JP2002535971A - Inducible expression system for use in plants - Google Patents

Abstract

  (57) [Summary] The present invention relates to a compound of formula (I) wherein R ¹And R^TwoRelates to the use of agriculturally acceptable hydrolysable esters (as defined herein). Control of the expression of plant genes is provided by an inducible promoter that requires the presence of an exogenous chemical for activation, which may include alcohol, where the agriculturally acceptable Hydrolysis of the appropriate ester results in the production of the alcohol. Formulations of these esters are also described and claimed.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to expression systems for use in plants, in particular expression systems utilizing exogenous chemicals as control mechanisms and the use of specific chemicals as said control substances.

[0002] Gene expression is controlled by a region upstream (5 ') of the protein coding region (commonly referred to as a "promoter"). A promoter can be constitutive, tissue-specific, developmentally programmable, or inducible.

[0003] Manipulation of crops to improve properties (eg, productivity or quality) requires the expression of foreign or endogenous genes in plant tissue. Thus, such genetic manipulation depends on the potency of the means to control gene expression as needed; for example, the potency and use of a suitable promoter effective in the plant. Advantageously, a variety of different promoters can be chosen, so that the most appropriate promoter for a particular gene, construct, cell, tissue, plant, or environment can be selected. A range of promoters are known to work in plants.

[0004] Particularly useful promoters in certain instances are promoters that can be induced by the application of exogenous chemical inducers. This means that the growth or development of a plant can be affected by the presence or absence of chemicals whose specific gene expression can be applied to the plant or seed (eg, by spraying or using known seed coating techniques). At a certain stage, it can be controlled. This is sometimes known as a gene "switch".

The gene under the control of an inducible promoter can be a gene that elicits the desired property or phenotype itself, or the inducible promoter can be, for example, (eg, a bacterial tet operator / repressor system and The expression of a repressor protein that suppresses target gene expression can be controlled by interacting with the operator sequence upstream of the target gene so as to prevent expression of the gene (known in the lac operator / repressor system). In yet another method, the gene under the control of an inducible promoter is, for example, barnase / barstar.
As in the system (in the absence of barstar, barnase suppresses or damages cells), it can express a protein that interacts with another protein to suppress its activity.

[0006] Gene switches of this type are known in a wide variety of applications. These include the development of reversible male sterility, a highly desirable feature in the production of hybrid plants, as described, for example, in WO 90/08830. Other applications of such promoters include germplasm protection, which requires the containment of certain crops (especially transgenic plants) and the control of native plants, and also WO 9
4/3619, including inhibition of germination prior to harvest.

[0007] Many organisms have mechanisms to metabolize chemicals, such as alcohols or ketones, for example, by the production of alcohol dehydrogenase enzymes. Since promoters may be inducible by the presence of a target alcohol or ketone, promoters of these systems may be useful in gene switches.

[0008] One such example is the fungal organism Anidulans, which expresses the enzyme alcohol dehydrogenase I (ADH1), encoded by the gene alcA, only when grown in the presence of various alcohols and ketones. Can be found in Induction is relayed via a regulatory protein encoded by the alcR gene and expressed continuously. Inducer (alcohol or ketone)
In the presence of, the regulatory protein activates the expression of the alcA gene. A regulatory protein also drives its own expression in the presence of an inducer. This means that under inducing conditions (ie, when alcohols or ketones are present), high levels of AD
It means that the H1 enzyme is produced. Conversely, the alcA gene and its product (
ADH1) is not expressed in the presence of the inducer. alcA expression and enzyme expression are also suppressed in the presence of glucose.

[0009] Thus, the alcA gene promoter is an inducible promoter, which is activated by an alcR regulatory protein (ie, by a protein / alcohol or protein / ketone combination) in the presence of an inducer. alcR
The gene and the alcA gene (including a representative promoter) were cloned and sequenced (Lockington RA et al., 1985, Gen.
e, 33: 137-149; Felenbook B et al., 1988, Gene, 7
3: 385-396; Gwyne et al., 1987, Gene, 51: 205-2.
16).

[0010] The alcohol dehydrogenase (adh) gene has been investigated in certain plant species. In corn and other cereals, the alcohol dehydrogenase gene is switched on by anaerobic conditions. The promoter region of the maize-derived adh gene contains the 300 bp regulator required for expression under anaerobic conditions. However, no equivalent of the alcR regulatory protein has been found in any plant. Therefore, the gene regulation system of the alcR / alcA type in plants is not known. Constitutive expression of alcR in plant cells does not result in activation of endogenous adh activity.

