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Genomics and Applied Biology, 2011, Vol.2 No.1
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(Ma et al., 2002).
Puccinellia tenuiflora
, an important salt-tolerant
herbage, is the main constructive species and pioneer
species of salinized grassland in North China; because
of its excellent cold-resistance, drought tolerance and
salt tolerance, it has become the precious material of
plant stress tolerant research (Li and Yang, 2004). In
this article, we cloned the
PutA Px
gene from
Puccinellia tenuiflora cDNA library, determined the
formula weight (32 kD) and isoelectric point (7.71) of
the encoded protein by using biosoftwares, which
showed the same number of nucleotide and formula
weightb as those of rice OsAPx3 and OsAPx4
(Teixeira et al., 2004; Miyazaki et al., 2003). And the
homology of amino acid sequence indicated that it had
a high similarity to graminea plants rice (89.7%),
barley (94.3%), wheat (94.2%), ryegrass ((73.7%),
corn (79.6%), respectively. The PutA Px encoded
protein had only one transmembrane domain and its
protein located on peroxisome, which was consistent
with the report of barley HvAPX1 (Shi et al., 2001).
PutA Px
was universally expressed in different tissues
(highest in ear, ear stem, anther and sheath, but lowest
in female flower). Antioxidative stress results of yeast
strain INYSc1inducing by galactose suggested that
overexpression
PutA Px
yeasts colonies showed better
antioxidative stress than the control under 8 mmol/L
H
2
O
2
, which meant that the
PutA Px
catalysed H
2
O
2
to
reduce into H
2
O and protected the tissues and cells,
thus it made
PutA Px
transformed yeast strains grow
better on SD medium with H
2
O
2
. All these evidence
demonstrated that the effects of acsorbate peroxidase
on resisting oxidative stress.
In this research, we successfully cloned acsorbate
peroxidase gene
PutA Px
and conducted preliminary
investigation on it, which would pave the way for
further understanding the mechanism of antioxidation.
3 Methods and Materials
3.1 Tested plant materials
Puccinellia tenuiflora
adult plants were obtained from
Anda Practice Base, Alkali Soil Natural
Environmental Science Center (ASNESC), Northeast
Forestry University, and identified by professor Jin
Zhuzhe.
3.2 Reagents
cDNA library of
Puccinellia tenuiflora
(deal with
150 mmol/L NaHCO
3
) was constructed by our team.
Escherichia coli
JM109 and yeast strain IVSc1 were
conserved in our lab. DNA extraction kit, pMD18
-
T
vector,
Taq
polymerase, T4 DNA ligase and restriction
endonucleases were bought from TAKARA. DNA
sequencing and primers were synthesized by
Invitrogen, Shanghai branch. Other reagents were all
domestic analytically pure.
3.3 Acquiring DNA fragment of target gene
Universal primers of vector PSK were designed by
Primer 5.0 (F1: 5'
-
GGATCCGGGCCCTTTATTCTA
-
3',
R1: 5'
-
GAGCTCGGGCCCTTACTTGCT
-
3'). The
full-length gene of ascorbate peroxidase of
Puccinellia
tenuiflora
Chinampoensis
Ohwi: PutAPx was amplified
used the cDNA library of plasmid PSK
-
46 as template.
PCR reaction system was 20
μ
L, containing ddH
2
O
12.0
μ
L, Pn 1.0
μ
L, Pm 1.0
μ
L, 10×Buffer 2.0
μ
L,
dNTP 2.0
μ
L, cDNA 1.0
μ
L and
Taq
polymerase 1.0
μ
L.
The PCR reaction conditions was following: an initial
denaturation step at 94°C for 2 min, followed by 30
cycles of 94
℃
for 30 s, 55
℃
for 30 s and 72
℃
for
1 min, and ending with a final extension at 72°C for
10 min, finishing at 4
℃
.
3.4 Extration and identification of recombinant
plasmid DNA
Plasmid DNA was extracted by boiling method
according to the Molecular Cloning (Sambrook et al.,
2002).
The extraction plasmid DNA was identified by
restriction endonuclease digestion
Hin
dШ/
Eco
RI, and
the digestion products were detected the by agarose
gel electrophoresis. After sequencing the positives
clone, it would be sent to sequence and named as
pT-PutA Px. The strain would be stored at
-
70
℃
.
3.5 Sequence analysis of
PutAPx
gene
By blasting the GenBank nucleotide data on NCBI
(
http://www.ncbi.Nlm.Nih.gov/gorf/gorf.html
),
we
analyzed ORF of the initiation site and terminator
domain and compared the similarities of
PutA Px
genes from
Puccinellia tenuiflora
,
Arabidopsis
thaliana
NP195226,
Oryza sativa
AK070842, barley
BAB6253,
Triticum aestivum
Linn.EF555121,