Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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THIOREDOXIN AND GRAIN PROCESSING
This invention relates to novel methods for grain processing to enhance
protein and starch
recovery, particularly in corn wet milling and soybean processing, as well as
novel
transgenic plants useful in such processes.
Thioredoxin (TRX) and thioredoxin reductase (TR) are enzymes that use NADPH to
reduce
disulfide bonds in proteins. Protein disulfide bonds play an important role in
grain
processing efficiencies, and in the quality of the products recovered from
grain processing.
Development of effective ways to eliminate or decrease the extent of protein
disulfide
bonding in grain would increase processing efficiencies. Additionally grain
performance in
livestock feed is also affected by inter- and intramolecular disulfide
bonding: grain
digestibility, nutrient availability and the neutralization of anti-nutritive
factors (e.g., protease,
amylase inhibitors etc.) would be increased by reducing the extent of
disulfide bonding.
Expression of transgenic thioredoxin and/or thiaredoxin reductase in corn and
soybeans
and the use of thioredoxin in grain processing, e.g., wet milling, is novel
and provides an
aJternativE method for reducing the disulfide bonds in seed proteins during
industrial
processing. The invention therefore provides grains with altered storage
protein quality as
well as grains that perform qualitatively differently from normal grain during
industrial
processing or animal digestion (both referred to subsequently as
"processing").
This method of delivery of thioredoxin and/or thioredoxin reductase eliminates
the need to
develop exogenous sources of thioredoxin and/or thioredoxin reductase for
addition during
processing. A second advantage to supplying thioredoxin and/or thioredoxin
reductase via
the grains is that physical disruption of seed integrity is not necessary to
bring the enzyme
in contact with the storage or matrix proteins of the seed prior to processing
or as an extra
processing step.
Three modes of thioredoxin utilization in grain processing are provided:
1 ) Expression and action during seed development to alter the composition and
quality of
harvested grain;
2) Expression (but no activity) during seed development to alter the quality
of the products
upon processing;
3) Production of thioredoxin and/or thioredoxin reductase in grain that is
used to alter the
quality of other grain products by addition during processing.
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The present invention thus provides:
- a method to increase efficiency of separation of starch and protein in a
grain milling
process, comprising steeping the grain at an elevated temperature in the
presence of
supplemental thioredoxin and/or thioredoxin reductase and separating the
starch and
protein components of the grain
- a method as mentioned hereinbefore wherein the grain includes grain from a
transgenic
plant wherein the transgene expresses thioredoxin and/or thioredoxin
reductase, particularly
a thermostable thioredoxin and/or thioredoxin reductase.
- a method as mentioned hereinbefore wherein the plant is selected from
dicotyledonous
or monocotyledonous plants, particularly from cereals and even more
particularly from com
(Zea mays) and soybean.
The invention further provides transgenic plants.
In particular, the invention provides:
- a plant comprising a heterologous DNA sequence coding for a thioredoxin
and/or
thioredoxin reductase stably integrated into its nuclear or plastid DNA
- a plant as mentioned before wherein the thioredoxin and/or thioredoxin
reductase is
thermostable
- a plant as mentioned before wherein the thioredoxin and/or thioredoxin
reductase is
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID
NO: 4, SEO ID NO: 5, SEO ID NO: 6, and SEQ ID NO: 7
- a plant as mentioned herein before wherein the plant is selected from corn
and soybean
The invention also provides:
- a plant expressible expression cassette comprising a coding region for a
thioredoxin
and/or thioredoxin reductase operably linked to promoter and terminator
sequences which
function in a plant
- a plant expressible expression cassette as mentioned before wherein the
thioredoxin
and/or thioredoxin reductase is thermostable
- a plant expressible expression cassette wherein the thioredoxin and/or
thioredoxin
reductase is selected from the group consisting of SEO ID NO: 1, SEQ ID NO: 2,
SEQ ID
NO: 3, SEO ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7
The invention further provides
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- method of producing grain comprising high levels of thioredoxin and/or
thioredoxin
reductase comprising transforming plants with an expression cassette as
mentioned before
-a method of producing grain comprising high levels of thioredoxin and/or
thioredoxin
reductase comprising
pollinating a first plant comprising a heterologous expression cassette
comprising a
transactivator-regulated promoter regulated and operably linked to a DNA
sequence coding
for a thioredoxin and/or thioredoxin reductase, with pollen from a second
plant comprising a
heterologous expression cassette comprising a promoter operably linked to a
DNA
sequence coding for a transactivator capable of regulating said transactivator-
regulated
promoter; and recovering grain from the plant thus pollinated
In addition the invention provides:
- the use of plants or plant material according to the invention as animal
feed.
The invention described herein is applicable to all grain crops, in particular
corn, soybean,
wheat, and barley, most particularly corn and soybean, especially corn.
Expression of
transgenic thioredoxin and/or thioredoxin reductase in grain is a means of
altering the
quality of the material (seeds) going into grain processing, altering the
quality of the
material derived from grain processing, maximizing yields of specific seed
components
during processing (increasing efficiency), changing processing methods, and
creating new
uses for seed-derived fractions or components from milling streams.
Wet-Milling
Wet milling is a process of separating the starch, protein and oil components
of grain, most
often cereals, for example corn. It is distinguished herein from dry milling,
which is simply
pulverizing grain. The first step in wet milling is usually steeping, wherein
the grain is
soaked in water under carefully controlled conditions to soften the kernels
and facilitate
separation of the components. The oil-bearing embryos float to the surface of
the aqueous
solution and are removed, and by a process of watering and dewatering,
milling, screening,
centrifuging and washing, the starch is separated from the protein and
purified. The key
difficulty is to loosen starch granules from the complicated matrix of
proteins and cell wall
material that makes up the endosperm of the grain. One reason for this
difficulty is believed
to be the presence of inter- or intramolecular disulfide bonds, which render
the protein
matrix less soluble and less susceptible to proteolytic enzymes and inhibit
release of the
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starch granules from the protein matrix in the grain. At present, the primary
means for
reducing these bonds is to steep the grain in the presence of sulfur dioxide,
but this is
costly, environmentally unfriendly, and not optimally effective.
Certain mutations exert beneficial effects on the protein matrix of corn
kernel endosperm
(floury and opaque), but impair kernel integrity. Transgenic thioredoxin
expression provides
some of these advantages without creating some of the kernel integrity
problems
associated with these mutations.
Past-harvest or processing-dependent activity of thioredoxin have equally
beneficial effects.
For example, in one embodiment, thioredoxin enzymes are targeted to and
accumulated in
cell compartments. Protein reduction occurs following physical disruption of
the seed. In
another embodiment, quiescent endosperm thioredoxin is activated upon
steeping. In a
preferred embodiment, the invention provides a plant expressing a transgenic
thermostable
thioredoxin and thioredoxin reductase, e.g. a thioredoxin and thioredoxin
reductase derived
from a hyperthermophilic organism, such that the thioredoxin and thioredoxin
reductase are
not significantly active except at high temperatures (e.g. greater than
50°C). In one
embodiment, the thermostable thioredoxin and thioredoxin reductase are
synergistic with
saccharification via expression of other thermostable enzymes in endosperm.
Feed applications
Expression of transgenic thioredoxin and/or thioredoxin reductase in grain is
also useful to
improve grain characteristics associated with digestibility, particularly in
animal feeds.
Susceptibility of feed proteins to proteases is a function of time and of
protein conformation.
Kernel cracking is often used in feed formulation as is steam flaking. Both of
these
processes are designed to aid kernel digestibility. Softer kernels whose
integrity can be
disrupted more easily in animal stomachs are desirable. Conformational
constraints and
crosslinks between proteins are major determinants of protease susceptibility.
Modifying
these bonds by increased thioredoxin expression thereby aids digestion.
Corn dry milling/ masa
Protein content and quality are important determinants in flaking grit
production and in masa
production. Reduction of disulfide bonds afters the nature of corn flour such
that it is
suitable for use as a wheat substitute, especially flours made from high-
protein white corn
varieties.
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Soybean crushing
Over half of the US soybean crop is crushed or milled, and the protein quality
in the
resulting low-fat soy flour or de-fatted soy flour (or grits) is important for
subsequent
processing. Protein yield and quality from soybean processing streams are
economically
important, and are largely dependent upon protein conformation. Increasing
thioredoxin
activity through expression of transgenic thioredoxin and/or thioredoxin
reductase increases
protein solubility, and thus increases yield, in the water-soluble protein
fractions. Recovery
is facilitated by aqueous extraction of de-fatted soybean meal under basic
conditions.
Enhancing thioredoxin activity through expression of transgenic thioredoxin
and/or
thioredoxin reductase also reduces the required pH for efficient extraction
and thereby
reduces calcium or sodium hydroxide inputs, as well as lowering the acid input
for
subsequent acid precipitation, allowing efficient recovery of proteins without
alkali damage,
and reducing water consumption and processing plant waste effluents (that
contain
substantial biological oxygen demand loads).
Protein redox status affects important functional properties supplied by soy
proteins, such
as solubility, water absorption, viscosity, cohesion/adhesion, gelation and
elasticity. Fiber
removal during soy protein concentrate production and soy protein isolate
hydrolysis by
proteases is enhanced by increasing thioredoxin activity as described herein.
Similarly, as
described for corn above, increasing thioredoxin activity through expression
of transgenic
thioredoxin and/or thioredoxin reductase enhances the functionality of enzyme-
active soy
flours and the digestibility of the soybean meal fraction and steam flaking
fraction in animal
feeds.
Modification of protein quality during seed development and during processing
both be
provided, although it is preferred that the transgenic thioredoxin and/or
thioredoxin
reductase be targeted to a cell compartment and be thermostable, as described
above, to
avoid significant adverse effects on storage protein accumulation will be
encountered as a
result of thioredoxin activity during seed development. Alternately, the
thioredoxin may be
added as a processing enzyme, as (in contrast to corn wet milling) breaking
the disulfide
bonds is not necessary until after grain integrity is destroyed (crushing and
oil extraction).
