Skip to main content
SpringerLink
Log in

Expression of the high molecular weight glutenin 1Ay gene from Triticum urartu in barley

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

In this study, we successfully expressed the active 1Ay subunit of Triticum urartu in barley by Agrobacterium-mediated transformation with a transformation efficiency of 19.9%. The results of SDS–PAGE revealed that the expressed proteins of 1Ay subunit were present at some grains of each of 46 original T0 plants, showing identical mobility to those of positive standards of T. urartu grain protein and bacteria expressional proteins. In the T2 generation, three homozygous lines, 2–28, 3–11, and 5–6, were identified. The results of scanning electron microscopy showed an increased amount of protein bodies in these transgenic lines. The main effects in the expression of the 1Ay subunits was a considerable increase in the glutenin content, but a decrease in the contents of gliadins while there were no effects in the contents of albumin, globulin and the total protein. We found that the gluten could not be washed out from the flour obtained from transgenic barley lines when using a Gluten index analyzer and a Farinograph indicating that the transgenic barley lines could not form dough. The lack of x-type HMW-GS and the reduction in number of subunit were inferred as the possible reasons for the inability to form gluten polymer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA (2008) High-throughput Agrobacterium-mediated barley transformation. Plant Methods 4:22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blake TK, Ullrich SE, Nilan RA (1982) Mapping of the Hor-3 locus encoding D hordein in barley. Theor Appl Genet 63:367–371

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Liu C, Shi B, Chai Y, Han N, Zhu M, Bian H (2017) Overexpression of HvHGGT enhances tocotrienol levels and antioxidant activity in barley. J Agric Food Chem 65:5181–5187

    Article  CAS  PubMed  Google Scholar 

  • Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Éva C, Téglás F, Zelenyánszki H, Tamás C, Juhász A, Mészáros K, Tamás L (2018) Cold inducible promoter driven Cre-lox system proved to be highly efficient for marker gene excision in transgenic barley. J Biotechnol 265:15–24

    Article  CAS  PubMed  Google Scholar 

  • Ferdous J, Whitford R, Nguyen M, Brien C, Langridge P, Tricker P (2017) Drought-inducible expression of Hv-miR827 enhances drought tolerance in transgenic barley. Funct Integr Genom 17:279–292

    Article  CAS  Google Scholar 

  • Gilhumanes J, Pistón F, Shewry PR, Tosi P, Barro F (2011) Suppression of gliadins results in altered protein body morphology in wheat. J Exp Bot 62:4203

    Article  CAS  Google Scholar 

  • Gu YQ, Anderson OD, Londeorë CF, Kong X, Chibbar RN, Lazo GR (2003) Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes. Genome 46:1084–1097

    Article  CAS  PubMed  Google Scholar 

  • Halford NG, Field JM, Blair H, Urwin P, Moore K, Robert L, Thompson R, Flavell RB, Tatham AS, Shewry PR (1992) Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality. Theor Appl Genet 83:373–378

    Article  CAS  PubMed  Google Scholar 

  • Harwood W (2014) A protocol for high-throughput Agrobacterium-mediated barley transformation. Methods Mol Biol 1099:251–260

    Article  CAS  PubMed  Google Scholar 

  • Hisano H, Sato K (2016) Genomic regions responsible for amenability to Agrobacterium-mediated transformation in barley. Sci Rep 6:37505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010

    Article  CAS  PubMed  Google Scholar 

  • Jiang Q, Wei Y, Wang F, Wang J, Yan Z, Zheng Y (2009) Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions. BMC Plant Biol 9:16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang Q, Ma J, Wei Y, Liu Y, Zheng Y (2012) Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding. BMC Plant Biol 12:73

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jones HD (2005) Wheat transformation: current technology and applications to grain development and composition. J Cereal Sci 41:137–147

    Article  CAS  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174

    Article  CAS  PubMed  Google Scholar 

  • Lafiandra D, D’Ovidio R, Porceddu E, Margiotta B, Colaprico G (1993) New data supporting high Mr glutenin subunit 5 as the determinant of quality differences among the pairs 5 + 10 vs. 2 + 12. J Cereal Sci 18:197–205

    Article  CAS  Google Scholar 

  • Lamacchia C, Shewry PR, Fonzo ND, Forsyth JL, Harris N, Lazzeri PA, Napier JA, Halford NG, Barcelo P (2001) Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. J Exp Bot 52:243–250

