Download HMW glutenin subunits in multiploid Aegilops species: composition

NOTES
HMW glutenin subunits in
multiploid Aegilops species:
composition analysis and
molecular cloning of coding
sequences
XIE Ruili, WAN Yongfang, ZHANG Yan
& WANG Daowen
Institute of Genetics, Chinese Academy of Sciences, Beijing 100101,
China
Correspondence should be addressed to Wang Daowen (e-mail:
[email protected])
Abstract The Aegilops genus contains species closely related
to wheat. In common with wheat, Aegilops species accumulate high molecular weight (HMW) glutenin subunits in their
endospermic tissue. In this study, we investigated the composition of HMW glutenin subunits in four multiploid Aegilops
species using SDS-PAGE analysis. Furthermore, by working
with Ae. ventricosa, we established an efficient genomic PCR
condition for simultaneous amplification of DNA sequences
coding for either x- or y-type HMW glutenin subunits from
polyploid Aegilops species. Using the genomic PCR condition,
we amplified and subsequently cloned two DNA fragments
that may code for HMW glutenin subunits in Ae. ventricosa.
Based on an analysis of the deduced amino acid sequences,
we concluded that the two cloned sequences encode one xand one y-type of HMW glutenin subunit, respectively.
Keywords: Aegilops, Ae. ventricosa, HMW glutenin subunit, molecular cloning.
The seed storage proteins of common wheat are
composed of three categories of polypeptides: high molecular weight (HMW) glutenin subunits, low molecular
weight (LMW) glutenin subunits and gliadins. HMW
glutenin subunits make up 10% of the total protein content
in the mature wheat grain, its composition and function
can have a profound influence on the baking quality of
wheat flour[1 3]. In hexaploid wheat (genome AABBDD),
three loci (Glu-1A, Glu-1B, Glu-1D), located on the long
arm of the homeologous group one chromosomes 1A, 1B
and 1D, respectively, contain genes encoding HMW glutenin subunits. Within each locus, there are two closely
linked genes, one coding for a higher molecular weight xtype subunit and the other for a lower molecular weight ytype subunit. Owing to gene silencing, in the Glu-1A locus,
the y-type subunit gene is not expressed in hexaploid
wheat. The remaining five genes are all active and each of
them possesses multiple alleles (including null). As a consequence, hexaploid wheat varieties usually express four
to five HMW glutenin subunits, and often differ in their
composition of HMW glutenin subunits.
Chinese Science Bulletin Vol. 46 No. 4 February 2001
Through genetic analysis, it was demonstrated in
1981 that the composition of HMW glutenin subunits in a
given common wheat variety could affect the baking quality of its flour[4]. Recent biotechnological investigations on
transgenic expression of the genes encoding the functionally superior HMW subunit (such as the 1Dx5 subunit
specified by the Glu-1D locus of the D genome) have not
only supported the conclusion drawn through genetic
analysis, but also provided direct examples for improving
wheat flour baking quality by changing HMW glutenin
subunit compositions[5 7]. At present, the number of functionally superior HMW subunits identified in common
wheat is very limited. This necessitates the search for
more and better subunits from alternative genetic resources. The Aegilops genus is closely related to the Triticum genus, to which common wheat belongs. Many Aegilops species possess either a D genome (Ae. tauschii,
DD) or a D genome component (multiploid Aegilops species)[8]. Cytogenetic analysis has shown that the D genome
component present in common wheat and in those multiploid Aegilops species is interrelated and may be originally derived from a common ancestor (Ae. tauschii). This
evolutionary relatedness points to the possibility that Aegilops species may contain genes coding for polypeptides
structurally similar among the HMW glutenin subunits of
common wheat. This supposition has been confirmed by
SDS-PAGE analysis of Aegilops seed storage proteins and
by the cloning of a gene from Ae. tauschii encoding a
polypeptide (1Dty subunit), which shows a high level of
amino acid sequence similarity to the 1Dy subunits specified by the Glu-1D locus[9 11]. In contrast to the studies
carried for Ae. tauschii, little is known about the composition of HMW glutenin subunits and the genes encoding
these polypeptides in polyploid Aegilops species (such as
Ae. crassa, Ae. juvenalis, Ae. vavilovii and Ae. ventricosa).
The primary structure (i.e. amino acid sequence) of
thus far published HMW glutenin subunits is very conserved, the amino acid sequence in both the N and C terminus is highly similar among the different subunits[12].