WO 93/21334 describes the production of transgenic plants, including for example systems such as gene switches. This document describes, inter alia, a first promoter operably linked to a regulatory sequence encoding a regulatory protein and an inducible promoter operably linked to a target gene (an inducible promoter in the presence of an effective exogenous inducer). Chemically inducible plant gene expression cassettes that are activated by regulatory proteins, whereby application of the inducer causes expression of the target gene). In particular, the alcR / alcA system is utilized in the construct. Exogenous chemical inducers applied in this case include Creather et al.
. Biochem. (1984) 225, 449-454) (e.g., butan-2-one (ethyl methyl ketone), cyclohexanone,
Acetone, butan-2-ol, 3-oxobutyric acid, propane-
2-ol, and ethanol).

[0012] For agricultural purposes, alcohol is commonly used as an exogenous chemical inducer. However, such chemicals are often volatile and are therefore difficult to handle in agricultural environments, as large amounts of chemicals can be lost during spraying.

[0013] The present invention provides the use of an agriculturally acceptable hydrolysable ester in controlling the expression of a plant gene. The control is provided by an inducible promoter that requires the presence of an exogenous chemical for activation, which may include an alcohol, where the hydrolysis of the agriculturally acceptable ester is performed. Decomposition results in the production of the alcohol.

In particular, agriculturally acceptable esters have the formula (I):

[0015]

Embedded image Wherein R ¹ is a lower alkyl, lower alkenyl, or lower alkynyl group, and R ² is an organic group such that R ² COOH is an agriculturally acceptable acid. is there. The hydrolysis of the compound of formula (I) is carried out according to formula (II):

[0016]

Embedded image This produces alcohol.

The term “agriculturally acceptable,” as used herein, refers to the ability of a compound to inhibit a particular soil or crop without causing unacceptable levels of soil damage or phytotoxicity in the crop. Means that it can be applied to

The expression “lower alkyl” as used herein is a C _1-6 alkyl group,
It preferably comprises C _1-4 alkyl groups, which may be straight or branched. The expressions "lower alkenyl" and "lower alkynyl" as used herein are, respectively, _C2-6 alkenyl and _C2-6 alkynyl, preferably _C2-4 alkenyl or _C2-4. It contains alkynyl groups, which can be straight-chain or branched.

Agriculturally acceptable esters, such as those of formula (I), for use in the present invention are suitably rearranged into the target plant. In this target plant, the gene regulatory system is in place and / or under environmental conditions or in the presence of a suitable catalytic moiety (eg, an enzyme or catalytic antibody), At the required time, it is hydrolyzed at a suitable rate to provide a sufficient amount of activated alcohol. These can vary depending on the nature of the plant species being treated, the gene expressed and the timing of the application of the ester.

Esters such as compounds of formula (I) are advantageous in that they are easier to handle than the corresponding alcohols. It has been found that these compounds can produce the desired effect on gene activation.

The compound should be applied before the required gene activation and for a time period sufficient to cause hydrolysis, which means that the plant growth stage where activation is required Should be reasonable depending on such factors. If the rate of hydrolysis is relatively slow, the time of application may be earlier to ensure that sufficient hydrolysis occurs before the plant enters the growth stage where gene activation is required. . If this is difficult, more rapidly hydrolyzing esters can be selected.

Alternatively, more than one ester with different rates of hydrolysis can be applied in one treatment. By choosing a combination of esters with different rates of hydrolysis, an effective "slow release" of the activated alcohol can be achieved, so that gene expression can be prolonged over a desired period of time. This means that repeated application of the chemical can be avoided and "one-off" treatment is possible.

Specific examples of alcohols of formula (II) include methanol, ethanol, propan-1-ol, propan-2-ol, butan-2-ol or but-3.
-En-2-ol.

Suitably, the alcohol of formula (II) is a lower alkyl alcohol, wherein the alkyl group has 1-4 carbon atoms and may be either branched or straight-chain . Preferred groups for R ¹ include ethyl, n-propyl and n-butyl.

A particularly preferred example of the compound of formula (II) is ethanol.

The exact nature of the R ² group is not critical, as long as the compound produces an agriculturally acceptable acid at a suitable rate in the particular target plant to which the compound of formula (I) is applied. The hydrolysis rate is determined, for example, by Mitchell et al., Pestic. Sci (
1995) 44: 49-58, and preferably by testing on all plant lines. What is suitable in any particular example depends on a variety of factors, including the nature of the gene expression to be controlled, the particular plant in which the gene is expressed, and other external conditions. The rate of hydrolysis should be sufficient so that the desired effect, eg, reversible male sterility, can be seen at a suitable time after application of the chemical inducer.

