CN1338000A - Inducible expression system for use in plants - Google Patents

Inducible expression system for use in plants Download PDF

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CN1338000A
CN1338000A CN99815969A CN99815969A CN1338000A CN 1338000 A CN1338000 A CN 1338000A CN 99815969 A CN99815969 A CN 99815969A CN 99815969 A CN99815969 A CN 99815969A CN 1338000 A CN1338000 A CN 1338000A
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alcohol
plant
immersion liquid
compound
ester
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E·D·克拉克
E·J·T·克里斯塔
I·杰普森
J·A·M·派尼
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SINZETA Ltd
Syngenta Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
    • A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
    • A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action

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Abstract

The use of an agriculturally acceptable hydrolysable ester such as a compound of formula (I) in which R<1> and R<2> are defined in the application, in the control of expression of a plant gene, said control being effected by an inducible promoter which requires for activation, the presence of an exogenous chemical which may comprise an alcohol, wherein hydrolysis of said agriculturally acceptable ester results in the production of said alcohol. Formulations of these esters are also described and claimed.

Description

The inducible expression system that is used for plant
The present invention relates to a kind of expression system that is used for plant, particularly a kind of exogenous chemical medicament that utilizes is as the expression system of controlling mechanism and some chemical purposes as described control agent.
Genetic expression is by the regional control of upstream, encoding histone zone (5 '), is commonly referred to " promotor ".Promotor can be composing type, and is tissue-specific, grow the programming type or induction type.
In order to improve performance (as productivity or quality), the manipulation of farm crop needs the external source in the plant tissue or the expression of allogenic gene.Therefore, the manipulation of this gene depends on the validity of method, with controlling gene expression as required; For example, depend on the validity and the purposes of effectively suitable promotor in plant.Selection with various different promoters is favourable, so that for special genes, and construct, cell, tissue, plant or environment can be selected optimum promotor.Known a series of promotor is effective in plant.
In some cases, especially effectively promotor is to use the inductive promotor by the exogenous chemical inductor.Under the situation that is with or without the chemical agent that is applied to plant or seed, this permission will be controlled the expression of special gene at the particular stage of plant-growth and growth, for example by spraying or using known seed coating technique.Sometimes, this is called as gene " switch ".
Gene under inducible promoter control may be to cause the performance that it is desired or the gene of phenotype, perhaps inducible promoter can be controlled the expression of repressor, this repressor suppresses target gene expression, for example interacts to stop expression of gene (for example known tet and lac operator gene/repressor system in bacterium) by the manipulation sequence upstream with target gene.In another is selected, gene under inducible promoter control can be expressed the albumen that reacts to each other with another albumen, to suppress its activity, for example in barnase/barstar system, do not having under the situation of barstar, barnase will suppress or cell killing.
Such gene switching is known in some application widely.These comprise the male sterile generation of reversible, promptly describe for example among the WO90/08830, produce the feature of camber expectation at hybrid plant.Other application of this promotor is included in the germplasm protection, and the control of strick precaution of wherein special farm crop, particularly transgenic plant and volunteer plant is necessary, and in the prevention of before the results that WO94/03619 describes, sprouting.
Many organisms have allows the mechanism of their metabolic chemistry preparations such as alcohol or ketone, for example the production by alcoholdehydrogenase.The promotor of these systems may be useful in gene switching, because promotor can be induced by the existence of target alcohol or ketone.
Such example can find in the fungal organism Aspergillus nidulans, and when only growing under it is having the situation of various alcohol or ketone, Aspergillus nidulans is expressed the alcoholdehydrogenase I (ADHI) by gene alcA coding.Can take over by the adjusting albumen of alcR genes encoding and induce and the expression of composing type ground.Under the situation that inductor (alcohol or ketone) is arranged, regulate albumen and activate the alcA expression of gene.Regulate albumen and also stimulate it self expression under the situation of inductor having.The high level that this means the ADHl enzyme (promptly when alcohol or ketone exist) under inductive condition produces.On the contrary, alcA gene and its product, ADHl does not express under the situation of inductor having.The expression of alcA and the production of enzyme are prevented having under the situation of glucose.
Thereby the alcA gene promoter is a kind of inducible promoter, and being is having under the situation of inductor the combination of albumen/alcohol or albumen/ketone (promptly by) to regulate the albumen activated by alcR.AlcR and alcA gene (comprising promotor separately) (Lockington RA et al, 1985, Gene, 33:137-149 have cloned and have checked order; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).
In the certain plants species, studied alcoholdehydrogenase (adh) gene.In corn or other cereal, they are anastomosing by anaerobic conditions.The promoter region that is derived from the adh gene of corn is included in the regulatory element of expressing necessary 300bp under the anaerobic conditions.Yet having been found that does not all have alcR to regulate proteic equivalent in any plant.Therefore, the alcR/alcA type of gene regulatory system is unknown in plant.The composition of alcR is expressed and is not caused the active activation of endogenous adh in the vegetable cell.
WO93/21334 has described production of transgenic plants, and this plant comprises the system as gene switching.This document has been described chemistry-inductive gene expression in plants box especially, this box comprises first promotor that effectively is connected on the adjusting sequence, this is regulated sequence encoding and regulates albumen, and inducible promoter is connected on the target gene effectively, under the situation of effective exogenous inductor, by regulating albumen activation-inducing type promotor, using of inductor causes target gene expression thus.Particularly, in construction, utilize the alcR/alcA system.The exogenous chemical inductor that is applied in this situation comprises that those pass through Creaser et al., and J.Biochem. (1984) 225,449-454 describe, as fourth-2-one (ethyl methyl ketone), pimelinketone, acetone, fourth-2-alcohol, 3-ketobutyric acid, propan-2-ol and ethanol.