The middle region of the primary sequence is named the
repetitive domain because this region consists of highly
repeated short amino acid sequence motifs. Different
HMW glutenin subunits usually differ in the length of the
repetitive domain, the type of the short amino acid sequence motifs contained, and the number of repeats for
individual motifs. In the x-type subunit, the repetitive
motifs are mainly of three types: GQQ, PGQGQQ and
GYYPTSP/SQQ. The presence of the GQQ motif is
unique to the x-type subunits. In the y-type subunit, the
repetitive motifs are also of three types: PGQGQQ,
GYYPTSLQQ and GHYPASLQQ. As a result of the conservation in terminal amino acid sequences, the nucleotide
sequences in the 5 and 3 ends of the DNA region
coding for HMW glutenin subunit are also highly homologous. This high level of nucleotide sequence homology
309
NOTES
facilitated the design of PCR primers for the amplification
of HMW glutenin subunit coding sequences[13 16]. However, this type of PCR analysis has so far limited to only a
few species of wheat and Ae. tauschii. Furthermore, there
is currently no report on simultaneous amplification of the
coding sequences for both x- and y-type subunits in single
PCR reactions.
We investigated the composition of HMW glutenin
subunits in four multiploid Aegilops species, and established an optimized PCR condition for simultaneous amplification of the coding sequences for both x- and y-type
subunits of Ae. ventricosa in single PCR reactions. Our
results contributed to the understanding of HMW glutenin
subunits and their genes in polyploid Aegilops species.
This knowledge may be an aid to the exploitation of Aegilops HMW glutenin subunits in wheat quality improvement in the future.
1 Materials and methods
( ) Plant materials. The seeds of the four polyploid species, Ae. crassa (DDD2D2McrMcr), Ae. juvenalis
(DDMjMjUU), Ae. vavilovii (DDMcrMcrSpSp) and Ae. ventricosa (DDMvMv), were provided by the Institute of Crop
Germplasm Resources of the Chinese Academy of Agricultural Sciences. Common wheat materials possessing
known HMW glutenin subunits were obtained from the
Long Ashton Research Station in UK.
( ) SDS-PAGE analysis. The endosperm proteins
were extracted from single seeds for SDS-PAGE analysis
according to the published method[17] . For each Aegilops
species, two accessions were analyzed. For each accession,
MW glutenin subunit composition was determined for
three seeds in independent experiments. Gel electrophoresis results were recorded using a digital camera.
( ) Genomic DNA extraction and genomic PCR
reactions. DNA extraction from the seedlings of Ae.
ventricosa was performed as previously described[18]. Two
pairs of PCR primers were designed according to the published nucleotide sequences of HMW glutenin genes (fig.
1). P1 (5
-ATGGCTAAGCGGC/TTA/GGTCCTCTTTG-3 ) and P2 (5 -CTATCACTGGCTG/AGCCGACAATGCG-3 ) were based on the conservation of terminal
nucleotide sequences in the coding region of HMW glutenin genes and for amplifying the complete coding region
of the gene encoding either x- or y-type subunits. P3 (5 ATCACCCACAACACCGAGCA-3 ) and P4 (5 -AGCTGCAGAGAGTTCTATCA-3 ) were based on the conservation of two stretches of nucleotide sequences located,
respectively, in the 5 and 3 untranslated region of
HMW glutenin genes and for amplifying the DNA fragment containing both the coding region and the untranslated regions. For both primer pairs (P1/P2, P3/P4), two
genomic PCR reactions were carried out. Except for the
DNA template and the nucleotide primers, the other com-
310
ponents (polymerase, nucleotides and buffer) required by
the PCR reaction were provided by either the kit (Taq
DNA polymerase PCR kit) from the Gibco BRL Company
or the kit (Advantage-GC Genomic PCR kit) from the
Clontech Company. PCR reactions using the Taq DNA
polymerase PCR kit in conjunction with P1/P2, P3/P4,
respectively, were conducted in 50 µL volume with 300 ng
genomic DNA, 0.2 mmol/L of each nucleotide and 20
µmol/L of each primer and 5 units of Taq polymerase. The
cycling parameters were: 95 for 5 min, followed by 34
cycles of 95 for 1 min, 55 for 1 min and 72 for 3
min, and a final extension at 72 for 10 min. PCR reactions using the Advantage-GC Genomic PCR kit in conjunction with P1/P2, P3/P4, respectively, were also conducted in 50 µL volume with 300 ng genomic DNA, 0.2
mmol/L of each nucleotide and 10 µmol/L of each primer
and 2.5 units of Taq polymerase. The cycling parameters
were: 94
for 1 min, followed by 34 cycles of 94 for
0.5 min, 68 for 3 min, and a final extension at 68 for
3 min. The PCR products were analyzed in 1% ethidium
bromide (EB) containing agarose gels. The results were
recorded using the Bio-Rad Gel Doc-1000 scanner.
