Subscribe Now Subscribe Today
Research Article
 

Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis



Zhuo Huang, Hai Long, Yu-Ming Wei and Ze-Hong Yan
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

In this study, PCR primers were designed base on the known genes in wheat and its relatives to isolate α-gliadin genes from Ae. sharonensis, one species of the Sitopsis section of the genus Aegilops, which was traditionally considered as the B-genome donor of tetraploid and hexaploid wheat. Three novel α-gliadin genes were obtained. Analyses of the nucleotide and deduced amino acids of the obtained genes indicated that they shared the high sequence identities and similar primary structures to the known α-gliadin genes. Further more, the extensive variations were found. A phylogenic analysis based on the multigene alignment of the deduced amino acid sequences showed that the α-gliadin genes derived from Ae. sharonensis and Ae. speltoides were significantly distinguished from those of diploid A and D genome progenitors of wheat, but clustered closed to some genes from tetra- and hexaploid wheats. Moreover, genes derived from Ae. sharonensis were more closed to wheat than from Ae. speltoides. This result suggested that the origin and formation of wheat B genome might be polyphyletic.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Zhuo Huang, Hai Long, Yu-Ming Wei and Ze-Hong Yan, 2010. Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis. Journal of Plant Sciences, 5: 256-262.

DOI: 10.3923/jps.2010.256.262

URL: https://scialert.net/abstract/?doi=jps.2010.256.262
 
Received: April 08, 2010; Accepted: June 05, 2010; Published: July 27, 2010



INTRODUCTION

For most of the human population, plant foods, especially cereal grains, provide the majority of nutritionally important proteins. In wheat, the most abundant storage proteins in endosperm are gliadins and glutenins, representing about 80% of the total protein in the wheat grain (Shewry et al., 1997). The gluten polymer is composed mainly of High-Molecular-Weight (HMW) and Low-Molecular-Weight (LMW) Glutenin Subunits (GS) linked by disulphide bonds (Shewry et al., 1989, 1992; MacRitchie, 1992). The genes coding for HMW-GS are located on the long arms of chromosomes 1A, 1B and 1D at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively (Payne et al., 1987). The LMW-GS are encoded by genes on the short arm of group-1 chromosomes at the Glu-A3, Glu-B3 and Glu-D3 loci (Singh and Shepherd, 1988). Gliadins are normally monomeric proteins, which are classified into three groups, α, γ and ω on the basis of their electrophoretic mobility in acidic polyacrylamide gel electrophoresis (Anderson and Greene, 1997; Metakovsky et al., 1984). Genes coding for most of the γ- and ω-gliadins are tightly clustered at three homoeologous loci, Gli-A1, Gli-B1 and Gli-D1, on the short arms of chromosomes 1A, 1B and 1D, respectively. The α-gliadins are encoded by tightly clustered genes at three homoeologous loci, Gli-A2, Gli-B2 and Gli-D2, on the short arm of each group-6 chromosome (Metakovsky et al., 1984; Metakovsky, 1991).

Although gliadins account for about 50% of the gluten proteins in wheat and the allele variation in wheat and its relatives has been widely evaluated (Pan et al., 2007; Zhuang et al., 2007; Xiong et al., 2008), their role in determining the mixing properties of dough are not well understood (Pistóna et al., 2006). This is because correlations between single gliadin and functional properties are difficult to determine due to the complex patterns and overlapping fractions present in polyploidy species. The well understand of the gliadin multigene families will not only provide the possibility to express and functionally investigate a single gliadin component or a group of similar gliadins in vitro, but also benefit to research the origin and evolution aspects of the gene families, which might also supply some information of genome evolution in wheat and its relatives.

The Sitopsis section of the genus Aegilops, including Ae. longissima (2n = 2x = 14, SlSl), Ae. sharonensis (2n = 2x = 14, SshSsh), Ae. searsii (2n = 2x = 14, SsSs), Ae. bicornis (2n = 2x = 14, SbSb) and Ae. speltoides (2n = 2x = 14, SS), were proposed as B genome donors of wheat (Sarkar and Stebbins 1956). In this article, the isolation and characterization of novel α-gliadin genes from Ae. sharonensis were reported. The comparison with known α-gliadin genes and their phylogenic relationship were also discussed.

MATERIALS AND METHODS

Plant Materials
The Ae.sharonesis (2n=2x=14, SshSsh) accession PI 584350 was used in this study and were kindly supplied by USDA-ARS.