Selection of thioredoxin and thioredoxin reductase for heterologous
expression:
Thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI) genes
are found in
eukaryotes, eubacteria as well as archea, including hyperthermophilic
organisms such as
Methanococcus jannaschii and Archaeoglobus fulgidus. Selection of a particular
gene
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depends in part on the desired application. For the methods of the present
invention,
preferred thioredoxins have the following characteristics:
1. Heat stability
Thioredoxin and related proteins from hyperthermophiles are found to have
increased
stability at high temperatures (>50°C) and relatively low activity at
ambient temperatures.
Expression of TRX and/or TR from hyperthermophiles, for example from archea
such as
Methanococcus jannaschii and Archaeoglobus fulgidus or other hyperthermophiles
is
preferred for expression during seed development, so that the thioredoxin
activity is not
markedly increased until the grain is steeped or processed at elevated
temperature. Most
grain processing methods involve, or are compatible with, a high temperature
step.
Thermostable thioredoxin and thioredoxin reductase is therefore preferred. By
thermostable is meant that the enzyme is preferentially active at high
temperatures, e.g.,
temperatures greater than 40°C, most preferably greater than
50°C, e.g. 45-60°C for wet
milling, or even higher, e.g., 45-95°C.
2. Substrate specificity
It is also possible to reduce undesirable effects on seed development by
selection of a
thioredoxin that acts preferentially on certain proteins such as the
structural protein in the
matrix and has low activity with essential metabolic enzymes. Various TRX's
have been
shown to differ in reactivity with enzymes that are under redox control. Thus
it is possible to
select a TRX that will primarily act on the desired targets, minimizing
undesirable side-
effects of over expression.
Suitable thermostabfe thioredoxins and thioredoxin reductases include the
following:
Sequence of thioredoxin from Methanococcus jannaschii, (SEQ ID N0:1;
gi~1591029)
MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMAVPTIVI
NGDVEFIGAPTKEALVEAIKKRL
Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID N0:2;
gi~2649903)(trx-1)
MPMVRKAAFYAtAVISGVLAAVVGNALYHNFNSDLGAQAKIYFFYSDSCPHCREVKPYVEEF
AKTHNLTWCNVAEMDANCSKtAQEFGIKYVPTLVIMDEEAHVFVGSDEVRTAIEGMK
Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID N0:3; gi~2649838)
(trx-2)
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MVFTSKYCPYCRAFEKVVERLMGELNGTVEFEVVDVDEKRELAEKYEVLMLPTLVLADGDE
VLGGFMGFADYKTAREAILEQISAFLKPDYKN
Sequence of thioredoxin from Archaeoglobus fulgidus (SECT ID N0:4; gi~2649295}
(trx-3)
MDELELIRQKKLKEMMQKMSGEEKARKVLDSPVKLNSSNFDETLKNNENVVVDFWAEWC
MPCKMIAPVIEELAKEYAGKVVFGKLNTDENPTIAARYGISAIPTLIFFKKGKPVDQLVGAMPK
SELKRWVQRNL
Sequence of thioredoxin from Archaeoglobus fulgidus (SEQ ID N0:5; gi~2648389)
(trx-4)
MERLNSERFREVIQSDKLVVVDFYADWCMPCRYISPILEKLSKEYNGEVEFYKLNVDENQD
VAFEYGIASIPTVLFFRNGKWGGFIGAMPESAVRAEIEKALGA
Sequence of thioredoxin reductase (trxB) from Methanococcus jannaschii (SEQ ID
N0:6:
gi~1592167)
MIHDTIIIGAGPGGLTAGIYAMRGKLNALCIEKENAGGRIAEAGIVENYPGFEEIRGYELAEKF
KNHAEKFKLPIIYDEVIKIETKERPFKVITKNSEYLTKTIVIATGTKPKKLGLNEDKFIGRGISYC
TMCDAFFYLNKEVIVIGRDTPAIMSAINLKDIAKKVIVITDKSELKAAESIMLDKLKEANNVEIIY
NAKPLEIVGEERAEGVKISVNGKEEIIKADGIFISLGHVPNTEFLKDSGIELDKKGFIKTDENCR
TNIDGIYAVGDVRGGVMQVAKAVGDGCVAMANIIKYLQKL
Sequence of thioredoxin reductase from Archaeogiobus fulgidus (SEQ ID N0:7;
gi~2649006) (trxB)
MYDVAIIGGGPAGLTAALYSARYGLKTVFFETVDPVSQLSLAAKIENYPGFEGSGMELLEKM
KEQAVKAGAEWKLEKVERVERNGETFTVIAEGGEYEAKAIIVATGGKHKEAGIEGESAFIGR
GVSYCATCDGNFFRGKKVIVYGSGKEAIEDAIYLHDIGCEVTIVSRTPSFRAEKALVEEVEKR
GIPVHYSTTIRKIIGSGKVEKVVAYNREKKEEFEIEADGIFVAIGMRPATDVVAELGVERDSM
GYIKVDKEQRTNVEGVFAAGDCCDNPLKQVVTACGDGAVAAYSAYKYLTS
The genes which encode these proteins for use in the present invention are
preferably
designed by back-translation using plant preferred codons, to enhance G-C
content and
remove detrimental sequences, as more fully described below. The activity of
the proteins
may be enhanced by DNA shuffling or other means, as described below. The
invention
therefore comprises proteins derived from these proteins, especially proteins
which are
substantially similar which retain thioredoxin or thioredoxin reductase
activity.
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For engineering thioredoxin expression in seeds for activity during grain
development,
promoters which direct seed-specific expression of TRX and TR are preferred,
as is
targeting to the storage so that the enzyme will have the desired effects on
storage
proteins, which may be desirable in some applications. In the present
invention, however, it
is more generally desirable to engineer thioredoxin and/or thioredoxin
reductase expression
in seeds for accumulation and inactivity during grain development. Several
strategies are
employed to create seeds that express, transgenic thioredoxin and/or
thioredoxin reductase
without having a significant impact on normal seed development, e.g.:
(i) To compartmentalize active thioredoxin or thioredoxin reductase such that
it does not
significantly interact with the target proteins, for example by targeting to
or expression in
amyloplasts. Plastid targeting sequences are used to direct accumulation in
the amyloplast.
Alternatively, the thioredoxin and/or thioredoxin reductase is targeted to an
extracellular
location in cell walls using secretion signals. Or finally, in the case of
monocots, expression
in cell types such as aleurone during seed development is used to keep the
thioredoxin
and/or thioredoxin reductase away from the storage components of the rest of
the
endosperm.
(ii) To engineer the expression of thioredoxin and/or thioredoxin reductase
from thermophilic
organisms. Enzymes which have little or no activity at ambient temperatures
(as high as
38-39°C in the field) are less likely to cause problems during
development. Preferably,
therefore, the enzymes are active primarily at high temperatures, e.g.,
temperatures greater
than 40°C, most preferably 45-60°C for wet milling, or even
higher, e.g., 45-95°C.
(iii) To place the thioredoxin and/or thioredoxin reductase under control of
an inducible
promoter, for example a chemically-inducible promoter, a wound-inducible
promoter, or a
transactivator-regulated promoter which is activated upon pollination by a
plant expressing
the transactivator.
(iv) To utilize thioredoxin having specific requirements for a particular
thioredoxin reductase,
such that activity of the thioredoxin or thioredoxin reductase is suitably
regulated via
availability of the appropriate thioredoxin reductase or thioredoxin,
respectively. For
example, the thioredoxin and thioredoxin reductase are expressed in different
plants, so
that the active combination is only available in the seed upon pollination by
the plant
expressing the complimentary enzyme. Alternatively, the thioredoxin or
thioredoxin
reductase is sequestered in the cell, for example in a plastid, vacuole, or
apoplast, as
described above, so that it does not become available until the grain is
processed.
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Methods of grain processing
The invention thus provides a novel method of enhancing separation of the
starch from the
protein matrix, using thioredoxin and/or thioredoxin reductase. In a first
embodiment,
thioredoxin activity is found to be useful in a variety of seed processing
applications,
including wet milling, dry milling, oilseed processing, soybean processing,
wheat processing
and flour/dough quality, most especially the wet milling of grains, in
particular corn.
Accordingly, the invention provides a method
~ to improve milling efficiency or increase milling yield,
~ to increase efficiency of separation of starch and protein,
~ to enhance yields of starch and soluble proteins from grain, or
~ to increase protein solubility in water or other solvents
comprising steeping grain in the presence of supplemental thioredoxin and/or
thioredoxin
and separating the starch and protein components of the grain.
Typically, steeping occurs before milling, but may occur afterwards, and there
may be more
than one milling or steeping step in the process method extraction and
increase protein
yield from seeds during the steep or points after steeping. Preferably, the
supplemental
thioredoxin and/or thioredoxin reductase is provided by expression of a
transgene in the
plant from which the grain is harvested.
The invention further provides:
~ the use of thioredoxin or thioredoxin reductase in a method to improve
milling efficiency
or increase milling yield of starches or proteins, for example in any of the
methods
described above
~ steepwater comprising an amount of thioredoxin and/or thioredoxin reductase
effective to
facilitate separation of starch from protein in grain
~ grain which has been exposed to thioredoxin an amount effective to
facilitate separation
of starch from protein; and
~ starch or protein which has been produced by the method described above.
The activity of the thioredoxin in the above method may be enhanced by
supplementing the
steepwater with thioredoxin reductase and/or NADPH. Other components normally
present
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in steepwater for wet milling may also be present, such as bacteria which
produce lactic
acid. Preferably, the steeping is carried out at a temperature of about
52°C for a period of
22-50 hours, so it is desirable that the thioredoxin is stable under these
conditions.
The grain may be a dicotyledonous seed, for example, an oil seed, e.g.,
soybean, sunflower
or canola, preferably soybean; or may be a monocotyledonous seed, for example
a cereal
seed, e.g., corn, wheat, oats, barley, rye or rice, most preferably com.
The thioredoxin may be any protein bearing thiol groups which can be
reversibly oxidized to
form disulfide bonds and reduced by NAPDH in the presence of a thioredoxin
reductase.
Preferably the thioredoxin is derived from a thermophilic organism, as
described above.
