Cloning And Characterization Of A Putative Endo-Polygalacturonase Cdna From Ripening Mango (Mangifera Indica Linn Cv. Nam Dok Mai)

The exo-polygalacturonase enzyme (EC 3.2.1.67) hydrolyses the terminal alpha (1-4) link between adjacent demethylated galacturonic acid residues while endopolygalacturonase (EC 3.2.1.15) hydrolyses the same linkage but randomly. In general, polygalacturonase (PG) tends to be absent or barely detectable in green fruit, its acitivty appears only with the onset of ripening and increases dramatically during ripening. Both endo and exo-PG were found in mango fruit with only the exo-PG increasing significantly during ripening.A cDNA for mango FG was made in order to study the expression of its ripening specific gene. Purified mRNA from ripening mango was used as a template for RT-FCR. Endo and exo-PG sequence from various sources obtained from EMBL data base were aligned. Conserved regions of the sequence were identified and used to design primer for PCR. PCR product were fractionated on an agarose gel and bands with appropriate sizes were eluted and ligated into a plasmid vector. One of the cDNA isolated had a size of 811 bp and encoded a 161 amino acids open reading frames, the deduced sequence of which was related by sequence similarly to plant PGs. A near match was to the endo-PG of kiwi fruit (acession No. P35336) which was 51% similar at the identity level and 67% similar when conserved substitution were allowed. This PG cDNA was used as a probe for hybridisation analysis of fruit RNA preparations.