Molecular cloning, heterologous expression, and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from the G genome of Triticum timopheevi

Abstract

A novel y-type high molecular weight (HMW) glutenin subunit gene from the G genome of Triticum timopheevi (2n = 4x = 28, AAGG) was isolated and characterized. Genomic DNA from accession CWI17006 was amplified and a 2200 bp fragment was obtained. Sequence analysis revealed a complete open reading frame including N- and C-terminal ends and a central repetitive domain encoding 565 amino acid residues. The molecular weight of the deduced subunit was 77 031, close to that of the x-type glutenin subunits. Its mature protein structure, however, demonstrated that it was a typical y-type HMW subunit. To our knowledge, this is the largest y-type subunit gene among Triticum genomes. The molecular structure and phylogenetic analysis assigned it to the G genome and it is the first characterized y-type HMW glutenin subunit gene from T. timopheevi. Comparative analysis and secondary structure prediction showed that the subunit possessed some unique characters, especially 2 large insertions of 45 (6 hexapeptides and a nonapeptide) and 12 (2 hexapeptides) amino acid residues that mainly contributed to its higher molecular weight and allowed more coils to be formed in its tertiary structure. Additionally, more a-helixes in the repeat domain of the subunit were found when compared with 3 other y-type subunits. We speculate that these structural characteristics improve the formation of gluten polymer. The novel subunit, expressed as a fusion protein in E. coli, moved more slowly in SDS–PAGE than the subunit Bx7, so it was designated Gy7*. As indicated in previous studies, increased size and more numerous coils and a-helixes of the repetitive domain might enhance the functional properties of HMW glutenins. Consequently, the novel Gy7* gene could have greater potential for improving wheat quality.