Identification Of HMW Glutenin Subunits In Wheat By Proteome Approaches

Wheat is one of the most important grain crops. Because of the abundant protein in endosperm, it become important protein source of food (accounting for about 38.4% in total grain proteins). It has showed that the composition and content of wheat storage proteins play a major role for viscoelasticity and extensibility of dough. Therefore, it is highly important to develop more powerful techniques for the separation and characterization of wheat glutenins, especially for the high molecular weight glutenin subunits (HMW-GS). The aim of this paper is to study on the proteome approaches for the identification of HMW-GS, mainly including mass spectrometry (MS), microsequencing as well as SDS-PAGE and high performance capillary electrophoresis (HPCE) methods. The main results were as the followings:· Characterization of HMW-GS and the N-terminal microsequencingHMW-GS from T. compaction L. and T. dicoccum wheat species were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and high performance capillary electrophoresis (HPCE), and all subunits showed to be a single band or peak on the electrophoretic patterns. These subunits identified were Bx6* and By22.1 (D154), Bx6.1 (D31), Bx13* (Club19) . The single subunit separated by SDS-PAGE was then spotted to polyvinylidence difluonde (PVDF), and sequenced using N-terminal Edman degradation microsequencing. It is apparent that the N-terminal sequences of HMW-GS were highly homologenous. Furthermore, the y-type subunits show more similarity than the x-type subunits, therefore they may have different evolutionary rates during evolution of the wheat prolamine gene family. In the first 20 N-terminal sequences, the variations were found at the locations of the sixth and twentieth amino acids. Based on previous work, our results showed that it is possible to use partial N-terminal sequences replacing the complete sequences for homologenous analysis of gluten genes. The development of N-terminal microsequencing method is important for the design of AS-PCR primer and coding gene cloning of desirable HMW-GS.· Two-dimentional electrophoresis (2-DE) analysis of wheat proteinsThe seed total protein and glutenins from T. aestivum L. were extracted and separated using 2-DE (IEF x SDS-PAGE) and the powerful electrophoresis method with high resolution for wheat storage protein analysis was developed. Our results showed that the good 2-DE patterns for different seed protein groups could be obtained by the extraction method used. The electrophoretic patterns and expression differences of glutenins from bread wheat cultivar NO.33 growing in Beijing andHeilongjiang were also investigated in this study.· Analysis of HMW-GS by Mass Spectrometry (MS)Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to detect different HMW-GS. Eighteen high sensitive MS profiles of HMW-GS from different Triticum species and their precise molecule weight were achieved. It was found that the rank of molecular weight sizes in A-PAGE is closer to the actual molecular weight after comparing with the results by SDS-PAGE. All of the determined molecular weights are close to those from calculated by gene sequences, which were all within the range of analytical error, except for Bxl4 subunit. Therefore, it could be concluded that HMW glutenin subunits lacked extensive protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, although the possibility of single sugar residues linked to some subunits could not be excluded. The results indicated that MALDI-TOF-MS is fast and simple without collecting the single subunit. In addition, the single subunits after SDS-PAGE were digested for PMF and PSD analyses. The clear patterns were obtained, and then the results were subjected to database searching against SWISS-PROT, TrEMBL and NCBI. Proteins had no good matched in the proteins databases. The possible reasons were discussed.This work primarily established the proteome approaches for HMW-GS analyses, which w...