KR20020013493A - Inducible expression system for use in plants - Google Patents

Abstract

In regulating plant gene expression, R ¹ and R ² are methods of using an agriculturally acceptable hydrolysable ester, such as a compound of formula (I), as defined in the application, Is carried out by an inducible promoter which is activated in the presence of water and produces said alcohol upon hydrolysis of said agriculturally acceptable ester. Preparations of such esters are also described and claimed.

Description

[0001] Description [0002] INDUCIBLE EXPRESSION SYSTEM FOR USE IN PLANTS [0003]

Expression of the gene is regulated by the 5 ' upper portion of the protein encoding portion, generally called the promoter. The promoter may be homeostatic, cell-specific, developmental-programmable or inducible.

Manipulation to improve the characteristics (such as productivity or quality) of an agricultural plant requires the expression of an exogenous or resistant gene in plant cells. This genetic manipulation thus depends on the availability of means for regulating gene expression as required: for example, on the availability and availability of appropriate promoters effective in plants. Having the selection of various kinds of promoters is advantageous in that it is possible to select a promoter most suitable for a specific gene, construct, cell, tissue, plant or environment. A series of promoters that are operable in plants are known.

A particularly useful promoter is a promoter sequence that is regulated by the application of an external chemical stimulus. This enables the regulation of gene expression in plant growth or in certain stages of development, for example by the presence or absence of chemicals applicable to plants or seeds, such as by application or using known seed coating techniques. These are also known as gene "switch" promoters.

A gene under the control of an inducible promoter may be an inducible promoter that regulates the expression of a gene of the desired characteristic or phenotype itself or an inhibitory protein that inhibits the onset of the target gene. For example, ( E. G. , The tet and lac operator / repressor systems of bacteria). Furthermore, alternatively, a gene under the control of an inducible promoter can express a protein that interferes with the activity of another protein to inhibit its activity. For example, a gene that inhibits or kills cells in the absence of Basta, (barnase / barstar) system.

These types of gene switches are known in a variety of applications. This includes the production of reversibly sterile males which are highly desirable in hybrid plant production, for example as described in WO 90/08830. Other applications of these promoters include the prevention of pathogen formation, the contamination of certain crop plants, particularly the protection from germ cell cytoplasm in transgenic plants, and the need for control of native plants and the need for harvesting - pregermination as described in WO 94/03619 Prevention.

Many organisms have mechanisms that can metabolize chemicals such as alcohol or ketones, such as the production of alcohol dehydrogenase enzymes. Promoters of such systems may be useful in gene switches as promoters that are induced by the presence of a target alcohol or ketone.

This example is found in the fungal organism Aspergillus nidulans , which expresses alcohol dehydrogenase I (ADH 1), which is encoded by the gene alc A when grown in the presence of various alcohols and ketones. Induction is accomplished through the regulator protein, which is expressed by the alc R gene, which is always expressed as an antagonist. In the presence of a derivative (alcohol or ketone), the regulatory protein activates the expression of the alc A gene. In the presence of derivatives, regulatory proteins also increase their expression. This means the high expression level of ADH 1 under the induction conditions (i.e. in the presence of alcohol or ketone). Conversely, the alc A gene and its protein ADH 1 are not expressed at the time of absence of the derivative. Expression of alc A and enzymes is also inhibited in the presence of glucose.

Thus, the alc A gene promoter is an inducible promoter that is activated by the alc R regulatory protein (i. E. , By protein / alcohol or protein / ketone combination) in the presence of the derivative. (Including each promoter), the alc R and alc A genes are homologously sequenced (Lockington RA et al, Gene, 33: 137-149; Felenbol B et al, 1988, Gene, 73: 385- 396; Gwynne et al, 1987, Gene, 51: 205-216).

Alcohol dehydrogenase ( adh ) Genes are being studied in certain plant species. In corn and other cereals, this gene is switched by anaerobic conditions. Corn adh The promoter portion of the gene contains a 300 bp regulatory element for expression in anaerobic conditions. But, alc RThe gene corresponding to the regulatory protein was not found in any plant. therefore alc R / alc AThe gene control system of the type is not known in plants. In plant cells alc ROf homeostasis is resistant adh It does not activate the activity.

WO 93/21334 describes the production of transgenic plants comprising such a system as a gene switch. This document describes in particular a primary promoter operably linked to a regulatory sequence encoding a regulatory protein and a second promoter operably linked to the target gene, wherein the application of the derivative is activated by a regulatory protein in the presence of an effective external derivative causing expression of the target gene Inducible plant gene expression cassette comprising an inducible promoter. In particular, the alc R / alc A system is used in constructs. The external chemical derivatives applied in this case are described by Creaser et al., J. Biochem. (Ethyl methyl ketone), cyclohexanone, acetone, butane-2 ol, 3-oxobutyl acid, propan-2-ol and ethanol .

