全 文 ：Received 20 Nov. 2003 Accepted 15 Mar. 2004
* Author for correspondence. E-mail: .
** Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1220-1225
Expression of Rice Glutelin Gene GluA-2 in Wheat Endosperm
CHEN Yu, LI Hui, LI Hong-Jie, LI Yi-Wen, ZHU Yin-Feng, QU Le-Qing*, JIA Xu**
(State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology,
The Chinese Academy of Sciences, Beijing 100101, China)
Abstract: Rice (Oryza sativa L.) glutelin accounts for about 80% of total seed storage protein. Glutelin
can be easily digested by human and contains high concentrations of lysine and other essential amino
acids. To improve the nutritional quality of wheat (Triticum aestivum L.), the cDNA sequence of rice
glutelin gene GluA-2 was introduced into common wheat through biolistic bombardment. Six hundreds of
immature embryos from wheat cultivar Bobwhite (T. aestivum cv. Bobwhite) were bombarded. Four transgenic
plants carrying GluA-2 gene were confirmed by PCR and Southern blotting analyses. Gene GluA-2 was
translated in three of the transgenic plants and their progeny, as indicated by SDS-PAGE analyses. The
expression of GluA-2 gene was not detected in one transgenic plant and the endogenous wheat high
molecular weight glutenin subunits Bx7 and By9 contents decreased greatly in both T0 and T1 generations.
Key words: rice glutelin gene GluA-2 ; wheat transformation; nutrient quality improvement
The main composition of endosperm storage proteins
in rice (Oryza sativa) is glutelins, which constitutes 60%-
80% of total endosperm proteins in mature rice kernels.
Glutelin can be easily consumed by human and contains
high concentrations of lysine (2.6%-3.5%, mol%) and es-
sential amino acids (33.6%-41.4%, mol%). Because of these
properties, glutelin has been recognized as one of the most
important sources of nutrition for human beings (Villareal
and Juliano, 1978).
Wheat (Triticum aestivum) is an important cereal crop,
which provides a major source of energy and proteins for
human being throughout the world. Gliadin and glutenin
are dominant parts of the storage proteins in wheat, ac-
counting for about 85% of total grain proteins. However,
wheat gliadin and glutenin can not be easily digested by
human because of the low solubility and unique structure
(Gianibelli et al., 2001). Moreover, lysine and other essen-
tial amino acids only account for 0.9% and 13.4% (mol%)
of wheat seed storage proteins (Shewry and Tatham, 1990;
Gianibelli et al., 2001). This reduces the use of wheat and
improvement of wheat nutritional quality is thus an impor-
tant objective in wheat breeding programs.
The use of cloned genes is an alternative approach to
improve nutritional composition of cereal crops to the time-
consuming conventional breeding methods. Genes that
originate from a crop may express themselves in another
crop, which allows prompt improvement of traits of interest.
Genes for soybean ferritin and phytoene synthase have
been transferred into rice, resulting in high contents of
iron and vitamine A in transgenic lines (Burkherdt et al.,
1997; Goto et al., 1999). In wheat, much attention has been
focused on improving b aking-qual i ty- re la ted
characteristics. A number of high molecular weight glute-
nin subunits (HMW-GS) genes isolated from common
wheat, such as 1Ax1, 1Dx5 and 1Dy10 conferring qualita-
tive and/or quantitative effects on baking quality, have
been transferred into various wheat cultivars and the bak-
ing quality of transgenic lines have been improved greatly
in most cases (Altpeter et al., 1996; Blechl and Anderson,
1996; Barro et al., 1997; Rook et al., 1997; Alvarez et al.,
2000). However, little information is available for the in-
crease of essential amino acid and digestible protein con-
tent through transgenic technique compared to improve-
ment of baking quality.
Rice glutelin gene GluA-2 belongs to a mini gene family.
