
-4
and 10
-5
fold with ddH
2
O. Then, aliquots of each
dilution were spotted onto solid yeast YPD medium