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Legume Genomics and Genetics (online), 2011, Vol. 2, No.1, 1-5
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4
2004). A proteome study based on 2-D gel electroph-
oresis followed by mass spectrometric found that 21
spots with more than 1.5-fold altered expression and
majority of the differentially expressed proteins be-
longed to the functional category of signal trans-
duction mechanisms and inorganic ion transport and
metabolism (Kumar Swami et al., 2011). This will
give an explanation that the isotypes of 78 kD (SBP)
most likely has a multiple roles and most likely
inducing a number of specific genes and signal trans-
duction that is leads to protection of cellular structures
from damage during dormancy and induce various
metabolic process associated with germination. Highly
expressed biotin isotypes in pea stem than plumules
and roots, probably playing a critical role in cellular
differentiation and plant growth. Similarly, it has been
reported that the amount of 49 kD isotypes was higher
in the stem than other investigated parts and play a
crucial role during germination of pea seed (Moustafa
et al., 2003).
2 Conclusion
The present report demonstrates that isotypes of 78 kD
streptavidin biotin-binding proteins (SBP) display a
specific pI range during seed dormancy and during
germination and in different tissues. Further studies on
their primary sequences and purification of these
isotypes may provide more detailed information on the
genetics, biochemical and physiological significance of
78 kD streptavidin binding protein in different plants.
3 Materials and methods
3.1 Sample preparation
Seeds of Alaska peas (
Pisum sativum
L. var. Alaska)
were imbibed for 10 hours and then germinated in
vermiculite in plastic boxes in a dark room for 72 hours
at 21 ~23 , as described before (Abe and Davies,
℃ ℃
1991; Abe and Davies, 1995). At each time point of
0 hours, 40 hours and 72 hours after germination, three
grams of intact embryonic tissue or separate plumules,
stems, and roots were dissected from the cotyledons,
collected on ice, and ground with a mortar and pestle
in 7 volumes of cytoskeleton-stabilizing buffer (CSB)
consisting of 5 mmol/LHEPES-KOH (pH 7.5), 10 mmol/L
Mg (OAc)
2
, 2 mmol/L of ethylenebis (oxyethylene-
nitrilo) tetraacetic acid, and 1 mmol/L phenylmethyl-
sulphonyl fluoride with the addition of 0.5% poly-
oxyethylene-10-tridecyl ether and homogenate was
filtered through Miracloth (Calbiochem, San Diego,
CA, USA).
3.2 Two-dimensional PAGE
An equal amount of protein (40 µg) from each sample
was precipitated with 4 volumes of acetone at
-
20
℃
overnight. The precipitate was washed with 80%
acetone, dissolved in 8 M urea, 0.5 % NP-40, 2% β-
mercaptoethanol, 0.8% Pharmalyte® 3-10 for IEF
(Amersham Pharmacia Biotech), and 0.01% BPB,
incubated at 39 for
℃
1 hour, applied onto a drystrip
gel (p
I
4-7, Amersham Pharmacia Biotech) equilibrated
with 8 M urea, 0.5% Triton-X100, 10 mmol/L DTT,
2 mmol/L acetic acid, and 0.01 mg/mL orange G and
subjected to IEF at 22 650 V h using a Multiphor II
2D-Electrophoresis System (Amersham Pharmacia
Biotech). The strip was immersed in 6 M urea, 0.05 M
Tris-HCl (pH 6.8), 30% glycerol, 1% SDS, and 16 mmol/L
DTT for 10 min, followed by 6 M urea, 0.05 M
Tris-HCl (pH 6.8), 30% glycerol, 1% SDS, and 0.01%
bromophenol blue for 10 min, both with gentle
shaking as described by Moustafa et al (2003).
3.3 Immunodetection of 78 kD biotin isotypes
The strip was placed onto a separating slab gel for
SDS-PAGE. After electrophoresis, the gels were blotted
onto a polyvinylidene fluoride membrane (Immo-
bilonTM Transfer Membranes, Millipore) and probed
with the biotinylated anti-rat Ig species-specific whole
antibody from sheep (Amersham Pharmacia Biotech)
as the primary antibody. The binding of the primary
antibody was detected with streptavidin-alkaline phos-
phatase conjugate (Amersham Pharmacia Biotech)
with 5-Bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium as substrates.
Authors' contributions
Mahmoud F.M. Moustafa conducted all the work.
Acknowledgments
This study was funded by Faculty of Science, South valley University in
Qena, and Ministry of Higher Education and the State for Scientific
Research in Egypt.
References
Abe S., and Davies E., 1991, Isolation of F-actin from pea stems: Evidence
from fluorescence microscopy, Protoplasma, 163: 51-61