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Genomics and Applied Biology 2014, Vol. 5, No. 2, 1-6
http://gab.biopublisher.ca
5
Figure 5 The analysis of argianse activity during seed
germination under 100 mM NaCl treatment in
Arabidopsis
thaliana
3 Materials and Methods
3.1 Materials and Treatments
Arabidopsis thaliana
ecotype Columbia seed was
preserved in the Alkali Soil Natural Environmental
Science Center (ASNESC), Northeast Forestry
Univeristy, and Harbin, China.
The seed was sterilized with 75 % ethanol and sodium
hypochlorite, and then was sown in glass containers
filled with 1/2 MS medium in the growth chamber
after stratification at 4
℃
for two days in darkness. For
experiments about analysis of expression level during
different developmental stages, RNA was extracted
from seed just after sterilization, 36-hour-old seeding,
3-week-old seeding. For experiments about analysis of
expression model under NaCl treatment, seed was
sown in glass container filled with 1/2 MS medium,
adding different
concentrations of
NaCl
(0,50,100,150,200) mM. For experiments about the
analysis of arginase genes under NaCl treatment
during seed germination, RNA was extracted from the
seed treated by 100 mM NaCl after culture different
time (0,12,24,36,48) h.
3.2 Arginase phylogeny
Members of the arginase in plants were obtained by
BLAST searches in NCBI. The cDNA of
ARGAH1
and
ARGAH2
was obtained from TIGR databases. A
total of 28 sequences were used for construction of the
phylogenetic tree (Figure 1).
3.3 Semi-quantitative reverse-transcription PCR
and real-time quantitative PCR
Total RNA was isolated from 0.2 g seed using a
modified method as described previously by Martin
(Martin
et al.,
2005), and treated by RNase-free
DNase(Takara) to remove genomic DNA. First-strand
cDNA was synthesized using Prime Script RT reagent
kit (Takara) from 1 μg total RNA, and 1 μl RNA was
added to 30 μl PCR mixture for SqRT-PCR. For
real-time quantitative PCR, a 20 μl mixture was used
with SYBR-green fluorescence (TransGen Biotech)
and the comparative
ᇞᇞ
CT method was used as
previously described (Yang
et al.,
2011). All the
primers were referred to Brauc (Brauc
et al.,
2012).
3.4 Enzyme assays
The method used to measure arginase activity is as
described by King (King and Gifford, 1997). Specific
operation will not be described.
Acknowledgements
This work was supported by specific fund for forest scientific
research in the public welfare (201404220) and Program for
Changjiang Scholars and Innovative Research Team (PCSIRT,
IRT13053).
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