U.S. patent application number 10/810160 was filed with the patent office on 2005-01-20 for value-added traits in grain and seed transformed with thioredoxin.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Buchanan, Bob B., Cho, Myeong-Je, Lemaux, Peggy G., Marx, Corina, Wong, Joshua.
Application Number | 20050015831 10/810160 |
Document ID | / |
Family ID | 32913365 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050015831 |
Kind Code |
A1 |
Cho, Myeong-Je ; et
al. |
January 20, 2005 |
Value-added traits in grain and seed transformed with
thioredoxin
Abstract
Compositions and methods of use are provided herein to make and
use transgenic plants with value-added traits.
Inventors: |
Cho, Myeong-Je; (Alameda,
CA) ; Lemaux, Peggy G.; (Moraga, CA) ;
Buchanan, Bob B.; (Oakland, CA) ; Wong, Joshua;
(San Francisco, CA) ; Marx, Corina; (Oakland,
CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
425 MARKET STREET
SAN FRANCISCO
CA
94105-2482
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
32913365 |
Appl. No.: |
10/810160 |
Filed: |
March 25, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10810160 |
Mar 25, 2004 |
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09538864 |
Mar 29, 2000 |
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6784346 |
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60126736 |
Mar 29, 1999 |
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60127198 |
Mar 31, 1999 |
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60169162 |
Dec 6, 1999 |
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60177740 |
Jan 21, 2000 |
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60177739 |
Jan 21, 2000 |
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Current U.S.
Class: |
800/278 |
Current CPC
Class: |
C12N 15/8242 20130101;
C12N 15/8261 20130101; A23V 2002/00 20130101; A23J 1/12 20130101;
A21D 13/064 20130101; C12N 15/8251 20130101; C07K 14/415 20130101;
A23K 10/30 20160501; C12N 9/0036 20130101; A23V 2002/00 20130101;
A23L 33/105 20160801; C12N 15/8245 20130101; Y02A 40/146 20180101;
A23V 2300/21 20130101 |
Class at
Publication: |
800/278 |
International
Class: |
A01H 001/00; C12N
015/82 |
Goverment Interests
[0002] This invention was made with Government support under Grant
9803835 from the U.S. Department of Agriculture. The Government has
certain rights to this invention.
Claims
1-31. (Canceled).
32. A transgenic monocot plant wherein at least part of said plant
comprises a recombinant nucleic acid comprising a promoter active
in said part operably linked to a nucleic acid molecule encoding a
thioredoxin h polypeptide wherein said promoter is a seed or grain
maturation-specific promoter.
33. The transgenic plant of claim 32 wherein said part is a
seed.
34. The transgenic plant of claim 32 wherein said part is a
grain.
35. (Canceled).
36. The transgenic plant of claim 32 wherein said promoter is
selected from the group consisting of rice glutelins, rice oryzins,
rice prolamines, barley hordeins, wheat gliadins, wheat glutelins,
maize zeins, maize glutelins, oat glutelins, sorghum kasirins,
millet pennisetins, rye secalins, and a maize embryo-specific
globulin promoter.
37. The transgenic plant of claim 36 wherein said barley hordein
promoter is selected from the group consisting of 131 hordein and D
hordein promoters.
38. The transgenic plant of claim 32 wherein said plant is selected
from the group consisting of rice, barley, maize, wheat, oat, rye,
sorghum, millet, triticale, turf grass and forage grass.
39. (Canceled).
40. The transgenic plant of claim 32 wherein said thioredoxin h is
barley, wheat, tobacco, rice, Brassica, Arabidopsis, Picea, or soy
bean thioredoxin h.
41. The transgenic plant of claim 32 wherein said recombinant
nucleic acid further comprises a nucleic acid molecule encoding a
signal peptide operably linked to said promoter and said nucleic
acid molecule encoding a thioredoxin protein.
42. The transgenic plant of claim 41 wherein said signal peptide
targets expression of the thioredoxin polypeptide to an
intracellular body.
43. The transgenic plant of claim 42 wherein said signal peptide is
selected from the group consisting of barley B1 hordein and D
hordein signal peptides.
44-76. (Canceled).
77. A transgenic seed or grain comprising a recombinant nucleic
acid comprising a promoter active in said seed or grain operably
linked to a nucleic acid molecule encoding thioredoxin h
polypeptide wherein said promoter is a seed or grain
maturation-specific promoter.
78. (Canceled).
79. The transgenic seed or grain of claim 77 wherein said promoter
is selected from the group 74 consisting of rice glutelins, rice
oryzins, rice prolamines, barley hordeins, wheat gliadins, wheat
glutelins, maize zeins, maize glutelins, oat glutelins, sorghum
kasirins, millet pennisetins, rye secalins, and a maize
embryo-specific globulin.
80. The transgenic seed or grain of claim 79 wherein said barley
hordein promoter is selected from the group consisting of B1
hordein and D hordein promoters.
81. The transgenic seed or grain of claim 80 wherein said seed or
grain is selected from the group consisting of rice, barley, maize,
wheat, oat, rye, sorghum, millet, and triticale seed or grain.
82. (Canceled).
83. The transgenic seed or grain of claim 77 wherein said
thioredoxin h is barley, wheat, tobacco, rice, soy bean, Brassica,
Picea, or Arabidopsis thioredoxin h.
84. The transgenic seed or grain of claim 77 wherein said
recombinant nucleic acid further comprises a nucleic acid molecule
encoding a signal peptide operably linked to said promoter and said
nucleic acid molecule encoding a thioredoxin protein.
85. The transgenic seed or grain of claim 84 wherein said signal
peptide targets expression of the thioredoxin polypeptide to an
intracellular body.
86. The transgenic seed or grain of claim 85 wherein said signal
peptide is selected from the group consisting of barley B1 hordein
and D hordein signal peptides.
87-111. (Canceled).
112. The transgenic plant of claim 32 wherein said plant is
wheat.
113. The transgenic plant of claim 112 wherein said thioredoxin is
wheat thioredoxin h.
114. The transgenic seed or grain of claim 81 wherein said seed or
grain is barley.
115. The transgenic seed or grain of claim 114 wherein said
thioredoxin is barley thioredoxin h.
116. The transgenic seed or grain of claim 81 wherein said seed or
grain is wheat.
117. The transgenic seed or grain of claim 116 wherein said
thioredoxin is wheat thioredoxin h.
118. The transgenic plant of claim 32 wherein said thioredoxin is
Arabidopsisthioredoxin h.
119. The transgenic plant of claim 32 wherein said thioredoxin is
soybean thioredoxin h.
120. The transgenic monocot seed or grain of claim 77 wherein said
thioredoxin is Arabidopsis thioredoxin h.
121. The transgenic monocot seed or grain of claim 77 wherein said
thioredoxin is soybean thioredoxin h.
122. The transgenic plant of claim 32 wherein said thioredoxin is
tobacco thioredoxin h.
123. The transgenic plant of claim 32 wherein said thioredoxin is
Brassica thioredoxin h.
124. The transgenic monocot seed or grain of claim 77 wherein said
thioredoxin is tobacco thioredoxin h.
125. The transgenic monocot seed or grain of claim 77 wherein said
thioredoxin is brassica thioredoxin h.
Description
[0001] This application claims the benefit of the filing date of
application Ser. No. 60/126,736, filed Mar. 29, 1999, pending;
application Ser. No. 60/127,198, filed Mar. 31, 1999 pending,
application Ser. No. 60/169,162, filed Dec. 6, 1999, pending;
application Ser. No. 60/177,740 filed Jan. 21, 2000, pending; and
application Ser. No. 60/177,739, filed Jan. 21, 2000, pending, all
of which are expressly incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0003] Thioredoxins are small (about 12 kDa) thermostable proteins
with catalytically active disulfide groups. This class of proteins
has been found in virtually all organisms, and has been implicated
in myriad biochemical pathways (Buchanan et al., 1994). The active
site of thioredoxin has two redox-active cysteine residues in a
highly conserved amino acid sequence; when oxidized, these
cysteines form a disulfide bridge (--S--S--) that can be reduced to
the sulfhydryl (--SH) level through a variety of specific
reactions. In physiological systems, this reduction may be
accomplished by reduced ferredoxin, NADPH, or other associated
thioredoxin-reducing agents. The reduced form of thioredoxin is an
excellent catalyst for the reduction of even the most intractable
disulfide bonds. Generally only one kind of thioredoxin is found in
bacterial or animal cells. In contrast, photosynthetic organisms
have three distinct types of thioredoxin. Chloroplasts contain a
ferredoxin/thioredoxin system comprised of ferredoxin,
ferredoxin-thioredoxin reductase and thioredoxins f and m, which
function in the light regulation of photosynthetic enzymes
(Buchanan, 1991; Scheibe, 1991). The other thioredoxin enzyme
system is analogous to that established for animals and most
microorganisms, in which thioredoxin (h-type in plants) is reduced
by NADPH and NADPH-thioredoxin reductase (NTR) (Johnson et al.,
1987a; Florencio et al., 1988; Suske et al., 1979). The reduction
of thioredoxin h by this system can be illustrated by the following
equation: 1
[0004] Thioredoxin is a component of two types of enzyme systems in
plants. Chloroplasts contain a ferredoxin/thioredoxin system
comprised of ferredoxin, ferredoxin-thioredoxin reductase and
thioredoxins f and m, that are involved in the light regulation of
photosynthetic enzymes (Buchanan, 1991; Scheibe, 1991). The other
enzyme system, the NADP-thioredoxin system or NTS, is analogous to
the system established for animals and most microorganisms, in
which thioredoxin (h-type in plants) is reduced by NADPH and
NADPH-thioredoxin reductase (NTR) (Johnson et al., 1987a; Florencio
et al., 1988; Suske et al., 1979). Thioredoxin h is widely
distributed in plant tissues and exists in mitochondria,
endoplasmic reticulum (ER) and cytosol (Bodenstein-Lang et al.,
1989; Marcus et al., 1991).
[0005] Plant thioredoxin h is involved in a wide variety of
biological functions. The presence of multiple forms of
thioredexoin h protein has also been reported in plant seeds
(Bestermann et al., 1983). In wheat, three different thioredoxin
have been characterized (Vogt and Follman, 1986). Thioredoxin h
functions in the reduction of intramolecular disulfide bridges of a
variety of low molecular-weight, cystine-rich proteins, including
thionins (Johnson et al., 1987b), protease inhibitors and
chloroform/methanol-soluble proteins (CM proteins or alpha-amylase
inhibitors) (Kobrehel et al., 1991). It is likely that cytoplasmic
thioredoxins participate in developmental processes: for example
thioredoxin h has been shown to function as a signal to enhance
metabolic processes during germination and seedling development
(Kobrehel et al., 1992; Lozano et al., 1996; Besse et al., 1996).
Thioredoxin h has also been demonstrated to be involved in
self-incompatibility in Phalaris coerulescens (Li et al., 1995) and
Brassica napus (Bower et al., 1996). Several functions have been
hypothesized for rice thioredoxin h, which is believed to be
involved in translocation in sieve tubes (Ishiwatari et al.,
1995).
[0006] The NTS has been shown to improve dough quality. The
improvement in dough strength and bread quality properties of
poor-quality wheat flour resulting from the addition of thioredoxin
(Wong et al., 1993; Kobrehel et al., 1994) may be attributable to
the thioredoxin-catalyzed reduction of intramolecular disulfide
bonds in the flour proteins, specifically the glutenins, resulting
in the formation of new intermolecular disulfide bonds (Besse and
Buchanan, 1997). Thus, the addition of exogenous thioredoxin
promotes the formation of a protein network that produces flour
with enhanced baking quality. Kobrehel et al., (1994) have observed
that the addition of thioredoxin h to flour of non-glutenous
cereals such as rice, maize and sorghum promotes the formation of a
dough-like product. Hence, the addition of exogenous thioredoxin
may be used to produce baking dough from non-glutenous cereals.
[0007] In addition, it has been shown that reduction of disulfide
protein allergens in wheat and milk by thioredoxin decreases their
allergenicity (Buchanan et al., 1997; del Val et al., 1999).
Thioredoxin treatment also increases the digestibility of the major
allergen of milk (.beta.-lactoglobulin) (del Val et al., 1999), as
well as other disulfide proteins (Lozano et al., 1994; Jiao et al.,
1992). Therefore, the manipulation of the NTS offers considerable
promise for production of nutraceutical and pharmaceutical
products. A more detailed discussion of the benefits of adding
exogenous thioredoxin to food products is presented in U.S. Pat.
No. 5,792,506 to Buchanan et al.
[0008] cDNA clones encoding thioredoxin h have been isolated from a
number of plant species, including Arabidopsis thaliana
(Rivera-Madrid et al., 1993; Rivera-Madrid et al., 1995), Nicotiana
tabacum (Marty and Meyer, 1991; Brugidou et al., 1993), Oryza
sativa (Ishiwatari et al., 1995), Brassica napus (Bower et al.,
1996), Glycine max (Shi and Bhattacharyya, 1996), and Triticum
aestivum (Gautier et al., 1998). More recently, two cDNA clones
encoding wheat thioredoxin h have been isolated and characterized
(Gautier et al., 1998). The Esherichia coli NTR gene has been first
isolated (Russel and Model, 1988) and the three-dimensional
structure of the protein has been analyzed (Kuriyan et al., 1991).
Some other NTR genes have been isolated and sequenced from
bacteria, fungi and mammals. Recently, Jacquot et al., (1994) have
reported a successful isolation and sequencing of two cDNAs
encoding the plant A. thaliana NTRs. The subsequent expression of
the recombinant A. thaliana NTR protein in E. coli cells (Jacquot
et al., 1994) and its first eukaryotic structure (Dai et al., 1996)
have also been reported.
[0009] Here we disclose value-added traits in transgenic grains,
such as barley (Cho et al., 1999b)., wheat, and sorghum,
overexpressing thioredoxin
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the thioredoxin h constructs used for
transformation.
[0011] FIG. 2 shows the thioredoxin activity profile of various
barley grains transformed with wheat thioredoxin gene (wtrxh).
[0012] FIG. 3 shows the effects of heat treatment on thioredoxin
activity of crude extracts from barley grains.
[0013] FIG. 4A-B shows a western blot analysis of extract from
segregating T.sub.1 barley grain of stable transformants containing
wtrxh. Panel A: lanes 1 and 6, control barley extract (cv. Golden
Promise); lane 2, bread wheat extract (Triticum aestivum, cv.
Capitole); lane 3, extract from GPdBhss BarWtrx 22; lane 4, extract
from GPdBhssBarWtrx 29; lane 5, extract from GPdBhBarWtrx 2. Panel
B: lane 1, GPdBhBaarWtrx 2; lane 2 control barley extract. W,
wheat; B, barley.
[0014] FIG. 5 shows western blot analysis of extracts of T.sub.1
T.sub.2 and T.sub.3 barley grain transformed with wtrxh. Forty
micrograms of soluble proteins extracted from 10-20 grains of each
line were fractionated by SDS/PAGE. Lane 1, wheat germ thioredoxin
h; lane 2, nontransgenic control of GP4-96; lane 3, null segregant
T.sub.2 grain of GPdBhssBarWtrx-29-11-10; lane 4, heterozygous
T.sub.1 grain of GPdBhssBarWtrx-29; lane 5, homozygous T.sub.2
grain of GPdBhssBarWtrx-29-3; lane 6, homozygous T.sub.2 grain of
GPdBhssBarWtrx-29-3-2; lane 7, prestained standards (aprotinin, 0.9
kDa; lysozyme, 17.8 kDa; soybean trypsin inhibitor, 30.6 kDa;
carbonic anhydrase, 41.8 kDa; BSA, 71 kDa).
[0015] FIG. 6 shows the nucleic acid sequence of the B1-hordein
promoter and the 57 base pair B1-hordein signal sequence
(underlined).
[0016] FIG. 7 shows the nucleic acid sequence of the D-hordein
promoter and the 63 base pair D-hordein signal sequence
(underlined).
[0017] FIG. 8A-C shows the effect of overexpressed thioredoxin h on
pullulanase activity in transgenic barley grain during germination
and seedling development. A homozygous line, GPdBhssBarWtrx-29-3,
and a null segregant, GPdBhssBarWtrx-29-11-10, were used for the
pullulanase assays. Panel A: Pullulanase was assayed
spectrophotometrically by measuring the dye released from red
pullulan substrate at 534 nm. Panel B: Pullulanase was separated on
native 7.5% polyacrylamide gels containing the red pullulan
substrate. Activity, identified by comparison with purified barley
pullulanase, is seen as clear areas that developed on incubating
the gel in 0.2 M succinate buffer, pH 6.0, for 1 hr at 37.degree.
C. Panel C: The gel in Panel B was scanned and analyzed by
integration of the activity bands.
[0018] FIG. 9A-D shows the change in the activity and abundance of
amylases in transgenic and null segregant barley grains during
germination and seedling development based on an activity gel.
Panel A: abundance of alpha-amylases in null segregant based on
western blot. Panel B: Total amylase activity in null segregant.
Panel C: abundance of alpha-amylases in thioredoxin overexpressing
grains. Panel D: total amylase activity in thioredoxin
overexpressed grains.
[0019] FIG. 10 shows the effect of overexpressed thioredoxin h on
the activity of the major form of alpha-amylase during germination
and seeding development. The size of the major alpha-amylase
activity band in FIG. 9 was estimated by its rate of mobility
during electrophoresis.
[0020] FIG. 11A-B shows the effect of overexpressed thioredoxin h
on the abundance of alpha-amylase A and B isozymes during
germination and seedling development. The figure represents western
blots of IEF gels developed for the null segregant and transgenic
barley grains. Panel A: Null segregant. Panel B: Transgenic with
thioredoxin overexpressed.
[0021] FIG. 12 depicts the DNA constructs used for wheat
transformation.
[0022] FIG. 13 shows the endosperm-specific expression of barley
D-hordein promoter sgfp(S65T) in transgenic wheat plants.
Transgenic endosperm is at the right, transgenic embryo is at the
left.
[0023] FIG. 14 shows the PCR analysis of genomic DNA from
transgenic wheat plants.
[0024] FIG. 15A-B shows wheat thioredoxin h-overexpressing wheat
lines screened by western blot analyses. Panel A: T.sub.0 wheat
lines. Panel B T.sub.3 homozygous line.
[0025] FIG. 16 shows the effect of thioredoxin reduction on
digestion of wheat glutenins by trypsin.
[0026] FIG. 17 shows the effect of thioredoxin reduction on
digestion of wheat glutenins by pancreatin.
[0027] FIG. 18 show the effect of NTR on the reduction of proteins
in extracts of transgenic wheat overexpressing thioredoxin h verses
a null segregant.
[0028] FIG. 19 shows the effect of overexpressed thioredoxin h on
allergenicity of proteins from wheat grain.
[0029] FIG. 20 shows the barley thioredoxin h nucleotide and amino
acid sequence (SEQ ID NO:24, SEQ ID NO:26, respectively).
[0030] FIG. 21 shows the effect of overexpressed wheat thioredoxin
h on the germination of null segregant and transgenic (homozygous)
barley grains.
[0031] FIG. 22 shows the relative redox status of protein fractions
in transgenic barley grain overexpressing wheat thioredoxin h in
comparison to the null segregant in dry and germination grain.
[0032] FIG. 23 shows the effect of glucose-6-phosphate
dehydrogenase on the reduction of proteins in extracts of
transgenic wheat grain overexpressing thioredoxin h in the presence
of glucose 6-phosphate and Arabidopsis NTR:+/-NTR.
[0033] FIG. 24 shows the effect of glucose-6-phosphate
dehydrogenase on the reduction of proteins in extracts of extracts
of null segregant derived from wheat grain overexpressing
thioredoxin h in the presence of glucose 6-phosphate and
Arabidopsis NTR:+/+NTR.
SEQUENCE LISTING
[0034] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids. Only one strand of each nucleic acid sequence is shown, but
it is understood that the complementary strand is included by any
reference to the displayed strand. SEQ ID NO:1 shows the nucleic
acid sequence of the barley B1-hordein promoter and signal
sequence. SEQ ID NO:2 shows the amino acid sequence of the barley
B1-hordein signal sequence. SEQ ID NO:3 shows the nucleic acid
sequence of the barley D-hordein promoter and signal sequence. SEQ
ID NO:4 shows the ammo acid sequence of the barley D-hordein signal
sequence. Other sequences are identified below.
SUMMARY OF THE INVENTION
[0035] The present invention provides recombinant nucleic acids
encoding thioredoxin and methods of use to produce transgenic
plants overexpressing thioredoxin. Indeed, given the powerful
reducing activity of thioredoxin, over-expression of this protein
in a plant cell would be anticipated to have a serious .
detrimental effect on the cell. However, the inventors have
discovered that thioredoxin can be expressed at a high level in
plants, particularly cereal grains, without affecting the viability
of the cells in which the protein is expressed, or the seeds
themselves. By way of example, in certain embodiments the inventors
have introduced a wheat thioredoxin gene (wtrxh) into wheat. Seeds
of the transgenic-wheat plants can show an increase thioredoxin
specific activity in comparison to non-transgenic-wheat plants.
[0036] The invention thus provides transgenic plants, wherein at
least a part of a plant has an elevated level of thioredoxin
protein and/or thioredoxin specific activity compared to the
homologous part of non-transgenic plants of the same species. The
level of thioredoxin specific activity in the parts of the
transgenic plants may be at least about two times greater than the
parts of non-transgenic plants of that species. While the invention
is applicable to any plant species, it will be particularly
beneficial as applied to the monocotyledons, for example cereal
crops including, but not limited to rice, barley, wheat, oat,
maize, rye, sorghum, millet, and triticale and the dicotyledons
including, but not limited to soybeans, lima beans, tomato, potato,
soybean, cotton, tobacco. In a preferred embodiment, thioredoxin
specific activity is increased in the seeds of the transgenic
plant.
[0037] Thioredoxin over-expression in a desired part of a plant,
for example, a seed, is achieved by use of a seed-specific promoter
operably linked to the thioredoxin coding sequence. In this
example, "seed-specific" indicates that the promoter has enhanced
activity in seeds compared to other plant tissues; it does not
require that the promoter is solely active in the seeds. However,
given the nature of the thioredoxin protein, it may be advantageous
to select a seed-specific promoter that in some cases causes little
or no protein expression in tissues other than seeds. In certain
embodiments, the seed-specific promoter that is selected is a seed
maturation-specific promoter. The use of promoters that confer
enhanced expression during seed maturation (such as the barley
hordein promoters) may result in even higher levels of thioredoxin
expression in the maturing seed.
