全 文 ：Received 23 Jul. 2003 Accepted 20 Nov. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (001AA241032).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 489-496
High-Molecular-Weight Glutenin Subunit Genes
in Decaploid Agropyron elongatum
FENG De-Shun, CHEN Fan-Guo, ZHAO Shuang-Yi, XIA Guang-Min*
(School of Life Sciences, Shandong University, Jinan 250100, China)
Abstract : Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski
were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of
the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit
genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other
two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences
suggested that the primary structure of the subunits encoded by the seven genes was very similar to that
of y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6
and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and
those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L.
(CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most
closely related to 1Dy; the AgeloG3 was to 1By; the AgeloG5, AgeloG6 and AgeloG7 were to 1Ay.
Key words: Agropyron elongatum ; HMW glutenin subunit; coding sequence; PCR; evolution
Agropyron elongatum (StStEeEbEx, 2n=70) is a decaploid
species in the Agropyron genus and is a good genetic re-
source for common wheat. However, few studies have been
conducted to compare the st ructure and function o f
homoeologous genes from wheat and A. elongatum. Com-
mon wheat cultivars have three Glu-1 loci presenting on
the long arms of chromosomes 1A, 1B and 1D, respectively,
encode high-molecular-weight (HMW) gluten in subunit
(Payne et al., 1987), each complex Glu-1 locus consists of
two closely linked genes which code different type of HMW
glutenin subunits, one with greater molecular weight, des-
ignated the x-type and the other with lower molecular weight,
designated the y-type (Harberd et al., 1986). They usually
con tain 3-5 HMW glu tenin s ubunits , and encoded by
genes at the Glu-1A, Glu-1B and Glu-1D loci. Complete
amino acid sequences of these proteins have indicated that
there are three distinct domains: a central hydrophobic re-
petitive domain flanked by non-repetitive N- and C- termi-
nal hydrophilic domain. The central repetitive domain com-
prises numbers of hexa- and nona- peptide motifs in the x-
and y- type subunits. Besides this, tripeptide motif is found
in x-type subunits. Difference in subunit size results mainly
from the number of hexapeptide and nonapeptide (Shewry
et al., 1989). The composition of HMW glutenin subunits
can influence the bread-making quality directly (Payne et
al., 1981), for example, the subunits 1Dx5 and 1Dy10 en-
coded by Glu-1D locus are positive to excellent bread-mak-
ing quality . Transgen ic experiments ind icated that
overexpression of certain subunit-coding sequence in wheat
improved baking quality (Barro et al., 1997). It was previ-
ously found that the coding sequences of HMW glutenin
subunits are not interrupted by introns and are highly con-
served at 5 and 3 terminal sequences, this makes it pos-
sible to clone unknown HMW glutenin subunits using PCR
directly from genomic DNA (Shewry et al., 1989; D’ Ovidio
et al., 1995). The isolated gene can also be used further to
study the relationship between structure and function of
protein through the expression of polypeptides in bacteria.
Some coding s equences for HMW glutenin subunit have
been cloned with PCR from common wheat, Tri ticum
tauschii, Aegilops ventricosa and Aegilops umbellulata
(D’ Ovidio et al., 1995; Mackie et al., 1996 ; Xie et al., 2001;
Liu et al., 2002).
Decaploid A. elongatum has many excellent characters
such as high content of seed protein and high resistance to
stres s. In China, a s eries of high quality hybrid cult ivars
were derived from sexual hybrid progen ies between A.
elongatum and common wheat, e. g. Xiaoyan 6 (Zhou et
al., 1995), and some somat ic hybrid lines were obtained
from progenies of T. aestivum with A. elongatum (Xia et
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004490
al., 2003). These somatic hybrid lines have higher protein
content and higher quality than their parental wheat, with
different HMW glutenin subunits which are absent in the
parental wheat (Xiang et al., 2001; Zhao et a l., 2003). But
study on the o rigin of excellen t HMW glu tenin subunits
indicated that the coding sequences of 1Bx14 and 1By15 in
hybrid lines of Xiaoyan 6 did not orig inate from A.
elongatum (Fan and Guo, 2000), and that of 1-4 subunits
in high quality somatic hybrid lines showed different elec-
trophoresis mobility from A. elongatum and parental wheat
(Zhao et a l., 2003). So it is necessary to investigate the
origin of these subunits and other storage proteins in these
hybrids through comparing their cod ing s equences be-
tween A. elongatum and wheat. Up to now, only the cod-
ing sequences of A, B, D, C, U and G genomes of wheat and
its related species have been reported (Allaby et al., 1999;
Liu et al., 2002).
In this study , we investigated some of HMW glutenin
subunit compositions in A. elongatum and the nuclear acid
sequences encoding those subunits of this species. The
results provided us not only a basic situat ion of the gene
structure but also the possibility to compare amino acid
sequence with that of wheat and other species, so as to
deduce the evolutionary relationship (Allaby et al., 1999).
