Page 5 - Genomics and Applied Biology

Genomics and Applied Biology, 2011, Vol.2 No.1
http://gab.sophiapublisher.com
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Shigeoka et al., 1980a; 1980b). In the past decade,
APX homologous genes of many plant has been
cloned and investigated. Ishikawa et al (1995) and
Kubo et al (1992) proved that cytoplasmic APXs of
spinach and Arabidopsis displayed inverse correlation
to strong light and MV, respectively. Lu et al (2007),
Wang et al (2009) and Ma et al (2002) also had found
that the expression of cytoplasmic APXs in rice, white
birch nursery stock and suaeda salsa enhanced under
salt stress induction. The results of searching nucleic
acid database showed that many APXs, such as grape
(Lin et al., 2006), cayenne pepper (Yoo et al., 2002),
pea (Mittler and Zilinskas, 1991) and so on, have the
same enzymatic characteristics and higher specificity
to ascorbic acid substrate but more prone to inactive
without substrate by comparing investigation of their
structure and enzyme kinetics (Yoshimura et al., 1998;
Nishikawa et al., 2003). Ascorbate peroxidase gene, as
the main enzymes for eliminating H2O2 generated
from salt stress in antioxidant system, has hitherto not
been reported in Puccinellia tenuiflora.
In present study, we cloned ascorbate peroxidase gene
from Puccinellia tenuiflora, preliminarily analyzed
the sequence, the tissue-specific expression and the
antioxidant capability by biology software, RT-PCR
and yeast over-expression, respectively, which would
pave the way for further investigating the mechanism
of antioxidation. It will engender great economic,
social and ecological benefits if it is used to breed
novel varieties of herbage with saline-alkali-resistance
and applied in developing saline and alkaline land.
1 Results and Analysis
1.1 Abtain PutA Px sequence
Plasmid PSK-46 in cDNA library was PCR by
universal primers F1 and R1 PutA Px vetor and the
products were validated in agarose gel. The results
showed that there was a fragment approximate 1000 bp
(Figure 1). And the target DNA fragment was recycled
by using DNA qiaquick gel extraction kit, was linked
to T-vector at 16℃. 16 hours later, the linked products
were tranformed into E. coli JM109 with Ampicillin
50 mg/L. The plasmid DNA of the positive clones T#1,
T#2, T#3 and T#4 were extracted by boiling method.
The DNA were identified by HindШ/Eco RI, and the
results displayed that three bandsof pMD18-T, about
2.7 kb, T#1,T#2,T#4, 700 bp and insert fragment, 400
bp, respectively, which were consistent with the target
gene fragment, however, the insert fragment of T#3 is
slightly smaller than others (Figure 2). The T#2 strain
was sequenced, and the recombination plasmid named
as pT-PutAPx.
Figure 1 PCR product of PutAPx gene
Note: M: Marker-λ/Hind Ⅲ; 1~3: PCR products
Figure 2 Identification of pMD18- T-PutApx confirmed by
enzyme digestion
Note: M: Marker-λ/HindⅢ; 1~4: pMD18- T-PutApx positive
clones (T#1, T#2, T#3 and T#4) confirmed by enzyme
digestion
1.2 Sequence analysis of PutA Px
1.2.1 Analysis of PutA Px promoter and terminator
sites and its deduced amino acids
Blasting the target gene sequence, 1,024 bp, in
GenBank at NCBI, the result showed that target gene
sequence was highly homologous with PutAPx gene
(>86%), and the score was up to 1,285. We presumed
that its encoded protein was very likely the ascorbate
peroxidase (Table 1). And then we analyzed the 1,024
bp fragment via network (http://www.ncbi.nlm.nih.
gov/gorf/gorf.html), the results revealed that promoter
site was at +74, terminator site was at +949 and ORF
is 876 bp, which contained 291 aa, and we estimated
that it was indeed PutA Px gene (Figure 3).