Alcohol is commonly used in agricultural applications. However, these chemicals are often very volatile and can be difficult to handle in an agricultural environment due to the loss of large amounts of chemicals during application.

The present invention relates to expression systems for use in plants, in particular to expression systems which utilize external chemicals as regulatory machinery, and to the use of certain chemicals as such modulating agents.

The present invention relates to the use of an agriculturally acceptable hydrolysable ester for the control of the expression of a plant gene, said regulation being carried out by an inducible promoter which requires the presence of an external chemical capable of containing alcohol for activation, Lt; / RTI > provides the use in which the hydrolysis of the acutely acceptable ester produces the alcohol.

In particular, agriculturally acceptable esters include the following compounds of formula (I)

Wherein R ¹ is a lower alkenyl, lower alkenyl or lower alkynyl group, and R ² is an agriculturally acceptable acid and is an organic group such as R ² COOH. The hydrolysis of the general formula (I) yields an alcohol of the general formula (II)

As used herein, " agriculturally acceptable " means a compound that can be applied without causing unacceptable level of soil damage or phytotoxicity of the crop in a particular soil or crop environment.

As used herein, " lower alkyl " includes a C _1-6 alkyl group, preferably a C _1-4 alkyl group, which may be linear or branched. As used herein, "lower alkenyl" and "lower alkynyl" are C _2-6 alkenyl and C _2-6 alkynyl groups, which may be linear or molecular chain, preferably C _2-4 alkenyl groups or C _{2 -4-} alkynyl group, respectively.

Agriculturally acceptable esters, such as the formula (I) used in the present invention, are suitably transferred to the location of the gene regulatory system in the target plant and / or the presence of a suitable catalytic moiety such as an environmental condition or an enzyme or catalytic antibody Lt; / RTI > is hydrolyzed at an appropriate rate to provide a sufficient amount of the active alcohol for the time required in the required portion of the plant. This depends on the nature of the plant species being treated, the gene being expressed, and the application time of the ester.

Esters, such as compounds of formula (I), have the advantage of being more manageable than the corresponding alcohols. These compounds have been found to produce the desired effect in terms of gene activation.

The compound must be applied over a period of time sufficient to hydrolyze prior to the required gene activation, depending on factors such as the growth stage of the plant that requires activation. If the hydrolysis rate is relatively slow, the application time may be earlier so that the hydrolysis occurs sufficiently until the activation of the gene necessary for the growth stage of the plant is required. If this is difficult, a more rapidly hydrolyzed ester can be selected.

Alternatively, one or more esters in a single treatment may be applied at different hydrolysis rates. With the choice of ester combinations of different hydrolysis rates, effective " slow release " of the active alcohol can extend gene expression for a desired period of time. This means that repeated applications of chemicals can be avoided and a "one-time" treatment is possible.

Specific examples of the general formula (II) include methanol, ethanol, propan-1-ol, propan-2-ol, butane-2-ol or butan-3-en-2-ol.

Suitably, the alcohol of formula (II) is a lower alkyl alcohol of alkyl having 1 to 4 carbon atoms which may be branched or linear. Preferred groups of R < ¹ > are ethyl, n-propyl and n-butyl.

An example of a particularly preferred compound of formula (II) is ethanol.

The exact nature of R ² does not matter as long as it produces an agriculturally acceptable acid at the appropriate rate in the particular target plant being applied. The rate of hydrolysis is described, for example, by G. Mitchell et al., Pestic. Sci (1995) 44: 49-58, and preferably in whole plant systems. Appropriate rates in any particular environment will depend on a variety of factors including the nature of the regulated gene expression, the particular plant in which the gene is expressed, and other external conditions. The rate of hydrolysis should be such that, for example, in the case of reversible infertile males, it is possible to produce the desired effect for the appropriate period after application of the chemical derivative.

R < ^{2 >} is, however, from the following general formula (III)

Can be selected as acids with useful agricultural effects produced. In particular, it can act as a derivative of an inducible promoter by itself. For example, it has been discovered that many acids, including 3-hydroxybutyric acid, 2-hydroxybutyric acid, pyruvic acid, and 3-oxobutyric acid, can act as derivatives in the alcR / alcA system (Creaser et al. remind).

