The low-molecular-weight glutenin subunit proteins of primitive wheats. III. The genes from D-genome species

The isolation and characterisation by DNA sequencing of two different low m olecular weight glutenin subunit (LMW-GS) genes from a genomic library deri ved from Triticum tauschii is described. These genes are similar (more than 90% similarity) but not identical to previously published LMW-GS gene sequ ences from cultivated wheats. A comparison of nucleotide sequence of the co ding regions revealed the presence of insertions and deletions preferential ly located in the region encoding the domains in the LMW-GS proteins rich i n proline and glutamine and the middle part of the C-domain. The signal seq uences, the amino-terminus and the remaining parts of the C-domain were con served between all the LMW-GSs compared. The differences detected between t he deduced amino-acid sequences in these three regions are only due to sing le nucleotide substitutions. The most important characteristic of all compa red LMW-GS genes is the conservation of eight cysteine residues that could be involved in potential secondary or tertiary structure and disulphide-bon d interactions. Comparisons between the 5' and 3' non-coding sequences of o ne of the isolated clones (LMW-16/10) with those of different prolamin gene s from wheat, barley and rye led to the distinction of five different gene families, and confirmed the evolutionary relationships determined previousl y for these genes mainly on the basis of the coding region. In particular, the LMW-GS sequences are more closely related to the B-hordein sequences th an to any other prolamin genes from wheat, barley and rye. Formal proof tha t the isolated genes coded for LMW-GSs, as defined by gel electrophoresis, was obtained by moving one of these genes (LMW-16/10) into a bacterial expr ession vector based on bacteriophage T7 RNA polymerase. The resulting plasm id directed the synthesis of large amounts of the mature form of the subuni t in Escherichia coli. This protein exhibited solubility characteristics id entical to those of the LMW-GSs and cross-reacted with antibodies reactive with these proteins.