The coding region of GluA-2 is 1 500 bp in length, which
encodes a 57 kD glutelin precursor. The contents of lysine
and essential amino acids are 2.42% and 33.86% (mol %) in
glutelin precursor, respectively. Although GluA-2 was
cloned more than ten years ago (Okita et al., 1989), this
gene has not been transferred into any crop. Since the
peptide encoded by the gene GluA-2 produces higher con-
tents of essential amino acids, GluA-2 might be useful to
improve wheat seed storage protein composition. In this
study, we introduced the cDNA sequence of rice glutelin
gene GluA-2 under the control of wheat endosperm
CHEN Yu et al.: Expression of Rice Glutelin Gene GluA-2 in Wheat Endosperm 1221
specific expression promoter Glu1-D1 into spring wheat
cultivar Bobwhite by biolistic bombardment. The expres-
sion of GluA-2 was confirmed by SDS-PAGE analyses. It
is expected that the overexpression of GluA-2 gene in
wheat endosperm will increase the essential amino acid
and digestible protein contents.
1 Materials and Methods
1.1 Plant materials
The spring wheat cultivar Bobwhite (Triticum aestivum
L. cv. Bobwhite) was grown in greenhouse set at 20-28
ºC/15-18 ºC day/night to provide immature embryos for
transformation. A wheat cultivar Chinese Spring (T.
aestivum cv. Chinese Spring) and a Japanese rice cultivar
Kinmzae (Oryza sativa L. cv. Kinmaze) were used as con-
trols in molecular characterization of transgenic plants.
1.2 Plasmid construction
The 1 645 bp long cDNA sequence of rice glutelin gene
GluA-2 was cloned by Okita (Okita et al., 1989) and cut by
EcoRⅠ, inserted into plasmid pBluscriptKS (Stragene, La
Jolla, CA, USA). The fragment released by cutting with
BamHⅠ and SalⅠ was placed at the downstream of wheat
endosperm specific expression promoter Glu1-D1
(Lamacchia et al., 2001) followed by 3 -untranslated re-
gion (terminator) of the nopaline synthase (Nos) and the
plasmid is designated pGluGluA-2 (Fig.1).
1.3 Wheat transformation
Wheat transformation was conducted following the pre-
viously published method (Liang et al., 2000) with slight
modification. The immature embryos of Bobwhite were
collected at 10-14 d after pollination, surface-sterilized with
70% (V/V) ethanol for 1 min and 5% (V/V) sodium
hypocholorite for 25 min, and rinsed with sterile-distilled
water for four times. The immature embryos were pre-cul-
tured on MS medium (Murashige and Skoog, 1962) supple-
mented with 2 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid)
for 3 d and then placed on high osmolarity medium (MS
medium with 0.5 mol/L maltose) for 6 h before bombard-
ment with the DNA-coated gold particles at 1 100 pound-
force per square inch (psi) using a BioRad PDS-1000/He
biolistic device (BioRad Laboratories Ltd., Hercules, CA,
USA). The gold particles were coated with a mixture of the
plasmids pGluGluA-2 and pAHC25 at 1:1 molar ratio. The
plasmid pAHC25 contains the marker genes Bar (encod-
ing phosphinothricin acetyltransferase) and UidA (GUS)
(encoding b-glucuronidase), each of which was under the
transcriptional control of a separate maize ubiquitin
(Ubi-1) promoter (Christensen et al., 1992) and terminated
by the Nos 3 untranslated region. Following bombardment,
the immature embryos were maintained on high osmolarety
medium for 18 h before being cultured on MS medium with
2 mg/L 2,4-D for 10 d . They were then cultured on selec-
tion medium, which was composed of MS salt, 5 mg/L
phosphinotricin (PPT), 2 mg/L 2,4-D, and 30 g sucrose pH
5.8 at 25 ºC in dark for 2-3 weeks. The embryogenic calli
were then transferred onto shoot regeneration medium (MS
basal medium except for half-strength of macro-salt and
supplemented with 1 mg/L 3-inositol-acetic-acid, 1 mg/L
Zeatin, 3 mg/L PPT) and cultured at 25 ºC with 16/8 light/
dark photoperiod. After 2-3 weeks growth, green shoots
were transferred on rooting medium (MS basal medium
except for half strength of macro salt supplemented with 3
mg/L PPT and 8% sucrose). Three weeks later, well-rooted
plantlets were transplanted into soil and then grown in
greenhouse.
