Cloning and characterization of a cDNA for 1-aminocyclopropane-1-carboxylate oxidase from papaya fruit.

摘要

A full-length cDNA clone encoding a putative 1-amino cyclopropane-1-carboxylate (ACC) oxidase from papaya (Carica papaya cv. Tainong 2) was amplified by PCR from cDNAs synthesized from messenger RNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete ORF encoding 310 amino acid residues. The deduced amino acid sequence shared homology (72 to 80%) with sequences of other plant ACC oxidases. No transit peptide was found, which suggests the protein is a cystolic enzyme. The 12 residues (P-5, A-27, G-32, H-39, H-177, D-179, L-195, Q-196, G-218, H-234, R-244 and S-246) are conserved among all ferrous ion and ascorbate requiring-enzymes. The putative ACC oxidase coding region from papaya was introduced into the expression vector pET-20b(+), and transformed into Escherichia coli BL21(DE3). A 38 kDa ACC oxidase protein was detected by Coomassie blue staining of SDS-PAGE. An enzyme assay and western blot immunoanalysis confirmed that the ACC oxidase cDNA clone can express active ACC oxidase in E. coli.