[0038] In an alternative embodiment, thioredoxin is overexpressed
in the root, stem, tuber, fruit, leaf, flower, pollen etc or any
one or more parts of a plant at the discretion of the
practitioner.
[0039] In one embodiment of the invention, the provided transgenic
plants comprise a recombinant nucleic acid molecule having a
structure: P-T, wherein P is a seed-specific promoter, and T is an
nucleic acid molecule encoding a thioredoxin polypeptide. In
particular embodiments, the seed-specific promoter is a barley
hordein gene promoter, such as a barley B1-hordein promoter, a
barley D-hordein promoter or a maize embryo specific globulin
promoter.
[0040] In another embodiment of the invention, the transgenic
plants comprise a recombinant nucleic acid molecule having a
structure: P-SS-T, wherein P is a seed-specific promoter, T is an
nucleic acid molecule encoding a thioredoxin polypeptide and SS is
a nucleic acid molecule that encodes a signal peptide that targets
expression of the thioredoxin polypeptide to an intracellular body,
and wherein P, SS and T are operably linked. Evidence presented
herein indicates that the presence of the signal peptide can
further enhance the level of thioredoxin expression in the
transgenic plants. Suitable signal peptides include, but are not
limited to, barley B1- and D-hordein signal peptides.
[0041] Parts of the transgenic plants overexpressing thioredoxin as
provided by the invention may be harvested for direct processing
into food products. For example, the seeds may be ground using
conventional means to produce flour. Alternatively, the seeds or
other plant parts may be used as a source of thioredoxin, which can
be extracted from the immature or mature transgenic plant by
standard protein extraction methods. Alternatively, crudely
processed seed material may be used directly as a source of
thioredoxin. Thus, another aspect of the invention is a method of
producing thioredoxin protein, the method comprising harvesting
thioredoxin from the seed of a transgenic plant having an elevated
level of thioredoxin in its seeds.
[0042] Accordingly, in another aspect the invention provides an
improved edible products for human and animal consumption, for
example increased digestibility and/or reduced allergenicity and
dough having increased strength and volume in comparison to dough
produced from non-transgenic plant of the same species.
[0043] In yet another aspect, the invention provides of methods of
making a transgenic plant having reduced allergenicity, increased
digestibility, increased redox state (increased SH:SS ratio), in
comparison to a non-transgenic plant of the same species.
[0044] In still yet another aspect, the invention provide a
transgenic plant comprising a nucleic acid encoding A. thaliana
NTR.
[0045] These and other aspects of the invention are further
illustrated by the following description and Examples.
DETAILED DESCRIPTION OF THE INVENTION
[0046] I. Definitions
[0047] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Lewin, Genes V published by Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.),
The Encyclopedia of Molecular Biology, published by Blackwell
Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology. a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8); Ausubel et al. (1987) Current Protocols in
Molecular Biology, Green Publishing; Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y.
[0048] In order to facilitate review of the various embodiments of
the invention, the following definitions are provided:
[0049] Thioredoxin protein or Thioredoxin polypeptide: A large
number of plant, animal, and microbial thioredoxin proteins or
polypeptides have been characterized, and the genes encoding many
of these proteins have been cloned and sequenced. The present
invention is preferably directed to the use of thioredoxin h
proteins, although other thioredoxin proteins may also be employed
to produce transgenic plants as described herein. Among the
thioredoxin h proteins from plants that have been described to date
are thioredoxin h proteins from Arabidopsis thaliana (Rivera-Madrid
et al., 1993; Rivera-Madrid et al., 1995), Nicotiana tabacum (Marty
and Meyer, 1991; Brugidou et al., 1993), Oryza sativa (ishiwatari
et al., 1995), Brassica napus (Bower et al., 1996), Glycine max
(Shi and Bhattacharyya, 1996), and Triticum aestivum (Gautier et
al., 1998). The amino acid sequences of these and other thioredoxin
h proteins, and the nucleotide sequence of cDNAs and/or genes that
encode these proteins, are available in the scientific literature
and publicly accessible sequence databases. For example, a cDNA
encoding thioredoxin h from Picea mariana is described in accession
number AF051206 (NID g2982246) of GenBank, and located by a search
using the Entrez browser/nucleotide sequence search of the National
Center for Biotechnology Information website, www.ncbi.nlm.nih.gov.
The cDNA encoding the Triticum aestivum thioredoxin h protein used
in the Examples described below is described on the same database
under accession number X69915 (NID g2995377).
[0050] The present invention may be practiced using nucleic acid
sequences that encode full length thioredoxin h proteins, as well
as thioredoxin h derived proteins that retain thioredoxin h
activity. Thioredoxin h derived proteins which retain thioredoxin
biological activity include fragments of thioredoxin h, generated
either by chemical (e.g. enzymatic) digestion or genetic
engineering means; chemically functionalized protein molecules
obtained starting with the exemplified protein or nucleic acid
sequences, and protein sequence variants, for example allelic
variants and mutational variants, such as those produced by in
vitro mutagenesis techniques, such as gene shuffling (Stemmer et
al., 1994a, 1994b). Thus, the term "thioredoxin h protein"
encompasses full length thioredoxin h proteins, as well as such
thioredoxin h derived proteins that retain thioredoxin h
activity.
[0051] Thioredoxin protein may be quantified in biological samples
(such as seeds) either in terms of protein level, or in terms of
thioredoxin activity. Thioredoxin protein level may be determined
using a western blot analysis followed by quantitative scanning of
the image as described in detail below. Thioredoxin activity may be
quantified using a number of different methods known in the art.
Preferred methods of measuring thioredoxin biological activity
attributable to thioredoxin h in plant extracts include NADP/malate
dehydrogenase activation (Johnson et al., 1987a,b) and reduction of
2',5'-dithiobis(2-nitrobenzoic acid) (DTNB) via NADP-thioredoxin
reductase (Florencio et al., 1988; U.S. Pat. No. 5,792,506). Due to
the potential for interference from non-thioredoxin h enzymes that
use NADPH, accurate determination of thioredoxin h activity should
preferably be made using partially purified plant extracts.
Standard protein purification methods (e.g.
(NH.sub.4).sub.2SO.sub.4 extraction or heat) can be used to
accomplish this partial purification. The activity of thioredoxin h
may also be expressed in terms of specific activity, i.e.,
thioredoxin activity per unit of protein present, as described in
more detail below.
[0052] In another embodiment, thioredoxin may be expressed in terms
of thioredoxin content, such as, mass/mass tissue (i.e., .mu.g/gram
tissue) or mass/mass soluble protein (i.e., .mu.g/mg soluble
protein)
[0053] Promoter: A regulatory nucleic acid sequence, typically
located upstream (5') of a gene that, in conjunction with various
cellular proteins, is responsible for regulating the expression of
the gene. Promoters may regulate gene expression in a number of
ways. For example, the expression may be tissue-specific, meaning
that the gene is expressed at enhanced levels in certain tissues,
or developmentally regulated, such that the gene is expressed at
enhanced levels at certain times during development, or both.
[0054] In a preferred embodiment, a transgene of the invention is
expressed in an edible part of a plant. By "edible" herein is meant
at least a part of a plant that is suitable for consumption by
humans or animals (fish, crustaceans, isopods, decapods, monkeys,
cows, goats, pigs, rabbits, horses, birds (chickens, parrots etc).
Accordingly, "edible" embraces food for human consumption and feed
for animal consumption and includes, for example, dough, bread,
cookies, pasta, pastry, beverages, beer, food additives,
thickeners, malt, extracts made from an edible part of plants,
animals feeds, and the like. An edible part of a plant includes for
example, a root, a tuber, a seed, grain, a flower, fruit, leaf etc.
The skilled artisan is aware that expression of the transgene is
effected in any tissue, organ or part of a plant by employing a
promoter that is active in the selected part of the plant the
transgene is to be expressed. In a preferred embodiment the
transgene is expressed in a seed, preferably under control of a
seed- or grain-specific promoter.
[0055] The expression of a transgene in seeds or grains according
to the present invention is preferably accomplished by operably
linking a seed-specific or grain-specific promoter to the nucleic
acid molecule encoding the transgene protein. In this context,
"seed-specific" indicates that the promoter has enhanced activity
in seeds compared to other plant tissues; it does not require that
the promoter is solely active in the seeds. Accordingly,
"grain-specific" indicates that the promoter has enhanced activity
in grains compared to other plant tissues; it does not require that
the promoter is solely active in the grain. Preferably, the seed-
or grain-specific promoter selected will, at the time when the
promoter is most active in seeds, produce expression of a protein
in the seed of a plant that is at least about two-fold greater than
expression of the protein produced by that same promoter in the
leaves or roots of the plant. However, given the nature of the
thioredoxin protein, it may be advantageous to select a seed- or
grain-specific promoter that causes little or no protein expression
in tissues other than seed or grain. In a preferred embodiment, a
promoter is specific for seed and grain expression, such that,
expression in the seed and grain is enhanced as compared to other
plant tissues but does not require that the promoter be solely
activity in the grain and seed. In a preferred embodiment, the
promoter is "specific" for a structure or element of a seed or
grain, such as an embryo-specific promoter. In accordance with the
definitions provided above, an embryo-specific promoter has
enhanced activity in an embryo as compared to other parts of a seed
or grain or a plant and does not require its activity to be limited
to an embryo. In a preferred embodiment, the promoter is
"maturation-specific" and accordingly has enhanced activity
developmentally during the maturation of a part of a plant as
compared to other parts of a plant and does not require its
activity to be limited to the development of a part of a plant.
[0056] A seed- or grain-specific promoter may produce expression in
various tissues of the seed, including the endosperm, embryo, and
aleurone or grain. Any seed- or grain-specific promoter may be used
for this purpose, although it will be advantageous to select a
seed- or grain-specific promoter that produces high level
expression of the protein in the plant seed or grain. Known seed-
or grain-specific promoters include those associated with genes
that encode plant seed storage proteins such as genes encoding:
barley hordeins, rice glutelins, oryzins, or prolamines; wheat
gliadins or glutenins; maize zeins or glutelins; maize
embryo-specific promoter; oat glutelins; sorghum kafirins; millet
pennisetins; or rye secalins.
[0057] The barley hordein promoters (described in more detail
below) are seed- or grain-specific promoters that were used in the
illustrative Examples.
[0058] In certain embodiments, the seed- or grain-specific promoter
that is selected is a maturation-specific promoter. The use of
promoters that confer enhanced expression during seed or grain
maturation (such as the barley hordein promoters) may result in
even higher levels of thioredoxin expression in the seed.
[0059] By "seed or grain-maturation" herein refers to the period
starting with fertilization in which metabolizable food reserves
(e.g., proteins, lipids, starch, etc.) are deposited in the
developing seed, particularly in storage organs of the seed,
including the endosperm, testa, aleurone layer, embryo, and
scutellar epithelium, resulting in enlargement and filling of the
seed and ending with seed desiccation.
[0060] Members of the grass family, which include the cereal
grains, produce dry, one-seeded fruits. This type of fruit, is
strictly speaking, a caryopsis but is commonly called a kernel or
grain. The caryopsis of a fruit coat or pericarp, which surrounds
the seed and adhere tightly to a seed coat. The seed consists of an
embryo or germ and an endosperm enclosed by a nucellar epidermis
and a seed coat. Accordingly the grain comprises the seed and its
coat or pericarp. The seed comprises the embryo and the endosperm.
(R. Carl Hoseney in "Principles of Cereal Science and Technology",
expressly incorporated by reference in its entirety).
[0061] Hordein promoter: A barley promoter that directs
transcription of a hordein gene in barley seeds or grains A number
of barley hordein genes and associated promoters have been
described and characterized, including those for the B-, C-, D-,
and Gamma-hordeins (Brandt et al., 1985; Forde et al., 1985;
Rasmussen and Brandt, 1986, S.o slashed.rensen et al., 1996). The
activities of these promoters in transient expression assays have
also been characterized (Entwistle et al., 1991; Muller and
Knudesen, 1993; S.o slashed.rensen et al, 1996). While any hordein
promoter may be employed for this invention, the specific Examples
provided describe the use of the promoter sequences from the
B.sub.1- and D-hordein genes of barley. The nucleic acid sequences
of the barley B.sub.1- and D-hordein genes are shown in SEQ ID
NOs:1 and 3, respectively and in FIGS. 6 and 7 (the promoter region
excludes those nucleotides that encode the hordein signal peptide
that is shown underlined). S.o slashed.rensen et al., (1996)
describes plasmids that comprise the B.sub.1- and D-hordein
promoters operably linked to a beta-glucuronidase gene (uidA; gus)
and the Agrobacterium tumefaciens nopaline synthase 3'
polyadenylation site (nos). These plasmids may be conveniently
utilized as sources of both the hordein promoters and the nos
polyadenylation site.
[0062] One of skill in the art will appreciate that the length of
the hordein promoter region may also be greater or less than the
sequences depicted in FIGS. 6 and 7. For example, additional 5'
sequence from the hordein gene upstream region may be added to the
promoter sequence, or bases may be removed from the depicted
sequences. However, any hordein promoter sequence must be able to
direct transcription of an operably linked sequence in plant seed
or grain. The ability of a barley hordein promoter to direct
transcription of a protein in a plant seed may readily be assessed
by operably linking the promoter sequence to an open reading frame
(ORF) that encodes a readily detectable protein, such as the gus
ORF, introducing the resulting construct into plants and then
assessing expression of the protein in seeds of the plant (see S.o
slashed.rensen et al., 1996). A hordein promoter will typically
confer seed-specific expression, meaning that expression of the
protein encoded by the operably linked ORF will generally be at
least about twice as high (assessed on an activity basis) in seeds
of the stably transfected plant compared to other tissues such as
leaves. More usually, the hordein promoter will produce expression
in seeds that is at least about 5 times higher than expression in
other tissues of the plant.
[0063] Functional homologs of the barley hordein promoters
disclosed herein may be obtained from other plant species, such as
from other monocots, including wheat, rice and corn. Such homologs
may have specified levels of sequence identity with the prototype
hordein promoters (e.g., at least 40% sequence identity). The
functional homologs retain hordein promoter function, i.e., retain
the ability to confer seed- or grain-specific expression on
operably linked ORFs when introduced into plants (Marris et al.,
1988; Mena et al., 1998). Accordingly, where reference is made
herein to a hordein promoter, it will be understood that such
reference includes not only nucleic acid molecules having the
sequences of the prototypical sequences disclosed herein (or
variations on these sequences), but also promoters from hordein
gene homologs. Also included within the scope of such terms are
molecules that differ from the disclosed prototypical molecules by
minor variations. Such variant sequences may be produced by
manipulating the nucleotide sequence of hordein promoter using
standard procedures such as site-directed mutagenesis or the
polymerase chain reaction. Preferably, the seed- or
grain-specificity of the promoter is retained. Examples of dicot
promoters that can be used include for example soybean glycinins
and con-glycinins, and kidney bean phaseolin promoters.
[0064] Signal peptide: As described in the Examples below, the
inventors have discovered that the level of expression of
thioredoxin in seed or grain can be enhanced by the presence of a
signal peptide. In one of the Examples described below, the B1
hordein signal peptide was utilized. In particular, it was
discovered that the expression of thioredoxin protein in seed or
grain is enhanced when the ORF encoding the protein is operably
linked to both a hordein promoter and a hordein signal sequence
encoding the signal peptide. (For convenience, the nucleic acid
sequence encoding a signal peptide is referred to herein as a
signal sequence.) While not wishing to be bound by theory, it is
proposed that the hordein signal peptide directs expression of the
thioredoxin protein to a protected subcellular location, such as a
vacuole or protein body. It is further proposed that proteins
directed to such vacuoles are protected from proteolysis during
certain stages of seed or grain maturation. In addition, the
sequestration of the thioredoxin protein to such a location may
also serve to protect the maturing seeds or grain from detrimental
effects associated with thioredoxin over-expression.
[0065] The hordein signal peptide typically comprises about the
first 15-25 amino acids of the hordein gene ORF, more usually about
18-21 amino acids. The nucleotide and amino acid sequences of the
hordein signal sequence and peptide of the prototypical barley B1-
and D-hordein genes are shown in SEQ ID NOS: 1-4 and FIGS. 6 and 7.
One of skill in the art will appreciate that while the B1-hordein
signal sequence and signal peptide are utilized in the examples
described below, the invention is not limited to these specific
sequences. For example, homologous sequences may be used as
effectively, as may sequences that differ in exact nucleotide or
amino acid sequences, provided that such sequences result in
enhanced levels of the encoded protein in immature seed or grain.
Typically, "enhanced expression" will be expression that is about
twice that observed with an equivalent construct lacking the signal
sequence. Accordingly, the term "hordein signal sequence" and
"hordein signal peptide" includes not only the particular sequences
shown herein, but also homologs and variants of these
sequences.
[0066] Furthermore, the invention is not limited to the use of
hordein signal peptides. Other signal peptides that serve to
localize the thioredoxin co-translationally or post-translationally
to a selected seed, grain or cell compartment may be employed.
Other such signal sequences include those associated with storage
proteins in maize, rice, wheat, soybeans, beans, and tobacco (see
for example: Bagga et al., 1997; Torrent et al., 1997; Wu et al.,
1998; Zheng et al., 1995; Grimwade et al., 1996; Conrad et al.,
1998; and Takaiwa et al., 1995.)
[0067] Starch: A polysaccharide made up of a chain of glucose units
joined by alpha-1,4 linkages, either unbranched (amylose) or
branched (amylopectin) at alpha-1,6-linkages.
[0068] Dextran: Any of a variety of storage polysaccharides,
usually branched, made of glucose residues joined by alpha-1,6
linkages.
[0069] Dextrin or Limit Dextrin: Any of a group of small soluble
polysaccharides, partial hydrolysis products of starch, usually
enriched in alpha-1,6-linkages.
[0070] Germination: A resumption of growth of a plant embryo in
favorable conditions after seed maturation and drying
(dessication), and emergence of young shoot and root from the
seed.
[0071] Allergen: An antigenic substance that induces an allergic
reaction in a susceptible host. Accordingly, a susceptible host has
an immune status (hypersensitivity) that results in an abnormal or
harmful immune reaction upon exposure to an allergen. In a
preferred embodiment, the transgenic grains of the invention have
reduced allergenicity in comparison to nontransgenic grains. The
immune reaction can be immediate or delayed; cell mediated or
antibody mediated; or a combination thereof. In a preferred
embodiment, the allergic reaction is an immediate type
hypersensitivity.
[0072] Digestion: By "digestion" herein is meant the conversion of
a molecule or compound to one or more of its components.
Accordingly, "digestibility" relates to the rate and efficiency at
which the conversion to one or more of its components occurs. In a
preferred embodiment a "digestible compound" is, for example, a
food, that is converted to its chemical components by chemical or
enzymatic means. For example, dextran is converted to dextrin,
polysaccharide, monosaccharides, limit dextrin etc; a protein is
converted to a polypeptides, oligopeptides, amino acids, ammonia
etc.; a nucleic acid is converted to oligonucleotides, nucleotides,
nucleosides, purine, pyrimidines, phosphates etc. In a preferred
embodiment, the transgenic grains of the invention have increased
digestibility, i.e. are more efficiently or rapidly digested in
comparison to nontransgenic grain.
[0073] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences is expressed in terms of
sequence identity (or, for proteins, also in terms of sequence
similarity). Sequence identity is frequently measured in terms of
percentage identity; the higher the percentage, the more similar
the two sequences are. As described above, homologs and variants of
the thioredoxin nucleic acid molecules, hordein promoters and
hordein signal peptides may be used in the present invention.
Homologs and variants of these nucleic acid molecules will possess
a relatively high degree of sequence identity when aligned using
standard methods.
[0074] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman (1981); Needleman and Wunsch
(1970); Pearson and Lipman (1988); Higgins and Sharp (1988);
Higgins and Sharp (1989); Corpet et al., (1988); Huang et al.,
(1992); and Pearson et al., (1994). Altschul et al., (1994)
presents a detailed consideration of sequence alignment methods and
homology calculations.
[0075] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., 1990) is available from several sources, including the
National Center for Biotechnology Information (NCBI, Bethesda, Md.)
and on the Internet, for use in connection with the sequence
analysis programs blastp, blastn, blastx, tblastn and tblastx. It
can be accessed at http://www.ncbi.nlm.nih.gov/BLAST. A description
of how to determine sequence identity using this program is
available at http://www.nchi.nlm.nih.gov/BLAST/blast.help.html.
[0076] Homologs of the disclosed protein sequences are typically
characterized by possession of at least 40% sequence identity
counted over the full length alignment with the amino acid sequence
of the disclosed sequence using the NCBI Blast 2.0, gapped blastp
set to default parameters. The adjustable parameters are preferably
set with the following values: overlap span=1, overlap
fraction=0.125, word threshold (T)=11. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched; however, the values may be
adjusted to increase sensitivity. Proteins with even greater
similarity to the reference sequences will show increasing
percentage identities when assessed by this method, such as at
least about 50%, at least about 60%, at least about 70%, at least
about 75%, at least about 80%, at least about 90% or at least about
95% sequence identity.
[0077] Homologs of the disclosed nucleic acid sequences are
typically characterized by possession of at least 40% sequence
identity counted over the full length alignment with the amino acid
sequence of the disclosed sequence using the NCBI Blast 2.0, gapped
blastn set to default parameters. A preferred method utilizes the
BLASTN module of WU-BLAST-2 (Altschul et al., 1996); set to the
default parameters, with overlap span and overlap fraction set to 1
and 0.125, respectively. Nucleic acid sequences with even greater
similarity to the reference sequences will show increasing
percentage identities when assessed by this method, such as at
least about 50%, at least about 60%, at least about 70%, at least
about 75%, at least about 80%, at least about 90% or at least about
95% sequence identity.
[0078] The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer amino acids than the protein encoded by the
sequences in the figures, it is understood that in one embodiment,
the percentage of sequence identity will be determined based on the
number of identical amino acids in relation to the total number of
amino acids. Thus, for example, sequence identity of sequences
shorter than that shown in the figures as discussed below, will be
determined using the number of amino acids in the longer sequence,
in one embodiment. In percent identity calculations relative weight
is not assigned to various manifestations of sequence variation,
such as, insertions, deletions, substitutions, etc.
[0079] In one embodiment, only identities are scored positively
(+1) and all forms of sequence variation including gaps are
assigned a value of "0", which obviates the need for a weighted
scale or parameters as described herein for sequence similarity
calculations. Percent sequence identity can be calculated, for
example, by dividing the number of matching identical residues by
the total number of residues of the "shorter" sequence in the
aligned region and multiplying by 100. The "longer" sequence is the
one having the most actual residues in the aligned region.
