Target identification method of plant source compound acting on insect 5-hydroxytryptamine receptor

Abstract

The invention discloses a method for identifying a plant source compound target acting on an insect 5-hydroxytryptamine receptor. Comprising the following steps: providing an insect 5-hydroxytryptamine receptor which is expressed in vitro and has a physiological function, determining the activity of the insect 5-hydroxytryptamine receptor, and identifying whether the insect 5-hydroxytryptamine receptor is a molecular target of a plant source compound or not by detecting whether the insect 5-hydroxytryptamine receptor can be directly activated or antagonized or not. According to the invention, a plurality of insect 5-hydroxytryptamine receptors can be expressed at the same time, a plurality of plant source compounds can be subjected to target identification at the same time, a result can be rapidly obtained within 180 seconds after testing, detection of excitation and antagonistic activity can be carried out at the same time, and the method has the advantage of being suitable for high-throughput screening of plant source compounds acting on insect 5-hydroxytryptamine receptors, and has a wide application prospect in the field of high-throughput screening of plant source compounds acting on insect 5-hydroxytryptamine receptors. Whether the plant source compound acts on the insect 5-hydroxytryptamine receptor or not is verified from the double angles of pharmacology and genetics, the result is reliable, the stable, efficient and non-toxic plant source insecticide is screened out, and the application prospect in pest control is wide.

Description

Technical Field

[0001] The invention relates to the field of agriculture, in particular to the application of insect 5-hydroxytryptamine receptors as molecular targets of plant-derived compounds, and specifically to a method for identifying plant-derived compound targets acting on insect 5-hydroxytryptamine receptors. Background Art

[0002] Botanical pesticides refer to pesticides derived from plants and developed using plant resources. Their active ingredients exist naturally in nature and are safe for non-target organisms, have specific modes of action, are not prone to drug resistance, and are environmentally friendly. Therefore, botanical pesticides are gradually becoming a new direction of pesticide development that people are paying attention to. Developing effective identification methods to identify the main action targets of botanical pesticides will help us understand their mechanism of action and develop more effective insecticides based on these active ingredients.

[0003] 5-HT is a biogenic amine that acts as an important neurotransmitter in invertebrates, controlling and regulating many physiological and behavioral processes including feeding, reproduction, movement and development. Although there has been some progress in the study of 5-HT and its receptors in insects, there are currently no insecticides targeting 5-HT receptors. 1a The receptor agonist 1-[(4-aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine, its derivatives were modified and synthesized, and it was found through biological testing that it could inhibit the growth of armyworms and the physiological activity of larvae, see "Design and Synthesis of Novel Insecticides Based on the Serotonergic Ligand 1-[(4-Aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine(PAPP)" Journal of Agricultural and Food Chemistry, 2010, 58, 5, 2624-2629. Therefore, the 5-hydroxytryptamine receptor is a potential target for insecticides. It is very important to develop a simple and efficient identification method for the study of plant-derived insecticides that may act on insect 5-hydroxytryptamine receptors, which is very important for the development of new pesticides. However, the existing identification technology has the problems of cumbersome identification procedures and single and incomplete evidence, making it difficult to verify the reliability of its results. The above problems need to be solved urgently. Summary of the invention

[0004] The present invention improves upon the shortcomings of the prior art and provides a method for identifying targets of plant-derived compounds that act on insect 5-hydroxytryptamine receptors. The present invention identifies the main target of the indole alkaloid gramine: insect 5-hydroxytryptamine type 1b receptor 5-HT 1b , antagonizing this receptor can achieve an anti-insect effect.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention discloses a method for identifying a target of a plant-derived compound acting on an insect 5-hydroxytryptamine receptor, comprising:

[0007] 1) Provide functional insect 5-HT receptors expressed in vitro and measure the functional activity of insect 5-HT receptors in the in vitro expression system;