For the purpose of agricultural, alcohol is usually as the exogenous chemical inductor.Yet this chemical agent is normally volatile, and therefore is difficult to handle under agricultural environment, because a large amount of chemical agent may be lost in spray process.
The invention provides the purposes of agricultural hydrolyzable ester in gene expression in plants control, described control is by the inducible promoter effect, this inducible promoter needs to activate, and can comprise the existence of the exogenous chemical medicament of alcohol, and the hydrolysis of wherein said agricultural ester causes the production of described alcohol.
Particularly, agricultural ester comprises the compound of formula (I),
Figure A9981596900061
R wherein 1Be low alkyl group, low-grade alkenyl or low-grade alkynyl, R 2Be organic group, R like this 2COOH is agricultural acid.The alcohol of the hydrolysis production (II) of formula (I) compound
Figure A9981596900062
The term of Shi Yonging " agricultural " means under not causing the unacceptable level that phytotoxicity in the farm crop or soil damage herein, compound can be applied to special soil or farm crop situation.
The phraseology of Shi Yonging " low alkyl group " comprises C herein 1-6Alkyl, preferred C 1-4Alkyl, it can be a straight or branched.The phraseology of Shi Yonging " low-grade alkenyl " and " low-grade alkynyl " comprise C respectively herein 2-6Alkenyl and C 2-6Alkynyl, preferred C 2-4Alkenyl and C 2-4Alkynyl, it can be a straight or branched.
To be used for agricultural ester of the present invention, transfer to aptly in the target plant suc as formula those of (I), wherein the Gene Handling system is positioned at suitable position and/or in envrionment conditions or suitable catalysed partial is arranged, under the situation as enzyme or catalytic antibody, under the speed that is being suitable for providing sufficient quantity activation alcohol, in required time and hydrolysis in the needed part of plant.These can be according to the natural characteristic of processing plant species, time that expressed genes and ester are used and changing.
Ester is favourable suc as formula the compound of (I), is that they are easy to handle than corresponding alcohol.It has been found that these compounds can produce desired effect according to gene activation.
Should before needed gene activation, in the competent time, use this compound, so that the hydrolysis generation, and according to rational factor, as needs activated plant growth phase.If the speed of hydrolysis is relatively slow, the time of application can be earlier, to guarantee having competent hydrolysis to take place in the plant-growth stage that needs gene activation.When having any problem, can select the ester of quicker hydrolysis.
Selectively, can in independent processing, use more than one ester with different hydrolysis rates.The combination of the ester by selecting to have different hydrolysis rates can obtain activating effectively " slowly discharging " of alcohol, surpasses desired period so that prolong genetic expression.This means that repeated application and " clicking (the one-shot) " processing that can avoid chemical agent are possible.
The specific embodiments of the alcohol of formula (II) comprises methyl alcohol, ethanol, third-1-alcohol, propan-2-ol, fourth-2-alcohol or fourth-3-alkene-2-alcohol.
Aptly, the alcohol of formula (II) is low-grade alkane alcohol, and wherein alkyl contains the 1-4 carbon atom and can be side chain, or linear.R 1Preferred group comprise ethyl, just-propyl group and just-butyl.
Formula (II) compound more preferred example is an ethanol.
R 2The accurate characteristic of group is unessential, as long as when it being applied to special target plant, under suitable speed, it produces agricultural acid and gets final product.Can use ordinary method, for example as G.Mitchell et al., Pestic.Sci (1995) 44:49-58 describes, and determines hydrolysis rate, and preferably uses the test with respect to the whole plants system.In any concrete example what be suit will depend on various factors, this factor comprises the characteristic of in check genetic expression, the concrete plant that gene is expressed therein and other outside conditions.Hydrolysis rate should be competent, to allow after chemical inducer is used, seeing desired effect in the suitable time, for example, reversible male sterile.
Yet, can select R 2, the acid of the formula of Sheng Chenging (III) like this
Figure A9981596900071
Has some useful agrochemicals effect.Particularly, it self can serve as the inductor of inducible promoter.For example, it has been found that many acid, comprise the 3-hydroxybutyric acid, 2-hydroxybutyric acid, pyruvic acid and 3-ketobutyric acid can serve as alcR/alcA system (Creaser etal., inductor supra.).
R 2Specific example comprise the alkyl of any replacement, the cycloalkyl that replaces arbitrarily, the alkenyl that replaces arbitrarily, the alkynyl that replaces arbitrarily, aryl that replaces or the heterocyclic radical that replaces arbitrarily arbitrarily.
The term of Shi Yonging " alkyl " comprises the alkyl chain of straight or branched herein, aptly, comprises and can reach 10 carbon atoms at most, preferred 1-6 carbon atom.Term " alkenyl " and " alkynyl " comprise containing and can reach 10 carbon atoms at most, the unsaturated straight or branched of preferred 2-6 carbon atom.Term " aryl " comprises phenyl and naphthyl.Term " heterocyclic radical " comprises containing and can reach 10 at most, preferably can reach 7 at most, can reach 3 the oxygen that is selected from most, the ring of sulphur or nitrogen-atoms.These rings can be that monocycle maybe can be the form of condensed ring system, and these can be fragrance or non-fragrance on characteristic.Term " halogen " or " halogen " comprise chlorine, fluorine, bromine and iodine.Term " alkoxyl group " relates to the alkyl defined above that is connected with Sauerstoffatom.