Isolation of α-Gliadin Genes
Genomic DNA was isolated using cetytrimethylammonium bomide (CTAB) procedure as reported by Murray and Thompson (1980). Based on the conserved sequences of the known α-gliadin genes, a pair of degenerate primers, α-F: 5’-G (G/C) TCAATACAAATCCA(C/T)CATG-3’ and α-R: 5’- TTCTCTTCTCAGTT (A/G) GTACC (A/G) -3’ was designed for the complete open reading frame (ORF) of α-gliadin genes. Polymerase Chain Reactions (PCR) were performed in 100 μL reaction volume, consisting of 4U ExTaqTM DNA polymerase (TaKaRa) with high fidelity, 10 μL PCR buffer (supplied with Taq DNA polymerase), 200 ng genomic DNA, 1.5 mM MgCl2 and 100 mM of each dNTP. PCR amplifications were conducted according to the following program: 95°C for 5 min denaturation followed by 35 cycles of 45s at 95°C, 45s at 57°C and 45s at 72°C. PCR products were separated in 1.5% agarose gels. The desired DNA fragments were cloned into the pMD18-T plasmid vector (TaKaRa) and several predominated clones were randomly selected for DNA sequencing. All the experiments were conducted in the laboratory of Triticeae Research Institute, Sichuan Agricultural University, during March, 2008 to May, 2009.

Sequence Analyses
The obtained sequences by PCR amplification and DNA sequencing were confirmed to be α-gliadin genes by using ORF finder and Blastp programs deposited in NCBI network (http://www.ncbi.nlm.nih/gov/). Sequence analyses were conducted by using the programs DNAMAN, BioEdit and MEGA 3.1 (Kumar et al., 2004).

RESULTS AND DISCUSSION

Isolation of α-Gliadin Genes from Ae. sharonesis
With genomic PCR using primers α-F and α-R, a DNA fragment of about 900 bp was amplified from the genomic DNA of Ae. sharonesis (Fig. 1). Ten positive clones with expected insert were randomly selected for DNA sequencing and three unique sequences, s47a-2, s47a-4 and s47a-11, were finally obtained. Analysis using ORFfinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) indicated that all three sequences contained a unique continuous ORF which were 930, 900 and 873 bp long, respectively.

Sequence Analysis
Sequence analysis indicated that three sequences could be translated into the proteins consisting of 309, 299 and 290 amino acid residues, respectively. Alignment of the deduced amino acid sequences showed that the obtained genes contained six domains (Fig. 2) as suggested by Anderson and Greene (1997). The leader sequences of the deduced α-gliadin of Ae. sharonesis encode a signal peptide consisting of 20 amino acid residues (Fig. 2). It was conserved among the three obtained genes.

The repetitive domain was composed of repeat motifs which were rich in proline and glutamine. S42-2 and S42-11 shared same repetitive domain. Comparing to S42-2 and S42-11, a deletion (six amino acids) and an insertion (7 amino acids) and several amino acid substitutions were present in S42-4 (Fig. 2).

Polyglutamine domains I and II were mainly composed of glutamines (Fig. 2). The Polyglutamine domain I in S42-2, S42-4 and S42-11 consisted of 31-32 amino acids and contained 23-26 glutamines (About 74-81%). The Polyglutamine domain II had a higher glutamine content and more variable than I. S42-2 had a longest Polyglutamine domain II with 29 amino acids, among which 28 were glutamines. The Polyglutamine domain II in S42-4 contained 19 amino acids and 17 were glutamines. However, S42-11 had only nine amino acids in this domain and among eight were glutamines.


Image for - Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis
Fig. 1: Amplification product obtained from genomic DNA of Ae. sharonesis using primers α-F and α-R

Image for - Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis
Fig. 2: Alignment and primary structure of the amino acid sequences of the α-gliadin genes obtained in this study

Table 1: Sequence similarity to the known α-gliadin genes
Image for - Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis
aPseudogene; bDirectly submitted to GenBank

The unique domains I and II were extremely conserved the three obtained α-gliadin genes (Fig. 2). Only nine amino acid substitutions were found. Six cysteines were in these two domains and four and two cysteines were in unique domains I and II, respectively.

Sequences Similarity and Phylogenic Analysis
Sequence comparison indicated that the obtained three α-gliadin genes show high degree similarities with the known α-gliadin genes from hexaploid, tetraploid and diploid wheat (Table 1). Phylogenic analysis using several known α-gliadin genes from different species was conducted. It indicated that the S47a-4 was close to the genes from Ae. speltoides, whereas S47a-2 and S47a-11 were clustered with genes from hexa- (AABBDD) and tetraploid (AABB ) wheats (Fig. 3).

