KR101816102B1 - Method of Screening Insecticides or Repellents by using a Gustatory Receptor - Google Patents

Abstract

The present invention relates to a method for screening a pest control agent using, for example, an insecticide or a repellent agent using a fruit receptors derived from a fruit fly, and more particularly to a method for screening a pest control agent comprising a step of selecting a substance that changes the activity of a taste receptor derived from a fruit fly Screening method.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for screening insecticides or repellents using taste receptors,

The present invention relates to a method for screening a pest control agent using, for example, an insecticide or a repellent agent using a fruit receptors derived from a fruit fly, and more particularly to a method for screening a pest control agent comprising a step of selecting a substance that changes the activity of a taste receptor derived from a fruit fly Screening method.

Taste is important to evaluate food quality. Dedicated sensory cells are dedicated to each taste pattern, and transmit sensory information directly to the high brain center to attract or avoid. Rapid and accurate detection of these compounds is an important defense in many herbivores because many bitter and other repellents are toxic.

In Drosophila, several types of membrane proteins participate in the awareness of unwanted chemicals. At least two transient receptor potential (TRP) channels, TRPA1 and TRPL, have the ability to sense unwanted chemicals. However, the detection of unwanted taste-stimulating chemicals is believed to be performed by members of the gustatory receptor (GR) family, which encodes 68 proteins. Insect taste receptors are not related to mammalian taste receptors, but G-protein coupled receptors have far-reaching implications for olfactory receptors (ORs) in fruit flies. It has been reported that the olfactory receptor complex of the heterodimer contains olfactory circle-opening-cation channels and that the insect fugitive receptor consists of a single GR subunit forming 18 non-selective cation channels.

Drosophila taste receptors are composed of several subunits, which respond to repellent chemicals. Based on the biodefense studies, the taste receptors of the three GR32a, GR33a and GR66a taste receptors have been extensively regulated and have a broad detection capability of the avoidant compounds. In addition, taste receptors such as GR8a, GR47a and GR93a are narrowed in scope and are required to sense L-carabanine, strychnine, and caffeine, respectively.

L-carabanine is a plant-derived analog of the amino acid L-arginine, which, when ingested, is introduced during protein synthesis instead of L-arginine and is therefore fatal to Drosophila and many other insects. Nevertheless, the minimal subunit composition of the L-carabanin receptor in response to the repellent compound or any other taste receptor complex is not known. These heteromultimeric taste receptors are also unclear in the presence of cation channels.

Under these technical backgrounds, the inventors of the present application have identified a full set of taste receptors involved in L-carabanin sensing and have used it to confirm that pest control agents, such as pesticides or repellants, can be screened, Thereby completing the invention.

The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .

It is an object of the present invention to provide a method for screening a ligand binding thereto via a complete set of taste receptors involved in L-carabanin detection, as disclosed by the inventors of the present application, as an insect control agent.

It is also an object of the present invention to provide a composition for screening a ligand comprising a complete set of taste receptors involved in L-carabanin sensing.

In order to achieve the above object, the present invention provides a method of screening an insecticide or a repellent agent comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor derived from a fruit fly; And (b) selecting a substance that increases or inhibits the activity of the taste receptor from the fruit fly as compared to the untreated control.

The present invention also provides a method of screening an insecticide comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor from a fruit fly; And

(b) selecting an insecticide to inhibit the activity of the taste receptor from the fruit fly compared to the untreated control.

The present invention also provides a method of screening for a repellent comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor from a fruit fly; And

(b) selecting, as a repellent agent, a substance that increases the activity of a taste receptor derived from a fruit fly compared to an untreated control group.

The present invention further provides a composition for screening a ligand binding to said taste receptor, comprising a taste receptor derived from a fruit fly, with an insect control agent.

The present invention can provide a significant target for screening for ligands binding thereto through a full set of novel taste receptors involved in L-carabanin sensing, insect pesticides or insect repellents using them, The screening method and the composition for screening according to the present invention can be usefully used for the discovery and development of pest control agents.