[0080] As will be appreciated by those skilled in the art, the
sequences of the present invention may contain sequencing errors.
That is, there may be incorrect nucleosides, frameshifts, unknown
nucleosides, or other types of sequencing errors in any of the
sequences; however, the correct sequences will fall within the
homology and stringency definitions herein.
[0081] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include one or more nucleic acid sequences that permit it to
replicate in one or more host cells, such as origin(s) of
replication. A vector may also include one or more selectable
marker genes and other genetic elements known in the art.
[0082] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, plant or animal cell, including transfection with
viral vectors, transformation by Agrobacterium, with plasmid
vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration and includes transient
as well as stable transformants.
[0083] Isolated: An "isolated" biological component (such as a
nucleic acid or protein or organelle) has been substantially
separated or purified away from other biological components in the
cell or the organism in which the component naturally occurs, i.e.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term embraces nucleic acids including
chemically synthesized nucleic acids and also embraces proteins
prepared by recombinant expression in vitro or in a host cell and
recombinant nucleic acids as defined below.
[0084] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary,
join two protein-coding regions in the same reading frame. With
respect to polypeptides, two polypeptide sequences may be operably
linked by covalent linkage, such as through peptide bonds or
disulfide bonds.
[0085] Recombinant: By "recombinant nucleic acid" herein is meant a
nucleic acid that has a sequence that is not naturally occurring or
has a sequence that is made by an artificial combination of two
otherwise separated segments of sequence. This artificial
combination is often accomplished by chemical synthesis or, more
commonly, by the artificial manipulation of of nucleic acids, e.g.,
by genetic engineering techniques, such as by the manipulation of
at least one nucleic acid by a restriction enzyme, ligase,
recombinase, and/or a polymerase. Once introduced into a host cell,
a recombinant nucleic acid is replicated by the host cell, however,
the recombinant nucleic acid once replicated in the cell remains a
recombinant nucleic acid for purposes of this invention. By
"recombinant protein" herein is meant a protein produced by a
method employing a recombinant nucleic acid. As outlined above
"recombinant nucleic acids" and "recombinant proteins" also are
"isolated", as described above.
[0086] Complementary DNA (cDNA): A piece of DNA that is synthesized
in the laboratory by reverse transcription of an RNA, preferably an
RNA extracted from cells. cDNA produced from mRNA typically lacks
internal, non-coding segments (introns) and regulatory sequences
that determine transcription.
[0087] Open reading frame (ORF): A series of nucleotide triplets
(codons) coding for amino acids without any internal termination
codons. These sequences are usually translatable into a
peptide.
[0088] Transgenic plant: As used herein, this term refers to a
plant that contains recombinant genetic material not normally found
in plants of this type and which has been introduced into the plant
in question (or into progenitors of the plant) by human
manipulation. Thus, a plant that is grown from a plant cell into
which recombinant DNA is introduced by transformation is a
transgenic plant, as are all offspring of that plant that contain
the introduced transgene (whether produced sexually or asexually).
It is understood that the term transgenic plant encompasses the
entire plant and parts of said plant, for instance grains, seeds,
flowers, leaves, roots, fruit, pollen, stems etc.
[0089] The present invention is applicable to both dicotyledonous
plants (e.g. tomato, potato, soybean, cotton, tobacco, etc.) and
monocotyledonous plants, including, but not limited to graminaceous
monocots such as wheat (Triticum spp.), rice (Oryza spp.), barley
(Hordeum spp.), oat (Avena spp.), rye (Secale spp.), corn (Zea
mays), sorghum (Sorghum spp.) and millet (Pennisetum spp). For
example, the present invention can be employed with barley
genotypes including, but not limited to Morex, Harrington ,
Crystal, Stander, Moravian III, Galena, Salome, Steptoe, Klages,
Baronesse, and with wheat genotypes including, but not limited to
Yecora Rojo, Bobwhite, Karl and Anza. In general, the invention is
particularly useful in cereals.
[0090] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified barley thioredoxin h protein preparation is one
in which the barley thioredoxin h protein is more enriched or more
biochemically active or more easily detected than the protein is in
its natural environment within a cell or plant tissue. Accordingly,
"purified" embraces or includes the removal or inactivation of an
inhibitor of a molecule of interest. In a preferred embodiment, a
preparation of barley thioredoxin h protein is purified such that
the barley thioredoxin h represents at least 5-10% of the total
protein content of the preparation. For particular applications,
higher protein purity may be desired, such that preparations in
which barley thioredoxin h represents at least 50% or at least 75%
or at least 90% of the total protein content may be employed.
[0091] Ortholog: Two nucleotide or amino acid sequences are
orthologs of each other if they share a common ancestral sequence
and diverged when a species carrying that ancestral sequence split
into two species, sub-species, or cultivars. Orthologous sequences
are also homologous sequences.
[0092] II. Production of Plants With Elevated Seed Thioredoxin
[0093] Standard molecular biology methods and plant transformation
techniques can be used to produce transgenic plants that produce
seeds having an elevated level of thioredoxin protein. The
following sections provide general guidance as to the selection of
particular constructs and transformation procedures.
[0094] a. Constructs
[0095] The present invention utilizes recombinant constructs that
are suitable for obtaining elevated expression of thioredoxin in
plant seeds relative to non-transformed plant seeds. In their most
basic form, these constructs may be represented as P-T, wherein P
is a seed-specific promoter and T is a nucleic acid sequence
encoding thioredoxin. In another embodiment, a peptide signal
sequence that targets expression of the thioredoxin polypeptide to
an intracellular body may be employed. Such constructs may be
represented as P-SS-T, wherein SS is the signal peptide. Nucleic
acid molecules that may be used as the source of each of these
components are described in the Definitions section above.
[0096] Each component is operably linked to the next. For example,
where the construct comprises the hordein D-promoter (P), the
hordein D-signal sequence (SS) encoding the hordein signal peptide,
and an open reading frame encoding, preferably, the wheat
thioredoxin h protein (T), the hordein promoter is linked to the 5'
end of the sequence encoding the hordein signal sequence, and the
hordein signal sequence is operably linked to the 5' end of the
thioredoxin open reading frame, such that C terminus of the signal
peptide is joined to the N-terminus of the encoded protein.
[0097] The construct will also typically include a transcriptional
termination region following the 3' end of the encoded protein ORF.
Illustrative transcriptional termination regions include the nos
terminator from Agrobacterium Ti plasmid and the rice alpha-amylase
terminator.
[0098] Standard molecular biology methods, such as the polymerase
chain reaction, restriction enzyme digestion, and/or ligation may
be employed to produce these constructs comprising any nucleic acid
molecule or sequence encoding a thioredoxin protein or
polypeptide.
[0099] b. General Principles of Plant Transformation
[0100] Introduction of the selected construct into plants is
typically achieved using standard transformation techniques. The
basic approach is to: (a) clone the construct into a transformation
vector; which (b) is then introduced into plant cells by one of a
number of techniques (e.g., electroporation, microparticle
bombardment, Agrobacterium infection); (c) identify the transformed
plant cells; (d) regenerate whole plants from the identified plant
cells, and (d) select progeny plants containing the introduced
construct. Preferably all or part of the transformation vector will
stably integrate into the genome of the plant cell. That part of
the transformation vector which integrates into the plant cell and
which contains the introduced P-T or P-SS-T sequence (the
introduced "thioredoxin transgene") may be referred to as the
recombinant expression cassette.
[0101] Selection of progeny plants containing the introduced
transgene may be made based upon the detection of thioredoxin or
NTR over-expression in seeds, or upon enhanced resistance to a
chemical agent (such as an antibiotic) as a result of the inclusion
of a dominant selectable marker gene incorporated into the
transformation vector.
[0102] Successful examples of the modification of plant
characteristics by transformation with cloned nucleic acid
sequences are replete in the technical and scientific literature.
Selected examples, which serve to illustrate the knowledge in this
field of technology include:
[0103] U.S. Pat. No. 5,571,706 ("Plant Virus Resistance Gene and
Methods");
[0104] U.S. Pat. No. 5,677,175 ("Plant Pathogen Induced
Proteins");
[0105] U.S. Pat. No. 5,510,471 ("Chimeric Gene for the
Transformation of Plants");
[0106] U.S. Pat. No. 5,750,386 ("Pathogen-Resistant Transgenic
Plants");
[0107] U.S. Pat. No. 5,597,945 ("Plants Genetically Enhanced for
Disease Resistance");
[0108] U.S. Pat. No. 5,589,615 ("Process for the Production of
Transgenic Plants with Increased Nutritional Value Via the
Expression of Modified 2S Storage Albumins");
[0109] U.S. Pat. No. 5,750,871 ("Transformation and Foreign Gene
Expression in Brassica Species");
[0110] U.S. Pat. No. 5,268,526 ("Overexpression of Phytochrome in
Transgenic Plants");
[0111] U.S. Pat. No. 5,780,708 ("Fertile Transgenic Com
Plants");
[0112] U.S. Pat. No. 5,538,880 ("Method For Preparing Fertile
Transgenic Corn Plants");
[0113] U.S. Pat. No. 5,773,269 ("Fertile Transgenic Oat
Plants");
[0114] U.S. Pat. No. 5,736,369 ("Method For Producing Transgenic
Cereal Plants");
[0115] U.S. Pat. No. 5,610,049 ("Methods For Stable Transformation
of Wheat").
[0116] These examples include descriptions of transformation vector
selection, transformation techniques and the construction of
constructs designed to express an introduced transgene.
[0117] c. Plant Types
[0118] The transgene-expressing constructs of the present invention
may be usefully expressed in a wide range of higher plants to
obtain seed- or grain-specific expression of selected polypeptides.
The invention is expected to be particularly applicable to
monocotyledonous cereal plants including barley, wheat, rice, rye,
maize, triticale, millet, sorghum, oat, forage, and turf grasses.
In particular, the transformation methods described herein will
enable the invention to be used with genotypes of barley including
Morex, Harrington, Crystal, Stander, Moravian III, Galena, Golden
Promise, Steptoe, Klages and Baronesse, and commercially important
wheat genotypes including Yecora Rojo, Bobwhite, Karl and Anza.
[0119] The invention may also be applied to dicotyledenous plants,
including, but not limited to, soybean, sugar beet, cotton, beans,
rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton,
flax, peanut, clover; vegetables such as lettuce, tomato,
cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage,
cauliflower, broccoli, Brussels sprouts, peppers; and tree fruits
such as citrus, apples, pears, peaches, apricots, and walnuts.
[0120] d. Vector Construction
[0121] A number of recombinant vectors suitable for stable
transformation of plant cells or for the establishment of
transgenic plants have been described including those described in
Weissbach and Weissbach, (1989), and Gelvin et al., (1990).
Typically, plant transformation vectors include one or more ORFs
under the transcriptional control of 5' and 3' regulatory sequences
and a dominant selectable marker with 5' and 3' regulatory
sequences. The selection of suitable 5' and 3' regulatory sequences
for constructs of the present invention is discussed above.
Dominant selectable marker genes that allow for the ready selection
of transformants include those encoding antibiotic resistance genes
(e.g., resistance to hygromycin, kanamycin, bleomycin, G418,
streptomycin or spectinomycin) and herbicide resistance genes (e.g,
phosphinothricin acetyltransferase).
[0122] e. Transformation and Regeneration Techniques
[0123] Methods for the transformation and regeneration of both
monocotyledonous and dicotyledonous plant cells are known, and the
appropriate transformation technique will be determined by the
practitioner. The choice of method will vary with the type of plant
to be transformed; those skilled in the art will recognize the
suitability of particular methods for given plant types. Suitable
methods may include, but are not limited to: electroporation of
plant protoplasts; liposome-mediated transformation; polyethylene
glycol (PEG) mediated transformation; transformation using viruses;
micro-injection of plant cells; micro-projectile bombardment of
plant cells; vacuum infiltration; and Agrobacterium mediated
transformation. Typical procedures for transforming and
regenerating plants are described in the patent documents listed at
the beginning of this section.
[0124] f. Selection of Transformed Plants
[0125] Following transformation, transformants are preferably
selected using a dominant selectable marker. Typically, such a
marker will confer antibiotic or herbicide resistance on the
seedlings of transformed plants, and selection of transformants can
be accomplished by exposing the seedlings to appropriate
concentrations of the antibiotic or herbicide. After transformed
plants are selected and grown to maturity to allow seed set, the
seeds can be harvested and assayed for over-expression of
thioredoxin.
[0126] III. Use of Plants, Seeds or Grains Expressing Elevated
Levels of Thioredoxin
[0127] In one embodiment, the transgene protein, for example
thioredoxin expressed in plants, especially seeds or grains, using
the methods described herein, is used in the production and
synthesis of thioredoxin. The thioredoxin transgene expressed by
the recombinant nucleic acid of the invention may be harvested at
any point after expression of the protein has commenced. When
harvesting from the seed or grain or other part of a plant for
example, it is not necessary for the seed or grain or other part of
the plant to have undergone maturation prior to harvesting. For
example, transgene expression may occur prior to seed or grain
maturation or may reach optimal levels prior to seed or grain
maturation. The transgene protein may be isolated from the seeds or
grain, if desired, by conventional protein purification methods.
For example, the seed or grain can be milled, then extracted with
an aqueous or organic extraction medium, followed by purification
of the extracted thioredoxin protein. Alternatively, depending on
the nature of the intended use, the transgene protein may be
partially purified, or the seed or grain may be used directly
without purification of the transgene protein for food processing
or other purposes.
[0128] For example, the addition of thioredoxin promotes the
formation of a protein network that produces flour with enhanced
baking quality. Kobrehel et al., (1994) have shown that the
addition of thioredoxin to flour of non-glutenous cereal such as
rice, maize, and sorghum promotes the formation of a dough-like
product. Accordingly, the addition of thioredoxin expressed in
seeds using the methods described herein find use in the production
of flour with improved baking quality such as increased strength
and/or volume.
[0129] The enhanced expression of thioredoxin also produces a seed
having an altered biochemical composition. For example, enhanced
thioredoxin expression produces seed with increased enzymatic
activity, such as, increased pullulanase and alpha-amylase A.
Enhanced thioredoxin expression also produces seed with early
alpha-amylase B activation. Pullulanase ("debranching enzyme") is
an enzyme that breaks down branched starch of the endosperm of
cereal seeds by hydrolytically cleaving alpha-1,6 bonds.
Alpha-amylases break down starch 1-4 linkages. Pullulanase and
amylases are enzymes fundamental to the brewing and baking
industries. Pullulanase and amylases are required to break down
starch in malting and in certain baking procedures carried out in
the absence of added sugars or other carbohydrates. Obtaining
adequate activity of these enzymes is problematic especially in the
malting industry. It has been known for some time that
dithiothreitol (DTT, a chemical reductant that reduces and
sometimes replaces thioredoxin) activates pullulanase of cereal
preparations (e.g., barley, oat, and rice flours). A method of
adequately increasing the activity of pullulanase and alpha-amylase
A and shortening the activation time of alpha-amylase B with a
physiologically acceptable system, leads to more rapid malting
methods and, owing to increased sugar availability, to alcoholic
beverages such as beers-with reduced carbohydrate content.
[0130] Accordingly, seeds or grains with enhanced thioredoxin
expression provide advantages in the production of malt and
beverages produced by a fermentation process. Enhanced pullulanase
and alpha-amylase A and earlier induction of alpha-amylase B in
grain increases the speed and efficiency of germination, important
in malting, where malt is produced having increased enzymatic
activity resulting in enhanced hydrolysis of starch to fermentable
carbohydrates, thereby, improving the efficiency of fermentation in
the production of alcoholic beverages, for example, beer and scotch
whiskey. Early alpha-amylase B activation would reduce the total
time for malting by about 20%. Enhanced fermentation processes also
find use in the production of alcohols that are not intended for
human consumption, i.e., industrial alcohols.
[0131] In another embodiment, seed or grains with enhanced
thioredoxin expression provide advantages in enhancing the onset
and efficiency of germination.
[0132] The overexpression of thioredoxin in seed or grains results
in an increase in the total protein. It also promotes the
redistribution of proteins to the most soluble albumin/globulin
fraction and the production of flour and other food products, feed,
and beverages with improved digestibility in comparison to edible
products made from non-transformed grains. Such edible products
find use in amelioration and treatment of food malabsorptive
syndromes, for example, sprue or catarrhal dysentery. Sprue is a
malabsorptive syndrome affecting both children and adults,
precipitated by the ingestion of gluten-containing foods. Edible
products that are more readily digested and readily absorbed avoid
or ameliorate the disease symptoms. Edible products with improved
digestibility also ameliorate or reduce symptoms associated with
celiac disease in which storage proteins that are not readily
digested in afflicated individuals result in inflammation of the GI
tract.
[0133] The expression of thioredoxin in seed grains results in the
production of foods and other edible products with reduced
allergenicity in comparison to edible products made from
non-transformed grains. Food allergies are a significant health and
nutrition problem (Lehrer et al., 1996). Up to 2% of adults and 8%
of children have a food allergy causing serious symptoms including
death. Wheat protein is one of the principal allergens. Food
allergies are defined by the American academy of Allergy and
Immunology Committee on Adverse Reactions to Food as "an
immunological reaction resulting from the ingestion of a food or a
food additive" (Fenema, 1996; Lasztity, 1996). Most true allergic
responses to food proteins appear to be caused by a type-I
imunolobulin E (IgE)-mediated hypersensitivity reaction (Sicherer,
1999). These responses may occur within minutes or a few hours
after eating the offending food (Furlong-Munoz, 1996). When the
offending food is injested by allergy-sensitive individuals the
body releases histamines and other biochemicals, resulting in itchy
eyes, rash or hives; runny nose; swelling of the lips, tongue, and
face; itching or tightness of the throat; abdominal pain; nausea;
diarrhea; and shortness of breath. Some individuals have severe,
anaphylactic reactions, resulting in approximately 135 deaths per
year in the United States. In the U.S. over 2,500 emergency rooms
visits per year are allergy-related. There is no cure for food
allergies, only avoidance of the food will prevent symptoms. For
example, patients with wheat allergy must avoid wheat- or
gluten-containing foods; wheat gluten is a very common ingredient
in many processed foods (Marx et al., 1999).
[0134] A feature common to many allergens is the presence of one or
more disulfide bonds that contribute to the resistance of allergens
to digestion (Astwood et al., 1996), allowing them to be mostly
intact when they react the small intestine where they are presented
to mucosal cells that mount an IgE immune response. The major
allergens were found to be insoluble storage proteins, gliadins and
glutenins. The soluble storage proteins, albumins and globulins
were considerably weaker (Buchanan et al., 1997). Allergenicity of
these proteins is substantially decreased after thioredoxin
treatment and disulfide bond reduction.
[0135] Edible products, for example, bread, cookies, dough,
thickeners, beverages, malt, pasta, food additives, including
animal feeds, made using the transgenic plants or parts of a
transgenic plant of the invention have decreased allergenicity and
accordingly can be used to in the treatment of an allergic
response. By "treatment" or "alleviating" symptoms herein is meant
prevention or decreasing the probability of symptoms.
[0136] Increased digestibility of seeds or grains also provides
wider consumption of grains by man and animals who otherwise can
not consume such grains. For example, sorghum is the world's fifth
leading grain in terms of metric tons after wheat, rice, maize, and
barley and third in production in the Untied States after maize and
wheat. The use of sorghum is constrained in part because of the
difficulty associated with the digestibility of its protein and
starch compared to other grains. This difficulty with the
digestibility of sorghum protein and starch has to do with the
structure of the seed and the manner in which the proteins are
associated with the starch. The digestibility of the starch flour
from sorghum cultivars is 15-25% lower in digestibility than, for
example, maize. Perhaps more notable is the fact that, unlike other
grains, the indigestibility of unprocessed sorghum flour increases
dramatically after boiling in water, a common practice in Africa. A
study with human subjects showed that protein digestibility in
cooked sorghum porridge can be as low as 46%, whereas the percent
digestibility for cooked wheat, maize, and rice was 81%, 73%, and
66% respectively (Mertz et al. 1984, MacLean et al. 1981).
Exogenous addition of reducing agents increases the digestibility
of the starch (Hamaker et al. 1987). However, the efficacy of
manipulating the thioredoxin system in vivo in the seed by
expressing increased amounts of thioredoxin in a manner which does
not adversely affect plant development or morphology had not
previously been demonstrated. Accordingly, the transgenic plants of
the invention provide wider use of seeds or grains as food sources
by increasing the digestibility of the starch and/or protein
component. The transgenic seeds or grains of the present invention
also provide the advantage of increasing the digestibility of food
products for human and feed for animals made of these grains
without the addition of exogenous reducing agents. In addition, the
increased digestibility results in greater utilization of the food
or feed, i.e., a human or animal consuming an edible product
comprising a transgenic seed or grain of the invention or an
extract thereof more efficiently absorbs nutrients and therefore
requires to consume less in comparison to a non-transgenic food
product. In another embodiment the transgenic seed, grain or
extracts thereof of the present invention and extracts or food
products thereof are used as a food or feed additives. For example,
an extract or flour or malt produced from a transgenic seed or
grain of the invention is added to a non-transgenic food or feed
product to improve the digestibility or decrease the allergenicity
of the nontransgenic food product or to improve the quality of the
total food product, such as, by increasing the strength and/or
volume of the food product.
[0137] Illustrative embodiments of the invention are described
below,
EXAMPLES
Example 1
Expression of Wheat Thioredoxin h (WTRXh) in Transgenic Barley
[0138] Four different DNA constructs were produced, each containing
a 384-bp wtrxh fragment encoding the 13.5-KDa WTRXh protein. The
four constructs are illustrated in FIG. 1 and described below. Each
construct comprised the 384-bp wtrxh fragment operably linked to a
seed-specific promoter (either the barley endosperm-specific
D-hordein or B1-hordein promoters or the maize embryo-specific
globulin promoter). An additional construct comprised the 384-bp
wtrxh fragment operably linked to the B1-hordein promoter and the
B1-hordein signal sequence (FIG. 6). The transformation vector used
included the bar gene, conferring resistance to bialaphos.
Twenty-eight independent regenerable barley lines were obtained
after bialaphos selection and all were PCR-positive for the bar
gene. The presence of the wtrxh gene was confirmed in the genome of
the 28 independent lines by PCR and DNA hybridization analyses. The
expression of the WTRXh protein was assessed by western blot
analysis, using purified wheat thioredoxin as a control. The WTRXh
expressed in transgenic barley had a molecular mass that differed
from native barley TRXh but was identical to WTRXh. The WTRXh was
found to be highly expressed in developing and mature seed of
transgenic barley plants although levels of expression varied among
the transgenic events. On average, higher expression levels were
observed in lines transformed with the DNA construct containing the
B1-hordein promoter plus the signal peptide sequence than the same
promoter without the signal peptide sequence. The WTRXh purified
from transgenic barley seed was confirmed to be biochemically
active.
