Note: Descriptions are shown in the official language in which they were submitted.
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USE OF A FORMULATION FOR CONTROLLING EXPRESSION OF A TARGET GENE
The present invention relates to the use of a formulation as a control
mechanism for
the expression of a target gene in an organism such as a plant, as well as to
certain
formulations useful in this way.
Gene expression is controlled by regions upstream (5') of the protein encoding
region,
commonly referred to as the "promoter". A promoter may be constitutive, tissue-
specific,
developmentally-programmed or inducible.
Manipulation of crop plants to improve characteristics (such as productivity
or
to quality) requires the expression of foreign or endogenous genes in plant
tissues. Such
genetic manipulation therefore relies on the availability of means to control
gene expression
as required; for example, on the availability and use of suitable promoters
which are effective
in plants. It is advantageous to have the choice of a variety of different
promoters so that the
most suitable promoter may be selected for a particular gene, construct, cell,
tissue, plant or
environment. A range of promoters are known to be operative in plants.
The term "inducible promoter" includes promoters which may be induced
chemically. Particularly useful promoters are promoter sequences which are
controlled by
the application of an external chemical stimulus. The external chemical
stimulus may be an
agriculturally acceptable chemical, the use of which is compatible with
agricultural practice
and is not detrimental to plants or mammals. This allows particular gene
expression to be
controlled at particular stages of plant growth or development, by the
presence or absence of
a chemical which can be applied to the plants or seeds, for example by
spraying or using
known seed coating techniques. These are also known as a gene "switch"
promoters.
The gene which is under the control of the inducible promoter may be the gene
which
gives rise to the desired characteristic or phenotype itself, or the inducible
promoter may
control expression of a repressor protein which inhibits expression of a
target gene, for
example by interacting with an operator sequence upstream of the target gene
so as to
prevent expression of the gene (for example as known in the bacterial tet and
lac
operator/repressor systems). In a further alternative, the gene under the
control of the
inducible promoter may express a protein which interacts with another protein
to inhibit the
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activity thereof, as for example in the barnase/barstar system which barnase
will inhibit or
kill cells in the absence of barstar.
Gene switches of this type are known in a wide variety of applications. These
include
the production of reversible male sterility, a feature which is highly
desirable in hybrid plant
s production as described for instance in WO 90/08830. Other applications of
such promoters
include in germplasm protection, where containment of particular crop plants,
in particular
transgenic plants, and the control of volunteers is necessary and also in the
prevention of
pre-harvesting sprouting as described in WO 94/03619.
Many organisms have mechanisms which allow them to metabolise chemicals such
1o as alcohols or ketones, for example by the production of alcohol
dehydrogenase enzymes.
The promoters of these systems may be useful in gene switches as the promoters
may be
inducible by the presence of the target alcohol or ketone.
One such example can be found in the fungal organism A nidulans which
expresses
the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when
it is
15 grown in the presence of various alcohols and ketones. The induction is
relayed through a
regulator protein encoded by the alcR gene which is constitutively expressed.
In the
presence of inducer (alcohol or ketone), the regulator protein activates the
expression of the
alcA gene: The regulator protein also stimulates expression of itself in the
presence of
inducer. This means that high levels of the ADHl enzyme are produced under
inducing
2o conditions (ie when alcohol or ketone are present). Conversely, the alcA
gene and its
product, ADH1, are not expressed in the absence of inducer. Expression of alcA
and
production of the enzyme is also repressed in the presence of glucose.
Thus, the alcA gene promoter is an inducible promoter, activated by the alcR
regulator protein in the presence of inducer (ie by the protein/alcohol or
proteinlketone
25 combination). The alcR and alcA genes (including the respective promoters)
have been
cloned and sequenced (Lockington RA et al, 1985, Gene, 33:137-149; Felenbok B
et al,
1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).