Thioredoxin and/or thioredoxin reductase for use in the instant invention is
suitably
produced in an engineered microbe, e.g. a yeast or Aspergillus, or in an
engineered plant
capable of very high expression, e.g. in barley, e.g., under control of a
promoter active
during malting, such as a high pl alpha-amylase promoter or other gibberellin-
dependent
promoters. The thioredoxin (in excreted or extracted form or in combination
with the
producer organism or parts thereof) is then added to the steepwater.
As an alternative or supplement to adding the thioredoxin to the steepwater,
the enzyme
can be expressed directly in the seed that is to be milled. Preferably, the
enzyme is
expressed during grain maturation or during a conditioning process.
Accordingly, in a further embodiment, the invention provides
~ a method of making thioredoxin on an industrial scale in a transgenic
organism, e.g., a
plant, e.g., a cereal, such as barley or corn, or a microorganism, e.g., a
yeast or
Aspergillus, for example a method comprising the steps of cultivating a
transgenic
organism having a chimeric gene which expresses thioredoxin, and optionally
isolating or
extracting the thioredoxin
~ a method of using transgenic plants that produce elevated quantities of
thioredoxin
during seed maturation or germination such that the quality of the proteins in
that seed
are affected by the endogenously synthesized thioredoxin during seed
development, or
during the steeping process, thereby eliminating or reducing the need for
conditioning
with exogenous chemicals or enzymes prior to milling
~ a method of making transgenic plants that produce etevated quantities of
thioredoxin
during seed maturation or germination such that the quality of the proteins in
that seed
are affected by the thioredoxin during seed development or during the steeping
process,
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thereby eliminating or reducing the need for conditioning with exogenous
chemicals or
enzymes prior to milling
~ a method for milling grain that uses transgenic seed containing thioredoxin,
that results in
higher starch and soluble protein yields.
Expression of thioredoxin and thioredoxin reductase in transgenic organisms
The invention further comprises a transgenic organism having in its genome a
chimeric
expression cassette comprising a coding region encoding a thermostable
thioredoxin or
thioredoxin reductase under operative control of a promoter.
Preferably, the transgenic organism is a plant which expresses a thioredoxin
and/or
thioredoxin reductase in a form not naturally occurring in plants of that
species or which
expresses thioredoxin at higher levels than naturally occur in a plant of that
species.
Preferably, the thioredoxin is expressed in the seed during seed development,
and is
therefore preferably under control of a seed specific promoter. Optionally,
expression of the
thioredoxin is placed under control of an inducible or transactivator-
regulated promoter, so
that expression is activated by chemical induction or hybridization with a
transactivator
when desired. The thioredoxin is suitably targeted to the vacuoles of the
plant by fusion
with a vacuole targeting sequence.
In the present invention, thioredoxin coding sequences are fused to promoters
active in
plants and transformed into the nuclear genome or the plastid genome. The
promoter is
preferably a seed specific promoter such as the gamma-zein promoter. The
promoter may
alternatively be a chemically-inducible promoter such as the tobacco PR-1 a
promoter; or
may be a chemically induced transactivator-regulated promoter wherein the
transactivator is
under control of a chemically-induced promoter; however, in certain
situations, constitutive
promoters such as the CaMV 35S or Gelvin promoter may be used. With a
chemically
inducible promoter, expression of the thioredoxin genes transformed into
plants may be
activated at an appropriate time by foliar application of a chemical inducer.
Alternatively, the thioredoxin coding sequence is under control of a
transactivator-regulated
promoter, and expression is achieved by crossing the plant transformed with
this sequence
with a second plant expressing the transactivator. In a preferred form of this
method, the
first plant containing the thioredoxin coding sequence is the seed parent and
is male sterile,
while the second plant expressing the transactivator is the pollinator.
Expression of
thioredoxin in seeds is achieved by interplanting the first and second plants,
e.g., such that
the first plant is pollinated by the second and thioredoxin is expressed in
the seeds of the
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first plant by activation of the transactivator-regulated promoter with the
transactivator
expressed by the transactivator gene from the second parent.
The invention thus provides a plant which expresses a thioredoxin and/or
thioredoxin
reductase, e.g. a thioredoxin and/or thioredoxin reductase not naturally
expressed in plants,
for example a plant comprising a heterologous DNA sequence coding for a
thioredoxin
stably integrated into its nuclear or plastid DNA, preferably under control of
an inducible
promoter, e.g., a chemically-inducible promoter, for example either operably
linked to the
inducible promoter or under control of transactivator-regulated promoter
wherein the
corresponding transactivator is under control of the inducible promoter or is
expressed in a
second plant such that the promoter is activated by hybridization with the
second plant;
wherein the thioredoxin or thioredoxin reductase is preferably thermostable;
such plant also
including seed therefor, which seed is optionally treated (e.g., primed or
coated) and/or
packaged, e.g. placed in a bag with instructions for use, and seed harvested
therefrom,
e.g., for use in a milling process as described above.
The transgenic plant of the invention may optionally further comprise genes
for enhanced
production of thioredoxin reductase and/or NADPH.
The invention further provides:
~ a method for producing thioredoxin comprising cultivating a thioredoxin-
expressing plant
as described above
a method for producing starch and/or protein comprising extracting starch or
protein from
seed harvested from a plant as described above; and
a method for wet milling comprising steeping seed from a thioredoxin-
expressing plant as
described above and extracting starch and/or protein therefrom.
The invention further provides:
~ a plant expressible expression cassette comprising a coding region for a
thioredoxin or
thioredoxin reductase, preferably a thioredoxin derived from a thermophilic
organism,
e.g., from an archea, for example from M. jannaschii or A. fulgidus, e.g., as
described
above, wherein the coding region is preferably optimized to contain plant
preferred
codons, said coding region being operably linked to promoter and terminator
sequences
which function in a plant, wherein the promoter is preferably a seed specific
promoter or
an inducible promoter, e.g., a chemically inducible or transactivator-
regulated promoter;
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for example a plastid or nuclear expressible expression cassette comprising a
promoter,
e.g., a transactivator-regulated promoter regulated by a nuclear
transactivator (e.g., the
T7 promoter when the transactivator is T7 RNA polymerase the expression of
which is
optionally under control of an inducible promoter)
~ a vector comprising such a plant expressible expression cassette
~ a plant transformed with such a vector; or
~ a transgenic plant which comprises in its genome, e.g., its nuclear or
plastid genome,
such a plant expressible expression cassette.
The invention also comprises a method of producing grain comprising high
levels of
thioredoxin or thioredoxin reductase comprising pollinating a first plant
comprising a
heterologous expression cassette comprising a transactivator-regulated
promoter regulated
and operably linked to a DNA sequence coding for a thioredoxin or thioredoxin
reductase,
the first plant preferably being emasculated or male sterile; with pollen from
a second plant
comprising a heterologous expression cassette comprising a promoter operably
linked to a
DNA sequence coding for a transactivator capable of regulating said
transactivator-
regulated promoter; recovering grain from the plant thus pollinated.
DEFINITIONS
In order to ensure a clear and consistent understanding of the specification
and the claims,
the following definitions are provided:
"Expression cassettg" as used herein means a DNA sequence capable of directing
expression of a gene in plant cells, comprising a promoter operably linked to
a coding
region of interest which is operably linked to a termination region. The
coding region
usually codes for a protein of interest but may also code for a functional RNA
of interest, for
example antisense RNA or a nontranslated RNA that, in the sense or antisense
direction,
inhibits expression of a particular gene, e.g., antisense RNA. The gene may be
chimeric,
meaning that at least one component of the gene is heterologous with respect
to at least
one other component of the gene. The gene may also be one which is naturally
occurring
but has been obtained in a recombinant form useful for genetic transformation
of a plant.
Typically, however, the expression cassette is heterologous with respect to
the host, i.e.,
the particular DNA sequence of the expression cassette does not occur
naturally in the host
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cell and must have been introduced into the host cell or an ancestor of the
host cell by a
transformation event.
A "nuclear exapression cassette" is an expression cassette which is integrated
into the
nuclear DNA of the host.
A "plastid expression cassette" is an expression cassette which is integrated
into the plastid
DNA of the host. A plastid expression cassette as described herein may
optionally
comprise a polycistronic operon containing two or more cistronic coding
sequences of
interest under control of a single promoter, e.g., a transactivator-regulated
promoter, e.g.,
wherein one of the coding sequences of interest encodes an antisense mRNA
which
inhibits expression of clpP or other plastid protease, thereby enhancing
accumulation of
protein expressed the other coding sequence or sequences of interest.
"Heterologous" as used herein means "of different natural origin". For
example, if a plant is
transformed with a gene derived from another organism, particularly from
another species,
that gene is heterologous with respect to that plant and also with respect to
descendants of
the plant which carry that gene.
"Homoplastidic" refers to a plant, plant tissue or plant cell wherein all of
the plastids are
genetically identical. This is the normal state in a plant when the plastids
have not been
transformed, mutated, or otherwise genetically altered. In different tissues
or stages of
development, the plastids may take different forms, e.g., chloroplasts,
proplastids,
etioplasts, amyloplasts, chromoplasts, and so forth.
An "inducible promoter" is a promoter which initiates transcription only when
the plant is
exposed to some particular external stimulus, as distinguished from
constitutive promoters
or promoters specific to a specific tissue or organ or stage of development.
Particularly
preferred inducible promoters for the present invention are chemically-
inducible or
transactivator-regulated promoters. Chemically inducible promoters include
plant-derived
promoters, such as the promoters in the systemic acquired resistance pathway,
for example
the PR promoters, e.g., the PR-1, PR-2, PR-3, PR-4, and PR-5 promoters,
especially the
tobacco PR-1 a promoter and the Arabidopsis PR-t promoter, which initiate
transcription
when the plant is exposed to BTH and related chemicals. See US Patent
5,614,395,
incorporated herein by reference, and WO 98/03536, incorporated herein by
reference.
