CN1179699A - Repellent compositions containing flavonoid aldehydes - Google Patents

Abstract

The present invention provides repellent compositions which contain falvonoid aldehydes such as cinnamic aldehyde, alpha -hexyl cinnamic aldehyde and/or coniferyl aldehyde are provided, together with methods for their use as repellents for pests including flies, cockroaches, aphids, silverleaf white flies, mosquitos, ticks, fleas, leafhoppers, thrips, two-spotted spider mites, snails, slugs, biting midges, earwigs, and moths.

Description

The repellant compositions that contains flavonoid aldehydes

Technical field

The present invention relates to flavonoid aldehydes as insects repellant.The present invention be by as culicifuge and be used for the cinnamic acid of pest such as aphid and thrips or jasmonal H should be used for illustrational. Background

Today, disease for example hemorrhagic heating, cockroach anaphylaxis, filthy flea disease, bubonic plague, ixodism toxication, Richettsia, spirillum, bilharzia disease and the sand fly heat of malaria, medium infection was still the reason that causes serious disease and many death between the resident in a lot of countries.The continuous growth that people pay close attention to for the protection endangered species, and public health broad-spectrum pesticide validity is decline trend, forcing scientists to go to seek can provide protection to avoid other economized form of disease media infringement.But for many more underdeveloped countries, the expense of agricultural chemicals is too high usually, and exists media to this serious day by day problem of the ever-increasing resistance of these compounds.

When other traditional media control method was infeasible, using repellant was a kind of goodish alternative method.For mammiferous media target body, it is not expensive that repellant suitably is applied on skin or the clothes, and is to reduce the practical methods of biting blood arthropod bite activity and preventing the media transmission.Repellant is that effectively vaccine then must must individually develop at each disease to the disease media of wide scope, and to have only the vaccine of a small part be effective for the disease by vection.Also can influence plant corpus by vector-borne disease.For example, Dutch elm disease has been destroyed the millions of elms of spreading all over the U.S..This disease is caused by fungi, and this fungi is propagated between tree and tree it attractive special caste by elm and causes disease to infect.Present preventive treatment is the limited success that obtained only.Therefore need a kind of to the effective preventive treatment of this destructive plant disease.

Cancellation use repellant in media control and prevention from suffering from the diseases normally is conceived to the problem aspect safety and the expense.For example, one of widely used repellant is the 6-2-2 repellant, and it comprises dimethyl phthalate, ethohexadiol and dihydropyrone, and ratio is 6: 2: 2.Dimethyl phthalate and dihydropyrone are still restriction and use, but in the producer's the requirement that Environmental Protection Agency USA in 1991 should be relevant, cancel the registration of all ethohexadiols.The act of doing this is because there is new data to show that it might have adverse influence to development of fetus.

Another example, one of the most effective culicifuge is Metadelphene (N, N-diethyl-1, a 3-methyl benzamide).During local the use, this material makes other repellant all overshadowed, and after it found nearly 40 years, it was still the topmost repellant of current use.But in recent years, the synthesis of derivatives of permethrin-a kind of Dalmatian chrysanthemum replaces Metadelphene in large quantities, is used for clothes and other textile fabric.As repellant, Metadelphene is efficiently, but it also may cause allergy and toxic effect, particularly when being recycled and reused on the skin with high concentration.The repellant preparation that contains 90% to 99% Metadelphene is considered to high concentration, but contain 50% or still less the repellant preparation of Metadelphene be considered to effective equally with 100% concentration.The Metadelphene of concentration 33% can provide 10 to 14 hours protection effectively.Yet minimum for possible ill-effect is reduced to for the product that contains the low concentration Metadelphene, suggestion loses no time to clean skin and water cleaning with gentle soap when no longer needing repellant.These suggestions are unsuitable for third world countries and military use usually.

More existing be locally applied to mammal and repel insects makes it not invade shelter and other regional repellant compositions.But the safety of many these class topical compositions is under suspicion.In addition, the effect of many these class topical compositions is limited, particularly in the zone that is subjected to the serious infringement of insect.The processing of the outside insect pest infestation of mammal, for example lice or crab are usually directed to the high poisoning of local application worm composition in skin or scalp.Usually can produce stimulation, and use for a long time and the unwholesome effect that causes also is known.Therefore need a kind of to the effective local application repellant compositions of mammalian safe.Prevent that the repellant compositions that insect enters from equally not having effect yet.Because its high toxicity, it also is unsafe that many known repellant compositions of this class use in airtight space, and particularly child may enter with pet and when contacting with it.Therefore need a kind of non-stimulated, avirulence and effective repellant compositions.

Annual millions of ornamental trees and shrubs of destruction of the insect pest infestation of trees and other woody plants and crops.Just part success of present processing, and can make that the agricultural product crop is valueless, because there is residual toxicity in they.Therefore, also need a kind of repellent trees and woody shrub insect pest infestation relative noresidue, effective method. Pertinent literature

U.S. Pat P5,093,326 discloses repellant compositions, and it comprises that the not full of ozonisation closes hydrocarbon, comprises obedient alkene.U.S. Pat P5,365,017 disclose the preparation of the genetically modified plants of the cycloartenol level with increase.The document relevant with the repellent preparation comprises people such as Reifenrath (1989), J.Am.Mosquito Control Association 5:45-61 and Reifenrath (1995) Cosmetics ﹠amp; Toiletries Magazme 110:85-93.

Summary of the invention

The invention provides the repellant compositions that contains flavonoid aldehydes and use these method for compositions.These repellant compositions contain the compound of structural formula shown in following (1) Wherein R represents-CH ₂OH or-CHO; N is from 0 to 3 integer; And each R ₁Represent OH independently or contain from 1 to 10 carbon atom and from 0 to 5 heteroatomic organic substituent, wherein at all R of described compound ₁Total no more than 15 of carbon atom in the substituting group and other atom; And R ₄Expression hydrogen or contain the organic substituent of 1 to 10 carbon atom.These compounds comprise natural compound such as cinnamic acid.The aldehyde that the same interested α of also having replaces is as jasmonal H (HCA).During use, compound administration on the surface, is wished repel insects and other insect part as skin, clothes, bark, habitat or the like.The present invention can be used to for example prevent because of contacting disease and the infection that causes with the insect vector that carries disease or other insect media.

Specific embodiments is described

The invention provides and obtain and/or keep the method and composition that a zone does not have insect or other insect basically.With the repellent insect and cause that the method for the organism of disease provides also have mammal, birds, fish and their habitat, and seed, nursery stock, plant or plant part such as fruit, their essentially no Pathogenic organismss for example fungi, insect and other insect and virus, bacterium, conveyor screw and other cause the organism of disease and inhale the myron insect.Interested surface is contacted with the flavonoid aldehydes of enough driving away the amount of insect or other insect.The amount of the repellant of being used will depend in part on the character on surface, and depend on formulations employed and specific compound to a certain extent, and therefore must determine best result with concrete insect or other insect experience ground.

The compositions and methods of the invention have several advantages to be better than existing composition and method.Main advantage is that this compound composition composition generally is to think safe (GRAS) and be that permission is used for food, for example, can find the aromatic aldehydes of purposes in preparation of the present invention, be the synthetic flavor enhancement (21CFR § 172.515) of GRAS as jasmonal H, cinnamic acid and vanillin.HCA generally used in the past in nineteen fifty, and still extensively was used in (soap, washing agent, creme, lotion, perfume) on the make-up preparation (fragrance raw material special topic, Food Cosmet.Toxicol.12:suppl., 915,1974) today.HCA obtains permission GRAS status (investigation of aroma extract manufacturer federation, seasoning one-tenth back application level, No.2569, the Fd.Technol. of FEMA in nineteen sixty-five, Champaign, 19:(part 2) 155,1965), and be used for food (21 CFR121,1164) by US FDA permission.European commission (1970) (European commission, natural and artificial flavoring substance, the part agreement in society and public health field, Strasbourg, List A (1), Series 1, no, 129, p.55,1970) in the synthetic flavoring substance of permission, comprise the HCA of 1ppm level.

The surfactant that can be used as emulsifier in preparation of the present invention, also as food additive, saponarin is (it also obtains the GRAS status) too as tween (polysorbate).In addition, the residual of preparation can try every possible means to solve.Because can to obtain short-term residual, thus this for the insect comprehensive regulation have huge interests the GRAS and the food of the open formulation components usage license.Pathogenic organisms is able to long-term control and can produces and compare the higher output of more healthy plant and host plant with non-transgenic plant.Especially aromatic aldehyde has positive organ and sense of smell performance, and these performances can be improved the fragrance and/or the stink of the product of processing in some cases, and can eliminate the unhappy smell relevant with many insects repellants.For example, the smell of jasmonal H (HCA) is described to the flower of some draft characteristics or the smell of jasmine sample (Technical DataSheet).