Alcohol dehydrogenase (adh) genes have been investigated in certain plant
species. In maize and other cereals they are switched on by anaerobic
conditions. The
30 promoter region of adh genes from maize contains a 300 by regulatory
element necessary for
expression under anaerobic conditions. However, no equivalent to the alcR
regulator protein
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has been found in any plant. Hence the alcRlalcA type of gene regulator system
is not
known in plants. Constitutive expression of alcR in plant cells does not
result in the
activation of endogenous adh activity.
WO 93/21334 describes the production of transgenic plants which include
such a system as a gene switch. This document specifically describes a
chemically inducible
plant gene expression cassette comprising a first promoter operatively linked
to a regulator
sequence which encodes a regulator protein, and an inducible promoter
operatively linked to
a target gene, the inducible promoter being activated by the regulator protein
in the presence
of an effective exogenous inducer whereby application of the inducer causes
expression of
l0 the target gene. In particular, the alcRlalcA system is utilised in the
constructs. Exogenous
chemical inducers which are applied in this case include those described by
Greaser et al., J.
Biochem. (1984) 225, 449-454) such as butan-2-one (ethyl methyl ketone),
cyclohexanone,
acteone, butan-2-ol, 3-oxobutyric acid, propan-2-of and ethanol.
For agricultural purposes, the application of simple alcohols may be preferred
as such
15 chemicals are relatively cheap and are likely to be readily degraded. They
therefore present
less of an environmental burden. However, such chemicals are often highly
volatile and
therefore difficult to handle in an agricultural context, as large volumes of
chemical may be
lost during spraying. ~ One of the problems associated with the use of the
known inducers is,
therefore, their ability to evaporate rapidly before they can be effectively
taken up by a plant.
2o It is therefore desirable to use a chemical inducer which may be taken up
by a plant
more effectively than the known inducers thereby allowing better control of
expression of a
target gene.
The present invention therefore seeks to overcome the problems associated with
the
prior art, by formulating the chemical inducer.
25 According to a first aspect of the present invention there is provided the
use of a
formulation comprising the components:
(a) a volatile chemical inducer;
(b) a polyethoxylated C,o Czo alcohol or a trisiloxane polyethoxylate and
(c) a diluent;
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for controlling expression of a target gene in amorganism having a chemically-
inducible gene
expression cassette comprising an inducible promoter operatively linked to the
target gene
wherein the inducible promoter is induced by the application to the organism
of (a) above.
Suitably, the organism is a plant such as a crop plant.
The diluent (c) may be, for example, water.
According to a second aspect of the present invention, there is provided a
method of
controlling expression of a target gene in an organism, such as a plant,
comprising
transforming the organism with a chemically-inducible plant gene expression
cassette
comprising an inducible promoter operatively linked to the target gene wherein
the inducible
to promoter is induced by the application to the organism of a formulation as
described above.
Suitably the expression system utilises a regulator protein.
Thus according to a third aspect of the present invention there is provided a
method
of controlling expression of a target gene in a plant comprising transforming
the plant with a
chemically-inducible plant gene expression cassette comprising a first
promoter operatively
linked to a regulator sequence which encodes a regulator protein, and an
inducible promoter
operatively linked to the target gene, the inducible promoter being activated
by the regulator
protein in the presence of a formulation as defined above, the method
comprising applying to
the plant a formulation as defined above, whereby application of the inducing
formulation
causes expression of the target gene.
2o The nature of component (a) in the above formulations depends upon the
character of
the inducible promoter present in the expression system. However, a particular
examples of
component (a) are a C,-C6 alcohol or a C3-C9 ketone, and preferably, ethanol
or propan-2-ol.
These components act as inducers for example, of the alc switch system as
described in
detail below.