However, R ² has the formula (III)

[0028]

Embedded image Can be selected to have some useful agrochemical effects.
In particular, it may itself act as an inducer of an inducible promoter. For example, 3
Many acids have been found that can act as inducers of the alcR / alcA system, including -hydroxybutyric acid, 2-hydroxybutyric acid, pyruvic acid and 3-oxobutyric acid (Creaser et al., Supra).

Particular examples of R ² are optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkynyl, Includes optionally substituted aryl or optionally substituted heterocyclyl.

As used herein, the term “alkyl” includes a straight or branched alkyl chain, suitably up to 10 carbon atoms, preferably up to 1-6 carbon atoms. Contains atoms. The terms "alkenyl" and "alkynyl" include unsaturated straight or branched chains containing up to 10 carbon atoms, preferably 2 to 6 carbon atoms. The term "aryl" includes phenyl and naphthyl. The term "heterocyclic" includes rings containing up to 10, preferably up to 7, atoms, of which up to 3 are selected from oxygen, sulfur or nitrogen. These rings may be single ring or in the form of a fused ring system, and they may be aromatic or non-aromatic in nature. The term “halo” or “halogen” includes chlorine, fluorine, bromine and iodine. The term "alkoxy" relates to an alkyl group, as defined above, attached to an oxygen atom.

Suitably, R ² is an optionally substituted C _1-10 alkyl group, which may be straight or branched. Preferred alkyl groups R ² are straight-chain and
It contains 8 carbon atoms, especially 5 carbon atoms.

Alkyl, alkenyl and alkynyl groups R^TwoA suitable optional substituent of is halo
, Nitro, cyano, oxo, optionally substituted aryl,
Transformed heterocyclyl, OR^Three, C (O)_pR^Three, S (O)_mR^Three, OCOR^Three, −
NR^FourC (O)_pR^Three, = NOH, NR^FiveR⁶, C (O) NR^FiveR⁶, C (O) NR^ThreeN
R^FiveR⁶, -CH = NOR^Three, P (O) R⁷R⁸Or P (O) OR⁷OR⁸, NR^ThreeC
ONR^FiveR⁶, -N = CR^FiveR⁶, S (O)_mNR^FiveR⁶Or -NR^ThreeS (O)_mR^Four,
−N = NR^ThreeIncluding one or more groups selected from: wherein each R^Three, R^Four, R^Five, R ⁶ , R⁷And R⁸Is independently hydrogen, alkyl, alkenyl, alkynyl, aryl
Or heterocyclyl, either of which is required by the functional group
And, in the case of aryl and heterocyclic groups, these are also
Alkyl, alkenyl or alkynyl groups; or R^FiveAnd R⁶ Refers to a ring which, together with the atoms to which it is attached, can be carbocyclic or heterocyclic.
P is 1 or 2 and m is 0, 1, 2 or 3.

As used herein, the term “functional group” refers to halo, cyano, nitro, oxo
SO, hydroxy, = NOR¹¹, C (O)_pR¹¹, OR¹¹, S (O)_mR¹¹, NR¹² R¹³, C (O) NR¹²R¹³, OC (O) NR¹²R¹³, -CH = NOR¹¹, -NR ¹² C (O)_nR¹¹, -NR¹¹CONR¹²R¹³, -N = CR¹²R¹³, S (O)_mNR ¹² R¹³Or -NR¹²S (O)_mR¹¹Where R¹¹, R¹²You
And R¹³Are independently selected from hydrogen or optionally substituted hydrocarbyl.
Selected or R¹²And R¹³Together, together with oxygen and nitrogen
Terror atom or S (O) R¹⁴Optionally further containing, optionally substituted
Wherein p is an integer of 1 or 2 and m is 0 or 1-3
And R¹⁴Is hydrogen or alkyl.

Suitable optional substituents for the hydrocarbyl group R ¹¹ , R ¹² or R ¹³ are halo, perhaloalkyl such as trifluoromethyl, mercapto, hydroxy,
Alkoxy, oxo, heteroaryloxy, alkenyloxy, alkynyloxy, alkoxyalkoxy, aryloxy (where the aryl group can be substituted by halo, nitro or hydroxy), cyano, nitro, amino, mono- or di-alkylamino , An alkylamide or S (O) _p R ¹⁴ , wherein m and R ¹⁴ are as defined above.