1 Materials and Methods
1.1 Plant materials
Agropyron elongatum (Host ) Nevski (Lophopyrum
elongatum; Thinopyrum ponticum; StStEeEbEx , 2n=70) and
Triticum aestivum L. (AABBDD, 2n=42) used in this study
are stored in our laboratory.
HMW glutenin subunits from hexaploid wheat cultivars
Chinese Spring (2+12 and 7+8), Jinan 177 (2+12 and 7+9)
and a high quality somatic hybrid lineⅡ-12 between Jinan
177 and A. elongatum with some HMW glutenin subunits
different from its parents were used as standards for ac-
cessing the electrophoresis mobility of the subunits in A.
elongatum (Zhao et al., 2003).
1.2 Extraction and SDS-PAGE analysis of glutenin sub-
units
Endospermic storage proteins prepared for SDS-PAGE
analysis were described previously (Zhao et al ., 2003).
Three individual seeds were examined for investigating the
composition of HMW glutenin subunits in each accession.
Total protein was fractionated by SDS-PAGE on 10% sepa-
rating gel (C=2.67%) and 3.7% stacking gel (C=2.67%). The
stacking gel buffer is 0.5 mol/L Tris-HCl (pH 6.8) and the
separating gel buffer is 0.025 mol/L Tris-HCl (pH 8.3). The
Tris -glycine buffer (0.025 mol/L Tris-HCl (pH 8.3), 0.192
mol/L glycine, 0.001% SDS) s ys tem was adop ted fo r
electrophoresis. A constant current of 15 mA was used to
run for 18 h at 4 ℃. After electrophoresis, the protein bands
were s tained for 5 h with 0.1% (W/V) Coomassie brilliant
blue G-250, 12.5% (W /V) trichlo roacetic acid, and then
distained with distilled water.
1.3 Amplification and cloning of the ORFs of glutenin
genes from A. elongatum
CTAB method was adopted for the ext ract ion of ge-
nomic DNA from leaves of A. elongatum according to
Murray and Thompson (1980) . For amplifing the ORFs of
glu tenin genes , two primers s pecific for HMW glutenin
genes were us ed : P1:(5-ATGGCTAAGCGGC/TTA/
GGTCCTCTTTG-3) and P2: (5-CTATCACTGGCTA/GGCC
GACAATGCG-3) (Xie et al., 2001; Liu et al., 2002).
A high-fidelity polymerase LA GC Taq with GC buffer
(TaKaRa, Dalian , China) was used instead of Taq poly-
merase in PCR in order to reduce the ris k of introducing
errors into the sequence. Protocol for PCR of the ORFs: the
denaturing step was at 95 ℃ for 3 min; basic cycling condi-
tions were 36 cycles, each with a 40 s denaturing step at 94
℃ and 4 min annealing and extension steps at 68 ℃, finally
kept at 68 ℃ fo r 10 min. PCR products were separated in
1.0% agarose gel.
1.4 Sequencing the cloned ORFs and sequence analysis
The purified PCR products were ligated into pUCm-T
vector (Sangon, Shanghai, China) and then t ransformed
into Escherichia coli DH10B competent cells. Following
the identification of positive clones, a set of subclones was
prepared using the nes ted deletion method fo llowed
Sambrook et al. (1989). DNA sequencing was performed by
commercial company (TaKaRa, Dalian, China and Gentech,
Shanghai, China). For sequence analysis, programs of the
NCBI and EBI networks were used.
2 Results
2.1 SDS-PAGE analysis
Gluten in s ubunits in the endos perm t is sue o f A.
elongatum and wheat Chinese Spring and cv. Jinan 177, as
well as somatic hybrid Ⅱ-12 were extracted and subjected
to SDS-PAGE analysis. Figure 1 illustrates that the number
of glutenin subunits in A. elongatum was much more than
that in common wheat and hybrid (Fig.1).
2.2 Cloning and sequencing the ORFs of glutenin sub-
unit genes from A. elongatum
Using degenerate primers P1 and P2, we specifically
amplified seven fragments in genomic PCR (Fig.2). They
were named AgeloG1, AgeloG2 , AgeloG3 , AgeloG4 ,
AgeloG5, AgeloG6 and AgeloG7. The first three fragments




FENG De-Shun et al.: High-Molecular-Weight Glutenin Subunit Genes in Decaploid Agropyron elongatum 495
Barro F, Rooke L, Bekes F, Gras P, Tatham A S, Fido R, Lazzeri
P A, Shewry P R, Barcelo P. 1997. Transformation of wheat
with high molecular weight subunit genes results in improved
functional properties. Nat Biotechnol, 15:1295-1299.
Belton P S. 1999. On the elasticity of wheat gluten. J Cereal Sci,
29:103-107.
Bustos A D, Rubio P, Jouve N. 2000. Molecular characterization
of the inactive allele of the gene Glu-A1 and the development
of a set of AS-PCR markers for HMW glutenins of wheat.
Theor Appl Genet, 100:1085-1094.