Component (b) of the formulation used as described above, preferably, a
polyethoxylated oleyl, lauryl, stearyl or cetyl alcohol. It is more preferably
a
polyoxyethylene-oleyl alcohol having a mean molar ethylene oxide content in
the range of 0
to 35 and more preferably in the range of 2 to 20. It is most preferably a
polyoxyethylene-
(2)-oleyl alcohol, a polyoxyethylene-(10)-oleyl alcohol or a polyoxyethylene-
(20)-oleyl
alcohol. Component (b) is, however, preferably a polyoxyethylene-(20)-oleyl
alcohol (the
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number in brackets indicates the mean ethylene oxide content per molecule).
Such products
are commercially available as BRIJ 92T"", BRIJ 97T"" and BRIJ 98T""
Preferably, component (b) of the formulation is at a concentration of about
0.5%
wdwt or less. It is preferably at a concentration between about 0.2% wt/wt and
0.5% wt/wt.
In an alternative embodiment, the formulation used in the methods of the
invention
includes as component (b), a hydrogen or methyl end-capped trisiloxane
polyethoxylate In
particular, component (b) is a methyl end-capped trisiloxane polyethoxylate.
The methyl
end-capped trisiloxane polyethoxylate preferably has a mean molar ethylene
oxide content of
between 4 and 12 per molecule and is most preferably 8 per molecule. Such
products are
to commercially available as SILWET 77T"" (SILWET is a trademark of Witco).
Preferably, the methyl end-capped trisiloxane polyethoxylate is at a
concentration of
about 0.5% wt/wt or less. It is preferably at a concentration between about
0.2% and 0.5%
wt/wt.
Component (a) of the formulation used in the above-described methods is
preferably
15 at a concentration of about 5% wtlwt or less. It is preferably at a
concentration between
about 2% and 5% wt/wt.
The inducible promoter according to a preferred embodiment of the third aspect
of
the present invention is the alcA inducible promoter sequence and the
regulator sequence
encodes the alcR regulator protein.
20 Component (c) of the formulation is preferably at a concentration between
about 90%
and 98% wt/wt.
Certain formulations used in accordance with the above-described methods are
novel
and provide a further aspect of the invention.
Thus, according to a fourth aspect of the present invention there is provided
an
25 agricultural formulation comprising the components:
(a) a volatile chemical inducer of an inducible promoter;
(b) a trisiloxane polyethoxylate; and
(c) a diluent.
As mentioned above, component (b) is suitably a hydrogen or a methyl end-
capped
3o trisiloxane polyethoxylate. and preferaably is a methyl end-capped
trisiloxane
polyethoxylate. Suitably, the methyl end-capped trisiloxane polyethoxylate has
a mean
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molar ethylene oxide content of between 4 and 12 per molecule, for example 8
per molecule.
Suitably, component (a) is at a concentration between about 2% and 5% wdwt.
According to a fifth aspect of the present invention, there is provided an
agricultural
formulation, comprising
(a) a C,-C6 alcohol inducer of an inducible promoter in an amount of less than
5%wt/wt;
(b) a polyethoxylated C,o-Czo alcohol; and
(c) water.
In this case, component (b) of the formulation is suitably a polyethoxylated
oleyl,
lauryl, stearyl or cetyl alcohol. and preferably a polyoxyethylene-oleyl
alcohol. Suitably, the
polyoxyethylene-oleyl alcohol has a mean molar ethylene oxide content in the
range of 2 to
20, such as a polyoxyethylene-(2)-oleyl alcohol, a polyoxyethylene-(10)-oleyl
alcohol or a
polyoxyethylene-(20)-oleyl alcohol. Again component (a), which is preferably
ethanol or
propan-2-ol, is suitably at a concentration between about 2% to less than 5%
wt/wt. The
concentration of component (B) is preferably about 0.5% wt/wt or less.
According to a sixth aspect of the present invention, there is provided an
agricultural
formulation comprising
(a) a C3-C9 ketone which is able to act as a chemical inducer of an inducible
promoter;
(b) a polyethoxylated C,o-Czo alcohol; and
(c) a diluent.