Aptly, R 2Be the C that replaces arbitrarily 1-10Alkyl, it can be linear or side chain.Preferred alkyl R 2Be linear and contain 3-8 carbon atom, particularly 5 carbon atoms.
Alkyl, alkenyl and alkynyl R 2Suitable the replacement arbitrarily comprise that one or more are selected from halogen, nitro, cyano group, oxo, the aryl that replaces arbitrarily, the heterocyclic radical that replaces arbitrarily,
OR 3, C (O) pR 3, S (O) mR 3, OCOR 3,-NR 4C (O) pR 3,=NOH, NR 5R 6, C (O) NR 5R 6, C (O) NR 3NR 5R 6,-CH=NOR 3, P (O) R 7R 8Or P (O) OR 7OR 8, NR 3CONR 5R 6,-N=CR 5R 6, S (O) mNR 5R 6Or-NR 3S (O) mR 4,-N=NR 3Group, each R wherein 3, R 4, R 5, R 6, R 7And R 8Be selected from hydrogen individually, alkyl, alkenyl, alkynyl, aryl or heterocyclic radical, its any can replacement arbitrarily by functional group, and with regard to aryl and heterocyclic radical group, can be by alkyl, alkenyl or alkynyl substituted; Or R 5And R 6With the atom that is attached thereto together, can form a ring in addition, this ring can be carbocyclic ring or heterocyclic radical; P is 1 or 2, and m is 0,1,2 or 3.
The term of Shi Yonging " functional group " refers to and comprises halogen herein, cyano group, and nitro, oxo, hydroxyl,
=NOR 11, C (O) pR 11, OR 11, s (O) mR 11, NR 12R 13, C (O) NR 12R 13, OC (O) NR 12R 13,-CH=NOR 11,-NR 12C (O) nR 11,-NR 11CONR 12R 13,-N=CR 12R 13, S (O) mNR 12R 13Or-NR 12S (O) mR 11R wherein 11, R 12And R 13The alkyl that is selected from hydrogen separately or replaces arbitrarily, or, R 12And R 13Form the ring that replaces arbitrarily together, this ring at random comprises other heteroatoms, as oxygen and nitrogen or S (O) R 14, wherein p is 1 or 2 integer, m is 0 or the integer of 1-3, and R 14It is hydrogen or alkyl.
Alkyl R 11, R 12And R 13Suitable any substituting group comprise halogen, fully halogenated alkyl such as trifluoromethyl, sulfydryl, hydroxyl, alkoxyl group, oxo, heteroaryloxy, alkenyloxy, alkynyloxy group, the alkoxyl group alkoxyl group, aryloxy (wherein aryl can be by halogen, and nitro or hydroxyl replace), cyano group, nitro, amino, single-or two-alkylamino, alkyl amide or S (O) pR 14, wherein m and R 14As top definition.
Alkyl, alkenyl or alkynyl R 2Going up the example that replaces arbitrarily is one or more oxos that are selected from; Alkoxy carbonyl is elementary alkoxy carbonyl particularly; Cyano group; Halogen, as chlorine, fluorine or bromine; With amino or single-or dialkyl amido or alkyl, the phenyl that replaces arbitrarily as methyl; OR 3, R wherein 3Be alkyl or the heterocyclic radical that replaces arbitrarily with halogen or alkyl; S (O) mR 11, wherein m is 0 or 2, R 11It is the alkyl or phenyl that replaces arbitrarily with alkyl; NR 5R 6Or C (O) NR 5R 6R wherein 5Be hydrogen, methyl or methoxy ethyl, R 6Be alkyl such as methyl, phenyl or benzyl stem, it uses halogen, and as fluorine or chlorine, alkyl such as methyl or trifluoromethyl or alkoxy carbonyl replace arbitrarily, and wherein moieties can have another alkoxy carbonyl, or R 6Be the thiazinyl of heterocyclic radical as replacing arbitrarily with alkyl and/or ethanoyl;-NR 4C (O) pR 3Wherein p is 2, R 3Be alkyl and R 4It is the alkyl that replaces arbitrarily with alkoxy carbonyl such as ethoxy carbonyl alkyl;-NR 3S (O) mR 4, R wherein 3Be hydrogen, R 4Be to use halogen, as any phenyl that replaces of chlorine, and m is 2; C (O) NR 3NR 5R 6, R wherein 3And R 5Be hydrogen, and R 6Be with halogen or alkoxyl group, as any phenyl that replaces of methoxyl group; S (O) mNR 5R 6, wherein m is 2, R 5Be hydrogen and R 6With any alkyl that replaces of one or more alkoxy carbonyls; Heterocyclic radical such as furyl, pyridyl, pyridyl or pyrazinyl, triazinyl, it is any can be to use alkyl, halogen, trihalogenmethyl, phenyl, the halobenzene base, cyano group or oxo replace arbitrarily.
Alkyl, alkenyl or alkynyl R 2Suitable especially substituting group comprise alkoxy carbonyl, the spy is that wherein alkoxyl group is a low alkyl group in addition; Two alkoxyl groups of alkoxyl group and particularly dialkyl group acetal form; Cyano group or the heterocyclic radical that replaces arbitrarily.Preferred substituted comprises, but is not restricted to elementary alkoxy carbonyl and dialkyl group acetal.R in substituted alkyl and formula (I) compound 1When identical, alkoxy carbonyl and dialkyl group acetal are to make us interested especially, because in hydrolysis, these produce more formula (II) inductor chemicals.