For plant, active component can be by will being responsible for one or more gene introduced plant cellular genome of synthetic various aldehydes, and by plant production.Because the compound of many formulas (1), particularly the compound of formula (3) and (4) is the GRAS food additive, therefore use genetically modified plants but not the local application repellant in plant corpus, can reduce feeding plant tissue or fieldwork person or animal, fish or the presumable any adverse side effect of birds partly.In addition, agricultural chemicals now for example can not be transferred to root and handle radicola, and the present invention has overcome this shortcoming.Equally, carry the insect media of disease or destruction target body by expeling and avoid infringement, can be significantly reduce and to die from the disease brought by insect or, for example the injury of being laid eggs in fruit to be caused by female Mediterranean fruitfly by the number of the target body animal or plant that activity injured of insect.

When being used to comprise people's animal, preparation of the present invention is nontoxic and non-stimulated to skin under working concentration.For example, the oral LD of rat of jasmonal H (HCA) ₅₀Be 3.1g/kg, through skin LD ₅₀Greater than 3g/kg (March 24,1971 for Moreno, O.M Report to RIFM).Under the situation of occlusion, not unhairing or the plucked son of exempting from were coated with 24 hours, the stimulation (Moreno) of moderate is arranged when finding HCA for pure compound.When with its petroleum jelly cream of 12% test, the plaster of being close to the people testee was tested (closed-patch test) after 48 hours, HCA does not produce stimulation, and does not produce susceptibility in the test of carrying out to greatest extent (Kligman (1966) J.Invest.Dermatol.47:393) on 25 people testees.Repeating of carrying out on 100 people testees damaged in the plaster test (insult patch test), and the 20%HCA in diethyl phthalate does not produce positive reaction.Can find the Jimbo of 18 kinds of aromatics to make the hypersensitivity data of table in the literature.Though cinnamic acid has positive reaction in people's test to greatest extent, HCA reaction in this test is negative.The plaster result of the test of 2% HCA on 100 eczemas and dermatitis patients is negative (0/100).4 patients to 2% cinnamic acid sensitivity do not see that the HCA to 2% has cross reaction.To the frequent report of cinnamic acid allergic may be because the amino acid on the albumen is rolled into a ball to react with aldehyde functional group stimulates.In replacement (for example hexyl of HCA), can reduce this reaction by the electrophilicity of creating steric hindrance and minimizing aldehyde carbon atom with respect to the big alkyl group on the α position of aldehyde radical.The cutaneous sensibility of the cinnamic acid of alpha-substituted is uncommon.Compare with cinnamic acid, the reaction of jasmonal H and amine very slowly or is not even reacted.In the experimental study of doing with cavy to greatest extent, Senma and partner's report thereof have so a kind of trend, that is, the number of the hydrocarbon of substituted alkyl group increases on the α position of cinnamic acid, and then the speed of sensitive response reduces.

This preparation also provides the effective control to multiple organism such as fungi and insect.Have and report that spore germination has the activity of inhibition to these compounds to C.botulinum.BowlesMiller，G.Food?Protection(1993)56：788-794。This many target body is renderd a service and has been reduced the demand that the plant that is intended to protect or animal are used medicament, and eliminates the needs of use of insecticide basically.Under special situation, for example destroy in animal or plant part or tissue and second kind of fungi or bacterial disease produce insect, it is of the present invention that this is particularly superior on the one hand.

Preparation of the present invention is shown in the following formula (1).Preferred preparation is shown in the following formula (2):

R wherein ₁Expression-CHO, R ₂Expression-OH or contain the organic substituent of 1 to 10 carbon atom, and R ₃Expression methoxyl group or contain the organic substituent of 1 to 10 carbon atom, R ₄Expression hydrogen or contain the organic substituent of 1 to 10 carbon atom.Interested especially is flavonoid aldehydes, particularly the aromatic series aldehydes.The example that is used in aromatic series aldehydes of the present invention is cinnamic acid (following (3)):

And coniferyl aldehyde (following (4)):

The compound of the analog that other compound of interest comprises formula (1) compound as replacing by alkyl such as hexyl or branched alkyl such as amyl group in the α position.Usually the group in the α position is C-5 to C-10.These compounds comprise jasmonal H and jasminal.The chemical structural formula of jasmonal H (HCA) is shown in following (5) The HCA title of chemical abstracts service (Chemical Abstracts Service (CAS)) is 2-(phenylmethylene) octanal, and the CAS registration number is [101-86-0].The chemical name of this compound is also referred to as 2-hexyl-3-phenyl-2-propionic aldehyde.The molecular formula of this compound is C ₁₅H ₂₀O, molecular weight are 216.3.HCA can be obtained by Firmenich, its product mainly and (Z) by (E)-cis-isomer (being up to 93.8%)-transisomer (being up to 6%) forms.A small amount of component wherein be octanal from aldol condensation (1-1.5%) (PersonalCommunication, June Burkhardt, Firmenich, Plainsboro, New Jersey).

These compounds can use separately or be used in combination with other activity or inert matter, and can use in the mode that the existing concentrate that is suitable for specific purposes most that contains this reactive compound, solution, suspension, powder or the like form is sprayed, pours into a mould, dips, injected.They also can or be dissolved in the suitable solvent with the form of for example dilute solution, as irrigating part of work or as independent using, being applied directly to rhizosphere.

During as blade spraying, though aldehydes can be processed separately, it also can put into practice by comprising emulsifier such as Tween 80.Operable other washing agent comprises that anionic detergent is as at U. S. application patent 4,978, those described in 686.Can be separately or other compound that is used in combination with washing agent comprise the saponarin that comes from multiple source, the saponarin that comes from Yucca schidigera or Yucca valida particularly.In general, be used in the washing agent of this preparation or other reagent repellent characteristic of flavonoid aldehydes that do not detract, but really can increase the substantial characteristic of this preparation (referring to, for example, U.S. Pat P4,477,361) and can improve the characteristic of agricultural chemicals, comprise fungicide characteristic (seeing below).The additional component such as the aqueous solution of polyacid salt such as sodium bicarbonate, sodium sulphate, sodium phosphate or sodium dihydrogen phosphate prepare liquid and can be included in this preparation, to increase the antifungal property of this preparation.During use, the emulsion of gained is diluted to debita spissitudo.

In preferred embodiments, this preparation comprises jasmonal H, cinnamic acid and/or coniferyl aldehyde, also comprises Tween 80 or saponarin as emulsifier in the preparation, and can comprise sodium bicarbonate.The preferred formulation that is used to drive away fly, mosquito, flea, tick, lice, cockroach, tetranychid, Bemisia argentifolii (silverleafwhite flies), aphid, leafhopper, thrips and ants at 2 is 10-5000ppm; The 100-2500ppm that is that is used for tick.Generally speaking, the aldehydes total amount that exists in preparation is 2% or still less.This preparation is effectively under the situation of not using antioxidant, though some special aldehydes contains inherent antioxidant properties, for example, coniferyl aldehyde.Alcohols such as di-alcohols comprise propane diols, are not that the effectiveness of increase preparation is required equally, and in fact may be harmful to plant.

This stability of formulation can be assessed by several different methods, comprises accelerated test, and interested in test preparation is exposed under the temperature that increases within a certain period of time.Formulation samples is taken out at a certain time interval, and carries out chemical analysis with method known to those skilled in the art, determines the speed and the character of decomposing.For example, HCA can analyze with gas-liquid chromatograph (GLC), adopt 30 meters nonpolar poly dimethyl silane capillary columns (for example, HP-1, Hewlett-Packard, or SPB-1, Supelco) and flame-ion detector (PersonalCommunication).Adopt helium as carrier gas (8ml/ minute), column temperature is about 240 ℃, (E)-retention time of cis-isomer (key component) is about 6.0 minutes, and (Z)-retention time of transisomer (a small amount of component) is about 6.3 minutes.

Comprise the effective dose of formula (3) and/or formula (4) and/or formula (5) compound compositions and the amount of useful other formula (1) compound, can adopt well known by persons skilled in the artly to be used for the scheme that the assessing compound repellent renders a service and to determine.The embodiment of this scheme is shown in hereinafter.These schemes also can be used for optimization and be used for each preparation of the included arbitrary compound of the employing formula (1) of specific pathogen or formula (5), and when the disease-resistant former effect that makes preparation reaches maximum, each preparation of optimization specific application makes phytotoxicity or cutaneous sensibility and other side effect for animal body reduce to minimum.