Particular examples of R < ² > include optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl or optionally substituted heterocyclyl, .

As used herein, " alkyl " includes linear or branched alkyl chains and suitably contains up to 10, preferably 1 to 6, carbon atoms. &Quot; Alkenyl " and " alkynyl " include unsaturated linear or branched chains suitably containing up to 10, preferably 1 to 6, carbon atoms. &Quot; Aryl " includes phenyl and naphthyl. &Quot; Heterocyclyl " contains up to 10, preferably up to 7, atoms, of which up to three include rings that are atoms selected from oxygen, sulfur or nitrogen. This can be in the form of a single ring or ring system fused, and these can be aromatic or non-aromatic in nature. &Quot; Halo " or " halogen " includes chlorine, fluorine, bromine and iodine. &Quot; Alkoxy " refers to alkyl as defined above linked to an oxygen atom.

R ² is an optionally substituted C _1-10 alkyl group, which may be suitably linear or branched. Preferred alkyl groups and R ² is linear and contains from 3 to 8, especially 5 carbon atoms.

Alkyl, alkenyl, and alkynyl groups suitable optional substituents on R ² are halogen, nitro, cyano, oxo, optionally substituted aryl, when an optionally substituted heterocyclic ^{dimethacrylate, OR 3, C (O)} P R 3 ^{_{, S (O) m R 3}} , OCOR 3, -NR 4 C (O) P R 3, = NOH, NR 5 R 6, C (O) NR 5 R 6, C (O) NR 3 NR 5 R 6 ^{, -CH = NOR 3, P (} O) R 7 R 8 , or ^{P (O) oR 7 oR 8} , NR 3 CONR 5 R 6, -N = CR 5 R 6, S (O) m NR 5 R 6 or _{^{-NR 3 S (O) m R}} 4, -N = NR ^3, and containing one or more groups selected from, in the ^{R 3, R 4, R 5} , R 6, R 7, and R ⁸ are independently Alkyl, alkenyl, alkynyl, aryl or heterocyclyl optionally substituted with a functional group; in the case of aryl and heterocyclyl, it may be substituted by an alkyl, alkenyl or alkynyl group; Or R < ⁵ > and R < ⁶ >, together with the atoms attached to them, may further form a ring which may be carbocyclic or heterocyclic; p is 1 or 2, and m is 0, 1, 2, or 3.

Herein, the "functional group (functional group)" is halo, cyano, nitro, oxo, ^{hydroxy, = NOR 11, C (O} ) P R 11, OR 11, S (O) m R 11, NR 12 R used in ^{13, C (O) NR 12} R 13, OC (O) NR 12 R 13, -CH = NOR 11, -NR 12 C (O) n R 11, -NR 11 CONR 12 R 13, -N = CR 12 _{^{R 13, S (O) m}} NR 12 R 13, -NR 12 S (O) m R ¹¹ includes, where R ^11, R ¹² and R ¹³ are independently selected from a hydrocarbon (hydrocarbyl substituted with hydrogen or optionally in Or R ¹² and R ¹³ together form an optionally substituted ring optionally containing an additional heteroatom such as oxygen and nitrogen or S (O) R ¹⁴ , wherein p is 1 or 2 , m is an integer of 1 to 3, and R ¹⁴ is hydrogen or alkyl.

Suitable optional substituents for the hydrocarbyl group R ¹¹ , R ¹² or R ¹³ include halogen, perhaloalkyl such as trifluoromethyl, mercapto, hydroxy, alkoxy, oxo, heteroaryloxy, (Wherein the aryl group may be substituted by halogen, nitro, or hydroxy), cyano, nitro, amino, mono- or di- alkylamino, alkylamido or, and S (O) _p R ¹⁴ include a, m, and R ¹⁴ is defined above.

