Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using
Agrobacterium-mediated
co-transformation. Two expression cassettes, consisting of separate DNA fragments containing
Glu-1Dy10
and hygromycin phosphotransferase II (
HPTII
) resistance genes, were introduced separately into
Agrobacterium tumefaciens
EHA105 for co-infection. Each EHA105 strain harboring
Glu-1Dy10
or
HPTII
was infected into rice calli at a 3: 1 ratio of
Glu-1Bx7
and
HPTII
. Among 290 hygromycin-resistant T
0
plants, we obtained 29 transgenic lines with both the
Glu-1Dy10
and
HPTII
genes inserted into the rice genome. We reconfirmed the integration of the
Glu-1Dy10
gene into the rice genome by Southern blot analysis. Transcripts and proteins of the
Glu-1Dy10
in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the
Glu-1Dy10
gene were successfully screened in the T
1
generation.
Introduction
Improvement of rice quality, especially that of rice flour is of great relevance to many Asian counties, where rice is widely cultivated, and rice flour has been used for many food products. However, dough made from rice lacks extensibility and elasticity. A probable cause is lack of proteins responsible for this trait in the rice endosperm. On the other hand, dough made from wheat flour is elastic and extensible making it suitable for many food products particularly for bread
[26]
. Transgenic rice with improved dough functionality by transformation of wheat gluten genes, such as high and low-molecular weight glutenin subunits, gliadins maybe substitute or complement wheat flour for making bread.
Wheat flour is different from other cereal flours, including rice. The unique processing properties of wheat flour result from the unusual biomechanical properties of the gluten proteins, which form a network conferring elasticity and extensibility to the dough
[5]
. Gluten proteins consist of monomeric gliadins and polymeric glutenins. Gliadins are single chain molecules which form only intra-chain disulphide bonds. In contrast, the glutenin subunits form both interand intra-chain disulphide bonds. The high molecular weight glutenin subunits (HMW-GS) of wheat play an important role in determining the functional properties of wheat dough
[19
,
20
,
27
,
29]
.
Bread wheat contains from three to six HMW-GS genes, with tightly linked pairs of genes encoding x- and y-type subunits being present at each of the Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively
[21]
. Allelic differences in the HMW-GS composition result in effects on the structures and properties of the glutenin polymers and hence on bread-making quality
[22
,
28]
.
Glu-1Dy10
, one of the HMW-GSs in the D1 chromosome, consists of 648 amino acids and five cysteine residues in the C-terminal domain. The central domain has an additional cysteine residue that is present close to the end of the C-terminus in the y-type subunits and, occasionally, closes to the N-terminus in the x-type subunits. The y-type subunits are highly cross-linked with x-type subunits and enhance mixing strength and tolerance of dough. In particular, HMW-GS 1Ax1 and 1Dx5+1Dy10, encoded by chromosomes 1A and 1D, respectively, are associated with strong dough and good bread-making quality.
Several HMW-GS genes have been shown to be functional when transformed into
Escherichia coli
[10]
, tobacco
[23]
, wheat
[1
,
2
,
5
,
6]
and tritordeum
[24]
.
The genetic engineering of transgenic plants in most crop species requires the use of selectable marker genes and selective agents, such as herbicides and antibiotics in order to minimize regeneration of non-transformed tissues. However, the presence of selectable marker genes in transgenic crops destined for field cultivation and human food leads to serious public concerns about the safety of transgenic crops, even though several risk-assessment reports
[14
,
25]
have shown that neither the genes nor their products are harmful to human or environmental health. Because combination of wheat gluten genes is important to make transgenic rice plants with good bread-making quality, transgene pyramiding of transgenic rice plants, which containing various wheat gluten genes is required. Repeated use of the same promoter and a polyadenylation signal for different selectable marker genes could result in transcriptional gene silencing
[12]
. Therefore, eliminating selectable marker genes is crucial for stacking multiple traits in a transgenic plant. Moreover, generating marker-free transgenic plants responds not only to public concerns over the safety of genetically engineered crops, but supports multiple transformation cycles for transgene pyramiding.