Examples of optional substituents on the alkyl, alkenyl or alkynyl group R ² are oxo; alkoxycarbonyl, especially lower alkoxycarbonyl; cyano;
Phenyl optionally substituted with alkyl such as amino or mono-alkylamino or di-alkylamino or methyl; OR ³ , wherein R ³ is halo or alkyl. S (O) _m R ¹¹ , wherein m is 0 or 2, and R ¹¹ is alkyl or phenyl optionally substituted with alkyl. NR ⁵ R ⁶ or C (O) NR ⁵ R ⁶ , wherein R ⁵ is hydrogen, methyl or methoxyethyl, and R ⁶ is optionally an alkyl such as methyl, a halo such as fluoro or chloro. Optionally substituted phenyl or benzyl, such as methyl or alkyl or trifluoromethyl or alkoxycar Cycloalkenyl (wherein the alkyl moiety may additionally may possess alkoxycarbonyl group) or a, or R ⁶ is heterocyclyl, such as triazinyl which is optionally substituted by alkyl and / or acetyl; -NR ⁴ C
(O) _p R ^3, where, p is 2, R ³ is alkyl and R ⁴ is alkyl optionally substituted with alkoxycarbonyl, such as ethoxycarbonyl alkyl; -NR ³ S (O) _m R ⁴ , where R ³ is hydrogen, R ⁴ is phenyl optionally substituted by halo such as chloro, and m is 2; C (O) NR ³ NR ⁵ R ^6, wherein, R ³ and R ⁵ are hydrogen and R ⁶ is phenyl which is optionally substituted by alkoxy, such as halo or ^{_{methoxy; S (O) m NR 5}} R ⁶ , wherein m is 2, R ⁵ is hydrogen, and R ⁶ is alkyl optionally substituted by one or more alkoxycarbonyl groups; furyl, pyridyl, pyridinyl or pyrazinyl, triazinyl; Like Roshikuriru (or any of these, alkyl, halo, trihalomethyl,
Phenyl, halophenyl, cyano or oxo, which may be optionally substituted).

Particularly suitable substituents for an alkyl, alkenyl or alkynyl group R ² are alkoxycarbonyl, in particular where the alkoxy group is a lower alkyl group; alkoxy and especially two groups in the form of a dialkylacetal An alkoxy group;
Includes cyano or optionally substituted heterocyclyl. Preferred substituents include, but are not limited to, a lower alkoxycarbonyl group and a dialkyl acetal. When the alkyl group of the substituent represents R ^{1 in} the compound of the formula (I),
In the same case, alkoxycarbonyl groups and dialkylacetals are of particular interest. Because, upon hydrolysis, they give rise to the more derivatized compounds of formula (II).

A specific aryl group for R ² is phenyl.

Suitable substituents for an aryl or heterocyclyl substituent on the cycloalkyl, aryl, and heterocyclyl group R ² and the alkyl, alkenyl, or alkynyl group R ² described above are halo. Haloalkyl; cyano; nitro; amino or mono-alkylamino or di-alkylamino; hydroxy; alkoxy, thioalkyl, alkyl or alkoxycarbonyl, wherein any of these alkyl moieties is, for example, halo, alkoxy, cyano, alkoxy. Optionally substituted by one or more groups selected from carbonyl, amino, mono- or di-alkylamino, aryl, or carboxylate or salt or ester thereof); cycloalkyl; Including the cyclyl.

Particularly suitable substituents for an aryl or heterocyclyl group R ² include lower alkoxy such as alkoxy, especially methoxy, alkyl, especially lower alkyl, alkoxycarbonyl, especially lower alkoxycarbonyl and halogen.

Certain subgroups of compounds of formula (I) include compounds of formula (1A)

[0041]

Embedded image Where R ¹ is as defined above for formula (I) and n is 2
And R ¹⁰ is an alkyl, alkenyl, or alkynyl group (any of which may be optionally substituted with a heteroatom), a cycloalkyl, heterocyclic or aryl group. Or R ¹⁰ is a cycloalkyl or aryl group of valence n.

In particular, R ¹⁰ is an alkyl or aryl group of valence n.

Particularly preferred compounds of formula (I) are: ethyl 2-n-pentyl-3-oxobutanoate (compound no. 49); triethyl 2-carboxyheptane-1,7-diate (compound no. 53) ;
And ethyl 2,4-dimethoxybenzoate (Compound No. 60).