Cassidy B G, Dvorak J, Anderson O D. 1998. The wheat low-
molecular-weight genes: characterization of six new genes and
progress in understanding gene family structure. Theor Appl
Genet, 96:743-750.
D’ Ovidio R, Porceddu E, Lafiandra D. 1994. PCR analys is of
genes encoding allelic variants of high-molecular-weight glute-
nin subunits at the Glu-D1 locus. Theor Appl Genet, 88:175-
180.
D’ Ovidio R, Masci S, Porceddu E. 1995. Development of a set of
oligonucleotide primers sp ecific for genes at the Glu-1 com-
plex loci of wheat. Theor Appl Genet, 91:189-194.
Fan S-H, Guo A-G. 2000. A study on the origin of HMW glutenin
subunit 14 and 15 in Xiao Yan 6. Acta Univ Agric Boreali-
Occidentalia , 28 (6):1-5. (in Chinese with English abstract)
Forde J, Malpica J M, Halford N G, Shewry P R, Anderson O D,
Greene F C, Miflin B J. 1985. The nucleotide sequence of a
HMW subunit gene located on chromosome 1A of wheat
(Triticum aestivum L.). Nucleic Acids Res, 13:6817-6832.
Harberd N P, Bartels D, Thompson R D. 1986. DNA restriction-
fragment variation in the gene family encoding high-molecu-
lar-weight (HMW) glutenin subunits of wheat. Biochem Genet,
24:579-595.
Liu Z -J, Zhang X-M, Wan Y- F, Liu K-F, Wang D-W. 2002.
Characterization of high-molecular-weight glutenin subunits
and t heir coding genes from Aegilops umbellulata. Acta Bot
Sin, 44:809-814.
Mackie A M, Sharp P J, Lagudah E S. 1996. The nucleotide and
derived amino acid sequence of a HMW glutenin gene from
Triticum tauschii and comparison with those from the D ge-
nome of bread wheat. J Cereal Sci, 24:73-78.
Murray M G, Thompson W F. 1980. The isolation of high mo-
lecular weight plant DNA. Nucleic Acids Res, 8:4321-4325.
Payne P I, Corfied K G, Holt L M. 1981. Correlations between
the inheritance of cert ain high-molecular-weight subunits of
glutenin and bread-making quality in progenies of six crosses
of bread wheat. J Sci Food Agr, 32:51-60.
Payne P I, Nightingale M A, Krattiger A F, Holt L M. 1987. The
(Managing editor: ZHAO Li-Hui)
relationship between HMW glutenin subunit composition and
the breadmaking qualit y of British-grown wheat varieties. J
Sci Food Agr, 40:51-65.
Rafalski J A. 1986. Structure of wheat γ-gliadin genes. Gene, 43:
221-229.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laboratory Manual, 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
Shewry P R, Halford N G, Tatham A S. 1989. The high-molecu-
lar-weight subunits of wheat, barley and rye: genetics , mo-
lecular biology, chemistry and role in wheat glutenin structure
and functionality. Miflin B J. Oxford Surveys of Plant Mo-
lecular and Biology. Vol. 6. Oxford: Oxford University Press.
163-219.
Shewry P R, Tatham A S, Fido R, Jones H, Barcelo P, Lazzeri P
A. 2001. Improving the end use properties of wheat by ma-
nipulating the grain protein composition. Euphytica, 119:45-
48.
Wan Y F, Wang D W, Shewry P R, Halford N G. 2002. Isolation
and characterization of five novel high-molecular-weight sub-
unit of glutenin genes from Triticum timopheevi and Aegilops
cylindrical. Theor Appl Genet, 104:828-839.
Xia G M, Xiang F N, Zhou A F, Wang H, He S X, Chen H M.
2003. Asymmet ric somatic hybridizat ion bet ween wheat
(Triticum aes tivum L.) and Agropyron elongatum (Hos t)
Nevski. Theor Appl Genet, 107:299-305.
Xiang F-N , Feng B-M, Xia G-M , Chen H-M . 2001. Agronomic
trait and protein component of F2 hybrid originated from in-
tergeneric somatic hybridization between Triticum aestivum
and Agropyron elongatum. Acta Bot Sin , 43:232-237.
Xie R L, Wan Y F, Zhang Y, Wang D W. 2001. HMW glut enin
subunits in multiploid Aegilops species: comp osition analy-
sis and molecular cloning of coding sequences. Chin Sci Bull,
46:309-313.
Zhao T-J, Quan T-Y, Xia G-M, Chen H-M. 2003. Glutenin and
SDS sedimentation analysis of the F5 somatic hybrids be-
tween Triticum aestivum L. and Agropyron elongatum. J
Shandong Univ (Nat Sci) , 38(3):112-116. (in Chinese with
English abstract)
Zhou H-P, Li B, Li Z-S. 1995. T he study of breeding blue-grain
gene translocation of wheat. Acta Bot Boreali-Occidentalia
Sin , 15:125-128. (in Chinese with English abstract)