KR102016007B1 - Insecticide Composition comprising inhibitors of Drosophila saponin receptor and saponin and Screening Method for inhibitors of Drosophila saponin receptor - Google Patents

Abstract

The present invention relates to an insecticide composition containing saponin and a Drosophila saponin receptor inhibitor as an active ingredient. The insecticide composition according to the present invention inhibits the saponin receptor function of pests and induces cell death by saponins, thereby causing both insect larvae / adults. It can be removed and is useful as a pesticide.

Description

Insecticide Composition comprising inhibitors of Drosophila saponin receptor and saponin and Screening Method for inhibitors of Drosophila saponin receptor}

The present invention relates to an insecticide composition comprising saponin and a Drosophila saponin receptor inhibitor and a method for screening a Drosophila saponin receptor inhibitor, and more particularly, an insecticide composition and a Drosophila saponin containing an inhibitor and a saponin that inhibit the Drosophila saponin receptor A method for screening a Drosophila saponin receptor inhibitor comprising the step of measuring the activity of a receptor.

Saponins are compounds found in many types of plants that, when dissolved in water, become amphiphilic glycosides. Quillaja saponaria husks are used as the main industrial source of triterpenoid saponins used as blowing agents (Pelah et al., Journal of Ethnopharmacology Vol. 81, pp. 407-409, 2002). Saponins also exhibit anti-viral and anti-inflammatory activity in humans and are widely used as dietary aids (Pelah et al., Journal of Ethnopharmacology Vol. 81, pp. 407-409, 2002). But for insects, saponins act as poisons. When larvae of Spodoptera littoralis are grown in medium containing quillaya saponin, it has been reported that larval mortality and longer larval seasons are observed when compared to those grown in normal medium (De Geyter et. al., Pest Technol Vol. 1, pp. 96-105, 2007). This means that saponin can be used as an insecticide. Rafi second quill Ria saponin derived from (Drosophila melanogaster) but has been reported that activating specific receptors (sensilla) of the lip end (labellum), Darling not activate any sense in praying (Ling, F. et al., Journal of Neuroscience Vol 34, pp. 7148-7164, 2014). This means that insects such as fruit flies can detect and avoid saponins for survival. However, molecular sensors capable of detecting saponins in insects are not known.

Preferences for food depend on taste, aroma and appearance. The taste buds of flies include the labellum, legs and internal organs. The lip end of the fruit fly is the most important taste bud, which includes 31 taste senses, each of which is divided into long (L- (, medium (I), and short (S)) bristle lengths (Liman, E). et al., Neuron Vol. 81, pp. 984-1000, 2014) Long and short types of sensory groups detect taste receptor neurons (GrNs) that detect four sugars, low concentrations of salt and water or osmotic molar concentrations. Liman, E. et al., Neuron Vol. 81, pp. 984-1000, 2014. Only S-types are known to detect bitter taste compounds and high concentrations of salts. Type sensory groups have two taste receptor neurons, one that detects sugars and low concentrations of salt and the other detects bitter taste compounds and high concentrations of salts (Liman, E. et al., Neuron Vol. 81, pp. 984-1000, 2014) .The taste sensory on the tip of the lip can determine whether fruit flies ingest or avoid certain foods. Additional taste receptors in the pharynx are known to enhance the chewing activity of foods identified as nutrients or to spit contaminated foods (Lee, Y. et al., Chemical senses Vol. 40, pp. 525-533, 2015; Joseph, RM et al. ELife Vol. 6, pp.e24992, 2017) .Evaluation of taste-stimulating substances by sensory organs in the legs can determine whether food is edible or poisonous. It is known to play an important role in the early decision of fruit flies to determine cognition.

In Drosophila, the gustatory receptor gene (Gr genes) family contains 60 members and is known to encode 68 receptor proteins through alternative splicing (Thorne, N. et al., Current Biology Vol. 14, pp. 1065-1079, 2004; Clyne, PJ et al., Science Vol. 287, pp. 1830-1834, 2000; Dunipace, L. et al., Current Biology Vol. 11, pp. 822-835, 2001). Molecular genetic analysis using the GAL4 / UAS reporter system has reported that the Gr gene family can be divided into sugar sensing groups and bitter taste sensing groups (Thorne, N. et al., Current Biology Vol. 14 , pp. 1065-1079, 2004; Wang, Z. et al., Cell Vol. 117, pp. 981-991, 2004; Weiss, LA et al., Neuron Vol. 69, pp. 258-272, 2011) , Taste receptor proteins that detect toxic substances are known to be expressed in taste receptor neurons that sense bitter taste (Weiss, LA et al., Neuron Vol. 69, pp. 258-272, 2011).

A knockout study of the Gr gene has revealed that so far, a total of nine Gr genes are needed to detect bitter taste. Of these, Gr32a, Gr33a and Gr66a are expressed in all bitter taste-sensing taste receptor neurons at the Drosophila lip labellum and have been reported to work to avoid various kinds of bitter taste compounds (Weiss, LA et. al., Neuron Vol. 69, pp. 258-272, 2011; Moon, SJ et al., Current Biology Vol. 19, pp. 1623-1627, 2009; Lee, Y. et al., Neuron Vol. 67, pp. 555-561, 2010). In comparison, six other genes are known to be required for the detection of compounds with a particular bitter taste (Lee, Y., et al., Proceedings of the National Academy of Sciences Vol. 106, pp. 4495-4500, 2009).

Under these technical backgrounds, the present inventors have made diligent efforts to find compositions that can enhance the effect of saponins as insecticides by finding and inhibiting saponin receptors in Drosophila. As a result, in the case of compositions containing inhibitors of Gr28b.c and saponins as active ingredients, It was confirmed that the function of the saponin taste receptor is suppressed and the effect as an insecticide is exhibited, thereby completing the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

It is an object of the present invention to provide a pesticide composition which exhibits a pesticidal effect by inhibiting the function of a saponin receptor.

