Studies On High Performance Capillary Electrophoresis Of Gliadins And High Molecular Weight Glutenin Subunits (HMW-GS) In Wheat(Triticum Aestivum L.)

It is known that wheat storage proteins, including gliadins and glutenins are responsible for bread-making quality, which are highly heterogeneous in composition. Thus, electrophoretic patterns of storage proteins could be used as genetic markers. Acid-polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-PAGE (SDS-PAGE) and high performance liquid chromatography (HPLC) are currently the routine methods to separate and characterize wheat storage proteins. In recent years, a new separation technique, high performance capillary electrophoresis (HPCE) has been developed. It has show great potential in the separation and characterization of proteins, peptides, DNA and so on. So far great progresses have been made in the research on crop storage proteins by capillary electrophoresis.This paper, on the base of A-PAGE and SDS-PAGE methods, studied on the separation of gliadins and glutenins by high performance capillary electrophoresis. By means of A-PAGE method, it was further proved that the electrophoretic composition of wheat gliadins is determined by genotypes and is reliable markers for wheat varietal identification and quality improvement. We also optimized the conditions of gliadin HPCE and developed a powerful HPCE technique for the separation and characterization of gliadins. The optimal conditions for gliadin HPCE were 0.1M phosphate-glycine (pH 2.50) buffer, containing 20% acetonitrile (ACN) and 0.05% Hydroxypropylmethyl-cellulose(HPMC); a 27 cm length capillary (50 u m i.d.) was used at 40癈 and 12.5 KV; samples were extracted by 30% ethanol. By using the method developed, four different wheat cultivars can be easily discriminated. Therefore, gliadin capillary electrophoresis is powerful and reliable tool for varietal identification.The high molecular weight glutenin subunit (HMW-GS) compositions of Triticum aestivum L., Triticum dicoccum L. and Aegilops squarrosa L. (Triticum tauschii L.) by SDS-PAGE and HPCE were investigated. The optimal conditions of HMW-GS capillary electrophoresis were 0.1M phosphate-glycine (pH2.50) buffer, containing 20%ACN and 0.05% HPMC; a 27 cm length capillary (50 urn i.d.) was used at 40C and 12.5 KV; samples were extracted by 50% 1-propanol, containing 1% dithiothreitol (DTT). In comparison with the results obtained from different cultivars, some HMW-GS such as 1Ax1, 1Ax5, 1Ax5, 1Bx7, 1By8, 1By9, 1Dx10 and!Dy12 can be identified. HPCE separation of good quality subunits 5+10 and poor quality subunits 2+12 was also studied. The result showed that it is easily to identify subunit 5 and 2.For the first time, HMW-GS of Aegilops squarrosa L. accessions were separated and characterized by HPCE. The results showed that some novel subunits were found, such as1Dy12.1, 1Dy12.2, 1DyT2 , and their HPCE patterns with excellent resolution were obtained. These novel HMW-GS present in Aegilops squarrosa L. are expected to be useful gene resources for wheat quality improvement.Our results showed that, in comparison with the traditional separation methods such as A-PAGE and SDS-PAGE, high performance capillary electrophoresis appears to be capable of rapid, high-resolution for the separation of wheat storage proteins and has great potential for wheat varietal identification and quality improvement.