Examples of substituents on the alkyl, alkenyl or alkynyl group R ² include oxo; Alkoxycarbonyl especially lower alkoxycarbonyl; Cyano, chlorine, fluorine or bromine and the liver is halogen; Amino or mono-or dialkylamino or alkyl, such as methyl; OR ^{3 wherein} R ³ is alkyl or heterocyclyl optionally substituted by halogen or alkyl; S (O) _m R < ¹¹ > wherein m is 0 or 2 and R < ¹¹ > is alkyl or phenyl optionally substituted with alkyl; R ⁵ is hydrogen, methyl or methoxyalkyl; R ⁶ is alkyl such as methyl, phenyl or benzyl optionally substituted with halogen such as fluorine or chlorine; or alkyl such as methyl having an additional alkoxycarbonyl group in or alkyl such as methyl, or alkoxycarbonyl with a triple, or R ⁶ is a heterocyclic dimethacrylate, such as alkyl and / or acetyl optionally thiazinyl (thiazinyl) substituted with NR ⁵ R ⁶ or C (O) NR ⁵ R ⁶ ; p is 2, R3 is alkyl, R ⁴ is ethoxycarbonyl-alkyl alkoxycarbonyl optionally the alkyl -NR ⁴ C (O) substituted by pR3, such as a; R ³ is hydrogen, R ⁴ is phenyl optionally substituted with halogen such as chlorine, m is 2, -NR ³ S (O) _m R ⁴ ; R ³ and R ⁵ is NR ³ NR ⁵ R ⁶ is hydrogen, R ⁶ is a C (O) optionally substituted by alkoxy or halogen, such as methoxyphenyl; m is 2, R ⁵ is hydrogen, R ⁶ is an S (O) alkyl optionally substituted with one or more alkoxycarbonyl _m NR ⁵ R ^6; Furyl, pyridyl, pyridinyl or pyrazinyl, triazinyl, which may optionally be substituted with alkyl, halogen, trihalomethyl, phenyl, halophenyl, cyano or oxo, And < / RTI >

Particularly suitable substituents for the alkyl, alkenyl or alkynyl group R ² are those in which the alkoxycarbonyl, especially the alkoxy group, is a lower alkyl group; Alkoxy and especially two alkoxy groups in dialkyl acetal form; Cyano or optionally substituted heterocyclyl. Preferred selectors include, but are not limited to, lower alkoxycarbonyl groups and dialkyl acetals. Of particular interest are alkoxycarbonyl groups and dialkyl acetals, because the alkyl group of the substituent is the same as R < ¹ > of the compound of formula (I), since hydrolysis of the compound further produces derivatives of formula (II).

The specific aryl group of R < ² > is phenyl.

Suitable optional substituents for the cycloalkyl, aryl and heterocyclyl groups R < ² > and the aryl or heterocyclyl substituents of the above-mentioned alkyl, alkenyl or alkynyl groups R < ² > include halogen; Haloalkyl; Cyano; Nitro; Amino or mono-or di-alkylamino; Hydroxy; Alkyl moieties include, for example, halogen, alkoxy, cyano, alkoxycarbonyl, amino, mono- or di-alkylamino, aryl or carboxylate or salts or esters thereof; Cycloalkoxy; Alkoxy, thioalkyl, alkyl, or alkoxycarbonyl, which may be optionally substituted with one or more groups selected from alkyl, alkoxy, aryloxy, or heterocyclyl.

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

A particular sub-group of compounds of formula (I) is a compound of formula (1A)

Wherein R ¹ is as defined above with respect to formula (I), n is an integer from 2 to 4, and R ¹⁰ is alkyl, cycloalkyl, heterocyclic group or aryl group optionally interposed therebetween, Alkenyl or alkynyl group, or R < ¹⁰ > is a cycloalkyl or aryl group of valency n.

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

Particularly preferred compounds of formula (I) include: - ethyl 2-n-pentyl-3-oxobutanoate (Compound No. 49); Triethyl 2-carboxyheptane-1,7-dioate (Compound No. 52); And ethyl 2,4-dimethoxybenzoate (Compound No. 60).

Of the compounds of formula (I) are the ethyl esters shown in Table 1.

The compound of formula (I) may also be a known compound which can be prepared from a known compound using a conventional method.

The compounds of the general formula (I) may be used either chemically in a target plant, or an enzyme introduced or expressed through genetic engineering to an enzyme or plant naturally present in the target plant, or an appropriate enzyme introduced or expressed through genetic engineering Catalytic antibody, or a portion of a catalytic antibody.

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

Suitable catalytic antibodies can be produced by standard techniques from analogs in the hydrolysis state of the tetrahedraester ester, for example when the appropriate phosphonate is used, the hydrolysis of the pro-drug ester of the chloramphenicol pro- (Ole K et al., 1998, J. Mol. Biol., 281: 501-511) and toxic removal of cocaine by methyl ester hydrolysis (Mets B et al., 1998, Proc. Nat. USA, 95: 10176-10181).

The metabolism of the compounds of the present invention was further investigated, and the results of the investigation are reported in the following examples. Without intending to be limited by the mechanistic considerations, for example, a representative compound of the present invention, compound 53, is metabolized in accordance with the following steps:

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

In a further aspect, the present invention provides a method of producing a protein comprising the steps of: (a) providing a first promoter operably linked to a regulatory sequence encoding a regulatory protein, and (b) an inducible promoter activated by a regulatory protein in the presence of an alcohol, A method for regulating the expression of a target gene in a plant transforming a plant with a chemically-inducible plant gene expression cassette comprising an ester which is hydrolyzed to form the alcohol as defined above (I) To plants to induce the expression of the target gene.