Fig. 1. A schematic representation of the positions of the four nucleotide primers (P1, P2, P3 and P4) in relation to the coding region of an
HMW glutenin gene. ATG and TAG denote the start and stop codon of
the coding region, respectively. The TATA box of the promoter region is
also shown. The diagram is not drawn to scale.
( ) Southern hybridization. To examine the
presence of DNA sequences bearing homology to HMW
glutenin genes in the PCR reactions, PCR products, after
agarose gel electrophoresis, were vacuum-blotted onto
Hybond N+ membrane (Amersham Pharmacia Biotech).
The resultant membrane was hybridized with a radioactive
probe, prepared using the Prime-A-gene ® kit (Promega)
and the plasmid DNA containing the coding sequence of
an x-type HMW glutenin subunit from Ae. tauschii
(AtDx2.5, Wan Yongfang and Wang Daowen, unpublished
result). The protocol of Southern hybridization and the
optimization of post hybridization washing conditions
were based on the published procedures[19]. The hybridization results were recorded using a phosphor-imager (Molecular Dynamics).
( ) Cloning of PCR products and nucleotide sequence analysis. The desired PCR products were recovered from agarose gels, then cloned into the pGEM-T
vector (Promega). Nucleotide sequence of positive clones
was determined by a commercial company (TaKaRa). For
nucleotide sequence comparison, the programs (such as
Chinese Science Bulletin Vol. 46 No. 4 February 2001
NOTES
Fig. 2. SDS-PAGE analysis of the composition of
HMW glutenin subunits in the four polyploid Aegilops species. Lanes 1 and 2, Ae. crassa (three subunits
detected); lanes 3 and 4, Ae. juvenalis (four subunits
detected); lane 5, common wheat (cv. Chinese Spring,
four subunits detected); lane 6, common wheat
(MG7249, five subunits detected,); lanes 7 and 8, Ae.
ventricosa (four subunits detected, the arrow indicated
subunits possessing an electrophoretic mobility slower
than that of the 1Dx2.2 subunit); lanes 9 and 10, Ae.
vavilovii (five subunits detected).
Fig. 3. Analysis of PCR products by
agarose gel electrophoresis (a) and Southern
hybridization (b). Lane 1, DNA markers
(kb); lane 2, empty track; lane 3, PCR products obtained with the primer pair P1/P2
and the Taq DNA polymerase PCR kit; lane
4, PCR products obtained with the primer
pair P3/P4 and the Taq DNA polymerase
PCR kit (* denotes non-specific amplification products); lane 5, PCR products obtained with the primer pair P1/P2 and the
Advantage-GC genomic PCR kit; lane 6,
PCR products obtained with the primer pair
P3/P4 and the Advantage-GC genomic PCR
kit. Long arrows indicate the fragments with
a size more than 1.8 kb, the short arrow
indicates the 1.0 kb product.
ORF Finder, Blast, etc.) in the NCBI network service were
used.
2 Results
( ) SDS-PAGE analysis. Fig. 2 shows the result of
SDS-PAGE analysis of HMW glutenin subunits in four
polyploid Aegilops species. Several preliminary conclu) Three HMW glutesions could be drawn from fig. 2:
nin subunits were expressed in the hexaploid Ae. crassa
species; ) in Ae. juvenalis, four subunits were detected
for each of the two accessions. However, the two accessions differed in the composition of the subunits they expressed. The electrophoretic mobility of the Jx1 subunit
was faster than that of 1Dx2.2, whereas that of the Jx2
subunit was similar to what was displayed by the 1Dx2.2
subunit. Considerable difference in electrophoretic mobil) In
ity also existed between the Jy1 and Jy2 subunits.
Ae. ventricosa, four subunits were detected with largest
one possessing an electrophoretic mobility similar to that
of the 1Dx2.2 subunit.
) In Ae. vavilovii, five subunits
were detected.
The findings described above demonstrated the following points: ) Except for Ae. ventricosa, there was no
correlation between the number of subunits expressed and
the ploidy level of the genome. For example, in theory, the
hexaploid species Ae. juvenalis should express six subunits. However, only four subunits were detected in the
SDS-PAGE analysis.