Figure 1 relates to the identification of taste receptors required for L-canavanine avoidance. FIG. 1A shows the 58 UAS-Gr RNAi baseline and 13 UAS-Ir RNAi gene screening for defects in L-cannabinin resistance. And UASDcr2 ( Dicer2 ) to enhance the effect of RNAi on Gr33a - GAL4 . The dotted line is a no preference.
Figure 1B relates to bi-directional selection analysis using two different RNAi lines. The control was UASDcr2 without RNAi transformation; It consists of Gr33a - GAL4 fruit flies. The RNAi stock number (VDRC) is listed in the bar. And five times for each genotype. Data mean ± SEM. ** p < 0.01 (ANOVA with post-hoc Tukey test).
Figure 1c depicts the expression of Gr98a1 & ^lt; RTI ID = 0.0 & ^gt; Lt; RTI ID = 0.0 &gt; alleles. &Lt; / RTI &gt; Arrowheads represent genomic PCR primers used to confirm deletion of Gr98b . 543 bp band appears in the control flies (flies control) without Gr98b ^1.
Figure 1d is a Gr98b ¹ relates to a two-way selection assays to test appears to be defective in L- Kana banin avoided. Gr98b as tested with the structure (rescue) of the ^first type traits (phenotype), Gr66a - GAL4, Gr8a - GAL4 and Gr98b - using the GAL4 Gr98b ¹ 0.0 &gt; Gr98b < / RTI &gt; And five times for each genotype. * p < 0.05 (ANOVA with post-hoc Tukey test).
Figure 1e shows the effect of Gr98b ¹ on bitter taste chemicals bidirectional selection analysis for testing the avoidance of flies. Drosophila flies were selected between 1 mM sucrose and 5 mM sucrose plus the following repellents: 0.5 mM Papavulin (PAP), 0.5 mM Strychnine (STR), 0.1 mM Denatron (DEN), 0.05 mM Berberine , 0.1 mM Lobel (LOB), 5 mM caffeine (CAF), 0.2% DEET and 0.5 mM quinine (QUIN). Each genotype was performed 4-7 times. All data means ± SEM.
Figure 2 relates to the dependence of L- carabanin on the action potential of Gr98a . Figure 2 is a control group (w ^1118), Gr98b ¹ and rescue flies to respond to 30 mM L- Kana banin; the action potential in the organoleptic S6 (Gr66a - - GAL4 / UAS Gr98b Gr98b 1) was induced. Figure 2b shows the average frequency of action potentials upon exposure to 30 mM L-cannabinin. The genotypes and senses (S3, S5 and S10) were tested and were performed 10-12 times for each genotype. Figure 2 c is a chemical indicating the bitter taste (1 mM papaverine (papaverine), 1 mM strychnine (strychnine), 1 mM Gardena benzethonium (denatonium), 0.1 mM berberine (Berberine), 1 mM Lovell Lin (lobeline), 10 mM The average frequency of action potentials induced by the S6 sensory response in response to caffeine, 0.2% diethyltoluamide (DEET), and 1 mM quinine was performed 10-21 times for each genotype. All data are mean ± SEM ** ** p <0.01 (ANOVA with post-hoc Tukey test).
Figure 3 relates to the ectopic expression of Gr8a and Gr98b in GRNs that sense bitter taste conferring L-carabanin sensitivity. Figure 3a Gr8a, Gr66a and Gr98b in Gr8a, Gr66a and Gr98b receptor expression patterns and respective label room (in the end of Paris) shows the location of expression of receptors. The reference is 50 μm. Figure 3b shows that the ectopic expression of Gr8a and Gr98b in GRNs (biter taste sensory), which senses bitter taste, imparts L- carabanin sensitivity to sensory receptors that do not normally respond to L- carabanine . The upper part of the figure shows the sensory depicting the ectopic expression experiment and the lower part shows the representative trace of the control group and UAS-Gr8a , UAS-Gr98b ; It was induced by 30 mM L- carabanine from Gr4a - GAL4 Drosophila I4 sensory. Figure 3c shows that the response frequency was induced by 30 mM L- cannabinin after ectopic expression of Gr8a and Gr98b in each sensory organ . UASGr8a , UAS - Gr98b Drosophila are negative controls. 4-14 times. ** p < 0.01 (Mann-Whitney U test). Medians and quartiles were indicated.
Figure 4 relates to the effect of ectopic expression of Gr8a, Gr66a and Gr98b in GRNs which react with sugars to low salt. Figure 4a is the top of depicts ectopic expression experiments of the Gr8a, Gr66a and Gr98b in GRNs to detect the sweet taste, and the lower 30 mM L- L3 from the organoleptic expressing Gr8a, Gr66a and Gr98b GRNs in which the representative trace detect sweetness It was induced by canavanine. Figure 4b shows the dose-dependent response of L-carabanine in GRNs sensing each sweetness. Without a GAL4 control Drosophila; shows a GAL4 (solid line) responses of the indicated receptors from Drosophila under the expression control of 3 Grs - (UAS - Gr8a, UAS - Gr66a, UAS - Gr98b dotted line) and Gr64f. 6-21 times. Figure 4c response frequency was induced by 30 mM L- cannabinin after ectopic expression of Gr8a , Gr66a and Gr98b in GRNs that detected each sweetness using Gr64f - GAL4 . Genotype: 1) green circles: 2X ( UAS - Gr66a , UAS - Gr98b ); Gr64f - GAL4 / +, 2) blue circles: 2X ( UAS - Gr8a , UAS - Gr98b ); Gr64f - GAL4 / +, 3) ocher circles: 2X ( UAS - Gr8a , UAS - Gr66a ); Gr64f - GAL4 / +, and 4) purple circles: 2X ( UAS - Gr8a , UAS - Gr66a , UAS - Gr98b ); Gr64f - GAL4 / +. Each genotype was performed 4-21 times. Medians and quartiles were indicated. * p <0.05, ** p <0.01 (Kruskal-Wallis test with Mann-Whitney U post hoc test). Figure 4d response frequency was induced by 30 mM L- cannabinin after ectopic expression of Gr8a , Gr66a, and Gr98b in GRNs that detected low salinity using Ir76b - GAL4 . Genotype: 1) blue circles: Ir76b - GAL4 / +, and 2) purple circles: UAS - Gr8a , UAS - Gr66a , UASGr98b / +; Ir76b - GAL4 / +. 8-14 times. Medians and quartiles were indicated. * p &lt; 0.05, ** p &lt; 0.01 (Mann-Whitney U test). In Figure 4e GRNs responsive to a sugar is a frequency response resulting from Gr8a, Gr66a and ectopic expression of receptors of Drosophila Gr98b. Each chemical was tested in 1 mM (papaverine: PAP, strychnine: STR, denatomium: DEN, berberine: BER, Lobelin: LOB) except caffeine (CAF; 6 to 9 times. Medians and quartiles were indicated.
Figure 5 relates to incentives for L-cannabinin induced by ectopic expression of sweetness-sensing GRNs. Gr8a , Gr66a and Gr98b in sweet- tinged GRNs are bi-directionally selective assay tests for attracting or avoiding L- carabanine in mis-expressing Drosophila. Gr is the s Gr66a ^ex83 heterologous in the mutant background. And five times for each genotype. Data mean ± SEM. * p &lt; 0.05 (ANOVA with post-hoc Tukey test).
Figure 6 relates to whole-cell voltage-fixed recording of S2 cell-expressing taste receptors. Cells were stimulated with 30 mM L-cannabinin. In Figures 6a and 6b , cells were transfected with pActin5c- GAL4 , pUAST- EGFP only (mock) or pActin5c- GAL4 , pUAST- EGFP plus pUAST- Gr8a , pUAST- Gr66a and pUAST- Gr98b (3 GRs). Figure 6 a is the current produced in response to a voltage step (-80 mV to +80 mV in 20 mV increments) of 500 ms duration obtained in the presence of L-cannabinin. Figure 6 b is an IV relationship diagram stimulated with L- carabanin using GR8a, GR66a and GR98b expressing cells. Fig 6 c The current density at +80 mV. Cells expressed Grs at the bottom of the graph and were recorded with or without L- carabanin stimulation. The recording numbers are shown in the figure. * p &lt; 0.05 (paired Student's t- test). The current-voltage traces in Figures 6d-f show that the two Grs expressions in S2 cells are not associated with L-canavanin-induced increases in current density. Fig. 6 d S2 cells expressing Gr8a and Gr66a (n = 10). Figure 6 e Gr8a and Gr98b expressing S2 cells (n = 11). Figure 6 f Gr66a and Gr98b expressing S2 cells (n = 6). Figure 6 g Effect of La ^{3+ in} the IV relationship. Cells were expressed GR8a, GR66a and GR98b and were recorded in the presence of L- carabanin . Figure 6 h Effect of La ³⁺ on the current density obtained at +80 mV. Cells were expressed in three GRs and stimulated with L- carabanin . ** p &lt; 0.05 (ANOVA with posthoc Tukey test). Figure 6 i Current densities stimulated with GR8a, GR66a and GR98b expressing cells and the following bitter taste chemicals: 30 mM L-cannabinin, 1 mM Papavellin (PAP), 1 mM streinin (STR), 1 mM denatron ), 100 [mu] M berberine (BER), 1 mM Lobelin (LOB), 5 mM caffeine (CAF) and 1 mM quinine (QUIN). The recording numbers are shown in the figure. * p <0.01 (ANOVA with post-hoc Tukey test). All error bars mean ± SEM.

Unless otherwise defined, 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.

While studying the complete set of taste receptors involved in bitter taste detection of insecticides such as L-carabanine, the inventors of the present application found that knockdown of Gr8a and Gr66a impairs L- cannabinis avoidance behavior and inhibits L- ( Gr98b ) that interferes with the expression of the gene ( Gr98b ). We removed Gr98b to eliminate repulsion and action potentials in response to L- cannabinin , and introduced Gr8a , Gr66a and Gr98b into sensory neurons that sensed sweetness or low salty taste, And the ability to respond to cannabinin. In addition, the ectopic expression of these taste receptors in taste-sensing taste neurons has been converted from avoiding the inherent L-carabanine of Drosophila to being more attractive. S2 tissue cultured cells showed that the ectopic expression of Gr8a , Gr66a and Gr98b gave L- carabanin- dependent currents.

Based on this, in one aspect, the present invention relates to a method for screening an insect pest controlling agent comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor from a fruit fly; And (b) selecting a substance that increases or inhibits the activity of the taste receptor from the fruit fly as compared to the untreated control.

