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by vortexing and centrifuged 10 min at 10000 rpm at 4
o
C. The two previous steps were
repeated. The pellet was washed twice with 70% ethanol, air dried and dissolved into 100
Pl of water (DEPC treated).
1.3 Oligonucletide Design and RT-PCR
Degenerated oligonucleotides were designed based on regions of high homology between
aligned PME-deduced amino acid sequences from
Lycopersicon esculentum
(Bird, et al.,
1993-1994; Pear et al., 1993; Bridges, et al. 1988; Ray, et al 1988),
Phaseolus. vulgaris
(Recourt et al., 1992; 1995),
Petunia inflata
(Um et al., 1994) and were synthesised.
First-strand cDNA was synthesised from 2 Pg of total RNA from mature fruit of
papaya. RNA was incubated in 20 Pl of 1 x first-strand buffer (50 mM Tris-HCl, pH 8,3, 75
mM KCl, and 3 mM MgCl2), 0.5 mM each dNTP and 100 ng of oligo(dt)17, 10 mM DTT
and 20 units of RNAs in at 65
o
C for 10 min and then placed on ice. 1 Pl of MMLV-RT
(200 units/Pl) was added and the reaction was incubated at 37
o
C for 1 h. Reaction was then
heated to 95
o
C for 5 min, and then placed on ice or stored at -20
o
C until further use.
1 Pl of first-strand reaction was used as a template in PCR. The reaction mixture
was composed of 10 Tris-HCl, pH8.3 50 mM KCl 1 mM MgCl2, 0.2 mM dNTPs, 100 pM
each PE1 and PE2 primers, and 0.2 Pl of Taq polymerase. The conditions for amplification
were 94
o
C for 4 min and 35 cycles of 94
o
C for 1 min, 55
o
C 1 min, 72
o
C for 1 min and
then 72
o
C for 7 min. Product was gel purified using Qiaex (Qiagen) and the product was
cloned in Bluscripts KSII. Cloned PCR product was sequenced and analysis was carried out
using the DNASTAR software.
1.4 Northern blotting analysis
Twenty micrograms of total RNA from papaya fruit was separated by glyoxal denaturation
agarose gel electrophoresis and transferred to nylon membrane (Hybond-N, Amersham),
according to the manufacturer's instructions. Membrane was probed with [D-
32
P]dCTP-
labelled insert DNA from CpPME (partial-length PCR clone). The probe was labelled by
Ridiprimer DNA labelled kit (Amersham) labelled the probe. The hybridisation was carried
out overnight at 65
o
C in 7% (w/v) SDS, 0.5 M phosphate buffer, 2 % (w/v), blocking
reagent (Boheringer) with approximately 50 ng of labelled probe. The blot was washed
twice in 2xSSC and 0.1 % (w/v) SDS at 65
o
C, twice in 0.1xSSC and 0.1 % (w/v) SDS at
65
o
C. Blot was exposed to film at -80
o
C overnight.
1.5 PME extraction and purification
Extraction of PME was as described in Fayyaz et al. (1994). Briefly, after thawing at 4
o
C
100 g of papaya pulp, previously frozen at -80
o
C were homogenised with 200 ml of 2 M
NaCl solution pH 8.0. After adjusting the pH to 8.0, the homogenate was incubated in a
cold room at 4
o
C for 5 hours under stirring condition. During the incubation period, the pH
of the homogenate was maintained at pH 8.0 by adding either 2 M NaOH or 2 M HCl. The
homogenate was centrifuged at 24000xg for 30 min at 4
o
C. Solid ammonium sulphate
sufficient to give 30 % precipitation was added to the extract with continuous stirring. The
extract was centrifuged at 24000xg for 30 min at 4
o
C. The precipitate was discarded and
solid ammonium sulphate was added to the supernatant to give 90 % saturation and was
allowed to stand for 4 hours at 4
o
C. The precipitate was centrifuged at 24000xg for 30 min