Previous study found that approximately 50% of the α-gliadin genes are pseudogenes (Anderson and Greene, 1997). Van Herpen et al. (2006) reported that the fraction of pseudogenes is much higher in diploid wheat species, which were 72% in A genome species (T. monococcum) and 95% in B genome species (Ae. speltoides and Ae. longissima). In this study, three α-gliadin genes were isolated from Ae.sharonesis, another species of Sitopsis section of the genus Aegilops.


Image for - Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis
Fig. 3: Phylogenic analyses of the α-gliadin genes of tetraploid and hexaploid wheats and their diploid progenitors. The genes with accession No. as DQ 002588, DQ002586, DQ002584, DQ002585, DQ002587, DQ002651 were derived from Aegilops speltoides; EF218658 and EF561272 were from Ae. tauchii; DQ401698 and DQ401700 were from T. monococcum; DQ401696 and M16496 were from T. urartu, DQ296195 was from T. durum; EF561274, K03075, X02540, EF569975, K03074 and X02538 were from T. aestivum, EF569978 and EF569971 were from T. sphaerococcum

However, no pseudogene was obtained from the ten randomly sequenced clones. Though the exact copy number of the α-gliadin genes in Ae. sharonesis has not been revealed and their sequences have not also been fully obtained, the results from the present study indicated that the percentage of the pseudogenes in Ae. sharonesis is lower than those in wheat and its relatives. This may result from different evolution process of these species.

The common wheat, T. aestivum, possesses three sets of homologous genomes, designated as AABBDD, of which the wild diploid Ae. tauschii and T. urartu or T. monococcum were the D and A genome donors (Huang et al., 2002). One or more species in the section Sitopsis of the genus Aegilops, were traditionally considered as the wild diploid B genome donor of wheat. The α-gliadin genes have been isolated from Ae. speltoides and Ae. longissima. But their relationship with those in wheat remains unknown. In this study, three novel genes were obtained from Ae. sharonensis. Phylogenic analysis indicated that the α-gliadin genes from A, D genomes or Sitopsis species were significant divergent from each other. This means the sequences of α-gliadin genes were genome-specific, similar to the previously found in another type of seed storage protein genes (Long et al., 2005, 2006). Further more, genes from Ae. sharonensis and Ae. speltoides were close to some from wheat with the ABD and AB genomes and the former seems closer than the latter. Though the exact chromosome location of these wheat α-gliadin genes is unknown, they apparently divergent from A or D genome. These results suggested that they might be derived from the B genome. Although Ae. speltoides was suggested as the exact B genome donor of polyploidy wheat more recently (Petersen et al., 2006). The results obtained in this study were not consistent with it, for that the α-gliadin genes in sharonensis seemed to be more closed to wheat than those from Ae. speltoides. This result suggested the polyphyly for the B genome which would come from intergenome recombination between different species in section Sitopsis of genus Aegilops. Further research need to isolate the α-gliadin genes from other species of section Sitopsis of the genus Aegilops and obtain more information of α-gliadin genes derived from A, B and D genomes of wheat. This may help to better understand the structure and relationship of α-gliadin multigene families.

ACKNOWLEDGMENTS

This study was supported by the Science and Technology Foundation (grants No. 200701) of Dujiangyan Campus, Sichuan Agricultural University, the Scientific Research Fund of Sichuan Provincial Education Department (grants No. 08ZA068) and the Key Technologies R and D Program of China (2006BAD01A02-23 and 2006BAD13B02).

REFERENCES

  1. Anderson, O.D. and F.C. Greene, 1997. The-gliadin gene family. II. DNA and protein sequence variation, subfamily structure and the role of group 6 and group 2 chromosomes in gliadins synthesis. Theor. Applied Genet., 95: 59-65.
    CrossRef  |  


  2. Huang, S., A. Sirikhachornkit, X. Su, J. Faris, B. Gill, R. Haselkorn and P. Gornicki, 2002. Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum-Aegilops complex and the evolutionary history of polyploid wheat. Proc. Nat. Acad. Sci. USA., 99: 8133-8138.
    PubMed  |  


  3. Kumar, S., K. Tamura and M. Nei, 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform., 5: 150-163.
    CrossRef  |  PubMed  |  Direct Link  |  


  4. Long, H., Y M. Wei, Z.H., Yan, B. Baum, E. Nevo and Y.L. Zheng, 2006. Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes. Sci. China Ser. C-Life Sci., 49: 322-331.
    CrossRef  |  


  5. Long, H., Y.M. Wei, Z.H. Yan, B. Baum, E. Nevo and Y.L. Zheng, 2005. Classification of wheat low-molecular-weight glutenin subunit genes and its chromosome assignment by developing LMW-GS group-specific primers. Theor. Applied Genet., 111: 1251-1259.
    CrossRef  |  PubMed  |  Direct Link  |  