) Different accessions of the same
Chinese Science Bulletin Vol. 46 No. 4 February 2001
species may differ in their composition of HMW glutenin
subunits. This was clearly shown by the two accessions of
Ae. juvenalis. ) In two of the four species analyzed, the
presence of the subunits possessing an electrophoretic
mobility similar to, or larger than, that of the 1Dx2.2 subunit was observed. However, it was not known if these
subunits were encoded by the D genome component in the
relevant Aegilops species.
( ) Genomic PCR reactions. Based on the above
SDS-PAGE analysis, we chose Ae. ventricosa as a model
species to establish an optimized PCR condition that may
permit simultaneous amplification of the coding sequences
for both x- and y-type subunits. Among the four different
PCR conditions tested, the reactions performed with the
Advantage-GC genomic PCR kit were generally better
than those with the Taq DNA polymerase PCR kit. In
agarose gel analysis of the PCR products derived from
reactions with the Advantage-GC genomic PCR kit, there
were more DNA fragments with a size in the range of 1.8
3.0 kb, and the yield of individual fragments was also
higher (fig. 3(a), lanes 5 and 6, arrows). In contrast, in
agarose gel analysis of PCR products obtained from reactions with the Taq DNA polymerase PCR kit, there were
no DNA fragments with a size in the range of 1.8 3.0 kb
(fig. 3(a), lanes 3 and 4). The two primer pairs affected the
amplification results obtained with the Advantage-GC
genomic PCR kit. In the reaction performed with P1/P2, a
higher yield was obtained for the DNA fragment with a
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NOTES
Table 1
accession numbera)
X12928
X61009
M22208
X13927
g543541
AF216868
X03041
X12929
U39229
AF216869
X61026
Comparison of the deduced, N-terminal amino acid sequences of Aevenx2.5 and Aeveny1.9 subunits
to those of published x- and y-type subunits
Aevenx2.5
Aeveny1.9
plant, subunit, type
wheat, Dx5, x-type
wheat, Ax1, x-type
wheat, Ax2*, x-type
wheat, Bx7, x-type
wheat, Bx17, x-type
rye,
x-type
wheat, Dy12, y-type
wheat, Dy10, y-type
Ae. tauschii,
y-type
rye,
y-type
wheat, By9, y-type
identity (%)
92
76
72
71
70
67
64
64
63
63
59
accession numbera)
X03041
X12929
U39229
AF216869
X61026
M22208
X13927
X61009
g543541
X12928
AF216868
plant, subunit,
wheat, Dy12,
wheat, Dy10,
Ae.tauschii,
rye,
wheat, By9,
wheat, Ax2*,
wheat, Bx7,
wheat, Bx17,
wheat, Ax1,
wheat, Dx5,
rye,
type
y-type
y-type
y-type
y-type
y-type
x-type
x-type
x-type
x-type
x-type
x-type
identity (%)
99
98
98
93
92
64
63
62
62
61
60
a) Except for g543541,which represents EMBO accession number for a protein sequence, the remaining accession numbers all represent nucleotide sequences.
higher molecular weight (approximately 2.5 kb) (fig. 3(a),
lane 5). In the case of P3/P4, the yield of the lower molecular weight DNA fragment (approximately 1.9 kb) was
higher (fig. 3(a), lane 6). Southern hybridization, with the
probe prepared the coding sequence for the AtDx2.5 subunit, revealed three cross-hybridizing DNA fragments in
the PCR products with a size in the range of 1.0 2.5 kb
(fig. 3(b)), confirming that he amplified DNA fragments
were indeed related to HMW glutenin genes.
( ) Cloning of PCR products and amino acid sequence analysis. The 2.5 and 1.9 kb PCR fragments
were cloned into the pGEM-T vector, yielding plasmids
pAeven2.5 and pAeven1.9, respectively. The 5 end sequence of the 2.5 and 1.9 kb inserts was determined. The
resultant nucleotide sequences (615 bps for the 2.5 kb
fragment, 537 bps for the 1.9 kb fragment) were translated
into amino acid sequences using the ORF Finder program.
