Isolation and Sequences Analysis of the α-gliadin Genes from Aegilops sharonensis
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.
Received: April 08, 2010;
Accepted: June 05, 2010;
Published: July 27, 2010
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
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.
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 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.
||Amplification product obtained from genomic DNA of Ae.
sharonesis using primers α-F and α-R
||Alignment and primary structure of the amino acid sequences
of the α-gliadin genes obtained in this study
|| Sequence similarity to the known α-gliadin genes
|aPseudogene; bDirectly submitted to
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.
||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.
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).
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