In some cases, can be intended to change in the preparation of its some special aspects by one or more other components (that is, the compound except that formula (1) or formula (5) compound) are added, thus the effect of increase preparation.For example, for a certain plant,, then can wish to reduce phytotoxicity effect (the phytotoxicity grade is 2 or still less, is preferably 1 or still less, sees below), or wish to increase the preparation repellent action, or both all have if phytotoxicity is big when using.Preferably, additional component can make and reduce to minimum to the side effect of plant corpus or animal body when increasing the preparation repellent action.Interested especially like this one or more component that is to use, it or they be increase anti-repelling properties and can make any side effect relevant reduce to minimum synergist simultaneously with it with specific formulation." synergist " is meant a kind of like this component, because its existence makes institute's phase effect greater than the adduction effect.When preserving or increasing institute's phase effect of preparation, can the concentration of one or more other preparation composition be made an amendment.Interestedly especially be, in preparation, add various ingredients, the concentration of one or more other composition in the given preparation is reduced, and keep the effectiveness of said preparation with it simultaneously basically.Other combination of components in this kind component and the preparation can mixed and/or finish in any suitable stage that this composition is used.

Preferred supplementary element is a saponarin because they can the substitution list surface-active agent as emulsifier, and under this external used concentration identical at least plant is had the effect of growth promoter.Saponarin is a compounds, and each is made up of the part of sapogenin part and sugar.Sapogenin may be steroids or triterpene, and sugar moieties can be glucose, galactose, pentose or methylpentose.S.Budavari，ed.，The?Merck?Index，11th?ed.，Merck?&?Co.，Inc.，Rahway，N.J.，1990，p.1328。Being used for saponarin of the present invention can or produce from the various plants position in multiple source or separate with methods known in the art, plant part comprises fruit, blade, seed and/or root, described source comprises the plant of many known generation saponarins, scope from yucca, Quillaia saponaria, american agave, Yan grass, Radix Glycyrrhizae, soybean, genseng and asparagus to aloe wood.The saponarin that together uses with the present invention is preferably nontoxic to people or higher mammal.Being used for saponarin of the present invention most preferably is avirulent food-grade product, its source form yucca plant.Preferred saponarin is from Yucca schidigera or Y.valida and their equivalent.Saponarin generally is by a kind of liquid extract that extracting process makes and the use gained of colding pressing.Also can use the fiber of yucca; With its Exposure to Sunlight, grind and the screening size typically.Usually the effective dose of saponarin is in 0.01 to 0.1% scope, and most preferably is 10 ° of Brix saponarin extract aqueous solution of about 0.01%v/v.

Known have on many structures a relevant saponarin, and the most changeable architectural feature is the glycosylation pattern.Saponarin also may contain additional modification, and for example Sa spills saponarin, and it is the saponarin that has the steroids that adheres to, and the structure of saponarin can be modified by any enzyme known in the art, chemistry and/or mechanical means.The saponarin that derives from Yucca schidiger contains the steroids saponarin, and the main sapogenin that this steroids saponarin has is sarsasapogenin and cautious announcement aglucon (tigogenin).When spilling the saponarin hydrolysis, Sa produces sarsasapogenin (sarsasapogenin 5-β, 20-β F, 22-δ F, 25-β F; Also be known as spirostan-3-β-01 and sarsa aglucon), glucose and galactose.The molecular formula of sarsasapogenin is C ₂₇H ₄₄O ₃Nobel，Park?S.，Agaves，Oxford?Univ，Press，NewYork，1994。Known have on the multiple structure a relevant saponarin, and the most changeable architectural feature is the glycosylation pattern.S.Budavari?ed.The?Merck?Index?11th?ed.，MERck?&?Co.，Inc.，Rahway，N.J.1990，p.1328。Saponarin also may contain additional modification, and for example Sa spills saponarin, and it is the saponarin that has the steroids that adheres to, and the structure of saponarin can be modified by any enzyme known in the art, chemistry and/or mechanical means.Usually, the effective dose of saponarin is about 0.01 to 3% and 10 ° of Brix saponarin extract aqueous solution of about 0.25%v/v preferably.10 ° of Brixs are the terms in the carbohydrate chemistry field.Brix degree equals the percentage by weight of sugar in solution.Hawley, ed., simple and clear chemical dictionary, 10th ed., Van NostrandReinhold, New York, 1981, P.149.

At preparation is to be used for preparing in the application that plantation is subject to the soil of the host plant that specific pathogen infects or other growth substrate, particularly be subjected to when infecting at growth substrate, preparation of the present invention directly can be added to rhizosphere or substrate and maybe it can be attached to a solid support or it is encapsulated in the slow-release material, to drive away harmful insect or other insect.When adopting solid support, should avoid using the material that can cause active aldehydes oxidation.The example of operable delivery systme comprises starch-glucan or the like.The example of other appropriate materials is referring to people's such as Yuan basis and application toxicology (1993) 20:83-87.

Except that the particular compound of aforementioned formula (1), (2), (3), (4) and (5), the precursor of any of these compound as long as this precursor under the effect of living things system, produces the compound identical with the said structure formula, then is considered to be equal to The compounds of this invention.Therefore precursor compound is applied to the plant corpus part or organizes to be equal to practical application of the present invention.The biotransformation of precursor compound becomes flavonoid aldehydes to have been described in for example U.S. Pat P5,149,715 and the list of references quoted in.Also can be referring to Casey and Dobb Enzyme Microb.Technol. (1992) 14:739-747.

The inventive method is to be introduced into surface interested by the repellant with q.s to implement to drive away insect or other insect.In addition, when interested surface be skin, fur, send out, during clothing or the like, use and to be undertaken by contacting with interested surface with preparation, preparation puts into practice for interested surface, thereby the preparation of repellent amount is stayed on the surface treated, and discharged with the speed of the insect that is enough to the repellent sensitivity or other insect.The preparation that contains repellant is normally introduced the surface by topical application.For example, with preparation wet or that do preparation is sprayed at the surface and/or is sprayed to lower surface when interested surface is fit to.The preparation that is fit to use is spray, sticker (stick) and repellent finish or paste.In some cases, can flood interested surface with the repellent preparation.In addition, preparation can be applied to rhizosphere with form wet or that do, there it can near evaporation root system, and act on mutually with the causal organism of consor in root system.In some cases, air can be imported root system, to increase evaporation process.For preventing that insect from invading an area, the present composition can be applied to the surface and/or on every side in this area, for example, composition can be applied to other inlet of door, window and building and/or the surface around these inlets.

When interested surface was plant or plant part, the existence of repellant can be the result of local application, and for example composition can be applied to crop through in the air, or as the achievement of genetic modification host plant, repellant can be synthetic by host plant.

The aldehydes of the present invention's aromatic series and aliphatic series can prepare by multiple synthetic method well known by persons skilled in the art.For example, referring to J.March ed., the senior organic chemistry of Appendix: reaction mechanism and structure, 2nd Ed., McGraw-Hill, New York, 1977.Cinnamic acid can prepare with being synthesized, oxidation (the Traynelis et al. by cinnamyl alcohol for example, american chemical federation magazine (1964) 86:298) or the condensation (BP GB 504,125) by styrene and formyl methylaniline.This alcohols also can separate from natural source.For example, cinnamic acid separates from rotten wooden fungi, the tough lead fungi of close suede (Stereumsubpileatum).Birkinshaw?et?al.，Biochem.J.(1957)66：188。Jasmonal H (HCA) can be obtained by rice, or presses USPN 5,055, and the method described in 621 is synthetic.

The method for optimizing that produces the required component of preparation of the present invention in plant is the mode through recombinant DNA, particularly, change the level of the compound of interest of at least a formula (1), (2), (3), (4) and/or (5) through the constructed genetically modified plants of technology known in the art by in interested plant tissue.These methods relate to and are used in expression cassette that function is arranged in the plant cell, transcribe with the rotaring intertranslating start regulatory region and translate with transcription termination region and transform interested plant cell, described expression cassette be as 5 ' the be operatively connected component of transcribing to 3 ' direction is contained in the plant cell, and transcribe with the rotaring intertranslating start control band open read frame 5 ' in be bonded to coding and can modulate on the dna sequence dna of production and/or be required for producing compound of interest.Because the result that related enzyme concentration changes in the compound of interest biosynthesis, the expression of therefore producing the required enzyme of this compound provides the increase of compound output.Interested especially is in for example production of Selective Control cinnamic acid and/or coniferyl aldehyde and/or HCA in leaf, root, fruit and the seed of plant tissue.The control of compound of interest level in plant, plant part, plant cell, the specified plant tissue and/or relevant with the moment of plant growing, need one or more gene or encoding gene or plurality of enzymes or enzymatic pathway or bunch (cluster), by modulating their expression, can be through producing one or more compounds of preparation of the present invention.