In this study, we produced marker-free transgenic rice expressing the wheat HMW-GS protein,
Glu-1Dy10
, without any herbicide or antibiotic resistance marker genes. The marker-free transgenic plant expressing
Glu-1Dy10
gene is critical material for generating transgenic plant advanced quality processing of bread and noodle without antibiotic markers.
Materials and Methods
- Cloning of the wheatGlu-1Dy10glutenin gene
′Jokyeong′ (
Triticum aestivum
L. cv. Jokyeong) was used for cloning the
Glu-1Dy10
gluenin gene. The
Glu-1Dy10
gene was amplified by polymerase chain reaction (PCR) of genomic DNA using the primers
Glu-1Dy10-CF (primer sequences
: 5′ - AGGGTACCGAGATGGCTAAGCGGCTGG - 3′) and
Glu-1Dy10-CR
(primer sequences: 5′- GATCTAGATCACTGGCTAGCCGACAATG -3′), which were designed from a sequence on GenBank (accession no. X12929). The PCR temperature cycling conditions were 4 min at 94°C, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 2 min, and a final extension at 72°C for 10 min. The amplification products were separated on a 1% agarose gel and visualized with EtBr. The amplified products were sub-cloned using a TOPO TA Cloning kit for sequencing (Invitrogen, Carlsbad, CA, USA).
- DNA constructs
To make a marker-free vector, we amplified promoter region of high-molecular weight glutenin gene (Genbank accession no. AY795083.1) from wheat cultivar ′Jokyeong′. And then amplified promoter was inserted into the
pBTEX
binary vector, which modified from pCAMBIA1300 binary vector. The
HPTII
expression cassette (
CaMV
35S promoter-
HPTII
gene-
CaMV
35S terminator) in the
pBTEX
binary vector was removed by
XhoI
and
EcoRI
restriction enzyme treatment. After klenow enzyme treatment for blunt ligation, the vector was self-ligated. Then, amplified the
Glu-1Dy10
gene with the
KpnI
and
XbaI
restriction enzyme sites was constructed into
pBTEX
binary vectors under the control of HMW (high-molecular weight) glutenin promoter (
Fig. 1
, upper panel). The positive selectable marker cassette for co-transformation was used by an empty
pBTEX
binary vector (
Fig. 1
, lower panel).
- Agrobacterium handling
Competent
Agrobacterium tumefaciens
EHA105 was transformed with
Glu-1Dy10
- cloned binary vector and an empty vector containing
HPTII
for the selectable marker using the freeze-thaw method
[7]
. T
0
plants were selected on YEP media containing kanamycin (50 mg/l). Transformation was confirmed by PCR amplification of plasmids mini-prepped from each
Agrobacterium
strain
[3]
.
- Rice co-transformation
Mature seeds of
Oryza sativa
L. subsp.
japonica
var. Dongjin were used to induce callus formation on callus induction (CI) medium [N6 salts
[9]
with vitamins, 2.5 g/l proline, 2 mg/l 2,4-D, 0.5 g/l casamino acid, 30 g/l sucrose and 2 g/l gelrite, pH 5.7]. After 21 days of incubation in the dark at 25°C, the scutellum-derived calli were excised and preincubated on CI medium for 1 week. Agrobacterial cells were grown on YEP solid medium containing antibiotics at 25°C for 2 days. And then, agrobacterial cells were resuspended in suspension medium (N
6
salts with vitamins, 2 mg/l 2,4-D, 0.5 g/l casamino acid, 30 g/l sucrose, and 10 g/l glucose, pH 5.7) with 200 μM acetosyringone as a final concentration. After two
Agrobacterium
cells were mixed in a 3:1 ratio of EHA105 with
Glu-1Dy10
gene expressing cassette and EHA105 with
HPTII
gene expressing cassette, the calli were transformed by swirling in the mixture of
Agrobacterium
cultures for 30 min. The calli were blotted on Whatman no. 1 paper and cocultivated on the cocultivation medium (N
6
salts with vitamins, 2 mg/l 2,4-D, 0.5 g/l casamino acid, 30 g/l sucrose, 10 g/l glucose, and 2 g/l gelrite, pH 5.2 with 200 μM acetosyringone as a final concentration). After 3 days, the calli were washed with liquid CI medium supplemented with 250 mg/l cefotaxime and 150 mg/l and placed on the selection medium (CI medium supplemented with 50 mg/l hygromycin, 250 mg/l cefotaxime). After selection and regeneration, the regenerated plantlets were acclimatized and grown in a greenhouse.