Preferably, in these formulations, component (a) is at a concentration between
about
2% and 5% wt/wt. As before, component (b) may be a polyethoxylated oleyl,
lauryl, stearyl
or cetyl alcohol, and is preferably a polyoxyethylene-oleyl alcohol, with a
mean molar
ethylene oxide content in the range of 2 to 20. Particular examples of
component (b) are
polyoxyethylene-(2)-oleyl alcohol, polyoxyethylene-(10)-oleyl alcohol and
polyoxyethylene-
(20)-oleyl alcohol.
A preferred embodiment of the present invention is a formulation comprising
the
3o components: a. ethanol; b. polyoxyethylene-(20)-oleyl alcohol and c. a
diluent wherein
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component a. is at a concentration of 2% wt/wt and component b. is at a
concentration of
0.5% wdwt.
An even more preferred embodiment of the present invention is a formulation
comprising the components: a: ethanol; b. methyl end-capped trisiloxane
polyethoxylate
having a mean molar ethylene oxide content of 8 and c. a diluent wherein
component a. is at
a concentration of 2% wdwt and component b. is in the concentration of 0.5%
wt/wt.
An advantage of the present invention is that the formulation is taken up by
the
organism such as the plant more effectively than when known inducers alone are
used. The
formulation according to the present invention therefore increases the
effectiveness of
1 o ethanol as an inducer of the alcAlalcR promoter.
It has been surprisingly found that the use of polyoxyethylene-oleyl alcohol
surfactants and/or methyl end-capped trisiloxane polyethoxylate adjuvants in
combination
with the known chemical inducers for the alcAlalcR promoter increases uptake
of the
formulation by a plant significantly, thereby allowing greater control of a
target gene which
may be operatively linked to the inducible promoter.
Other additives, such as antibacterial compounds, dispersants, wetter
compounds and
anti-evaporants may be added.
The invention is particularly applicable in the context of a plant gene
expression
system comprising
(i) a first promoter operatively linked to a regulator sequence which encodes
a
regulator protein; and
(ii) an inducible promoter operatively linked to a target gene, the inducible
promoter
being activated by the regulator protein in the presence of an effective
exogenous
inducer, which is the formulation defined above, whereby application of the
inducer causes expression of the target gene.
Such gene expression systems may be contained within a plant cell as well as
plant
tissue or a plant comprising plant cells as defined above and plants, or
seeds, derived
therefrom.
The term "volatile chemical inducer" means any chemical which is capable of
inducing a chemically inducible promoter system and which may evaporate
rapidly before it
can be effectively taken up by a plant.
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The term "C,-C6 alcohol" includes methanol, ethanol, n-propanol, isopropanol,
n-
butanol, iso-butanol, sec- butanol, tent-butanol, n-pentanol, n-hexanol and
cyclohexanol and
unsaturated analogues thereof.
The term "C3-C9 ketone" includes acetone, butanone, pentanone, hexanone,
cyclohexanone, heptanone, octanone, nonanone and aromatic ketones such as
acetophenone.
The term "target gene" with reference to the present invention means any gene
of
interest. It may comprise any gene which is required to be introduced into a
plant in order to
modify the characteristics thereof. The target gene may be an endogenous plant
gene or a
foreign gene, and may be a single gene or a series of genes. The target gene
sequence may
1 o encode at least part of a functional protein or an antisense sequence. An
inducible promoter,
therefore, when linked to an endogenous or foreign gene and introduced into a
eukaryote by
transformation, provides a means for the external regulation of expression of
that gene.
The term "expression cassette", which is synonymous with terms such as
"construct"
"hybrid" and "conjugate" - includes a gene of interest directly or indirectly
attached to an
15 inducible promoter. An example of an indirect attachment is the provision
of a suitable
spacer group such as an intron sequence intermediate the promoter and the
target gene. The
same is true for the term "fused" in relation to the present invention which
includes direct or
indirect attachment. Such constructs also include plasmids and phage which are
suitable for
transforming a cell of interest. The expression cassette of the present
invention may also
20 comprise additional components such as a regulator sequence operatively
linked to a further
promoter, the inducible promoter being activated by the regulator protein in
the presence of
an exogenous inducer.