Chemically-inducible promoters also include receptor-mediated systems, e.g.,
those derived
from other organisms, such as steroid-dependent gene expression, copper-
dependent gene
expression, tetracycline-dependent gene expression, and particularly the
expression system
utilizing the USP receptor from Drosophila mediated by juvenile growth hormone
and its
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agonists, described in PCT/EP 96/04224, incorporated herein by reference, as
well as
systems utilizing combinations of receptors, e.g., as described in PCT/EP
96/00686,
incorporated herein by reference. Chemically inducible promoters may be
directly linked to
the thioredoxin gene or the thioredoxin gene may be under control of a
transactivator-
regulated promoter while the gene for the transactivator is under control of a
chemically
inducible promoter. See generally, C. Gatz, "Chemical Control of Gene
Expression", Annu.
Rev. Plant Physiol. Plant Mol. Biol. (1997) ~: 89-1 Og, the contents of which
are
incorporated herein by reference. Transactivator regulated promoters are
described more
fully below, and may also be induced by hybridization of a plant comprising
the thioredoxin
gene under control of a transactivator-regulated promoter with a second plant
expressing
the transactivator.
An "isolated DNA molecule" is a nucleotide sequence that, by the hand of man,
exists apart
from its native environment and is therefore not a product of nature. An
isolated nucleotide
sequence may exist in a purified form or may exist in a non-native environment
such as, for
example, a transgenic host cell.
A ° rotein" as defined herein is the entire protein encoded by the
corresponding nucleotide
sequence, or is a portion of the protein encoded by the corresponding portion
of the
nucleotide sequence.
An "isolated ~ rotein" is a protein that is encoded by an isolated nucleotide
sequence and is
therefore not a product of nature. An isolated protein may exist in a purified
form or may
exist in a non-native environment, such as a transgenic host cell, wherein the
protein would
not normally expressed or would be expressed in a different form or different
amount in an
isogenic non-transgenic host cell.
A "plant" refers to any plant or part of a plant at any stage of development,
and is
specifically intended to encompass plants and plant material which have been
damaged,
crushed or killed, as well as viable plants, cuttings, cell or tissue
cultures, and seeds.
"DNA shuffling" is a method to introduce mutations or rearrangements,
preferably randomly,
in a DNA molecule or to generate exchanges of DNA sequences between two or
more DNA
molecules, preferably randomly. The DNA molecule resulting from DNA shuffling
is a
shuffled DNA molecule that is a non-naturally occurring DNA molecule derived
from at least
one template DNA molecule. The shuffled DNA encodes an enzyme modified with
respect
to the enzyme encoded by the template DNA, and preferably has an altered
biological
activity with respect to the enzyme encoded by the template DNA.
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In its broadest sense, the term "ss.~bstantially similar", when used herein
with respect to a
nucleotide sequence, means a nucleotide sequence corresponding to a reference
nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having
substantially the same structure and function as the polypeptide encoded by
the reference
nucleotide sequence, e.g. where only changes in amino acids not affecting the
polypeptide
function occur. Desirably the substantially similar nucleotide sequence
encodes the
polypeptide encoded by the reference nucleotide sequence. The percentage of
identity
between the substantially similar nucleotide sequence and the reference
nucleotide
sequence desirably is at least 80%, more desirably at least 85%, preferably at
least 90%,
more preferably at least 95%, still more preferably at least 99%. Sequence
comparisons are
carried out using a Smith-Waterman sequence alignment algorithm (see e.g.
Waterman,
M.S. Introduction to Computational Biology: Maps, sequences and genomes.
Chapman &
Hall. London: 1995. ISBN 0-412-99391-0, or at htt :/
hto.usc.edu/software/seaalNindex.html}. The IocaIS program, version 1.16, is
used with
following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2,
extended-gap
penalty: 2.
A nucleotide sequence "substantially similar" to reference nucleotide sequence
hybridizes
to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaP04, 1
mM EDTA at 50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more
desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with
washing in 1X SSC,
0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate
(SDS), 0.5 M NaP04,
1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C,
preferably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with
washing in 0.1X
SSC, 0.1 % SDS at 50°C, more preferably in 7% sodium dodecyl sulfate
(SDS), 0.5 M
NaP04, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at
65°C.
The term "substantially similar", when used herein with respect to a rotein,
means a protein
corresponding to a reference protein, wherein the protein has substantially
the same
structure and function as the reference protein, e.g. where only changes in
amino acids not
affecting the polypeptide function occur. When used for a protein or an amino
acid
sequence the percentage of identity between the substantially similar and the
reference
protein or amino acid sequence desirably is at least 80%, more desirably 85%,
preferably at
least 90%, more preferably at least 95%, still more preferably at least 99%.
A "transactivator" is a protein which, by itself or in combination with one or
more additional
proteins, is capable of causing transcription of a coding region under control
of a
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corresponding transactivator-regulated promoter. Examples of transactivator
systems
include phage T7 gene 10 promoter, the transcriptional activation of which is
dependent
upon a specific RNA polymerase such as the phage T7 RNA polymerase. The
transactivator is typically an RNA polymerase or DNA binding protein capable
of interacting
with a particular promoter to initiate transcription, either by activating the
promoter directly or
by inactivating a repressor gene, e.g., by suppressing expression or
accumulation of a
repressor protein. The DNA binding protein may be a chimeric protein
comprising a binding
region (e.g., the GAL_4 binding region) linked to an appropriate
transcriptional activator
domain. Some transactivator systems may have multiple transactivators, for
example
promoters which require not only a polymerase but also a specific subunit
(sigma factor) for
promoter recognition, DNA binding, or transcriptional activation. The
transactivator is
preferably heterologous with respect to the plant.
Modification of Microbial Genes to Optimize Nuclear Exioression in Plants
If desired, the cloned thioredoxin genes described in this application can be
modified for
expression in transgenic plant hosts. For example, the transgenic expression
in plants of
genes derived from microbial sources may require the modification of those
genes to
achieve and optimize their expression in plants. In particular, bacterial ORFs
that encode
separate enzymes but which are encoded by the same transcript in the native
microbe are
best expressed in plants on separate transcripts. To achieve this, each
microbial ORF is
isolated individually and cloned within a cassette which provides a plant
promoter sequence
at the 5' end of the ORF and a plant transcriptional terminator at the 3' end
of the ORF.
The isolated ORF sequence preferably includes the initiating ATG colon and the
terminating STOP colon but may include additional sequence beyond the
initiating ATG
and the STOP colon. In addition, the ORF may be truncated, but still retain
the required
activity; for particularly long ORFs, truncated versions which retain activity
may be
preferable for expression in transgenic organisms.
By "plant promoter" and "plant transc~tional terminator" it is intended to
mean promoters
and transcriptional terminators which operate within plant cells. This
includes promoters
and transcription terminators which may be derived from non-plant sources such
as viruses
(examples include promoters and terminators derived from the Cauliflower
Mosaic Virus or
from opine synthase genes in Agrobacterium Ti or Ri plasmids).
In some cases, modification to the ORF coding sequences and adjacent sequence
will not
be required, in which case it is sufficient to isolate a fragment containing
the ORF of interest
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and to insert it downstream of a plant promoter. Preferably, however, adjacent
microbial
sequences left attached upstream of the ATG and downstream of the STOP colon
should
be minimized or eliminated. In practice, such construction may depend on the
availability of
restriction sites.
In other cases, the expression of genes derived from microbial sources may
provide
problems in expression. These problems have been well characterized in the art
and are
particularly common with genes derived from certain sources such as Bacillus.
The
modification of such genes can be undertaken using techniques now well known
in the art.
The following problems are typical of those that may be encountered:
1. Colon Usagg
The preferred colon usage in plants differs from the preferred colon usage in
certain
microorganisms. Comparison of the usage of colons within a cloned microbial
ORF to
usage in plant genes (and in particular genes from the target plant) will
enable an
identification of the colons within the ORF which should preferably be
changed. Typically
plant evolution has tended towards a strong preference of the nucleotides C
and G in the
third base position of monocotyledons, whereas dicotyledons often use the
nucleotides A or
T at this position. By modifying a gene to incorporate preferred colon usage
for a particular
target transgenic species, many of the problems described below for GC/AT
content and
illegitimate splicing will be overcome.
2. GC/AT Content
Plant genes typically have a GC content of more than 35%. ORF sequences which
are rich
in A and T nucleotides can cause several problems in plants. Firstly, motifs
of ATTTA are
believed to cause destabilization of messages and are found at the 3' end of
many short-
lived mRNAs. Secondly, the occurrence of polyadenylation signals such as
AATAAA at
inappropriate positions within the message is believed to cause premature
truncation of
transcription. In addition, monocotyledons may recognize AT-rich sequences as
splice sites
(see below).
3. S~le uences Adjacent to the Initiating Methionine
Plants differ from microorganisms in that their messages do not possess a
defined
ribosome binding site. Rather, it is believed that ribosomes attach to the 5'
end of the
message and scan for the first available ATG at which to start translation.
Nevertheless, it
is believed that there is a preference for certain nucleotides adjacent to the
ATG and that
expression of microbial genes can be enhanced by the inclusion of a eukaryotic
consensus
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translation initiator at the ATG. Clontech (1993/1994 catalog, page 210) have
suggested
the sequence GTCGACC~GTC (SEQ ID NO: 8) as a consensus translation initiator
for
the expression of the E. coli uidA gene in plants. Further, Joshi {NAR 15:
6643-6653
(1987)) has compared many plant sequences adjacent to the ATG and suggests the
consensus TAAACAAT GCT (SEQ ID NO: 9). In situations where difficulties are
encountered in the expression of microbial ORFs in plants, inclusion of one of
these
sequences at the initiating ATG may improve translation. In such cases the
last three
nucleotides of the consensus may not be appropriate for inclusion in the
modified sequence
due to their modification of the second AA residue. Preferred sequences
adjacent to the
initiating methionine may differ between different plant species. A survey of
14 maize genes
located in the GenBank database provided the following results:
Position Before the Initiating ATG in 14 Maize Genes:
-10 -9 ~ -7 ~ ~ -4 -3 -2-1
C 3 8 4 6 2 5 6 0 10 7
T 3 0 3 4 3 2 1 1 1 0
A 2 3 1 4 3 2 3 7 2 3
G 6 3 6 0 6 5 4 6 1 5
This analysis can be done for the desired plant species into which the
thioredoxin or
thioredoxin reductase genes are being incorporated, and the sequence adjacent
to the ATG
modified to incorporate the preferred nucleotides.
