Page 9 - PGTv3no2

Plant Gene and Trait 2012, Vol.3, No.2, 6 -12
http://pgt.sophiapublisher.com
10
within chloroplasts are extremely sensitive to H2O2.
When the H2O2 level rose to a certain degree to trigger
foreign gene over-expression, consequently higher
antioxidant enzyme activities of TS occurred at 100
mmol/L NaCl, especially APX activity had a substantial
increase at 100 mmol/L NaCl. The different increasing
value of APX activity and other antioxidant enzyme
activities suggest that APX might be responsible for
the fine modulation of ROIs signaling during stress,
and that of CAT might be responsible for removal of
excess ROIs (Mittler, 2002). Further intensification of
the NaCl stress caused decreases of antioxidant
enzyme activities in both sweetpotato plants, because
cytoplasmic membrane was destructed seriously and
antioxidant enzymes were inactivated. But the degrees
of decline were moderate in transgenic sweetpotato
and sharp in no-transgenic sweetpotato.
The MDA content and the decline range of the
chlorophyll of TS were lower than that of in the NS
under NaCl stress. These results were agreed to the
results of the root length. These correlated with
improvement of the enzymes activities. The MDA
content and the decline range of the chlorophyll of TS
increased a little with increasing a little of the enzyme
activities under the 50 mmol/L NaCl concentration
level. This might due to the response time to NaCl
stress signal. With the enzyme activities improved
quickly under the 100 mmol/L NaCl concentration
level, The MDA content of TS decreased a little and
the decline range of the chlorophyll of TS did not
increased so much, consequently here it became
evident that ROS cleaning system is a complex
balance process and the high enzyme activities could
not cleaned out the whole of the MDA.
All of the above results indicate that the tolerant
improvement of transgenic sweetpotato to salt stress
is accompanied with the enhancement of antioxidative
capacity. With the development of research, transgenic
plants with salt tolerance would be applied in the
saline land and improve the ecological environment.
3 Materials and Methods
3.1 Materials
Transgenic sweetpotato plants with CuZnSOD and
APX genes and sweetpotato plants cv. Yulmi as control
(Lim et al., 2007) grew under normal growth conditions
until 6~7 leaf stage. And then, their stems with 5
leaves were treated with solutions containing 0 (as
control), 50 mmol/L, 100 mmol/L and 150 mmol/L
NaCl and maintained for 5 days with 3 replicates, each
replicate consisting of 5 plants.
3.2 Analysis of PCR
The genomic DNA of the TS and NS leaves was
extracted according to modified CTAB method (Li et
al., 2007). The specific primers for the PCR analysis
were designed according to the sequences of NPT Ⅱ,
SOD and APX. A 750 bp product appro- ximately was
amplified with the NPTⅡ primer (5'- GAGGCTATT
CGGCTATGACTG-3', 5'-ATCG GGAGCGGCGATA
CCGTA-3'); a 752 bp product was amplified with the
APX primer (5'-ATGGGAAAATCTTACCCAACTG
TTA-3', 5'-TTAGGCTTCA GCAAATCCAAGCTC-3'),
as well as the SOD primer (5'-ATGGTGAAGGCTGA
AGCTGTTCTT-3', 5'-C TATCCTCGCAAACCAAT
ACCG- 3') with a 458 bp product. PCR amplification
reactions were initially incubated at 94℃ for 5 min,
followed by 30 cycles at 94℃ for 1 min, 62 ℃ (NPT
Ⅱ , SOD) or 60℃ (APX) for 1 min, and 72℃ for 1
min, at last 72℃ for 5 min. The reaction products
were analyzed by 1.2% agarose gel electrophoresis.
3.3 Physiological indexes assays
The third fully expanded leaf from the top of the NS
and TS plants was collected to assay for SOD, APX,
CAT, POD activities and MDA content, respectively.
Leaf samples (1 g) were homogenized in ice cold 0.1
M phosphate buffer (pH=7.8 for SOD and CAT
extraction, pH=7.0 for APX, POD and MDA extraction)
containing 1% PVP (polyvinyl pyrrolidone) with
pre-chilled pestle and mortar. Each homogenate was
transferred to centrifuge tubes and was centrifuged
at 4℃ for 15 min at 12 000×g. The supernatant was
used for enzyme activity assay.
SOD activity was measured by recording the decrease
in absorbance of superoxidenitro blue tetrazolium
complex by the enzyme (Beauchamp and Fridovich,
1971). Absorbance was recorded at 560 nm and one
unit of SOD activity was defined as that which
inhibited 50% of the reaction rate under these