This enzyme or this fermentoid can be in biosynthesis pathway or degradation pathway, and can promptly, regulate intrinsic or endogenous plant expression of gene respectively to raising or downward modulation.Intrinsic plant gene is to be born gene for the host plant gene group.The endogenous plant gene be present in the gene in the interested host plant gene group, it may be intrinsic plant gene or the gene (for example viral gene) that exists owing to the result of plant infection, or mixes the gene of Plant Genome naturally.Host plant also can be modified or introduce one or more by the traditional plant breeding method by recombination method is the gene of the codase of external source for host plant, and described enzyme control compound of interest level and/or be enzyme on the route of synthesis of one or more compound of formula (1), (2), (3), (4) or (5)." regulation and control of gene expression " are meant in the production of transcribing, translating and/or translating control gene of interest product on the level of back.The level of compound of interest is by regulating and control one or more endogenous gene or genetically modified expression is controlled, and one or more synthetic required enzyme of compound interested of these gene codes.

The method of the gene expression of regulation and control in plant corpus is known in the art.The change or the external application compound that plant corpus are carried out growth conditions can influence expression of gene.On molecular level, expression of gene depends on basically that the adjustment structure gene coding region expresses transcribes, translates and stop the control zone.Plant signal by utilize regulating these control zones or directly to the operation of recombinating of these control zones can be regulated and control the expression that (for example) controls the encoding gene of the required enzyme of cinnamic acid level.In the genetically modified application that the plant corpus external source is provided, this transgenosis will comprise the control zone, and this control zone is to select and design obtains the level of required tissue and/or gene expression and time.Suitable, the control zone may be (born) of homology or nonhomologous (non-born) to interested gene." homology " its meaning be the control zone be derived from or basically similar in appearance to usually with the related control zone of interested gene." nonhomologous " its meaning is that the control zone is derived from different nucleotide sources or sequence or is different from the control zone relevant with interested gene usually basically.For example, if compare with the control zone, the sequence of this codase is non-homogeneous on the source, in order to make this gene in interested plant cell, expression be arranged, then must operationally be provided at has transcribing and rotaring intertranslating start regulatory region or promotor of function in these plant cells, make it to be connected with coded sequence.In plant corpus, there be transcribing of function to comprise that those exist host plant body or other floristics gene with the rotaring intertranslating start signal, and direct composition or optionally expression in the host plant body.

Interested especially is this genoid control zone, its optionally be adjusted in plant corpus, plant part, plant cell, special plant tissue structural gene expression and/or be relevant with the moment of plant growing.Those control zones known in the art preferably, particularly transcripting controling area or promotor, it can be used for the expression of gene of modulating-coding enzyme, and described enzyme be in plant, plant part, plant cell, specific plant tissue control formula (1), (2), (3), (4) compound level required and/or be relevant with the moment of plant growing.For example, show that in fruit the different promotors of expressing types are described in U.S. Pat PN4,943,674 and U.S. Pat PN 5,175,095 in; Situation in seed is described among the U.S. Pat PN 5,315,001; And the situation in quick developmental tissue and tender shoots is described among the U.S. Pat PN 5,177,011.

Selective Control cinnamic acid and/or coniferyl aldehyde and/or the HCA biosynthesis in plant in interested tissue, also plant cell is transformed with the expression cassette of coding DNA that comprises structural gene, wherein structural gene is synthetic cinnamic acid and/or coniferyl aldehyde and/or HCA is required, and can increase the structural gene of one or more enzymes of these aldehydes levels in interested tissue.Interested especially is that those can be by the substrate that sees usually in the plant cell, and metabolism goes out the encoding gene of one or more enzymes of the required precursor compound of interested cinnamic acid and/or coniferyl aldehyde and/or HCA biosynthesis.The transgene expression of more preferably at least a formula (1), (2), (3), (4) or (5) compound.

Can prepare the DNA construct of expressing gene of interest, this DNA construct provides expression cassette is integrated in the genome of host plant.Integration can transformation system known in the art agrobacterium (Argobacterium) for example, electroporation or finish by high speed particle mediated transformation method.According to this application, one of saponarin or interested other compound can preferentially be expressed in interested tissue and/or special organelle.Tissue specificity is finished by the transcriptional regulatory district that use has required expression condition.Enzyme is transferred to specific born of the same parents' device can be finished by using suitable translocation peptide.The method of the specifically expressing of the tissue of the DNA construct of having described already and born of the same parents' device is known in the art.

For adjusting and the expression of confirming gene of interest, there are many technology can determine to be present in required dna sequence dna in the plant cell and whether have been integrated in the genome and transcribed out.These technology such as Northern blotting can be used for detecting the mRNA of the required enzyme of coding.Expression can further detect by the enzyme analysis activity or to the protein immunoassay.More preferably, the compound of interest level that is present in the host plant body is to measure with methods known in the art.For example, required phenotype is that as determined with interested gene expression, the content of saponarin increases in interested plant tissue, and/or compares with check plant, the HCA level increase in plant host cell.

For one or more compound with this preparation is incorporated in the target body organism, the target body organism is exposed at least a composition of this repellent preparation, thereby produces the host plant that a kind of encoding gene of the enzyme to control compound of interest level is expressed.At least a composition of repellent preparation can be expressed by plant host, and the seedbed is applied to plant host outside other composition of optional repellent preparation, comes to induce in combination required repellent effect like this.

Can prepare a kind of genetically modified plants, the accumulation flavonoid aldehydes that these genetically modified plants have an increase for example cinnamic acid and coniferyl aldehyde ability with self-protection effect that anti-plant insect is provided or be used to be used as extraction flavonoid aldehydes natural sources and subsequently as repellant.

The accumulation of flavonoid aldehydes can obtain by downward modulation specified plant expression of gene, and gene wherein is the encoding gene that causes the further metabolism of required aldehydes or move away the enzyme of metabolic intermediate from required aldehydes.Under the situation of cinnamic acid, for example, accumulation relates to downward adjusting cinnamic acid 4-hydroxylase (CA4H) and cinnamyl-alcohol dehydrogenase (CAD).CA4H moves away some cinnamic acids from cinnamic acid, produces right-coumaric acid, and right-coumaric acid itself is the metabolism intermediate.Reduce the accumulation that the CA4H activity is not enough to cause cinnamic acid separately, because CAD can change into cinnamyl alcohol with cinnamic acid apace, cinnamyl alcohol is incorporated lignin gradually into or is accumulated with glucosides then.The activity that reduces CA4H and CAD simultaneously can cause the increase that is changed by cinnamic acid to cinnamic acid metabolism and reduce the conversion of cinnamic acid to cinnamyl alcohol.Some cinnamic acids are incorporated lignin gradually into, but cinnamic acid (or free or as glucosides) also is accumulate to above normal level, particularly when the biosynthesis of cinnamic acid increases.This situation occurs in phenylalanine ammoniacalyase (PAL; First and rate-limiting step in general phenylpropionic acid metabolism, Hahlbrock andScheel (1989) Annu.Rev.Plant Physiol.Plant Mol.Biol.40:347-369) active when high, the situation of a kind of abiogenesis in plant corpus, it be to comprise encroached on by fungi and with injured the extensive irritant reaction that insect eats the mechanical wounding of stinging.

The CAD activity that is suppressed in the genetically modified plants body has been proposed as the method (WO93/05159) of synthesizing and improve thus the eupepsy of forage crop that reduces lignin in plant corpus.Though these experiments show the existing qualitative change of the biosynthesis of lignin, but need not to do quantitative change, but clear or make the people recognize a kind of like this what one hopes or wishes for, that is, the accumulation cinnamic acid can be used as the method for increase to insect and other pest repelling.

Cloned some plant CA4H and CAD gene already, and its sequence can obtain in gene pool (GeneBank).These are included between different floristics the Gene Partial for conservative nucleotide sequence, can directly be used in (antisense or sense orientation) on the plant expression vector, in order to the expression that suppresses corresponding endogenous gene (for example, Pear, Deng people The Plant CellAntisense Res.and Develop (1993) 3:181-190, Napoli waits people ThePlant Cell (1990) 2:279-289).More preferably, these conservative gene orders can be used for isolating from the floristic cDNA library that is intended to modify CA4H and CAD cDNA clone.Resulting cDNA clone or part wherein are introduced into plant expression vector (antisense or sense orientation) afterwards and are used to transform interested plant.DNA construct according to the present invention preferably includes the sequence of at least 50 bases, this sequence and endogenous CD4H or CAD dna homolog.