R 2Special aryl be phenyl.
Cycloalkyl, aryl and heterocyclic radical R 2And abovementioned alkyl, alkenyl or alkynyl R 2On aryl or suitable any substituting group of heterocyclic radical comprise halogen; Alkylhalide group; Cyano group; Nitro, amino or single-or two-alkylamino; Hydroxyl; Alkoxyl group, alkylthio, alkyl or alkoxy carbonyl, wherein these arbitrarily moieties can be usefulness, for example one or more halogens that are selected from, alkoxyl group, cyano group, alkoxy carbonyl, amino, single-or two-alkylamino, aryl or carboxylicesters or its salt or ester; Cycloalkyl; Or any replacement of the group of heterocyclic radical.
Aryl or heterocyclic radical R 2Specially suitable substituting group comprise alkoxyl group, lower alkoxy particularly, as methoxyl group, alkyl, particularly low alkyl group, alkoxy carbonyl, particularly elementary alkoxy carbonyl and halogen.
The special subunit of formula (I) compound is the compound of formula (IA) R wherein 1With respect to formula (I) definition, n is the integer of 2-4, and R according to top 10Be alkyl, alkenyl or alkynyl, it is any can at random use heteroatoms, cycloalkyl, heterocyclic radical or aryl insert, or R 10Be that valency is cycloalkyl or the aryl of n.
Particularly, R 10Be that valency is the alkyl or aryl of n.
The particularly preferred compound of formula (I) comprises: 2-just-amyl group-ethyl 3-oxobutanoate (No. 49 compound); 2-carboxyl heptan-1,7-diacid (dioate) triethyl (No. 53 compound); With 2,4-dimethoxybenzoic acid ethyl ester (No. 60 compound).
The example of formula (I) compound is an ethyl ester, lists in table 1.
Table 1
Figure A9981596900102
Figure A9981596900121
Figure A9981596900131
Figure A9981596900141
Formula (I) compound or compound known can use ordinary method to prepare them from known compound.
Formula (I) compound may be in the target plant chemical hydrolysis, perhaps by naturally occurring enzyme in the target plant, perhaps introduce by genetic engineering technique, and the enzyme of in plant, expressing, or by suitable catalytic antibody or by in the genetic engineering technique introduced plant, and the catalytic activity part of the enzymically hydrolyse catalytic antibody of in plant, expressing.
Suitable enzyme comprises, but is not restricted to esterase and lipase.
Use standard technique, can produce suitable catalytic antibody from the analogue of tetrahedron ester hydrolysis transition state, for example, when using suitable phosphoric acid ester, for the hydrolysis Ole K et al. of paraxin precursor-medicine ester, 1998, J.Mol.Biol., 281:501-511 for the detoxifcation of Cocaine, uses the hydrolysis of methyl ester, Mets B etal., 1998, Proc.Nat.Acad.Sci.USA, 95:10176-10181.
People have further studied the metabolism of The compounds of this invention, and the result of these researchs is open in following embodiment.Be not restricted to the reason of mechanism, believe representative compounds for example of the present invention, compound 53 is metabolic according to following schema:
This metabolic product is an ethanol, and it can serve as above-described chemical inducer.
In another aspect, the invention provides the method for expression of target gene in a kind of controlling plant, wherein said plant transforms with chemistry-inductive gene expression in plants box, this box comprises first promotor that effectively is connected on the adjusting sequence, this is regulated sequence encoding and regulates albumen, and inducible promoter is connected on the target gene effectively, this inducible promoter is that alcohol is being arranged, under the situation as formula defined above (II) compound, by regulating the albumen activated, described method comprises ester is applied to described plant, this ester hydrolysis, form described alcohol, as formula defined above (I) compound, to cause target gene expression.
Aptly, as described above, regulate sequence encoding alcR albumen, and inducible promoter is the alcA promoter sequence.
In the place of needs or expectation, can also transform plant, it is expressed or overexpression enzyme or catalytic antibody or its catalytic activity fragment like this, and the compound of its hydrolyzing type (I) is to form formula (II) compound.
In some cases, preferably must with those under the situation that does not have enzyme or other parts for inactive enzyme by through engineering approaches in plant, because their effective forces in the target transformed the seed only.
Can be with the coding lytic enzyme, the nucleotide sequence of antibody or antibody fragment is included in the construct, this construct comprises adjusting albumen and/or the target gene that effectively is connected on the inducible promoter, or they may be present in the independently construct that is used for common-conversion plant.Yet this system is new.
Thereby, in one aspect of the method, provide a kind of gene expression in plants system, comprise
(i) effectively be connected to first promotor of regulating on the sequence, this is regulated sequence encoding and regulates albumen,
(ii) effectively be connected to the inducible promoter on the target gene, this inducible promoter is under the situation that the effective exogenous inductor of previously defined formula (I) is arranged, and by regulating the albumen activated, using of inductor causes target gene expression whereby; With
The (iii) sequence of proteins encoded, this albumen influences ester under the control of another promotor, be corresponding alcohol suc as formula (I) compound hydrolysis, and this another promotor allows its expression in plant tissue.
