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The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly

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Abstract

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

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Abbreviations

A-PAGE:

Acidic polyacrylamide gel electrophoresis

LMW-GS:

Low molecular weight glutenin subunits

LC-MS/MS:

Ion trap liquid chromatography-electrospray ionization tandem mass spectrometry

RP-HPLC:

Reversed-phase high-performance liquid chromatography

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SNPs:

Single-nucleotide polymorphisms

InDels:

Insertions/deletions

References

  • Allaby RG, Banerjee M, Brown TA (1999) Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat. Genome 42:296–307

    Article  PubMed  CAS  Google Scholar 

  • An X, Zhang Q, Yan Y, Li Q, Zhang Y, Wang A et al (2006) Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.). Theor Appl Genet 113:383–395

    Article  PubMed  CAS  Google Scholar 

  • Anderson OD, Greene FC (1997) The α-gliadin gene family II: DNA and protein sequence variation, subfamily structure, and origins of pseudogenes. Theor Appl Genet 95:59–65

    Article  CAS  Google Scholar 

  • Anderson OD, Litts JC, Gautier MF, Greene FC (1984) Nucleic acid sequence and chromosome assignment of a wheat storage protein gene. Nucleic Acids Res 12:8129–8144

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Bartels D, Thompson RD (1986) Synthesis of mRNAs coding for abundant endosperm proteins during wheat grain development. Plant Sci 46:117–125

    Article  CAS  Google Scholar 

  • Bittner C, Grassau B, Frenzel K, Baur X (2008) Identification of wheat gliadins as an allergen family related to baker's asthma. J Allergy Clin Immun 121:744–749

    Article  PubMed  CAS  Google Scholar 

  • Colot V, Robert LR, Kavanagh TA, Bevan MW, Thompson RD (1987) Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. Embo J 6:3559–3564

    PubMed Central  PubMed  CAS  Google Scholar 

  • Cornell HJ, Wills-Johnson G (2001) Structure-activity relationships in coeliac–toxic gliadin peptides. Amino Acids 21:243–253

    Article  PubMed  CAS  Google Scholar 

  • D'Aloisio E, Paolacci AR, Dhanapal AP, Tanzarella OA, Porceddu E, Ciaffi M (2010) The protein disulfide isomerase gene family in bread wheat (T. aestivum L.). BMC Plant Biol 10:101

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R et al (2001) Brachypodium distachyon: a new model system for functional genomics in grasses. Plant Physiol 127:1539–1555

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Faris JD, Zhang Z, Fellers JP, Gill BS (2008) Micro-colinearity between rice, Brachypodium and T. monococcum at the wheat domestication locus Q. Funct Integr Genomic 8:149–164

    Article  CAS  Google Scholar 

  • Gao LY, Ma WJ, Chen J, Wang K, Jing L, Wang SL (2010) Characterization and comparative analysis of wheat high molecular weight glutenin subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS. J Agric Food Chem 58:2777–2786

    Article  PubMed  CAS  Google Scholar 

  • Gibbon BC, Larkins BA (2005) Molecular genetic approaches to developing quality protein maize. Trends Genet 21:227–233

    Article  PubMed  CAS  Google Scholar 

  • Gu YQ, Wanjugi H, Coleman-Derr D, Kong XY, Anderson OD (2010) Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins. Funct Integr Genomics 10:111–122

    Article  PubMed  CAS  Google Scholar 

  • Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:1

    Article  CAS  Google Scholar 

  • Huo N, Lazo GR, Vogel JP, You FM, Ma YQ, Hayden DM et al (2008) The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences. Funct Integr Genomics 8:135–147

    Article  PubMed  CAS  Google Scholar 

  • International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768

    Article  CAS  Google Scholar 

  • Kasarda DD, D'Ovidio R (1999) Deduced amino acid sequence of an alpha-gliadin gene from spelt wheat (spelta) includes sequences active in celiac disease. Cereal Chem 76:548–551

    Article  CAS  Google Scholar 

  • Kawaura K, Mochida K, Ogihara Y (2005) Expression profile of two storage-protein gene families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags. Plant Physiol 139:1870–1880

    Article  PubMed Central  PubMed  Google Scholar 

  • Kellogg EA (2001) Evolutionary history of the grasses. Plant Physiol 125:1198–1205

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kimura M (1977) Preponderance of synonymous changes as evidence for the neutral theory of molecular evolution. Nature 267:275–276

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Mohan A, Balyan HS, Gupta PK (2009) Orthology between genomes of Brachypodium, wheat and rice. BMC Res Notes 2:93