Recombinant DNA molecules can be by being operably connected to carrier useful dna fragmentation can be used to do Plant Transformation with formation plasmid.Can guide the carrier of rna expression to be called as " expression vector " in this article by the cloned segment of gene.This expression vector contains the control element that comprises promotor.Be used in that the typical carriers of expressing gene is known in the art in the higher plant, and comprise the carrier that Agrobacterium tumefaciens (Agrobacterium tumefeciens) Ti-plasmids is derived, these derivative vectors are described in Methods in Enzymology (1987) 153:253-277 by people such as Rogers.Be used to provide the common promotor of introducing the strong constructive expression of gene be cauliflower mosaic virus (CaMV) 35S promoter (can derive from Pharmacia, Piscataway, NJ).Can use composition promotor (as CaMV35S) maybe can induce or grow the promotor of adjusting (as promotor) from PAL gene or CA4H or CAD gene.Use the composition promotor will influence the function of all parts of plant corpus easily, and use inducibility or grow the promotor of regulating, the advantage that then has antisense or have adopted RNA only can under its required tissue and condition, express.In the present invention, it is preferred using the promotor of growing adjusting, because the biosynthetic downward modulation effect of known phenylpropionic acid class can produce bad side effect (Elkind, people such as Y. 1990) Proc.Nat.Acad.Sci. (1990) 87:9057-9061 in the growth of the genetically modified plants that contain allos PAL gene.

There are many different method for transformation to can be used for conduct floristic customary transformation widely.A kind ofly relate to the use agrobacterium for DNA being transferred to the special effective method of dicotyledon.In the method, interested gene is inserted between the T-DNA edges of regions, and this T-DNA district cuts into little recombinant plasmid (for example encode neomycin phosphotransferase II or phosphatidyl threonyl (phosphinothricin) transacetylase) with selectable marker gene.Then this recombinant plasmid is introduced in the agrobacterium host by conversion or three parental generation mating.The plant tissue (for example leaf dish) of this agrobacterium strain of carrying gene of interest by will be suitable cell therewith transforms plant tissue from co-incubation.Cell transformed screens with suitable screening reagent in tissue culture, and plant corpus regenerate subsequently (referring to people Science (1985) 227:1229-1231 such as Horsch.R.B.).Other has been used in the method for transformed plant cells, and the method for the transformation of other more ticklish crop comprises biolistics and electroporation (detailed protocol is referring to people such as Sanford (1993) Methods in Enzymology 217:483-509 especially; And Potter (1993) Methods in Enzymology 217:461-478).

In case produce genetically modified plants, determine the level of the inhibitory enzyme activity that in different transformant, reached with regard to traditional enzyme analysis of available CA4H and CAD.The transformant that may have only sub-fraction to produce has the accumulation that enough low enzyme residual activity removes to cause flavonoid aldehydes, and does not produce some bad side effects in growing process.For this reason, the method for optimizing of producing required transformant with CA4H that suppresses and CAD is respectively these two kinds of genes to be introduced different transformant, passes through the sexual hybridization of standard afterwards with they combinations.This makes that the combination of a large amount of gene inhibition levels can be simultaneously evaluated.

The another kind of way of excessive generation flavonoid aldehydes is to use a class plant gene in genetically modified plants, and these plant genes invest the synthetic specific flavonoid aldehydes ability of microbial hosts.The microorganism of gained can be used for producing in flavonoid aldehydes or the microorganism prepared product that living or non-work natural transportation system as flavonoid aldehydes at fermentation system.Saccharomycete, particularly Saccharomyces cerevisiae is the preferred organism of this order, because it has done the genetic engineering modified (people such as Faulkener of high level expression PAL, (1994) Gene 143:13020,), and it is (people such as Urban, (1994) Eur.J.Biochem 222:843-850) that function is arranged that the cinnamic acid 4-hydroxylase of plant had been presented in the saccharomycete already.

The ability that is produced cinnamic acid by phenyl alanine has been introduced in the expression of PAL.Need two other enzyme steps to produce cinnamic acid from phenyl alanine.At plant corpus, these steps are by the enzyme cinnamate: CoA ligase (CL) and the catalysis of cinnhyl CoA reductase (CCoAR) institute, but 4-coumaric acid CoA ligase (4CL) also can be used as substrate (Knobloch and Hahlbrock (1977) Arch.Biochem.Biophys.184:237-248) with cinnamic acid, and 4CL can be used for replacing CL.Done enough to describe (GeneBank) in detail more than 20 clones' PAL gene with more than 6 4CL genes, be enough to be beneficial to their uses when enforcement is of the present invention.The gene of CCoAR obtains by using the standard gene clone technology to separate the cDNA clone, and described cDNA is as the probe sequence of being derived by N-terminal amino acids sequence or peptide segment or purifying protein.CCoAR from following material purifying come out and done the Partial Feature description: soybean culture (people (1976) Eur.J.Biochem such as Wengenmater, 65:529-536; Luderitz and Grisebach (1981) Eur.J.Biochem, 119:115-124), dragon spruce cambium juice (Luderitz and Grisebach supra), the willow xylem (people (1984) Eur.J.Biochem such as Sarni, 139:259-265) and the xylem in eucalyptus (Eucalyptusgunnii) differentiation people (1994) Plant Physiol.106:625-632 such as () Goffner.The purifying method for optimizing is the method for people such as Goffner (the same), because this method can obtain the single protein belt on the SDS-polyacrylamide gel, this band can be made protein sequence analysis.

(Becker and Guarante (1991) Methods in Enzymol 194:182-187), introduces the gene that clones the expression vector of standard and transforms microbial hosts, preferred yeast for transformation technology by standard such as electroporation.Determine the functional expression of the gene of genetic engineering modified mistake with the enzyme analysis of standard, and select the bacterial classification of maximum output with the analysis of flavonoid aldehydes.Because flavonoid aldehydes has antimicrobial characteristic, so it preferably uses such expression vector, promptly only can cause the gene expression of introducing or chemoattractant be had the expression vector of response in the later stage of growth circulation.Also might wish in fixing full cell reactor people (1987) Biotechnology and Bioengineering 30:1067-1072 such as (for example) Evans, to grow, avoid aldehydes in medium, to accumulate with this through the microbial hosts of genetic engineering modified mistake.

Target body insect and other insect are meant that those are the insect and the insect of disease organism media, and disease organism such as consor are in the lip-deep fungi of a plant part, and plant then is the person of bringing out of fungi.The person of bringing out is meant the nutrients that the plant secretion fungi is required.Fungi is as follows with the example of the plant corpus part of its consor.Alternaria on the fruit, Fusarium (Fusariumsp.) on flower, root and the blade and Fusarium (Fusarium spp.) and the aspergillus (Aspergillus) on root and the blade.Fusarium causes that the dimension pipe of annual vegetables and flowers, perennial herb ornamental plants, raise crop and sensitive plant is withered.Different plants is subjected to the particular type of fungi or plants infringement.Verticillium (a cotton yellow wheel branch spore and a big beautiful spore of taking turns) causes that the withered and consor of dimension pipe is on root, flower and blade.In addition, followingly also constitute the target body organism; Many spores Rust (Phragmidium spp); Rose bivalve bacterium (Diplocaopan rosae); Monofilament shell bacterium (Sphaerotheca tannosa), Oibiapsis sicula; Phytophthora (Phytophthorataraesitica); Phytophthora infestan (Phytophthora infestans); The handle rust belongs to (Pucciniaspp); Alternaria (Alternaria sp); Susaium spp; Gray botrytis (Botrytiscinera); Sclerotinite (Sclerotinia homoeocarca); Tricophytonmentagrophpytes; Dutch elm disease bacterium (Ceratocystis ulmi) and oak wilt bacterium (C.fagacearum).Ceratocystis causes that the dimension pipe is withered, mainly sets.Also comprise blue-green algae (Cyanobacteria).The media of these diseases of available preparation repellent of the present invention comprises beetle and wasp.The target body organism comprises insect, particularly orthoptera; Thrips, it comprises aquatic weevil (water weevil) and thrips; And Homoptera, it comprises aphid such as root aphid and tree louse, leafhopper, aleyrodid, giant coccid, thrips, cicada, caterpillar such as Anticarsia caterpillar, the moth-eaten moth of apple, leaf roll snout moth's larva and a red-spotted lizard.Other target body organism comprises arachnids (particularly tetranychid), fly class (housefly), cockroach, Gastropoda, moth class and bedbug (bed bug (Cimex lectularis)) and kindred class (chicken bedbug (Haematosiphon indorus Duges)) and European dove bedbug (Cimexcolumbarius Jerjus) and Oeciacus (Oeciains vicarius Hrovath).

Interested equally especially is the infringement of avoiding being subjected to radicola by the repellent radicola on grape.Use for this, this preparation need be released into the root system of plant that radicola perches usually.When using with solid form or microencapsulation form, the dosage that the typical case uses is the magnitude of 1% to 35% (w/w benchmark), and the maximum load that is intended to determine is the function of selected shell matter.With technique of analytical chemistry decision and optimization rate of release.For qualitative, can use the GC technology to decide the amount of the aldehydes that disengages.The outturn sample of packing (Pelleting) and selected earth mixture and in different times sampling to measure release conditions.In addition, also can analyze the escaping gas that preparation from then on discharges.Irrigate the activity of application in order to measure blade face and drip irrigation, also preparation can be assessed with GC method well known by persons skilled in the art through the stability of certain hour.The methyl alcohol or the alcohol extraction liquid that also can prepare this preparation are used for the HPLC analysis.Yet the method for the insect of preferred repellent radicola and other root growth provides for example system response of foliage applying, and the preparation of foliage applying is transferred to root system.Need determine the time that this class is used for concrete plant empirically because by blade face to the current of root be shift required.Usually, this be flowing under the following situation the fastest: in time-division between the lights for example cool time, at night or in early in the morning time with before or after fruit growth or during development of plants.