To determine the receptor required for the L- Kana banin detected, Gr2a (v102185), Gr5a ( v13730), Gr8a (v31104), Gr9a (v15446), Gr10a (v39237), Gr10b (v31151), Gr21a (v104122), Gr22a ( v106736), Gr22b (v107792), Gr22c (v7249), Gr22e (v9389), Gr22f (v102860), Gr23a (v40852), Gr28a (v100938), Gr28b (v101727), Gr32a (v47956), Gr33a (v42802), Gr36a ( v48018), Gr36b (v8062), Gr36c (v3872), Gr39a (v8685), Gr39b (v33215), Gr43a (v39518), Gr47b (v4594), Gr57a (v45879), Gr58a (v1703), Gr58b (v9565), Gr58c ( v29137), Gr59a (v31107), Gr59b (v101219), Gr59c (v3530), Gr59d (v2766), Gr59e (v31110), Gr59f (v18989), Gr61a (v106007), Gr63a (v108203), Gr64a (v103342), Gr64b ( v42517), Gr64c (BL36734), Gr64d (v29422), Gr64e (v109176), Gr64f (v105084), Gr66a (v14820), Gr68a (v13380), Gr77a (BL38236), Gr85a (v47992), Gr89a (v8253), Gr92a ( v44408), Gr93a (v13569), Gr93b (v12160), Gr93c (v109794), Gr93d (v6813), Gr94a (v9537), Gr97a (v4395), Gr98a (v1300), Gr98b (v1302 and v101040), Gr98c (BL36735), Gr98d (v43 98), IR7a (v108171), IR47a (v11812), IR56a (v5010), IR56b (v4704), IR56d (v6112), IR94e (v33066), IR20a (v8658), IR94a (v7566), IR94c (v6817), IR94h ( As a result of screening for taste receptors and ion receptors such as IR60b (v1563), IR60b (v12089), IR67c (v37261) and IR94f (v109702), the taste receptors derived from Drosophila have two or more selected from the group consisting of Gr8a, Gr66a and Gr98b It was confirmed that the heterogeneous polymorphism including the subunit consisting of Gr8a, Gr66a and Gr98b was responsive to L-carabanine.

The Gr8a subunit is represented by the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 10, the Gr66a subunit is represented by the amino acid sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 11, The amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 12.

In one embodiment, a taste receptor comprising a subunit consisting of Gr8a, Gr66a and Gr98b may be introduced into a taste-gating neuron responsive to a candidate substance and then expressed to be used for insecticidal or repellent screening. For example, the nucleotide coding for the taste receptor can be cloned into a known expression vector and transduced into a taste neuron for expression.

The vector means an expression vector capable of expressing a desired protein in a suitable host cell, and comprising a necessary regulatory element operably linked to the expression of the gene insert. In addition to expression regulatory elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal, enhancer, and the like, a signal sequence or leader sequence for membrane targeting or secretion may be prepared variously according to the purpose. The promoter of the vector may also be constitutive or inducible. In addition, the vector includes a selectable marker for selecting a transformant containing the vector, and includes a replication origin when the vector is a replicable expression vector.

This introduction refers to the introduction of foreign DNA into the cells by transfection or transduction. Transfection and transduction can be carried out by a variety of methods known in the art.

A gene encoding a taste receptor comprising a subunit consisting of Gr8a, Gr66a and Gr98b used in the present invention can be cloned into a suitable host cell by cloning and various host microorganisms such as Escherichia coli, insect cells or animal cells For example, neurons, in particular S2 cells, may be used.

The candidate substance may be, for example, a compound that changes the activity of a taste receptor comprising a subunit consisting of Gr8a, Gr66a and Gr98b, for example an organic or inorganic low molecular weight, a biological molecule such as a peptide, But are not limited to, extracts prepared from their analogs, bacteria, plants, fungi, and the like.

Based on the taste receptor containing the subunit consisting of Gr8a, Gr66a and Gr98b, a substance whose activity is increased or suppressed in the taste receptor derived from a fruit fly treated with a candidate substance as compared to the untreated control is referred to as an example of an insect control agent For example, they can be screened for pesticides or repellents.

Based on this, the present invention relates to a method of screening an insecticide comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor from a fruit fly; And (b) selecting an insecticide for inhibiting the activity of the taste receptor from the fruit fly compared to the untreated control.

The present invention also relates to a screening method of a repellent agent comprising the steps of: (a) treating a candidate substance to a Gustatory Receptor from a fruit fly; And (b) selecting, as a repellent agent, a substance that increases the activity of a taste receptor derived from a fruit fly compared to an untreated control.

In the present invention, a pest means an object that directly or indirectly damages a human, livestock, or cultivated plant, or causes economic loss. In terms of control, pesticides are drugs that actively kill insect pests, while repellents are drugs that are used with the aim of chasing insects.

For example, if an increase in activity is observed in a taste receptor derived from a fruit fly derived from a candidate substance as compared with an untreated control, it means that the candidate substance is felt to have a strong bitter taste and insects or insects may avoid the candidate substance Therefore, it can be selected as a repellent to prevent access to insects or pests. In addition, if the inhibitory effect of the candidate substance on the taste receptors derived from the fruit fly is compared with the untreated control group, it is possible that the candidate substance can be ingested into the body without recognizing the candidate substance. It can be selected as an insecticide to be exterminated. In the examples of the present invention, it was confirmed whether or not the Drosophila-derived taste receptor according to the present invention can be used as an insect repellent screening system through L-carabanin.

If the activity of the taste receptor is changed by the combination of the taste receptor containing the subunit composed of Gr8a, Gr66a and Gr98b of the insect with the candidate substance, the candidate substance is selected as an insecticide or a ligand usable as a repellent, . In this case, the activity of the taste receptor may be determined by measuring the mRNA expression level, or the amount of the protein may be confirmed by an immunoassay using an antibody that specifically binds to the taste receptor. In the examples of the present invention, The change in current due to channel formation in the taste receptor recognized as a ligand was measured and confirmed.

In another aspect, the present invention relates to a composition for screening a ligand binding to the taste receptor, comprising a taste receptor derived from a fruit fly, with an insect control agent. The configuration related to this is as described above in detail. The composition according to the present invention may further comprise reagents known in the art used for screening.

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

[Example]

Example 1 Screening for Receptors for L-carnabinine Detection

All Drosophila stocks were maintained in conventional corn agar molasses at 12 h light / 12 h dark cycles at 25 ° C and 60% humidity. 70FLP , 70I-SceI / CyO , 2) UAS - Dcr 2, and 3) UAS - mCD8 :: GFP from Bloomington Stock Center. UAS - Gr8a , UAS - Gr66a , Gr33a - GAL4 and Gr8a - GAL4 fruit flies were described above. Gr66a - GAL4 Drosophila was presented by H. Amrein as a gift. Gr98b - GAL4 and Gr64f - GAL4 were provided to J. Carlson. Drosophila stocks were obtained from the Vienna Drosophila RNAi Center and Bloomington Stock Center for RNAi gene screening. Stock numbers are as follows: Gr2a (v102185), Gr5a ( v13730), Gr8a (v31104), Gr9a (v15446), Gr10a (v39237), Gr10b (v31151), Gr21a (v104122), Gr22a (v106736), Gr22b (v107792 ), Gr22c (v7249), Gr22e (v9389), Gr22f (v102860), Gr23a (v40852), Gr28a (v100938), Gr28b (v101727), Gr32a (v47956), Gr33a (v42802), Gr36a (v48018), Gr36b (v8062 ), Gr36c (v3872), Gr39a (v8685), Gr39b (v33215), Gr43a (v39518), Gr47b (v4594), Gr57a (v45879), Gr58a (v1703), Gr58b (v9565), Gr58c (v29137), Gr59a (v31107 ), Gr59b (v101219), Gr59c (v3530), Gr59d (v2766), Gr59e (v31110), Gr59f (v18989), Gr61a (v106007), Gr63a (v108203), Gr64a (v103342), Gr64b (v42517), Gr64c (BL36734 ), Gr64d (v29422), Gr64e (v109176), Gr64f (v105084), Gr66a (v14820), Gr68a (v13380), Gr77a (BL38236), Gr85a (v47992), Gr89a (v8253), Gr92a (v44408), Gr93a (v13569 ), Gr93b (v12160), Gr93c (v109794), Gr93d (v6813), Gr94a (v9537), Gr97a (v4395), Gr98a (v1300), Gr98b (v1302 and v101040), Gr98c (BL36735), Gr98d (v4398), IR7a (v108171), IR47a (v11812 ), IR56a (v5010), IR56b (v4704), IR56d (v6112), IR94e (v33066), IR20a (v8658), IR94a (v7566), IR94c (v6817), IR94h (v1563), IR60b (v12089), IR67c (v37261 ), IR94f (v109702). The line numbers starting with V represent the lines purchased from the Vienna Drosophila RNAi Center (VDRC), and their sequence can be found at http://stockcenter.vdrc.at/control/vtlibrary/.