An example of a compound of formula (I) is an ethyl ester as shown in Table 1.

[0045]

[Table 1] Compounds of formula (I) are either known compounds or can be prepared from known compounds using conventional methods.

The compound of formula (I) can be chemically hydrolyzed in the target plant, or introduced into the plant by a naturally occurring enzyme, or introduced into the plant by genetic engineering, in the plant. It can be enzymatically hydrolyzed by the expressed enzyme or by a suitable catalytic antibody, or by the catalytically active portion of the catalytic antibody introduced into the plant by genetic manipulation and expressed in the plant.

[0047] Suitable enzymes include, but are not limited to, esterases and lipases.

Suitable catalytic antibodies can be generated from analogs of the tetrahedral ester hydrolysis transition state by standard techniques (eg, when the appropriate phosphonate is used)
For the hydrolysis of prodrug esters of chloramphenicol, see Ole
K et al., 1998, J. Am. Mol. Biol. 281: 501-511, and for the detoxification of cocaine by methyl ester hydrolysis, see Mets B et al.
998, Proc. Nat. Acad. Sci. USA, 95: 10176-1.
0181).

The metabolism of the compounds of the invention has been further investigated, and the results of these investigations are reported in the examples herein below. Without being limited to the consideration of the mechanism,
For example, a representative compound of the present invention (Compound 53) is represented by the following scheme:

[0050]

Embedded image Is considered to be metabolized.

The product of this metabolism is ethanol, which can act as a chemical inducer, as described above.

[0052] In a further aspect, the invention provides a method for controlling expression of a target gene in a plant, wherein the plant is operably linked to a regulatory sequence encoding a regulatory protein. A chemically inducible plant gene expression cassette comprising a promoter, and an inducible promoter operably linked to the target gene;
Upon transformation, the inducible promoter is activated by a regulatory protein in the presence of an alcohol as defined above (eg, a compound of formula (II)). This method involves hydrolyzing and expressing the above defined formula (
Applying an alcohol-forming ester such as the compound of I) to the plant.

As noted above, the regulatory sequence suitably encodes an alcR protein, and the inducible promoter is an alcA promoter sequence.

If necessary or desired, the plant may also comprise an enzyme or a catalytic antibody or a catalytically active antibody thereof that hydrolyzes a compound of formula (I) to form a compound of formula (II). The fragment can be transformed to express or overexpress it.

[0055] Enzymes that must be engineered in plants or that are inactive in the absence of other parts may be preferred in some circumstances because they are only effective in the target transformed seeds. .

The nucleic acid sequence encoding a hydrolase, antibody or antibody fragment may be included in a construct comprising a target gene operably linked to a regulatory protein and / or an inducible promoter, or they may be included in the plant May be on a separate construct used to co-transform E. coli. However, such systems are novel.

Thus, in a further aspect, there is provided a plant gene expression system comprising: (i) a first promoter operably linked to a regulatory sequence encoding a regulatory protein, (ii) a target gene Operably linked inducible promoter, wherein said inducible promoter is activated by a regulatory protein in the presence of an effective exogenous inducer of formula (I) as defined above, whereby said inducible promoter is The application of the substance
An inducible promoter that causes expression of the target gene; and (iii) a protein that results in the hydrolysis of an ester, such as a compound of formula (I), to the corresponding alcohol under the control of an additional promoter that allows expression in plant tissue. Array encoding.

The target gene may include any gene required to be introduced into a plant in order to modify the characteristics of the plant summarized above. The target gene can be an endogenous plant gene or a foreign gene, and can be a single gene or a series of genes. The target gene sequence encodes at least a portion of a functional protein or antisense sequence.

[0059] Any transformation method suitable for the target plant or plant cell may be used, including recombinant T
Includes infection with Agrobacterium tumefaciens containing the i plasmid, electroporation, microinjection of cells and protoplasts, microprojectile transformation, and pollen tube transformation. The transformed cells can then, where appropriate, be regenerated into whole plants in which the new nuclear material is stably integrated into the genome. Both transformed monocots and dicots can be obtained in this manner.

Examples of genetically modified plants that can be produced include crops, cereals, fruits and vegetables, such as: canola, sunflower, tobacco, sugar beet, cotton, soybean, corn, wheat, barley, rice, sorghum, Tomato, mango, peach, apple, pear,
Strawberries, bananas, melons, potatoes, carrots, lettuce, cabbage, onions.