    Article  CAS  PubMed  Google Scholar 

  • Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mackie AM, Lagudah ES, Sharp PJ, Lafiandra D (1996) Molecular and biochemical characterisation of HMW glutenin subunits from T. tauschii and the D genome of hexaploid wheat. J Cereal Sci 23:213–225

    Article  CAS  Google Scholar 

  • Mrizova K, Jiskrova E, Vyroubalova S, Novak O, Ohnoutkova L, Pospisilova H, Frebort I, Harwood W, Galuszka P (2013) Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility. PLoS ONE 8:e79029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Osborne TB (1907) The proteins of the wheat kernel. Carnegie Institution of Washington, Washington

    Book  Google Scholar 

  • Payne PI (1983) Catalogue of alleles for the complex gene loci, GluA1, Glu-B1 and Glu-D1 which code for high-molecular-weight subunits of glutenin in hexaploid wheat. Cereal Res Commun 11:29–35

    Google Scholar 

  • Payne PI (1987) Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Annu Rev Plant Biol 38:141–153

    Article  CAS  Google Scholar 

  • Payne P, Corfield K, Blackman J (1979) Identification of a high-molecular-weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theor Appl Genet 55:153–159

    Article  CAS  PubMed  Google Scholar 

  • Payne PI, Corfield KG, Holt LM, Blackman JA (1981) Correlations between the inheritance of certain high molecular weight subunits of glutenin and breadmaking quality in progencies of six crosses of bread wheat. J Sci Food Agric 32:51–60

    Article  CAS  Google Scholar 

  • Payne PI, Nightingale MA, Krattiger AF, Holt LM (2010) The relationship between HMW glutenin subunit composition and the bread-making quality of British-grown wheat varieties. J Sci Food Agric 40:51–65

    Article  Google Scholar 

  • Piston F, Shewry PR, Barro F (2007) D hordeins of Hordeum chilense: a novel source of variation for improvement of wheat. Theor Appl Genet 115:77–86

    Article  PubMed  Google Scholar 

  • Sallaud C, Meynard D, Van BJ, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408

    Article  CAS  PubMed  Google Scholar 

  • Santamaria M, Diaz-Mendoza M, Perez-Herguedas D, Hensel G, Kumlehn J, Diaz I, Martinez M (2018) Overexpression of HvIcy6 in barley enhances resistance against Tetranychus urticae and entails partial transcriptomic reprogramming. Int J Mol Sci 19:697

    Article  CAS  PubMed Central  Google Scholar 

  • Shewry P, Halford N (2002) Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot 53:947–958

    Article  CAS  PubMed  Google Scholar 

  • Shewry PR, Jones HD (2005) Transgenic wheat: where do we stand after the first 12 years? Ann Appl Biol 147:1–14

    Article  CAS  Google Scholar 

  • Shewry PR, Halford NG, Tatham AS (1992) High molecular weight subunits of wheat glutenin. J Cereal Sci 15:105–120

    Article  CAS  Google Scholar 

  • Shewry PR, Tatham AS, Barro F, Barcelo P, Lazzeri P (1995) Biotechnology of breadmaking: unraveling and manipulating the multi-protein gluten complex. Bio/Technology 13:1185

    CAS  Google Scholar 

  • Shewry PR, Halford NG, Lafiandra D (2003a) Genetics of wheat gluten proteins. Adv Genet 49:111–184

    Article  CAS  PubMed  Google Scholar 

  • Shewry PR, Halford NG, Tatham AS, Popineau Y, Lafiandra D, Belton PS (2003b) The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. Adv Food Nutr Res 45:219–302

    Article  CAS  PubMed  Google Scholar 

  • Shewry PR, Halford NG, Tatham AS, Popineau Y, Lafiandra D, Belton PS (2003c) The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. Adv Food Nutr Res 45:219–302

    Article  CAS  PubMed  Google Scholar 

  • Shibata D, Liu YG (2000) Technical focus-Agrobacterium-mediated plant transformation with large DNA fragments. Trends Plant Sci 5:354–357

    Article  CAS  PubMed  Google Scholar 

  • Shou H, Frame BR, Whitham SA, Kan W (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208

    Article  CAS  Google Scholar 

  • Tang Y-L, Yang W-Y, Tian J-C, Chen F (2008) Effect of HMW-GS 6 + 8 and 1.5 + 10 from synthetic hexaploid wheat on wheat quality traits. Agric Sci China 7:1161–1171

