Cloning and characterization of an apple (Malus domestica [L.] Borkh) calmodulin gene

Abstract

A full-length calmodulin cDNA clone was isolated and used to monitor steady-state calmodulin mRNA levels in apple (Malus domestica [L.] Borkh) plant organs. Changes in calmodulin mRNA levels were observed in response to wounding. Genomic Southern analysis revealed that calmodulin is encoded by a small number of genes and that numerous calmodulin-related genes are present. The nucleotide sequence for one calmodulin gene, including approximately 900 bp of 5′ upstream DNA encompassing the startpoint of transcription, was determined. The coding sequence appeared to be interrupted by a single intron. Other than the common TATA box, there was no other significant homology between the 5′ flanking region and those of other known calmodulin genes. However, two sequence motifs (AATATTTTAATT and CCCCTCCC), which exhibit similarities to previously identified binding sites for important nuclear factors (AT-1, AP-2 respectively) are present in the upstream region of the apple calmodulin gene. To detect changes in gene-specific transcript abundance, a genomic fragment corresponding to the 5′-untranslated leader sequence of the calmodulin gene was used to probe northern blots containing total RNA isolated from plantlets at various times after wounding. The hybridization pattern obtained with this probe was almost the same as the one obtained with the full-length cDNA probe.