RNAi gene screening was conducted to address the potential needs of different taste receptors to obtain full set of information for the 58 available UAS-Gr RNAi lines. In addition, thirteen genes encoding Ionotropic Receptors (IRs) expressed in GRNs were knocked down. Dicer ( UAS-Dcr2) and Gr33a-GAL4 driver expressing these RNAi lines were crossed to Drosophila and expressed in gustatory sensory neurons (GRNs) responsive to repellents. All progeny appeared viable and healthy.

To assess L-carabine avoidance, two-way selective behavioral analysis was performed. Given the choice between 5 mM sucrose mixed with 1 mM sucrose and 30 mM L-cannabinin, wild-type flies strongly avoided high sugar with L-cannabinin. As expected, the knockdown of Gr8a or Gr66a markedly reduced the avoidance of L-cannabinin (Fig. 1a). In addition, RNAi-mediated suppression of one other receptor ( Gr98b ) also found a significant reduction in L- canavanine evasion (Fig. 1a; line v101040). The introduction of other UAS - Gr or UAS-Ir RNAi lines did not affect the avoidance of L-carabanin (Fig. 1a). The UASGr98b RNAi line (v1302), which produced the same phenotype in line 1, was further tested (Fig. 1b; v101040). Thus, GR98b was found to be important as an additional candidate receptor for detecting L-carabanine.

Example 2: Detection of L-carabanin Gr98b Mutation of

To confirm the role of L- cannabinin-repressed GR98b , a mutation was made by end-to-end homologous recombination (Figure 1c). A Gr98b mutant ( Gr98b ¹ ) was generated from the ends-out homologous recombination. To obtain the DNA construct for homologous recombination, the Genomic DNA template was used from the homologous gene w ¹¹¹⁸ Drosophila and the 3 kb arms located at 5 'and 3' were amplified by PCR using Gr 98b locus target. The primers used for the 5 'arm are 5'-GGTGGCTTAGGTGCTGCCATTAC-3' (SEQ ID NO: 4) and 5'-TTGGGTGAGTTCTGAAAACTAAC-3 '(SEQ ID NO: 5). The primers used for the 3 'arm are 5'-TCTGAAACGCAATCAATTGCTA-3' (SEQ ID NO: 6) and 5'-GTAGCCCAATATCACAATTC-3 '(SEQ ID NO: 7). Two arms were subcloned (BestGene Inc., Chino Hills, Calif.) With the pw35 vector of transgenic Drosophila produced by germ cell transformation and the transgene was mobilized to generate the homologous recombination described above. Through Genomic PCR in combination with the following primers it was confirmed alleles ^{Gr98b 1: 5'-TCTCCTGGCCAGAGCCTTTCCATA-3} '( SEQ ID NO: 8) and 5'-TGCTGCATTATCATGACGAACTCGG-3' (SEQ ID NO: 9).

UAS - in order to create a Gr98b transgenic Drosophila, and w ¹¹¹⁸ using a Hi-fidelity PCR kit (Roche) - was Gr98b amplifying cDNA from a cDNA library derived labellar, the cDNA clone was in pUAST vector. CDNA cloning of the DNA sequence was confirmed, and the transformed Drosophila was generated by BestGene Inc. (Chino Hills, Calif.). The Gr98b1 and UASGr98b Drosophila were hybridized to w ¹¹¹⁸ Drosophila for the fifth generation.

The GR98b ¹ mutation deleted the region encoded by N-terminal 233 of the 403 residue. Mutant Drosophila can be homozygous and fertile. GR98b ¹ Drosophila failed to avoid L- cannabinin , consistent with RNAi experiments (Fig. 1d). L - cannabinavir was rescued from the Gr98b ¹ Drosophila by the wild type Gr98b transgene ( UAS - Gr98b ) under the control of Gr66a ( Gr66a - GAL4 ), Gr8a (Gr8a - GAL4 ) or Gr98b ( Gr98b - GAL4 ) promoter (Fig. 1d). The action potential by L- cannabinin is destroyed by Gr98b ¹ Drosophila and defects are rescued by the expressed wild type Gr98b transgene ( UAS - Gr98b ) using Gr66a-GAL4 . Unlike the effect of detecting L- cannabinin , the Gr98b ¹ Drosophila strongly inhibited papaverine, strychnine, denatonium, berberine, lobelin , caffeine, DEET and quinine and observed electrophysiological responses 2c). These data indicate that GR98b is required for the detection of L-carabanine and are narrowly tuned.

Cell culture and transfection

S2 cells (Ajou University) were supplemented with 10% FBS (Invitrogen, Carlsbad, CA) and 50 units / ml penicillin-streptomycin in a 25 ° C T-25 flask (Thermo, Waltham, Mass.) To prepare Schneider's insect medium Welgene, Gyeongsan-si, Republic of Korea). 2 of the fixed patch (patch clamp) to carry out the experiment, pActin5c- GAL4, pUAST- EGFP and X-tremeGENE HP DNA transfected plasmid Gr (pUAST- Gr8a, pUAST- Gr66a and pUAST- Gr98b) using infection reagent (Roche) Or 3 times, and cells were transfected 24 hours later. At this time, the gene sequences of Gr8a, Gr66a and Gr98b are SEQ ID NOS: 10, 11 and 12, respectively, derived from Drosophila melanogaster . The transfection mixture consists of 4 μl of the transfection reagent and 1.3 μg of total DNA. The transfection cocktail and cells were cultured in serum-free medium for 12 hours, then transferred to serum medium and cell culture was continued for 24 hours to express taste receptor and EGFP.

S2 Patch-Clamp experiments in S2 cells

Gr- and EGFP -cultured S2 cells placed on the coverslips were transferred to a chamber located on the stage of an inverted microscope (IX71, Olympus). Whole cell currents were measured using an Axon 200B amplifier at -60 mA holding potential. bath solution contains the usual Ringer's solution (in mM): 140 NaCl, 5 KCl, 5 HEPES, 2 pyruvic acid sodium salt, 1.25 KH ₂ PO ₄ , 2 CaCl ₂ , 2 MgCl ₂ and 10 D-glucose . The pipette solution contains (in mM): 140 KCl, 5 EGTA-2K, 10 HEPES and 10 D-glucose (pH 7.2). After fire polishing, electrodes were taken from borosilicate glass with 2-4Ω resistance. Following the whole cell array, currents were recorded in the presence of L-cannabinin by applying voltage pulses to hyperpolarizing and depolarizing using a voltage ramp of +80 and -80 at -60 mA holding potential and 20 mV step. All data were recorded at room temperature using an Axopatch-200B amplifier (Axon Instruments, Foster City, Calif.) And filtered at 5 kHz to quantify the currents with a Digidata 1440A converter (Axon Instruments, Foster City, CA). Command potential and data collection were adjusted with pClamp 10.2 software (Axon instruments). Data analysis of whole cells was performed using Clampfit 10.2. The current density was normalized to the cell capacitance.