Another object of the present invention is to provide a method for screening a substance which inhibits saponin receptor function.

In order to achieve the above object, the present invention provides a pesticide composition comprising an inhibitor of Gr28b.c, which is a saponin and a saponin receptor.

The present invention also provides a method of treating a drug, comprising: (a) treating a candidate with Gr28b.c, a saponin receptor; And (b) selecting a substance that reduces the activity of Gr28b.c as an insecticide, as compared to the untreated control.

The screening method of the insecticide composition according to the present invention may inhibit the activity of the saponin receptor Gr28b.c to increase cell death induced by saponins, and may screen for substances that exhibit insecticidal effects, and are screened by the present method. The composition comprising the prepared substance and saponin as an active ingredient is useful for inducing cell death by saponins and showing insecticidal effects on larvae and adults.

Figure 1 shows the behavioral avoidance and electrophysiological responses of Drosophila to saponins.
1A: Schematic diagram showing the basic molecular structure of saponin.
1B: A graph measuring binary food choice analysis according to the concentration of saponin.
1C: Tip recording analysis of tip reactivity according to saponin concentration in S6 sensor.
1D is a graph of action potentials induced by saponins measured at 1C.
1E: A graph of sugar inhibition reaction using 100 mM sugar and saponin by concentration in L4 sensor.
1F: It is a graph which measured action potential induced by saponin measured in 1E.
The asterisk indicates that the change was significant compared to the control group, and the statistical meaning is the analysis of two data sets (* P <0.05, ** P <0.01) using single factor ANVOVA with Scheffe's analysis as a post hoc test. The error bars represent SEMs.
It illustrates the composition and the hybridization relationship between the components required for the implementation of the present invention.
Figure 2 shows the results of identifying the Gr28b gene as a fruit fly saponin receptor.
2A: As a result of dual food selection analysis using 21 Gr gene variants and 5% saponin, at least four times were performed.
2B: Tip recording of the control group and Gr28b ^Mi Drosophila in S6 sensory organs, with stimulants 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone ), 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobelin, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine and 0.1 mM berberine berberine) was used.
2C: Rejection test results of saponin concentrations in control group and Gr28b ^Mi Drosophila.
2D: 15, 30, 60 and 240 minutes the control group and the food containing 5% saponin was measured by the control group and Gr28b ^Mi Drosophila.
The asterisk indicates that the change was significant compared to the control group, and the statistical meaning is the analysis of two data sets (* P <0.05, ** P <0.01) using single factor ANVOVA with Scheffe's analysis as a post hoc test. The error bars represent SEMs.
Figure 3 is the result of testing the saponin reactivity by sensory sensory groups.
3A: the results of analysis of PER in the control group and the Gr28b ^Mi Drosophila leg.
3B: PER analysis of control and Gr28b ^Mi Drosophila mouth (proboscis). Drosophila was first injected with 2% sugar water, and then mixed radish mixed with 5% saponin in 2% sugar water was tested four times.
3C: Mapping analysis of control group stimulated with 5% saponin and all sensory phases of Gr28b ^Mi Drosophila.
3D: Saponin-induced action potentials were measured in the S6 sensory group of the control group and Gr28b ^Mi Drosophila.
The asterisk indicates that the change was significant compared to the control group, and the statistical meaning is the analysis of two data sets (* P <0.05, ** P <0.01) using single factor ANVOVA with Scheffe's analysis as a post hoc test. The error bars represent SEMs.
4 is a result of analyzing the structure of the Gr28b genome, confirming that the Gr28b.c gene is a saponin receptor.
4A is a structural diagram of a Gr28b dielectric. The Gr28b genome produces five different proteins through selective seaming. Arrowheads indicate P-element insertions and each arrow indicates the position of the primer for RT-PCR.
RT-PCR results of the Gr28b genome of the control and Gr28b ^Mi Drosophila using the expression primers in 4B 4A. tubulin was used as a control.
4C: Results of dual food selection analysis for saponins when each Gr28b isoform was introduced using the GAL4 / UAS system. Forms a to e were derived from the Gr28b.a-Gal4 system prepared in the Gr28b ^Mi Drosophila background. Four to six experiments were performed.
4D: Measurement of action potentials induced by saponin in each transgenic Drosophila S6 sensory organ.
4E: Representative tracking results for 4D samples.
4F: Results of measuring action potentials induced by saponin in each transgenic Drosophila I3 or L6 sensory organ.
4G: Representative tracking results for 4F samples.
The asterisk indicates that the change was significant compared to the control group, and the statistical significance is the analysis of two data sets (* P <0.05, ** P <0.01) using single factor ANVOVA with Scheffe's analysis as a post hoc test. The error bars represent SEMs.
5 is a result confirming the toxicity of larvae and adults by saponin.
5A: Survival rate according to saponin concentration of control larvae.
5B is a graph measuring survival rate according to the saponin concentration of Gr28b ^Mi larvae.
5C is a graph measuring the pupation rate according to the saponin concentration of the control larvae.
5B is a graph measuring the pupation rate according to the saponin concentration of Gr28b ^Mi larvae.
The asterisk indicates that the change was significant compared to the control group, and the statistical significance is the analysis of two data sets (* P <0.05, ** P <0.01) using single factor ANVOVA with Scheffe's analysis as a post hoc test. The error bars represent SEMs.
6 is a result of measuring the saponin repellent action when the expression of the fruit flies saponin receptor protein was inhibited using Gr28b RNAi.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, to determine the insecticide effect of the fruit fly saponin receptor.