- PCR analysis of T0plants
PCR was performed with the GeneAmp System 9700 (Applied Biosystems, Foster City, CA, USA) with a gene-specific primer set (
Glu-1Dy10
; forward 5′-CGCAAGACAATATGAGCAAAC- 3′, reverse 5′-GTTGCCTTTGTCCTGTGTGCT- 3′,
HPTII
; forward 5′-CGCTTCTGCGGGCGATTT-3′, reverse 5′-CCCATTCGGACCGCAAGGA -3′) and EF Taq DNA polymerase (Solgent Co. Seoul, South Korea). Each reaction mixture (30 μM) consisted of 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl
2
, 40 mM KCl, 250 μM dNTPs, and 1 U Taq DNA polymerase. Amplified products were separated on a 1% agarose gel, stained with EtBr, and visualized with a UV illuminator.
- Southern hybridization analysis
Rice genomic DNA was prepared by the CTAB extraction method
[30]
. Aliquots of 5 μg of purified DNA were digested with restriction endonuclease (
EcoRI
), size-fractionated on a 0.8% agarose gel, and the DNA was transferred to a nylon membrane through capillary blotting in 10× SSC (Gene Screen, DuPont, Wilmington, DE, USA). The blots were la-labeled using AlkPhos Direct (Amersham, GE Healthcare. Piscataway, NJ, USA) according to the manufacturer′s instructions. After hybridization, the filters were washed for 30 min at 55°C to remove unlabelled probe. Subsequently, CD-star Detection Reagent (Amersham, GE Healthcare. Piscataway, NJ, USA) was used to detect and generate signals.
- RNA extraction and RT-PCR analysis
T
1
generation seeds were frozen in liquid nitrogen and then ground to powder using a mortar and pestle. Total RNA was extracted using a method reported previously [33]. The isolated RNA preparations were then reverse-transcribed with oligo-dT primer and a First Strand cDNA Synthesis kit for RT-PCR (Roche Co., Basel, Switzerland) with gene-specific primers. The primers were as follows:
Glu-1Dy10
forward 5′-CGCAAGACAATATGAGCAAAC- 3′,
Glu-1Dy10
reverse 5′-GTTGCCTTTGTCCTGTGTGCT -3′;
OsActin
primers were used as internal standards for mRNA expression profiling
[17
,
32]
. The PCR conditions consisted of initial denaturation at 94°C for 4 min, followed by 30 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min. The experiments were repeated three times and all produced similar results. The OsActin control primers were 5′- GGAACTGGTATGGTCAAGGC -3′ and 5′- AGTCTCATGGATACCCGCAG -3′
[8]
.
- Protein extraction and Western blot
T
1
generation seeds were frozen in liquid nitrogen and then ground to powder using a mortar and pestle. Total storage proteins in the rice endosperm were extracted with 50mM Tris-HCl (pH 8.0) containing 2% SDS, 50% of 1-propanol and 1% of dithiothreitol, as described
[4]
. Amount of extracted total proteins was measured by Nanodrop Spectrophotometer (ND-1000, Thermo Fischer Scientific, Wilmington DE, USA). Western blot analysis was performed as described
[18]
.
Results and Discussion
- DNA construction andAgrobacteriumtransformation for marker-free transgenic rice
- Co-transformation experiments were conducted using two expression cassettes containing separate linear DNA fragments with theGlu-1Dy10and hygromycin resistance (HPTII) genes. The amplifiedGlu-1Dy10gene from genomic DNA ofTriticum aestivumcv. Jokyeong was constructed into thepBTEXmarker-free vector, which was changed with the HMW promoter after removing hygromycin resistance gene (HPTII) and the cauliflower mosaic virus promoter (CaMV35S) (Fig. 1A, upper panel). An emptypBTEXvector was used as the positive selectable marker cassette for co-transformation(Fig. 1B, lower panel). The two expression binary vectors were separately introduced into A. tumefaciens EHA105 strain for plant transformation. Each binary vector was rescued from the EHA105 strain harboringGlu-1Dy10and theHPTII, and then theHPTIIandGlu-1Dy10genes were validated by PCR analysis with specific primers.