Example 3: L-cannabinin reactivity to GRNs sensing bitterness

The label room (the tip of the mouth of the fly), which is the main taste of Drosophila, consists of taste palate (sensory) and is divided into three categories according to length and location: Long (L), intermediate (I) . Gr66a is widely expressed in GRNs that sense the bitter taste of S-type and I-type senses, while Gr8a expression is restricted to a subset of Gr66a -expressing GRNs that respond to L-cannabinin. To elucidate the expression pattern of Gr98b , Gr98b - GAL4 ; GFP staining of UAS - mCD8 :: GFP flies was performed. Expression of the Gr98b - GAL4 reporter was detected in the GRNs of the I1, S1, S3, S5, S6, S7, S10, and S11 sensory senses (Fig. Consistent with its role in detecting L-cannabinin, it includes all of the sensory receptors that respond to L-cannabinin, and the S-type sensory receptors containing GRNs express Gr8a-GAL4 .

To elucidate whether Gr8a, Gr66a and Gr98b are generally sufficient to confer L-carabanin sensitivity to GRN that does not respond to L-cannabinin, Gr8a and Gr98b are eukaryotic to Gr- galabanin-insensitive Gr66a -expressing GRNs Lt; / RTI &gt; The mis-expression of Gr8a in expression of Gr66a did not confer L-carabanin sensitivity on GRNs insensitive to L-carabanine. S2 and I-type sensory cells under the control of Gr33a-GAL4 with a strong L -cannabanin response, we found the introduction of Gr8a and Gr98b into S2 and I-type senses insensitive to L- cannabinin (Fig. 3b, 3c ). These results indicate that Gr8a and Gr98b are essential components of the functional L-carabanin receptor.

Example 4: Giving L-cannabinin reactivity to GRNs sensing sweetness

It was possible that additional unwanted taste receptors could be involved in L-carabanin sensing, since GRNs sensing bitter taste in S2 and I-type sensory receptors also express other taste receptors. One of the wild-type L-type sensory receptors containing four GRNs responded to sugar and did not respond to L-carabanin. To provide more robust evidence that Gr8a , Gr66a , and Gr98b are sufficient for L-carabanin sense, the three taste receptors were mixed expressed in sugar-activated GRNs of L-type sensory using Gr64f - GAL4 ). This operation was found to give GRNs that sense sweetness and the ability to respond strongly to L-carnabinin (Figs. 4b and 4c). Only two ectopic expressions of the three taste receptors were insufficient to confer significant L-carabanin responsiveness (Figure 4c). However, in 12 out of 90 recordings, the co-expression of two taste receptors ( Gr8a and Gr66a ) caused a small L-cannabanin-induced response in the L- cannabine sense (Fig. 4c). It was found that Gr8a , Gr66a and Gr98b were ectopically expressed in Ir76b - GAL4- positive GRNs that sense a low salty taste, and that these GRNs impart remarkable L-cannabin responsiveness (Fig. 4d). Ectopic expression of Gr8a , Gr66a, and Gr98b did not induce responsiveness in various bleaching tests (Fig. 4e).

Imaging

Immunostaining of whole mount Drosophila labella was performed using rabbit anti-GFP (1: 1000, molecular probe) primary antibodies and goat anti-rabbit Alexa488 (1: 400, molecular probe) secondary antibodies. Labella was dissected from the head and fixed with 4% paraformaldehyde diluted in PBS-T (1x PBS for 20 min and 0.2% TritonX-100) for 20 min and washed three times with PBS-T. The labela was incubated in blocking solution (5% heat-inactivated goat serum in PBS-T) for 30 minutes and incubated with primary antibodies diluted in blocking solution overnight at 4 ° C. The tissues were washed three times with PBS-T and incubated with secondary antibodies diluted in blocking solution for 1 hour at room temperature. After washing three times with PBS-T, the sample was fixed with Vectashield (Vector Laboratories, Burlingame, CA) and observed with a Zeiss LSM700 confocal microscope (Jena, Germany).

Two-way choice behavioral assay

For each assay in the binary food selection assay, the flies were placed in hunger ~ 50 Drosophila (3-6 days old) and 72-well microtiter dishes for 18 hours. Each alternate well was filled with a combination of 1% agarose with one of the two test mixtures. The avoidance of bitter chemicals was analyzed by comparing the preferences for the indicated concentrations of 1 mM sucrose and 5 mM sucrose + repellent. In order to measure the activation effect of the sugar GRNs on L-cannabinin, preferences for 1 mM sucrose versus 1 mM sucrose + 30 mM L-cannabinin were tested.

To monitor food intake, a blue dye (Brilliant Blue FCF, 0.125 mg / ml) was added to one test mixture and a red dye (sulforhodamine B, 0.2 mg / ml) was added to another test mixture. Drosophila was allowed to ingest for 90 minutes at room temperature in the dark, then the fruit flies were frozen at -20 ° C. The number of blue (N _B ), red (N _R ) or purple (N _MIX ) fruit flies was calculated with a dissection microscope and the preference index (PI) _B N _R ) / (N _R + N _B + N _MIX ) or (N _R -N _B ) / (N _R + N _B + N _MIX ). A PI of 1.0 and -1.0 represents a perfect preference for one or another food. A PI of 0 indicates no preferred food.

Tip recordings

For tip recording, eclosion was maintained and fresh food was kept in fresh food for one day. An animal (Drosophila) was fixed by inserting a glass capillary reference electrode filled with Ringer's solution extending from the abdomen and abdomen to the head. Labela sensilla was stimulated with a recording electrode (10-20 μm tip diameter) containing 1 mM potassium chloride (KCl) or tastants dissolved in 30 mM tricholine citrate. The recording electrodes were connected to an amplifier (TastePROBE, Syntech, Hilversum, The Netherlands) and taste responses were collected and combined with a 100-3000 Hz bandpass filter and amplified using a signal interface (Syntech). The input was turned on to the speaker to facilitate audio monitoring. Active potentials were recorded at a 12-kHz sampling rate and amplitude-based spikes were classified and quantified using the Autospike 3.1 Software package (Syntech).

Example 5: Induction of behavioral induction of L-carabanine

Since the expression of Gr8a , Gr66a and Gr98b was sufficient to confer L-carabanin sensitivity to GRNs sensing sweetness, it was tested whether the ectopic expression of the taste receptor was inducing L-cannabinin. The control Drosophila ( w ¹¹¹⁸ ) strongly preferred 1 mM sucrose rather than 1 mM sucrose mixed with 30 mM L-cannabinin (FIG. 5). In contrast, Gr66a ^ex83 The mutants did not have a preference for 1 mM sucrose. The Gr66a ^ex83 Drosophila expressing two taste receptors ( Gr8a / Gr66a, Gr8a / Gr98b or Gr66a / Gr98b ), which are sweet taste sensing GRNs, were more indifferent to sucrose alone than to the sucrose to which L- ^carabanin was added ). However, the introduction of three taste receptors ( Gr8a / Gr66a / Gr98b ) caused considerable incentive to foods containing L- carabanine (Fig. 5).