In the present invention, when the expression of the Drosophila saponin receptor is suppressed, the sensitivity to saponin decreases, and the Drosophila ingests a large amount of saponin, so that the insects do not grow from larvae to adulthood.

That is, in one embodiment of the present invention, when a food containing saponin was provided to the larva from which the Drosophila saponin receptor Gr28b.c was removed, it was confirmed that the survival rate and solubility mull were significantly reduced compared to the control group (FIG. 5). .

Therefore, in one aspect, the present invention relates to an insecticide composition comprising an inhibitor of Gr28b.c, which is a saponin and a saponin receptor.

In the present invention, the inhibitor of Gr28b.c may be any one having a property of inhibiting the activity of Gr28b.c, but is preferably applicable to a living body, but is not limited thereto, for example, Gr28b. antibodies or aptamers that specifically bind to .c; It may be siRNA specific to the Gr28b.c gene, RNAi or shRNA including a shRNA, and more preferably RNAi represented by SEQ ID NO: 3, but is not limited thereto.

The present invention screened the Gr gene family, a Drosophila taste receptor gene, to confirm that the Gr28b gene is important for the detection of saponin in Drosophila (FIG. 3), and five kinds of taste produced by selective seaming of the Gr28b genome. It was confirmed that the Gr28b.c protein is a Drosophila saponin receptor among the receptor proteins (Gr 28b.a to Gr28b.e) (FIG. 4).

That is, the present invention first revealed that, among the 68 taste receptor proteins, the Gr28b.c protein, which is expressed at the fruit fly lip labellum, functions as a receptor in the detection of saponin in fruit flies.

Thus, in another aspect, the present invention provides a method for treating a candidate substance, comprising: (a) treating a candidate with Gr28b.c, a saponin receptor; And (b) selecting a substance that reduces the activity of Gr28b.c as an insecticide, as compared to the untreated control.

In the screening method, candidates of step (a) include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts or plasma, and the compounds are novel compounds. Or a well-known compound, preferably obtained from a library of synthetic or natural compounds.

Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like.

In one embodiment, the Gr28b.c taste receptor can be introduced into taste neurons in response to candidates and then expressed and used for insecticide or repellent screening. For example, the nucleotide encoding the taste receptor can be cloned into a known expression vector and transduced into taste neurons to express it.

The vector is an expression vector capable of expressing a protein of interest in a suitable host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert. In addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. In addition, the promoter of the vector may be constitutive or inducible. In addition, the vector includes a selectable marker for selecting a transformant including the vector, and in the case of a replicable expression vector, includes a replication origin.

The introduction refers to the introduction of foreign DNA into the cell by transfection or transduction. Transfection and transduction can be performed by several methods known in the art.

The gene encoding the Gr28b.c taste receptor used in the present invention can be cloned and expressed in a suitable host cell. As the host cell, various microorganisms such as Escherichia coli, insect cells or animal cells may be used, for example, nerves. Cells, in particular S2 cells, can be used.

Such candidates include, for example, compounds that change the activity of the Gr28b.c receptor, for example organic or inorganic small molecules, biological molecules such as peptides, proteins, nucleic acids, or analogs thereof, bacteria, plants, fungi and the like. It may be an extract prepared from, but is not limited thereto.

Based on the Gr28b.c taste receptor, compared to the untreated control, a substance whose activity is increased or inhibited in the taste receptor treated with the candidate can be selected as a pest control agent, for example, an insecticide or a repellent.

In the present invention, the pest refers to a target that can directly or indirectly damage humans, livestock, or cultivated plants, or cause economic loss. In terms of control, pesticides refer to drugs that actively kill pests, and repellents refer to drugs used for the purpose of chasing pests.

For example, if an increase in activity occurs in a taste receptor treated with a candidate compared to an untreated control, the candidate may feel a strong bitter taste, which means that insects or pests may evade the candidate, Alternatively, it can be screened as a repellent that prevents pest access. In addition, if there is activity inhibition in the taste receptor treated with the candidate substance as compared to the untreated control group, this may cause the candidate substance to be ingested by ingestion without recognition of the candidate substance. Therefore, such a candidate substance is an insecticide that fights insects or pests. Can be selected. In the embodiment of the present invention, it was confirmed whether the fruit fly-derived taste receptor according to the present invention can be used as a pest control screening system through saponin.

The candidate substance may be selected as a ligand that can be used as a pest control agent, for example, an insecticide or a repellent agent, when the activity of the taste receptor is changed by the binding of the Gr28b.c taste receptor and the candidate substance of the insect. At this time, the change in activity of the taste receptor can be confirmed by measuring mRNA expression level, or by using an antibody that specifically binds to the taste receptor to determine the amount of protein by immunoassay. The change in current generated by channel formation in taste receptors recognized as ligand was measured and confirmed.

In the present invention, the insects are mosquitoes (Culcidae), such as mosquitoes (Ceratopogonidae), tsetse flies (tsetse fly), black flies (Simuliidae), sand flies (sand fly), bed bugs (bed bug), needle norinjae (assassin bug ), Fleas, (louse), mite (mite) and mite (tick) may be selected from the group consisting of, but is not limited thereto.