[0139] A. Materials and Methods
[0140] Plant Materials for Transformation
[0141] A two-rowed spring cultivar of barley, Golden Promise, was
grown in growth chambers as described previously (Wan and Lemaux
1994; Lemaux et al., 1996).
[0142] Construction of Wheat Thioredoxin h Expression Vectors and
DNA Sequencing
[0143] Expression vectors were constructed containing the wheat
thioredoxin h gene (wtrxh) driven by the barley endosperm-specific
B1- or D-hordein promoter or the maize embryo-specific globulin
promoter.
[0144] The plasmids were constructed as follows.
[0145] (1) pDhWTRXN-2: A 384-bp wtrxh coding region was amplified
by PCR from pTaM13.38 (Gautier et al., 1998). This plasmid
contained a cDNA of wtrxh, which was used as a template, creating
XbaI and SacI sites with the following primers Wtrxh1
(5'-atatctagaATGGCGGCGTCGGCGGCGA) (SEQ ID NO:5) and Wtrxh2R
(5'-atagagctcTTACTGGGCCGCGTGTAG) (SEQ ID NO:6), respectively (FIG.
1). Small letters in the primer denote a restriction enzyme site
for subcloning of the DNA fragment containing the wtrxh gene;
underlined letters denote wtrxh sequences. The ATG initiation codon
for wtrxh expression was included in the Wtrxh1 primer. PCR
reactions were performed on a thermocycler (MJ Research Inc.,
Watertown, Mass.) using recombinant Taq DNA polymerase (Promega.
Madison, Wis.) in a 100-.mu.l reaction volume. The reaction buffer
contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl.sub.2,
0.1% Triton-X-100, and 50 .mu.M of each deoxyribonucleoside
triphosphate. PCR conditions utilized 25 cycles of 94.degree. C.
for 1 min, 55.degree. C. for 1 min and 72.degree. C. for 2 min,
with a final extension step at 72.degree. C. for 7 min. The wtrxh
fragment, which was amplified with the primers Wtrxh1 and Wtrxh2R,
was purified from a 0.7% agarose gel using a QIAquick.RTM. gel
extraction kit (Qiagen Inc., Chatsworth, Calif.), digested with
XbaI and SacI and ligated into XbaI/SacI-digested pUC19 to generate
the pWTRXh-1 plasmid. Nucleotide sequences of the PCR-amplified
wtrxh coding region fragment were determined by the
dideoxynucleotide chain termination method using Sequenase
according to manufacturer's instructions (United States
Biochemical, Cleveland, Ohio) with double-stranded plasmid
templates and regularly spaced primers
[0146] pDhWTRXN-2 was made by replacing the uidA gene in pDhGN-2
(containing barley endosperrn-specific D-hordein promoter (FIG. 7)
and nos 3' terminator) with the XbaI/SacI fragment containing the
wtrxh coding sequence from pWTRXh-I, which contains the
PCR-amplified wtrxh coding sequence in pUC19. To construct pDhGN-2
a 0.4-kb D-hordein promoter was amplified by PCR from pDII-Hor3
(S.o slashed.renson et al., 1996; Cho et al., 1999a). This plasmid
contained the D-hordein promoter sequence, which was used as a
template, creating Sphl and XbaI sites with the following primers:
Dhor1 (5'-ggcgcatgcgaattcGAATTCGATATCGATCTTCGA-3') (SEQ ID NO:23)
and Dhor2 (5'-aactctagaCTCGGTGGACTGTCAATG-3') (SEQ ID NO:12),
respectively. Small letters in the primers contain restriction
enzyme sites for subcloning of the DNA fragment containing the
D-hordein promtoer; underlined letters denote D-hordein promoter
sequences. The PCR amplified D-hordein promoter fragment was
digested with Sphl and XbaI and repalced with the cauliflower
mosaic 35S (CaMV 35S) promoter in p35SGN-3 to generate the pDhGN-2
plasmid. p35SGN-3 was made by ligating the 3.0-kb Sphl-EcoRl
fragment containing the CaMV 35S promoter, uidA
(beta-glucuronidase, gus) gene and nos into the Sphl/EcoRl-digested
pUC18.
[0147] (2) pdBhWTRX-1: The construction of pdBhWTRXN-1 started by
using pBhWTRXN-1. pBhWTRXN-1 was made by replacing the uidA gene in
pBhGN-1, which contains uidA driven by the barley
endosperm-specific B1-hordein promoter and terminated by the nos 3'
terminator, with the XbaI/SacI fragment from pWTRXh-1, which
contains the wtrxh coding sequence. The 120-bp HindIII-5'
B1-hordein flanking region was deleted from the pBhWTRXN-1 and
religated to make the pdBhWTRXN-1 construct.
[0148] (3) pdBhssWTRXN3-8: Primers Bhor7
(5'-GTAAAGCITTAACAACCCACACATTG) (SEQ ID NO:7) and BhorWtrxh1R
(5'-CCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT) (SEQ ID NO:8) containing
HindIII and Acyl sites, respectively, were used for amplification
of a 0.49-kb B1-hordein 5'-region, which included the B1-hordein
signal peptide sequence (FIG. 6). A .lambda.2-4/HindIII plasmid
containing a genomic clone of B1-hordein (Brandt et al., 1985; Cho
and Lemaux, 1997) was used as a template for the amplification. The
primer BhorWtrxh1R is an overlapping primer, which contains the
wtrxh coding sequence (underlined) and a partial signal peptide
sequence from the B1-hordein promoter, but lacks the ATG initiation
codon for wtrxh. pdBhssWTRXN3-8 was made by replacing the D-hordein
promoter (FIG. 7) in pDhWTRXN-2 with the 0.49-kb PCR-amplified
HindIII/Acyl fragment, which contains the B1-hordein promoter, its
signal peptide sequence and the junction region from the 5' trxh
gene. Thus, construct pdBhssWTRXN3-8 contains the barley
endosperm-specific B1-hordein promoter with its signal peptide
sequence (FIG. 6), wtrxh , and nos (FIG. 1). The signal peptide
sequence containing the ATG initiation codon was directly combined
with the sequence of wtrxh, with no extra amino acid sequences
being introduced between the two. This ensures that the WTRXh
protein has a precise cleavage site in the lumen of the endoplasmic
reticulum (ER). The authenticity of a PCR-amplified fragment from
the chimeric product was confirmed by DNA sequencing.
[0149] (4) pGlb1WTRXN-1: The 1.42-kb HindIII/BamHI fragment
containing the maize embryo-specific globulin promoter from the
ppGlb1GUS plasmid (Liu and Kriz, 1996) was ligated into pBluescript
II KS(+) to create HindIII and XbaI sites. pGlbWTRXN-1 was made by
restricting pDhWTRXN-2 with HindIII and XbaI in order to remove the
0.49-kb HindIII/XbaI barley D-hordein promoter from the pDhWTRXN-2.
In place of the 0.49-kb HindIII/XbaI D-hordein promoter fragment
(FIG. 7), the 1.42-kb HindIII/,XbaI maize globulin promoter was
ligated into the HindIII/XbaI digested pDhWTRXN-2 to form the
pGlbWTRXN-1 plasmid.
[0150] Stable Barley Transformation
[0151] Stable transgenic lines of barley expressing WTRXh driven by
the B1-hordein promoter with and without the signal peptide
sequence (FIG. 6), by the D-hordein promoter (FIG. 7) and by the
maize globulin promoter were obtained following modifications of
published protocols (Wan and Lemaux 1994; Lemaux et al., 1996; Cho
et al., 1998a-c). Whole immature embryos (IEs) (1.0-2.5 mm) were
aseptically removed, placed scutellum-side down on DC
callus-induction medium containing 2.5 mg/L 2,4-D and 5 .mu.M
CuSO.sub.4 (Cho et al., 1998a-c). One day after incubation at
24.+-.1.degree. C. in the dark, the IEs were transferred
scutellum-side up to DC medium containing equimolar amounts of
mannitol and sorbitol to give a final concentration of 0.4 M. Four
hours after treatment with the osmoticum, the IEs were used for
bombardment. Gold particles (1.0 .mu.m) were coated with 25 .mu.g
of a 1:1 molar ratio of pAHC20 (Christensen and Quail, 1996) and
one of the following plasmids, pdBhWTRXN-1, pdBhssWTRXN3-8,
pDhWTRXN-2 and pG1bWTRXN-1. The microprojectiles were bombarded
using a PDS-1000 He biolistic device (Bio-Rad, Inc., Hercules,
Calif.) at 1100 psi. Bombarded IEs were selected on DC medium with
5 mg/L bialaphos for 2 to 3 months. Bialaphos-resistant callus was
transferred onto an intermediate culturing medium (DBC2; Cho et
al., 1998a-c), containing 2.5 mg/L 2,4-D, 0.1 mg/L BAP and 5.0
.mu.M CuSO.sub.4, between the selection [DC medium plus bialaphos
(Meiji Seika Kaisha, Ltd., Yokohama, Japan)] and regeneration (FHG
medium; Hunter, 1988) steps. The culturing after callus induction
and selection on DC medium were carried out under dim light
conditions (approximately 10 to 30 .mu.E, 16 h-light) (Cho et al.,
1998a-c). Regenerated shoots were transferred to Magenta boxes
containing rooting medium (callus-induction medium without
phytohormones) containing 3 mg/L bialaphos. When shoots reached the
top of the box, plantlets were transferred to soil in the
greenhouse.
[0152] Cytological Analysis
[0153] For cytological analysis of transgenic barley plants.
healthy root meristems were collected from young plants grown in
the greenhouse. After pre-treatment at 4.degree. C. in saturated
1-bromonaphthalene solution overnight, root meristems were fixed in
1:3 glacial acetic acid:ethanol and stored at 4.degree. C. Root
meristems were hydrolyzed in 1 M HCl at 60.degree. C. for 5-7 min,
stained in Feulgen solution and squashed on a glass slide in a drop
of 1% aceto-carmine. Chromosomes were counted from at least five
well-spread cells per plant.
[0154] Herbicide Application
[0155] To determine herbicide sensitivity of T.sub.0 plants and
their progeny, a section of leaf blade at the 4- to 5-leaf stage
was painted using a cotton swab with 0.25% (v/v) Basta.TM. solution
(starting concentration 200 g/L phophinothricin, Hoechst AG,
Frankfurt, Germany) plus 0.1 % Tween 20. Plants were scored 1 week
after herbicide application.
[0156] Polymerase Chain Reaction (PCR) and DNA Blot
Hybridization
[0157] Total genomic DNA from leaf tissues was purified as
described by Dellaporta (1993). To test for the presence of wtrxh
in genomic DNA of putatively transformed lines, 250 ng of genomic
DNA was amplified by PCR using one of two primer sets:
1 Set 1: Wtrxh1 (5'-ATATCTAGAATGGCGGCGTCGGCGGCGA) (SEQ ID NO:5) and
Wtrxh2R (5'-ATAGAGCTCTTACTGGGCCGCGTGTAG); (SEQ ID NO:6) or Set 2:
Wtrxh4 (5'-CCAAGAAGTTCCCAGCTGC) (SEQ ID NO:11) and Wtrxh5R
(5'-ATAGCTGCGACAACCCTGTCCTT). (SEQ ID NO:19)
[0158] The presence of bar was determined using the primer set:
2 BAR5F (5'-CATCGAGACAAGCACGGTCAACTTC3') (SEQ ID NO:13) and BAR1R
(5'-ATATCCGAGCGCCTCGTGCATGCG) (SEQ ID NO:14) (Lemaux et al.,
1996).
[0159] Amplifications were performed with Taq DNA polymerase
(Promega, Madison, Wis.) in a 25-.mu.l reaction (Cho et al.,
1998a-c). Twenty-five microliters of the PCR product with loading
dye were subjected to electrophoresis in a 1.0% agarose gel with
ethidium bromide and photographed using exposure to UV light.
Presence of 0.4- and 0.14-kb fragments was consistent with intact
and truncated wtrxh fragments, respectively; an internal 0.34-kb
fragment was produced from the bar gene with bar primers.
Homozygous lines for wtrxh were screened by PCR and western blot
analysis in T.sub.2 or T.sub.3 plants.
[0160] For DNA hybridization analysis, 10 .mu.g of total genomic
DNA from leaf tissue of each line was digested with HindIII and
SacI, separated on a 1.0% agarose gel, transferred to Zeta-Probe GT
membrane (Bio-Rad, Hercules, Calif.) and hybridized with a
radiolabeled wtrxh-specific probe following the manufacturer's
instructions. The wtrxh-containing 0.4 kb XbaI-SacI fragment from
pDhWTRXN-9 was purified by QIAEX gel extraction kit (QIAGEN,
Chatsworth, Calif.) and labeled with .sup.32P-dCTP using random
primers
[0161] Western Blot Analysis
[0162] Western blot analysis was performed on seeds from selected
transgenic lines as well as from control barley seeds from
non-transgenic Golden Promise grown under the same conditions as
the transgenic plants and from control wheat seeds of a durum wheat
cultivar, cv. Monroe, or a bread wheat cultivar cv. Capitale. Whole
seeds were ground to a fine powder with a mortar and pestle under
liquid nitrogen. Ten to 20 seeds were used for each sample; the
volume of extraction buffer (50 mM Tris HCl or phosphate buffer, pH
7.8, 0.5 mM phenylmethyl sulfonyl fluoride [PMSF], 1 mM EDTA)
varied from 2 to 4 ml depending on the number of seeds used and the
viscosity of the extract. Grinding was continued for an additional
minute after buffer addition; the mixture was then centrifuged at
14,000.times.g for 10 minutes and the supernatant solution was
saved as the albumin-globulin fraction that contained the
thioredoxin.
[0163] SDS-PAGE of the albumin-globulin fraction was performed in
12-17% polyacrylamide gradient gels at pH 8.5 (Laemmli, 1970). From
each sample equal amounts of protein (.about.40 .mu.g) quantitated
according to Bradford (1976) were diluted 1:2 v/v in Laemmli sample
buffer, boiled for 3 minutes, loaded onto gels and subjected to
electrophoresis at a constant current of 15 mA. Proteins were
transferred to nitrocellulose at a constant voltage of 40 V for 4
hours at 4.degree. C. using a Hoefer Transphor Transfer Unit
(Alameda, Calif.). Nitrocellulose was blocked with 5% powdered milk
in TBS for 2 hours at room temperature (RT), incubated in primary
antibody for 4 hours at RT and in secondary antibody for 1 hour at
RT. Primary antibody was wheat anti-thioredoxin h II Ab (Johnson et
al., 1987b) diluted 1 to 500; secondary antibody was goat
anti-rabbit alkaline phosphatase (Bio-Rad, Hercules Calif.) diluted
1:3000. Blots were developed in NBT/BCIP alkaline phosphatase color
reagent (according to Bio-Rad instructions); gels were stained with
Coomassie blue to assure transfer. Images were scanned using a
Bio-Rad GelDoc 1000 (Hercules, Calif.) and analyzed using Bio-Rad
Multi Analyst, version 1.0.2. All bands were scanned over the same
area, using a rectangle of comparable density as background;
results were expressed as % of volume scanned. The number shown
represents the percent of the total volume (pixel
density.times.area of scanned band).
[0164] WTRXh Activity Measurements
[0165] Preparation of Materials for Extraction.
[0166] Mature grains from various heterozygous and homozygous
transgenic lines served as starting materials for the assay.
Heterozygous lines with a D-hordein promoter were: GPDhBarWtrx-5,
GPDhBarWtrx-9-1, and GPDhBarWtrx-9-2. Heterozygous lines with a
B-hordein promoter and no signal sequence were: GPdBhBarWtrx-2, -5,
-9, -19 and GPdBhBarWtrx-20. Heterozygous lines with a B-hordein
promoter plus a signal sequence were: GPdBhssBarWtrx-2, -7,
GPdBhssBarWtrx-29, GPdBhssBarWtrx-20, GPdBhssBarWtrx-14,
GPdBhssBarWtrx-22. Homozygous lines with a signal sequence were:
GPdBhssBarWtrx-2-17, GPdBhssBarWtrx-2-17-1, GPdBhssBarWtrx-29-3 and
GPdBhssBarWtrx-29-3-2. Control materials included a non-transformed
tissue culture derived line, 4-96, a transformed line containing
only bar, GPBar-l, and null segregant lines, GPdBhssBarWtrx-29-11
and GPdBhssBarWtrx-29-11-10, derived from line
GPdBhssBarWtrx-29.
[0167] Preparation of (NH.sub.4).sub.2SO.sub.4 Extracts for Gel
Filtration
[0168] Approximately fifteen grams of barley grains were ground to
powder in a coffee grinder and extracted with 80 ml (1:4 w/v) of
buffer [(50 mM Tris-HCl buffer, pH 7.9, 1 mM EDTA, 0.5 mM PMSF
(phenylmethysulfonyl fluoride)], 2 mM e-amino-n caproic acid, 2 mM
benzamidine-HCl) by stirring for 3 hrs at 4.degree. C. The slurry
plus the rinse was subjected to centrifugation at 25,400.times.g
for 20 min, the supernatant solution was decanted through glass
wool, pellets were resuspended in a small volume of buffer and then
clarified by centrifugation as before. The supernatant fractions
were combined, an aliquot was removed and the remainder was
subjected to acidification by adjusting the pH from 7.83 to 4.80
with 2 N formic acid; denatured proteins were removed by
centrifugation as above prior to assay. The pH of the acidified
supernatant solution was readjusted to 7.91 with 2 N NH.sub.4OH and
an aliquot was removed for assay. Powdered (NH.sub.4).sub.2SO.sub.4
was added to a final concentration of 30% and the sample was
stirred for 20 min at 4.degree. C., followed by centrifugation as
described above. The pellet was discarded. Additional
(NH.sub.4).sub.2SO.sub.4 was added to bring the decanted
supernatant solution to 90% saturation; the sample was stirred for
16 hrs at 4.degree. C., followed by centrifugation as described
above.
[0169] The supernatant solution was discarded, the 30-90%
(NH.sub.4).sub.2SO.sub.4 pellets were re-suspended in 30 mM
Tris-HCl, pH 7.9 buffer and then subjected to centrifugation at
40,000.times.g for 15 min to clarify. The resulting supernatant
(30-90% (NH.sub.4).sub.2SO.sub.- 4 fraction) was added to dialysis
tubing (6,000-8,000 MW cut-off) and exposed to solid sucrose at
4.degree. C. to obtain a 10-fold reduction in volume. An aliquot (1
ml) of the clarified and concentrated 30-90%
(NH.sub.4).sub.2SO.sub.4) sample was saved and the remaining sample
was applied to a pre-equilibrated (30 mM Tris-HCl, pH 7.9, 200 mM
NaCl) Sephadex G-50 superfine column (2.5.times.90 cm;.about.400 mL
bed volume) with a peristaltic pump at a flow rate of 0.5 mL/min.
Protein was eluted with the same buffer at the same flow rate; one
hundred fifty drop-fractions were collected. Selected fractions
were used to measure absorbance at 280 nm using a Pharmacia Biotech
Ultrospec 4000 and to assay for TRXh activity following the
NADP-MDH activation protocol (see below). Active fractions were
pooled, stored at 4.degree. C., and then assayed for total NADP-MDH
activation activity.
[0170] Preparation of Heat-Treated Extracts
[0171] Approximately 10 grams of barley grains were ground to
powder for about 30 sec in a coffee grinder and extracted by
shaking for 1 hr at room temperature in 50 mL buffer as above. The
slurry plus the rinse was subjected to centrifugation at
27,000.times.g for 20 min and the supernatant solution decanted
through glass wool. A 20 mL aliquot of each sample was heated at
65.degree. C. until sample temperature reached 60.+-.1.degree. C.
(.about.10 min). The sample was held at 60.degree. C. for 10
additional min, followed by cooling in an ice/water bath. The
cooled sample was centrifuged and the supernatant solution was
concentrated by sucrose as above and stored at -20.degree. C.
Frozen samples were thawed and clarified by centrifugation at
14,000 rpm for 10 min at 4.degree. C. Total TRXh activity was
estimated on the concentrated, supernatant fractions.
[0172] NADP-Malate Dehydrogenase Activation Assay
[0173] Thioredoxin h activity was assayed as previously described
(Florencio et al., 1988; Johnson et al., 1987a). Fifty to 120 .mu.l
of extract (depending on activity) was preincubated with DTT, and
0.16 to 0.32 .mu.l of the pre-incubation mixture was used for the
NADP-MDH assay. Control assays were conducted on identical
fractions in the absence of NADP-MDH. Western blot analysis was
conducted as described above except that 10 to 20 %
SDS-polyacrylamide gels were used for electrophoresis and transfer
to nitrocellulose paper was for 4 hrs at 40 V.
[0174] Sequential Extraction of Multiple Protein Fractions
[0175] Ten grams of barley grain were sequentially extracted for
albumin (H.sub.2O-soluble), globulin (salt-soluble), hordeins
(alcohol-soluble) and glutelins (Shewry et al., 1980). Barley
powder was stirred with 0.5 M NaCl for 1 h at 25.degree. C. to
remove salt-soluble proteins. Two sequential hordein fractions were
extracted from the residue with 50% propanol in the absence
(hordein-I) and presence (hordein-II) of 2% (v/v)
2-mercaptoethanol. Glutelins were extracted from the residue with
0.05 M borate buffer, pH 10, containing 1% (v/v) 2-mercaptoethanol
and 1% (v/v) sodium dodecylsulphate.
[0176] In Vitro Monobromobimane (mBBr) Labeling of Proteins
[0177] Immature, mature, or germinating seeds from nontransformed
and transgenic plants were ground in 100 mM Tris-HCl buffer, pH
7.9. Reactions were carried out following the protocol of Kobrehel
et al., (1992). Seventy microliters of the buffer mixture
containing a known amount of protein was either untreated or
treated with DTT to a final concentration of 0.5 mM. After
incubation for 20 min, 100 nmol of mBBr was added, and the reaction
was continued for another 15 min. To stop the reaction and
derivatize excess mBBr, 10 .mu.l of 10% SDS and 100 .mu.l of 100 mM
2-mercaptoethanol were added. The samples were applied to a 15%
SDS-PAGE gel. Fluorescence of mBBr was visualized by placing gels
on a light box fitted with a UV light source (365 nm). Protein
determination was carried out by the Bradford dye binding method
(Bradford 1976) using bovine serum albumin or gamma globulin as
standards.