Preparation of the present invention, particularly those contain the preparation of HCA, also can be used for handling: grape, with repellent thrips, nematode and leaf roll snout moth's larva; Rose is with repellent thrips and cotton bollworm; Ox is with the repellent soft ticks; The people is with mosquito repellent; Apple is with the moth-eaten moth of repellent apple; Animal is with the repellent flea; The cockroach dried rhizome of rehmannia of dwelling is with repellent or eliminate cockroach and grow; And corn, with repellent root aphid (root aphid).

Except handling host plant, also available preparation of the present invention is handled insect or other insect that seed is encroached on seed with repellent and/or is used as the disease organism media.Can dust with powder preparation (inorganic material that can adsorb this preparation is referring to U.S. Patent application 4,978,686) or in plant substrate such as vermiculite, mix.The seedling that is become by the seed growth of handling under aseptic environments is the influence that is not subjected to fungi and insect.In addition, seedling also can be handled with this preparation.In some cases, when weak tender shoots may show phytotoxic symptom, need to adjust the preparation of handling, thereby reduce any phytotoxicity relevant with processing.Treatment agent also can be used to control the pollination time of flowering plant.For example, for preventing or postponing pollination, preparation is used with the amount that is enough to repellent honeybee and other insect pollinator.By regulating the residual of preparation, people can control the time span that suppresses pollination.On the other hand, for plant, no matter whether cross pollination is the required of insemination, if insect pollinator should be avoided using during this period this preparation by preparation institute repellent,

In order to determine the susceptibility of concrete insect to the anti-repelling properties of desired composition, adopt in the body or in vitro test, compare in test the target body insect towards behavior, for example near " bait " food that has or do not exist test preparation.Preparation effect through a period of time can be by prolonging observing time up to having only minority test insect (being less than 50% approximately) to walk near the repellent of bait.For disease carrying insect, 90% or bigger scattering ratio normally suitable.For common tedious insect, 80% degree that is reduced to is (for example in the garden or feeding ground) that is fit to.This preparation also needs assessment its be used in phytotoxicity and epidermis susceptibility on the plant, when particularly being used in human skin and/or clothes; Contact dermatitis and olfactory sensitivity adopt cutaneous sensibility well known by persons skilled in the art to test and monitor.Equally, phytotoxicity detects and can adopt method known to those skilled in the art to carry out.The ordinal ranking that phytotoxicity can toxicity seriousness increases is as follows: the 0-plant is without any symptom; The change palm fibre (not having other symptom) that the 1-hypocotyl is very slight; The 2-plant has some withered, and following blade is dying, and skeleton has some to become palm fibre; The whole strain plant of 3-is withered, and blade is dying, and hypocotyl has external and inherent symptom; The necrosis of 4-root, plant is dying.Preferably, formulations employed has 2 grades or lower phytotoxicity grade, more preferably 1 grade or lower.

The formulation components that is intended to do special applications can followingly be determined: at first, assess a concentration range, this scope, is assessed this component respectively and with interested other combination of components of giving customization agent to maximum activity (dose response curve) is provided then from given component non-activity.At each prescription and component, determine of anti-repelling properties and/or phytotoxicity and/or the epidermis influence of concrete preparation then for given insect or other insect and host with or without the serial dilutions of interested any annexing ingredient.Best employing technology well known by persons skilled in the art is calculated with external or in vivo studies.Determine to provide the preparation of following effect: 90% scattering ratio, and/or be 2 or lower to the phytotoxicity grade of plant, be preferably 1 or lower, animal and poultry there are not contact dermatitis basically.

Following example is with explanation but not provides in the mode that limits.

Embodiment

The embodiment scheme that provides is hereinafter used following products: (1) cinnamic acid, from Spectrum Chemical Co., New Jerscy, USA; (2) coniferyl aldehyde is from ADNI Chemical Co., VF; (3) sodium bicarbonate and Tween 80, from SpectrumChemical Co., New Jerscy, USA.

Embodiment 1

Fly class (housefly)The purpose of this test is assessment cinnamic acid and the jasmonal H repellent activity to fly class (housefly).The female fly of 20 2-3 ages in days is put in the cage of 62 * 62 * 34cm, and cage has 325 purpose top mesh to allow the circulation (Carolina BiologicalSupplies) of air.Bait adds that by sweet milk (Carnation) (90%) glucose (10%) and dyestuff (bromophenol blue 0.01%) and 1 milliliter preparation are made, and be put in the 3.5cm culture dish, and place in the pine cage (Carolina Biological Supplies), crib has one, and to bore a diameter down from the top be the 1cm hole, with the convenient approaching cage that contains bait.The culture dish that the diameter 3.5cm of 5 ml waters is housed is put in and supplies with moisture in the cage.After 24 hours, fly is shifted out, and crush on filter paper, check the existence of dyestuff, the vigor of food is got in the dyestuff indication.Fly more than 10% enters gets food, thinks that then preparation lacks repellent activity.

Embodiment 2

Cockroach (Groton bug)

The purpose of this test is the repellent activity of assessment cinnamic acid to cockroach (Groton bug).The adult of 50 pupas and body long 1.5 to 3.5cm is put in the cage of 62 * 62 * 34cm, and cage has 325 purpose top mesh to allow the circulation of air.The inner surface wall of cage is escaped to avoid cockroach with the mixture process of mineral oil and oil glue (2: 3) from floor 5cm to 10cm.Cockroach was fed 48 hours with dog food (Purina), milk powder and water, made it to conform.Two Whatman filter paper " C " (fix with staple, and soak with each preparation of 1 milliliter by 4 * 4cm) doublings twice.Filter paper is subsequently in air drying.Filter paper is put in the cube of two 4cm * 4cm * 4cm, each on ground (end) layer with an independent 0.75cm so that enter.Conceal box for two and be put in 14cm bottom, cage floor far away.After 20 hours, will conceal box and remove, and the cockroach of will and conceal removes and counts.Cockroach greater than 10% enters to conceal thinks then in the box that preparation lacks repellent activity.

Embodiment 3

Aphid (aphis fabae)

The purpose of this test is the repellent activity of assessment cinnamic acid preparation to the black soya bean aphid.In the greenhouse, allow beet (Beta vulgaris) in the compost of 7.5mm diameter basin, grow.When plant reaches tri-leaf period, select eight strain plants at random.In dividing other test, four strain plants are made the cinnamic acid of following processing: 50ppm; Cinnamic acid prescription (the NaHCO of 50ppm ₃+ Tween 80); NaHCO ₃Tween 80; And blank formula.Processing is a foliage applying, and the material that sprays 5 milliliters with manual sprayer (Gilmour) becomes fine mist.Four strain plants are not then handled in addition, and water spray is only used in a strain.Handled with the plant of end reason and put into two row respectively, A or B handle or are untreated, and in 60 * 60 * 30cm boxes and baskets, cage band 325 purpose screen clothes are to allow the circulation of air.At 4,8 and 24 hours, the aphid quantity on counting and recording processing and untreated (A and B are capable) plant.

Embodiment 4

Bemisia argentifolii (tetranychus telarius)

The purpose of this test is the anti-repelling properties of assessment cinnamic acid to Bemisia argentifolii.In the greenhouse, allow cotton in the compost of 7.5mm diameter basin, grow.When plant reaches tri-leaf period, select eight strain plants at random.In dividing other test, four strain plants are made the cinnamic acid of following processing: 50ppm; Cinnamic acid prescription (the NaHCO of 50ppm ₃+ Tween 80); NaHCO ₃Tween 80; And blank formula.Processing will be a foliage applying, and the material that sprays 5 milliliters with manual sprayer (Gilmour) becomes fine mist.Four strain plants are not then handled in addition, and water spray is only used in a strain.Handled with the plant of end reason and put into two row respectively, A or B handle or are untreated, and in 60 * 60 * 30cm boxes and baskets, cage band 325 purpose screen clothes are to allow the circulation of air.At 4,8 and 24 hours, the Bemisia argentifolii quantity that exists on counting and recording processing and untreated (A and B are capable) plant.At 48 hours, count and write down the ovum number on each row plant.

