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. 2013 Mar 11;14(3):5650-67.
doi: 10.3390/ijms14035650.

Proteogenomic Characterization of Novel x-Type High Molecular Weight Glutenin Subunit 1Ax1.1

Miguel Ribeiro  1 , Emmanuelle Bancel, Annie Faye, Mireille Dardevet, Catherine Ravel, Gérard Branlard, Gilberto Igrejas
Affiliations

Proteogenomic Characterization of Novel x-Type High Molecular Weight Glutenin Subunit 1Ax1.1

Miguel Ribeiro et al. Int J Mol Sci. .

Abstract

Analysis of Portuguese wheat (Triticum aestivum L.) landrace 'Barbela' revealed the existence of a new x-type high molecular weight-glutenin subunit (HMW-GS) encoded at the Glu-A1 locus, which we named 1Ax1.1. Using one-dimensional and two-dimensional electrophoresis and mass spectrometry, we compared subunit 1Ax1.1 with other subunits encoded at the Glu-A1 locus. Subunit 1Ax1.1 has a theoretical molecular weight of 93,648 Da (or 91,508 Da for the mature protein) and an isoelectric point (pI) of about 5.7, making it the largest and most acidic HMW-GS known to be encoded at Glu-A1. Specific primers were designed to amplify and sequence 2601 bp of the Glu-A1 locus from the 'Barbela 28' wheat genome. A very high level of identity was found between the sequence encoding 1Ax1.1 and those encoding other alleles of the locus. The major difference found was an insertion of 36 amino acids in the central repetitive domain.

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Figures

Figure 1
Figure 1
Reduced and alkylated glutenin subunit patterns of selected bread wheat accessions analyzed by SDS–PAGE using a 12% gel for both high molecular weight-glutenin subunits (HMW-GS) and low molecular weight-glutenin subunits (LMW-GS). The HMW-GS are labeled according to the nomenclature of Payne and Lawrence [18]. Lane 1, ‘Fukihokomugi’; lane 2, ‘Barbela 28’; lane 3, ‘PI 355’; lane 4, ‘Carala’; lane 5, ‘211 12 04’; lane 6, ‘13-21’; lane 7, ‘Konini’. The arrowhead points to the Glu-A1 allele 1.1. Sizes (in daltons) of protein molecular weight markers are shown on the left.
Figure 2
Figure 2
Two-dimensional electrophoresis pattern (IEF × SDS-PAGE) of the HMW-GS of wheat line ‘Barbela 28’. The arrowhead points to the Glu-A1 allele 1.1. One-dimensional SDS-PAGE separations are shown to the left of the two-dimensional separations.
Figure 3
Figure 3
Overlap of two-dimensional patterns of HMW-GS from ‘Barbela 28’ wheat line and other extracts (A) with the variety ‘Carala’ and (B) with an equal mixture from varieties ‘Carala’ and ‘Atlas 66’. Subunits 1.1, 1, 2 and 2* are indicated.
Figure 4
Figure 4
MALDI-TOF mass spectra of the two high molecular weight glutenin subunits 1Ax1 and 1Ax1.1. (A) Individual mass traces and (B) Overlaid mass traces.
Figure 5
Figure 5
Alignment of 1Ax1.1 (GenBank® accession no. JN172932.1) and 1Ax1 (GenBank® accession no. X61009.1) HMW-GS nucleotide coding sequences. The identity between the two sequences is 95%.
Figure 5
Figure 5
Alignment of 1Ax1.1 (GenBank® accession no. JN172932.1) and 1Ax1 (GenBank® accession no. X61009.1) HMW-GS nucleotide coding sequences. The identity between the two sequences is 95%.
Figure 6
Figure 6
Alignment of the predicted primary structure of 1Ax1.1 and 1Ax1 HMW-GS. The dashes indicate the deletion of 36 amino acids at the end of the central repetitive domain at position +771 relative to the 1Ax1.1 sequence. Substitutions are indicated by colons.
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References

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