Molecular characterization of the HMW glutenin genes Dt x1.5 + Dt y10 from Aegilops tauschii and their PCR-mediated recombinants

Abstract

The high molecular weight (HMW) glutenin subunits, D t x1.5 + D t y10, are special types of storage proteins found in Aegilops tauschii that are never found in common wheat (Triticum aestivum). This study reports the characterization of the complete open reading frames (ORFs) of the HMW glutenin genes, D t x1.5 and D t y10, using a restrict-enzyme based method named the ‘restricted deletion method’ (RDM). The D t x1.5 and D t y10 were found to have an identical structure compared with the other published HMW glutenin genes. Comparison of the deduced protein sequences also indicated that the D t y10 in Ae. tauschii differed from its counterpart Dy10 in common wheat, by having insertions and deletions in the central repetitive domain. This result confirms the two subunits with same mobility in SDS-PAGE are different types of HMW glutenin subunits. In addition, four PCR-mediated recombinants of the D t x1.5 and D t y10 genes were amplified using a PCR program with shorter extension time. The recombinants had a similar structure to their corresponding natural genes, but a significantly different central repetitive domain. Western blot analysis exhibited a normal expression of the recombinants in E. coli. In addition to its usefulness for studying structure and function of the HMW glutenin subunits, the PCR-mediated recombination may provide an efficient method to generate novel HMW glutenin genes for wheat breeding.