Agriculture Reference
In-Depth Information
Figure 1b. Growth pattern of very early stage of wild-type and the
R3-1
mutant. The
cultures were maintained for 14 days.
Crude Protein Extraction
Leaves from the 1
st
, 2
nd
and 3
rd
position under strong sunlight were
harvested at the late vegetative growth stage, covered with foil, frozen in
liquid N
2
immediately after harvest, and stored at -80
℃
. All operations were
performed at 0 to 4
℃
under infra-red light as a safe-light using a Noctovision
system (NVR 2015, Nihon Electric Co., Hamamatsu, Japan), unless otherwise
indicated.
Proteins were extracted from the pea leaves by centrifugation, as described
by Struglics et al. (1993), with some minor modifications. Leaf residues of
wild type and
R3-1
from -80
℃
were ground 30 times with a prechilled mortar
and pestle into a pea homogenization buffers containing 30 mM Mops (pH
7.3), 3 mM EDTA, 25 mM cystein, and 0.3 M manitol, using 5 volumes of
buffer g
-1
fresh weight, and the homogenates were squeezed through a double
layers sheet of nylon cloth. The chloroplasts were sedimented by
centrifugation at 4, 000 rpm (4, 000 x g) for 5 min. The resulting supernatant
was centrifuged at 10, 000 rpm (10,000 x g) for 10 min to remove cell debris,
and the supernatant was centrifuged at 105,000 xg for 30 min. The supernatant
