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Wang K.,Murdoch University | Wang K.,Capital Normal University | Islam S.,Murdoch University | Ma J.,Murdoch University | And 7 more authors.
Hereditas | Year: 2014

Wheat bread-making quality is mainly determined by glutenin proteins in the grain, which exist in a wide range of variable alleles with differential influence on processing attributes. A recently identified allele, Bx7 over-expression (Bx7oe), has been showing highly significant positive effects on wheat dough strength over the normally expressed Bx7 allele. SDS-PAGE and normal RP-HPLC procedures failed to separate the two alleles. In the current study, an extensively optimised MALDI-TOF based procedure and a refined DNA based marker for efficiently differentiating Bx7oe from normal Bx7 allele were established. Results indicated that the MALDI-TOF procedure is cost effective, high throughput, and proven reliable, while the refined PCR marker only amplifies Bx7oe allele, a clear advantage over the previously developed codominant marker. © 2015 The Authors.

Wang K.,Murdoch University | Wang K.,Capital Normal University | Wang K.,Chinese Academy of Agricultural Sciences | Ma J.,Murdoch University | And 7 more authors.
International Journal of Mass Spectrometry | Year: 2015

The wheat seed storage protein plays a key role in determining the processing quality. The disulfide bonds formed between cysteine residues in these proteins are critical in the formation of the unique rheological properties of wheat dough, which is the physical basis of bread making. Determining the number of cysteine residues in a particular protein, especially in high molecular weight glutenin subunits (HMW-GS), is an important task in evaluating wheat glutenin effects on end-product quality. In the current study, we established a fast method to accurately measure the number of cysteine residues in the HMW-GS. An alkylation reagent, 4-vinylpyridine (4-vp), was used to treat the proteins during extraction. For every cysteine residue in a protein, this treatment increases its molecular mass value by 105.14 Da, which can be accurately determined by MALDI-TOF equipment. Based on the changes of the molecular mass value caused by 4-vp treatment, the number of cysteine residues in a protein can be reliably determined. This method is also confirmed to be useful in studying non-glutenin proteins such as lupin seed storage proteins. It is expected that this method will speed up the process of selecting desirable HMW-GS in wheat breeding. © 2015 Elsevier B.V. All rights reserved.

Peng Y.,Huazhong Agricultural University | Peng Y.,Murdoch University | Peng Y.,Australia Export Grains Innovation Center | Yu K.,Huazhong Agricultural University | And 8 more authors.
PLoS ONE | Year: 2015

High molecular weight glutenin subunits (HMW-GSs) are key determinants for the end-use quality of wheat. Chinese wheat landraces are an important resource for exploring novel HMW-GS genes to improve the wheat baking quality. Two novel Glu-1Dy HMW-GSs (designated as 1Dy12.6 and 1Dy12.7) were identified and cloned from two Chinese wheat landraces Huazhong830 and Luosimai. The 1Dy12.6 and 1Dy12.7 subunits were deposited as the NCBInr Acc. No KR262518, and KR262519, respectively. The full open reading frames (ORFs) of 1Dy12.6 and 1Dy12.7 were 2022 bp and 1977 bp, encoding for proteins of 673 and 658 amino acid residues, respectively. Each contains four typical primary regions of HMW-GSs (a signal peptide, N-and C-terminal regions, and a central repetitive region). Their deduced molecular masses (70,165 Da and 68,400 Da) were strikingly consistent with those identified by MALDI-TOF-MS (69,985Da and 68,407 Da). The 1Dy12.6 is the largest 1Dy glutenin subunits cloned in common wheat up to date, containing longer repetitive central domains than other 1Dy encoded proteins. In comparison with the most similar active 1Dy alleles previously reported, the newly discovered alleles contained a total of 20 SNPs and 3 indels. The secondary structure prediction indicated that 1Dy12.6 and 1Dy12.7 have similar proportion of α-helix, β-turn, and β-bend to those of 1Dy10 (X12929). The phylogenetic analysis illustrated that the x-and y-type subunits of glutenins were well separated, but both 1Dy12.6 and 1Dy12.7 were clustered with the other Glu-1Dy alleles. Our results revealed that the 1Dy12.6 and 1Dy12.7 subunit have potential to strengthen gluten polymer interactions, and are valuable genetic resources for wheat quality improvement. © 2015 Peng et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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