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Molecular Entomology 2013, Vol.4, No.3, 13-21
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18
triticlae seed extract. These data shows that
proteinaceous extract of different plant species shows
different specificity toward different insect species
which is an important step in developing molecules
for production of insect resistant transgenic plants
(Valencia et al., 2000; Bandani et al., 2009).
Overall, these data are in agreement with the finding
of the other researcher (Valencia et al., 2000) which
shown that a dose dependent inhibition of the
Amaranthus cruentus
against α-maylase of coffee
berry borer,
Hypothenemus hampei
. Mehrabadi et al.
(2012) showed that triticale seed extract affect
E.
integriceps
salivary enzyme as well as midgut
enzymes. Also, they found that triticale seed extract is
resistant to the insect enzymes so they concluded that
thiticale seed extract has potential to be investigated
further in order to determine its potential application
in integrated pest management program (IPM).
Gel assays using native gel confirmed results obtained
from spectrophotometric assay showing the greatest
effect of the wheat seed extract on the carob
α-amylase than protease. Also, these results confirmed
that the effect of seed extracts on the digestive enzyme
was in a dose dependant manner because as dose is
increased either enzyme band/s was/were disappeared
or intensity of the enzyme band/s was/were decreased.
In conclusion, different plant species produces
different proteins with different specificity toward
insect digestive enzymes that these proteins could be
used in plant protection strategies. Specificity of
inhibitors is an important issue since the introduced
inhibitors must not adversely affect the plant’s own
enzymes as well as non target organisms including
mammals’ enzymes.
4 Materials and Methods
4.1 Insects rearing
A population of
E. ceratoniae
was collected from
Chandab Region, Varamin, Tehran province, Iran. The
larvae were reared on artificial diet under laboratory
conditions at (29.6 ± 5) , with a 16:8 h photoperiod
℃
and (75 ± 5)% RH as described by Norouzi et al.
(2008) with some modifications.
4.2 Sample preparation
Dissection of the insect gut and sample preparation
was done based on Kazzazi et al. (2005). Briefly,
fourth larval instar gut was removed by dissection
under a light microscope. Then, the removed gut
placed in pre-cooled homogenizer. Homogenization
was done in ice cold distilled water and samples were
centrifuged at 15 000 g for 15 min at 4 , the
℃
℃
supernatant was removed and stored at −20 for
subsequent use.
4.3 α-amylase and Protease assays
α-amylase activity of gut was assayed by the
dinitrosalicilic acid (DNS) procedure (Bernfeld, 1955),
using 1% soluble starch solution as substrate as
described by Bandani et al. (2009). One unit of
α-amylase activity was defined as the amount of
enzyme required to produce 1 mg maltose in 30 min at
35 . A standard curve of absorbance against amount
℃
of maltose released was constructed to enable
calculation of the amount of maltose released during
α-amylase assay. A blank without substrate but with
α-amylase extract and a control containing no
α-amylase extract with substrate were run
simultaneously with reaction mixture. All assays were
performed in triplicates and with three replications.
General protease assay was done according to the
methods of Elpidina et al. (2001) and Gatehouse et al.
(1999), with slight modification. Briefly, 10 µL
enzyme extract and 50 µL substrate solution
(Azocasein 2%) were mixed with 40 µL 20mM
Glycine-NaOH buffer (pH 10). After 60 min
incubation, 100 µL 30% trichloroacetic acid (TCA)
was added to the reaction mixture, and kept it at 4
℃
for 30 min, followed by centrifugation at 15 000 g for
15 min to precipitate non hydrolysis substrate. 100 µL
1 M NaOH was added to 100 µL supernatant and the
absorbance at 405 nm was measured.
4.4 Seed protein extraction
Wheat and triticale seed proteins were extracted
according to Baker (1987) and Melo et al. (1999).
Seed was powdered thoroughly, and then 30 grams of
powdered seeds from each plant separately was mixed
with a solution of 0.1 M NaCl and stirred for 3 h,
followed by centrifugation at 8 000 g for 30 min. The
pellet was discarded, and the supernatant was placed
at 70 for 20 min to inactivate enzymes within the
℃
seeds. Seed protein was extracted using a saturation of
70% ammonium sulfate followed by centrifugation at
8 000 g for 30 min at 4 . The pellet containing the
℃