  6. MacRitchie, F., 1992. Physicochemical properties of wheat proteins in relation to functionality. Adv. Food Nutr. Res., 36: 1-87.
    Direct Link  |  


  7. Metakovsky, E.V., 1991. Gliadin allele identification in common wheat II. J. Gen. Breed., 45: 325-344.


  8. Metakovsky, E.V., A.Y. Novoselskaya and A.A. Sozinov, 1984. Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis. Theor. Applied Genet., 69: 31-37.
    CrossRef  |  


  9. Murray, M.G. and W.F. Thompson, 1980. Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res., 8: 4321-4326.
    CrossRef  |  PubMed  |  Direct Link  |  


  10. Pan, D., L. Hong, L. Wei, Q. Peng-Fei, W. Yu-Ming and Y.L. Zheng, 2007. Genetic diversity of storage proteins in Triticum polonicum L. J. Plant Sci., 2: 416-424.
    CrossRef  |  Direct Link  |  


  11. Payne P.I., J.A. Seekings, A.J. Worland, M.G. Jarvis and L.M. Holt, 1987. Allelic variation of glutenin subunits and gliadins and its effect on bread making quality in wheat: Analysis of F5 progeny from Chinese Spring x Chinese Spring (Hope 1A). J. Cereal Sci., 6: 103-118.
    CrossRef  |  


  12. Pistona, F., G. Doradob, A. Martina and F. Barro, 2006. Cloning of nine γ-gliadin mRNAs (cDNAs) from wheat and the molecular characterization of comparative transcript levels of γ-gliadin subclasses. J. Cereal Sci., 43: 120-128.


  13. Petersen, G., O. Seberg, M. Yde and K. Berthelsen, 2006. Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B and D genomes of common wheat (Triticum aestivum). Mol. Phylogenet. Evol., 39: 70-82.
    CrossRef  |  PubMed  |  


  14. Reeves C.D. and T.W. Okita, 1987. Analyses of alpha/beta-type gliadin genes from diploid and hexaploid wheats. Gene, 52: 257-266.
    PubMed  |  


  15. Shewry, P.R., N.G. Halford and A.S. Tatham, 1989. The High Molecular Weight Subunits of Wheat, Barley and Rye: Genetics, Molecular Biology, Chemistry and Role in Wheat Gluten Structure and Functionality. In: Oxford Surveys of Plant Molecular and Cell Biology, Miflin B.J. (Ed.). Vol. 2, Oxford University Press, Oxford, ISBN: 019854202X, pp: 163-219


  16. Sarkar, P.G. and L. Stebbins, 1956. Morphological evidence concerning the origin of the B genome in wheat. Am. J. Bot., 43: 297-304.
    Direct Link  |  


  17. Shewry, P.R., A.S. Tatham and P. Lazzeri, 1997. Biotechnology of wheat quality. J. Sci. Food Agric., 73: 397-406.
    Direct Link  |  


  18. Singh, N.K. and K.W. Shepherd, 1988. Linkage mapping of genes controlling endosperm storage proteins in wheat. Theor. Applied Genet., 75: 642-650.
    CrossRef  |  


  19. Sumner-Smith, M., J.A. Rafalski, T. Sugiyama, M. Stoll and D. Soell, 1985. Conservation and variability of wheat alpha/beta-gliadin genes. Nucleic Acids Res., 11: 3905-3916.
    PubMed  |  


  20. Van Herpen, T.W., S.V. Goryunova, J. van der Schoot, M. Mitreva and E. Salentijn 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.
    PubMed  |  


  21. Wang, H.Y., Y.M. Wei, H.Y. Ze and Y.L. Zheng, 2007. Isolation and analysis of alpha-gliadin gene coding sequences from Tritcum durum. Agric. Sci. China, 6: 25-32.
    CrossRef  |  


  22. Xiong, L.J., W. Li, Y.M. Wei and Y.L. Zheng, 2008. Variation of high-molecular-weight glutenin subunits and gliadin in T. aestivum ssp. macha. Asian J. Biol. Sci., 1: 19-25.
    CrossRef  |  Direct Link  |  


  23. Zhuang, P.P., J.R. Wang, Y.M. Wei and Y.L. Zheng, 2007. Evaluation of the storage protein variations and agronomic performance in Persian wheat (Triticum carthlicum Nevski). Int. J. Agric. Res., 2: 528-536.
    CrossRef  |  Direct Link  |  


  24. Shewry, P.R., N.G. Halford and A.S. Tatham, 1992. High molecular weight subunits of wheat glutenin. J. Cereal Sci., 15: 105-120.
    CrossRef  |  


©  2022 Science Alert. All Rights Reserved