The moderately regulatory sequence encodes the alcR protein described above and the inducible promoter is the alcA promoter sequence.

If necessary or desired, the plant may also be used to express or overexpress enzymes or catalytic antibodies or catalytically active fragments thereof which hydrolyze compounds of general formula (I) to form compounds of general formula (II) Can be transformed.

In the absence of enzymes or other parts to be manipulated and inserted into plants, inactive enzymes may be effective in the target transformed seeds and therefore may be desirable in some circumstances.

The nucleic acid sequence encoding the enzyme, antibody or antibody fragment may be contained in a construct containing a target gene operably linked to a regulatory protein and / or an inducible promoter, or may be a co-tranform distinct construct Lt; / RTI > Such systems are new.

Thus, a further aspect provides a plant gene expression system comprising:

(I) a primary promoter operably linked to a regulatory sequence encoding a regulatory protein;

(Ii) an inducible promoter operably linked to the target gene, wherein the application is activated by a regulatory protein in the presence of an effective external inducing agent of the general formula (I) above causing expression of the target gene; And

(Iii) a sequence encoding a protein that affects the degradation of an ester, such as a compound of formula (I), to the corresponding alcohol under the control of an additional promoter that causes expression in plant tissue.

The target gene may comprise any gene that needs to be introduced into a plant to modify the trait. The target gene may be a resistant plant gene or an external gene, and a single gene or a series of genes. The target gene sequence encodes at least a portion of the functional protein or antisense.

The method of infection, the method of electrotransmission, the method of fine introduction into cells and plasma cells, the microprojectile impact method, the method of bacterial impact, especially the "fiber" method, the method of infection with Agrobacterium tumefaciens containing Ti recombinant plasmid, Any transformation method suitable for the target plant or plant cell, including " whisker " methods, and pollen tube transformation, can be selected. Transformed cells are regenerated as whole plants when new nucleic acid materials are stably introduced into the genome. Both the transformed cotyledon and the dicotyledonous plant can be obtained in this way.

Examples of genetically modified plants that can be made are: field crops, grains, fruits and vegetables: canola, sunflower, tobacco, modern, cotton, beans, corn, wheat, barley, rice, sorghum, tomatoes, peaches, , Strawberries, bananas, melons, potatoes, carrots, lettuce, cabbage, and onions.

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

In this process, preferred examples of the compounds of formula (I) are mentioned above.

The agriculturally acceptable esters of the present invention, such as the compounds of general formula (I), may be applied as a form of a suitably agriculturally acceptable composition, in the form of a diluent or carrier. Such a composition forms a further aspect of the present invention.

The concentration of agriculturally acceptable esters such as the compounds of formula (I) of the present invention is preferably 5% w / w or less. And preferably a concentration of 2% to 5% weight / weight.

Suitable carriers or diluents vary according to the particular nature of the compounds of general formula (I) which are apparent to the person skilled in the art and which are used. For example, if the compound of formula (I) is an oil, the presence of an emulsifier to be sprayed in an aqueous solution may be necessary. Emulsifiers are well known to those skilled in the art, and specific examples are partially hydrolyzed polyvinyl acetate (PVA) or Tween ^TM .

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

Thus, 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 amount of composition depends to a large extent on the mixture of the various constituents. Suitably, however, the emulsifier will be present in the composition in an amount of from 1 to 5% w / w, preferably 2.5% w / w.

Thus, examples of compositions of the present invention include:

Composition 1

1.5% The compound of the present invention (for example, Compound 53)

2.5% PVA

Balance water

Composition 2

1.5% The compound of the present invention (for example, Compound 53)

5% acetone

0.05% Tween-20 ^TM

Balance water

Composition 3

3.0% The compound of the present invention (for example, Compound 53)

2.5% PVA

Balance water

Composition 4

3.0% The compound of the present invention (for example, Compound 53)

5% acetone / water

0.05% Tween-20 ^TM

Balance water

Alternative compositions may be used, analogous to those described in co-pending UK application number 9902236.0. In particular, the compositions comprise the following components:

(a) an agriculturally acceptable ester such as a compound of formula (I);

(b) a polyethoxylated C ₁₀ -C ₂₀ alcohol or trisiloxane polyethoxylate; And

(c) Thinner

The diluent (c) may be water, for example.

