Molecular characterization of HMW-GS 1Dx3t and 1Dx4t genes from Aegilops tauschii and their potential value for wheat quality improvement

Abstract

Two x-type high molecular weight glutenin subunits (HMW-GS) in Aegilops tauschii, 1Dx3(t) and 1Dx4(t) were identified by SDS-PAGE and MALDI-TOF-MS. Their complete coding sequences were isolated by AS-PCR. 1Dx3(t) and 1Dx4(t) genes consist of 2535 bp and 2508 bp and encode 845 and 836 amino acid residues, respectively. The deduced molecular masses of 1Dx3(t) and 1Dx4(t) gene products are 87655.26 Da and 86664.24 Da, respectively, well corresponding to the molecular masses measured by MALDI-TOF-MS. A total of 18 SNPs were identified between 1Dx3(t) and 1Dx4(t). Comparing with 1Dx5 subunit, 1Dx3(t) had a six amino acid insertion at 146-151 while the 1Dx4(t) had a nine amino acid deletion when compared with 1Dx3(t) subunit. The authenticity of the cloned 1Dx3(t) and 1Dx4(t) genes were confirmed by successful expression of their ORFs in E. coli. Comparison and phylogenetic tree based on the amino acid and nucleotide sequences confirmed that 1Dx3(t) was most closely related to 1Dx5 subunit that is widely accepted as a superior subunit for bread-making property. The secondary structure prediction demonstrated that 1Dx3(t) subunit has significantly high α-helix and β-strand contents, suggesting it might have positive effects on dough quality.

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

“…It was shown earlier that Dx3 shows sequence homologies with Dx subunits from Aegilops tauschii (Tausch's goatgrass), which has contributed the D genome in wheat [5], [9]. However, the M r of Dx3 found in this study (87,207) clearly differs from the protein M r s calculated from the Dx3 t (87,655) and Dx4 t (86,666) genes of Aegilops tauschii [35]. Thus, the unique primary structure of wheat Dx3 remains to be elucidated.…”

A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

“…Comparison of the peptide list derived from the MS/MS data of subunit 6 with that of the sequence from the database resulted in an MS/MS sequence coverage of 68.4%, which strongly suggests a sequence match between subunit 6 and the Bx sequence from durum wheat (Table 2). Since no gene or protein sequence was found for subunit 3, in Table 2 we used the subunit 3 MALDI-TOF MW determined by Gao et al (2010), which was determined from the sequence of subunit 4 t (815 amino acids) from Aegilops tauschii based on their good MW agreement (Wang et al, 2012). Also here, the identified peptide list derived from the MS/MS data of subunit 3 at least suggested a strong sequence homology between subunits 3 and 4 t ( Table 2).…”

Molecular cloning and characterization of six novel HMW‐GS genes from Aegilops speltoides and Aegilops kotschyi

“…According to Tam as et al (2002), glutenin subunits may act as chain extenders or chain terminators, affecting polymer size and thus dough strength and extensibility. Gao et al (2012) gave a plausible explanation for the weak strength of H45 by suggesting that the extra cysteine found in H45 sequences might act as a chain terminator. However, there is some evidence that in most cases, HMW glutenin subunits with extra numbers of cysteine residues are positively associated with improved processing quality in bread wheat varieties (Lafiandra et al 1993(Lafiandra et al , B ek es et al 1995.…”

“…Numerous novel HMW-GS have been identified and characterized from Ae. tauschii (D t D t ; Lu et al 2005, Wang et al 2012, Aegilops umbellulata (UU; Liu et al 2003), Aegilops caudata (CC; Wan et al 2000, Liu et al 2003, Aegilops searsii (S s S s ; Sun et al 2006) and Aegilops cylindrica (CCDD; Wan et al 2002). However, less study has been conducted on HMW-GS in Aegilops speltoides (SS, 2n = 2x = 14) and Aegilops kotschyi (UUSS, 2n = 4x = 28).…”