In the present invention, the mosquito is Aedeomyia ( Aedesomyia ), Aedes ( Aedes ) (including Aedes aegypti ), Anopheles ( Anopheles ) ( Anopheles Gambia ( Anopheles gambiae ) and Anopheles annulipes ), Armigeres genus, Ayurakitia genus, Bironella genus, Borichinda genus, Chagacia Chagasia genus, Coquillettidia genus, Culex genus, Culiseta genus, Deinocerites genus, Eretmapodites genus, Ficalbia in, Galindo also Maia (Galindomyia) in, seahorses and Ethiopia (Haemagogus) in Hayes, only California (Heizmannia), A Horde posted ah (Hodgesia) speed, iso Stoke Maia (Isostomyia) in John belki California (Johnbelkinia) in a key Mia (Kimia) in Lima, Tooth (Limatus) in, Lucia (Lutzia) , Malaya (Malaya) in only Sonia (Mansonia) in Maori go'el Dia (Maorigoeldia), A beauty Maia (Mimomyia) in sludge Leone (Onirion) in, Opie Pécs (Opifex) in ortho grape Maia (Orthopodomyia ), A program Thoreau Fora (Psorophora), A runchyo Maia (Runchomyia) in, Sarbanes Tess (Sabethes), A Shan say Ana (Shannoniana), A topographic Maia (Topomyia) genus Toxoplasma Lynch Tess (Toxorhynchites), A Trichoprosopon genus, Tripteroides genus, Udaya genus, Uranotaenia genus, Verrallina genus, Wyeomyia genus and zeus Diagnostics Maia be being selected from the group consisting in (Zeugnomyia), but the embodiment is not limited thereto.

In the present invention, the mosquito on the back may be selected from the group consisting of Culicoides genus, Leptoconops , Forcipomyia genus, but is not limited thereto. .

In the present invention, the body fly is Glossina austeni , Glossina morsitans , Glossina pallidipes , Glossina swynnertoni , Glossina fusca fusca , Glossina fuscipleuris , Glossina frezili , Glossina haningtoni , Glossina haningtoni , Glossina Glossina longipennis , Glossina medicorum , Glossina nashi , Glossina niGrofusca niGrofusca , Glossina severini severini , Glossina schwetzi , Glossina tabaniformis , Glossina vanhoofi , Glossina caliginea , Glossina caliginea , Glossina caliginea

Ski festival Fu ski festival (Glossina fuscipes fuscipes), global seed and Fu ski festival mareutiniyi (Glossina fuscipes martinii), global seed or sold Keraton sold Mosquera (Glossina pallicera pallicera), global seed or sold Mosquera news Ponte Adige (Glossina pallicera newsteadi) , Glossina palpalis palpalis , Glossina palpalis gambiensis and Glossina tachinoides , but may be characterized in that it is selected from the group consisting of It is not limited to this.

In the present invention, the black fly is Simulium damnosum , Simulium neavei , Simulium nelivi , Simulium callidum , Simulium metallicum , Simulium metallic Ozraqueum ( Simulium ochraceum ), Simulium colombaschense , Simulium pruinosum and Simulium posticatum may be selected from the group consisting of, but is not limited thereto. It is not.

In the present invention, the sand fly, but may be characterized as being selected from the group consisting of genus Lucho MAIA (Lutzomyia) in play and a bottoming mousse (Phlebotomus), but is not limited to such.

In another aspect, the present invention relates to a composition for screening a ligand that binds to the taste receptor with a pest control agent, including the taste receptor. The configuration related thereto is as described above in detail. The composition according to the invention may further comprise reagents known in the art for use in screening.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Experimental Method

The method used in this experiment is as follows.

Stock of fruit flies

From Bloomington Stock Centor, Gr2a ¹ , Gr8a ¹ , Gr10a ¹ , Gr22e ¹ , Gr22f ¹ , Gr23a ¹ , Gr28b ^Mi , Gr33a ¹ , Gr36b ¹ , Gr36c ¹ , Gr47a ¹ , Gr58b ¹ , Gr59a ¹ , Gr66a ^ex83 , Gr77a ¹ , Gr93a ³ , Gr93d ¹ , Gr87a ¹ and Gr98b ¹ were obtained. ΔGr32a ²⁷ was obtained from H. Amerin, UAS-Gr28b ¹⁹ : Gr28b ^Mi ; UAS-Gr28b.a, Gr28b ^Mi ; UAS-Gr28b.b, Gr28b ^Mi ; UAS-Gr28b.c, Gr28b ^Mi ; UAS-Gr28b.d and Gr28b ^Mi ; UAS-Gr28b.e was obtained from K.Kang, and we used w ¹¹¹⁸ as a wild type control.

compound

Sucrose (Sucrose, cat.No .: 57-50-1), sulfohodamine B (sulforhodamine B, cat.No .: 3520-42-1), saponin, cat.no .: 8047-15- 2), caffeine (caffeine, cat.No.:58-08-2), strychnine, cat.No.:1421-86-9, quinine, cat.No.:6119-47-7 , Lobeline (cat.No.:134-63-4), papaverine (papaverine, cat.No.:61-25-6), denatonium (denatonium, cat.No.:6234-33-6 ), Umbelliferone (cat.No.:93-35-6), and coumarin (catmarin No.:91-64-5) are the Sigma-Aldrich Company (St. Louis, MO, USA)). Brilliant blue FCF (cat. No.:027-12842) and berberine (cat. No.:020-05502) were purchased from Wako Pure Chemical Industry, Ltd (Osaka, Japan).

Binary food choice assay

Dual food selection assays were performed in the same manner as known in the literature (Moon, SJ et al., Current Biology Vol. 16, pp. 1812-1817, 2006; Meunier, N. et al., J. Neurobiol. Vol. 56, pp. 139-152, 2003). More specifically, first 40-50 fruit flies aged 3-6 days were starved in 1% agarose for 18 hours, followed by 1% agarose and red die (sulfohodamine B, 0.2mg / ml). Each concentration of saponin + 5mM sucrose mixture and 1mM sucrose solution containing 1% agarose and blue die (brilliant blue FCF, 0.125mg / ml) was applied in a zigzag form. Drosophila were transferred to 72-well microtiter dishes, incubated in dark chambers, and then sacrificed at -20 ° C, and the color of the fruit fly embryos was red, blue or violet die. The preference index (P.I) was calculated using the following formula:

(N _B -N _R ) / (N _R + N _B + N _P ) or (N _R -N _B ) / (N _R + N _B + N _P ), depending on the die / food combination.

