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Plant Gene and Trait 2012, Vol.3, No.2, 6
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plants expressing both CuZnSOD and APX provide
stronger protection to methyl viologen (MV)-induced
oxidative stress than transgenic tobacco plants
expressing CuZnSOD, MnSOD or APX, which was
relative with SOD and APX scavenging ROS at the
same time in different path (Kwon et al., 2002). There
were many reports indicated that transgenic sweetpotato
plants expressing the genes of CuZnSOD and APX in
chloroplasts improved its tolerance to drought, chilling,
high temperature and sulfur dioxide stress responses
(Lim et al., 2007; Li and Deng, 2007).
The foreign gene cannot express without high-effective
expression promoter. The constitutive promoter CaMV
was used in the most transgenic researches; however,
this kind of constitutive promoter drives the gene to
express regardless of time and space. For example,
Kasuga et al (1999) reported that with the help of
CaMV 35S promoter, it can drive the expression of the
DREB1A
gene related to drought resistance resulted in
severe growth retardation under normal growing
conditions. The stress-inducible promoter has the
transcriptional activity because of the signal stimulation,
comparing to tissue and organ-specific promoter, it
can drive the target gene up-regulation expression
only after the plants was stimulation by signal, so that
it not only does not result in waste of resources in
plants, but also improve resistance of plant. Kim et al
(2003) reported that the isolation of a strong oxidative
stress-inducible promoter of POD (SWPA2) from
sweetpotato and then characterized that it was more
effective to drive resistant gene expression in transgenic
tobacco plants. The application of SWPA2 enhanced
tolerance to environmental stresses of transgenic
potato and sweetpotato plants (Tang et al., 2006; Lim
et al., 2007; Ahmad et al., 2008). Therefore, the
stress-inducible promoter might be useful for the
development of stress-tolerant transgenic plants.
In this Study, some physiological indexes of leaves in
transgenic sweetpotato (
Ipomoea batatas
L. cv. Yulmi),
which harbors two genes CuZn superoxide dismutase
(CuZnSOD) and ascorbate peroxidase (APX) genes
with the stress-inducible SWPA2 promoter were
evaluated under different concentrations of NaCl
treatment, which would provide a basis for breeding
stress-tolerant sweetpotato plants.
1 Results and Analysis
1.1 PCR analysis of transgenic plants
PCR analyses were performed to determine integration
of SOD, APX, and neomycin phosphotransferase
Ⅱ
(NPT
Ⅱ
) genes in the genomic DNA of transgenic
plants. The result showed that the predicted 458 bp,
752 bp and 700 bp internal fragments for the SOD,
APX, and NPT
Ⅱ
, respectively, were amplified in the
transgenic plant, indicating that those genes were
integrated into the genome (Figure 1).
Figure 1 PCR analysis of transgenic plant
Note:
Ⅰ
: PCR analysis of NPT
Ⅱ
gene;
Ⅱ
: PCR analysis
of APX gene;
Ⅲ
: PCR analysis of SOD gene
NS: Non-transgenic plant; P: Positive control (Plasmid
pSSA-K); TS: transgenic plant
1.2 Anti-oxidation enzyme assays
The enzyme activities of SOD, APX, CAT and POD
showed approximately the same trend in transgenic
sweetpotato under NaCl stress (Figure 2). The levels
of SOD, APX, CAT and POD activities in leaves of
TS were always higher than those in NS under the
same NaCl stress, which indicated that excessive
expression of SOD-APX gene were also raised POD
and CAT activities of the antioxidant enzyme system,
thus strengthen the ability to withstand attack of
superoxide radical, etc. Physiological indexes were no
remarkable differences without NaCl stress between
TS and NS, which was concerned with the stress-
inducible promoter that did not start the expression of
foreign genes. With the increase of the concentration
of the Nacl, the enzyme activity in TS improved
quickly, and reached the peak value in 100 mmol/L
NaCl concentration. We observed remarkable differences
under 100 mmol/L NaCl concentration between TS
and NS (P<0.01). In the case of the APX activity, this
exhibited a significant difference (P<1%) from NS at
100 mmol/L NaCl, approximately 4
-
fold higher than