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 cDN A clones, designated as carnation flower maturation-induced (CFMI), were ve rified 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 th e clones, CFMI-5, showed high sequence similarity to the cysteine proteinas e inhibitor gene, predicted to be involved in newer maturation, The full le ngth cDNA clone CFMI-5 was 531 nucleotides (nts) long and consisted of an o pen reading frame of 294 nucleotides, encoding a 98 amino acid protein, 12 nucleotides of S-untranslated region and 3'-untranslated region (225 nts) w ith a poly(A)(+) tail. The predicted CFMI-5 amino acid sequence had a conse rved sequence Gln-Val-Val-Ala-Gly, which corresponds to the active site of proteinase inhibition. Northern blot analysis revealed tissue-specific expr ession of CFMI-5 transcripts, as the transcripts were expressed preferentia lly in petals and styles, A PCR-based cDNA subtraction method, termed suppr ession subtractive hybridization, was identified as a rapid method to scree n differentially expressed genes in a short time.