Target gene can comprise any gene that needs in the introduced plant, so that modify its characteristic as mentioned above.Target gene can be exogenous plant gene or foreign gene, and can be term single gene or series of genes.Target-gene sequence is encoding to small part functional protein or antisense sequences.
Can use any method for transformation that is suitable for target plant or vegetable cell, comprise with the Agrobacterium tumefaciems that contain the Ti-plasmids of recombinating and infecting, electroporation, the micro-injection of cell and protoplastis, particle conversion and pollen tube transform.Then, cell transformed can be regenerated in whole plants in suitable situation, in whole plants, is attached in the genome new nucleus material settling out.Can obtain the unifacial leaf and the dicotyledons that transform in this way.
The embodiment of the genetically modified plant that can produce comprises farm crop, cereal, and fruits and vegetables is as canola, Sunflower Receptacle, tobacco, beet, cotton, soybean, corn, wheat, barley, rice, Chinese sorghum, tomato, mango, peach, apple, pears, strawberry, banana, muskmelon, potato, Radix Dauci Sativae, romaine lettuce, Caulis et Folium Brassicae capitatae, onion.
The present invention further provides the vegetable cell that comprises gene expression system of the present invention.This gene expression system can stably be attached in the Plant Genome by transforming.The present invention also provides the plant tissue that comprises this cell or plant and from its deutero-plant or seed.
In this method the preferred embodiment of employed formula (I) compound be above-described those.
The agricultural ester of the present invention, suitable suc as formula (I) compound with agricultural composition, use with the form of diluent or carrier combination.This composition forms another aspect of the present invention.
The concentration preferably approximately 5%wt/wt of agricultural ester or concentration still less in the preparation.When the concentration of about 2%-5%wt/wt is preferred.
Suitable carrier or thinner are conspicuous for the technician, and will change according to the special characteristics of institute's use formula (I) compound.For example, if formula (I) compound is an oils, it may need the existence of emulsifying agent so that make its atomizing in the aqueous solution.Emulsifying agent is known in the art, and special example is polyvinyl acetate of partial hydrolysis (PVA) or tween TM
Organic solvent or thinner also can exist as acetone.
Therefore, preferred compositions comprises agricultural ester, and suc as formula the compound of (I), emulsifying agent is as PVA and thinner, as water.The relative quantity of component depends on various components compatibility each other on sizable degree.Yet the amount of the emulsifying agent that exists in composition is 1-5%w/w, preferably approximately 2.5%w/w aptly.
Thereby the embodiment of preparation of the present invention comprises following:
Preparation 1
1.5% The compounds of this invention (for example compound 53)
2.5％PVA
The water of surplus
Preparation 2
1.5% The compounds of this invention (for example compound 53)
5% acetone
0.05% tween 20 TM
The water of surplus
Preparation 3
3.0% The compounds of this invention (for example compound 53)
2.5％PVA
The water of surplus
Preparation 4
3.0% The compounds of this invention (for example compound 53)
5% acetone
0.05% tween 20 TM
The water of surplus
The composition selected that may use with in our relevant therewith UK Patent Application the 9902236.0th, describe those are similar.Particularly, said composition will comprise component:
(a) agricultural ester is suc as formula the compound of (I);
(b) C of polyethoxylated 10-C 20Alcohol or trisiloxanes polyethoxylated and
(c) thinner.
Thinner (c) can be, for example, and water.
The component of above-mentioned preparation (b) is preferred, the oleyl alcohol of polyethoxylated, lauryl alcohol, stearyl alcohol or hexadecanol.
More preferably contain 0-35, more preferably polyoxyethylene-the oleyl alcohol of the molar average ethylene oxide content of 2-20.Polyoxyethylene-(2)-oleyl alcohol most preferably, ethylene oxide-(10)-oleyl alcohol or ethylene oxide-(20)-oleyl alcohol.Component (b), yet, preferred polyoxyethylene-(20)-oleyl alcohol (the average ethylene oxide content of the numeral per molecule in the bracket).The commercial this product that uses is BRIJ92 TM, BRIJ97 TMAnd BRIJ98 TM
Preferably, the component in the preparation (b) is about 0.5%wt/wt or littler concentration.The concentration of preferably approximately 0.2%wt/wt-0.5%wt/wt.
Can select in the embodiment, preparation comprises component (b), the trisiloxanes polyethoxylated of hydrogen or methyl blocking.Particularly, component (b) is the trisiloxanes polyethoxylated of methyl blocking.The trisiloxanes polyethoxylated per molecule of this methyl blocking preferably contains 4-12, most preferably 8 molar average ethylene oxide content.This product of commercial is SILWET 77 TM(SIL WET is the trade mark of Witco).
Preferably, the trisiloxanes polyethoxylated of methyl blocking is about 0.5%wt/wt or littler concentration.The concentration of preferably approximately 0.2%wt/wt-0.5%wt/wt.
The concentration of the preferred 90%-98%wt/wt of component in the preparation (c).
Other additive that can be included in this preparation comprises dispersion agent, and antimicrobial compounds also can add wetting agent compound and anti--evaporation agent.
Now, by the special description the present invention of embodiment.