Preference index 1.0 or 1.0 means complete selectivity for either saponin plus 5 mM sucrose or 1 mM sucrose, respectively (for 1 mM sucrose, P.I is 1.0). A preference index of 0.0 means that no food is particularly preferred between the two food combinations.

Tip recording assay

Terminal analysis was performed by the method of Tanimaura. The average action potential (spike / sec) was abandoned and only spikes causing 50-550 ms were counted. Analysis was carried out using a known method using saponin at each concentration and 1 mM potassium chloride (KCl) or 30 mM tricholine citrate (TCC) (Moon, SJ et al., Current Biology Vol. 16, pp. 1812-1817, 2006). 1 mM potassium chloride (KCl) or 30 mM tricholine citrate (TCC) acts as electrolyte in terminal analysis. First, 3-7 days old fruit flies were fixed on ice, and then a reference glass electrode filled with Ringer's solution was inserted into the back of the chest and removed from the mouth. The sensory organs in the labial palp were stimulated with a recording pipette (10-20 μm in diameter) with a taste stimulant dissolved in a buffer. The recording electrode was connected to a pre-amplifier (Taste PROBE, Synthech, Germany) to amplify the signal. The recorded action potentials were obtained at a 12 kHz sampling rate and analyzed by Autospike 3.1 software (Synthech).

Ingestion assay

Without hunger, 3-6 days old Drosophila were fed with foods labeled with 1% (w / v) Brilliant Blue FCF and containing 1% saponin for a defined time. After feeding, the fruit flies were sacrificed by freezing at -20 ° C, then 3 male and 3 female fruit flies were transferred to 1.5 ml Eppendorf tubes with 200 μl PBST (1X PBS with 0.2% triton X-100), respectively. After grinding, 800 μl of PBST was mixed and centrifuged at 13,00 rpm and 4 ° C. for 5 minutes to obtain a supernatant, and then the supernatant was injected into a spectrogram cuvette.

The absorbance of the solution containing only PBS at 630 nm was measured by blank, and the digestion index (I.I) was calculated by the following equation.

II = (OD _saponin-diet -OD _control-diet ) / (OD _control-diet )

Each experiment was repeated four or more times.

Survival assay

Twenty two to four day old fruit flies (10 males, 10 females) were incubated in a cornmeal medium containing saponin at 60% humidity at 25 ° C, and then counted after 12 h. Transferred to fresh medium.

The experiment was performed four or more times each.

PER assay

Twenty three to six days old fruit flies were starved for 18 hours in 1% agarose, and then two different fixation methods were used for PER analysis. First, in order to test the taste receptor on the leg side, the fruit fly wings were fixed on the slide glass and then recovered for at least 1 hour in a humidifier. For stimulation of the labellum, each fruit fly was fixed to a yellow pipette tip, and then the tip was cut to expose the head. 2% sucrose was given as a starting material for stimulation, and fruit flies that did not respond to sucrose were excluded from the experiment. A 1.0% saponin and 2.0% sucrose mixture was used as an experimental stimulant, and was touched to the front limb or lip end of Drosophila by immersing it in a Kim-wipe paper wick. In the same way, the stimulus was exposed once more. Ten or more fruit flies were used in one experiment, and each experiment was performed four or more times.

Pupation assay

Three to six days old female fruit flies were laid for 12 hours to obtain larvae. After one day, 20 larvae were transferred to a medium containing or without saponins at a defined concentration. The pupation rate was measured every 24 hours.

Statistical analysis

All error bars represent standard error of means (SEMs). To compare different data sets, except for survival analysis, a single factor ANVOVA with Scheffe's analysis as a post hoc test was performed, with an asterisk indicating statistical significance (* P <0.05, ** P <0.01). In the survival analysis, statistics were applied after Kaplan-meier analysis, and log-rank values were calculated by comparing each experiment.

Example 1 Confirmation of Saponin Avoidance Mechanism of Drosophila

When performing a dual food selection assay based on 1 mM sucrose and 5 mM sucrose in Drosophila, all Drosophila selected 5 mM sucrose (FIG. 1B), but when 5 mM sucrose was contaminated with saponin, it was proportional to the concentration of saponin. It was confirmed that the selection ratio was reduced. In addition, the concentration of more than 5% did not induce more avoidance of fruit flies, so it was confirmed that saponin acts as a repellent such as caffeine and strickin in fruit flies.

Next, as a result of measuring the action potential occurring at the end of the dendritic end in the Drosophila S6 sensor, it was confirmed that the action potential occurs even at 0.1% concentration of saponin and even 10 spikes occur at 0.01% (FIG. 1C & D).

Most bitter substances are known to play two roles in suppressing sugar-response. In order to confirm that saponin also suppressed the sugar-response, 100mM sucrose generated ~ 103 spikes in the L4 sensory organ, and after saponin was administered by concentration, it was confirmed that the spike decreased in proportion to the concentration ( 1E & F).

Example 2. Identification of Saponin Receptors in Drosophila

As shown in FIG. 2A, as a result of performing dual food selection analysis with 5% saponin-containing food based on 21 Gr gene deletion variants, it was confirmed that the Gr28b ^Mi variant did not perform saponin avoidance (FIG. 2A). The variant was confirmed to show a normal reaction in the well known 11 bitter substances other than saponin (FIG. 2B). In addition, the variant showed a failure to avoid in 5% saponin, but showed a normal response in the saponin of 10% concentration (Fig. 2C) was found to be due to the sugar inhibition phenomenon of saponin (Fig. 1E).