The component (b) of the composition is preferably a polyethoxylated oleyl, lauryl, stearyl or cetyl alcohol. More preferably a polyoxyethylene-oleoyl alcohol having an average molar ethylene oxide content in the range of 0 to 35, more preferably in the range of 2 to 20. Most preferred are polyoxyethylene- (2) -oleyl alcohols, polyoxyethylene- (10) -oleyl alcohols or polyoxyethylene- (20) -oleyl alcohols. However, component (b) is preferably polyoxyethylene- (20) -oleyl alcohol (the number in parentheses indicates the average ethylene oxide content per molecule). These are commercially available as BRIJ 92 ^TM , BRIJ 97 ^TM , and BRIJ 98 ^TM .

Preferably, the composition (b) of the composition is at a concentration of 0.5% weight / weight or less. Preferably between 0.2% w / w and 0.5% w / w.

In an alternative embodiment, hydrogen or methyl end-capped trisiloxane polyethoxylate is included as component (b) of the formulation used in the process of the present invention. In particular, component (b) is a methyl end-capric trisiloxane polyethoxylate. The methyl end-capric trisiloxane polyethoxylate preferably has an average molar ethylene oxide content of from 4 to 12 per molecule, most preferably 8 per molecule. These are commercially available as SILWETT 77 ^TM (SILWETT is a trademark of Witco).

Preferably, the trisiloxane polyethoxylate in the methyl end-cap is at a concentration of about 0.5% weight / weight or less. And preferably between 0.2% and 0.5% weight / weight.

Component (c) of the formulation is preferably between about 90% and 98% weight / weight.

Other additives such as antimicrobial compounds, dispersants, wetting compounds and anti-volatilizing agents may be added.

The invention will be described in detail in the examples.

Example 1

Chemical testing of soil growing plants

Following hydroponic solution preliminary screening, a compound test was performed in a 1.5 "pot of 4-week alccat tobacco homozygote conjugate line 30 in a greenhouse." Undetected "leaf samples were separated as a control. The leaf pieces were incubated with 200 μl of 250 mM Tris pH 8.0 and the supernatant was collected, centrifuged and recovered and stored overnight at 4 ° C. The compound was dissolved in 200 mg / ml in 50% acetone 50% water with 0.05% Tween-20 added, Unless otherwise noted, 5 ml of solution is applied roughened to each soil of the two plants of the duplicate treatment test. After 22 and 44 hours, the leaf sample is separated The cat protein was quantitated using a CAT ELISA kit from Boehringer Manheim and the total protein was determined by Bradford crystallization. The results are shown in Table 2 W becomes, CAT value of 1 0-5000ng / g, 2 is 5001-10000ng / g, 3 corresponds to 10001-15000ng / g.

Compound No. CAT value 0 hours 22 hours 44 hours One 0 One 0 (2.5 mls) 2 0 One 0 3 0 One 2 (2.5 mls) 4 0 0 One 5 0 0 3 8 One One One 9 One 0 2 10 2 0 One 12 One 0 2 13 0 0 0 14 0 One 0 15 0 One One 16 One One 6 17 One 2 One 18 0 One One 19 0 One One 20 0 0 One 21 0 0 One 22 0 0 One 23 0 0 One 24 One 0 One 25 0 0 One (2.5 mls) 26 0 0 0 28 0 0 One 30 0 2 2 32 0 0 2 33 One One One (2.5 mls) 35 0 0 One 36 0 0 One 38 0 0 3 39 0 0 One 40 0 0 0 (2.5 mls) 41 One 0 One 42 2 One One 43 0 0 2 44 One 0 0 45 One 0 2 46 2 0 One 47 3 0 2 5 mls of preparation 0 0 0 2.5 mls of preparation 0 0 0 5% ethanol One 8 10 5% butane-1-o1 One One 3 dH20 One 0 0

Example 2

Further processing of soil growing plants

In this experiment, the compound was dissolved in a 200 mg / ml solution in 50% acetone 50% water at 0.05% Tween-20. They were diluted with 1.5% and 0.1% solutions. A " not induced " leaf sample from the seedling was isolated as a control. The leaf pieces were pulverized in 200 μl of 250 mM Tris pH 8.0, the supernatant was recovered, centrifuged and recovered and stored overnight at 4 ° C. 5 ml of the solution is applied to each soil of the two plants of the duplicate treatment test roots. After 22 and 65 hours the leaf samples were separated and extracted as in Example 1 and the supernatant was stored at 4 ° C overnight. The samples were quantitated using catheter kit from Boehringer Mannheim, cat protein, and the total protein was determined by Bradford crystals. The results are shown in Table 3, and the CAT values represent the values set in Table 2, respectively.