1.4 PCR analysis
Oligonucleotide primers were designed according to
the coding sequence of gene GluA-2 (GenBank accession
number M28156). The sequences of the primers were for-
ward 5-ATTAGGCCAGAGCACTAGTCAATG-3 and re-
verse 5-CTTGTATTGGAGGGGAGTGAATG-3. The am-
plification was programmed in a PTC-100 thermocycler (MJ
Research Inc. Watertown, MA, USA) at 94 ºC for 5 min to
allow denature of DNA, followed by 35 cycles of 94 ºC for
1 min, 57 ºC for 1 min, and 72 ºC for 2 min. The final exten-
sion step was performed at 72 ºC for 10 min. The PCR prod-
ucts were separated on a 0.8% agarose gel.
1.5 Southern blotting analysis
Genomic DNA was isolated from leaf tissue of transgenic
plants by using CTAB method (Rogers and Bendlich, 1994)
. Twenty microgrammes of DNA was digested with BamH
Ⅰ. Digested DNA was electrophoretically on a 0.8%
Fig.1. Map of plasmid pGluGluA-2. Nos, nopaline synthase.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041222
agarose gel and transferred onto Hybond N+ membrane
(Amersham, Buckinghamshire, England) according to the
instructions of the manufacturer. The 1 645 bp cDNA frag-
ment of gene GluA-2 was labeled with a-32P-dCTP by ran-
dom primer labeling method (Primer G labeling system,
Promega, Madison, Wisconsin , USA). Hybridization and
washing steps were carried out as previously described
(Sambrook et al., 1989).
1.6 SDS-PAGE analysis
Half mature grain was grinded by mortar and pestle and
extracted by 400 mL of extraction buffer (62.5 mmol/L Tris-
HCl, pH 6.8, 2% SDS, 20% glycerol, 0.05% bromophenol
blue, and 5% mercaptoethanol). Following incubation for
at least 3 h at room temperature, the extracts were boiled
for 5 min and centrifuged for 5 min at 12 500 r/min. Total
proteins were separated by SDS-PAGE method following a
method described by Laemmli (1970).
2 Results
2.1 PCR and Southern blotting analysis
Six hundreds of immature embryos from Bobwhite were
bombarded and 14 PPT-resistant plantlets were obtained.
To examine the presence of gene GluA-2, DNA from leaves
of all the resistant plants were amplified by PCR reaction,
using primers specific for gene GluA-2. Four of them (BT-
5-2, BT-7-2, BT-9A and BT-11) exhibited the polymorphic
band 1 381 bp in length (data not shown). PCR analysis
was conducted on all available T1 progeny plants to deter-
mine the presence of gene GluA-2. For each sample, PCR
reactions were repeated three times. A plant was consid-
ered as a transgenic plant when the diagnostic 1 381 bp
fragments were amplified at least in two reactions (Table 1,
Fig.2).
Genomic DNA was extracted from the four T0 transgenic
plants and digested with BamHⅠ. BamHⅠ has a cleavage
site between cDNA sequence of gene GluA-2 and Nos
terminator. The products digested with BamHⅠ allow de-
tection of the integration pattern of GluA-2. Multiple hy-
bridization fragments similar in size to the plasmid used for
transformation (6.0 kb) or longer were detected in DNA
samples from the T0 and T1 transgenic plants, which con-
firmed that the GluA-2 gene was integrated into the
gonomic of all of the four transgenic plants and their
progeny, and the copy number was estimated over five
(Fig.3). The smaller hybridization fragments indicated the
presence of truncated or rearranged forms of the gene
GluA-2.
2.2 SDS-PAGE analysis
The SDS-PAGE analysis on the four wheat transgenic
plants and their progeny indicated that a major band was
present in T0 and T1 seeds of the three transgenic plants
(BT-5-2, BT-7-2 and BT-11), which was absent in non-trans-
formed Bobwhite and Chinese Spring controls. The mo-
lecular weight of this additional band was about 57 kD, which
Table 1 PCR analysis of individual transgenic plants of T1
progenies
T1 progeny PCR-positive* PCR-negative*
BT5-2 8 12
BT7-2 7 8
BT9A 3 2
BT11 6 2
*, a plant that exhibited the polymorphic band 1 381 bp in length
was considered as positive to PCR amplification, or negative when
this band was not amplified.
