Isolation and Characterization of a Tomato Acid Phosphatase Complementary DNA Associated with Nematode Resistance

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Abstract
The tomato (Lycopersicon esculentum) acid phosphatase-1 (Apase-1(1), EC 3.1.3.2) isozyme variant, genetically linked to the root-knot nematode resistance locus (Mi) on chromosome 6, has been purified by a rapid procedure from tomato cell suspension cultures. Peptide fragments of the purified enzyme were generated from trypsin and Lys-C endoprotease digests and separated by reverse-phase high-performance liquid chromatography. Amino acid sequences derived from the purified peptide fragments represented >50% of the total amino acid content of the protein and enabled the construction of degenerate oligonucleotide probes that were used to screen a tomato cell culture complementary DNA library. Clones corresponding to full-length coding sequences for Apase-1 have been isolated and sequenced. Southern blot analysis of DNA isolated from a number of tomato cultivars shows that the Apase-1(1) gene (aps1) is present at one copy per genome and that genotypes containing the aps1(1) allele have restriction fragment length polymorphisms that distinguish them from cultivars having the aps1(+) allele. Segregation analysis demonstrates that the restriction fragment length polymorphisms are associated with the aps1 locus. Tomato Apase-1(1) is also found to have significant homology at the amino acid sequence level to a class of vegetative storage proteins characterized in soybean.