Normal, saponin-containing foods were ingested in the control and Gr28b ^Mi variants for 15, 30, 60, and 240 minutes, and then digestion efficiency was measured. Normal fruit flies consumed only normal foods, whereas Gr28b ^Mi variants contained normal and saponin-containing foods. It was confirmed that the intake without distinguishing food (Fig. 2D)

Example 3 Confirmation of Saponin Receptor Location in Drosophila

In order to identify which organs in the Drosophila taste organs are important for saponin detection, a proboscis extension response (PER) assay was performed.

As a result of showing sucrose or sucrose + saponin on the leg or labella of the fruit fly, as shown in FIG. 3A, the control group and Gr28b ^Mi Drosophila responded only to sucrose, while saponin was presented to the mouth. As shown in FIG. 3B, the control group did not expand its mouth, whereas the Gr28b ^Mi Drosophila showed a response. This means that Gr28b is essential for detecting saponin in the mouth.

Saponin reactivity was tested for all sensory groups present in the mouth for a more detailed analysis, and it was confirmed that S1, S3, S5, S6, S7 and S10 sensory groups showed strong reactivity (FIG. 3C).

Example 4. Identification of Gr28b.c as a Drosophila Saponin Receptor

The Gr28b gene consists of five genes (Gr28b.a, Gr28b.b, Gr28b.c, Gr28b.d, and Gr28b.e), and five kinds of proteins are produced by selective stitching based on three common exons. (FIG. 4A). First, it was confirmed by RT-PCR that the Gr28b ^Mi variant expresses nothing of the Gr28b gene (FIG. 4B).

The UAS-Gr28b isoform was used to identify which protein in the Gr28b isoform is the saponin receptor in the Gr28b.a-GAL4 system. When the Gr28b.c protein is recovered by UAS, the avoidance mechanism by saponin is restored. Confirmation (FIG. 4C-E).

Example 5 Effect of Saponin as an Insecticide

As disclosed in Example 1, it was confirmed that saponin can be used as a repellent. It is generally known that repellents can also be used as insecticides. In order to confirm the effect of saponin as a killing agent, the survival rate of normal and Gr28b ^Mi variant larvae in a medium containing 0, 0.5, 2.5, 5.0 and 7.5% saponin was analyzed. As shown in FIGS. 5A and B, 2.5 and It was confirmed that the survival rate of the mutant larvae at 5% saponin concentration was reduced.

In addition, 1 week-old larvae of normal and Gr28b ^Mi variants were cultured in saponin medium at different concentrations. As a result, 93.8% of normal larvae succeeded in solubilization, whereas less than 50% Gr28b ^Mi variant larvae were soluble in 5.0% and 7.5% saponin medium. It was confirmed that the success (Fig. 5C & D).

Log-rank and median life span (LT50) values of saponin calculated from Kaplan-Meier survival rate table Line control-diet 0.5% saponin 2.5% saponin 5.0% saponin 7.5% saponin LT ₅₀ (hrs) control Gr28 b ^Mi control Gr28b ^Mi control Gr28b ^Mi control Gr28b ^Mi control Gr28b ^Mi NA NA NA NA 96.0 ± 5.77 72.0 ± 4.85 60.0 ± 1.33 36.0 ± 2.63 48.0 ± 2.74 36.0 ± 2.39 Log-rank control Gr28b ^Mi control Gr28b ^Mi control Gr28b ^Mi control Gr28b ^Mi control Gr28b ^Mi a 0.307 0.056 0.092 <10 ^-3 <10 ^-3 <10 ^-3 <10 ^-3 <10 ^-3 <10 ^-3 b 0.307 0.092 0.145 <10 ^-3 <10 ^-3 <10 ^-3 <10 ^-3 <10 ^{-3 <10-3}

a and b mean control flies and Gr28b ^Mi variant flies that received control food

Example 6 Effect of Gr28b.c Inhibitors and Saponin-Containing Agents as Pesticides

RNAi, used in this experiment, causes small RNA molecules to turn inactive cells into inactive cells in the cell, preventing the mRNA from expressing proteins. It is initiated by the endogenous hydrolase in Dicer enzyme that cleaves double stranded RNA into oligonucleotides. In order to inhibit the protein expression as described above using the Gr28b RNAi line using the experimental group to reduce the mRNA level before the protein synthesis, and the control effect on the saponin of the control group was tested by the method of Example 4.

As a result, in the experimental group in which the expression level of the Drosophila receptor protein was reduced as shown in FIG. 6, it was confirmed that the repelling effect on saponin was significantly reduced (n = 4, * P = 0.0411, FIG. 6).

