Characterization Of Novel Glutenin Subunits In Wheat Related Species And Their Coding Gene Cloning And Molecular Evolutionary Analysis

The glutenins are the major components of wheat storage proteins, which consist of high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS). It is well known that the composition and content of HMW-GS and LMW-GS could influence the flour processing and bread-making quality. Previous investigations showed that there were a lot of novel allelic variations in the seed storage protein compositions in Glu-1D~t locus of Aegilops tauschii. (DD, 2n=2x=14) and Glu-A3 locus of Triticum monococcum L. (A~mA~m , 2n=2x=14). Thus, separation and identification of novel glutenin genes from wheat related species are highly important. In this study, some specific HMW-GS and LMW-GS from Aegilops tauschii and Triticum monococcum were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimentional electrophoresis (2-DE), high performance capillary electrophoresis (HPCE), reversed-phase high performance liquid chromatographic (RP-HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). And then allele specific PCR primers were designed to amplify, clone and sequence these specific glutenin genes, and phylogenetic analysis of storage protein gene family and secondary structure predication of glutenin were carried out The HMW-GS genes cloned were further characterized by expressing in Escherichia coli. The main results were as the followings:1. Separation and identification of specific HMW-GS in Aegilops tauschiiThree novel HMW-GS in Aegilops tauschii accessions T16, T132 and T128 were detected by SDS-PAGE, named as 1Dx 1.6~t, 1Dx3~t and 1Dx5.2~t, respectively. Some new subunits combination such as 1Dx1.6~t+1Dy12.4~t, 1Dx3~t+1Dy10.1~t and 1Dx5.2~t +1Dy12~t, were also found. The novel subunits were further characterized by IEF× SDS-PAGE, HPCE and RP-HPLC. On the 2-DE patterns, a majority of subunits were divided into a few components except for the subunits 1Dy12~t and 1Dy10.1~t. In particular, single lDx subunits were usually separated into 2 to 4 protein dots while the multiple peaks of single HMW-GS under acidic CE conditions were detected, suggesting that these subunits may exist post-translational modifications (PTMs). The analysis of HMW-GS from Aegilops tauschii through RP-HPLC indicated that the...