4. Removal of ill~qitimate Sglice Sites
Genes cloned from non-plant sources and not optimized for expression in plants
may also
contain motifs which may be recognized in plants as 5' or 3' splice sites, and
be cleaved,
thus generating truncated or deleted messages. These sites can be removed
using the
techniques described in patent application WO 97/02352, hereby incorporated by
reference.
Techniques for the modification of coding sequences and adjacent sequences are
well
known in the art. In cases where the initial expression of a microbial ORF is
low and it is
deemed appropriate to make alterations to the sequence as described above,
then the
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construction of synthetic genes can be accomplished according to methods well
known in
the art. These are, for example, described in the published patent disclosures
EP 0 385
962, EP 0 359 472 and WO 93/07278. In most cases it is preferable to assay the
expression of gene constructions using transient assay protocols (which are
well known in
the art) prior to their transfer to transgenic plants.
A major advantage of plastid transformation is that plastids are generally
capable of
expressing bacterial genes without substantial modification. Codon adaptation,
etc. as
described above is not required, and plastids are capable of expressing
multiple open
reading frames under control of a single promoter.
Construction of Plant Transformation Vectors and Selectable Markers
Numerous transformation vectors are available for plant transformation, and
the genes of
this invention can be used in conjunction with any such vectors. The selection
of vector for
use will depend upon the preferred transformation technique and the target
species for
transformation. For certain target species, different antibiotic or herbicide
selection markers
may be preferred. Selection markers used routinely in transformation include
the nptll gene
which confers resistance to kanamycin and related antibiotics (Vieira &
Messing, Gene 19:
259-268 (1982); Bevan et aL, Nature 3Q4:184-187 (1983)), the bar gene which
confers
resistance to the herbicide phoSphlnothricin (White et al., Nucl Acids Res 1~:
1062 (1990),
Spencer et aL Theor Appl Genet 7~: 625-631 (1990)), the hpt gene which confers
resistance
to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol _4: 2929-
2931 ), the dhfr
gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2 7 :
1099-1104
(1983)); and the mannose-6-phosphate isomerase gene which confers the ability
to
metabolize mannose, as described in US 5767378.
(Requirements for Constructign of Plant Expression Cassettes
Gene sequences intended for expression in transgenic plants are firstly
assembled in
expression cassettes behind a suitable promoter and upstream of a suitable
transcription
terminator. These expression cassettes can then be easily transferred to the
plant
transformation vectors described above.
1. Promoter Selection
The selection of promoter used in expression cassettes will determine the
spatial and
temporal expression pattern of the transgene in the transgenic plant. Selected
promoters
will express transgenes in specific cell types (such as leaf epidermal cells,
mesophyll cells,
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root cortex cells) or in specific tissues or organs (roots, seeds, leaves or
flowers, for
example) and this selection will reflect the desired location of biosynthesis
of the
thioredoxin. In the present invention, seed specific promoters are preferred.
Alternatively,
the selected promoter may drive expression of the gene under a light-induced
or other
temporally regulated promoter. A further alternative is that the selected
promoter be
inducible by an external stimulus, e.g., application of a specific chemical
inducer, or by
hybridization with a second plant line, providing the possibility of inducing
thioredoxin
transcription only when desired.
2. Transcrir~tionai Terminators
A variety of transcriptional terminators are available for use in expression
cassettes. These
are responsible for the tem~ination of transcription beyond the transgene and
its correct
polyadenylation. Appropriate transcriptional terminators and those which are
known to
function in plants and include the CaMV 35S terminator, the tml terminator,
the nopaline
synthase terminator, the pea rbcS E9 terminator. These can be used in both
monocotyledons and dicotyledons.
3. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the
genes of this
invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly
in
monocotyledonous cells. For example, the introns of the maize Adh 1 gene have
been
found to significantly enhance the expression of the wild-type gene under its
cognate
promoter when introduced into maize cells. Intron 1 was found to be
particularly effective
and enhanced expression in fusion constructs with the chloramphenicol
acetyltransferase
gene (Callis et al., Genes Develop 1: 1183-1200 (1987)). In the same
experimental system,
the intron from the maize bronzel gene had a similar effect in enhancing
expression. Intron
sequences have been routinely incorporated into plant transformation vectors,
typically
within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also
known to
enhance expression, and these are particularly effective in dicotyledonous
cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "SZ-
sequence"), Maize
Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown
to be
CA 02353794 2001-06-O1
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effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:
8693-8711 (1987);
Skuzeski et al. Plant Molec. Biol. 1_~; 65-79 (1990)).
4. Targeting of the Gene Product Within the Cell
Various mechanisms for targeting gene products are known to exist in plants
and the
sequences controlling the functioning of these mechanisms have been
characterized in
some detail. Aminoterminal sequences are responsible for targeting to the ER,
the
apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant
Cell _2: 769-
783 (1990)). Additionally, aminoterminal sequences in conjunction with
carboxyterminal
sequences are responsible for vacuolar targeting of gene products (Shinshi et
aI. Plant
Molec. Biol. 14: 357-368 (1990)). By the fusion of the appropriate targeting
sequences
described above to transgene sequences of interest it is possible to direct
the transgene
product to any organelle or cell compartment. The signal sequence selected
should include
the known cleavage site and the fusion constructed should take into account
any amino
acids after the cleavage site which are required for cleavage. In some cases
this
requirement may be fulfilled by the addition of a small number of amino acids
between the
cleavage site and the transgene ATG or alternatively replacement of some amino
acids
within the transgene sequence.
The above-described mechanisms for cellular targeting can be utilized not only
in
conjunction with their cognate promoters, but also in conjunction with
heterologous
promoters so as to effect a specific cell targeting goal under the
transcriptional regulation of
a promoter which has an expression pattern different to that of the promoter
from which the
targeting signal derives.
Examples of Expression Cassette Construction
The present invention encompasses the expression of thioredoxin genes under
the
regulation of any promoter that is expressible in plants, regardless of the
origin of the
promoter.
Furthermore, the invention encompasses the use of any plant-expressible
promoter in
conjunction with any further sequences required or selected for the expression
of the
thioredoxin or thioredoxin reductase gene. Such sequences include, but are not
restricted
to, transcriptional terminators, extraneous sequences to enhance expression
(such as
introns [e.g. Adh intron 1 ], viral sequences [e. g. TMV-S2]), and sequences
intended for the
targeting of the gene product to specific organelles and cell compartments.
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Various chemical regulators may be employed to induce expression of the
thioredoxin or
thioredoxin reductase coding sequence in the plants transformed according to
the present
invention. In the context of the instant disclosure, "chemical regulators"
include chemicals
known to be inducers for the PR-1 a promoter in plants (described in US
5,614,395), or
close derivatives thereof. A preferred group of regulators for the chemically
inducible genes
of this invention is based on the benzo-i,2,3-thiadiazole (BTH) structure and
includes, but is
not limited to, the following types of compounds: benzo-1,2,3-
thiadiazolecarboxylic acid,
benzo-1,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole,
benzo-1,2,3-
thiadiazolecarboxylic acid amide, benzo-1,2,3-thiadiazolecarboxylic acid
hydrazide, benzo-
1,2,3-thiadiazole-7-carboxylic acid, benzo-1,2,3-thiadiazole-7-thiocarboxylic
acid, 7-cyano-
benzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylic acid amide,
benzo-1,2,3-
thiadiazole-7-carboxylic acid hydrazide, alkyl benzo-1,2,3-
thiadiazolecarboxylate in which
the alkyl group contains one to six carbon atoms, methyl benzo-1,2,3-
thiadiazole-7-
carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-
1,2,3-thiadiazole-
7-carboxylate, benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide,
and suitable
derivatives thereof. Other chemical inducers may include, for example, benzoic
acid,
salicylic acid (SA), polyacrylic acid and substituted derivatives thereof;
suitable substituents
include lower alkyl, lower alkoxy, lower alkylthio, and halogen. Still another
group of
regulators for the chemically inducible DNA sequences of this invention is
based on the
pyridine carboxylic acid structure, such as the isonicotinic acid structure
and preferably the
haloisonicotinic acid structure. Preferred are dichloroisonicotinic acids and
derivatives
thereof, for example the lower alkyl esters. Suitable regulators of this class
of compounds
are, for example, 2,6-dichloroisonicotinic acid (INA), and the lower alkyl
esters thereof,
especially the methyl ester.
Constitutive Expression: the Actin Promoter
Several isoforms of actin are known to be expressed in most cell types and
consequently
the actin promoter is a good choice for a constitutive promoter. In
particular, the promoter
from the rice Actl gene has been cloned and characterized (McElroy et al.
Plant Cell 2_:
163-171 (1990)). A 1.3 kb fragment of the promoter was found to contain all
the regulatory
elements required for expression in rice protoplasts. Furthermore, numerous
expression
vectors based on the Actl promoter have been constructed specifically for use
in
monocotyledons (McElroy et aI. Mol. Gen. Genet. 2~1: 150-160 (1991 )). These
incorporate
the Actl-intron 1, Adhl 5' flanking sequence and Adhl-intron 1 (from the maize
alcohol
dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing
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highest expression were fusions of 35S and the Acti intron or the Actt 5'
flanking sequence
and the Actl intron. Optimization of sequences around the initiating ATG (of
the GUS
reporter gene) also enhanced expression. The promoter expression cassettes
described by
McElroy et al. (Mol. Gen. Genet. 2~: 150-160 (1991 )) can be easily modified
for the
expression of genes of the invention and are particularly suitable for use in
monocotyledonous hosts. For example, promoter containing fragments can be
removed
from the McEiroy constructions and used to replace the double 35S promoter in
pCGN1761 ENX, which is then available for the insertion or specific gene
sequences. The
fusion genes thus constructed can then be transferred to appropriate
transformation
vectors. In a separate report the rice Actl promoter with its first intron has
also been found
to direct high expression in cultured barley cells (Chibbar et al. Plant Celi
Rep. 12: 506-509
(1993)).
constitutive Expression' the Ubiquitin Promoter
Ubiquitin is another gene product known to accumulate in many cell types and
its promoter
has been cloned from several species for use in transgenic plants (e.g.
sunflower - Binet et
al. Plant Science 79: 87-94 (1991 ), maize - Christensen et al. Plant Mofec.