Vector constructs expressing the Glu-1Dy10 (upper panel) and hygromycin phosphotransferase II (HPTII) (lower panel) genes in the binary vectors. HMW pro, high-molecular weight promoter; OCS-ter, octopine synthase terminator; CaMV 35S, cauliflower mosaic virus promoter; 35S-ter, 35S terminator; RB, right border; LB, left border.
- Generation of marker-freeGlu-1Dy10transgenic rice plants
Each EHA105 strain harboring
Glu-1Dy10
expression vector or
HPTII
expression vector was cultured in YEP medium for plant transformation. The cultured cells were resuspended to OD600 =0.1 in AAM medium
[11]
, and each
Glu-1Dy10
and
HPTII
cell was added at a 3:1 ratio. These mixed cells were co-infected into rice calli. The transformed calli were selected in hygromycin medium because we co-infected EHA 105 cells containing
HPTII
gene to calli. We obtained 290 independent hygromycin-resistant T
0
plants through
Agrobacterium
-mediated co-transformation system. Genomic DNA from 290 independent T
0
plants was extracted and insertion of
HPTII
and
Glu-1Dy10
genes was analyzed using PCR analysis with gene specific primers. As shown in
Figure 2
,
HPTII
gene in all of T
0
plants was amplified, but no PCR products in ′Dongjin′ used as negative controls were detected. Next, we investigate the insertion of
Glu-1Dy10
gene in T
0
plants. Among 290 independent transgenic lines, only 29 T
0
plants contained
Glu-1Dy10
gene (
Fig. 2)
. This result means that 29 transgenic lines harbored both
Glu-1Dy10
and
HPTII
genes. And the co-transformation frequency was 10% in our experimental system (
Table 1
). In a previous report, co-transformation frequency in rice was about from 2% to 14%
[13]
. This result indicates that transformation efficiency is dependent on rice cultivar and the experimental conditions. Although the generating of marker- free plants based on the
Agrobacterium
-mediated co-trans-formation using two different expression cassettes was need more time consuming and effort, this method could be efficiently produce marker-free transgenic rice plants.
Identification of T0 plants by gene specific primer sets. Glu-1Dy10 (upper panel) and HPTII (lower panel) genes were amplified using Glu-1Dy10 and HPTII specific primer sets, respectively. SM, molecular marker; ‘Dongjin′ (Korean rice cultivar), non-transgenic plant; 1-23, co-transformed transgenic lines. Genomic DNAs from each plant were used as the template for Glu-1Dy10 and HPTII specific amplification. The reaction products of the sample plant were analyzed by electrophoresis on a 1.0% agarose gel.
Co-transformation efficiency calculated during regeneration in rice-transformation experiments
Co-transformation efficiency calculated during regeneration in rice-transformation experiments
We performed Southern blot analysis to validate integration of
Glu-1Dy10
genes and guess segregation ratio of the marker-free plant in T
1
plants. One or multi signal bands were detected in 29 selected T
0
plants lines (
Fig. 3
). These results indicating that 29 selected transgenic lines were independent transgenic rice plants.
Southern hybridization analysis of Glu-1Dy10 gene from T0 plants. The 1.35 kb fragment of HMW promoter was amplified by PCR using specific primer sets as the probe.