    Article  Google Scholar 

  • Travella S, Ross S, Harden J, Everett C, Snape J, Harwood W (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789

    Article  CAS  PubMed  Google Scholar 

  • Waines JG, Payne PI (1987) Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum, T. urartu, and the A genome of bread wheat (T. aestivum). Theor Appl Genet 74:71–76

    Article  CAS  PubMed  Google Scholar 

  • Wan Y, Wang D, Shewry P, Halford N (2002) Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica. Theor Appl Genet 104:828–839

    Article  CAS  PubMed  Google Scholar 

  • Wieser H (2007) Chemistry of gluten proteins. Food Microbiol 24:115

    Article  CAS  PubMed  Google Scholar 

  • Xia L, Geng H, Chen X, He Z, Lillemo M, Morris CF (2008) Silencing of puroindoline a alters the kernel texture in transgenic bread wheat. J Cereal Sci 47:331–338

    Article  CAS  Google Scholar 

  • Yang Q, Deng M, Zhang LL, Zhang XW, Wang LN, Chen H, Ma J, Qi PF, Jiang QT, Lan XJ (2016) A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation. Plasmid 87–88:58–64

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFD0100100), the Key Research and Development Program of Sichuan Province (2018NZDZX0002), the International Science and Technology Cooperation project of Sichuan Province, China (2016HH0057).

Author information

Author notes

  1. Qiang Yang and Siyu Li have contributed equally to this paper.

Authors and Affiliations

  1. Triticeae Research Institute, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China

    Qiang Yang, Siyu Li, Xiaoyu Li, Jian Ma, Jirui Wang, Pengfei Qi, Guoyue Chen, Zhien Pu, Wei Li, Xiujin Lan, Mei Deng, Yuming Wei, Youliang Zheng & Qiantao Jiang

  2. John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK

    Wendy Harwood

  3. CSIRO Agriculture and Food, Black Mountain, Canberra, ACT, 2601, Australia

    Zhongyi Li

  4. The Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810008, Qinghai, China

    Bao-Long Liu

  5. Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, T1J 4B1, Canada

    Zhenxiang Lu

Authors
  1. Qiang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siyu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaoyu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jian Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jirui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pengfei Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Guoyue Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhien Pu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wendy Harwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zhongyi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Bao-Long Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xiujin Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Mei Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Zhenxiang Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Yuming Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Youliang Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Qiantao Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YQ carried out the experiments and wrote the draft; LSY did the barley transformation; LXY and MJ finished the SDS–PAGE detection; WJR and QPF designed the plant expression vector; CGY, PZE and LW extracted DNA for the experiment and finished the PCR detection; Wendy Harwood guided the barley transformation; LZY conducted data analysis; DM did the qRT-PCR; LXJ and LZX contributed to Southern blotting; WYM reviewed the manuscript; ZYL contributed to the improvement of the research program; JQT revised the manuscript and conducted data analysis. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Qiantao Jiang.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig.

 1. Barley transformation. a The immature grains for embryo isolation. b Isolated immature embryo. c Induction of embryogenic callus. d Regeneration of transgenic shoots. e Rooting of transgenic plants. f A T0 plant in soil. g Transgenic barley growing in greenhouse. h Gus staining of transgenic lines (right) compared to non-transgenic controls (left). (TIFF 879 kb)

Supplementary Fig.

 2. PCR analysis of the transgenic barley T0 plants. Lanes 1 to 9 represent some of the transgenic barley lines; the CK- indicates the genomic DNA of non-transgenic barley ‘Golden promise’, and the CK + indicates the plasmid DNA of pCAMBIA1302-Glu-D1-1Ay. (TIFF 213 kb)

Supplementary Fig.

 3. Scanning electron microscopy images of dried barley grains. a donor barley ‘Golden promise’; b transgenic barley line 2-28; c transgenic barley line 3-11; d transgenic barley line 5-6. Yellow arrows: large starch granule A type. Blue arrows: B type starch granule. Red arrows: protein matrices. Bars indicating 20 um are shown on each micrograph. (TIFF 758 kb)

Supplementary material 4 (DOCX 16 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Q., Li, S., Li, X. et al. Expression of the high molecular weight glutenin 1Ay gene from Triticum urartu in barley. Transgenic Res 28, 225–235 (2019). https://doi.org/10.1007/s11248-019-00117-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-019-00117-6

Keywords

Search

Navigation