Methodology for determining glutenin subunit composition of isogenic wheat lines varying in the number of high molecular weight glutenin subunits.

摘要

The aim of this work was to devise an accurate procedure for quantitating the glutenin subunit composition of a set of isogenic wheat lines. These wheat lines contained variable numbers of HMW-GS, thus providing a series having a wide range in the ratio of LMW/HMW-GS.; In order to obtain the polymeric (glutenin) protein for its analysis, several procedures were used to remove first the monomeric proteins (albumins, globulins, and gliadins). These procedures included dimethyil sulfoxide (DMSO) extraction, propanol-water (1:1) extraction, and collection of the polymeric protein peak from SE-HPLC using a fraction collector. Three different methods were evaluated to quantify the LMW/HMW-GS: (1) reversed-phase high-performance liquid chromatography (RP-HPLC); (2) size-exclusion high-performance liquid chromatography (SE-HPLC); and (3) SDS-PAGE of reduced glutenin. The functional properties of flours from the isogenic wheat lines were also measured using a 10 g Mixograph.; Of the three preparation procedures, extractions by both DMSO and propanol-water resulted in removal of some polymeric protein, thus leading to changes in the measured LMW/HMW-GS ratio. With fraction collection of the polymeric protein using SE-HPLC, a more quantitative recovery of glutenin was obtained but this fraction was also shown to include HMW albumins.; Of the procedures used, the best for quantitation of individual glutenin subunits is RP-HPLC. However, the procedure that is recommended for quantitation of the LMW/HMW-GS ratio is the one involving fraction collection of the glutenin followed by reduction, running samples on SE-HPLC, and quantitating the two peaks corresponding to HMW-GS and LMW-GS. The HMW albumins elute in a separate peak and can also be quantitated. The best correlation with Mixograph dough development time was with the LMW/HMW-GS ratio evaluated by the recommended procedure, tending to support this as being the best procedure. The method is accurate and can be used as a basis for development of more rapid procedures to quantify glutenin groups.