The term "expression system" means that the system defined above can be
expressed
in an appropriate organism, tissue, cell or medium. In this regard, the
expression system of
25 the present invention may comprise one or more expression cassettes and may
also comprise
additional components that ensure the increased expression of the target gene
by use of the
inducible promoter.
Any transformation method suitable for the target plant or plant cells may be
employed, including infection by Agrobacterium tumefaciens containing
recombinant Ti
3o plasmids, electroporation, microinjection of cells and protoplasts,
microprojectile
bombardment, bacterial bombardment, particularly the "fibre" or "whisker"
method, and
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pollen tube transformation. The transformed cells may then in suitable cases
be regenerated
into whole plants in which the new nuclear material is stably incorporated
into the genome.
Both transformed monocot and dicot plants may be obtained in this way.
Reference may be
made to the literature for full details of the known methods.
Examples of genetically modified plants which may be produced include
dicotyledonous and monocotyledonous plants such as field crops, cereals,
fruits and
vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, Soya,
maize, wheat, barley,
rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries,
bananas, melons,
potatoes, carrot, lettuce, cabbage, onion.
to Various preferred features and embodiments of the present invention will
now be
described by way of non-limiting example and with reference to the
accompanying drawings
in which:-
Figure 1 shows CAT induction by foliar spray using ethanol alone (2% wt/wt)
and
ethanol (2% wt/wt) in combination with BRIJ 92, 97 and 98T"" (0.5% wt/wt)
formulations (5
repeats/ treatment);
Figure 2 shows the relationship of increasing SILWET L-77T"' adjuvant
concentration (0.2% wt/wt and 0.5% wt/wt) with 2% wdwt ethanol and ethanol (2%
wt/wt)
alone to CAT activity in a glasshouse leaf spray test;
Figure 3 shows the induction of Alc-GUS oilseed rape by application of 5%
wt/wt
2o ethanol and 0.5% wt/wt formulation to the upper leaf surface of oilseed
rape leaves ('T' _
target leaf, 'A' = adjacent leaf from the same plant); and
Figure 4 shows the structure of Silwett L77T"".
The Table below gives details of the components of each surfactant/ adjuvant
described in the Examples.
Surfactant/ Components Manufacturer
adjuvant
BRIJ 92T"" Polyoxyethylene-(2)-oleyl alcohol ICI Surfactants
BRIJ 97T"" Polyoxyethylene-(10)-oleyl alcohol ICI Surfactants
3o BRIJ 98T"" Polyoxyethylene-(20)-oleyl alcohol ICI Surfactants
SILWET L-77TM Methyl end-capped trilsiloxane polyethoxylate (8) -
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84% Allyloxypolyethyleneglycol methyl ether - 16% Witco
(structure shown in Figure 4)
EXAMPLE 1
Component Amount
a. Ethanol 1.99g
b. BRIJ 92T"" 0.51 g
c. Water 97.86g
99.86g of a 20% wt/wt ethanol solution (20.01 g ethanol in 980.56g water) were
mixed in a vial with O.SIg Brij 92T"". The sample was shaken before use.
EXAMPLE 2
Component Amount
a. Ethanol 2.OOg
is b. BRIJ 97T"" 0.52g
c. Water 98.OSg
100.O5g of a 20% wt/wt ethanol solution (20.O1g ethanol in 980.56g water) were
mixed in a vial with 0.52g Brij 97T"". The sample was shaken before use.
2o EXAMPLE 3
Component Amount
a. Ethanol 1.99g
b. BRIJ 98T"" O.SOg
c. Water 97.9g
99.90g of a 20% wt/wt ethanol solution (20.01 g ethanol in 980.56g water) were
mixed in a vial with O.SOg Brij 98T"". The sample was shaken before use.