Compound No. CAT protein value 1.5% root roots 0 hours 22 hours 65 hours 17 0 3 One 50 0 2 One 51 0 One 3 51 0 One 3 52 0 2 One 53 0 3 8 54 0 One 2 55 0 One One 56 0 2 One 57 0 2 5 58 0 One 0 59 0 One One 61 0 One One 62 0 One 2 63 0 One One 64 0 One One 65 0 0 One ethanol 0 3 6 Butan-1-ol 0 3 5 Preparation 0 0 0 dH20 0 0 0

Example 3

Determination of the time course of the expression switch after induction

To determine the expression time function of the alc switch after induction of ester ovule, various compounds were applied to the 5 week alccate homozygous line 30 plant in a 1.5 " pot of soil or greenhouse. The compounds were grown in 50% acetone The leaf samples were separated into a " not induced " control from the seedlings.The leaf pieces were frozen in dry ice / ethanol and stored in -70 < RTI ID = ° C. 5 ml of the solution was applied as root seed or leaf spray to up to 8. The leaf samples were separated at various times and frozen in dry ice / ethanol and stored at -70 ° C. All After the samples were prepared, the leaf pieces were pulverized in 200 μl of 250 mM Tris pH 8.0, the supernatant was recovered, centrifuged and recovered and stored at 4 ° C. overnight. The cat protein was quantitated using a Mannheim CAT ELISA kit and the total protein was determined by Bradford crystal. The compounds tested and the results are shown in Tables 4, 5, 6, 7, 8, 9 and 10. The results of the shaded form are the results obtained in an independent but similar test.

time Compound 51 CAT value 05102648981441930510264898144193051026489814419301022 Time 264865 hours 98 0.5% 49 injection 0.5% 49 S0.5 49% S0.5 49% S0.5 49% S0.5 49% S0.5 49% S0.5 49% S1.5 49% S1.5 49% S1.5 49% S1.5 49% S1.5 49% S1.5 49% S1.5 49% S1.5 49% S0.5% 49 Root wetting 0.5% 49% R0.5% 49% R0.5% 49% R0.5 49% R0.5 49% R0.5 49% R0.5 49% R1.5% 49 R1.5% 49 R1.5% Root 1.5% 49 R1.5% 49 R1.5% Root 1.5% 49 R 0000000000001000001214400111334

time Compound No. 57 CAT value t = 26t = 48t = 0t = 5t = 10t = 26t = 48t = 0t = 5t = 10t = 26t = .5% 30% .5% 30 S.5% 30 S.5% 30 S.5% 30 S1.5% 30S1.5% 30S1.5% 30S1.5% 30S1.5% 30S.5% 30 root 30% RH 30% 0.5% 30% R0.5% 30% R0.5% 30% R0.5% 30% R0.5% 30% R1.5% 30% 30R 00011000000012240121253

Time Compound No. 53 CAT value 0 0.5% 51 injection 05 0.5% 51 S 010 0.5 51% S 126 0.5% 51 S 148 0.5% 51 S 198 0.5% 51 1144 0.5 0.5% 51 1193 0.5 51% S 1.5 1.5% S 05 1.5% 51 S 110 1.5% 51 S 226 1.5% 51 S 148 1.5% 51 S 198 1.5% 51 S 1144 1.5% 51 S 0193 1.5% S 10 0.5% 51 Root wetting 05 0.5% 51 R 110 0.5% 51 R 126 0.5% 51 R 148 0.5% 51 R 198 0.5% 51 R 3144 0.5% 51 R 2193 0.5% 51 R 00 1.5% 51 R 010 1.5% 51 R 122 1.5% Roots 426 1.5% 51 R 448 1.5% 51 R 865 hr 1.5% Root 898 1.5% 51 R 5

time Compound No. 60 CAT value t = 0 t = 10 t = 26 t = 48 t = 144 t = 0 t = 1022 time t = 26 t = 4865 time t = 98 0.5% 57 Root wetting 0.5% 57 R0.5 57% R0.5 57% R0.5 57% R1.5 57% R1.5% 57 R1.5% Root 1.5% 57 R1.5 57 R1.5% Roots 1.5% 57 R 011258011114

time Compound 52 CAT value t = 0t = 10t = 26t = 48t = 98t = 144t = 193t = 0t = 10t = 26t = 48t = 98t = 144t = 193t = 0t = 10t = 26t = 48t = 98t = 144t = 193t = 26t = 4865 hours t = 98 0.5% 50% 0.5% 50S0.5% 50S0.5% 50S0.5% 50S0.5% 50S0.5% 50S1.5% 50S1.5% 50S1.5% 50S1.5% 50S1.5% 50S1.5% 50S1.5% 50S0.5% 50 Root wetting 0.5% 50R0.5% 50R0.5% 50R0.5% 50R0.5% 50R0.5% 50R1.5% 50R1.5% 50R1.5% roots 1.5% 50R1. 5% 50R1.5% Root 1.5% 50R 0001000000000001121110021211

