Isolation and Characterization of a cDNA Encoding the Cysteine Proteinase Inhibitor, Induced upon Flower Maturation in Carnation Using Suppression Subtractive Hybridization

The suppression subtractive hybridization (SSH) method was used to isolate differentially expressed genes during carnation flower maturation. Five cDNA clones, designated as carnation flower maturation-induced (CFMI), were verified as flower maturation-induced cDNAs. Sequence analysis of five CFMI (CFMI-5, CFMI-6, CFMI-7, CFMI-9, and CFMI-10) clones revealed that one of the clones, CFMI-5, showed high sequence similarity to the cysteine proteinase inhibitor gene, predicted to be involved in flower maturation. The full length cDNA clone CFMI-5 was 531 nucleotides (nts) long and consisted of an open reading frame of 294 nucleotides, encoding a 98 amino acid protein, 12 nucleotides of 5′-untranslated region and 3′-untranslated region (225 nts) with a poly(A)⁺ tail. The predicted CFMI-5 amino acid sequence had a conserved sequence Gln-Val-Val-Ala-Gly, which corresponds to the active site of proteinase inhibition. Northern blot analysis revealed tissue-specific expression of CFMI-5 transcripts, as the transcripts were expressed preferentially in petals and styles. A PCR-based cDNA subtraction method, termed suppression subtractive hybridization, was identified as a rapid method to screen differentially expressed genes in a short time.