Example 6: GR8a / GR66a / GR98b-dependent L-carabanin current in S2 cells

In order to test whether Gr8a , Gr66a and Gr98b could form L- canavanine -active cationic channels, the three taste receptors were co-expressed in Drosophila S2 cells and whole-cell voltage-clamp recording Respectively. Cells were fixed at -60 mV holding potential and a voltage ramp was applied from -80 to +80 mV. Using a physiological bath solution, it was found that the addition of 30 mM L-cananavin produces a rectifier current slightly outside with a -0.88 ± 1.89 mV reversal potential (FIGS. 6a and 6b). However, no current was produced when all combinations of two taste receptors ( Gr8a / Gr66a, Gr8a / Gr98b or Gr66a / Gr98b ) were expressed (Figures 6c to 6f). The induction currents for L-cannabinin were completely inhibited by La ³⁺ , a broad spectrum cationic inhibitor (Fig. 6g, 6h). Consistent with the Invivo data, there were no other repellents in the test to derive conductivity in S2 cells expressing GR8a, GR66a and GR98b .

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

<110> Kookmin University Industry Academy Cooperation Foundation          Industry-Academic Cooperation Foundation, Yonsei University          University of California at Santa Barbara <120> Method of Screening Insecticides or Repellents by using a          Gustatory Receptor <130> 302 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 516 <212> PRT <213> Drosophila melanogaster <400> 1 Met Ser Gly His Leu Gly Arg Val Leu Gln Phe His Leu Arg Leu Tyr   1 5 10 15 Gln Val Leu Gly Phe His Gly Leu Pro Leu Pro Gly Asp Gly Asn Pro              20 25 30 Ala Arg Thr Arg Arg Arg Leu Met Ala Trp Ser Leu Phe Leu Leu Ile          35 40 45 Ser Leu Ser Ala Leu Val Leu Ala Cys Leu Phe Ser Gly Glu Glu Phe      50 55 60 Leu Tyr Arg Gly Asp Met Phe Gly Cys Ala Asn Asp Ala Leu Lys Tyr  65 70 75 80 Val Phe Ala Glu Leu Gly Val Leu Ala Ile Tyr Leu Glu Thr Leu Ser                  85 90 95 Ser Gln Arg His Leu Ala Asn Phe Trp Trp Leu His Phe Lys Leu Gly             100 105 110 Gly Gln Lys Thr Gly Leu Val Ser Leu Arg Ser Glu Phe Gln Gln Phe         115 120 125 Cys Arg Tyr Leu Ile Phe Leu Tyr Ala Met Met Ala Ala Glu Val Ala     130 135 140 Ile His Leu Gly Leu Trp Gln Phe Gln Ala Leu Thr Gln His Met Leu 145 150 155 160 Leu Phe Trp Ser Thr Tyr Glu Pro Leu Val Trp Leu Thr Tyr Leu Arg                 165 170 175 Asn Leu Gln Phe Val Leu His Leu Glu Leu Leu Arg Glu Gln Leu Thr             180 185 190 Gly Leu Glu Arg Glu Met Gly Leu Leu Glu Tyr Ser Arg Phe Ala         195 200 205 Ser Glu Thr Gly Arg Ser Phe Pro Gly Phe Glu Ser Phe Leu Arg Arg     210 215 220 Arg Leu Val Gln Lys Gln Arg Ile Tyr Ser His Val Tyr Asp Met Leu 225 230 235 240 Lys Cys Phe Gln Gly Ala Phe Asn Phe Ser Ile Leu Ala Val Leu Leu                 245 250 255 Thr Ile Asn Ile Arg Ile Ala Val Asp Cys Tyr Phe Met Tyr Tyr Ser             260 265 270 Ile Tyr Asn Asn Val Ile Asn Asn Asp Tyr Tyr Leu Ile Val Pro Ala         275 280 285 Leu Leu Glu Ile Pro Ala Phe Ile Tyr Ala Ser Gln Ser Cys Met Val     290 295 300 Val Val Pro Arg Ile Ala His Gln Leu His Asn Ile Val Thr Asp Ser 305 310 315 320 Gly Cys Cys Ser Cys Pro Asp Leu Ser Leu Gln Ile Gln Asn Phe Ser                 325 330 335 Leu Gln Leu Leu His Gln Pro Ile Arg Ile Asp Cys Leu Gly Leu Thr             340 345 350 Ile Leu Asp Cys Ser Leu Leu Thr Arg Met Ala Cys Ser Val Gly Thr         355 360 365 Tyr Met Ile Tyr Ser Ile Gln Phe Ile Pro Lys Phe Ser Asn Thr Tyr     370 375 380 Met Cys Gly Ala Thr Ala Cys Gly Ala Ala Thr Cys Gly Ala Thr Thr 385 390 395 400 Gly Cys Cys Thr Cys Gly Gly Cys Cys Thr Gly Ala Cys Cys Ala Thr                 405 410 415 Cys Cys Thr Gly Gly Aly Thr Thr Gly Cys Ala Gly Thr Cys Thr Thr             420 425 430 Cys Thr Ala Ala Cys Thr Cys Gly Aly Thr Gly Gly Cys Cys Thr         435 440 445 Gly Thr Thr Cys Cys Gly Thr Gly Gly Gly Cys Ala Cys Cys Thr Ala     450 455 460 Cys Ala Thr Gly Ala Thr Cys Thr Ala Thr Ala Gly Cys Ala Thr Cys 465 470 475 480 Cys Ala Gly Thr Thr Thr Ala Thr Ala Cys Cys Ala Ala Ala Gly Thr                 485 490 495 Thr Cys Ala Gly Cys Ala Ala Thr Ala Cys Cys Thr Ala Thr Ala Thr             500 505 510 Gly Thr Ala Gly         515 <210> 2 <211> 527 <212> PRT <213> Drosophila melanogaster <400> 2 Met Ala Gln Ala Glu Asp Ala Val Gln Pro Leu Leu Gln Gln Phe Gln   1 5 10 15 Gln Leu Phe Phe Ile Ser Lys Ile Ala Gly Ile Leu Pro Gln Asp Leu              20 25 30 Glu Lys Phe Arg Ser Arg Asn Leu Leu Glu Lys Ser Arg Asn Gly Met          35 40 45 Ile Tyr Met Leu Ser Thr Leu Ile Leu Tyr Val Val Leu Tyr Asn Ile      50 55 60 Leu Ile Tyr Ser Phe Gly Glu Glu Asp Arg Ser Leu Lys Ala Ser Gln  65 70 75 80 Ser Thr Leu Thr Phe Val Ile Gly Leu Phe Leu Thr Tyr Ile Gly Leu                  85 90 95 Ile Met Met Val Ser Asp Gln Leu Thr Ala Leu Arg Asn Gln Gly Arg             100 105 110 Ile Gly Glu Leu Tyr Glu Arg Ile Arg Leu Val Asp Glu Arg Leu Tyr         115 120 125 Lys Glu Gly Cys Val Met Asp Asn Ser Thr Ile Gly Arg Arg Ile Arg     130 135 140 Ile Met Leu Ile Met Thr Val Ile Phe Glu Leu Ser Ile Leu Val Ser 145 150 155 160 Thr Tyr Val Lys Leu Val Asp Tyr Ser Gln Trp Met Ser Leu Leu Trp                 165 170 175 Ile Val Ser Ala Ile Pro Thr Phe Ile Asn Thr Leu Asp Lys Ile Trp             180 185 190 Phe Ala Val Ser Leu Tyr Ala Leu Lys Glu Arg Phe Glu Ala Ile Asn         195 200 205 Ala Thr Leu Glu Glu Leu Val Asp Thr His Glu Lys His Lys Leu Trp     210 215 220 Leu Arg Gly Asn Gln Glu Val Pro Pro Leu Asp Ser Ser Gln Pro 225 230 235 240 Pro Gln Tyr Asp Ser Asn Leu Glu Tyr Leu Tyr Lys Glu Leu Gly Gly                 245 250 255 Met Asp Ile Gly Ser Ile Gly Lys Ser Ser Val Ser Gly Ser Gly Lys             260 265 270 Asn Lys Val Ala Pro Val Ala His Ser Met Asn Ser Phe Gly Glu Ala         275 280 285 Ile Asp Ala Ala Ser Arg Lys Pro Pro Pro Pro Leu Ala Thr Asn     290 295 300 Met Val His Glu Ser Glu Leu Gly Asn Ala Ala Lys Val Glu Glu Lys 305 310 315 320 Leu Asn Asn Leu Cys Gln Val His Asp Glu Ile Cys Glu Ile Gly Lys                 325 330 335 Ala Leu Asn Glu Leu Trp Ser Tyr Pro Ile Leu Ser Leu Met Ala Tyr             340 345 350 Gly Phe Leu Ile Phe Thr Ala Gln Leu Tyr Phe Leu Tyr Cys Ala Thr         355 360 365 Gln Tyr Gln Ser Ile Pro Ser Leu Phe Arg Ser Ala Lys Asn Pro Phe     370 375 380 Ile Thr Val Ile Val Leu Ser Tyr Thr Ser Gly Lys Cys Val Tyr Leu 385 390 395 400 Ile Tyr Leu Ser Trp Lys Thr Ser Gln Ala Ser Lys Arg Thr Gly Ile                 405 410 415 Ser Leu His Lys Cys Gly Val Val Ala Asp Asp Asn Leu Leu Tyr Glu             420 425 430 Ile Val Asn His Leu Ser Leu Lys Leu Leu Asn His Ser Val Asp Phe         435 440 445 Ser Ala Cys Gly Phe Phe Thr Leu Asp Met Glu Thr Leu Tyr Gly Val     450 455 460 Ser Gly Gly Ile Thr Ser Tyr Leu Ile Ile Leu Ile Gln Phe Asn Leu 465 470 475 480 Ala Ala Gln Gln Ala Lys Glu Ala Ile Gln Thr Phe Asn Ser Leu Asn                 485 490 495 Asp Thr Ala Gly Leu Val Gly Ala Ala Thr Asp Met Asp Asn Ile Ser             500 505 510 Ser Thr Leu Arg Asp Phe Val Thr Thr Thr Met Thr Pro Ala Val         515 520 525 <210> 3 <211> 403 <212> PRT <213> Drosophila melanogaster <400> 3 Met Val Ala Gln Lys Ser Arg Leu Leu