Isolation and promoter characterization of barley gene Itr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutant lys3a endosperm

Abstract

The gene Itr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library of Hordeum vulgare L. The gene has no introns and presents in its 5'-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the 'copia-like' retro-transposon Bare-1. Functional analysis of the Itr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system the Itr1 promoter retains its endosperm specifity and the trans-regulation mediated by the Lys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer full GUS expression and for endosperm specifity. In protoplasts derived from the lys3a mutant, Risø 1508, GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.