Cloning and characterisation of a putative endo-polygalacturanase cDNA from ripening mango (Mangifera indica Linn cv. Nam Dok Mai)
1999
Speirs, J. | Chaimanee, P. | Bungaruang, L. | Lertwikoon, N. | Suntornwat, O.(Silpakorn Univ., Nakhon Pathom (Thailand). Faculty of Science. Dept. of Chemistry)
Exo-polygalacturonase enzyme (EC 3.2.1.67) hydrolyses the terminal alpha(1-4) link between adjacent demethylated galacturonic acid residues while endo-polygalacturonase (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 activity appears only with the onset of reipening and increases dramatically during ripening. Both endo and exo-PG were found in mango fruit with only the exo-PG increased significantly during ripening. A cDNA for a mango P.G. was made in order to study the expression of its ripening specific gene. Purified mRNA from ripening fruit was used as a template for RT-PCR. Endo and exo-PG sequences from various sources obtained from EMBL data base were aligned. Conserved regions of the sequence were identified and use to design primers for PCR. PCR products fractionated on an agarose gel and bands with appropriate sizes were eluted and ligated into a plasmid vector. One of the cDNAs isolated had a size of 811 bp and encoded with 161 amino acids open reading frame, the deduced sequence of which was related by sequence similarity to plant endo-PGs. The neareset match was to the endo-PG of kiwifruit (Acession No. L12019) which was 50.9 percent similar at the identity level and 66.5 percent similar when conserved substitutions were allowed. This PG cDNA was used as a probe for hybridisation analysis of fruit RNA preparations.
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