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

                         SEQUENCE LISTING <110> Kookmin University Industry Academy Cooperation Foundation   <120> Insecticide Composition comprising inhibitors of Drosophila        saponin receptor and saponin and Screening Method for inhibitors        of Drosophila saponin receptor <130> P17-B289 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 470 <212> PRT <213> Drosophilla Gr28b.c <400> 1 Met Asp Ile Glu Met Ala Lys Glu Pro Val Asn Pro Thr Asp Thr Pro 1 5 10 15 Asp Ile Glu Val Thr Pro Gly Leu Cys Gln Pro Leu Arg Arg Arg Phe             20 25 30 Arg Arg Phe Val Thr Ala Lys Gln Leu Tyr Glu Cys Leu Arg Pro Val         35 40 45 Phe His Val Thr Tyr Ile His Gly Leu Thr Ser Phe Tyr Ile Ser Cys     50 55 60 Asp Thr Lys Thr Gly Lys Lys Ala Ile Lys Lys Thr Ile Phe Gly Tyr 65 70 75 80 Ile Asn Gly Ile Met His Ile Ala Met Phe Val Phe Ala Tyr Ser Leu                 85 90 95 Thr Ile Tyr Asn Asn Cys Glu Ser Val Ala Ser Tyr Phe Phe Arg Ser             100 105 110 Arg Ile Thr Tyr Phe Gly Asp Leu Met Gln Ile Val Ser Gly Phe Ile         115 120 125 Gly Val Thr Val Ile Tyr Leu Thr Ala Phe Val Pro Asn His Arg Leu     130 135 140 Glu Arg Cys Leu Gln Lys Phe His Thr Met Asp Val Gln Leu Gln Thr 145 150 155 160 Val Gly Val Lys Ile Met Tyr Ser Lys Val Leu Arg Phe Ser Tyr Met                 165 170 175 Val Leu Ile Ser Met Phe Leu Val Asn Val Leu Phe Thr Gly Gly Thr             180 185 190 Phe Ser Val Leu Tyr Ser Ser Glu Val Ala Pro Thr Met Ala Leu His         195 200 205 Phe Thr Phe Leu Ile Gln His Thr Val Ile Ala Ile Ala Ile Ala Leu     210 215 220 Phe Ser Cys Phe Thr Tyr Leu Val Glu Met Arg Leu Val Met Val Asn 225 230 235 240 Lys Val Leu Lys Asn Leu Ala His Gln Trp Asp Thr Arg Ser Leu Lys                 245 250 255 Ala Val Asn Gln Lys Gln Arg Ser Leu Gln Cys Leu Asp Ser Phe Ser             260 265 270 Met Tyr Thr Ile Val Thr Lys Asp Pro Ala Glu Ile Ile Gln Glu Ser         275 280 285 Met Glu Ile His His Leu Ile Cys Glu Ala Ala Ala Thr Ala Asn Lys     290 295 300 Tyr Phe Thr Tyr Gln Leu Leu Thr Ile Ile Ser Ile Ala Phe Leu Ile 305 310 315 320 Ile Val Phe Asp Ala Tyr Tyr Val Leu Glu Thr Leu Leu Gly Lys Ser                 325 330 335 Lys Arg Glu Ser Lys Phe Lys Thr Val Glu Phe Val Thr Phe Phe Ser             340 345 350 Cys Gln Met Ile Leu Tyr Leu Ile Ala Ile Ile Ser Ile Val Glu Gly         355 360 365 Ser Asn Arg Ala Ile Lys Lys Ser Glu Lys Thr Gly Gly Ile Val His     370 375 380 Ser Leu Leu Asn Lys Thr Lys Ser Ala Glu Val Lys Glu Lys Leu Gln 385 390 395 400 Gln Phe Ser Met Gln Leu Met His Leu Lys Ile Asn Phe Thr Ala Ala                 405 410 415 Gly Leu Phe Asn Ile Asp Arg Thr Leu Tyr Phe Thr Ile Ser Gly Ala             420 425 430 Leu Thr Thr Tyr Leu Ile Ile Leu Leu Gln Phe Thr Ser Asn Ser Pro         435 440 445 Asn Asn Gly Tyr Gly Asn Gly Ser Ser Cys Cys Glu Thr Phe Asn Asn     450 455 460 Met Thr Asn His Thr Leu 465 470 <210> 2 <211> 1595 <212> DNA <213> Drosophilla Gr28b.c DNA <400> 2 atggacattg aaatggccaa ggagccggtg aatccaacgg atactccgga tatagaagtg 60 actcccggtc tatgccagcc cttgcgtcgt agatttcggc gatttgttac cgccaaacag 120 ctatacgagt gcctgcgccc ggtgttccat gtgacctaca tccacggact cacctccttc 180 tacattagtt gcgatactaa aaccggaaag aaagccatca agaaaaccat tttcggctac 240 atcaatggaa tcatgcacat cgccatgttt gtctttgcct atagccttac gatttataac 300 aattgcgagt cggtggctag ttacttcttt cgatctcgca ttacctattt cggggatttg 360 atgcagatag tgagtggatt cattggagtt actgtcatct acctgactgc ctttgtccca 420 aaccatcgat tggagcgttg ccttcagaag tttcacacca tggacgtgca actccagacg 480 gtgggagtga agatcatgta cagcaaggtg ctgcgattta gctacatggt cctgatctcc 540 atgttccttg taaacgtact ctttaccggc ggcacctttt cggttctcta ttcctcggaa 600 gtggcgccca ccatggccct gcacttcacc ttcctcatcc agcacacggt catcgccatt 660 gccattgcgc tcttcagttg cttcacatat ctggtggaga tgcgactggt gatggtcaat 720 aaggtgttga agaacctagc ccatcaatgg gacacccgaa gcctcaaggc agtgaatcaa 780 aaacagcgct ctctacaatg tctcgattca ttttccatgt acaccattgt aaccaaggat 840 cctgcggaga ttatacagga gtccatggag atacatcatc tcatttgcga ggcagctgcc 900 acggctaaca aatattttac ctaccaattg ctgaccatta tatccatagc atttctgatc 960 atcgttttcg atgcatacta tgttctggag acgctactgg gaaaatcgaa gcgcgaaagc 1020 aaattcaaaa ctgtggaatt tgtgacattt ttctcgtgtc aaatgatttt gtatctgatc 1080 gccataattt ccattgtcga gggaagtaat cgagccatca aaaagagcga gaaaactgga 1140 ggcatagtgc actccctact caataaaacc aaaagtgctg aggtcaagga gaaactgcag 1200 caattctcca tgcagttgat gcatctgaaa attaatttta ctgcagctgg tctgttcaac 1260 atcgaccgca cattgtattt cacgatcagc ggggccttga ccacttatct catcatcttg 1320 ctgcagttca catccaattc cccgaacaat ggttatggga atggcagctc ttgctgtgag 1380 accttcaata atatgacgaa tcatacgctt tagatgtgct atcgatgtca ttacaatatg 1440 ggggatggaa atcgaaggaa gtcgatggag tcggtgaaat ggtattgaat acgtccagca 1500 gaatgtgtat tttttgtaaa tgttattgtt gttgctaaaa aaaagtgttt tctgtcaata 1560 taaaccaacg attggaaata aatttttaat cgttt 1595 <210> 3 <211> 368 <212> DNA <213> RNAi <400> 3 gctgcagttt ctccttgacc tcagcacttt tggttttatt gagtagggag tgcactatgc 60 ctccagtttt ctcgctcttt ttgatggctc gattacttcc ctcgacaatg gaaattatgg 120 cgatcagata caaaatcatt tgacacgaga aaaatgtcac aaattccaca gttttgaatt 180 tgctttcgcg cttcgatttt cccagtagcg tctccagaac atagtatgca tcgaaaacga 240 tgatcagaaa tgctatggat ataatggtca gcaattggta ggtaaaatat ttgttagccg 300 tggcagctgc ctcgcaaatg agatgatgta tctccatgga ctcctgtata atctccgcag 360 gatccttg 368