The deduced amino acid sequences were compared with
those of published HMW glutenin genes using the Blast
program. The deduced N-terminal amino acid sequence
(composed of 204 amino acids) of the 2.5 kb fragment
showed higher than 90% identity with those of x-type
subunit genes, whereas its identity with those of the y-type
subunit was comparatively lower (around 70%). This suggests that the subunit specified by the 2.5 kb fragment was
an x-type subunit, for which a tentative name Aevenx2.5
was therefore given. A limited analysis of the amino acid
sequence in the repetitive domain of the Aevenx2.5 subunit revealed the presence of three types of repeated, short
amino acid sequence motifs, namely, GQQ, PGQGQQ and
GYYPTS/PQQ. The existence of the repeated tripeptide
GQQ in the Aevenx2.5 subunit further confirmed that this
subunit was an x-type subunit. The deduced N terminal
amino acid sequence (composed of 179 amino acids) of
the 1.9 kb fragment exhibited more than 90% identity with
those of y-type subunit genes. In contrast, its identity with
those of the x-type subunit was less than 70%. This sug-
312
gests that the subunit specified by the 1.9 kb fragment was
a y-type subunit, for which a tentative name Aeveny1.9
was subsequently given. A preliminary analysis of the
amino acid sequence in the repetitive domain of the
Aeveny1.9 subunit showed the presence of two types of
repeated, short amino acid sequence motifs, namely,
PGQGQQ and GYYPTS/PQQ. The absence of the repeated tripeptide GQQ in the Aeveny1.9 subunit provided
additional evidence, supporting that this subunit was a ytype subunit (see table 1).
3 Discussion
Many agronomically important genes, such as the
mildew resistance genes Pm2, Pm12, Pm13, Pm19 and the
fertility restoration gene Rf6, are originally derived from
the genome of Aegilops species[20 23]. These Aegilops
genes have contributed to the genetic improvement of
common wheat varieties. Based on these examples, the
potential value of the Aegilops HMW glutenin subunits in
wheat quality breeding merits a thorough investigation.
Some investigations on the HMW glutenin subunits and
their coding genes in Ae. tauschii have been conducted[9, 10]. A preliminary evaluation of the genetic materials
derived from a common wheat × Ae. tauschii cross has
already shown that some progeny lines possess quality
properties superior to those of the common wheat parent[24].
In this study, we analyzed HMW glutenin subunit
composition of four multiploid Aegilops species. In common with bread wheat varieties, multiple subunits (at least
three) were expressed in the seeds of different Aegilops
species. Because it was observed that there was generally
no correlation between the number of subunits expressed
and the ploidy level of the species investigated, we deduced that null alleles and/or gene silencing might also
affect the expression of HMW glutenin subunit genes in
Aegilops species. Owing to a limited number of accessions
analyzed, our finding on the difference of HMW glutenin
Chinese Science Bulletin Vol. 46 No. 4 February 2001
NOTES
subunit composition between different accessions of a
single species requires further investigation.
Although PCR reactions have been employed to
study HMW glutenin genes in common wheat and Ae.
tauschii materials, successful attempts in simultaneous
amplification of the coding sequences for both x- and ytype subunits in single PCR reactions have not been reported by previous investigators. By designing two pairs
of PCR primers and testing four different PCR conditions,
we found a PCR condition that gave rise to the amplification of both x- and y-type subunit genes in single PCR
reactions. There were three different DNA fragments (2.5,
1.9 and 1.0 kb, respectively) in the PCR reactions conducted with the Advantage-GC genomic PCR kit. Southern hybridization indicated that they all possessed nucleotide homology to HMW glutenin genes. DNA cloning and
amino acid sequence analysis confirmed that the 2.5 kb
fragment represented the coding sequence for an x-type
subunit (Aevenx2.5) and the 1.9 kb fragment for a y-type
subunit (Aeveny1.9). However, the identity of the 1.0 kb
fragment remains to be determined. The PCR reactions
performed with the Advantage-GC genomic PCR kit were
generally more efficient in terms of product yield. Considering that the Advantage-GC genomic PCR kit was developed especially for genomic PCR reactions using GC-rich
templates and that the coding sequence of HMW glutenin
genes was generally rich in GC content, it was natural to
find that this kit performed better than the Taq DNA
polymerase PCR kit in our experiments.
Because of the limited sequence information generated in this study, it is not known at this stage how different
the two Ae. ventricosa subunits are from the subunits published previously in overall amino acid sequence. Considering that Ae. ventricosa is a tetraploid species with two
component genomes (D and MV), further studies are needed to find out to which genome(s) the 2.5 and 1.9 kb fragments belong. The simultaneous amplification of the coding sequences for both Aevenx2.5 and Aeveny1.9 subunits
in single PCR reactions demonstrates that the PCR condition established in this study is highly efficient, and will be
useful in further molecular genetic studies on HMW glutenin subunit in the multiploid Aegilops species.
Acknowledgements This work was supported by a special fund for
biotechnology research from the Chinese Academy of Sciences (Grant
No. STZ-3-11). The nucleotide sequences for the Aeveny1.9 and
Aevenx2.5 subunit have been deposited in the EMBO database with the
accession numbers AF226698 and AF226699, respectively.
5.
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