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Larré C, Penninck S, Bouchet B, Lollier V, Tranquet O, Denery-Papini S et al (2010) Brachypodium distachyon grain: identification and subcellular localization of storage proteins. J Exp Bot 61(6):1771–1783

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Laudencia-Chingcuanco DL, Vensel WH (2008) Globulins are the main seed storage proteins in Brachypodium distachyon. Theor Appl Genet 117:555–563

    Article  PubMed  CAS  Google Scholar 

  • Li XH, Ma WJ, Gao LY, Zhang YZ, Wang AL, Ji KM (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

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Li QY, Ji KM, Zhang YZ, An XL, He ZH, Xia XC et al (2009) Characterization of monoclonal antibodies specific to wheat glutenin subunits and their correlation with quality parameters. Can J Plant Sci 89:11–19

    Article  CAS  Google Scholar 

  • Li X, Wang K, Wang S, Gao L, Xie Z, Hsam S et al (2010) Molecular characterization and comparative transcriptional analysis of LMW-m-type genes from wheat (Triticum aestivum L.) and Aegilops species. Theor Appl Genet 121:845–856

    Article  PubMed  CAS  Google Scholar 

  • Nei M, Gojobori T (1986) Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3:418–426

    PubMed  CAS  Google Scholar 

  • Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497

    Article  PubMed  CAS  Google Scholar 

  • Shewry PR, MiXin BJ, Kasarda DD (1984) The structural and evolutionary relationships of the prolamin storage proteins of barley, rye and wheat. Philosl T Roy Soc L 304:297–308

    Article  CAS  Google Scholar 

  • Shewry PR, Tatham AS, Halford NG (1999) The prolamins of the Triticeae. In: Shewry PR, Casey R, Shewry PR, Casey R (eds) Seed proteins. Kluwer Academic Publishers, Dordrecht

    Chapter  Google Scholar 

  • Van Herpen T, Goryunova SV, van der Schoot J, Mitreva M, Salentijn E, Vorst O et al (2006) Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes. BMC Genomics 7:1–13

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Wang K, Han XF, Dong K, Gao LY, Li HY, Ma WJ et al (2010) Characterization of seed proteome in Brachypodium distachyon. J Cereal Sci 52:177–186

    Article  CAS  Google Scholar 

  • Wang SL, Wang K, Chen GX, Lv DW, Han XF, Yu ZT et al (2012) Molecular characterization of LMW-GS genes in Brachypodium distachyon L. reveals highly conserved Glu-3 loci in Triticum and related species. BMC Plant Biol 12:221

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Wieser H (2001) Comparative investigations of gluten proteins from different wheat species. III. N-terminal amino acid sequences of α-gliadins potentially toxic for coeliac patients. Eur Food Res Technol 213:183–186

    Article  CAS  Google Scholar 

  • Xie Z, Wang C, Wang K, Wang S, Li X, Zhang Z et al (2010) Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.). Theor Appl Genet 121:1239–1251

    Article  PubMed  CAS  Google Scholar 

  • Xu JH, Messing J (2008) Organization of the prolamin gene family provides insight into the evolution of the maize genome and gene duplications in grass species. Proc Natl Acad Sci U S A 105:14330–14335

    Article  PubMed Central  PubMed  Google Scholar 

  • Xu JH, Messing J (2009) Amplification of prolamin storage protein genes in different subfamilies of the Poaceae. Theor Appl Genet 119:1397–1412

    Article  PubMed  CAS  Google Scholar 

  • Yan Y, Hsam SLK, Yu JZ, Jiang Y, Zeller FJ (2003) 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

    Article  CAS  Google Scholar 

  • Yue SJ, Li H, Li YW, Zhu YF, Guo JK, Liu YJ et al (2008) Generation of transgenic wheat lines with altered expression levels of 1Dx5 high-molecular weight glutenin subunit by RNA interference. J Cereal Sci 47:1–161

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was financially supported by grants from the Chinese Ministry of Science and Technology (2009CB118303), the National Natural Science Foundation of China (31271703 and 31101145), and Key Project of National Plant Transgenic Genes of China (2011ZX08009-003-004 and 2011ZX08002-004).

Author information

Authors and Affiliations

  1. College of Life Science, Capital Normal University, 100048, Beijing, China

    G. X. Chen, D. W. Lv, W. D. Li, S. Subburaj, Z. T. Yu, Y. J. Wang, X. H. Li & Y. M. Yan

  2. Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, 100081, Beijing, China

    K. Wang & X. G. Ye

  3. State Agriculture Biotechnology Centre, Western Australian Department of Agriculture and Food, Murdoch University, Perth, WA, 6150, Australia

    Wujun Ma

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  1. G. X. Chen
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  2. D. W. Lv
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  3. W. D. Li
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  5. Z. T. Yu
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Corresponding authors

Correspondence to Wujun Ma or Y. M. Yan.