EXAMPLE 4
3o Component Amount
a. Ethanol 2.02g
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b. SILWET L-77T"" 0.50g
c. Water 99.27g
1 O 1.30g of a 20% wt/wt ethanol solution (20.01 g ethanol in 980.56g water)
were
mixed in a vial with 0.50g Silwet L77T"". The sample was shaken before use.
EXAMPLE 5
In order to see the effect of the above formulations (see Figures l and 2) on
8 week
old Alc-CAT ('Alc' refers to the CaMV35S:A1cR, AIcA/CaMV35S:CAT system)
tobacco
plants, the plants were hand-sprayed to run-off on the adaxial leaf surface on
the V5 leaf with
1o an adjuvant-(or surfactant) ethanol mixture as described in Examples 1 to 4
above. The
plants were left for 72 h under glasshouse growth conditions with root water
irngation and
the target leaf sampled and assayed for CAT (chloramphenical acetyl
transferase) activity
using a CAT ELISA kit (Boehringer Mannheim). Plants sprayed with ethanol alone
were
used as controls. 5 repeats per treatment were used (error bars indicate the
standard
deviation).
It can be seen that the use of polyoxyethylene-(20)-oleyl alcohol (BRIJ 98T""
) as a
surfactant allows greater expression of the CAT reporter gene than the use of
ethanol alone.
The use of a methyl end-capped trisiloxane ethoxylate with a mean molar
ethylene oxide
content of 8 per molecule (SILWET L-77T"") in combination with ethanol is,
however, able to
2o increase expression of the CAT gene to an even greater extent than
polyoxyethylene-(20)-
oleyl alcohol (BRIJ 98T"") in combination with ethanol. In particular, the use
of BRIJ 98T"" or
SILWET L-77T"" , each at 0.2% wt/wt and at 0.5% wt/wt, significantly increased
reporter
gene expression and uptake in AIcCAT tobacco plants when applied as a mixture
with'4C-
ethanol (at concentrations varying between about 1 % to 5% wt/wt) to the upper
surface of the
leaves. Phosphoimage studies were used to detect '4C-ethanol uptake.
It was found that the use of ethanol alone resulted in a 2.1 % uptake by the
tobacco
plants. The addition of increasing concentrations of SILWET L-77T"" from 0.2%
to 0.5%
wt/wt resulted in a 61 % to 78% increase in CAT expression compared to ethanol
alone at a
concentration of 5% wt/wt. In '4C-ethanol formulation studies with BRIJ98T"",
a 68%
increase in ethanol uptake was seen compared to ethanol alone.
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Significant reporter gene activation was also seen with these formulations in
potato
and oil seed rape.
EXAMPLE 6
Homozygous 8-week-old soil-grown transgenic Alc-GUS (CaMV 35S:A1cR,
AIcA/CaMV 35S:CAT construct) oilseed rape plants were hand-sprayed to run-off
on the
adaxial leaf surface on the 5 ~' leaf from the apex with an adjuvant-ethanol
mixture (S
wt/wt ethanol and 0.5 % wt/wt adjuvant). The plants were left for 72 h under
glasshouse
growth conditions with root water irrigation. Target leaves were sampled and
assayed for
to GUS ((3-Glucuronidase) activity and values normalised against protein
concentration. Plants
sprayed with ethanol without formulation were used as controls ('Ethanol
alone'). Five
replicate plants were used per treatment. As can be seen from Figure 3, error
bars indicate
standard deviation, 'CaMV 35S/10' signifies a tenth of the GUS activity in a
constitutive
control plant containing the cauliflower mosaic virus driving the GUS reporter
gene,
'Silwett' signifies Silwett L77~' and 'EtOH' signifies a 5 % wt/wt ethanol
leaf spray
treatment without formulation.
Other modifications of the present invention will be apparent to those skilled
in the
art without departing from the scope of the present invention.