Embodiment 1 tests chemicals on the soil growing plant
After prescreen hydroponiv solution, compound is used for this test, this test be greenhouse 1 1/2 " in the basin, 4 weeks big alccat tobacco carry out on isozygotying 30 kind of plant that are.The sample of leaf is taken off contrast as " not inducing " from plant.Pulverize blade in 200 μ l250mM Tris pH8.0, centrifugal and recovery supernatant liquor is 4 ℃ of overnight storage.Compound dissolution is containing 50% acetone of 0.05% tween 20,50%dH 2O200mg/ml solution is then with 1/10 solution that dilutes to prepare 2%.Unless other regulation is arranged, 5ml solution is applied in the soil of per 2 kind of plant of handling in duplicate as the root immersion liquid.After 22 and 44 hours, remove the sample of leaf, store supernatant liquor according to said extracted and at 4 ℃.By the cat protein quantification of use Boehringer Mannheim CAT ELISA test kit analytic sample, and by the definite total protein level of Bradford mensuration.The result lists in table 2, and wherein the 1CAT value equals detecting of 0-5000ng/g, and 2 equal 5001-10, and 000ng/g detects, and 3 equal 10,001-15,000ng/g.
Compound number The CAT value
0 hour 22 hours 44 hours
????1 ????0 ????1 ????0
(2.5 milliliters) ????2 ????0 ????1 ????0
????3 ????0 ????1 ????2
(2.5 milliliters) ????4 ????0 ????0 ????1
????5 ????0 ????0 ????3
????8 ????1 ????1 ????1
????9 ????1 ????0 ????2
????10 ????2 ????0 ????1
????12 ????1 ????0 ????2
????13 ????0 ????0 ????0
????14 ????0 ????1 ????0
????15 ????0 ????1 ????1
????16 ????1 ????1 ????6
????17 ????1 ????2 ????1
????18 ????0 ????1 ????1
????19 ????0 ????1 ????1
????20 ????0 ????0 ????1
????21 ????0 ????0 ????1
????22 ????0 ????0 ????1
????23 ????0 ????0 ????1
????24 ????1 ????0 ????1
????25 ????0 ????0 ????1
(2.5 milliliters) ????26 ????0 ????0 ????0
????28 ????0 ????0 ????1
????30 ????0 ????2 ????2
????32 ????0 ????0 ????2
????33 ????1 ????1 ????1
(2.5 milliliters) ????35 ????0 ????0 ????1
????36 ????0 ????0 ????1
????38 ????0 ????0 ????3
????39 ????0 ????0 ????1
????40 ????0 ????0 ????0
(2.5 milliliters) ????41 ????1 ????0 ????1
????42 ????2 ????1 ????1
????43 ????0 ????0 ????2
????44 ????1 ????0 ????0
????45 ????1 ????0 ????2
????46 ????2 ????0 ????1
????47 ????3 ????0 ????2
5 ml of formulation ????0 ????0 ????0
2.5 ml of formulation ????0 ????0 ????0
5% ethanol ????1 ????8 ????10
5% fourth-1-alcohol ????1 ????1 ????3
????dH 2O ????1 ????0 ????0
Another processing of embodiment 2 soil growing plants
In this experiment, compound dissolution is containing 50% acetone of 0.05% tween 20,50%dH 2Form 200mg/ml solution among the O.These are diluted to 1.5% and 0.1% solution.Take of the contrast of the sample of leaf from seedling as " not inducing ".In 200 μ l 250mM TrispH8.0, pulverize blade, centrifugal, reclaim supernatant liquor and 4 ℃ of overnight storage.5ml solution is applied in the soil as duplicate per 2 kind of plant handled of root immersion liquid.After 22 and 65 hours, from handled per two kind of plant, remove the sample of leaf, extract, and store supernatant liquor at 4 ℃ according to the description among the embodiment 1.Quantitative by the catabolic protein of using Boehringer MannheimCAT ELISA test kit analytic sample, and by the definite total protein level of Bradford mensuration.Compound of being tested and result list in table 3, and wherein the value of CAT value representative is listed in table 2.
Table 3
Compound number CAT albumen value
1.5% immersion liquid 0 hour 22 hours 65 hours
????17 ????0 ????3 ????1
????50 ????0 ????2 ????1
????51 ????0 ????1 ????3
????51 ????0 ????1 ????3
????52 ????0 ????2 ????1
????53 ????0 ????3 ????8
????54 ????0 ????1 ????2
????55 ????0 ????1 ????1
????56 ????0 ????2 ????1
????57 ????0 ????2 ????5
????58 ????0 ????1 ????0
????59 ????0 ????1 ????1
????61 ????0 ????1 ????1
????62 ????0 ????1 ????2
????63 ????0 ????1 ????1
????64 ????0 ????1 ????1
????65 ????0 ????0 ????1
Ethanol ????0 ????3 ????6
Fourth-1-alcohol ????0 ????3 ????5
Preparation ????0 ????0 ????0
????dH 2O ????0 ????0 ????0
Determining of the time course that switch was expressed after embodiment 3 induced
In order to determine after ester is induced, the time course that the alc switch is expressed, with 5 weeks of some compound administration in greenhouse 1 1/2pots big alccat tobacco to isozygoty be on the blade or soil of 30 strain plants.Compound dissolution is being contained 50% acetone of 0.05% tween 20,50%dH 2Among the O to form 150mg/ml or 50mg/ml solution.Form 1.5% or 0.5% solution with 1/10 dilution then.Take of the contrast of the sample of leaf from seedling as " not inducing ".Freezing blade in dry ice/ethanol, and-70 ℃ of storages.Use 5ml solution as root immersion liquid or leaf sprays, to 8 strain plants.At the sample of each time point except that defoliation, and freezing in dry ice/ethanol ,-70 ℃ of storages.When results with after storing all samples, in 200 μ l250mM Tris pH8.0, extract blade, centrifugal, reclaim supernatant liquor and 4 ℃ of overnight storage.By the cat protein quantification of use Boehringer Mannheim CAT ELISA test kit analytic sample, and by the definite total protein level of Bradford mensuration.Compound of being tested and result be at table 4, lists in 5,6,7,8,9 and 10.List among the superincumbent embodiment 1 of CAT unit, each successive digitized representation increase progressively 5, the scope of 000ng/g.The result who represents with ghost form is at different batches, but obtain in the same test.