[0178] Assay of Pullulanase and its Inhibitor
[0179] To measure pullulanase activity, grain was germinated in a
dark chamber and retained for up to 5 days at 25.degree. C. as
described (Kobrehel et al., 1992.; Lozano et al., 1996.). A set of
plates from each line was removed for extract preparation each day.
Cell-free endosperm extracts were prepared from lots of 10-20
germinated grains of equivalent root and coleoptile length within a
given cohort. Endosperm was separated from the embryo and other
tissues and added to Tris-HCl buffer (50 mM, pH 7.9) supplemented
with 1 mM EDTA and 0.5 mM PMSF (1:3 to 1:6, wt/vol ratio of tissue
to buffer depending on developmental stage). After grinding in a
mortar on ice, the sample was clarified by centrifugation (10 min
at 24,000.times.g); the supernatant fraction was recovered and
stored in 0.5-ml aliquots -80.degree. C. for pullulanase
spectrophotometric or gel assays.
[0180] Pullulanase activity was determined spectrophotometrically
at 37.degree. C. by measuring dye released after 30 min at 534 nm
using red pullulan (Megazyme, Bray, Ireland) as substrate in 50 mM
citrate-phosphate buffer (pH 5.2) (Serre et al., 1990). Pullulanase
also was assayed on native activity gels of 7.5% acrylamide, 1.5 mm
thickness, containing 1% red pullulan (Furegon et al., 1994.). Gels
were scanned using a Bio-Rad Gel Doc 1000 and analyzed using
Bio-Rad MULTI ANALYST, version 1.0.2. Pullulanase inhibitor
activity was determined on fractions heated to inactivate
pullulanase (70.degree. C. for 15 min) by measuring their ability
to inhibit added purified barley malt pullulanase. Endogenous
pullulanase activity was shown to be completely eliminated by this
heat-treatment while the inhibitor activity was not affected (Macri
et al., 1993; MacGregor et al., 1994).
[0181] Alpha-Amylase Activity in Barley Grain Overexpressing
Thioredoxin h
[0182] Amylase activity from the null segregant and homozygous
barley grains was analyzed during germination and early seedling
growth by using gels containing starch. Native polyacrylamide
electrophoresis gels [6% acrylamide, 1.5 mm thick] were prepared
and developed according to the method of Laemmli (1970) except that
SDS was omitted from all solutions. The separating gel contained
0.5% soluble starch (Lintner potato starch, Sigma Chemical Co., St.
Louis, Mo.). Lyophilized samples were dissolved in distilled
H.sub.2O and mixed 1:1 with a buffer consisting of 0.25 M Tris-HCl,
pH 6.8, 50% glycerol, 0.04% bromophenol blue, and 3 mM CaCl.sub.2.
Fifty micrograms of sample protein were loaded in each lane.
Electrophoresis was carried out at 80 milliamps per gel at
4.degree. C. until the dye front was at the edge of the gel
(usually 4 to 5 hours). After electrophoresis, the gels were
incubated in 100 ml of 0.1 M succinate buffer, pH 6.0, for 1-2
hours at 37.degree. C. The gels were then stained for 5 min in a
solution containing 2.5 mM I.sub.2 and 0.5 M KI. Gels were washed
in distilled H.sub.2O. Except for the white regions containing
amylase activity, gels were stained dark blue.
[0183] Isoelectricfocusing (IEF)
[0184] For determination of alpha-amylase isozyme patterns,
extracts from both dry and germinating grain of transformed and
control (untransformed) barley were separated by electrophoresis at
4.degree. C. [1.0 mm thick, pH 3-10 isoelectric focusing (IEF)
polyacrylamide gels, using the X cell II system (NOVEX, San Diego,
Calif.)]. Cathode buffer contained 20 mM arginine, and 20 mM
lysine; anode buffer was 7 mM phosphoric acid. Samples were mixed
1:1 and 2.times. IEF sample buffer pH 3-10 (NOVEX). After sample
application (20 .mu.g/lane) gels were developed at constant voltage
[100 V for 1 hr, 200 V for an additional 1 hr, and 500 V for 30
min]. IEF standards (Bio-Rad) were used to determine the pH
gradient of the gels.
[0185] Multiple Antibody Probing of IEF Gels
[0186] Western blot analysis of alpha-amylase isozymes was
performed using a Mini Trans-Blot Electrophoretic Transfer Cell
(Bio-Rad). Seed extracts from the null segregant and homozygous
lines overexpressing wheat thioredoxin h were separated by IEF gels
as described above. Proteins were transferred to nitrocellulose at
a constant voltage of 100 V for 1 hr at 4.degree. C. using 0.75%
acetic acid as blotting buffer. Nitrocellulose was blocked with 5%
powdered milk in Tris buffer solution (20 mM Tris-HCl, pH 7.5,
supplemented with 0.15 M NaCl) for 1 hr at room temperature,
incubated with primary antibody for 4 hours at room temperature and
then with secondary antibody for 1 hour at room temperature.
Primary antibody was anti-barley alpha-amylase B diluted 1:1000;
secondary antibody was goat anti-rabbit alkaline phosphatase
(Bio-Rad) diluted 1:3000. Blots were developed in NBT/BCIP alkaline
phosphatase color reagent (according to Bio-Rad instructions)
thereby rendering the cross-reacted alpha-amylase bluish-purple. To
achieve full identity of isozyme pattern, blots were probed a
second time with another primary antibody, anti-alpha-amylase A
(diluted 1:1000) and the secondary antibody (as above). This time
blots were developed in Naphthol Phosphate/Fast Red alkaline
phosphatase color reagent (according to Bio-Rad instructions) which
gave a pink stain to the alpha-amylase A. The blot shown was
subject to this dual probing procedure.
[0187] B. Results and Discussion
[0188] Production of Transgenic Plants
[0189] One day after bombardment, the whole embryos were
transferred onto DC medium with 5 mg/L bialaphos. At transfer to
the second selection plate (5 mg/L bialaphos), all material from
individual callusing embryos was broken into small pieces (2-4 mm)
using forceps and maintained separately. During the subsequent two
to five selection passages on 5 mg/L bialaphos (at 10-20 d
intervals). callus pieces showing evidence of more vigorous growth
were transferred to new selection plates. During the second round
of selection, some pieces of callus were inhibited in growth and in
some cases pieces turned brown. In general, transformed tissues
were observed after three or more rounds of selection. The
bialaphos-resistant tissues were transferred onto an intermediate
medium, DBC2 or DBC3 (Cho et al., 1998a-c) with bialaphos (5 mg/L),
and grown for 1 to 2 months before regeneration on FHG medium
containing 3 mg/L bialaphos. Green plantlets were transferred into
Magenta boxes containing 3 mg/L bialaphos. Twenty-eight independent
putatively transformed, regenerable lines were produced after
bialaphos selection (shown in Table 1).
3TABLE 1 Transgenic Barley Lines Transformed with Wheat Thioredoxin
h Gene. DNA PCR Plasmids for (T.sub.0 leaf) TRXh Expression
Bombardment Transgenic Barley Line bar wtrxh in T.sub.1 seeds
Ploidy Comments pdBhWTRXN-1 + GPdBhBarWTRX-1 + + n.d. Tetraploid
pAHC20 GPdBhBarWTRX-2 + + + Tetraploid GPdBhBarWTRX-3 + + + Diploid
GPdBhBarWTRX-5 + + + Tetraploid Sterile GPdBhBarWTRX-16 + - n.d.
Tetraploid GPdBhBarWTRX-17 + + n.d. Tetraploid GPdBhBarWTRX-19 + +
+ Diploid GPdBhBarWTRX-20 + + + Diploid GPdBhBarWTRX-22 + + +
Diploid GPdBhBarWTRX-23 + + + Diploid pdBhssWTRXN3-8 +
GPdBhssBarWTRX-1 + - - Diploid pAHC20 GPdBhssBarWTRX-2 + + +
Diploid Homozygous GPdBhssBarWTRX-3 + + - Diploid GPdBhssBarWTRX-7
+ + + Diploid GPdBhssBarWTRX-9 + + n.d. Tetraploid
GPdBhssBarWTRX-11 + + - Diploid GPdBhssBarWTRX-13 + + + Tetraploid
GPdBhssBarWTRX-14 + + + Diploid GPdBhssBarWTRX-20 + + + Tetraploid
GPdBhssBarWTRX-21 + + n.d. Tetraploid Sterile GPdBhssBarWTRX-22 + +
+ Tetraploid GPdBhssBarWTRX-29 + + + Diploid Homozygous pDhWTRXN-2
+ GPDhBarWTRX-5 + + + Tetraploid pAHC20 GPDhBarWTRX-7 + + + Diploid
GPDhBarWrRX-8 + + + Diploid GPDBhBarWTRX-9 + + + Diploid Homozygous
GPDBhBarWTRX-22 + + + Diploid Sterile pGlbWTRXN-1 + GPGlbBarWTRX-1
+ + + Diploid pAHC20 *n.d.: not determined
[0190] Analysis of T.sub.0 Plants and their Progeny
[0191] PCR analysis was performed using two sets of WTRXh primers
and one set of BAR primers (see FIG. 1). PCR amplification resulted
in 0.4-kb intact wtrxh or 0.14 kb truncated wtrxh and 0.34-kb
internal bar fragments from transgenic lines. Of the 28 lines
tested, 28 yielded bar fragments from T.sub.0 leaf tissue and 26
produced PCR-amplified fragments for wtrxh, giving a 93%
co-transformation frequency. Nine lines were transformed with
pdBhWTRXN-1, eleven with pdBhssWTRXN-8, five with pDhWTRXN-2 and
one with pG1bWTRXN-1 (see Table 1). Three lines (GPdBhBarWtrx-5,
GPdBhssBarWtrx-21 and GPDhBarWtrx-22) were sterile. Seeds of
T.sub.1 plants and their progeny from selected wtrxh-positive lines
were planted in order to screen for homozygous lines. Homozygous
lines and null segregants were obtained from GPdBhssBarWtrx-2, -29
and GPDhBarWtrx-9 (see Table 1).
[0192] Cytological Analysis of Transgenic Plants
[0193] Chromosomes were counted in root meristem cells of
independently transformed T.sub.0 barley plants. Out of 28
independent transgenic lines examined. 17 lines had the normal
diploid chromosome complement (2n=2x=14), while the remaining 11
lines were tetraploid (2n=4x=28) (see Table 1).
[0194] Characterization and Content of WTRXh Produced in Transgenic
Seed
[0195] As discussed above, several stably transformed barley lines
were obtained that express wheat thioredoxin h. As seen in FIG. 2,
the stable introduction of the wtrxh linked to the B1-hordein
promoter with the signal peptide sequence resulted in greatly
enhanced expression of active WTRXh in transgenic barley seed.
[0196] Analysis by western blot of soluble protein fractions of the
three lines in which the thioredoxin gene was linked to a signal
sequence (GPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and
GPdBhssBarWtrx-7) showed differences in the level of expression
(shown in Table 2). Line GPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and
GPdBhssBarWtrx-7, respectively, showed 22 times, 10 times and 5.5
times more WTRXh protein than nontransformed control seeds. The
analyses showed that the thioredoxin content of the null segregant
(GPdBhssBarWtrx-29-11) was approximately half that of the
corresponding control. The three lines generated from the construct
in which the thioredoxin gene was not associated with a signal
sequence were also compared to nontransformed control barley seed
and they exhibited the following increases in TRXh levels as
indicated by the western blot analyses: GPDhBarWtrx-9: 12 times;
GPDhBarWtrx-5: 6.3 times; GPdBhBarWtrx-2: 6.4 times. When probed on
Western Blots, the transgenic lines show two bands while the
control barley generally shows only one and in some cases a second
minor band. Furthermore, the tissues from the transgenic lines were
characterized by a band that did not correspond to either of the
barley bands but did correspond to wheat thioredoxin h. These data
indicate that the protein introduced by transformation is wheat
thioredoxin h.
4TABLE 2 Western Blot Analyses of Overexpression of Wheat
Thioredoxin h in Barley. % Fold Increase Barley Line Volume Scanned
(or Decrease) Non-Transformed Control: Golden Promise 1.46 1.0
Transformed with Signal Sequence: GPdBhssBarWtrx-22 32.44 22
GpdBhssBarWtrx-29 14.62 10 GpdBhssBarWtrx-7 7.99 5.5 Transformed
without Signal Sequence: GPDhBarWtrx-9 17.69 12 GPDhBarWtrx-5 9.20
6.3 GPdBhBarWtrx-2 9.29 6.4 Null Segregant: GPdBhssBarWtrx-29-11-10
0.93 (0.64)
[0197] The Wheat Thioredoxin h in Barley Grains is Biologically
Active
[0198] Because of interference from other enzymes that oxidize
NADPH, the activity of TRXh cannot be accurately assayed in crude
extracts, thereby necessitating its partial purification. Partially
purified extracts of the different transgenic and control lines
were prepared from 15 grams of seed using ammonium sulfate
fractionation and gel filtration chromatography. Activity was
measured with an NADP-MDH activation assay. Profiles based on these
assays show that the activity of TRXh in the transformed seed is
much higher than in the nontransformed control (see FIG. 2). The
activity results are summarized in Table 3.
[0199] Total WTRXh activity from the seeds of two lines transformed
with the B1-hordein promoter and the signal sequence
(GPBhssBarWtrx-3; GPdBhssBarWtrx-29) is about 4- to 10-fold higher,
respectively, than that of control, nontransformed seed. Total
activity from a line transformed with the D-hordein promoter
without the signal sequence (BGPDhBbarWtrx-5) is only slightly
higher (1.25-fold) than that of the nontransformed control (see
Table 3). In the transgenics, the specific activity of thioredoxin
is generally about 0.128 A.sub.340 nm/min/mg protein or about two
fold over null segregants.
5TABLE 3 Summary of Total Buffer-Extracted Protein and Total
Thioredoxin Activity from Active Fraction after Gel Filtration.
Specific Total Total Activity, Activity, Barley Line Protein, mg
A.sub.340/min A.sub.340/min/mg Control (GP 4-96) 102.6 (1.00)* 7.4
(1.00)* 0.064 (1.00)* GPDhBarWtrx-5 171.2 (1.67) 9.2 (1.2) 0.054
(0.8) GpdBhssBarWtrx-29 149.1 (1.45) 72.0 (9.7) 0.483 (7.5)
GpdBhssBarWtrx-3 231.3 (2.25) 27.7 (6.4) 0.794 (12.4) *Numbers in
brackets are fold increase over that of the control.
[0200] The transformed barley grains analyzed so far appear to have
more total buffer-extracted protein than control, nontransformed
seed (Table 3).
[0201] The transformed grains have a thioredoxin content of at
least about 10-15 .mu.g thioredoxin/mg soluble protein(about 2-8
.mu.g thioredoxin/mg tissue) or about two-fold higher than the null
segregant.
[0202] Because of the tediousness of the (NH.sub.4).sub.2SO.sub.4
procedure and the requirement for large quantities of seed, the
original extraction procedure was modified to include a heat
treatment step. This change was based on the fact that E. coli
WTRXh is stable after treatment at 60.degree. C. for 10 min (Mark
and Richardson, 1976). Results on WTRX from two different
transgenic barley seeds (GPdBhBarWtrx-3, GPdBhssBarWtr-29) showed
no significant difference in activity between the heat treated and
non-heat treated extracts (FIG. 3). In addition heat-treatment
decreased the endogenous, nonspecific activity in this assay,
thereby increasing the reliability of the measurements.
[0203] Ten different barley lines (transformed and nontransformed)
were extracted using the heat-treatment step and assayed with the
NADP-MDH assay; the results are summarized in Table 4. In general,
total WTRXh activities in seeds from lines transformed with the
B-hordein promoter and signal sequence linked to wtrxh are much
higher (4- to 35-fold) than in seeds from lines transformed with
the same promoter without signal sequence linked to wtrxh or in
seeds from the nontransformed control (Table 4). At this point it
is not known whether all expressed wheat WTRXh in barley seeds is
heat stable.
6TABLE 4 Relative Total Thioredoxin Activity in Different
Transgenic Barley Lines. Total Total Specific Line Designation
Protein (%) Activity (%) Activity (%) Non-transgenic control GP4-96
100 100 100 Bar Gene Only GPBar-1 92 120 131 Without Signal
Sequence GPdBhBarWtrx-1 101 192 190 GPdBhBarWtrx-22 113 151 133
GPdBhBarWtrx-23 118 180 153 With Signal Sequence GPdBhssBarWtrx-2
137 1650 1203 GPdBhssBarWtrx-14 122 1723 1418 GPdBhssBarWtrx-20 147
440 299 GPdBhssBarWtrx-22 154 3470 2245 GPdBhssBarWtrx-29 108 1316
1219 One hundred percent of (a) total protein, mg; (b) total
activity, nmol/min; and (c) specific activity, nmol/min/mg protein
of the non-transgenic control are: (a) 116.4; (b) 157.38 (c) 1.52,
respectively
[0204] Of the stably transformed lines that expressed wheat
thioredoxin h, on average, its level was found to be higher in
transformants that had the signal peptide-containing constructs
than to those that did not (Table 4). Western blot analysis of
soluble protein fractions from heterozygous mixtures of seeds from
three of the lines, GPdBhssBarWtrx-7, GPdBhssBarWtrx-29, and
GPdBhssBarWtrx-22 showed 5.5 times, 22 times, and 10 times more
thioredoxin h, respectively, than nontransformed control grain
(Table 2). The thioredoxin content of the null segregant
(GPdBhssBarWtrx-29-11-10) was about half that of the corresponding,
nontransformed control.
[0205] Extracts from barley typically showed one immunologically
reactive band (identified by B in FIG. 4A, lanes 1 and 6) but in
some transfers showed a second faint, faster moving band (FIG. 4B,
lane 2). Tissues from transgenic lines overexpressing wtrxh were
characterized by a band that did not correspond to either of the
two counterparts in barley, but rather to thioredoxin h from wheat.
The difference between the overexpressed 13.5-kDa wheat and the
endogenous 13.1-kDa barley thioredoxin h is particularly pronounced
in the barley line transformed with the nontargeted thioredoxin h
gene (FIG. 4A, line 5 and FIG. 4B, lane 1). Repeated analyses of
the various transgenic lines by SDS/PAGE led to the conclusion that
the band identified in FIGS. 4A-B by W corresponds to the bread
wheat wtrxh introduced by barley. Independent biochemical assays
with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Florencio et al.,
1988.) confirmed the ability of barley NTR to reduce wheat
thioredoxin h (data not shown).
[0206] Because of their value in assessing biochemical attributes
of the grain, homozygous wtrxh lines were identified and analyzed
by Western blot. The two lines identified as homozygous showed both
enhanced expression of thioredoxin h relative to that of their
heterozygous parents and nontransformed controls. Analysis of
GPdBhssBarWtrx-29-3 is shown in FIG. 5. It is noted that
demonstration of the thioredoxin h present in the nontransgenic
control and null segregant grains (not apparent in the exposure
shown in FIG. 4) required conditions that led to overexposure of
the enriched transgenic preparations. Thioredoxin in the parent
heterozygous grain was shown to be biochemically active.
[0207] Pullulanase and Pullulanase Inhibitor Activity in Barley
Grain Overexpressing Thioredoxin h
[0208] Pullulanase is an amylolytic enzyme present in cereal grain,
which has a disulfide inhibitor protein (Macri et al., 1993.;
MacGregor et al., 1994.), the activity of which is linked to
thioredoxin (Wong et al., 1995.). Thioredoxin reduced by NADPH via
NTR, reduces the disulfide bonds of the inhibitor, allowing the
targeted pullulanase enzyme to be active. Because of this
relationship, it was of interest to determine the activity of
pullulanase in the thioredoxin h-overexpressing transformants.
[0209] Spectrophotometric assays (FIG. 8A) of extracts from
transformed grain of a homozygous line (GPdBhssBarWtrx-29-3)
overexpressing thioredoxin h showed a 3- to 4-fold increase in
pullulanase activity on the fifth day after initiation of
germination relative to its null segregant. Confirmatory results
were obtained in a separate experiment with native activity gels.
The increase in activity was apparent either when gels were viewed
directly (FIG. 8B) or when the activity on the gels was assessed by
scanning and integrating the clarified bands (FIG. 8C). A
homozygous line isolated from a different, independent
transformation event (GPdBssBarWtrx-2-1-15) showed a similar
response (data not shown). The transgenic plants expressed an
pullulanase activity of about 1-2 Absorbance units at 534 nm/30
min/mg protein, which is about two-fold higher than null
segregants.
[0210] Pullulanase inhibitor activity was determined on fractions
heated to inactivate pullulanase (70.degree. C. for 15 min) by
measuring the inhibition of the fractions on added purified barley
malt pullulanase. The endogenous pullulanase activity was shown to
be completely eliminated by this heat treatment whereas inhibitor
activity was not affected (Macri et al., supra; MacGregor et al.,
supra). Analysis of comparable grain extracts revealed that the
pullulanase inhibitor was inactive on the fourth and fifth days
after water addition in both the transformant and null segregants.
These results thus demonstrate that the increase in pullulanase
activity observed after the third day is not caused by enhanced
inactivation of the inhibitor in the transgenic grain. It is
possible that thioredoxin acts either by increasing the de novo
synthesis of pullulanase (Hardie et al., 1975.) or by lowering the
binding of the mature enzyme to the starchy endosperm. There is
evidence that some of the pullulanase of the mature endosperm is
present in bound form and can be solubilized by reducing conditions
(Sissons et al., 1993.; Sissons et al., 1994.).
[0211] Alpha-Amylase Activity in Barley Grain Overexpressing
Thioredoxin h
[0212] Alpha-amylase, also an amylolytic enzyme that is induced by
gibberellic acid like pullulanase, has long been considered key to
germination. The synthesis of the major (B) and minor (A) forms of
this enzyme are known to be triggered by the hormone, gibberellic
acid (GA). In addition, alpha-amylase activity is increased in
vitro by the reductive inactivation of its disulfide inhibitor
protein by thioredoxin h (in the presence of NADPH and
NADP-thioredoxin reductase). The present results with transformed
barley seeds show that, like pullulanase, thioredoxin h expression
alters alpha-amylase activity. In this case, the appearance of the
enzyme during germination is accelerated and its abundance and
activity are increased.
[0213] FIG. 9A-D shows the early increase in both the abundance and
activity of alpha-amylase (A+B forms) during gemination and
seedling development. Based on the antibody response in western
blots, alpha-amylase was first detected 3 days after the onset of
germination in the transgenic grain FIG. 9C) whereas the enzyme did
not appear until the fourth day in the null segregant (FIG. 9A).