The present invention further provides a plant cell comprising the gene expression system according to the present invention. This gene expression system can be stably integrated into the plant genome by transformation. The present invention also provides plant tissues or plants containing such cells, and plants or seeds derived therefrom.

Preferred examples of the compounds of the formula (I) used in this method are the compounds mentioned above.

The agriculturally acceptable esters of the present invention, such as the compounds of formula (I), are suitably applied in the form of an agriculturally acceptable composition in combination with a diluent or carrier. Such compositions form a further aspect of the present invention.

The concentration of the agriculturally acceptable ester in the formulation is preferably no more than about 5% (w / w). Concentrations between about 2% (w / w) and 5% (w / w) are preferred.

[0065] Suitable carriers or diluents will be apparent to those skilled in the art and may be used according to the formula
It will vary depending on the particular properties of the compound of (I). For example, a compound of formula (I)
When oil, it is emulsified to allow it to be sprayed with an aqueous solution
The presence of the agent may be required. Emulsifiers are well known in the art, and
Can be partially hydrolyzed polyvinyl acetate (PVA) or Tween ^TM It is.

An organic solvent or diluent such as acetone may also be present.

Accordingly, preferred compositions comprise an agriculturally acceptable ester such as a compound of formula (I), an emulsifier such as PVA and a diluent such as water. The relative amounts of these components are largely determined by the mutual miscibility of the various components. However, suitably, the emulsifier is present in the composition in an amount of 1-5% (w / w), preferably about 2.5% (w / w).

Examples of formulations of the present invention include the following: (Formulation 1) 1.5% of a compound of the invention (eg, compound 53) 2.5% of PVA balance water (formulation 2) 1.5% of a compound of the invention (eg, compound 53) 5% acetone 0.05% of Tween-20 ^™ equilibrium water (Formulation 3) 3.0% of a compound of the invention (eg, compound 53) 2.5% PVA equilibrium water (formulation 4) 3.0% of a compound of the invention (e.g., compound 53) 5% acetone / H ₂ O 0.05% of Tween-20 ^TM equilibrium water No.

An alternative composition that can be used is described in Applicants' co-pending UK Patent Application 990
Similar to the compounds described in 2236.0. In particular, these compounds, the following components: (a) Formula agriculturally acceptable ester such as a compound of (I); (b) a polyethoxylated C ₁₀ -C ₂₀ alcohol or trisiloxane polyethoxylate, And (c) a diluent.

The diluent (c) is, for example, water.

[0071] Component (b) of the above formulation is preferably a polyethoxylated oleyl alcohol, lauryl alcohol, stearyl alcohol or cetyl alcohol. More preferred are polyoxyethylene-oleyl alcohols having an average molar content of ethylene oxide in the range 0-35, more preferably in the range 2-20. Most preferred are polyoxyethylene- (2) -oleyl alcohol, polyoxyethylene- (10) -oleyl alcohol or polyoxyethylene- (20) -oleyl alcohol. However, component (b) is preferably polyoxyethylene- (20) -oleyl alcohol (the numbers in parentheses indicate the average ethylene oxide content per molecule). Such a product is called BRIJ9
2 ^™ , commercially available as BRIJ97 ^™ and BRIJ98 ^™ .

Preferably, component (b) of the formulation is at a concentration of no more than about 0.5% (w / w). Concentrations between about 0.2% (w / w) and 0.5% (w / w) are preferred.

In an alternative embodiment, the formulation comprises hydrogen or methyl end-capped trisiloxane polyethoxylate as component (b). In particular, component (b) is a methyl end-capped trisiloxane polyethoxylate. Methyl end-capped trisiloxane polyethoxylates preferably have an average ethylene oxide molar content of between 4 and 12 per molecule, and more preferably 8 per molecule. Such products are available from SILWET77 ^™ (
(SILWET is a registered trademark of Witco).

[0074] Preferably, the methyl end-capped trisiloxane polyethoxylate is at a concentration of about 0.5% (w / w) or less. Concentrations between about 0.2% and 0.5% (weight / weight) are preferred.

[0075] Component (c) of the formulation is preferably at a concentration between about 90% and 98% (w / w).

Other additives that may be included in the formulation include dispersants, antimicrobial compounds, wetting compounds, and anti-evaporants may also be added.

The present invention will now be described in detail by way of examples.

