Hormonal control of alpha-amylase gene expression in barley. Studies using a cloned CDNA probe.

Abstract

Starting with an enriched alpha-amylase mRNA preparation, cDNA was synthesized by reverse transcription and was cloned at the Pst I site of plasmid vector pBR322 by the "G-C tailing" procedure. Clones containing alpha-amylase cDNA sequences were identified among the recombinant clones by in vitro translation of hybrid-selected mRNA followed by immunoprecipitation of translation products with antiserum to alpha-amylase. The longest alpha-amylase cDNA clone isolated was 630 base pairs long and was characterized by digestion with restriction enzymes. Using this clone as a labeled nucleic acid hybridization probe, we observed that multiple bands of DNA with alpha-amylase sequences were present in restriction digests of barley embryo DNA. This indicates the presence of a family of barley alpha-amylase or alpha-amylase-like genes. Analysis of RNA blots with the cloned alpha-amylase hybridization probe indicated that alpha-amylase mRNA is synthesized de novo in barley aleurones after addition of gibberellic acid. The gibberellic acid-induced accumulation of alpha-amylase mRNA is blocked by cycloheximide which suggests the requirement of a newly synthesized protein factor for efficient expression of alpha-amylase gene.