The onset of activity (based on the activity gel) followed a
similar pattern (FIG. 9B and FIG. 9D). The mobility of the enzyme
in the activity gel also reflected the early induction of activity
in the transgenic grain (FIG. 10). That much of this increase in
activity seen early on was due to the B (a gibberellic acid-linked
form) is supported by FIG. 11. Here, one can also see that the
level of the minor A form of the enzyme (also gibberellic acid
dependent) was increased in grain overexpressing thioredoxin h.
Again, the appearance of significant levels of the major (B form)
alpha-amylase enzyme was advanced by 1 day.
[0214] Germination of Barley Grains Overexpressing Thioredoxin
h
[0215] All operations were carried out at 25.degree. C. (unless
otherwise specified below) under conditions described by Kobrehel
et al., 1992 and Lozano et al. 1996. Grains were surface sterilized
by continuous stirring in 0.25% bleach for 30 min: Bleach was
removed by extensive washing with sterilized distilled water.
Thirty sterilized null segregant (GPdBhssBarWtrx-29-22-10, in which
the transgene was removed by crossing with a self-polinated plant
from the same line) and thirty sterilized homozygous
(GPdBhssBarWtrx-29-3) seeds were placed in each of a series of
plastic Petri dishes (12.5 cm diameter) fitted with three layers of
Whatman #1 filter paper moistened with 15 ml sterile distilled
water. Plates were wrapped with aluminum foil and grain was
germinated in a dark chamber at 20.degree. C. for up to 7 days. One
plate was read at each time point shown in FIG. 21. Percent
germination, in the first day (from the start of incubation up to
24 hours), was determined by observing the emergence of the
radicle. On the subsequent days, percent germination represents
seedling growth as determined by measuring the length of coleoptile
and roots of the germinated grains.
[0216] The results, shown in FIG. 21, indicate that germination in
transgenic barley overexpressing wheat thioredoxin h is detected
about 16 hours after the onset of incubation in about 25-30% of the
seeds. In contrast, no germination in the null segregant was
detected at 16 hours but is first detected 8 hours later, on Day 1.
Therefore, in the transgenic germination is advanced about 8 hours.
However, on Day 1 germination was detected in approximately 70% or
about twice the number of transgenic grains in comparison to their
null segregant counterparts. It is interesting to note that the
onset of germination in the transgenics parallels the onset of the
detection of alpha amylase as shown in FIG. 10.
[0217] Sequential Extraction of Grain Proteins from Transgenic
Barley Grains.
[0218] Isolated endosperm from 10 dry grains or seedlings
(germinated as described above) were ground with mortar and pestle
4.degree. C. with 3 ml Tris-HCl buffer as indicated below. The
separate mixtures of homozygous GPdBhssBarWtrx-29-3 and null
segregant GPdBhssBarWtrx-29-22-10 grains were placed in a 5-ml
screw-top centrifuge tube. Grains were mechanically shaken for 30
minutes and then centrifuged for 10 min at 24,000.times.g. The
supernatant fraction (buffer-soluble) was decanted and saved for
analysis and the residue was extracted sequentially with the
following solvents for the indicated times: [1] 0.5 M NaCl (30
min); [2] water (30 min); [3] 2.times.50% propanol (2 hr); [4]
2.times.50% propanol+2% 2-mercaptoethanol (MET) (2 hr); and [5] 0.5
M borate buffer, pH 10, containing 1% SDS and 2% 2-mercaptoethanol
(2 hr). Supernatant fractions of all extracts were determined for
volume and protein content (by Coomassie dye binding method), then
were stored at -20.degree. C. until use. By convention, the
fractions are designated: [1] albumin/globulin (buffer/salt/water);
[2] Hordein I (propanol); [3] Hordein II (propanol+MET); and [4]
glutelin (Borate/SDS/MET) (Shewry et al., 1980). These fractions
were used to determine, protein content, the distribution of
proteins between the water soluble and insoluble fractions, the
total extractable protein, and reduction with NADPH.
[0219] To determine the in vivo redox status of protein from
transgenic barley grain during germination and seedling
development, the extraction procedure was repeated except that 2 mM
mBBr was included in the Tris grinding buffer and the grinding was
under liquid nitrogen. The mBBr derivatized proteins were
electrophoresed on SDS-polyacrylamide gels (1.5 mm thickness,
10-20% gels, pH 8.5 (Laemmli, 1970). Gels were developed for 16 hr
at a constant current of 8 mA. Following electrophoresis, gels were
placed in 12% (w/v) trichloroacetic acid and soaked for 4 to 6 hr
with one change of solution to fix the proteins; gels were then
transferred to a solution of 40% methanol/10% acetic acid for 8 to
10 hr with agitation to remove residual mBBr. The fluorescence of
mBBr (both free and protein bound mBBr), was visualized by placing
gels on a light box fitted with an ultraviolet light source (365
nm). Following removal of the excess (free) mBBr, images of gels
were captured by Gel Doc 1000 (Bio-Rad).
[0220] To ascertain the equivalent protein amount of loaded
extracts, SDS-gels were stained with Coomassie Brilliant Blue G-250
in 10% acetic acid for 30 min, and destained in 10% acetic acid for
30 min with the aid of a microwave oven. Protein stained gels were
captured by Gel Doc 1000 as above.
[0221] The quantification of fluorescence (pixel.times.mm.times.mm)
and protein (optical density.times.mm.times.mm) on gels were
carried out by a software program for image
analysis--Multi-Analyst, version 1.0 (Bio-Rad). Relative reduction
was expressed as the ratio of fluorescence to protein.
[0222] The results of two experiments shown in Table 5, Table 6,
and Table 7 demonstrate an increase in the total protein on a
percent grain and a percent weight basis in the transgenic barley
as compared to the null segregant. The transgenic have a
thioredoxin content that is at least two-fold higher (10-15
.mu.g/mg soluble protein; 2-8 .mu.g/gram tissue) than the null
segregant. The data indicate that this increase in total
extractable protein is the result in redistribution of the protein
to the most soluble albumin/globulin fraction. The redistribution
of the protein to the soluble fraction increase in the transgenics
is at least 5% higher than the controls.
7TABLE 5 Protein Content of Various Fractions in Transgenic Barley
Grain Overexpressing Wheat Thioredoxin h Experiment I* Null
Segregant Homozygous Protein Fraction mg/seed mg/gram mg/seed
mg/gram Albumin/Globulin 0.462 12.25 0.546 13.58 Hordein I 0.239
6.34 0.322 8.01 Hordein II 0.136 3.61 0.094 2.34 Glutelin 0.110
2.92 0.097 2.41 Total Extractable Protein 0.947 25.12 1.059 26.34
*Weight per 10 seeds is 0.377 and 0.402 full null segregant and
homozygous line of transgenic barley
[0223]
8TABLE 6 Protein Content of Various Fractions in Transgenic Barley
Grain Overexpressing Wheat Thioredoxin h Experiment II** Null
Segregant Homozygous Protein Fraction mg/seed mg/gram mg/seed
mg/gram Albumin/Globulin 0.691 20.03 1.044 27.12 Hordein I 0.373
10.81 0.368 10.03 Hordein II 0.254 7.36 0.240 6.23 Glutelin 0.066
1.91 0.062 1.61 Total Extractable Protein 1.384 40.11 1.732 44.99
*Weight per 10 seeds is 0.377 and 0.402 for null segregant and
homozygous line of transgenic barley
[0224]
9TABLE 7 Percent Increase of Extractable Protein in Homozygous Line
%/(grain basis %/mass basis Experiment I 12 4.9 Experiment II 25
12
[0225] Analysis of the relative redox status (SH:SS) of protein
fractions in transgenic and null segregant barley grains during
germination and as dry grains are shown in FIG. 22. In dry
transgenic grain, the greatest increase in reduction relative to
the null segregant was observed in the hordein I fraction. This
increase was paralleled by decreases in the relative redox status
in the hordein II and glutelin fractions while the relative redox
status of the albumin/globulin fraction was unchanged. The relative
redox status of the transgenic in comparison to the null segregant
is at least 5:1.
[0226] During germination, the albumin/globulin fraction
progressively increases, reaching a relative redox ratio of about
1.5 on Day 4. The relative redox status of the hordein II and
glutelin fractions also increased during germination but only
reached parity with the null segregant. In contrast the relative
redox status of the hordein I fraction was highly variable.
[0227] According to the above example, other types of plants, are
transformed in a similar manner to produce transgenic plants
overexpressing thioredoxin, such as transgenic wheat, described
below, rice, maize, oat, rye sorghum (described below), millet,
triticale, forage grass, turf grass, soybeans, lima beans, tomato,
potato, soybean, cotton, tobacco etc. Further, it is understood
that thioredoxins other than wheat thioredoxin or thioredoxin h can
be used in the context of the invention. Such examples include
spinach h; chloroplast thioredoxin m and f, bacterial thioredoxins
(e.g., E. coli) yeast, and animal and the like.
Example 2
Transgenic Wheat Grain Overexpressing Thioredoxin h and Arabidopsis
NTR
[0228] A. Materials and Methods
[0229] Plant Materials
[0230] Spring cultivar of wheat, Bobwhite, Anza and Yecora Rojo,
were grown in the greenhouse as described previously (Wan and
Lemaux 1994; Lemaux et al. 1996). Ten- to 14-day-old germinating
plants of a winter-wheat cultivar, Karl, were incubated at
4.degree. C. for 45 to 60 days in the dark for vernalization
treatment.
[0231] Wheat Expression Vectors
[0232] For wheat transformation, synthetic green fluorescent
protein gene [sfgp(S65T)], wheat thioredoxin h (wtrxh) or
Arabidopsis ntr expression vectors driven by barley
endosperm-specific B.sub.1- or D-hordein were constructed as
follows:
[0233] (1) pDhSSsGFPN3-4: the chimeric DNA construct containing the
D-hordein promoter-signal sequence-sgfp(S65T)-nos was obtained
using a modified method of site-directed mutagenesis by PCR (Cho
and Lemaux 1997). The three-primer strategy was used. A shorter
fragment of 0.5-kb DHORSS was produced by PCR in the first reaction
using primers, Dhor4 (5'-agaaagcttggtaccCTTCGAGTGCCCGCCGAT-3'; SEQ
ID NO:9) and DhorSSsGFP1 R
(5'-GAACAGCTCCTCGCCCTTGCTCACAGCGGTGGTGAGAGCCACGAGGGC-3'; SEQ ID
NO:10), with the template pHor3-1 containing a genomic clone of D
hordein (S.o slashed.rensen et al., 1996), and this first PCR
product (megaprimer) was diluted 50 times. DhorSSsGFP1R is an
overlapping primer which contain the sgfp(S65T) coding sequence and
a partial signal peptide sequence (underlined) from the D-hordein
promoter. For the second PCR reaction, five .mu.l of the diluted
megaprimer (DHORSS), twenty ng of template (pAct1IsGFP-1; Cho et
al., 2000) and 40 pmol of external primers [Dhor4 and Nos1R
(5'-cggaattcGATCTAGTAACATAGATGACA-3': SEQ ID NO:17)] were mixed to
a final volume of 100 .mu.l in 1.times. PCR buffer; pAct1IsGFP-1
contains synthetic gfp gene [sgfp(S65T)] (Chiu et al., 1996)
controlled by the rice actin1 promoter and its intron and
terminated by nos. The resulting chimeric PCR product was digested
with HindII and EcoRI and ligated into the HindII/EcoRI-digested
pBluescript II KS(+) vector, further confirmed by DNA sequencing of
the PCR-amplified fragment [D-hordein promoter with its signal
peptide sequence plus the junction region with the 5' sgfp(S65T)],
and used for stable transformation of wheat.
[0234] (2) pDhWTRXhN-2: the 384-bp wtrxh coding region was
amplified by PCR utilizing the plasmid pTaM13.38 (Gautier et al.,
1998) containing cDNA clone of wtrxh gene as a template to create
XbaI and SacI sites with primers Wtrxh1
(5'-atatctagaATGGCGGCGTCGGCGGCGA-3'; SEQ ID NO:5) and Wtrxh2R
(5'-atagagctcTTACTGGGCCGCGTGTAG-3'; SEQ ID NO:6), respectively
(FIG. 12); small letters contain a restriction enzyme site for
subcloning of the DNA construct containing the wtrxh gene and
underlined letters indicate the wtrxh sequences. The ATG initiation
codon for wtrxh expression was included in the Wtrxh1 primer. PCR
reactions were performed on a thermocycler (MJ Research Inc.,
Watertown, Mass.) using recombinant Taq DNA polymerase (Promega,
Madison, Wis.) in a 100-.mu.l reaction volume. The reaction buffer
contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl.sub.2,
0.1% Triton-X-100, and 50 .mu.M of each deoxyribonucleoside
triphosphate. PCR conditions were 25 cycles of 94.degree. C. for 1
min, 55.degree. C. for 1 min and 72.degree. C. for 2 min, with a
final extension step at 72.degree. C. for 7 min. The wtrxh fragment
amplified with primers Wtrxh1 and Wtrxh2R was purified from a 0.7%
agarose gel using QIAquick.RTM. gel extraction kit (Qiagen Inc.,
Chatsworth, Calif.), digested with XbaI and SacI and ligated into
XbaI/SacI digested pUC19 to generate the pWTRXh-1 plasmid.
Nucleotide sequences of the PCR-amplified wtrxh coding region were
determined by dideoxynucleotide chain termination method using
Sequenase according to manufacturer's instructions (United States
Biochemical, Cleveland, Ohio) with double-stranded plasmid
templates and regularly spaced primers. pDhWTRXN-2 was made by
replacing the uidA gene in pDhGN-2 (containing barley
endosperm-specific D-hordein promoter and nos 3' terminator; M.-J.
Cho, unpublished) with the XbaI/SacI fragment containing wtrxh
coding sequence from the pWTRXh 1.
[0235] (3) pdBhssWTRXhN3-8: primers Bhor7
(5'-GTAAAGCTTTAACAACCCACACATTG-3- '; SEQ ID NO:7) and BhorWtrxh1R
(5'-CCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT-3'; SEQ ID NO:8) containing
HindIII and Acyl sites, respectively, were used for amplification
of 0.49-kb B.sub.1-hordein 5' region including the B.sub.1-hordein
signal peptide sequence using the .lambda.2-4/HindIII plasmid
containing genomic clone of B.sub.1-hordein (Brands et al., 1985;
Cho et al., 1997) as a template. The primer BhorWtrxhIR is an
overlapping primer containing the wtrxh coding sequence
(underlined) and a partial signal peptide sequence from the
B.sub.1-hordein promoter without the ATG initiation codon for
wtrxh. pdBhssWTRXhN3-8 was made by replacing the D-hordein promoter
in pDhWTRXN-2 with the 0.49-kb PCR-amplified HindIII/Acyl fragment
containing B.sub.1-hordein promoter with its signal peptide
sequence plus the junction region with the 5' wtrxh. Thus,
construct pdBhWTRXN3-8 contains the barley endosperm-specific
B.sub.1-hordein promoter with its signal peptide sequence, wtrxh
and nos (FIG. 12). The signal peptide sequence containing the ATG
initiation codon was directly combined with the sequence of the
wtrxh gene (Gautier et al., 1998), without having extra amino acid
sequences between the two, in order to make WTRXh, protein provide
a precise cleavage site in the lumen of endoplasmic reticulum (ER).
The PCR-amplified fragment of the chimeric product was confirmed by
DNA sequencing.
[0236] (4) pKBhssWTRXN-2: pBhor-1 was digested with Sphl and SacI
in order to obtain the 0.55-kb 5'-flanking region of B.sub.1-barley
hordein promoter. The 0.55-kb Sphl/SacI fragment was ligated into
pSPORT 1 (GIBCO BRL, Gaithersburg, Md.) to make pSPBhor-4.
pdBhssWTRN3-8 was digested with HindIII/EcoRI and the HindIII/EcoRI
fragment containing the 0.43-kb barley endosperm-specific
B.sub.1-hordein promoter plus its signal peptide sequence, wrxh and
nos was ligated into the HindIII/EcoRI-digested pSPBhor-4 to
generate the pSPBhssWTRXN-4 plasmid. In order to remove ampicillin
resistance gene, the 1.3-kb Sphl/EcoRI fragment of pSPBhssWTRXN-4
was ligated into Sphl/EcoRI-digested pJKKmf(-) containing kanamycin
resistance gene to form pKBhssWTRXN-2. Thus, the
kanamycin.sup.r-backbone construct, pKBhssWTRXN-2, contains the
0.55-kb 5'-flanking region of the B.sub.1-barley hordein promoter
plus its signal peptide sequence, wrxh and nos (FIG. 12).
[0237] (5) pDhAtNTR-4: pDhAtNTR-4 was made by replacing the wtrxh
gene in pDhWTRXN-2 (described above) with the PCR-amplified
XbaI/SacI fragment containing Arabidopsis ntr coding sequence from
pAtNTR (a gift from Dr. S. Y. Lee). Primers, AtNTR1
(5'-ggtctagaATGGAAACTCACAAAACC-3'; SEQ ID NO:18) and AtNTR2R
(5'-gggagctcTCAATCACTCTTACCCTC-3'; SEQ ID NO:20), were used for
amplification of the 1.009-Kb XbaI/SacI fragment containing
0.993-Kb Arabidopsis ntr coding sequence; small letters contain a
restriction enzyme site for subcloning of the DNA construct
containing Arabidopsis ntr gene and underlined letters indicate the
Arabidopsis ntr sequences. The Arabidopsis ntr fragment was
purified from a 0.7% agarose gel using QIAquick.RTM. gel extraction
kit, digested with XbaI and SacI and ligated into
XbaI/SacI-digested pDhWTRXN-2 to generate the pDhAtNTR-4 plasmid.
Nucleotide sequences of the PCR-amplified Arabidopsis ntr coding
region were determined by DNA sequencing.
[0238] Stable Wheat Transformation
[0239] Stable transgenic lines of wheat transformed with
pDhSSsGFPN3-4, pdBhssVVTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR4 were
obtained using highly regenerative, green tissues as transformation
targets. Highly regenerative tissues have a high percentage of
totipotent cells capable of sustained cell division and competent
for regeneration over long period. In order to induce highly
regenerative green tissues, whole immature embryos (IEs; 1.0-2.5
mm) were aseptically removed, placed scutellum side down on DBC3
medium (callus-induction medium containing 1.0 mg/L
2,4-dichlorophenoxyacetic acid, 0.5 mg/L BAP and 5.0 .mu.M
CuSO.sub.4; Cho et al., 1998a-c). Five to 7 days after initiation,
germinating shoots and roots were removed by manual excision. After
3 weeks of incubation at 24.+-.1.degree. C. under dim light
conditions (approximately 10 to 30 .mu.E, 16 h-light), highest
quality tissues from the scutellum was selected and maintained on
DBC3 medium. Alternatively, highly regenerative, green tissues were
obtained from daughter tissues, oval-shaped tissues with highly
embryogenic structures which were emerged at the base of
germinating shoots or from the outside layer of the tissues near
the base of germinating shoots. Seven to 14 days after initiation,
daughter tissues (2-4 mm in length) were isolated from germinating
IEs by manual excision and transferred to fresh DBC3 medium. After
an additional 3- to 4-week incubation, the tissues were selected
again, broken into 2 to 4 pieces of about 3 to 5 mm in size and
transferred onto fresh medium. The tissues were maintained on fresh
medium, subculturing at 3- to 4-week intervals.
[0240] Only good quality tissues were selected for bombardment. The
highly regenerative tissues (preferably about 3 to 4 mm in size)
were transferred for osmotic pretreatment to DBC3 medium containing
equimolar amounts of mannitol and sorbitol to give a final
concentration of 0.4 M. Four hours after treatment with the
osmoticum, the tissues were bombarded as previously described (Wan
and Lemaux 1994; Lemaux et al. 1996). Gold particles (1.0 .mu.m)
were coated with 25 .mu.g of a 1:1 or 1:2 molar ratio of a mixture
of pAct1lHPT-4 (or pUbilNPTII-1) and and one of 4 plasmids,
pDhSSsGFPN3-4, pdBhssWTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR-4,
followed by bombardment using a PDS-1000 He biolistic device
(BioRad, Inc., Hercules, Calif.) at 600 or 900 psi. The plasmid
pAct1 lHPT-4 contains the hygromycin phosphotransferase (hpt)
coding sequence under control of the rice actin1 promoter (Act1),
its intron and the nos 3' terminator (Cho et al., 1998a-c).
pUbilNPTII-1 contains the neomycin phosphotransferase (nptl) gene
under control of the maize ubiquitin promoter and first intron and
terminated by nos. Sixteen to 18 hr after bombardment, the
bombarded tissues were placed to DBC3 medium without osmoticum and
grown at 24.+-.1.degree. C. under dim light.
[0241] Following the initial 10- to 14-day culturing period, each
regenerative tissue was broken into 1 to 3 pieces depending on
tissue size and transferred to DBC3 medium supplemented with 20-25
mg/L hygromycin B (Boehringer Mannheim, Mannheim, Germany) for
selection for hpt or 30 mg/L G418 (Sigma, Saint Louis, Mo.) for
nptII. Three weeks after the first round of selection, the cultures
were transferred to fresh DBC3 medium containing 30 mg/L hygromycin
B or 40 mg/L G418. From the third round selection, the tissues were
subcultured and maintained on DBC3 medium containing 30 mg/L
hygromycin B or 40 mg/L G418 at 3- to 4-week intervals. After the
fourth or fifth round of selection, surviving tissues were
transferred to DBC3 medium without selective agent. Following the
identification of green tissues with sufficient regenerative
structures on DBC3, the tissues were plated on solid regeneration
medium without selective agent and exposed to higher intensity
light (approximately 45-55 .mu.E). After four weeks on regeneration
medium (callus-induction medium without phytohormones), the
regenerated shoots were transferred to Magenta boxes containing the
same medium without selective agent. When the shoots reached the
top of the box plantlets were transferred to the soil.