Biol. 12: 619-632
(1989)). The maize ubiquitin promoter has been developed in transgenic monocot
systems
and its sequence and vectors constructed for monocot transformation are
disclosed in the
patent publication EP 0 342 926. Further, Taylor et al. (Plant Cell Rep. 12:
491-495 (1993))
describe a vector (pAHC25) which comprises the maize ubiquitin promoter and
first intron
and its high activity in cell suspensions of numerous monocotyledons when
introduced via
microprojectile bombardment. The ubiquitin promoter is suitable for the
expression of
thioredoxin genes in transgenic plants, especially monocotyledons. Suitable
vectors are
derivatives of pAHC25 or any of the transformation vectors described in this
application,
modified by the introduction of the appropriate ubiquitin promoter and/or
intron sequences.
Root SRgcific Ex reP ssion
Another desirable pattern of expression for the thioredoxins and thioredoxin
reductases of
the instant invention is root expression, for example to enhance extraction of
starches and
sugars from root crops such as sugarbeets and potatoes. A suitable root
promoter is that
described by de Framond (FEBS 290: 103-106 (1991 )) and also in the published
patent
application EP 0 452 269. This promoter is transferred to a suitable vector
such as
pCGN1761 ENX for the insertion of a thioredoxin or thioredoxin reductase gene
and
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WO 00/36126 PCT/EP99J09986
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subsequent transfer of the entire promoter-gene-terminator cassette to a
transformation
vector of interest.
Wound Inducible Promoters
Wound-inducible promoters may also be suitable for the expression of
thioredoxin genes,
which are activated upon harvest. Numerous such promoters have been described
(e.g. Xu
et al. Plant Molec. -Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1:
151-158 (1989),
Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant
Molec. Biol. 22:
129-142 (1993), Wamer et al. Plant J. ~_3: 191-201 (1993)) and all are
suitable for use with
the instant invention. Logemann ef al, describe the 5' upstream sequences of
the
dicotyledonous potato wun 1 gene. Xu et aL show that a wound inducible
promoter from the
dicotyledon potato (pint) is active in the monocotyledon rice. Further,
Rohrmeier 8~ Lehle
describe the cloning of the maize INip1 cDNA which is wound induced and which
can be
used to isolated the cognate promoter using standard techniques. Similarly,
Firek et aI. and
Warner et al, have described a wound induced gene from the monocotyledon
Asparagus
officinalis which is expressed at local wound and pathogen invasion sites.
Using cloning
techniques well known in the art, these promoters can be transferred to
suitable vectors,
fused to the thioredoxin genes of this invention, and used to express these
genes at the
sites of plant wounding.
Expression with Plastid Targetina
Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) have described the
successful
use of a chloroplast transit peptide for import of a heterologous transgene.
This peptide
used is the transit peptide from the rbcS gene from Nicotiana plumbaginifolia
(Poulsen et al.
Mol. Gen. Genet. 2Q~: 193-200 (1986)). Using the restriction enzymes Dral and
Sphl, or
Tsp5091 and Sphl the DNA sequence encoding this transit peptide can be excised
from
plasmid prbcS-8B and manipulated for use with any of the constructions
described above.
The Dral-Sphl fragment extends from -58 relative to the initiating rbcS ATG
to, and
including, the first amino acid (also a methionine) of the mature peptide
immediately after
the import cleavage site, whereas the Tsp5091-Sphl fragment extends from -8
relative to the
initiating rbcS ATG to, and including, the first amino acid of the mature
peptide. Thus, these
fragments can be appropriately inserted into the polylinker of any chosen
expression
cassette generating a transcriptional fusion to the untranslated leader of the
chosen
promoter (e.g. 35S, PR-1 a, actin, ubiquitin etc.), whilst enabling the
insertion of a
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thioredoxin or thioredoxin reductase gene in correct fusion downstream of the
transit
peptide.
Transformation of Dicotyledons
Transformation techniques for dicotyledons are well known in the art and
include
Agrobacterium-based techniques and techniques which do not require
Agrobacterium.
Non-Agrobacterium techniques involve the uptake of exogenous genetic material
directly by
protoplasts or cells. This can be accomplished by PEG or electroporation
mediated uptake,
particle bombardment-mediated delivery, or microinjection. Examples of these
techniques
are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et
aL, Mol. Gen.
Genet. 1~9: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986),
and Klein et
al., Nature ~: 70-73 (1987). In each case the transformed cells are
regenerated to whole
plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for
transformation of
dicotyledons because of its high efficiency of transformation and its broad
utility with many
different species. The many crop species which are routinely transformable by
Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape,
potato, soybean,
alfalfa and poplar see, e.g. EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO
87/07299
(Brassica), or US 4,795,855 (poplar). Agrobacterium transformation typically
involves the
transfer of the binary vector carrying the foreign DNA of interest (e.g.
pCIB200 or
pCIB2001 ) to an appropriate Agrobacterium strain which rnay depend of the
complement of
virgenes carried by the host Agrobacterium strain either on a co-resident Ti
plasmid or
chromosomally (e.g. strain CIB542 for pCIB200 and pC1B2001 (Uknes et aL Plant
Cell ~:
159-169 (1993)). The transfer of the recombinant binary vector to
Agrobacterium is
accomplished by a triparental mating procedure using E. coli carrying the
recombinant
binary vector, a helper E coli strain which carries a plasmid such as pRK2013
and which is
able to mobilize the recombinant binary vector to the target Agrobacterium
strain.
Alternatively, the recombinant binary vector can be transferred to
Agrobacferium by DNA
transformation (Hbfgen & Willmitzer, Nucl. Acids Res. 16: 9877(1988)).
Transformation of the target plant species by recombinant Agrobacterium
usually involves
co-cultivation of the Agrobacterium with explants from the plant and follows
protocols well
known in the art. Transformed tissue is regenerated on selectable medium
carrying the
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antibiotic or herbicide resistance marker present between the binary plasmid T-
DNA
borders.
Transformation of dicots may also be carried out using biolistics.
Particularly preferred for
soybean transformation is the method described in US 5024944.
Transformation of Monocot~don~
Transformation of most monocotyledon species has now also become routine.
Preferred
techniques include direct gene transfer into protopiasts using PEG or
electroporation
techniques, and particle bombardment into callus tissue. Transformations can
be
undertaken with a single DNA species or multiple DNA species (i.e. co-
transformation) and
both these techniques are suitable for use with this invention. Co-
transformation may have
the advantage of avoiding complex vector construction and of generating
transgenic plants
with unlinked loci for the gene of interest and the selectable marker,
enabling the removal of
the selectable marker in subsequent generations, should this be regarded
desirable.
However, a disadvantage of the use of co-transformation is the less than 100%
frequency
with which separate DNA species are integrated into the genome (Schocher et
al.
Biotechnology 4: 1093-1096 (1986)).
Patent applications EP 0 292 435, EP 0 392 225 and WO 93/07278 describe
techniques for
the preparation of callus and protoplasts from an Mite inbred line of maize,
transformation of
protoplasts using PEG or electroporation, and the regeneration of maize plants
from
transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and
Fromm et
aL (Biotechnology $: 833-839 (1990)) have published techniques for
transformation of
A188-derived maize line using particle bombardment. Furthermore, international
patent
application WO 93/07278 and Koziei et aL (Biotechnology 11: 194-200 (1993))
describe
techniques for the transformation of Mite inbred lines of maize by particle
bombardment.
This technique utilizes immature maize embryos of 1.5-2.5 rnm length excised
from a maize
ear 14-15 days after pollination and a PDS-1000He Biolistics device for
bombardment.
Maize may also be transformed by Agrobacterium, e.g., using the methods
described in
Ishida et al., 1996; High efficiency transformation of maize (Zea mays L.)
mediated by
Agrobacterium tumefaciens, Nature Biotechnology 14, 745-750.
Transformation of rice can also be undertaken by direct gene transfer
techniques utilizing
protoplasts or particle bombardment. Protoplast-mediated transformation has
been
described for Japonica types and Indica-types (Zhang ef aL, Plant Cell Rep 7:
379-384
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WO 00/36126 PCT/EP99/09986
- 28 _
(1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al.
Biotechnology g: 736-740
(1990)). Both types are also routinely transformable using particle
bombardment (Christou
et al. Biotechnology ~: 957-962 (1991 )).
Patent application EP 0 332 581 describes techniques for the generation,
transformation
and regeneration of Pooideae protoplasts. These techniques allow the
transformation of
Dactylis and wheat. Furthermore, wheat transformation was been described by
Vasil et al.
(Biotechnology ~: 667-674 (1992)) using particle bombardment into cells of
type C long-
term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558
(1993)) and
Weeks et al. (Plant Physiol. Q: 1077-1.084 (1993)) using particle bombardment
of
immature embryos and immature embryo-derived callus. A preferred technique for
wheat
transformation, however, involves the transformation of wheat by particle
bombardment of
immature embryos and includes either a high sucrose or a high maltose step
prior to gene
delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length)
are plated
onto MS medium with 3% sucrose (Murashige & Skoog, Physiology Planetarium 15:
473-
497 (1962)) and 3 mg/I 2,4-D for induction of somatic embryos which is allowed
to proceed
in the dark. On the chosen day of bombardment, embryos are removed from the
induction
medium and placed onto the osmoticum (i.e. induction medium with sucrose or
maltose
added at the desired concentration, typically 15%). The embryos are allowed to
plasmolyze
for 2-3 h and are then bombarded. Twenty embryos per target plate is typical,
although not
critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is
precipitated
onto micrometer size gold particles using standard procedures. Each plate of
embryos is
shot with the DuPont Biolistics~ helium device using a burst pressure of -1000
psi using a
standard 80 mesh screen. After bombardment, the embryos are placed back into
the dark
to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are
removed from
the osmoticum and placed back onto induction medium where they stay for about
a month
before regeneration. Approximately one month later the embryo explants with
developing
embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter
NAA, 5 mg/liter
GA), further containing the appropriate selection agent (10 mg/I Basta in the
case of
pCIB3064 and 2 mg/I methotrexate in the case of pSOG35). After approximately
one
month, developed shoots are transferred to larger sterile containers known as
"GA7s" which
contained half-strength MS, 2% sucrose, and the same concentration of
selection agent.