- Transcript and protein analysis ofGlu-1Dy10gene in the co-transformed rice plants
We used HMW glutenin promoter to express
Glu-1Dy10
because
Glu-1Dy10
expression in rice endosperm is important for rice flour quality. Total RNAs from randomly selected two-copy inserted T
1
transgenic seeds (13, 16, 29 lines) were extracted, and
Glu-1Dy10
gene transcript level was examined by semi-quantitative RT-PCR. The
Glu-1Dy10
transcripts were successively expressed in the T
1
generation transgenic seeds, whereas
Glu-1Dy10
expression in ′Dongjin′ was not detected (
Fig. 4
). OsActin expression was used as a quantitative control. And we analyzed the protein expression of
Glu-1Dy10
by Western blot with an anti x-type HMW specific antibody. The 11 transgenic plants (2, 6, 7, 9, 15, 18, 20, 22, 24, 26, 28) which were shown abnormal morphologies comparing with ′Dongjin′ were removed. After total proteins were extracted from wheat (′Jokyeong′ cultivar), ′Dongjin′ and transgenic plants, 0.5 μ g of wheat and 40 μ g of total protein extract of transgenic plants were used for SDS-PAGE. The immunospecificity of the anti- x-type HMW specific antibody was verified by in vivo experiment. Although the used antibody was x-type HMW specific, protein bands of
Glu-1Dy10
were well detected in transgenic plants. However, the level of protein expression was not depended on their inserted copy number (
Fig. 5
). No protein bands were detected in one copy-inserted lines and muti-copies inserted lines. We guess that the expression levels of
Glu-1Dy10
were too low to detect signal in case of the one-copy inserted lines. In case of Multi-copies inserted lines, this phenomenon may be related to homology- dependent gene silencing in plants
[2]
. Genetic engineering of plants sometimes results in transgene silencing after integration into the genome, which may relate to a defense mechanism against foreign DNA expression
[15
,
31]
. Homology-dependent gene silencing has attracted considerable interest because it may be detrimental to genetic engineering and also because of its usefulness as a tool to study the mechanisms involved in detecting and inactivating exogenous DNA
[15
,
16]
.
Transcript analysis of the Glu-1Dy10 gene from T1 seeds. RT-PCR was performed with Glu-1Dy10 T1 seed transcripts to measure Glu-1Dy10 mRNA expression. OsActin was used as a control. The reaction products of the sample plant were analyzed by electrophoresis in a 1.0% agarose gel.
Protein expression analysis of Glu-1Dy10 gene from T1 seeds. Western blotting was performed with an anti x-type HMW specific antibody. Total protein extracts of 0.5 μ g of wheat and 40 μ g of transgenic plants and ‘Dongjin′ were used for SDS-PAGE. CN, copy number of the Glu-1Dy10 integrated in transgenic plants.
- Selection of marker-free plants harboringGlu-1Dy10gene in the T1generation
To select
Glu-1Dy10
marker-free plants harboring only the
Glu-1Dy10
gene,
72
T
1
generation seeds of the transgenic plant 13 were planted in soil and genomic DNA was extracted from leaves of plantlets after 4 weeks. Insertion of the
Glu-1Dy10
and
HPTII
genes was investigated by PCR analysis with
Glu-1Dy10
and
HPTII
specific primers, respectively. As shown in
Fig. 6
, most of the transgenic lines harbored both the
Glu-1Dy10
and
HPTII
genes, and some inserted only the
HPTII
gene. However, transgenic 1, 7, 10, 14 and 21 lines contained only the
Glu-1Dy10
gene (
Fig. 6
). This result shows that marker-free plants containing only the
Glu-1Dy10
gene were successfully screened at the T
1
generation. Finally, we produced marker-free transgenic rice plants harboring
Glu-1Dy10
gene. This marker-free transgenic plant harboring
Glu-1Dy10
will become useful material to optimize transgenic rice plants, which has advanced quality processing of bread and noodle by crossing with genetically engineered rice plants with other gluten genes.
PCR analysis of T1 progenies to select marker-free transgenic plant containing Glu-1Dy10 gene. Dongjin, non-transgenic plant as negative control; 1-24, T1 progeny lines from T0 plants containing both Glu-1Dy10 and HPTII genes. The reaction products of the sample plant were analyzed by electrophoresis in a 1.0% agarose gel.
Acknowledgements
This work was supported by a grant from the Department of Functional Crop, NICS, RDA (No. PJ008717), Republic of Korea. We would like to thanks sincerely to Dr. Paul Scott’s assistance of USDA-ARS for offering anti-x-type HMW specific antibody.
Altpeter F.
,
Vasil V.
,
Srivastava, V. l Vasil I. K.
1996
Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat
Nat Biotechnol
14
1155 -
1159
DOI : 10.1038/nbt0996-1155
Alvarez M. L.
,
Guelman S.
,
Halford N. G.
,
Lusting S.