Embodiment 5

Leafhopper class (Cicadellidae)

The purpose of this test is the anti-repelling properties of assessment cinnamic acid to leafhopper.In the greenhouse, allow cotton in the compost of 7.5mm diameter basin, grow.When plant reaches tri-leaf period, select eight strain plants at random.In dividing other test, four strain plants are made the cinnamic acid of following processing: 50ppm; Cinnamic acid prescription (the NaHCO of 50ppm ₃+ Tween 80); NaHCO ₃Tween 80; And blank formula.Processing is a foliage applying, and the material that sprays 5 milliliters with manual sprayer (Gilmour) becomes fine mist.Four strain plants are not then handled in addition, and water spray is only used in a strain.Handled with the plant of end reason and put into two row respectively, A or B handle or are untreated, and in 60 * 60 * 30cm boxes and baskets, cage band 325 purpose screen clothes are to allow the circulation of air.At 4,8 and 24 hours, the leafhopper quantity that exists on counting and recording processing and untreated (A and B are capable) plant.At 48 hours, count and write down the ovum number on each row plant.

Embodiment 6

Thrips (thrips)

The purpose of this test is the anti-repelling properties of assessment cinnamic acid to thrips.In the greenhouse, allow cotton in the compost of 7.5mm diameter basin, grow.When plant reaches tri-leaf period, select eight strain plants at random.In dividing other test, four strain plants are made the cinnamic acid of following processing: 50ppm; Cinnamic acid prescription (the NaHCO of 50ppm ₃+ Tween 80); NaHCO ₃Tween 80; And blank formula.Processing is a foliage applying, and the material that sprays 5 milliliters with manual sprayer (Gilmour) becomes fine mist.Four strain plants are not then handled in addition, and water spray is only used in a strain.Handled with the plant of end reason and put into two row respectively, A or B handle or are untreated, and in 60 * 60 * 30cm boxes and baskets, cage band 325 purpose screen clothes are to allow the circulation of air.At 4,8 and 24 hours, the thrips quantity that exists on counting and recording processing and untreated (A and B are capable) plant.At 48 hours, count and write down the ovum number on each row plant (folial gap place).

Embodiment 7

2 tetranychids (tetranychus telarius)

The purpose of this test is the anti-repelling properties of assessment cinnamic acid to leafhopper.In the greenhouse, allow cotton in the compost of 7.5mm diameter basin, grow.When plant reaches tri-leaf period, select eight strain plants at random.In dividing other test, four strain plants are made the cinnamic acid of following processing: 50ppm; Cinnamic acid prescription (the NaHCO of 50ppm ₃+ Tween 80); NaHCO ₃Tween 80; And blank formula.Processing is a foliage applying, and the material that sprays 5 milliliters with manual sprayer (Gilmour) becomes fine mist.Four strain plants are not then handled in addition, and water spray is only used in a strain.Handled with the plant of end reason and put into two row respectively, A or B handle or are untreated, and in 60 * 60 * 30cm boxes and baskets, cage band 325 purpose screen clothes are to allow the circulation of air.At 4,8 and 24 hours, 2 tetranychid quantity that exist on counting and recording processing and untreated (A and B are capable) plant.At 48 hours, count and write down the ovum number on each row plant (folial gap place).

Embodiment 8

Mosquito (Aedes aegypti) External anti-repelling properties test method

The purpose of this test is the anti-repelling properties of assessment cinnamic acid to mosquito.With 20 roughly the female mosquito of not sucking blood of four ages in days introduce in the chamber.Four milliliters test preparation is drawn and is put on 16cm Whatman2 number round filter paper and in air drying.The test paper of test usefulness is positioned in the ventilation intake case.Make CO ₂Via water from the ventilation intake end in odorimetry case air channel by going in the gateway bubbling; And the fan assembly of use minimum speed.Catch was opened 5 minutes, closed and calculate and write down the number of mosquito afterwards.23% Metadelphene is used for being used as positive controls. The anti-repelling properties test method, the field test

The purpose of this experiment is the activity of bioanalysis cinnamic acid as the mosquito repellant.Cut the ring of two diameter 18cm and two diameter 16cm from the 1mm of screen material nylon mosquito cage keeper.Handling ring (16cm) is dipped in 1 ml of formulation: cinnamic acid (2%) is in 2% Tween 80 and 6%NaHCO ₃In, make it afterwards in air drying 2 hours.Ten female mosquitos of not feeding (Aedes aegypti) (5 to 7 age in days) from the mosquito control research laboratory of KearneyAgricultural Center are introduced into (contrast and processing carton) in one pint of mosquito carton of two Kearney (Fischer).Each carton all covers with untreated 18cm circular screen and seals this pint carton with fringing, and the lid part is removed.The 16cm that handles was encircled on the pin that is put in the adult male volunteer (cleaning with perfumed soap and water in advance), untreated 16cm ring is put on its another pin.One pint of mosquito carton flushes placement, makes one side of screen cloth contact 5 minutes with the gauze of pin.Mosquito does not directly contact with compound.After 5 minutes, the counting 10 mosquito numbers of sucking blood/be satiated with food of merely hitting the results are shown in table 1, and are as follows.Assessing 30 insects altogether, have only two ends to be driven away by the cinnamic acid preparation, is 19 and contrast in (being untreated) group.

Table 1

The mosquito anti-repelling properties

ProGuard scheme Kearney mosquito research laboratory

(the mosquito number of sucking blood/being satiated with food in 10 mosquitoes)

Test 1 test 2 tests 3 total cinnamic acid preparations ¹0,/10 2,/10 0,/10 2/30 contrast (end reason) 5,/10 7,/10 7,/10 19/30 ¹Cinnamic acid (2%), 2% Tween 80 and 6%NaHCO ₃The aqueous solution scheme similar to top description be used to test jasmonal H.

Embodiment 9

The Lepidoptera anti-repelling properties of laying eggs

The purpose of this experiment is the expeling power of decision cinnamic acid to the beet armyworm adult.Build a covering device, this device import forces air flow stream to be crossed in the flight cage and handles or untreated pot plant.Five strain tomato plant were handled with the chemical formulation and the component of 5 milliliters of variable concentrations in tri-leaf period; Be put in afterwards in this cage.In addition five strain tomatos then compare with 5 milliliters water spray, also are put in the cage afterwards.In cage, discharge 40 beet armyworms to be laid eggs (Spodoptera exigna) adult.Open the exhaust fan on this device, and with the cage of speed straight line airflow passes as the chemicals gas evaporator.After 24 hours, on processing plant, be untreated plant and cage wall, assess the egg situation of laying eggs.

Embodiment 10

The steam test of radicola-anti-repelling properties

The purpose of this test is the steam anti-repelling properties of assessment cinnamic acid to radicola.The rhizomatic piece of grape that will have the radicola ovum (n-30) that can live is positioned in the dish of 50 * 9mm, and its inner surface is to handle through 400 milliliters of known products concentrates.Chemical substance is not placed directly on the root, so the absorption of root or metabolism will not be factors of influence.This dish is shut and sealed with adhesive tape.After 7 days, these dishes are opened, determine whether insect can hatch and base oneself upon and grow, or do not have the radicola of new hatching to flee from root and death in root.

Embodiment 11

At genetically modified plants body overexpression flavonoid aldehydes

The poly+RNA for preparing 20 micrograms, and synthetic cDNA.This part is cloned into (a commercial available cloning vector) in λ-ZAP II carrier.At least screen 500,000 recombinants with oligonucleotide probe, described oligonucleotide probe is by following two kinds of sequences Design: the clone CA4H and the CAD gene conservative sequence that derive from GeneBank; Reach the protein peptide sequence that obtains by the host plant purifying of wanting.Select strong hybrid strain, and be used for studying this cDNA library.To its sequence of clonal analysis of gained, with guarantee with suitable gene order with antisense or have justice direction introduced plant expression cassette in.According to disclosed method, with this antisense with there is the construct of justice to introduce in the Agrobacterium tumefaciens LBA4404 by directly transforming.Tobacco (kind Samsun) leaf dish transforms people (1985) Science 227:1229-1231 such as () Horsch with very perfect open method.The plant that contains CA4H or CAD construct confirms with polymerase chain reaction (PCR) (PCR), and selects and be used for further analysis.

Adopt very perfect open analytical method, be used for determining CA4H and CAD enzymic activity from the vegetable material of that transform or unconverted check plant.The activity of selecting CA4H and CAD is reduced to 20% the plant that is lower than the being seen activity of check plant, is used for further analysis.Have the plant of low CA4H activity and plant hybridization, and screen the filial generation of two kinds of gene constructs of heredity by PCR with low CAD activity.The separating method of employing standard is analyzed the output of flavonoid aldehydes of plant of the CAD activity of the CA4H have inhibition and inhibition.Afterwards, adopt arbitrary suitable embodiment, test produces the plant of flavonoid aldehydes the repellent of insect or other insect is renderd a service, and for example uses the method for embodiment 3, the ability of test transgene cotton repellent aphid.

