Abstract

β-Galactosidases have been proposed as key hydrolytic enzymes involved in cell wall degradation and rapid softening during postharvest processing and storage of papaya. To genetically improve the shelf life of papaya, we identified a softening-related β-Galgene in fruit pulp at the 50% pericarp yellowing stage and isolated a novel β-Galpromoter region. An online database search predicted that the core promoter motifs and cis-acting elements in the isolated sequence were related to phytohormones, particularly to ethylene and stress responsiveness. GUS staining revealed different patterns of transient GUS expression in papaya organs driven by the putative promoter, with the highest level in the fruit pulp followed by the embryo and the root. Further, such expression was wound inducible in vascular tissues. Co-transformation of the two T-DNAs was performed, mediated by Agrobacterium tumefaciensharboring a plant expression vector with the fruit pulp-specific promoter and an inverted repeat β-Gal cassette in one T-DNA and marker genes in the other T-DNA. A total of 24 regenerated plantlets were obtained, one of which was identified to be co-transformed using GUS staining, PCR assay and Southern blotting.

Key words: β-Gal, Carica papaya, fruit softening, gene silencing, marker-free transgenic plants, two-T-DNA transformation.

Abbreviation

ACS, 1-Aminocyclopropane-1-carboxylate synthase; ACO, -aminocyclopropane-1-carboxylate oxidase; β-GAL, β-galactosidase; EST, expressed sequence tags; IRB, inverted repeat β-Gal; IPCR, inverse PCR; CTAB,cetyltrimethylammonium bromide; MS, Murashige and Skoog.