,
Reggiardo M. I.
,
Ryabushkina N.
,
Shewry P.
,
Stein J.
,
Vallejos R. H.
2000
Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits
Theor Appl Genet
100
319 -
327
DOI : 10.1007/s001220050042
An G.
,
Evert P. R.
,
Mitra A.
,
Ha S. B.
,
Gelvin S.B.
,
Schilperoort R.A.
1988
Plant molecular biology manual
Kluwer Academic Publishers
Boston, MA
Binary vectors
A3/1 -
A3/19
Araki E.
,
Ikeda M. T.
,
Ohgihara Y.
,
Toyoda A.
,
Yano H.
2008
Development of transgenic rice (Oryza sativa L.) expressing wheat high- and low-molecular-weight glutenin subunit proteins
Breed Sci
58
121 -
128
DOI : 10.1270/jsbbs.58.121
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
DOI : 10.1038/nbt1197-1295
Blechl A. E.
,
Anderson O. D.
1996
Expression of a novel highmolecular-weight glutenin gene in transgenic wheat
Nat Biotechnol
14
875 -
879
DOI : 10.1038/nbt0796-875
Chen H.
,
Nelson R. S.
,
Sherwood J. L.
1994
Enhanced recovery of transformants of agrobacterium tumefaciens after freeze-thaw transformation and drug selection
Biotechniques
16
664 -
669
Cho J. I.
,
Ryoo N.
,
Ko S.
,
Lee S. K.
,
Lee J.
,
Jung K. H.
,
Lee Y. H.
,
Bhoo S. H.
,
Winderickx J.
,
An G.
,
Hahn T. R.
,
Jeon J. S.
2006
Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)
Planta
224
598 -
611
DOI : 10.1007/s00425-006-0251-y
Chu C. C.
,
Wang C. S.
,
Sun C. S.
,
Hsu C.
,
Yin K. C.
,
Chu C. Y.
,
Yun. B. F.
1975
Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources
Sci China Math
18
659 -
668
Galili G.
1989
Heterologous expression of a wheat high-molecular-weight glutenin gene in Escherichia coli
Proc Natl Acad Sci USA
86
7756 -
7760
DOI : 10.1073/pnas.86.20.7756
Hiei Y.
,
Ohta S.
,
Komari T.
,
Kumashiro T.
1994
Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA
Plant J
6
271 -
282
DOI : 10.1046/j.1365-313X.1994.6020271.x
Komari T.
,
Hiei Y.
,
Saito Y.
,
Murai N.
,
Kumashiro T.
1996
Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers
Plant J
10
165 -
174
DOI : 10.1046/j.1365-313X.1996.10010165.x
Kuiper H. A.
,
Kleter G. A.
,
Noteborn H. P.
,
Kok E. J.
2001
Assessment of the food safety issues related to genetically modified foods
Plant J
27
503 -
528
DOI : 10.1046/j.1365-313X.2001.01119.x
Kumpatla S. P.
,
Chandrasekharan M. B.
,
Iyer L. M.
,
Li G.
,
Hall T. C.
1998
Genome intruder scanning and modulation systems and transgene silencing
Trends Plant Sci
3
97 -
104
DOI : 10.1016/S1360-1385(97)01194-1
Matzke M. A.
,
Matzke J. M.
,
Eggleston W. B.
1996
Paramutation and transgene silencing: a common response to invasive DNA?
Trends Plant Sci
1
382 -
388
DOI : 10.1016/S1360-1385(96)80313-X
McElroy D.
,
Zhang W.
,
Cao J.
,
Wu R.
1990
Isolation of an efficient actin promoter for use in rice transformation
Plant Cell
2
163 -
171
DOI : 10.1105/tpc.2.2.163
Park S. K.
,
Jung Y. J.
,
Lee J. R.
,
Lee Y. M.
,
Jang H. H.
,
Lee S. S.
,
Park J. H.
,
Kim S. Y.
,
Moon J. C.
,
Lee S. Y.
,
Chae H. B.
,
Shin M. R.
,
Jung J. H.
,
Kim M. K.
,
Kim Y. Y.
,
Yun D. J.
,
Lee G. O.
,
Lee S. Y.