Embodiment 12

In microorganism system, produce flavonoid aldehydes

Produce a cDNA library with extraction from the RNA of the tobacco stem in six ages in week.The poly+RNA for preparing 20 micrograms, and synthetic cDNA.This part is cloned into (a commercial available cloning vector) in λ-ZAP II carrier.At least screen 500 with oligonucleotide probe, 000 recombinant, described oligonucleotide probe is by following sequences Design: adopt people's such as Goffner scheme Plant Physiol. (1994) 106:625, the CCoAr protein peptide sequence that is purified into by the tobacco stem tissue in six ages in week.Select strong hybrid strain, and be used for studying this cDNA library.With its sequence of clonal analysis that obtains, guarantee the cDNA of total length is inserted in the evaluation of body and the CCoAR gene order introducing Yeast expression carrier pNTL8110 that will be suitable people (1994) Gene 143:13-20 such as () Faulkner.Coded sequence (the PAL of the red winter spore of class yeast (Rhodpsporidiumtoruloides) phenylalanine ammoniacalyase; GeneBank RHDPAL district) and parsley 4-coumaric acid: the coded sequence (4CL of CoAl ligase; The GeneBankPC4CL1AA district) introduces the yeast expressed carrier that is equal to like the homogeneous phase.By very perfect open method (Becker, and Guarente (1991) Methods inEnzymology 194:182-187; Simon, (1993) Method in Enzymol.217:478-483), adopt this PAL, 4CL and CCoAR construct to transform the Saccharomyces cerevisiae bacterium with electroporation.Transformant is screened with lacking leucic Min. medium.The conversion bacterial strain that has all three kinds of gene constructs confirms with PCR, and selects and be used for further analysis.

Adopt very perfect open determination method, be used for from transforming or the extract of unconverted control strain is determined the enzymic activity of PAL, 4CL and CCoAR.The activity of selecting PAL, 4CL and CCoAR is used for further analysis apparently higher than the bacterial strain that detects the background activity in control strain.With the open method of standard, analyze the bacterial strain of selecting, select those bacterial strains of the cinnamic acid of producing significant quantity, be used for the optimization fermentation condition.Afterwards, with described any method, the effectiveness of test gained plant on repel insects.

Embodiment 13

HCA is as the activity of insect repellent

The purpose of this experiment is to determine whether jasmonal H is a kind of effective insect repellent.After FDA carries out the negative skin irritatin test of rabbit, on the skin of 2 to 4 male subject, estimate.With 1ml HCA obliterating on a forearm.Be with the untreated hand of gloves protect; and with it simultaneously; the arm of handling is exposed to about 30 minutes time interval is equipped with a large amount of (2; 000-4; 000) cage of the mosquito that do not suck three minutes, up to there being two places to bite (have two places to bite or respectively have a place to bite) two continuous in-tests an in-test.Time interval of using and be known as " guard time " when having two places to bite.To Aedes aegypti, HCA is decided to be 3 grades (121-180 minute duration).To anopheles quadrimaculatus (Anopheles quadrimaculatus Say), HCA is decided to be 2 grades (31-60 minutes).

Also on the clothes of handling, made HCA to the Aedes aegypti Evaluation on effect.In these trials, women's mercerized cotton stockings have been adopted.Mensuration part on the ankle is soaked with HCA, in an amount equivalent to per 2 feet 3.3g.These stockings are deployed on the frame, in a row are hung on indoor then.First test was carried out in processing in back 24 hours.Stockings are through on the arm, and the processing section is in the centre of forearm.Untreated hand gloves protect, and will wear stockings and arm and be exposed to test cage one minute.Bite if obtain 5 places, processing then is considered to invalid.Bite if be less than 5 places, then will expose and carry out continuously every day, up to the 14th day, afterwards with weekly or fortnightly interval carry out.HCA is decided to be 4 grades (term of validity was greater than 10 days) in this test, and is identical with the value of Metadelphene.

These tests confirm that cinnamic acid of the present invention and jasmonal H preparation are effective repellants to mosquito.

All documents of mentioning in this manual with apply for a patent the indication field relevant with the present invention in technical staff's technical merit.All documents and apply for a patent and all incorporate this paper into as a reference, this incorporate into to be equal to indicate each independent file or apply for a patent specifically and individually be incorporated herein by reference.

The present invention has now narrated fully, for those skilled in the art, can make many changes and correction under the spirit and scope that do not depart from the appended claims claim of the present invention.

Claims

Modification according to the 19th of treaty

1. the composition of a repellent insect, described repellant compositions are included as at least a flavonoids aldehyde of the concentration that is enough to provide repellent insect fragrance.

2. according to the composition of claim 1, wherein said flavonoids aldehyde is cinnamic acid, jasmonal H and/or coniferyl aldehyde.

4. according to arbitrary composition of claim 1-3, wherein said composition also comprises saponarin.

5. near method from repellent insect such as the insect body of mammals, described method comprises not cause the arbitrary composition of skin irritant amount local application according to claim 1-4.

6. according to the method for claim 5, wherein said composition forms is selected from by spray, sticker, repellent finish and paste.

7. method according to claim 5 or claim 6, wherein said mammal is selected from people, ox and sheep.

8. arbitrary method according to claim 5-7, wherein said mammal is that people and described insect are flea or mosquito.

9. arbitrary method according to claim 5-7, wherein said mammal is that ox and described insect are soft ticks.

10. method near the repellent insect plant, said method comprises arbitrary composition of using according to claim 1-4, and wherein said plant is selected from grape, rose, apple and corn.

11. according to the method for claim 10, wherein said plant is that grape and described insect are thrips, nematode, radicola and leaf roll snout moth's larva.

12. the method according to claim 10, wherein said plant are rose and described insect is thrips or melon aphid.

13. the method according to claim 10, wherein said plant are apple tree and described insect is the moth-eaten moth of apple.

14. the method according to claim 10, wherein said plant are corn and described insect is the root aphid.

15. one kind prevents that insect from invading the method in a zone, described method be according to claim 1-2,4 and the composition of one of 18-20 be applied near the described intrusion.

16. according to the method for claim 15, wherein said insect is a cockroach.

17. one kind prevents to set the method that is subjected to insect infringement, described method comprises: with described trunk with contact with arbitrary composition of 18-21 according to claim 1-2,4.

18. the composition of a repellent insect, described composition comprise saponarin and are at least a flavonoids aldehyde of the concentration that is enough to provide repellent insect fragrance.

19. according to the composition of claim 18, wherein said flavonoids aldehyde is selected from cinnamic acid, jasmonal H and coniferyl aldehyde.

20. the composition of a repellent insect, described repellant compositions comprise at least a of the jasmonal H of the concentration that is enough to provide repellent insect fragrance and coniferyl aldehyde.

21. a method that prevents that trees from being encroached on by insect, described method comprises: described trunk is contacted with one group of composition, and said composition is included as at least a flavones aldehyde that is enough to provide the concentration of keeping away insect.

Claims (17)

1. the composition of a repellent insect, described repellant compositions are included as at least a flavonoids aldehyde of the concentration that is enough to provide repellent insect fragrance.

2. according to the composition of claim 1, wherein said flavonoids aldehyde is cinnamic acid, jasmonal H and/or coniferyl aldehyde.

3. according to the composition of claim 1 or 2, wherein said concentration is 10-5000ppm for total composition.

4. according to arbitrary composition of claim 1-3, wherein said composition also comprises saponarin.

5. near method from repellent insect such as the insect body of mammals, described method comprises not cause the arbitrary composition of skin irritant amount local application according to claim 1-4.

6. according to the method for claim 5, wherein said composition forms is selected from by spray, sticker, repellent finish and paste.

7. method according to claim 5 or claim 6, wherein said mammal is selected from people, ox and sheep.

8. arbitrary method according to claim 5-7, wherein said mammal is that people and described insect are flea or mosquito.

9. arbitrary method according to claim 5-7, wherein said mammal is that ox and described insect are soft ticks.

10. method near the repellent insect plant, said method comprises arbitrary composition of using according to claim 1-4, and wherein said plant is selected from grape, rose, apple and corn.

11. according to the method for claim 10, wherein said plant is that rose and described insect are thrips, nematode, radicola and leaf roll snout moth's larva.

12. the method according to claim 10, wherein said plant are rose and described insect is the melon aphid.

13. the method according to claim 10, wherein said plant are apple tree and described insect is the moth-eaten moth of apple.

14. the method according to claim 10, wherein said plant are corn and described insect is the root aphid.

15. one kind prevents that insect from invading the method in a zone, described method is that the composition according to one of claim 1-4 is applied near the described intrusion.

16. the method for claim 15, wherein said insect is a cockroach.

17. a method that prevents that forest from being encroached on by insect, said method comprises: with the trunk of described tree with contact according to the arbitrary composition of claim 1-4.