Additional information

G. X. Chen, D. W. Lv, and W. D. Li contributed equally to this work.

Electronic supplementary material

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Supplementary Table 1

Protein identification from SDS-PAGE bands in B. distachyon by LC-MS-MS (DOC 89 kb)

Supplementary Table 2

The positions of SNPs and InDels identified among 5 α-gliadin genes and other 19 genes from GenBank, including EF561278 (T. aestivum), DQ234066 (T. aestivum subsp. spelta ), HM120221 (T. aestivum ), EF569971 (T. aestivum subsp. sphaerococcum), EU018299 (T. aestivum), FJ441092 (T. monococcum), EF218656 (A. tauschii), GQ131532 (A. tauschii), EU849650 (Thinopyrum intermedium), HM452956 (Lophopyrum elongatum), EU849641 (L. elongatum), HM452983 (Eremopyrum bonaepartis), HM452981 (E. bonaepartis), HM452974 (Pseudoroegneria spicata), HM452970 (P. spicata), GU168577 (Dasypyrum breviaristatum), EU186107 (D. breviaristatum), HM585045 (Agropyron desertorum), and EU026395 (Australopyrum retrofractum). (DOC 93 kb)

Supplementary Table 3

The average number of each toxicity epitopes in α-gliadins from Brachypodium, Triticum, and related genomes. (DOC 81 kb)

Supplementary Table 4

A total of 530 α-gliadin genes from different Triticum related species used in this study (DOC 103 kb)

Supplementary Fig. 1

Mutliple alignment of the deduced amino acid sequences of 25 α-gliadin genes, including 6 cloned from Brachypodium and other 19 genes from Triticum and related species deposited in GenBank: EF561278, DQ234066, HM120221, EF569971, and EU018299 from T. aestivum; FJ441092 from T. monococcum; EF218656 and GQ131532 from A. tauschii; EU849650 from T. intermedium; HM452956 and EU849641 from L. elongatum; HM452983 and HM452981 from E. bonaepartis; HM452974 and HM452970 from P. spicata; GU168577 and EU186107 from D. breviaristatum; and HM585045 from A. desertorum and EU026395 from A. retrofractum. Black frames indicate the position of cysteine residues and dashes represent the deletions. The red frames of the aligned sequences show the position of the peptides, which had activity in CD (in the N-terminal domain) and associated with adenovirus type 12 infections (in the C-terminal domain). (DOC 170 kb)

Supplementary Fig. 2

Comparison of the 5′ flanking DNA sequences of 23 α-gliadin genes: four sequences from B. distachyon, include one sequence from diploid Bd3-1 and Bd21, respectively, two from tetraploid genotypes B347, and one from hexaploid Bd19; four sequences from Chinese Spring (CS); three sequences from T. monococcum L.; three sequences from A. comosa (MM), three sequences from A. umbellulata (UU), two sequences from A. markgrafii (CC), and three sequences from A. uniaristata (NN), respectively; −300 element, GCN motif, TATA box, and CAAT box are enclosed in a red box. In all genes, dot and dashes indicate the same sequences with Bd3-1 and deletion. (DOC 210 kb)

Supplementary Fig. 3

Specificity of qRT-PCR amplification. a The standard curve of the α-gliadin genes and housekeeping genes, including the amplification efficiencies and R 2 values (coefficient of determination). b Dissociation curves of the α-gliadin genes showing single peaks (each including three technical replicates for each of 12 cDNA pools from developmental stages). c Agarose gel (2 %) showing the amplification of a single specific PCR product of expected size for the α-gliadin genes and housekeeping genes. (DOC 1544 kb)

Supplementary Fig. 4

Multiple alignment of the deduced amino acid sequences of α-gliadin gene (FJ159430) from wheat and prolamins (Bradi1g50300.1, Bradi2g39920.1, Bradi1g50205.1, Bradi1g50200.1, Bradi2g39940.1, Bradi2g38530.1, and Bradi1g50307.1) from B. distachyon, which were blast and obtained from the B. distachyon genome project Websites (http://www.brachypodium.org/). (DOC 4028 kb)

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Chen, G.X., Lv, D.W., Li, W.D. et al. The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly. Funct Integr Genomics 14, 149–160 (2014). https://doi.org/10.1007/s10142-013-0353-0

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