[0242] Polymerase Chain Reaction (PCR) and DNA Hybridization
[0243] Total genomic DNA from leaf tissues was purified as
described (Dellaporta, 1993). To test tor the presence of wtrxh in
genomic DNA of putatively transformed lines, 500 ng of genomic DNA
was amplified by PCR using either of two primer sets, Wtrxh1
(5'-ATATCTAGAATGGCGGCGTCGGCGGCGA-- 3'; SEQ ID NO:5) and Wtrxh2R
(5'-ATAGAGCTCTTACTGGGCCGCGTGTAG-3'; SEQ ID NO:6) or Wtrxh4
(5'-CCAAGAAGTTCCCAGCTGC-3'; SEQ ID NO:11) and Wtrxh5R
(5'-ATAGCTGCGACAACCCTGTCCTT-3'; SEQ ID NO:19). The presence of hpt
and nptII was tested by using each of the primer sets, HPT6F
(5'-AAGCCTGAACTCACCGCGACG-3'; SEQ ID NO:21) plus HPT5R
(5'-AAGACCAATGCGGAGCATATAC-3'; SEQ ID NO:22) (Cho etal., 1998a-c)
and NPT1F (5'-CAAGATGGATTGCACGCAGGTTCT-3'; SEQ ID NO:15) plus NPT2R
(5'-ATAGAAGGCGATGCGCTGCGAAT-3'; SEQ ID NO:16). Amplifications were
performed with Taq DNA polymerase (Promega, Madison, Wis.) in a
25-.mu.l reaction (Cho et al., 1998a-c). Twenty-five .mu.l of the
PCR product with loading dye was electrophoresed on a 1.0% agarose
gel with ethidium bromide and photographed using exposure to UV
light. Presence of 0.4- and 0.14 kb fragments was consistent with
an intact and truncated wtrxh fragments, repectively; 0.81-kb hpt
and 0.76-kb nptII fragments for the pAct1lHPT-4 and pUbilNPTII-1
plasmids, were produced with hpt and nptII primers, respectively.
Homozygous lines for wtrxh were screened using T.sub.1, T.sub.2 or
T.sub.3 plants by PCR anlaysis.
[0244] GFP Expression Detection by Fluorescence Microscopy
[0245] GPF expression was monitored at higher magnification using a
Nikon Microphot-5A fluorescent microscope equipped with a Nikon
B-2A filter block containing a 450-490 excitation filter and a
BAS20 emission barrier filter (Cho et al., 2000).
[0246] Western Blot Analysis
[0247] Western blot analysis was performed on seeds from selected
transgenic wheat lines as well as from control counterparts grown
under the same conditions. Thioredoxin h purified from seeds of a
bread wheat cultivar, cv. Capitole, was used as a reference. Whole
seeds were ground to a fine powder with a mortar and pestle under
liquid nitrogen. Ten seeds were used for each sample; the volume of
extraction buffer [50 mM Tris HCl or phosphate buffer, pH 7.8, 0.5
mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM EDTA] varied from 2
to 4 ml depending on the number of seeds used and the viscosity of
the extract. Grinding was continued for an additional min after
buffer addition, the preparation was centrifuged at 14,000.times.g
for 10 min and the supernatant solution was saved as the soluble
(albumin-globulin) fraction. SDS-PAGE of the soluble fraction was
performed in 12-17% polyacrylamide gradient gels at pH 8.5
(Laemmli, 1970). Equal amounts of protein (40 .mu.g) of each sample
quantitated according to Bradford (1976) were diluted 1:2 v/v in
Laemmli sample buffer, boiled for 3 minutes, loaded onto gels and
subjected to electrophoresis at a constant current of 15 mA.
Proteins were transferred to nitrocellulose at a constant voltage
of 40 V for 4 hours at 4.degree. C. using a Hoefer Transphor
Transfer Unit (Alameda, Calif.) (all at 25.degree. C.).
Nitrocellulose was blocked with 5% powdered milk in TBS for 2
hours, incubated in primary antibody for 4 hours and in secondary
antibody for 1 hour. The primary antibody was wheat
anti-thioredoxin h II (Johnson et al., 1987b) diluted 1 to 500;
secondary antibody was goat anti-rabbit alkaline phosphatase
(Bio-Rad, Hercules, Calif.) diluted 1:3000. Blots were developed in
NBT/BCIP alkaline phosphatase color reagent (Bio-Rad, Hercules,
Calif.). Images were scanned using a Bio-Rad GelDoc 1000 (Hercules,
Calif.) and analyzed using Bio-Rad Multi Analyst, version
1.0.2.
[0248] B. Results and Discussion
[0249] Construction of Expression Vectors
[0250] To overexpress sGFP(S65T), WTRXh and AtNTR in wheat seed,
five expression constructs containing wtrxh driven by
endosperm-specific hordein promoters, pDhSSsGFPN3-4, pDhWTRXN-2,
pdBhssWTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR-4, were made. Out of
five constructs, four (pDhSSsGFPN3-4, pdBhssWTRXhN3-8,
pKBhssWTRXN-2 or pDhAtNTR-4; FIG. 12) were used for stable
transformation of wheat.
[0251] Production of Transgenic Plants
[0252] Highly regenerative tissues (at least 1 tissue, preferably
50, and most preferably 500 of 3-4 mm in length) were bombarded and
cultured on DBC3 medium for the first 10 to 14 days in the absence
of selection. For the second transfer (1st round selection),
selection was on DBC3 medium supplemented with 25-30 mg/L
hygromycin B for hpt selection or 30 mg/L G418 for nptII selection.
At the second round selection, DBC3 medium with 30 mg/L hygromycin
B or 40 mg/L G418 was used. From the 4th transfer (3rd round
selection) onward, the selection pressure was maintained at the
same level. In general, hygromycin- or G418-resistant tissues with
some green sectors were observed at the third round selection.
Putative transgenic calli with green sectors were maintained and
proliferated on the same medium without selective agent from after
the fourth or fifth round of selection, until the green sectors
formed fully developed regenerative structures. Green regenerative
tissues were regenerated on regeneration medium and the plantlets
transferred to soil approximately 3 to 4 weeks after growth on the
same medium of the Magenta boxes. To date using this transformation
protocol, we obtained two independent Bobwhite lines, four
transgenic Anza lines, two transgenic Yecora Rojo lines transformed
with pdBhssWTRXhN3-8, one Bobwhite line transformed with
pKBhssWTRXN-2 and one Yecora Rojo line transformed with pDhAtNTR-4
(Table 8). We also obtained two independent Bobwhite lines
transformed with pDhSSsGFPN3-4 (data not shown).
[0253] Endosperm-Specific Expression of Barley Hordein Promoter in
Transgenic Wheat
[0254] Expression of GFP driven by barley D-hordein promoter was
found specifically in the endosperm tissue of developing wheat
grains; GFP expression was not observed in immature embryo tissues
(FIG. 13)
[0255] Analysis of T.sub.0 Plants and their Progeny
[0256] PCR analysis was performed using two sets of WTRXh primers
and one set of AtNTR primers. PCR amplification resulted in 0.4-kb
intact wtrxh or 0.14-kb truncated wtrxh (FIG. 14) and 0.5-kb
internal Atntr fragments from transgenic lines. Seeds of T.sub.1
and their progeny from some wtrxh-positive lines were planted in
order to screen homozygous lines. Homozygous lines and null
segregants were obtained from AZHptWTR-1, AZHptWTR-21 and
YRHptWTR-1 (Table 8). Other lines are currently being screened for
homozygous lines.
[0257] Characterization of Wheat Thioredoxin h Produced in
Transgenic Grain
[0258] Of the stably transformed lines that expressed wheat
thioredoxin h, on average, its level was found to be higher in
transformants. Western blot analysis of soluble protein fractions
from heterozygous mixtures of seeds from three of these lines,
AZHptWTR-1, AZHptWTR-21 and YRHptWTR-1, showed approximately 5
times, 20 times, and 30 times more thioredoxin h, respectively,
than nontransformed control grain (FIG. 15A). The thioredoxin
content of the null segregant (YRHptWTR-1-2-1 to -3) was similar to
that of the corresponding, nontransformed control (FIG. 15A and
B).
10TABLE 8 Summary of Transformation Experiments for Three Wheat
Cultivars: Bobwhite, Anza and Yecora Rojo DNA PCR WTRXh or NTR
Cultivars/Plasmids for Transgenic (T.sub.0 leaf) expression in
T.sub.1 bombardment wheat lines hpt wtrx ntr seeds Comments
BW/pAct1IHPT-4 + BWHptWTR-1 + + n.d. pdBhssWTRXhN3-8 BWHptWTR-3 + -
n.d. BWHptWTR-4 + + n.d. BWHptWTR-5 + - n.d. AZ/pACT1IHPT-4 +
AZHptWTR-1 + + + homozygous pdBhssWTRXhN3-8 AZHptWTR-11 + + +
AZHptWTR-13 + + n.d. AZHptWTR-21 + + + homozygous YR/pACT1IHPT-4 +
YRHptWTR-1 + + + homozygous pdBhssWTRXhN3-8 YRHptWTR-2 + - n.d.
YRHptWTR-8 + + n.d. BW/pUbiINPTII-1 + BWNptBhWTR- + + n.d.
pKBhssWTRN-2 10 YR/pAct1IHpt-4 + YRHptAtNTR-1 + + n.d. pDHAtNTR-4
BW, AZ and YR represent Bobwhite, Anza, Yocora Rojo, respectively
n.d.: not determined
Example 3
Effect of Thioredoxin Reduction on Digestion of Wheat Glutenins by
Trypsin and Pancreatin
[0259] Sequential Extraction of Grain Proteins from Transgenic
Wheat Grains
[0260] Transgenic grain (YRHptWTR-1-1) and null segregant
(YRHptWTR-1-2) grain were ground with a coffee grinder at room
temperature. Ground powder from 10 grams of each line was placed in
a 250-ml screw-top centrifuge bottle and 60 ml of each extraction
solution indicated below was added. The mixture was shaken
mechanically and then centrifuged for 30 min at 5,000.times.g. The
supernatant fraction was decanted and saved for analysis, and the
residue was mixed with the next solution. The powdered grain was
extracted sequentially with the following solvents for the
indicated times: [1] 2.times.0.5 M NaC1 (30 min); [2] 2.times.70%
ethanol (2 hr); [3] 2.times.0.1 M acetic acid (2 hr). Supernatant
fractions of all extracts were analyzed for protein by the
Coomassie dye binding method (Bradford, 1976) and then were stored
at -20.degree. C. until use. By convention, the fractions are
designated: [1] albumin/globulin (water/salt-water); [2] gliadin
(ethanol); and [3] glutenin (acetic acid) (Kruger et al., 1988;
Shewry et al., 1986). These fractions were used for digestion and
skin tests in Example 5, below.
[0261] Digestion of Glutenins
[0262] For reduction of glutenins extracted as above from
non-transgenic green house plants, 4.2 .mu.g NTR, 2.4 .mu.g
thioredoxin (both from E. coli), and 1 mM NADPH were added to 240
.mu.g of target protein and incubated in a 37.degree. C. water bath
for 45 minutes. NTS (NTR/thioredoxin/NADPH) treated and untreated
glutenins were incubated in 100 .mu.l of simulated intestinal fluid
(SIF) (Board of Trustees (ed.), 1995, Simulated Gastric Fluid, TS.,
pp 2053, The United States Pharmacopeia, 23, The National Formulary
18, United States Pharmacopeial Convention, Inc., Rockville, Md.)
as described below. SIF contained 5 .mu.g trypsin (or 20 .mu.g
pancreatin), 48.9 mM monobasic potassium phosphate, and 38 mM
sodium hydroxide. After addition of the enzyme, the pH was brought
to 7.5 with 0.2 M sodium hydroxide. Digests were incubated in a
37.degree. C. water bath for 0, 20, 60, or 80 minutes. To stop the
reaction, 100 mM PMSF and leupeptin (1 .mu.g/ml) was added for
trypsin digests and 1 N HCl for pancreatin digests. SDS-PAGE
analysis of the digested samples was performed in 8-16% gradient
gels as described by Laemmli (1970). Gels of 1.5 mm thickness were
developed for 16 hr at a constant current of 7 mA. SDS gels were
stained with Coomassie brilliant blue R-250 in 10% acetic acid for
30 min, and destained in 10% acetic acid for 30 min with the aid of
a microwave oven. Protein stained gels were captured by Gel Doc
1000. The quantification of protein (optical
density.times.mm.times.mm) on the gels was carried out with a
software program for image analysis-Multi-Analyst, version 1.0
(Bio-Rad). Relative digestion was expressed as the percentage of
zero time undigested protein.
[0263] The results shown in FIGS. 16 and 17 demonstrate that
thioredoxin reduction results in enhanced susceptibility of
glutenins to protease digestion by trypsin and pancreatin,
respectively. The most pronounced effects were observed with
trypsin where about 55% of protein remained at 60 minutes
post-digestion in the NTS treated sample in comparison to about
90-95% of the starting protein remained in the non-NTS treated
sample. In the trypsin digestions, proteolysis progressed for 60
minutes and apparently plateaued. In the pancreatin digests,
proteolysis progressed less rapidly. At 80 minutes post-pancreatin
treatment, about 60% of the starting proteins remained in the NTS
treated sample in comparison to 95% protein remaining in the
non-NTS sample. Thus the transgenic grains of the present invention
are more susceptible to digestion and are hyperdigestible. The
increase in the digestibility is at least 5% in the transgenic
plants in comparison to the non-transgenic grains.
Example 4
Effect of NTR on the Reduction of Proteins in Extracts of Wheat
Grains Overexpressing Thioredoxin h
[0264] In Vitro Reduction of Proteins by NADPH or NTR or NADPH
& NTR
[0265] Aliquots of the albumin/globulin fraction from the
homozygous lines overexpressing thioredoxin h as described in
Example 2 and null segregant lines were used. The reaction was
carried out in 30 mM Tris-HCl buffer, pH 7.9. As indicated the
treatments were: (i) control, (ii) 1.25 mM NADPH, (iii) 3.0 .mu.g
Arabidopsis NTR, (iv) NADPH & NTR combined, and (v) 5 mM
dithiothreitol (DTT). The above reagents were added to 70
microliters of this buffer containing 60 .mu.g of protein. Total
reduction by dithiothreitol (DTT) was achieved by boiling for 5
min. After incubation for 60 min at 37.degree. C., 100 nmoles of
mBBr were added and the reaction was continued for another 15 min
at room temperature. To stop the reaction and derivatize, excess
mBBr, 10, .mu.l of 100 mM MET was added. The reduced samples, after
adding 25 .mu.l of 4.times. Laemmli sample buffer, were analyzed as
described by mBBr/SDS-PAGE (Kobrehel, K. et al. 1992).
[0266] The results shown in FIG. 18 indicate that the
albumin/globulin proteins in the homozygous transgenics
overexpressing thioredoxin h are more efficiently reduced than the
albumin/globulin fraction of grain from their null segregant
counterparts.
Example 5
Effect of Overexpressed Thioredoxin h on Allergenicity of Proteins
From Wheat Grain
[0267] The following protocol was approved by the appropriate
committees at both the University of California-Davis (Animal Use
and Care Administrative Advisory Committee, effective Jan. 21,
1999-Jan. 21, 2000) and the University of California-Berkeley
(Animal Care and Use Committee, effective May 1, 1999-Apr. 30,
2000).
[0268] Dogs from the UC-Davis sensitized Dog Colony (Ermel et al.
1997) that were sensitized to commercial whole wheat grain extract
(Bayer), were selected as strong reactors from two groups: 1) 2
year-old, designated "young dogs," and 2) 7 year-old, "old dogs."
Before starting the skin tests, each animal received an intravenous
injection of 5 ml sterile saline solution containing 0.5% Evans
Blue (0.2 ml/kg). After 5 min, skin tests were performed by 100
.mu.l intradermal injections of log dilutions of each wheat protein
fraction in PBS buffer on the ventral abdominal skin. The quantity
of protein injected ranged from 33 pg to 10 .mu.g. The fractions
tested were: 1) salt water-soluble (albumins and globulins); 2)
ethanol-soluble (gliadins); acid acetic-soluble (glutenins). After
20 min, length and width of wheal areas were measured by a blinded
reader. The total area was calculated as an ellipse
(.pi./4.times.L.times.W). Protein allergenicity of the null
segregant (control) and the homozygous wheat lines was obtained by
comparison of the total wheal area generated by the different
dilutions of each extract.
[0269] The responses of the animals are shown in FIG. 19 and
indicate that the proteins obtained from the transgenic wheat are
less allergenic that the protein obtained from the null segregant.
For each fraction tested, both young and old animals were less
responsive to proteins from transgenic wheat. The allergenicity
with the transgenics were decreased at least 5% in comparison to
nontransgenic controls. The allergencity in the young dogs was more
substantially reduced, ranging from 20 to 32% decrease. In
contrast, the allergenicity in older animals was reduced by 8 to
23%.
[0270] To demonstrate the hypoallergenicity of malt produced from
the transgenic wheat grain, malt is produced according to standard
protocols known in the art from the transgenic seeds. Extracts of
the malt are produced according to the above procedure. Young and
old sensitized dogs, as described above, are injected intravenously
with about 5 ml sterile saline solution containing 0.5% Evans Blue
(0.2 ml/kg). After about 5 min, skin tests are performed by 100
.mu.l intradermal injections of log dilutions of each malt protein
fraction in PBS buffer on the ventral abdominal skin. The quantity
of protein injected is about 33 .mu.g to 10 .mu.g. The fractions
are as described above. After about 20 min, the length and width of
the wheal areas are measured by a blinded reader and the total area
is calculated as an ellipse. Malt protein allergenicity of malt
produced from a null segregant (control) and malt from homozygous
wheat lines are obtained by comparison of the total wheal area as
described above. The allergenicity in the young dogs is more
substantially reduced, and range from about 20-30% decrease. The
older animals allergenicity is reduced by about 5-20%.
[0271] Accordingly, a food product such as beer produced from the
hypoallergenic malt also is hypoallergenic.
Example 6
Transgenic Sorghum Expressing Barley Thioredoxin h
[0272] A. Seed Digestibility
[0273] Seeds from ten major cultivars of Sorghum vulgare are
screened for a thioredoxin-dependent increase in digestibility of
constituent proteins using simulated gastric (pepsin), and
intestinal (pancreatin) fluids. The cultivars are representative of
those grown in the United States, Australia and different parts of
Africa.
[0274] Albumin, globulin, kafirin. and glutelin protein fractions
are isolated according to their differential solubilities. Seed, 3
g, is ground in a coffee grinder, extracted sequentially with 30 ml
of: [1] 0.5 M NaCl, [2] 60% (v/v) 2-propanol, and [3] 0.1 M sodium
borate buffer, pH 10, on a shaker at 25.degree. C. for 30 min, 4
hours, and 4 hours, respectively. The extracted fractions
correspond, respectively, to [1] albumin plus globulin [2] kafirin,
and [3] glutelin. Total kafirins or cross-linked kafirins are
extracted with 60% 2 propanol plus 1% 2-mercaptoethanol (Shull et
al., 1992). Each suspension is clarified by centrifugation at
10,000.times.g for 20 min at 4.degree. C.; three successive
extractions are performed with the salt solution followed by two
water washes. The remaining extractions are repeated twice.
Resulting supernatant solutions are pooled and the digestibility of
each fraction is tested on the same day as isolation.
[0275] Aliquots of individual sorghum protein fractions are reduced
either with the NADP/thioredoxin or the NADP/glutathione system
prior to digestion and the results compared with untreated control
preparations. Alternatively, total protein extracted with sodium
myristate, a nonreducing detergent that solubilize wheat gliadins
and glutenins in a biochemically active form (Kobrehel and Buchuk,
1978) can be tested for digestibility. Reduction of the disulfide
bonds of proteins is performed using mBBr/SDS-PAGE as previously
described (del Val et al., 1999) in a volume of 100 .mu.l with
either: (i) the NADP/thioredoxin system, consisting of 5 .mu.l of
25 mM NADPH, 8 .mu.l of 0.3 mg/ml E. coli thioredoxin and 7.mu.l of
0.3 mg/ml E. coli NTR; or (ii) the NADP/glutathione system composed
5 .mu.l of 25 mM NADPH, 10 .mu.l of 30 mM glutathione and 15 .mu.l
of 0.1 mg/ml glutathione reductase. Reactions are carried out in a
30 mM physiological buffered saline (PBS) solution containing 50
.mu.g of each protein. The reaction mixtures are incubated at
4.degree. C. overnight or at 37.degree. C. and 55.degree. C. for 15
min (Kobrehel et al., 1992; del Val et al., 1999). The temperature
found to work best is used for subsequent experiments. For complete
reduction, samples are incubated in PBS with 5 .mu.l 100 mM DTT and
boiled 5 min. Protein fractions (albumin-globulin, kafirin,
glutelin: 240 .mu.g protein) is subjected to simulated digestion,
either untreated or reduced with NADP/thioredoxin or
NADP/glutathione, by pepsin (gastric simulation) or
trypsin/chymotrypsin/carboxypeptidase (pancreatin: intestinal
simulation).
[0276] Pepsin Assay
[0277] Each fraction, 500 .mu.g of protein, is added to 100 .mu.l
of simulated gastric fluid [0.32% pepsin (w/v) and 30 mM NaCl
adjusted to pH 1.2 with HCl] (Astwood et al., 1996). The reaction
mixture is incubated for up to 60 min at 37.degree. C. and stopped
with 0.375-fold volume of 160 mM Na.sub.2CO.sub.3 to give neutral
pH. The protein mixture is subjected to SDS-PAGE and stained for
protein with Coomassie blue as described below.
[0278] Pancreatin Assay
[0279] Each fraction, 500 .mu.g protein, is added to 100 .mu.l of
simulated intestinal fluid (1% porcine pancreatin (w/v), 48.9 mM
monobasic potassium phosphate and 38 mM NaOH adjusted to pH 7.5
with NaOH) (see United States Pharmacopeai, 1995). The reaction
mixture is incubated for up to 60 min at 37.degree. C. and stopped
with {fraction (1/10)} volume of 100 mM phenylmethyl sulfonyl
fluoride (PMSF) plus 1 .mu.g/ml leupeptin. The protein mixture is
subjected to SDS-PAGE and stained with Coomassie blue as described
below.
[0280] Two widely grown cultivar showing the most improved
susceptibility to proteolytic and starch digestion after reduction
by the thioredoxin system are used for the transformation work.
[0281] B. Isolation and Digestibility of Starch
[0282] Starch Granule Isolation
[0283] Starch granules from dry mature sorghum grain are extracted
as described (Sun and Henson 1990). Sorghum grain is washed with
distilled water and steeped for 48 h in 20 mM Na-acetate buffer, pH
6.5, containing 0.02% NaAzide. Softened kernels are ground first
with a motar and pestle and then with a VirTis homogenizer for 6
min at 80% full speed and the grist passed through two sieves (250
and 75 .mu.m). Crude starch that passes through both sieves is
purified by centrifugation (60.times.g for 2.5 min) through a layer
of 65% (w/v) sucrose. Pelleted starch granules are recentrifuged
one or two times under the same conditions and resuspended in 20 mM
sodium acetate buffer, pH 6.5 containing 0.02% sodium azide.
