Isolation, Characterization and Farinograph Analysis of Novel HMW-GSs from Dasypyrum villosum

Abstract

Dasypyrum villosum carrying many novel HMW-GS alleles is an important genetic resource for wheat protein improvement.In this study,we isolated six HMW-GS genes from D.villosum TA10220(1.0–1.7 kb,GenBank accession numbers: KF887414–KF887419),which were substantially smaller than those from common wheat,using a pair of specific primers.An in-frame stop codon was found in the coding sequences of KF887418 and KF887419 and thus these genes might be pseudogenes.The comprehensive analysis of deduced amino acid sequence,and phylogenetic and evolutionary analyses of full sequence,N- and C-terminal domains revealed that KF887414 was closely related to y-type HMW-GS,but KF887415–KF887419 had structural characteristics of both x- and y-types.DNA fragments of KF887414–KF887417 were subcloned into the pEASY-E2 expression vector and expressed in Escherichia coli Rosetta-gami B(DE3)cell under IPTG induction.The four genes were successfully expressed in E.coli system according to SDS-PAGE analysis(both the expressed protein and HMW-GS isolated from seed)and western-blotting assay.The fusion protein was purified and recovered by His-Trap affinity chromatography and low temperature cryodesiccation,and then integrated into the control flour by using a 4 g Micro-dough LAB Farinograph.Results showed that the four HMW-GSs originated from D.villosum had positive effects on dough quality property.