Table 4
Hour Compound 51 The CAT value
05 10 26 48 98 144 193 05 10 26 48 98 144 193 05 10 26 48 98 144 193 0 10 22 hours 26 48 65 hours 98 1.5% 1.5%49 immersion liquid of 0.5%49 spray, 0.5%49 spray 0.5%49 spray, 0.5%49 spray 0.5%49 spray, 0.5%49 spray 0.5%49 spray, 0.5%49 spray 1.5%49 sprays, 1.5%49 spray 1.5%49 sprays, 1.5%49 spray 1.5%49 sprays, 1.5%49 1.5%49 immersion liquid of spray 1.5%49 immersion liquid of 1.5%49 immersion liquid of 1.5%49 immersion liquid of 1.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 0.5%49 immersion liquid of 1.5%49 sprays, 1.5%49 sprays ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????1 ????0 ????0 ????0 ????0 ????0 ????1 ????2 ????1 ????4 ????4 ????0 ????0 ????1 ????1 ????1 ????3 ????3 ????4
Table 5
Hour 65 hours t=98 of t=0 t=5 t=10 t=26 t=48 t=0 t=5 t=10 t=26 t=48 t=0 t=5 t=10 t=26 t=48 t=8 22 hours t=26 t=48 of t=0 t=10 1.5% 1.5%30 immersion liquid of 0.5%30 immersion liquid .5%30 of No. 57 compound .5%30 spray .5%30 spray .5%30 spray .5%30 spray .5%30 spray 1.5%30 spray 1.5%30 sprays, 1.5%30 spray 1.5%30 sprays, 1.5%30 spray .5%30 root immersion liquid root immersion liquid .5%30 root immersion liquid .5%30 root immersion liquid 1.5%30 immersion liquid of 1.5% 1.5%30 immersion liquid of 1.5%30 immersion liquid of .5%30 1.5%30 immersion liquid of root immersion liquid CAT value 00011000000012240121253
Table 6
Hour No. 53 compound c AT value
0 0.5%51 spray 05 0.5%51 sprays, 0 10 0.5%51 spray 1 26 0.5%51 sprays, 1 48 0.5%51 spray 1 98 0.5%51 sprays, 1 144 0.5%51 spray 1 193 0.5%51 sprays, 00 1.5%51 spray 05 1.5%51 sprays, 1 10 1.5%51 spray 2 26 1.5%51 sprays, 1 48 1.5%51 spray 4 48 1.5%51 immersion liquid of 0 10 1.5%51 immersion liquid of 1 98 1.5%51 spray 00 1.5%51 immersion liquid of 2 193 0.5%51 immersion liquid of 3 144 0.5%51 immersion liquid of 1 98 0.5%51 immersion liquid of 1 48 0.5%51 immersion liquid of 1 26 0.5%51 immersion liquid of 1 10 0.5%51 immersion liquid of 05 0.5%51 immersion liquid of 10 0.5%51 immersion liquid of 1 144 1.5%51 sprays, 0 193 1.5%51 sprays, 1.5% 4 26 1.5%51 immersion liquid in 1 22 hours, 1.5% 8 98 1.5%51 immersion liquid 5 in 8 65 hours
Table 7
Hour No. 60 compound The CAT value
65 hours t=98 of t=0 t=10 t=26 t=48 t=98 t=144 22 hours t=26 t=48 of t=0 t=10 1.5%57 of 1.5%57 immersion liquid of 1.5%57 immersion liquid of 1.5%57 immersion liquid of 0.5%57 immersion liquid of 0.5%57 immersion liquid of 0.5%57 immersion liquid of 0.5%57 immersion liquid of 0.5%57 immersion liquid of 0.5%57 immersion liquid, 1.5%57 immersion liquid of 1.5%57 1.5%57 immersion liquid ????0 ????1 ????1 ????2 ????5 ????8 ????0 ????1 ????1 ????1 ????1 ????1 ????4
Table 8
Hour No. 52 compound The CAT value
65 hours t=98 of t=0 t=10 t=26 t=48 t=98 t=144 t=93 t=0 t=10 t=26 t=48 t=98 t=144 t=193 t=0 t=10 t=26 t=48 t=98 t=144 t=193 22 hours t=26 t=48 of t=0 t=10 1.5%50 1.5%50 immersion liquid of 0.5%50 spray, 0.5%50 spray 0.5%50 spray, 0.5%50 spray 0.5%50 spray, 0.5%50 spray 0.5%50 spray, 1.5%50 spray 1.5%50 sprays, 1.5%50 spray 1.5%50 sprays, 1.5%50 1.5%50 immersion liquid of 1.5% 1.5%50 immersion liquid of 1.5%50 immersion liquid of spray 1.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 0.5%50 immersion liquid of 1.5%50 sprays, 1.5%50 sprays ????0 ????0 ????0 ????1 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????1 ????1 ????2 ????1 ????1 ????1 ????0 ????0 ????2 ????1 ????2 ????1 ????1
Table 9
Hours 05 10 26 48 98 144 193 05 10 26 48 98 144 193 05 10 26 48 98 144 193 0 10 26 48 98 1.5% immersion liquid of ethanol ethanol 0.5% spray ethanol, 0.5% spray ethanol 0.5% spray ethanol, 0.5% spray ethanol 0.5% spray ethanol, 0.5% spray ethanol 0.5% spray ethanol, 0.5% spray ethanol 1.5% spray ethanol, 1.5% spray ethanol 1.5% spray ethanol, 1.5% spray ethanol 1.5% spray ethanol, 1.5% spray ethanol 1.5% immersion liquid ethanol of 1.5% immersion liquid ethanol of 1.5% immersion liquid ethanol of 1.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 0.5% immersion liquid ethanol of 1.5% spray ethanol, 1.5% spray ethanol CAT value 02221111024542210246444304976
Table 10
Hour ????dH 2O ????dH 2O root immersion liquid dH 2O root immersion liquid dH 2O root immersion liquid dH 2O root immersion liquid dH 2O root immersion liquid dH 2O root immersion liquid dH 2O root immersion liquid dH 2The immersion liquid of O root CAT value 00000000
????0 ????5 ????10 ????26 ????48 ????98 ????144 ????193
The preparation hour
