Isolation and expression of a new high molecular weight glutenin subunit gene at the Glu-D-1-2 locus from Aegilops tauschii


Two new y-type HMW-GSs in Ae. tauschii, 1Dy12.1*t and 1Dy12.2t with the mobility order of 1Dy12.2t > 1Dy12.1*t > 1Dy12.1t>1Dy12, were identified by both SDS-PAGE and MALDI-TOF-MS. Molecular cloning and sequencing showed that the genes encoding subunits 1Dy12.1*t and 1Dy12.2t had identical nucleotide acid sequences with 1,947 bp encoding a mature protein of 627 residues. Their deduced molecular weights were 67,347.6 Da, satisfactorily corresponding to that of 1Dy12.2t subunit determined by MALDI-TOF-MS (67,015.7 Da), but was significantly smaller than that of the the 1Dy12.1*t subunit (68,577.1 Da). Both subunits showed high similarities to 1Dy10, suggesting that they could have a positive effect on bread-making quality. Interestingly, the expressed protein of the cloned ORF from accessions TD87 and TD130 in E. coli co-migrated with subunit 1Dy12.2t, but moved slightly faster than 1Dy12.1*t on SDS-PAGE. The expressed protein in transgenic tobacco seeds, however, had the same mobility as the 1Dy12.1*t subunit, as confirmed by both SDS-PAGE and Western blotting. Although direct evidence of phosphoprotein could not be obtained by specific staining method, certain types of post-translational modifications (PTMs) of the 1Dy12.1*t subunit could not be excluded. We believe PTMs might be responsible for the molecular weight difference between the subunits 1Dy12.1*t and 1Dy12.2t.


  1. Agrawal, G.K., Thelen, J.J. 2005. Development of a simplified economical polyacrylamide gel staining protocol for phosphoproteins. Proteomics 5:4684–4688.

    CAS  Article  Google Scholar 

  2. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acid Res. 12:8711–8721.

    CAS  Article  Google Scholar 

  3. Dong, H., Cox, T.S., Sears, R.G., Lookhart, G.L. 1991. High molecular weight glutenin genes: Effects on quality in wheat. Crop Sci. 31:974–979.

    Article  Google Scholar 

  4. Flavell, R.B., Doldsbrough, A.P., Robert, L.S., Schnick, D., Thompson, R.D. 1989. Genetic variation in wheat HMW glutenin subunits and the molecular basis of bread-making quality. Bio/Tech 7:1281–1285.

    Google Scholar 

  5. Hickman, D.R., Roepstorff, P., Shewry, P.R., Tatham, A.S. 1995. Molecular weights of high molecular weight subunits of glutenin determined by mass spectrometry. J. Cereal Sci. 22:99–103.

    CAS  Article  Google Scholar 

  6. Hsam, S.L.K., Kieffer, R., Zeller, F.J. 2001. Significance of Aegilops tauschii glutenin genes on breadmaking properties of wheat. Cereal Chem. 78:521–525.

    CAS  Article  Google Scholar 

  7. Lamacchia, C., Shewry, P.R., Fonzo, N.D., Forsyth, J.L., Harris, N., Lazzeri, P.A., Napier, J.A., Halford, N.G., Barcelo, P. 2001. Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. J. Exp. Bot. 52:243–250.

    CAS  Article  Google Scholar 

  8. Li, X.H., Zhang, Y.Z., Gao, L.Y., Wang, A.L., Ji, K.M., He, Z.H., Appels, R., Ma, W.J., Yan, Y.M. 2007. Molecular cloning, heterologous expression and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from G genome of Triticum timopheevi. Genome 50:1130–1140.

    CAS  Article  Google Scholar 

  9. Li, X.H., Ma, W., Gao, L.Y., Zhang, Y.Z., Wang, A.L., Ji, K.M., Wang, K., Appels, R., Yan, Y. 2008. A novel chimeric LMW-GS gene from the wild relatives of wheat Ae. kotschyi and Ae. juvenalis: evolution at the Glu-3 loci. Genetics 180:93–101.

    CAS  Article  Google Scholar 

  10. Matheis, G. 1999. Phosphorylation of food proteins with phosphorus oxychloride improvement of functional and nutritional properties: A review. Food Chem. 39:13–26.

    Article  Google Scholar 

  11. Payne, P.I., Corfield, K.G. 1979. Subunit composition of wheat glutenin proteins, isolated by gel filtration in a dissociating medium. Planta 145:83–88.