Time 051026489814419305102648981441930510264898144193010264898 Ethanol Etoh 0.5% Injection Etoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% SEtoh 0.5% Root wetting Etoh 0.5% REtoh 0.5% REtoh 0.5% REtoh 0.5% REtoh 0.5% RETOh 0.5% RETOh 0.5% RETOh 1.5% RETOh 1.5% RETOh 1.5% RETOh 1.5% RETOh 1.5% R CAT value 02221111024542210246444304976

time dH ₂ 0dH ₂ 0 Root wetting dH ₂ 0 RdH ₂ 0 RdH ₂ 0 RdH ₂ 0 RdH ₂ 0 RdH ₂ 0 RdH ₂ 0 R CAT value 00000000 0510264898144193 Formulation time 0510264898144191930510264898144193 Injection SSSSSSS Root wetting RRRRRRR 0000000000001110

These results show that the derivation time function can be changed in comparison with the alcohol using the ester according to the present invention. This leads to the preferred induction profile with the choice of suitable ester.

Claims (17)

In the use of an agriculturally acceptable hydrolysable ester for the modulation of the expression of a plant gene, said regulation is carried out by an inducible promoter which requires the presence of an external chemical capable of containing an alcohol for activation, Wherein the hydrolysis of the as possible ester produces the alcohol. The process according to claim 1, wherein the agriculturally acceptable hydrolyzable ester is a compound of formula (I) Wherein R ¹ is a lower alkyl group and R ² is an organic group which upon hydrolysis produces an agriculturally acceptable acid of the general formula R ₂ COOH; And wherein said promoter is required for its activation in the presence of an alcohol of formula (II). 3. The method of claim 2, general formula (Ⅱ) compounds in which the alkyl group R ¹ having from 1 to 4 carbon atoms, branched or linear, lower (lower) alkyl alcohol in which the number of applications. 4. The compound of claim 2 or 3 wherein R < ² > is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, Heterocyclyl. 5. The method of claim 4, wherein R < ^{2 >} is an optionally substituted C _1-10 alkyl group. 6. The method of claim 5 wherein R < ² > is substituted with an alkoxycarbonyl group or a dialkyl acetal. 7. The compound 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 2,4-dimethoxybenzoate. A method for regulating the expression of a target gene in a plant, the method comprising: contacting a plant with a primary promoter operably linked to a regulatory sequence encoding the regulatory protein; and an inducible promoter operably linked to the target gene A plant is transformed with a chemically-inducible plant gene expression cassette comprising a promoter, the method comprising applying an agriculturally acceptable hydrolyzable ester to the plant to produce the alcohol upon hydrolysis to produce the expression of the target gene A method for regulating the expression of a target gene in a plant. 9. The method of claim 8, wherein the regulatory sequence encodes the alcR protein and the inducible promoter is an alcA promoter sequence. 10. The method according to claim 8 or 9, wherein the plant is transformed to express or overexpress an enzyme or a catalytic antibody or a catalytically active fragment thereof that hydrolyzes the ester to form the alcohol. Plant gene expression systems, including: (I) a primary promoter operably linked to a regulatory sequence encoding a regulatory protein; (Ii) an inducible promoter operably linked to the target gene, wherein the application is activated by a regulatory protein in the presence of an exogenous derivative alcohol as defined above that is effective to cause expression of the target gene; And (Iii) a sequence that encodes a protein that undergoes hydrolysis of the above ester under the control of an additional promoter that allows its expression in the plant. 12. A plant cell containing the gene expression system according to claim 11. Plant tissues or plants containing cells according to claim 12 and plants or seeds derived therefrom. An agriculturally acceptable ester as defined in claim 1 in combination with a diluent or carrier. The composition of claim 14, wherein the agriculturally acceptable ester is a compound of formula (I) as defined in claim 2. 16. The composition of claim 14 or 15 further comprising an emulsifier. 17. The composition of claim 16, wherein the emulsifier is PVA.