Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

Abstract

A cDNA library was derived from the poly(A)⁺ RNA of young tomato leaves. The library was cloned in a λgt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5′ end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)⁺ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.