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and pellet dissolved in 0.02 M, sodium phosphate buffer at pH 7.5. The enzyme solution
was dialysed for 36 hours against several changes of 15 volumes of 0.02 M, sodium
phosphate buffer solution, pH7.5. The dialysed solution was clarified by centrifugation at
24000xg for 30 min and pellet was discarded. The enzyme solution was concentrated by
ultrafiltration using an Amicon system. The concentrated enzyme was applied to a CM-
Sephadex C-50 column (2x6x37 cm) which had been previously equilibrated with 0.02 M
pH 7.5 sodium phosphate buffer. The column was washed with the equilibration buffer and
the enzyme was eluted by using 500 ml of linear gradient of 0.1 M NaCl in 0.02 M, pH 7.5
sodium phosphate buffer. Fraction were collected and assayed from protein and PE activity.
The active fractions obtained from the previous step were combined and concentrated using
Amicon filter. The concentrate sample were applied to a of Sephadex G-100 (2x6x65 cm )
column, equilibrated with 0.02 M sodium phosphate buffer pH 7.5 containing 0,2 M NaCl
and 0.02 sodium aside. The enzyme was eluted with the same buffer, until the absorbency
at 280 nm of the effluent was negligible. Active enzyme fractions were pooled,
concentrated as above. Pectinmethylesterase activity was assayed using the method of H.
Fayyaz et al. (1993).
1.6 Southern Blotting
Total genomic DNA was isolated by CTAB method (Auto e data...) 10 Pg of DNA were
digested with restriction enzymes BamHI, EcoRI and HindIII (Boheringer), separated on
0.8% agarose gels, and transferred to Nitro-cellulose membranes according to the
manufacturer's (Amersham) instructions. Membranes were probed with gel-purified, >D-
32
P@dCTP-labeled insert DNA from cpPME1 (partial-length RT-PCR clones), under the
conditions described above for RNA-blot hybridisation, washed in 5x SSC and 0.1% SDS
at 65
o
C and 0.2x SSC and 0.1 SDS at 65
o
C and exposed to film with one intensifying
screen at -80
o
C for overnight.
1.7 Phylogenetic Analysis
The deduced amino acid sequence of cpPME1 and pPME2 were aligned to 10 amino acids
sequences of pectinmethylesterase gene. Homologies between the deduced amino acid
sequences of PME were determined using Clustal V multiple-sequence alignment software.
The sequences were: 6 from tomato fruit PME, 3 from
Phaseolus. vulgaris
PME and 1
from
Petunia inflata
PME. The PME phylogenetic tree was inferred from the aligned
sequences using the maximum parsimony algorithm of the DNASTAR software.
2. Results and Discussion
A characteristic feature during the ripening of papaya fruit is softening. Softening is the
result of the structural changes in the cell wall caused by the activity of hydrolases (Hubert
1983). Pectinmethylesterase, an enzyme that catalyses demethylation of the C
6
carboxyl
group of galacturonosyl residues, may play an important role in determining the extent to
which pectin is accessible to degradation by polygalacturonase. Indeed, it has been
suggested that the increased susceptibility of tomato fruit cell walls to polygalacturonase
action during ripening is due to the action of pectinmethylesterase (Koch et al., 1989).
In
Carica papaya,
fruit softens differentially in relation to the position of the tissue.
Based on carotenoid development, Paul and Chen (1983) considered that papayas ripen