    CAS  Article  Google Scholar 

  12. Pfluger, L.A., D’Ovidio, R., Margiotta, B., Pena, R., Mujeeb-Kazi, A., Lafiandra, D. 2001. Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats. Theor. Appl. Genet. 103:1293–1301.

    CAS  Article  Google Scholar 

  13. Robert, L.S., Thompson, R.D., Flavell, R.B. 1989. Tissue-specific expression of a wheat high molecular weight glutenin in transgenic tobacco. Plant Cell 1:569–578.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Salzano, A.M., Crescenzi, M. 2005. Mass spectrometry for protein identification and the study of post translational modifications. Ann. Ist. Super. Sanità. 41:443–450

    PubMed  Google Scholar 

  15. Shewry, P.R., Halford, N.G., Tatham, A.S. 1992. The high molecular weight subunits of wheat glutenin. J. Cereal Sci. 15:105–120.

    CAS  Article  Google Scholar 

  16. Yan, Y., Hsam, S.L.K., Yu, J.Z., Jiang, Y., Zeller, F.J. 2003a. Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by Sodium Dodecyl Sulphate (SDS-PAGE), Acid Polyacrylamide Gel (A-PAGE) and Capillary Electrophoresis. Euphytica 130:377–385.

    CAS  Article  Google Scholar 

  17. Yan, Y., Hsam, S.L.K., Yu, J.Z., Jiang, Y., Ohtsuka, I., Zeller, F.J. 2003b. HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (Triticum spelta L.) progenitors. Theor. Appl. Genet. 107:1321–1330.

    CAS  Article  Google Scholar 

  18. Yan, Y., Yu, J., Jiang, Y., Hu, Y., Cai, M., Hsam, S.L.K., Zeller, F.J., 2003c. Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers. Electrophoresis 24:1429–1436.

    CAS  Article  Google Scholar 

  19. Yan, Y., Zheng, J., Xiao, Y., Yu, J., Hu, Y., Cai, M., Li, Y., Hsam, S.L.K., Zeller, F.J. 2004a. Identification and molecular characterization of a novel y-type Glu-Dt1 glutenin gene of Aegilops tauschii. Theor. Appl. Genet. 108:1349–1358.

    CAS  Article  Google Scholar 

  20. Yan, Y., Jiang, Y., Sun, M., Yu, J., Xiao, Y., Zheng, J., Hu, Y., Cai, M., Li, Y., Hsam, S.L.K., Zeller, F.J. 2004b. Rapid identification of HMW glutenin subunits from different hexaploid wheat species by acidic capillary electrophoresis. Cereal Chem. 81:561–566.

    CAS  Article  Google Scholar 

  21. Zhang, Q., Dong, Y.M., An, X.L., Wang, A.L., Zhang, Y.Z., Li, X.H., Xia, X.C., He, Z.H., Yan, Y.M. 2008a. Characterization of HMW glutenin subunits in common heat and related species by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). J. Cereal Sci. 47:252–261.

    CAS  Article  Google Scholar 

  22. Zhang, Y.Z., Li, X.H., Wang, A.L., An, X.L., Zhang, Q., Pei, Y.H., Gao, L.Y., Ma, W.J., Appels, R., Yan, Y.M. 2008b. Novel x-type HMW glutenin genes from Aegilops tauschii and their implications on the wheat origin and evolution mechanism of Glu-D1-1 proteins. Genetics 178:23–33.

    CAS  Article  Google Scholar 

  23. Zhang, Y.Z., Li, Q.Y., Yan, Y.M., Zheng, J.G., An, X.L., Xiao, Y.H., Wang, A.L., Pei, Y.H., Wang, H.B., Hsam, S.L.K., Zeller, F.J. 2006. Molecular characterization and phylogenetic analysis a novel glutenin gene (Dy10.1t) from Aegilops tauschii. Genome 49:735–745.

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Y. Yan.

Additional information

Communicated by F. Békés and R.A. McIntosh

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Cite this article

Zhang, Y., An, X., Li, X. et al. Isolation and expression of a new high molecular weight glutenin subunit gene at the Glu-D-1-2 locus from Aegilops tauschii. CEREAL RESEARCH COMMUNICATIONS 37, 449–457 (2009).

Download citation


  • Aegilops tauschii
  • common wheat
  • HMW-GS
  • protein post-translational modifications