????0 ????5 ????10 ????26 ????48 ????98 ????144 ????193 ????0 ????5 ????10 ????26 ????48 ????98 ????144 ????193 The immersion liquid of aerosol spray agent aerosol spray agent aerosol spray agent aerosol spray agent root immersion liquid root immersion liquid root immersion liquid root immersion liquid root immersion liquid root immersion liquid root immersion liquid root ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????0 ????1 ????1 ????1 ????0
These results show, with the alcohol contrast, ester that can the application of the invention changes the inductive time course.Therefore, the suitable selection of ester will cause the favourable form of inducing.

Claims (17)

1. the purposes of agricultural hydrolyzable ester in gene expression in plants control, described control is finished by inducible promoter, this inducible promoter need activate in the presence of the exogenous chemical medicament that can comprise alcohol, and the hydrolysis of wherein said agricultural ester causes the generation of described alcohol.
2. according to the purposes of claim 1, wherein agricultural hydrolyzable ester is the compound of formula (I)
Figure A9981596900021
R wherein 1Be low alkyl group, R 2Be organic group, it can be in hydrolysis, and the generation chemical formula is R 2The agricultural acid of COOH; And described promotor needs the existence of formula (II) alcohol to be used for its activation.
Figure A9981596900022
3. according to the purposes of claim 2, formula wherein (II) compound is a low-grade alkane alcohol, alkyl R wherein 1Contain 1-4 carbon atom and can be side chain, or linear.
4. according to the purposes of claim 2 or claim 3, R wherein 2Be the alkyl that replaces arbitrarily, the cycloalkyl of any replacement, the alkenyl that replaces, the alkynyl of replacement, any aryl that replaces or the heterocyclic radical that replaces arbitrarily arbitrarily arbitrarily.
5. according to the purposes of claim 4, R wherein 2Be the C that replaces arbitrarily 1-10Alkyl.
6. according to the purposes of claim 5, R wherein 2Replace with alkoxy carbonyl or dialkyl group acetal.
7. according to the purposes of aforementioned any one claim, its Chinese style (I) compound be 2-just-amyl group-ethyl 3-oxobutanoate; 2-carboxyl heptan-1,7-diacid triethyl; Or 2,4-dimethoxybenzoic acid ethyl ester.
8. the method for expression of target gene in the controlling plant, wherein said plant transforms with chemistry-inductive gene expression in plants box, this box comprises first promotor that effectively is connected on the adjusting sequence, described adjusting sequence encoding is regulated albumen and is connected to inducible promoter on the target gene effectively, this inducible promoter activates by regulating albumen under the situation that alcohol is arranged, described method comprises agricultural hydrolyzable ester is applied to described plant, produce described alcohol when this is esterolytic, so that cause target gene expression.
9. method according to Claim 8, the adjusting sequence wherein alcR albumen of as previously described, encoding, and inducible promoter is the alcA promoter sequence.
10. according to Claim 8 or the method for claim 9, wherein transform plant, so that its is expressed or overexpression enzyme or catalytic antibody or its catalytic activity fragment, its hydrolyzable ester is to form described alcohol.
11. a gene expression in plants system comprises
(i) effectively be connected to first promotor of regulating on the sequence, this is regulated sequence encoding and regulates albumen,
(ii) effectively be connected to the inducible promoter on the target gene, under the situation that previously defined effective exogenous inductor alcohol is arranged, by regulating albumen activation-inducing type promotor, using of alcohol causes target gene expression whereby; With
The (iii) sequence of proteins encoded, this albumen is finished the hydrolysis of above-mentioned ester under the control of another promotor, and this another promotor allows its expression in plant tissue.
12. vegetable cell that comprises the gene expression system of claim 11.
13. comprise the plant tissue of cell of claim 12 or plant and from its deutero-plant or seed.
14. a Pestcidal compositions comprises the agricultural ester with claim 1 definition of diluent or carrier combination.
15. according to the composition of claim 14, wherein agricultural ester is formula (I) compound of definition in the claim 2.
16. according to the composition of claim 14 or claim 15, it further comprises emulsifying agent.
17. according to the composition of claim 16, emulsifying agent wherein is PVA.
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