Fig.2. PCR analysis of T1 generation of transgenic line. Lanes
M, 1 kb ladder (MBI Fermentas); N, Bobwhite; P, cDNA of
GluA-2; 1-4, T1 progeny of transgenic plant BT-9A; 5-12 ,T1
progeny of transgenic plant BT-11.
Fig.3. Southern blot of T1 generation of transgenic line. Lanes
M, 1 kb ladder; N, genomic DNA of Bobwhite digested with
BamHⅠ; P, cDNA of gene GluA-2; 1-2 , genomic DNA of
BT9A T1 transgenic plants digested with BamHⅠ; 3-4, ge-
nomic DNA of BT-11 digested with BamHⅠ.
CHEN Yu et al.: Expression of Rice Glutelin Gene GluA-2 in Wheat Endosperm 1223
was similar to that of the rice glutelin precursor (Fig.4).
Based on this result, we can draw a conclusion that glute-
lin was synthesized in seeds of T0 and T1 transgenic plants,
and the expression of GluA-2 gene was almost at the same
level (Table 2). The bands of 37-39 kD and 20-22 kD were
not observed in wheat seed protein extract. The expres-
sion of HMW-GS 1Bx7 and 1By9 genes might be partially
or completely suppressed by the introduction of gene
GluA-2 in one transgenic plant (BT-9A) and the suppres-
sion can be transmitted to its progeny (Fig.5).
3 Discussion
Gene GluA-2 for rice glutelin was transferred through
biolistic bombardment into wheat cultivar Bobwhite and
expressed in wheat endosperm, as indicated by the pres-
ence of the glutelin similar in size to the 57 kD rice glutenin
precursor (Fig.4). The plants that have been verified to
carry gene GluA-2 are being increased to realize geneti-
cally uniform lines. The analysis in later generations will
allow understanding whether gene GluA-2 is helpful to
increase the content of essential amino acids in wheat.
In rice, glutelin is synthesized in endoplasmic reticu-
lum as a 57-kD precursor and then processed into two
mature subunits with a molecule mass of 37-39 kD and
21-23 kD. Sindhu et al. (1997) introduced pea legumin
gene into rice. The expression pattern of legumin gene in
rice remains the same as that in pea. The 60 kD precursor
was synthesized and processed into a 40-kD acid and a 20-
kD basic subunit. This might be that the glutelin and legu-
min share the same ancestor of 11S globulin and similar
post-translation processing site. However, the acid and
basic subunits were not observed in seed extracts of
transgenic wheat plants in present study. This may be
caused by the fact that the post-translation process of
wheat storage protein is unlike that of rice glutelin.
Therefore, the 57 kD precursor can not be divided in wheat
endosperm.
Currently, little is known about the precise organiza-
tion of the HMW-GS and low molecular weight glutenin
subunits (LMW-GS) in glutenin polymers. It is widely ac-
cepted that HMW subunits form a disulphide bonded
network. The LMW-GS acts as branches and possibly pro-
vides some cross-links (Shewry and Tatham, 1990). The
introduced HMW-GS gene, such as 1Ax1, dramatically in-
creased the dough strength (Altpeter et al., 1996). However,
Popineau et al. (2001) reported that the overexpression of
HMW-GS 1Dx5 gene in transgenic lines resulted in diffi-
culty for dough formation. Rhelogical analysis of glutens
isolated from these lines indicated that the connectivity of
gluten network had been greatly increased for 10 to 100
folds. Rice glutelin is hexamers composed of six identical
subunits, which are formed by acid and basic subunits.
The acid and basic glutelin subunits contain six and two
Cysteine residues. The acid subunit has one intramolecu-
lar disulfide bond and is linked to the basic subunit with
an intermolecular disulfide bond (Huebner et al., 1990) . It
is not known whether the same structure would be formed
in wheat endosperm because the acid and basic subunits
were not observed. However, the glutelin may primarily be
Fig.4. SDS-PAGE analysis of the seeds from transgenic plants.
Lanes K, Kinmaze; M, protein marker (Sabc bioengineer Co.,
Luoyang, China); N, Bobwhite; T0 and T1, transgenic plants; 1
and 2, BT5-2; 3 and 4, BT7-2; 5 and 6, BT-11.
