Suppression of alpha-amylase gene expression by antisense oligodeoxynucleotide in barley cultured aleurone layers.

Abstract

Antisense oligodeoxynucleotides (ODNs) have been applied to regulate gene expression using cell-free media or animal cells. Here we demonstrate the specific inhibition of barley alpha-amylase gene expression by synthetic antisense ODNs. In a cell free system using wheat-germ extracts, 5 microM of a 20-mer antisense ODN prevented the synthesis of the polypeptide corresponding to the predetermined length of alpha-amylase translated in vitro, whereas there was no effect on other protein synthesis. Furthermore, in cultured aleurone cells, alpha-amylase activity was efficiently decreased by addition of ODNs. At the concentrations higher than 5 microM, antisense ODN inhibited alpha-amylase gene expression almost completely. These results imply that ODN could transport into the cultured aleurone cells crossing the cell membrane, and regulate specific gene expression. This simple model system could be applicable not only for the analysis of the alpha-amylase multigene family in barley but also for studying functions of cryptic genes in higher plant.