2009
Heat-shock and redox-dependent functional switching of an h-type arabidopsis thioredoxin from a disulfide reductase to a molecular chaperone
Plant Physiol
150
552 -
561
DOI : 10.1104/pp.109.135426
Payne P. I.
,
Corfield K. G.
,
Blackman J. A.
1979
Identification of a high-molecular-weight subunit of glutenin whose presence correlates with breadmaking quality in wheats of related pedigree
Theor Appl Genet
55
153 -
159
DOI : 10.1007/BF00295442
Payne P. I.
,
Corfield K. G.
,
Holt L. M.
,
Blackman J. A.
1981
Correlations between the inheritance of certain high-molecular-weight subunits of glutenin and breadmaking quality in progenies of six crosses of bread wheat
J Sci Food Agric
32
51 -
60
DOI : 10.1002/jsfa.2740320109
Payne P. I.
,
Law C. N.
,
Mudd E. E.
1980
Control by homologous group 1 chromosomes of the high-molecular- weight subunits of glutenin, a major protein of wheat endosperm
Theor Appl Genet
58
113 -
120
Payne P. I.
,
Nightingale M. A.
,
Krattiger A. F.
,
Holt L. M.
1987
The relationship between HMW glutenin subunit composition and the bread-making quality of Britishgrown wheat varieties
J Sci Food Agric
40
51 -
65
DOI : 10.1002/jsfa.2740400108
Roberts L. S.
,
Thompson R. D.
,
Flavell R. B.
1989
Tissue-specific expression of a wheat high-molecular-weight glutenin gene in transgenic tobacco
Plant Cell
1
569 -
578
DOI : 10.1105/tpc.1.6.569
Rooke L.
,
Barro F.
,
Tatham A. S.
,
Fido R.
,
Steele S.
,
Bekes F.
,
Gras P.
,
Martin A.
,
Lazzeri P. A.
,
Shewry P. R.
,
Barcelo P.
1999
Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes
Theor Appl Genet
99
851 -
858
DOI : 10.1007/s001220051305
Ramessar K.
,
Peremarti A.
,
Go´mez-Galera S.
,
Naqvi S.
,
Moralejo M.
,
Muñoz P.
,
Capell T.
,
Christou P.
2007
Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics
Transgenic Res
16
261 -
280
DOI : 10.1007/s11248-007-9083-1
Sangtong V.
,
Moran D. L.
,
Chikkwamba R.
,
Wang K.
,
Woodman-Clikeman W.
,
Long M. J.
,
Lee M.
,
Scott M. P.
2002
Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize
Theor Appl Genet
105
937 -
945
DOI : 10.1007/s00122-002-1036-8
Shewry P. R.
,
Tatham A. S.
,
Fido R.
,
Jones H.
,
Barcelo P.
,
Lazzeri P. A.
2001
Improving the end use properties of wheat by manipulating the grain protein composition
Euphytica
119
45 -
48
DOI : 10.1023/A:1017590321267
Tatham A. S.
,
Shewry P. R.
1985
The conformation of wheat gluten proteins. The secondary structures and thermal stabilities of alpha-, beta-, gamma- and omega- gliadins
J Cereal Sci
3
103 -
113
DOI : 10.1016/S0733-5210(85)80021-7
Thompson B. G.
,
Anderson R.
,
Murray R. G.
1980
Unusual polar lipids of Micrococcus radiodurans strain Sark
Can J Microbiol
26
1408 -
1411
DOI : 10.1139/m80-234
Vaucheret H.
,
Béclin C.
,
Elmayan T.
,
Feuerbach F.
,
Godon C.
,
Morel J. B.
,
Mourrain P.
,
Palauqui J. C.
,
Vernhettes S.
1998
Transgene-induced gene silencing in plants
Plant J
16
651 -
659
DOI : 10.1046/j.1365-313x.1998.00337.x
Volkov R. A.
,
Panchuk I. I.
,
Schoffl F.
2003
Heatstress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR
J Exp Bot
54
2343 -
2349
DOI : 10.1093/jxb/erg244
Zhiwu Li.
,
Harold N. T.
2005
Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch
Biotechniques
38
872 -
876
DOI : 10.2144/05386BM05