Fig.5. SDS-PAGE analysis of seeds from BT-9A T0 and T1
progeny. Lanes B, Bobwhite; CS, Chinese Spring; T0, transgenic
plants; T1, progeny; 1-2, seeds of BT-9A; 3-8, seeds of BT-9A.
Table 2 Densitometer analysis of relative Glutelin transgene
expression in T0 and T1 generations of the transgenic plants
Plants Generation
No. of seeds
Glutelin/Bx7*
Glutelin/
examined Dx5*
BT5-2 T 0 5 1.42 2.83
T 1 9 1.23 2.16
BT7-2 T 0 8 1.33 3.56
T 1 11 1.20 3.32
BT11 T 0 4 1.06 3.17
T 1 10 1.01 3.21
*, average from density scan of SDS-PAGE gels. The density value
of Glutelin was divided by the density value of HMW-GS and back-
ground value.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041224
present as small polymer in wheat endosperm. So the glu-
telin may deposit in the network that was built by wheat
HMW-GS other than join the network construction.
Gene silence has been frequently documented in
transgenic studies. Alvarez et al. (2000) transferred genes
1Ax1 and 1Dx5 conferring HMW-GS into common wheat.
In transgenic plants with high copy numbers (20-50) of
the transgenes, all endogenous HMW-GS genes were
silenced. In two of four plants having low copy numbers
(1-3) transgenes, endogenous 1Ax2 gene did not express,
and no gene silence was observed in other two transgenic
plants. Altpeter et al. (1996) transformed genes HMW-GS
1Ax1 and 1Dx5 into wheat cultivar Bobwhite and specu-
lated that the integration of transgene could result in en-
dogenous 1Ax2* gene silence. Since both transgenes and
endogenous genes 1Bx7 and 1By9 were suppressed at
the same time in one transgenic plant, it resembles co-
suppression that mutual inactivation of transgene and ho-
mologous gene occurs. Vaucheret (1993) indicated that only
partial homology in promoter or coding sequence was
needed for co-suppression. However, de Carvalho et al.
(1995) concluded that the transgene expression level was
very high when co-suppression occurred. In the present
study, the expression of gene GluA-2 was not detected in
some transgenic plants of T0 and T1 generations. The ho-
mology between GluA-2 and HMW-GS 1Bx7 and 1By9
were about 25%, which was measured by DNAMAN soft-
ware (Lynnon Biosoft Co. USA). In this study, the Glu1-
D1 promoter was amplified from HMW-GS gene 1Dx5
promoter. When gene UidA under the control of Glu1-D1
promoter was introduced into common wheat, UidA was
specifically expressed in wheat endosperm and no gene
silence was observed (Lamacchia et al., 2001). Based on
these results, the gene silence in the present study might
result from the homology between gene GluA-2 and genes
Bx7 and By9 and gene GluA-2 insertion site in wheat
genome. Furthermore, the gene silence can be inherited to
T1 progeny, since genes 1Bx7 and 1By9 were completely
silenced.
The transgenic progenies reported in this study pro-
vide materials to study relationship between baking-qual-
ity and HMW glutenin subunits composition in wheat.
Wheat HMW-GS appears to have quantitative and quali-
tative effects on baking quality. For example, HMW-GS
genes 1Ax1, 1Dx5 and 1Dy10 have been proved to be
associated with good bread making quality. While HMW-
GS genes 1Ax2 and 1Ay12 are considered to be respon-
sible for poor bread baking quality. However, there is not
a clear resolution for the relationships because of lacking
enough mutants (Gianibelli et al., 2001). Backcrossing this
HMW-GS 1Bx-7 and 1By-9 gene mutants with Bobwhite
wheat allows the production of near isogenic lines with
different genes for glutenin subunits, with which the rela-
tionship between grain baking quality and HMW-GS Bx-7
and By-9 subunits can be elucidated clearly.
Acknowledgements: We thank Dr. WANG Dao-Wen
(Institute of Genetics and Developmental Biology, The
Chinese Academy of Sciences) for kindly providing wheat
endosperm specific promoter Glu1-D1.
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