Example 1 Testing of Chemicals in Soil-Growing Plants After preliminary screening with hydroponiv solution, compounds were placed in 11/2 inch pots in a greenhouse. 4 weeks old alcc
used in tests performed on at tobacco homozygous strains. Leave sample
These plants were removed as "non-derivatized" controls. Leaves 200μ
1 in 250 mM Tris pH 8.0, centrifuged and the supernatant was collected and stored at 4 ° C. overnight. 200 mg / ml of these compounds
50% acetone 50% dH ₂ O containing 0.05% tween-20 in solution
Dissolved in. This was then diluted 1/10 to give a 2% solution. Unless otherwise stated, 5 ml of the solution was applied as a root dip to the soil of each of the two plants to be repeated. After 22 and 44 hours, leaf samples were removed, extracted as above, and the supernatant was stored at 4 ° C. Boe sample
cat using the CAT ELISA kit from Ringer Mannheim.
Analyze for protein quantification and measure total protein levels in Bradfor
d Determined by the measurement method. The results are shown in Table 2, where the CAT value is between 0 and 50.
Equivalent to detection of 00 ng / g, 2 is equivalent to detection of 5001-10000 ng / g, and 3 is equivalent to detection of 10,000-15,000 ng / g.

[0079]

[Table 2] Example 2 Further Treatment of Soil-Growing Plants In this experiment, the compound was used as a 200 mg / ml solution in 0.05% tween.
It was dissolved in 50% acetone 50% dH ₂ in O containing n-20. 1.5% of these
And diluted to 0.1% solution. Leaf samples were removed from seedlings as "non-induced" controls. Leaves were removed from 200 μl of 250 mM Tris pH8
. Triturated in 0, centrifuged and the supernatant was collected and stored at 4 ° C overnight. 5 ml of the solution was applied as a root dip to the soil of each of the two plants to be treated repeatedly. After 22 and 65 hours, leaf samples were removed from each of the two treated plants, extracted as described in Example 1, and the supernatant was stored at 4 ° C. These samples were collected from Boehringer Mannhe.
Analyzed for cat protein quantification using im's CAT ELISA kit, and total protein levels were determined by Bradford assay. The compounds tested and the results are shown in Table 3, where the CAT scores represent the values stated for Table 2.

[0080]

[Table 3] Example 3 (Measurement of time course of expression of switch after induction) To measure the time course of expression of alk switch after induction from ester,
Some compounds were applied to soil or leaves of a 5-week-old alcat homozygous strain in 30 plants in a 11/2 inch pot in a greenhouse. These compounds are
Tween-20 as either a 50 mg / ml or 50 mg / ml solution.
Was dissolved in 50% acetone and 50% dH ₂ O containing 0.05%. These are then diluted 1/10 to a 1.5% or 0.5% solution. Leave sample
Remove from seedlings as "uninduced" control. Leaf pieces were frozen in dry ice / ethanol and stored at -70 ° C. 5 ml of this solution was applied to up to eight plants as either a root dip or a leaf spray. Leaf samples were removed at various time points and frozen in dry ice / ethanol,
And stored at -70 ° C. Once all samples were harvested and stored, they were extracted in 200 μl of 250 mM Tris pH 8.0, centrifuged, and the supernatant was collected and stored at 4 ° C. overnight. Bo these samples
cat using the CAT Elisa kit from Ehringer Manheim
Analyze for protein quantification and measure total protein levels in Bradfor
d Determined by the measurement method. The compounds tested and the results are shown in Tables 4, 5, 6, 7,
8, 9 and 10. The CAT units are as shown in Example 1 above, with each successive number representing an increasing range of 5,000 ng / g. The results, shown in shaded form, were obtained in separate but similar tests.

[0081]

[Table 4]

[0082]

[Table 5]

[0083]

[Table 6]

[0084]

[Table 7]

[0085]

[Table 8]

[0086]

[Table 9]

[0087]

[Table 10] These results indicate that the time course of the induction can be modified by using the esters according to the invention compared to alcohol. Thus, proper selection of the ester results in a favorable induction profile.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 Inventor Crystal, Ivan James Turner, UK RG 426, EA Berkshire, Bracknell, Jerott's Hill Research Station (72) Inventor Jepson, IAN RG 426, EA Berkshire, Bracknell, Jerot's Hill Research Station (72) 72) Inventor Payne, Jacqueline Anne Marie UK Rakkuneru, Jefferies Charlottesville Hill research station F-term (reference) 2B030 AB04 CA15 CA17 CA19 4B024 AA07 BA07 CA01 DA01 FA02 GA11 GA17 4B065 AA88X AB01 AC20 BA02 CA05 CA27 CA47 CA53