Patent application WO 94/13822 describes methods for wheat transformation and
is hereby
incorporated by reference.
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Plastid Transformation
Plastid transformation technology is extensively described in U.S. Patent Nos.
5,451,513,
5,545,817, and 5,545,818, all of which are hereby expressly incorporated by
reference in
their entireties; in PCT application nos. WO 95/16783 and WO 98/11235, which
are hereby
incorporated by reference in its entirety; and in McBride et al. (1994) Proc.
Natl. Acad. Sci.
USA 91, 7301-7305, which is also hereby incorporated by reference in its
entirety; The
basic technique for chloroplast transformation involves introducing regions of
cloned plastid
DNA flanking a selectable marker together with the gene of interest into a
suitable target
tissue, e.g., using biolistics or protoplast transformation (e.g., calcium
chloride or PEG
mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting
sequences,
facilitate homologous recombination with the plastid genome and thus allow the
replacement or modification of specific regions of the plastome. Initially,
point mutations in
the chloroplast 16S rRNA and rpsl2 genes conferring resistance to
spectinomycin and/or
streptomycin were utilized as selectable markers for transformation (Svab, Z.,
Hajdukiewicz,
P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530, hereby
incorporated by
reference; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45, hereby
incorporated by
reference). This resulted in stable homoplasmic transformants at a frequency
of
approximately one per 100 bombardments of target leaves. The presence of
cloning sites
between these markers allowed creation of a plastid targeting vector for
introduction of
foreign genes (Staub, J.M., and Maliga, P. (1993) EMBOJ. 12, 601-606, hereby
incorporated by reference). Substantial increases in transformation frequency
were
obtained by replacement of the recessive rRNA or r-protein antibiotic
resistance genes with
a dominant selectable marker, the bacterial aadA gene encoding the
spectinomycin-
detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab, Z., and Maliga,
P. (1993)
Proc. Nafl. Acad Sci. USA 90, 913-917, hereby incorporated by reference).
Previously, this
marker had been used successfully for high-frequency transformation of the
plastid genome
of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991)
Nucl. Acids
Res. 19, 4083-4089, hereby incorporated by reference). Other selectable
markers useful
for plastid transformation are known in the art and encompassed within the
scope of the
invention. Typically, approximately 15-20 cell division cycles following
transformation are
required to reach a homoplastidic state.
Plastid expression, in which genes are inserted by homologous recombination
into all of the
several thousand copies of the circular plastid genome present in each plant
cell, takes
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advantage of the enormous copy number advantage over nuclear-expressed genes
to
permit expression levels that can readily exceed 10% of the total soluble
plant protein.
However, such high expression levels may pose potential viability problems,
especially
during early plant growth and development. Similar problems are posed by the
expression
of bioactive enzymes or proteins that may be highly deleterious to the
survival of transgenic
plants and hence if expressed constitutively may not be introduced
successfully into the
plant genome. Thus, in one aspect, the present invention has coupled
expression in the
nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under control
of the
chemically inducible PR-1 a promoter (US 5,614,395 incorporated by reference)
of tobacco
to a chloroplast reporter transgene regulated by T7 gene 10
promoter/terminator
sequences. For example, when plastid transformants homoplasmic for the
maternally
inherited uidA gene encoding the (3-glucuronidase (GUS) reporter are
pollinated by lines
expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry
both
transgene constructs but do not express the GUS protein. Synthesis of large
amounts of
enzymatically active GUS is triggered in plastids of these plants only after
foliar application
of the PR-1 a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S
methyl ester
(BTH).
BRIEF DESCRIPTION OP THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID N0:1protein sequence of thioredoxin from Methanococcus
jannaschii
SEQ ID N0:2protein sequence of thioredoxin from Archaeoglobus
fulgidus (trx-1 )
SEQ ID N0:3protein sequence of thioredoxin from Archaeoglobus
fulgidus (tnc-2)
SEQ ID N0:4protein sequence of thioredoxin from Archaeoglobus
fulgidus (trx-3)
SEQ ID N0:5protein sequence of thioredoxin from Archaeoglobus
fulgidus (tnc-4)
SEQ ID N0:6protein sequence of thioredoxin reductase from Methanococcus
jannaschii
(trxB)
SEQ ID N0:7 protein sequence of thioredoxin reductase from Archaeoglobus
fulgidus
(tncB)
SEQ ID N0:8 Clontech sequence
SEQ ID N0:9 Joshi sequence
EXAMPLES
The invention is further described by reference to the following detailed
examples. These
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examples are provided for purposes of illustration only, and are not intended
to be limiting
unless otherwise specified.
Standard recombinant DNA and molecular cloning techniques used here are well
known in
the art and are described, for example, by Sambrook et al. (1989) Molecular
Cloning and by
Ausubel et al. (1994) Current Protocols in Molecular Biology.
Example 1: Transformation of maize with heat-stable thioredoxin
A gene expressing the neat-stable thioredoxin from Methanococcus jannaschii,
having the
sequence
MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPOKAMEYGIMAVPTIVI
NGDVEFIGAPTKEALVEAIKKRL (SEQ ID N0:1)
is prepared using maize preferred codons as described in US patent 5625136,
under
control of the seed-specific gamma-zein promoter, and the expression cassette
incorporated between the T-DNA boundaries of the pGIGUP plasmid. The T-DNA of
this
plasmid contains a plant expressible bar gene driven by the ubiquitin promoter
(Christensen
et al., Plant Mol. biol. 18: 875-689, 1992) to provide resistance to
phosphinothricin. It also
contains the GUS gene (beta-glucuronidase) with an intron in the N-terminal
codon of the
coding sequence driven by a chimeric promoter (SMAS) derived from the octopine
and
mannopine synthase genes (a trimer of the octopine synthase promoter upstream
activating
sequence with a domain of the mannopine synthase gene, Ni et al., Plant J. 7:
661-676,
1995). This intron-GUS gene expresses GUS activity in plant cells but not in
Agrobacterium. Alternatively, the heat-stable thioredoxin from Methanococcus
jannaschii is
cloned into the plasmid pNOV117 which contains a plant expressible-pmi gene
driven by
the maize ubiquitin promoter for selection on mannose (Christensen ef al.,
1992, Joersbo et
al., 1998).
Strain A. tumefaclens LBA4404 (pAL4404, pSB1) is used in these experiments.
pAL4404 is
a disarmed helper plasmid. pSBi is a wide host range plasmid that contains a
region of
homology to pGIGUP and pNOV117 and a 15.2 kb Kpnl fragment from the virulence
region
of pTiBo542 (Ishida et al., 1996; High efficiency transformation of maize (Zea
mays L.)
mediated by Agrobacterium tumefaciens, Nature Biotechnology 14, 745-750). The
introduction of the plasmid pGIGUP or pNOV117 by electroporation into LBA4404
(pAL4404, pSB1) results in a cointegration of pGIGUP or pNOV117 and pSBI. The
T-DNA
of pNOV117 contains a mannose-6-phosphate isomerase gene driven by the
ubiquitin
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promoter to provide the ability to metabolize mannose, as well as the
thioredoxin gene
described above.
Agrobacterium is grown for 3 days on YP medium (5g/l yeast extract, 10g/I
peptone, 5g/I
NaCI, 15 g/l agar, pH 6.8) supplemented with 50 mgh spectinomycin and 10 mg/I
tetracycline. Bacteria are collected with a loop and suspended in N6 liquid
medium at a
density ranging from 108 to 5 109cells/ml. Agrobacterium cells can also be
collected from an
overnight culture in YP medium and resuspended in N6 liquid medium. For 1 L of
medium
add: 4g powdered N6 salts (Sigma, St. Louis, MO), 30g sucrose, 100 mg myo-
inositol, 2 mg
glycine, 1 mg thiamine, 0.5 mg pyrodoxine HCL, 0.5 mg nicotinic acid, 2 mg 2,4-
D (from
stock solution [1 mg/mLj made by dissolving 2,4-D in dilute KOH). Adjust to pH
6.0 with 1 M
KOH, add 3 g Gelrite, and autoclave.
Maize immature embryos are obtained approximately 10 to 14 days after self-
pollination.
The immature zygotic embryos are divided among different plates containing
medium
capable of inducing and supporting embryogenic callus formation at about 25
immature
embryos per plate.
The immature embryos are inoculated either on the plate or in liquid with
Agrobacterium
having a Ti plasmid comprising a selectable marker gene. The immature embryos
are plated
on callus initiation medium containing silver nitrate (10 mg/I) either prior
or immediately after
inoculation with Agrobacterium. Approximately 25 immature embryos are placed
onto each
plate. 16 to 72 hours after inoculation, immature embryos are transferred to
callus initiation
medium with silver nitrate and cefotaxim. Selection of transformed cells is
carried out as
follows:
Mannose is used to select transformed cells in vitro. This selection can be
applied as low as
1 g/L 2 to 20 days after inoculation and maintained for a total of 2-12 weeks.
The
embryogenic callus so obtained is regenerated in the presence or absence of
mannose on
standard medium of regeneration. All plants are tested by the chlorophenol red
(CR) test for
tolerance to mannose. This assay utilizes a pH sensitive indicator dye to show
which cells
are growing in the presence of mannose. Cells that grow produce a pH change in
the media
and turn the indicator Chlorophenol Red (CR) yellow from red. Plants
expressing the
tolerance to mannose are easily identified in this test. Plants positive by
the CR test are
assayed by PCR for the presence of the mannose gene. Plants which are positive
for PCR
test are analyzed by Southern blot.
The regenerated plants are assayed for expression of the thioredoxin. The
plants are
developmentally normal. Corn grain from progeny plants derived from the
highest
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expressing event is assayed in a small scale wet milling process and starch
extractability is
measured compared to corn of the same genotype without the thioredoxin
transgene. Corn
expressing the thioredoxin gene exhibits substantially greater starch
availability in the wet-
milling process than the isogenic non-transformed corn.