Ala Arg Ala Phe Pro Tyr Leu   1 5 10 15 Asp Ile Phe Ser Val Phe Ala Leu Thr Pro Pro Pro Gln Ser Phe Gly              20 25 30 His Thr Pro His Arg Arg Leu Arg Trp Tyr Leu Met Thr Gly Tyr Val          35 40 45 Phe Tyr Ala Thr Ala Ile Leu Ala Thr Val Phe Ile Val Ser Tyr Phe      50 55 60 Asn Ile Ile Ale Ile Asp Glu Glu Val Leu Glu Tyr Asn Val Ser Asp  65 70 75 80 Phe Thr Arg Val Met Gly Asn Ile Gln Lys Ser Leu Tyr Ser Ile Met                  85 90 95 Ala Ile Ala Asn His Leu Asn Met Leu Ile Asn Tyr Arg Arg Leu Gly             100 105 110 Gly Ile Tyr Lys Asp Ile Ala Asp Leu Glu Met Asp Met Asp Glu Ala         115 120 125 Ser Gln Cys Phe Gly Gly Gln Arg Gln Arg Phe Ser Phe Arg Phe Arg     130 135 140 Met Ala Leu Cys Val Gly Val Trp Met Ile Leu Met Val Gly Ser Met 145 150 155 160 Pro Arg Leu Thr Met Thr Ala Met Gly Pro Phe Val Ser Thr Leu Leu                 165 170 175 Lys Ile Leu Thr Glu Phe Val Met Ile Met Gln Gln Leu Lys Ser Leu             180 185 190 Glu Tyr Cys Val Phe Val Leu Ile Ile Tyr Glu Leu Val Leu Arg Leu         195 200 205 Arg Arg Thr Leu Ser Gln Leu Gln Glu Glu Phe Gln Asp Cys Glu Gln     210 215 220 Gln Asp Met Leu Gln Ala Leu Cys Val Ala Leu Lys Arg Asn Gln Leu 225 230 235 240 Leu Leu Gly Arg Ile Trp Arg Leu Glu Gly Asp Val Gly Ser Tyr Phe                 245 250 255 Thr Pro Thr Met Leu Leu Leu Phe Leu Tyr Asn Gly Leu Thr Ile Leu             260 265 270 His Met Val Asn Trp Ala Tyr Ile Asn Lys Phe Leu Tyr Asp Ser Cys         275 280 285 Cys Gln Tyr Glu Arg Phe Leu Val Cys Ser Thr Leu Leu Val Asn Leu     290 295 300 Leu Leu Pro Cys Leu Leu Ser Gln Arg Cys Ile Asn Ala Tyr Asn Cys 305 310 315 320 Phe Pro Arg Ile Leu His Lys Ile Arg Cys Thr Ser Ala Asp Pro Asn                 325 330 335 Phe Ala Met Leu Thr Arg Gly Leu Arg Glu Tyr Ser Leu Gln Met Glu             340 345 350 His Leu Lys Leu Arg Phe Thr Cys Gly Gly Leu Phe Asp Ile Asn Leu         355 360 365 Lys Tyr Phe Gly Gly Leu Leu Val Thr Ile Phe Gly Tyr Ile Ile Ile     370 375 380 Leu Ile Gln Phe Lys Val Gln Ala Ile Ala Ala Asn Arg Tyr Lys Lys 385 390 395 400 Val Val Asn             <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggtggcttag gtgctgccat tac 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ttgggtgagt tctgaaaact aac 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tctgaaacgc aatcaattgc ta 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtagcccaat atcacaattc 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tctcctggcc agagcctttc cata 24 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tgctgcatta tcatgacgaa ctcgg 25 <210> 10 <211> 1027 <212> DNA <213> Drosophila melanogaster <400> 10 ggtcctgcg ttccatgggc tgccgttgcc gggcgatggg aatccggcca ggaccaggag gcgtctgatg 120 gcatggagcc tgttcctgct catttcgctg agtgccctcg tactcgcgtg cctctttagc 180 ggcgaggagt tcctctatcg cggcgacatg ttcggctgtg ccaatgatgc ccttaaatac 240 gtattcgccg aattgggcgt gctggccata tatctggaga cgctgagcag ccagcggcat 300 ttggccaact tctggtggct gcacttcaag ttgggcggcc aaaaaacggg cttggtgagc 360 ctgcgcagtg agttccagca gttttgtcgc tatctgatat tcctgtacgc catgatggcc 420 gccgaagtgg cgatccattt gggattgtgg cagttccaag cgctcaccca acatatgttg 480 ctcttttgga gcacctatga gccgctcgtg tggctgacgt atctgcgcaa tctgcagttc 540 gtactgcact tggagctgct cagggagcag ctgaccggct tggaacgcga aatgggtctg 600 ctggcggagt actcgcgatt tgctagcgaa acgggtcgga gttttcctgg attcgaaagt 660 ttcctgcgcc gacgactagt gcagaagcag cgcatctata gccatgtgta tgacatgctc 720 aaatgtttcc agggtgcctt caacttctcc attctcgccg tcctgctgac catcaacata 780 cgcatcgccg tggactgcta cttcatgtac tacagcatct acaacaatgt gattaacaac 840 gattactacc taatcgttcc cgccttgctc gagattcccg ccttcatata cgcttcgcag 900 agctgcatgg tcgtggtgcc caggatcgcc caccagctgc ataatatagt caccgattcc 960 ggttgctgca gctgtcccga tctctccctg cagattcaga acttttcact gcaactcctg 1020 catcagc 1027 <210> 11 <211> 1584 <212> DNA <213> Drosophila melanogaster <400> 11 atggcgcagg cggaggacgc agtgcaacca ctattgcagc agttccagca actgttcttc 60 atatccaaga tagctggaat tctgccacag gatctcgaga agtttcgatc taggaatctg 120 ctggagaaat cccgtaatgg catgatttac atgctgagta ctttaatact ctacgttgtg 180 ctctataata ttttgatata ttcctttgga gaggaggacc gcagcctaaa ggcctcgcag 240 agcaccttga ctttcgtgat tggcttgttc ctgacctata tcggtctgat tatgatggtc 300 tccgaccagt tgaccgcgtt acgaaaccag ggtagaattg gagagcttta cgagcgcatc 360 cgtctggtgg atgagcgcct ttacaaagag gggtgtgtta tggacaacag tacaattgga 420 cggcgcatac gaattatgct gatcatgacg gtcatctttg agttgtccat tttggtgagc 480 acctatgtca agctggtgga ctatagtcaa tggatgtcct tgttatggat agtgtccgcc 540 attcccacgt tcatcaacac gctagacaag atctggttcg ctgtttcgtt atatgcgttg 600 aaagaacgct tcgaggccat aaacgccacc ctagaggaac tggtggacac gcacgagaag 660 cataagctgt ggctgcgagg caatcaagag gttccgcctc ctctggacag ctcccagccg 720 cctcagtatg acagcaacct ggagtatctg tacaaggaac taggaggtat ggacataggt 780 tccattggca agagttcagt gtctggttcg gggaaaaaca aagtagcacc agtggcccac 840 tccatgaact cctttggtga agcaattgat gcggccagca ggaagcctcc accgcctccc 900 ctggccacta acatggtcca tgaaagcgag ctgggaaatg ccgctaaggt agaggagaaa 960 ctaaacaacc tgtgccaggt gcacgacgag atctgtgaaa tcggaaaagc tttgaacgag 1020 ctgtggagct atcccattct atctctaatg gcctatggtt ttctgatttt cactgctcaa 1080 ctttatttcc tctactgcgc tacacagtac caatcgatac catcgctttt ccgttccgcc 1140 aagaatccct tcatcactgt tatagttcta agttatacgt ctggaaaatg cgtgtacctc 1200 atctacctga gttggaaaac gtcgcaggcc tccaagcgca caggaatcag tctgcacaaa 1260 tgtggcgtgg tggccgatga taatcttctc tacgaaattg ttaaccacct atcgctaaaa 1320 ttgctcaacc actcggtgga cttttcggct tgcggcttct ttaccctgga catggaaaca 1380 ttgtatggtg tgagtggcgg gatcactagc tacctgatca tcctgattca gttcaatttg 1440 gccgcccagc aggccaaaga ggctatacag acgttcaact cgcttaatga caccgccggc 1500 ttggttggtg ccgccaccga tatggataat attagctcca cgctgcgtga tttcgtcacc 1560 acgaccatga caccggcggt ctaa 1584 <210> 12 <211> 1212 <212> DNA <213> Drosophila melanogaster <400> 12 atggtggccc agaagagccg tctcctggcc agagcctttc catatctcga catcttttcc 60 gtgtttgctc tcacaccacc gccccaaagt tttggccaca ctccacatag acgtcttaga 120 tggtacttga tgactggcta tgtgttctat gccaccgcga tccttgcgac ggtcttcatt 180 gtgtcctact ttaacattat agccattgac gaagaggtct tggagtacaa tgtgtctgat 240 tttaccagag tgatgggtaa tattcaaaag agcctgtatt cgattatggc catcgccaac 300 catctcaata tgctcatcaa ttatcgccgg cttggcggaa tctacaagga cattgccgat 360 ttggagatgg acatggacga ggcatcgcag tgctttggtg gccaaagaca acgctttagt 420 ttccgctttc gcatggccct ctgtgtgggc gtgtggatga ttctgatggt gggttcaatg 480 ccccgattaa ccatgacggc catgggtcct tttgtcagca cgctattgaa gatcttaacc 540 gagttcgtca tgataatgca gcagctgaag agtctggagt attgcgtctt tgtactcatc 600 atttatgagt tggtcctgcg gttgcgccgc acacttagtc agctgcagga ggagttccag 660 gactgcgagc agcaggacat gcttcaagct ctatgtgtgg ctctgaaacg caatcaattg 720 ctattgggac ggatttggag gctggagggc gatgtgggca gctacttcac cccgacaatg 780 ttgctgctgt tcctctacaa tggtctgacc atccttcaca tggtcaactg ggcgtacatc 840 aacaagttcc tctacgattc gtgttgtcag tatgaacggt tcttagtttg ctccacgctg 900 gt; tttccacgaa tcctgcataa gattcgctgc acatccgcgg atcccaattt tgccatgttg 1020 accagaggat tgcgggagta ctccctgcaa atggaacacc tgaagttgcg tttcacatgc 1080 ggcgggttat ttgacatcaa tttgaaatac tttggcgggt tgcttgtcac catttttgga 1140 tatatcataa tcctcataca attcaaagtg caggcgattg ctgcgaatag atacaaaaaa 1200 gtggttaatt aa 1212