[0284] Starch Digestion
[0285] Starch digestibility is measured based on enzymatic
hydrolysis using porcine pancreatic alpha-amylase (Type VI-B, Sigma
Chemical Co., St. Louis, Mo.). Incubation mixtures containing 2%
(w/v) starch, 0.5% (w/v) BSA, 0.02% (w/v) azide, 25 mM NaCl, 5 mM
CaCl.sub.2, and 10 units of alpha-amylase in 10 mM sodium phosphate
buffer, pH 6.9, are incubated 37.degree. C. Aliquots (50 to 100
.mu.l) of reaction mixture is periodically removed for
determination of glucose and total reducing sugars released from
starch granules. Reducing sugar concentration is measured by the
dinitrosalicylic acid method (Bernfeld, 1955) and total starch
content by the enzymatic procedure of McClear et al., (1994).
[0286] Reduction of Protein on Starch Granules
[0287] Aliquots of the isolated 2% (w/v) starch are incubated with
the NTS system to reduce the proteins on the surface of the granule
as described above (Examples 3 and 4). Following reduction, the
starch granules are tested for digestibility by alpha-amylase
(McCleary et al., 1994) and stimulated intestinal fluid (Board of
Trustees 1995)
[0288] C. Production of Stably Transformed Sorghum Lines and
T.sub.1 Plants Containing Barley trxh
[0289] Using a cDNA library from scutellum tissues of barley
(constructed by R. Schuurink, UCB), a full-length gene for
thioredoxin h (trxh; FIG. 20) was isolated and characterized
(Calliau, del Val, Cho, Lemeaux, Buchanan, unpublished). The
full-length cDNA clone has been placed into expression vectors with
the hordein promoters plus the targeting sequence as described (Cho
et al., unpublished) is used for sorghum transformation. This
vector, pdBhssBTRXN-2, contains the 0.43-kb B.sub.1-hordein
promoter plus its signal sequence, barley trxh (btrxh) and nos.
[0290] Sorghum is transformed by the methods of Cho et al,(1998b,
1999b, 1999c, 1999d, 2000) to give rise to highly regenerative
green tissues. These tissues contain multiple, light-green, shoot
meristem-like structures, which were characterized as such in
barley because they expressed a gene associated with maintenance of
the shoot meristematic state, a knofted I homologue (Zhang et al.,
1998). Target tissues such as these highly regenerative tissues,
which a high percentage of totipotent cells capable of sustained
cell division and competent for regeneration over long period,
represent a high-quality target tissue for transformation. They can
be maintained for more than a year with minimal loss in
regenerability (Cho et al., 1998b, 1999b, 1999c, 1999d, 2000; Kim
et al., 1999; Ha et al., 2000). In addition, the result from
genomic DNA methylation analyses (Zhang et al. 1999b) showed that
barley plants regenerated from these highly regenerative tissues
were less variable in terms of methylation pattern polymorphism and
agronomic performance than those regenerated from callus maintained
in the embryogenic state.
[0291] Media developed for the other cereals and grasses are
utilized for optimizing the response of the sorghum variety, TX430,
to produce high quality, green regenerative tissues with sorghum
similar to those observed with other cereals and grasses. Such
tissues have been used successfully for stable transformation with
all varieties tested. Briefly, this method, the development of
green, regenerative tissues, involves the initiation of embryogenic
cultures from immature embryos of cultivar TX430. The medium giving
the highest quality tissue is D'BC2 and DBC3 (Cho et al., 1998a-c,
1999d). Such media, containing copper, maltose, and cytokinins have
been found to improve the quality and long-term regenerability of
tissue from other cereal and grasses. Tissue developed on this
medium is used as transformation targets using bombardment.
[0292] The desired DNA construct(s) containing barley trxh are
introduced into target cells via bombardment. Selection to identify
transformants is via bialaphos, kanamycin, or other appropriate
selection agents according to published procedures (Cho et al.,
1998a-c; Lemaux et al. 1999). Small portion of putatively
transformed calli are analyzed by PCR (Cho et al., 1998a-c) for
barley trxh and transformed tissue is manipulated to regenerate
plants (Cho et al, 1998a-c). Leaf tissue is tested for resistance
to the selective agent, if possible, and as appropriate is analyzed
by PCR for the transgene(s). Plants are grown to maturity to obtain
T.sub.1 seeds and homozygous T.sub.2 plants.
[0293] D. Determination of Amounts and Activity of TRXh in Stably
Transformed Sorghum
[0294] The activity of the barley thioredoxin h from the different
production systems (targeted vs. nontargeted, i.e, with or without
the signal sequence, respectively) and obtained with different
fractionation procedures, as described above, is assayed using the
DTNB [2',5'-dithiobis (2-nitrobenzoic acid)] method (Florencio et
al., 1988) as described (Cho et al., 1999e). The NTR and
thioredoxin controls are prepared from wheat grains as described by
Johnson et al., (1987a, b).
[0295] Western Blot Analysis
[0296] Western blots are performed on extracts from selected
transgenic lines as well as control seeds. Lots of 10 to 20 intact
seeds are processed and analyzed for content of TRXh and NTR by
SDS-PAGE and western blot procedures (Cho et al., 1999e).
[0297] Preparation of Seed Extract, Heat Treatment and Column
Chromatography
[0298] Extracts are prepared, heat treated, and fractionated by
column chromatography as described by Cho et al., (1999e).
[0299] Measurement of Thioredoxin h Activity
[0300] Thioredoxin h is assayed by the chloroplast NADP-malate
dehydrogenase procedure as adapted for barley (Cho et al.,
1999).
[0301] Protein Determination
[0302] Protein is determined or measured according to Bradford
(1976) using the Coomassie blue method with gamma-globulin as a
standard. Protein content is confirmed by measuring total nitrogen
in an automated gas analyzer or by standard micro-kjeldahl
procedure.
[0303] E. Measurements in Changes in Abundance and Redox State of
Endosperm Proteins
[0304] Transgenic sorghum seeds overexpressing barley thioredoxin h
are the staring material used to demonstrate that increased levels
of this protein cause altered digestibility. Preliminary mBBr
measurements are also made with the genetically engineered grain.
Changes in the redox state of endosperm protein are determined
using the mBBr/SDS-PAGE procedure (Krobehel et al., 1992). As the
major indigenous storage proteins in sorghum are known to be
insoluble, propanol as well as the different aqueous endosperm
extracts are monitored in the grain. Residues are extracted
sequentially, as described above (A. Seed Digestibility) for the
various protein fractions. Supernatant fractions of each extract is
analyzed for protein and fluorescence by the mBBr/SDS-PAGE
technique.
[0305] Dry grain, 1 g, from transgenic and null segregant lines are
ground with a mortar and pestle in liquid nitrogen. When the liquid
nitrogen evaporates, 3-6 ml of 30 mM Tris-HCl, pH 7.9 buffer
containing 1 mM EDTA and 1 mM mBBr is added and mixed for 1 min.
After thawing the extract is incubated 15 min, centrifuged (10 min
at 12,000.times.g), extracted sequentially with salt, propanol, and
borate solutions as described above (A. Seed Digestibility). Sixty
.mu.g protein samples are loaded onto a 10-20% SDS-polyacrylamide
gradient gel as described above. Following electrophoresis (1 h,
constant current of 30 mA), gels are soaked for 2 h in 12% (w/v)
trichloroacetic acid and transferred to a solution containing 40%
methanol and 10% acetic acid for 12 h to remove excess mBBr. Gels
are scanned for fluorescence with a UV light source (365 nm) and
stained for protein with Coomassie blue.
[0306] F. Measurements of Change in Digestibility and Solubility of
Endosperm Proteins in T.sub.1 Heterozygous and T.sub.2 Homozygous
Sorghum Grain
[0307] In parallel with the in vitro experiments (Ori et al.,
1995), the extent that in vivo thioredoxin-mediated reduction
contributes to the digestibility and solubility of sorghum
endosperm proteins is determined. The extent of solubilization of
protein is measured using the ratio of the soluble to the insoluble
protein in the transgenic, relative to a null segregant. Extracts
are prepared in parallel without mBBr labeling and tested for
susceptibility to digestion by simulated gastric and intestinal
fluids are described above (Example 3). The proteins from the
different transgenic grain also are reduced with thioredoxin and
glutathione as described above (A. Seed Digestibility).
[0308] G. Measurements of Change in Digestibility of Starch in
T.sub.1 Heterozygous and T.sub.2 Homozygous Sorghum Grain
[0309] As in the case of the kafirin storage proteins, the ability
of the overexpressed thioredoxin h to enhance the digestibility of
starch with alpha-amylase is determined. The starch is isolated
from both transgenic and null segregant lines and its digestibility
tested in vitro with alpha-amylase as described above (B. Isolation
and Digestibility of Starch). Because of their association with
starch granules, an increase in the digestibility of the kafirin
proteins is accompanied by an increase in the digestibility of the
starch.
[0310] H. Thioredoxin h Overexpressed in Sorghum to Improve
Digestibility of Grain Protein
[0311] The above-noted digestibility of the different protein
fractions (albumin/globulin, kafirin, glutelin) is tested with
simulated gastric and intestinal fluids. The results from the
transgenic grain overexpressing barley TRXh is compared to those
with the null segregant to demonstrate improvement in digestibility
in the transgenic grain.
Example 7
Improvement of Dough Quality
[0312] In U.S. application Ser. No. 08/211,673 (expressly
incorporated by reference), dough quality was improved by reducing
the flour proteins using the NADP/thioredoxin system. Without being
bound by theory, reduced thioredoxin specifically breaks
intramolecular sulfur-sulfur bonds that cross-link different parts
of a protein and stabilize its shape. When these cross-links are
broken the protein can unfold and supposedly link with other
proteins in dough, creating an interlocking lattice that forms an
elastic network. The dough rises because the network helps trap
carbon dioxide produced by yeast during the fermentation process.
It was proposed that the reduced thioredoxin reduced the gliadins
and glutenins in flour letting them recombine in a way that
strengthened the dough. Reduced thioredoxin facilitated their
forming a protein network during dough making. Treatment of
intermediate or poor quality wheat flour (Apollo cultivar) with E.
coli thioredoxin, NADP-thioredoxin reductase, and NADPH showed
dough strengthening (higher farinograph measurements) and improved
loaf volume and viscoelasticity in comparison with untreated flour.
Higher farinograph measurements of dough correspond to improved
dough strength and improved baked good characteristics such as
better crumb quality, improved texture and higher loaf volume.
[0313] Wheat Bread Baking Studies and Farinograph Measurements
[0314] The baking tests are carried out by using a computer
operated PANASONIC bread maker to demonstrate improved quality of
dough made using flour prepared from the transgenic seeds of the
present invention.
[0315] Composition of bread:
[0316] Control:
11 Flour*: 200 gm (dry) Water: 70% hydratation Salt (NaCl): 5.3 g
Yeast: 4.8 g (S. cerevisiae) (dry yeast powder)
[0317] *Flour samples are obtained from transgenic and
non-transgenic wheat (cv. Thesee, Apollo, Arbon, and other animal
feed grade and other grades having from poor to good baking
quality), sorghum, corn, and rice.
[0318] Experimental Conditions
[0319] Flour and salt are weighed and mixed
[0320] The volume of water needed to reach a hydration of 70% was
put into the bread maker.
[0321] The mixture of flour and salt is added to the water and the
baking program is started by the computer. The complete program
lasts about 3 hrs 9 min and 7 secs.
[0322] Yeast is added automatically after mixing for 20 min and 3
secs.
[0323] The program operating the Panasonic apparatus is:
12 Mixing Segments Duration Conditions Heating Mixing 00:00:03 T1
off Mixing 00:05:00 T2 off Mixing 00:05:00 T1 off Rest 00:10:00 TO
off Mixing 00:17:00 T2 off Mixing 00:07:00 T1 off Rest 00:30:00 TO
to reach 32.degree. C. Mixing 00:00:04 T1 32.degree. C. Rest
01:15:00 TO 32.degree. C. Baking 00:14:00 TO to reach 180.degree.
C. Baking 00:26:00 TO 180.degree. C. Mixing Conditions: TO = no
mixing (motor at rest) T1 = normal mixing T2 = alternately 3 second
mixing, 3 second rest
[0324] After the dough is formed, farinograph readings are taken as
described in U.S. application Ser. No. 08/211,673. Bread loaf
volume is measured at the end of the baking, when bread loaves
reach room temperature. Farinograph readings of dough produced from
flour made from transgenic wheat seeds of the invention are at
least about 10-20% higher and are maintained about 40% longer than
dough produced from flour made from non-transgenic seeds. Bread
produced from flour made from transgenic seeds of the invention has
at least about 5% and up to about 20% increased volume in
comparison to bread produced from flour made from non-transgenic
seeds. Bread-like products made from transgenic flour of cereals
that normally produce a nonglutenous flour, for example, rice, hold
together and hold gas better than products produced from the flour
of their nontransgenic counterparts. They also show at least a 3%
increase in loaf volume when compared to their nontransgenic
counterparts.
Example 8
Effect of Glucose-6-Phosphate Dehydrogenase on Reduction of
Proteins in Exacts of Homozygous vs. Null Segregant Wheat Grain
Overexpressing Thioredoxin h
[0325] Samples were from the salt-soluble fractions (albumin and
globulin) of the transgenic and null segregant wheat grain
overexpressing wheat thioredoxin h. Reactions were carried out in
30 mM Tris-HCl buffer, pH 7.9, in a final volume of 100 .mu.l. The
complete reaction mixture contained 10 .mu.mol glucose-6-phosphate,
0.25 .mu.mol NADP, 2 units glucose-6-phoshate dehydrogenase (Bakers
Yeast, Type XV, Sigma, St. Louis, Mo.), plus or minus 1.5 .mu.g NTR
(Arabidopsis), and 80 .mu.g protein. Other treatments, where
omission of one or two component(s) of the NADPH generating system,
were as indicated. The negative control was the extracted protein
alone. As a positive control NADPH was used in place of
NADP/glucose-6-phoshate/glucose-6-phosphate dehydrogenase.
[0326] After incubation at 37.degree. C. for 60 min, 100 nmol mBBr
was added tot he reaction mixture, and the reaction was continued
for 15 min. Ten .mu.l of 100 mM 2-mercaptoethanol was added to stop
the reaction and derivatize excess mBBr. An appropriate amount of
4.times. Laemmeli sample buffer was added and the samples were
applied onto 10-20% polyacrylamide gel in the presence of SDS.
Electrophoresis was carried out at room temperature at 7 mA/gel for
16 hours. Flourescence of sulfhydryl containing proteins on gels
was captured by Gel Doc 1000 (Blo-Rad), protein was stained by
0.025% Coomassie Brilliant Blue G-250 in 10% acetic acid.
[0327] For visualizing the effect of glucose-6-phosphate
dehydrogenase (FIG. 23): in the presence of NTR, comparison of
lanes 2 vs. 4 (-NADP) and lanes 5 vs 7 (+NADP) (+NTR gel on the
left); in the absence of NTR, compare lanes 1 vs. 3 (-NADP) and
lanes 2 vs. 4 (+NADP) (-NTR gel on the right). The maximal increase
in reduction effected by glucose-6-phosphate dehydrogenase was
observed in the presence of NTR, without NADP (lane 2 vs. lane 4,
gel on the left). Note also the greater reduction of NTR in lane 4
vs. lane 2.
[0328] With the null segregant (FIG. 24), note the greater
reduction of NTR in the presence of glucose-6-phosphate
dehydrogenase (lane 4 vs. lane 2) but a lower extent of the
reduction of the smaller target proteins (lane 4) compared to the
corresponding treatment (lane 4) with the transgenic extract (FIG.
23).
[0329] This invention has been detailed both by example and by
description. It should be apparent that one having ordinary skill
in the relevant art would be able to surmise equivalents to the
invention as described in the claims which follow but which would
be within the spirit of the foregoing description and examples. It
should be realized that those equivalents and various modifications
as may be apparent to those of skill in the art to which the
invention pertains also fall within the scope of the invention as
defined by the appended claims. All herein cited patents, patent
applications, publications, references, and references cited
therein are hereby expressly incorporated by reference in their
entirety.
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Sequence CWU 1
1
25 1 486 DNA Artificial Sequence barley B1-hordein promoter and
signal sequence 1 aagctttaac aacccacaca ttgattgcaa cttagtccta
cacaagtttt ccattcttgt 60 ttcaggctaa caacctatac aaggttccaa
aatcatgcaa aagtgatgct aggttgataa 120 tgtgtgacat gtaaagtgaa
taaggtgagt catgcatacc aaacctcggg atttctatac 180 tttgtgtatg
atcatatgca caactaaaag gcaactttga ttatcaattg aaaagtaccg 240
cttgtagctt gtgcaaccta acacaatgtc caaaaatcca tttgcaaaag catccaaaca
300 caattgttaa agctgttcaa acaaacaaag aagagatgaa gcctggctac
tataaatagg 360 caggtagtat agagatctac acaagcacaa gcatcaaaac
caagaaacac tagttaacac 420 caatccacta tgaagacctt cctcatcttt
gcactcctcg ccattgcggc aacaagtacg 480 attgca 486 2 19 PRT Artificial
Sequence barley B1-hordein signal protein 2 Met Lys Thr Phe Leu Ile
Phe Ala Leu Leu Ala Ile Ala Ala Thr Ser 1 5 10 15 Thr Ile Ala 3 497
DNA Artificial Sequence barley D-hordein promoter and signal
sequence 3 cttcgagtgc ccgccgattt gccagcaatg gctaacagac acatattctg
ccaaaacccc 60 agaacaataa tcacttctcg tagatgaaga gaacagacca
agatacaaac gtccacgctt 120 cagcaaacag taccccagaa ctaggattaa
gccgattacg cggctttagc agaccgtcca 180 aaaaaactgt tttgcaaagc
tccaattcct ccttgcttat ccaatttctt ttgtgttggc 240 aaactgcact
tgtccaaccg attttgttct tcccgtgttt cttcttaggc taactaacac 300
agccgtgcac atagccatgg tccggaatct tcacctcgtc cctataaaag cccagccaat
360 ctccacaatc tcatcatcac cgagaacacc gagaaccaca aaactagaga
tcaattcatt 420 gacagtccac cgagatggct aagcggctgg tcctctttgt
ggcggtaatc gtcgccctcg 480 tggctctcac caccgct 497 4 20 PRT
Artificial Sequence barley D-hordein signal protein 4 Ala Lys Arg
Leu Val Leu Phe Val Ala Val Ile Val Ala Leu Val Ala 1 5 10 15 Leu
Thr Thr Ala 20 5 28 DNA Artificial Sequence primer 5 atatctagaa
tggcggcgtc ggcggcga 28 6 27 DNA Artificial Sequence primer 6
atagagctct tactgggccg cgtgtag 27 7 26 DNA Artificial Sequence
primer 7 gtaaagcttt aacaacccac acattg 26 8 34 DNA Artificial
Sequence primer 8 ccgacgccgc tgcaatcgta cttgttgccg caat 34 9 33 DNA
Artificial Sequence primer 9 agaaagcttg gtacccttcg agtgcccgcc gat
33 10 48 DNA Artificial Sequence primer 10 gaacagctcc tcgcccttgc
tcacagcggt ggtgagagcc acgagggc 48 11 19 DNA Artificial Sequence
primer 11 ccaagaagtt cccagctgc 19 12 27 DNA Artificial Sequence
primer 12 aactctagac tcggtggact gtcaatg 27 13 25 DNA Artificial
Sequence primer 13 catcgagaca agcacggtca acttc 25 14 24 DNA
Artificial Sequence primer 14 atatccgagc gcctcgtgca tgcg 24 15 24
DNA Artificial Sequence primer 15 caagatggat tgcacgcagg ttct 24 16
23 DNA Artificial Sequence primer 16 atagaaggcg atgcgctgcg aat 23
17 29 DNA Artificial Sequence primer 17 cggaattcga tctagtaaca
tagatgaca 29 18 26 DNA Artificial Sequence primer 18 ggtctagaat
ggaaactcac aaaacc 26 19 23 DNA Artificial Sequence primer 19
atagctgcga caaccctgtc ctt 23 20 26 DNA Artificial Sequence primer
20 gggagctctc aatcactctt accctc 26 21 21 DNA Artificial Sequence
primer 21 aagcctgaac tcaccgcgac g 21 22 22 DNA Artificial Sequence
primer 22 aagaccaatg cggagcatat ac 22 23 36 DNA Artificial Sequence
primer 23 ggcgcatgcg aattcgaatt cgatatcgat cttcga 36 24 369 DNA
Barley misc_feature (1)...(369) thioredoxin h 24 atggcggcgt
cggcaacggc ggcggcagtg gcggcggagg tgatctcggt ccacagcctg 60
gagcagtgga ccatgcagat cgaggaggcc aacaccgcca agaagctggt ggtgattgac
120 ttcactgcat catggtgcgg accatgccgc atcatggctc cagttttcgc
tgatctcgcc 180 aagaagttcc caaatgctgt tttcctcaag gtcgacgtgg
atgaactgaa gcccattgct 240 gagcaattca gtgtcgaggc catgccaacg
ttcctgttca tgaaggaagg agacgtcaag 300 gacagggttg tcggagctat
caaggaggaa ctgaccgcca aggttgggct tcacgcggcg 360 gcccagtaa 369 25
122 PRT Barley SITE (1)...(122) thioredoxin h 25 Met Ala Ala Ser
Ala Thr Ala Ala Ala Val Ala Ala Glu Val Ile Ser 1 5 10 15 Val His
Ser Leu Glu Gln Trp Thr Met Gln Ile Glu Glu Ala Asn Thr 20 25 30
Ala Lys Lys Leu Val Val Ile Asp Phe Thr Ala Ser Trp Cys Gly Pro 35
40 45 Cys Arg Ile Met Ala Pro Val Phe Ala Asp Leu Ala Lys Lys Phe
Pro 50 55 60 Asn Ala Val Phe Leu Lys Val Asp Val Asp Glu Leu Lys
Pro Ile Ala 65 70 75 80 Glu Gln Phe Ser Val Glu Ala Met Pro Thr Phe
Leu Phe Met Lys Glu 85 90 95 Gly Asp Val Lys Asp Arg Val Val Gly
Ala Ile Lys Glu Glu Leu Thr 100 105 110 Ala Lys Val Gly Leu His Ala
Ala Ala Gln 115 120
* * * * *
References