Example 2: Transformation of maize with heat-stable thioredoxln and
thioredoxin
reductase
Using the procedures described in Example 1, maize is co-transformed with
genes for both
thioredoxin and thioredoxin reductase from M. jannaschii, described above.
Both genes are
under control of the seed specific gamma zein promoter. The two genes are
linked and
placed between the right and left borders of the pGIGUP or pNC?V117 plasmid to
enhance
the likelihood that both genes will be incorporated into the chromosome of the
plant as a
single insert.
The regenerated plants are assayed for expression of the thioredoxin and
thioredoxin
reductase. The plants are developmentally normal. Corn grain from progeny
plants derived
from the highest expressing event is assayed in a small scale wet milling
process and
starch extractability is measured compared to corn of the same genotype
without the
thioredoxin/thioredoxin reductase transgenes. Corn expressing the thioredoxin
and
thioredoxin reductase genes exhibits substantially greater starch availability
in the wet-
milling process than the isogenic non-transformed corn.
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1
LISTING
<110> Navartis AG
<120> Thioredoxi.n and Grain processing
<130> S-30758/A
<140> 09/213,208
<141> December 17, 1998
<160> 9
<170> Patentln Ver. 2.2
<210> 1
<211> 85
<212> PRT
<213> Methanococcus jannaschii
<400> 1
Met Ser Lys Val Lys Ile Glu Leu Phe Thr Ser Pro Met Cars Pro His
1 5 10 15
Cps Pro Ala Ala Lys Arg Val Val Glu Glu Val Ala Asn Glu Met Pro
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Asp Ala Val Glu Val Glu Tyr Ile Asn VaI Met Glu Asn Pro Gln Lys
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Ala Met Glu Tyr Gly Ile Met Ala Val Pro Thr Ile Val Ile Asn Gly
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Asp Val Glu Phe Ile Gly Ala Pro Thr Lys Glu Ala Leu Val Glu Ala
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Ile Lys Lys Arg Leu
<210> 2
<211> lI9
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Met Pro Met Val Arg Lys Ala Ala Phe Tyr Ala Ile Ala Val Ile Ser
1 5 10 15
Gly Val Leu Ala Ala Val Val Gly Asn Ala Leu Tyr His Asn Phe Asn
20 25 30
Ser Asp Leu Gly Ala Gln Ala Lys Ile Tyr Phe Phe Tyr Ser Asp Ser
35 40 45
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2
Cys Pro His Cps Arg Glu Val Lys Pro Tyr Val Glu Glu Phe Ala Lys
50 55 60
Thr His Asn Leu Thr Trp Cys Asn Val Ala Glu Met Asp Ala Asn Cps
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Ser Lys Ile Ala Gln Glu Phe Gly Ile Lys Tyr Val Pro Thr Leu Val
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Ile Met Asp Glu Glu Ala His Val Phe Val Gly Ser Asp Glu Val Arg
100 105 110
Thr Ala Ile Glu Gly Met Lys
115
<210> 3
<211> 93
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1 5 10 15
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20 25 30
Val Val Asp Val Asp Glu Lys Arg Glu Leu Ala Glu Lys Tyr Glu Val
35 40 45
Leu Met Leu Pro Thr Leu Val Leu Ala Asp Gly Asp Glu Val Leu Gly
50 55 60
Gly Phe Met Gly Phe Ala Asp Tyr Lys Thr Ala Arg Glu Ala Ile Leu
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Glu Gln Ile Ser Ala Phe Leu Lys Pro Asp Tyr Lys Asn
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<210> 4
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Val Lys Leu Asn Ser Ser Asn Phe Asp Glu Thr Leu Lys Asn Asn Glu
35 40 45
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3
Asn Val Val Val Asp Phe Trp Ala Glu Trp Cars Met pro CAS Lys Met
50 55 60
Ile Ala Pro Val Ile Glu Glu Leu Ala Lys Glu Tyr Ala Gly Lys Val
65 70 75 80
Val Phe Gly Lys Leu Asn Thr Asp Glu Asn Pro Thr Ile Ala Ala Arg
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<210> 5
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1 5 10 15
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20 25 30
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35 40 45
Val Glu Phe Tyr Lys Leu Asn Val Asp Glu Asn Gln Asp Val Ala Phe
50 55 60
Glu Tyr Gly Ile Ala Ser Ile Pro Thr VaI Leu Phe Phe Arg Asn Gly
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<213> Methanococcus jannaschii
<400> 6
Met Ile His Asp Thr Ile Ile Ile Gly Ala Gly Pro Gly Gly Leu Thr
CA 02353794 2001-06-O1
WO 00/36126 PCT/EP99/09986
4
1 5 10 15
Ala Gly Ile 'I~r Ala Met Arg Gly Lys Leu Asn Ala Leu Cys Ile Glu
20 25 30
Lys Glu Asn Ala Gly Gly Arg Ile Ala Glu Ala Gly Ile Val Glu Asn
35 40 45
Tyr Pro Gly Phe Glu Glu Ile Arg Gly err Glu Leu Ala Glu Lys Phe
50 55 60
Lys Asn His Ala Glu Lys Phe Lys Leu Pro Ile Ile Tyr Asp Glu Val
65 70 7S g0
Ile Lys Ile Glu Thr Lys Glu Arg Pro Phe Lys Val Ile Thr Lys Asn
85 90 95
Ser Glu 'I~rr Leu Ttlr Lys Thr Ile Val Ile Ala Thr Gly Thr Lys Pro
100 105 110
Lys Lys Leu Gly Leu Asn Glu Asp Lys Phe Ile Gly Arg Gly Ile Ser
115 120 125
Tyr Cys Thr Met Cps Asp Ala Phe Phe Tyr Leu Asn Lys Glu Val Ile
130 135 140
Val Ile Gly Arg Asp Thr Pro Ala Ile Met Ser Ala Ile Asn Leu Lys
145 150 155 160
Asp Ile Ala Lys Lys Val Ile Val Ile Thr Asp Lys Ser Glu Leu Lys
165 170 175
Ala Ala Glu Ser Ile Met Leu Asp Lys Leu Lys Glu Ala Asn Asn Val
180 185 190
Glu Ile Ile Tyr Asn Ala Lys Pro Leu Glu Ile Val Gly Glu Glu Arg
195 200 205
Ala Glu Gly Val Lys Ile Ser Val Asn Gly Lys Glu Glu Ile Ile Lys
210 215 220
Ala Asp Gly Ile Phe Ile Ser Leu Gly His Val Pro Asn Thr Glu Phe
225 230 235 240
Leu Lys Asp Ser Gly Ile Glu Leu Asp Lys Lys Gly Phe Ile Lys Thr
245 250 255
Asp Glu Asn Cps Arg Thr Asn Ile Asp Gly Ile Tyr Ala Val Gly Asp
260 265 270
Val Arg Gly Gly Val Met Gln Val Ala Lys Ala Val Gly Asp Gly Cars
275 280 285
Val Ala Met Ala Asn Ile Ile Lys Tyr Leu Gln Lys Leu
290 295 300
CA 02353794 2001-06-O1
WO 08/36126 PCT/EP99/09986
<210> 7
<211> 300
<212> PRT
<213> Archaeoglobus fulgidus
<400> 7
Met Tyr Asp Val Ala Ile Ile Gly Gly Gly Pro Ala Gly Leu Thr Ala
1 5 10 15
Ala Leu Tyr Ser Ala Arg Tyr Gly Leu Lys Thr Val Phe Phe Glu Thr
20 25 30
Val Asp Pro Val Ser Gln Leu Ser Leu Ala Ala Lys Ile Glu Asn Tyr
35 40 45
Pro Gly Phe Glu Gly Ser Gly Met Glu Leu Leu Glu Lys Met Lys Glu
50 55 60
Gln Ala Val Lys Ala Gly Ala Glu Trp Lys Leu Glu Lys Val Glu Arg
65 70 75 80
Val Glu Arg Asn Gly Glu 'I'rlr Phe Thr Val Ile Ala Glu Gly Gly Glu
85 90 95
Tyr Glu Ala Lys Ala Ile Ile Val Ala Thr Gly Gly Lys His Lys Glu
100 105 110
Ala Gly Ile Glu Gly Glu Ser Ala Phe Ile Gly Arg Gly Val Ser Tyr
115 120 125
Cars Ala Thr Cps Asp Gly Asn Phe Phe Arg Gly Lys Lys Val Ile Val
130 135 140
Tyr Gly Ser Gly Lys Glu Ala Ile Glu Asp Ala Ile Tyr Leu His Asp
145 150 155 160
Ile Gly Cys Glu Val Thr Ile Val Ser Arg Ttlr Pro Ser Phe Arg Ala
165 170 175
Glu Lys Ala Leu Val Glu Glu Val Glu Lys Arg Gly Ile Pro Val His
180 185 190
Tyr Ser Thr Ttlr Ile Arg Lys Ile Ile Gly Ser Gly Lys Val Glu Lys
195 200 205
Val Val Ala Tyr Asn Arg Glu Lys Lys Glu Glu Phe Glu Ile Glu Ala
210 215 220
Asp Gly Ile Phe Val Ala Ile Gly Met Arg Pro Ala Thr Asp Val Val
225 230 235 240
Ala Glu Leu Gly Val Glu Arg Asp Ser Met Gly Tyr Ile Lys Val Asp
245 250 255
CA 02353794 2001-06-O1
WO 00/36126 PCT/EP99/09986
6
Lys Glu Gln Arg Thr Asn Val Glu Gly Val Phe Ala Ala Gly Asp Cps
260 265 270
Cys Asp Asn Pro Leu Lys Gln Val Val Thr Ala Cys Gly Asp Gly Ala
275 280 285
Val Ala Ala Tyx- Ser Ala Tyr Lys Tyr Leu 'hhr Ser
290 295 300
<210> 8
<211> 13
<212> I~1A
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 8
gtcgaccatg gtc 13
<210> 9
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 9
taaacaatgg ct 12