Claims (10)

A screening method of an insect control agent comprising the steps of:
(a) treating a candidate substance with a heterozygote comprising a subunit of Gr8a, Gr66a and Gr98b which is a Gustatory Receptor derived from a fruit fly; And
(b) selecting, as an insect control agent, a substance that increases or inhibits the activity of the taste receptor from the fruit fly, as compared to the untreated control.
A method for screening an insecticide comprising the steps of:
(a) treating a candidate substance with a heterozygote comprising a subunit of Gr8a, Gr66a and Gr98b which is a Gustatory Receptor derived from a fruit fly; And
(b) selecting an insecticide to inhibit the activity of the taste receptor from the fruit fly compared to the untreated control.
A screening method for a convulsant comprising the steps of:
(a) treating a candidate substance with a heterozygote comprising a subunit of Gr8a, Gr66a and Gr98b which is a Gustatory Receptor derived from a fruit fly; And
(b) selecting, as a repellent agent, a substance that increases the activity of a taste receptor derived from a fruit fly compared to an untreated control group.
delete 4. The method according to any one of claims 1 to 3,
Wherein the Gr8a subunit has the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 10.
4. The method according to any one of claims 1 to 3,
Wherein the Gr66a subunit has the amino acid sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 11.
4. The method according to any one of claims 1 to 3,
Wherein the subunit of Gr98b has the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 12.
4. The method according to any one of claims 1 to 3,
Wherein the taste receptors derived from the fruit flies are expressed in neurons.
4. The method according to any one of claims 1 to 3,
Wherein the activity of the taste receptor is measured through a change in current generated by the formation of a channel in a taste receptor that recognizes the candidate substance as a ligand.
A composition for screening a ligand binding to said taste receptors, comprising a heteropolymer comprising subunits of Gr8a, Gr66a and Gr98b, which are taste receptors derived from fruit flies, with an insect control agent.

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