Claims (14)

An inhibitor of Gr28b.c represented by the amino acid sequence of SEQ ID NO: 1, which is a saponin receptor, a pesticide composition comprising RNAi of SEQ ID NO.
delete delete delete delete The insecticide composition according to claim 1, wherein the insecticide composition exhibits a pesticidal effect by inhibiting the taste-accepting capacity of saponins of the pest.
delete delete 7. The method of claim 6 wherein the pests are mosquitoes (Culcidae), such as mosquitoes (Ceratopogonidae), tsetse flies (tsetse fly), black flies (Simuliidae), sand flies (sand fly), bed bugs (bed bug), needle norinjae (assassin insecticide composition, characterized in that it is selected from the group consisting of bugs, fleas, louses, mites and ticks.
The genus of claim 9, wherein the mosquitoes are of the genus Aedeomyia , genus Aedes (including Aedes aegypti ), genus Anopheles ( Anopheles gambia). ( Including Anopheles gambiae ) and Anopheles annulipes , Armigeres genus, Ayurakitia genus, Bironella genus, Borichinda genus, Chagacia (Chagasia), A kokwil retina Dia (Coquillettidia) genus Culex (Culex), A Cooley theta (Culiseta), A Day No-Kerry Tess (Deinocerites) genus, Yerevan bit Mapo de Tess (Eretmapodites), A Pical via (Ficalbia ), Galindomyia genus, Haemagogus genus, Heizmannia genus, Hodgesia genus, Isostomyia genus, Johnbelkinia ) Genus, Kimia genus, Limatus genus, Lutzia genus , Malaya (Malaya) in only Sonia (Mansonia) in Maori go'el Dia (Maorigoeldia), A beauty Maia (Mimomyia) in sludge Leone (Onirion) in, Opie Pécs (Opifex) in ortho grape Maia (Orthopodomyia ), A program Thoreau Fora (Psorophora), A runchyo Maia (Runchomyia) in, Sarbanes Tess (Sabethes), A Shan say Ana (Shannoniana), A topographic Maia (Topomyia) genus Toxoplasma Lynch Tess (Toxorhynchites), A Trichoprosopon genus, Tripteroides genus, Udaya genus, Uranotaenia genus, Verrallina genus, Wyeomyia genus and zeus Pesticide composition, characterized in that selected from the group consisting of Zeugnomyia
The insecticide composition according to claim 9, wherein the mosquito is selected from the group consisting of Culicoides genus, Leptoconops and Forcipomyia genus.
The method of claim 9, wherein the body fly is Glossina austeni , Glossina morsitans , Glossina pallidipes , Glossina swynnertoni ), Glossina fusca fusca , Glossina fuscipleuris , Glossina frezili , Glossina haningtoni , Glossina fusca Glossina longipennis , Glossina medicorum , Glossina nashi , Glossina niGrofusca niGrofusca , Glossina severini Glossina severini , Glossina schwetzi , Glossina tabaniformis , Glossina vanhoofi , Glossina caliginea , Glossina Foo
Ski festival Fu ski festival (Glossina fuscipes fuscipes), global seed and Fu ski festival mareutiniyi (Glossina fuscipes martinii), global seed or sold Keraton sold Mosquera (Glossina pallicera pallicera), global seed or sold Mosquera news Ponte Adige (Glossina pallicera newsteadi) , global seed and VITAL less VITAL less (Glossina palpalis palpalis), global seed and VITAL less Diet N-Sys (Glossina palpalis gambiensis) and global seed or waterfall noise des pesticide composition being selected from the group consisting of (Glossina tachinoides).
The method of claim 9, wherein the black fly is Simulium damnosum , Simulium neavei , Simulium callidum , Simulium metalum , Simulium metallicum Insecticide composition, characterized in that it is selected from the group consisting of (Ceulum Simulium ochraceum ), Simulium colombaschense , Simulium pruinosum and Simulium posticatum .
10. The method of claim 9, wherein the sand fly Lucho MAIA (Lutzomyia) in play and a bottoming mousse pesticide composition being selected from the group consisting in (Phlebotomus).

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