Claims (18)

[Claims] 1. Use of an agriculturally acceptable hydrolysable ester other than γ-butyrolactone in the control of the expression of a plant gene, the control comprising the presence of an exogenous chemical for activation. Use provided by an inducible promoter in need, wherein the exogenous chemical may comprise an alcohol, wherein hydrolysis of the agriculturally acceptable ester results in the production of the alcohol. 2. The use according to claim 1, wherein said agriculturally acceptable hydrolysable ester is a compound of the following formula (I): Wherein R ¹ is a lower alkyl group, and R ² has the formula R ² C
An organic group that gives rise to the agriculturally acceptable acid of OOH; and said promoter requires the presence of an alcohol of formula (II) for its activation: use. 3. Use according to claim 2, wherein said compound of formula (II) is a lower alkyl alcohol, wherein said alkyl group R ¹ is
The use, having 4 carbon atoms and which can be either branched or linear. 4. Use according to claim 2 or 3, wherein the R ²
Is an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aryl, or an The use, which is heterocyclyl, optionally substituted. 5. The use according to claim 4, wherein said R ² is an optionally substituted C _1-10 alkyl group. 6. The use according to claim 5, wherein said R ² is substituted by an alkoxycarbonyl group or a dialkyl acetal. 7. Use according to any one of claims 1 to 6, wherein the compound of formula (I) is ethyl 2-n-pentyl-3-oxobutanoate; triethyl 2-carboxyheptane-1,7-dioate; or ethyl 2,
Use which is 4-dimethoxybenzoate. 8. A method for controlling the expression of a target gene in a plant, comprising:
Here, the plant uses a chemically inducible plant gene expression cassette comprising a first promoter operably linked to a regulatory sequence encoding a regulatory protein and an inducible promoter operably linked to a target gene. Has been transformed,
Wherein the inducible promoter is activated by the regulatory protein in the presence of an alcohol, the method comprising applying to the plant an agriculturally acceptable hydrolysable ester other than γ-butyrolactone; Wherein the ester forms the alcohol upon hydrolysis thereof, thereby causing expression of the target gene. 9. The method of claim 8, wherein said regulatory sequence comprises:
Encodes the alcR protein as described above, and wherein the derived protein
The method, which is a cA promoter sequence. 10. The method of claim 8 or claim 9, wherein the plant is transformed, such that the plant hydrolyzes the ester to form the alcohol. A method wherein the catalytic antibody or a catalytically active fragment thereof is expressed or overexpressed. 11. A plant gene expression system, wherein the plant gene expression system comprises:
(I) a first promoter operably linked to a regulatory sequence encoding a regulatory protein; (ii) an inducible promoter operably linked to a target gene, wherein the inducible promoter is an effective exogenous inducible alcohol. An inducible promoter that is activated by a regulatory protein in the presence of, whereby application of said alcohol causes expression of said target gene; and (iii) a sequence encoding a protein that results in ester hydrolysis. A sequence that allows expression in plant tissue under the control of a promoter,
A plant gene expression system comprising: 12. A plant cell comprising the gene expression system according to claim 11. 13. A plant tissue or plant and a plant or seed derived therefrom, comprising the cell of claim 12 containing the gene expression system of claim 11. 14. An agricultural composition, said agricultural composition comprising an agriculturally acceptable ester as defined in claim 1 in combination with a diluent or carrier. Composition. 15. The composition according to claim 14, wherein the agriculturally acceptable ester is a compound of formula (I) as defined in claim 2. 16. The composition according to claim 14, further comprising an emulsifier. 17. The composition according to claim 16, wherein said emulsifier is PVA. 18. A method for controlling the expression of a target gene in a plant, wherein the plant is operable with a first promoter and a target gene operably linked to a regulatory sequence encoding a regulatory protein. Has been transformed with a chemically inducible plant gene expression cassette containing an inducible promoter operably linked, wherein the inducible promoter is activated by the regulatory protein in the presence of alcohol, and wherein The plant is further transformed such that the plant expresses or overexpresses an enzyme that hydrolyzes an ester to form an alcohol or a catalytic antibody or catalytically active fragment thereof; The method comprises applying to the plant an agriculturally acceptable hydrolysable ester, wherein the ester is a hydrolyzable ester thereof. A method wherein upon degradation, the alcohol is produced, resulting in expression of the target gene.