Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana

Abstract

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage lambda gt11 library of cDNA from Arabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)+ RNA from leaves of A. thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence of Arabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP of A. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochrome c peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochrome c peroxidase are conserved in the amino acid sequence of Arabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3' untranslated region of the cDNA.