The cloning of calmodulincDNAs from barley and Arabidopsis and expression of barley calmodulin in transgenic tobacco

Abstract

Calmodulin (CaM) is a calcium-binding protein found in eukaryotes, and appears to be essential for growth and development. CaM is thought to play a pivotal role in modulating biochemical pathways in conjunction with transient calcium fluxes into the cytoplasm. In order to better understand calcium-regulated signal transduction pathways in plants at the molecular level, cDNA sequences of CaM and CaM-like proteins were cloned and characterized from higher plants.

A barley (Hordeum vulgare L.) leaf cDNA library in lambda phage gt10 was initially screened with a cam-specific synthetic, 14 nucleotide oligonucleotide probe. A full-length cDNA encoding barley CaM was isolated. Using barley CaM cDNA as heterologous DNA probes, three cDNAs were isolated from an Arabidopsis thaliana cDNA library. Two cDNAs encoded CaM isoforms and one cDNA encoded a CaM-like protein (ACaMLP). Northern hybridization analysis reveal differential transcript accumulation of CaM isoforms and calmodulin-like protein in Arabidopsis total RNA.

In order to examine effects of CaM action in vivo, tobacco leaf discs (Nicotiana tabacum, cv. Xanthi) were transformed with barley CaM expression vectors driven by a 35-cauliflower mosaic virus promoters. Homozygous R1 plants were bred and were found to contain one- to two-fold increase of CaM protein as compared to normal tobacco. Some aberrant phenotypes were found and are discussed.