[0008] 2) Determine the agonistic effect of the plant-derived compound on the insect 5-hydroxytryptamine receptor of step 1). If the insect 5-hydroxytryptamine receptor can be directly activated by the plant-derived compound in reaction system A, it is determined to be the in vitro receptor of the plant-derived compound;

[0009] 3) Determine the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1). If the response of the insect 5-hydroxytryptamine receptor to 5-hydroxytryptamine in reaction system B is inhibited by the plant-derived compound to be tested, then it is determined to be the in vitro receptor of the plant-derived compound to be tested;

[0010] 4) Through bioassay, a mutant insect strain resistant to the plant-derived compound to be tested is obtained, and its in vivo receptor is determined. Combined with the in vitro receptor determined in step 2) or 3), the target of the plant-derived compound to be tested is determined.

[0011] As a further improvement, the in vitro expression system of step 1) of the present invention comprises: a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

[0012] As a further improvement, the method for determining the functional activity of insect 5-hydroxytryptamine receptors in step 1) of the present invention is: in an in vitro expression system, an agonist 5-hydroxytryptamine is applied, which has an obvious calcium ion flow process within 180 seconds.

[0013] As a further improvement, the method for determining the agonist effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor in step 1) in step 2) of the present invention is: applying equal concentrations of the plant-derived compound to be tested and 5-hydroxytryptamine, there is an obvious calcium ion flow process within 180 seconds, and the reaction system A includes: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

[0014] As a further improvement, the method for determining the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1) in step 3) of the present invention is: applying a fixed high concentration of the plant-derived compound to be tested while applying 5-hydroxytryptamine, which can significantly inhibit its response to 5-hydroxytryptamine within 180 seconds, and the reaction system B includes: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

[0015] As a further improvement, if the result of either step 2) or 3) of the present invention can determine the in vitro receptor of the plant-derived compound to be tested, the other step does not need to be performed.

[0016] As a further improvement, the mutant insect strain in step 4) of the present invention is a strain that completely loses the function of 5-hydroxytryptamine receptors.

[0017] As a further improvement, the target of the plant-derived compound to be tested in step 4) of the present invention refers to a receptor that is consistent with the in vitro receptor determined in step 2) or 3) and the in vivo receptor determined in step 4).

[0018] As a further improvement, the insects described in the present invention are agricultural pests, horticultural pests, disease-carrying insects or parasites including the model organism Drosophila melanogaster.

[0019] As a further improvement, the plant-derived compound to be tested described in the present invention is an organic small molecule, an inorganic small molecule, a polysaccharide, a peptide, a protein, a nucleic acid or any one or more combinations thereof.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention discloses a method for identifying a target of a plant-derived compound that acts on an insect 5-hydroxytryptamine receptor, comprising providing an insect 5-hydroxytryptamine receptor that is expressed in vitro and has a physiological function, determining the activity of the insect 5-hydroxytryptamine receptor, identifying whether the insect 5-hydroxytryptamine receptor is a molecular target of the plant-derived compound by detecting whether the insect 5-hydroxytryptamine receptor can be directly activated or antagonized, and clarifying the specific target and molecular mechanism of the plant-derived compound in combination with genetic evidence.

[0022] 1. The present invention identifies a molecular target of the plant-derived alkaloid gramine: insect 5-HT 1b Receptor. As an antagonist, rutin inhibits the agonist activity of the endogenous agonist 5-hydroxytryptamine, resulting in insecticidal activity;

[0023] 2. The present invention provides a simple and easy-to-operate identification method that can simultaneously express multiple insect 5-hydroxytryptamine receptors, simultaneously identify multiple plant-derived compounds, and quickly obtain results within 180 seconds after the test. Considering that plant-derived compounds have agonist or antagonist activity, the present invention can also detect agonist and antagonist activity at the same time, and has the advantage of being suitable for high-throughput screening of plant-derived compounds that act on insect 5-hydroxytryptamine receptors;

[0024] 3. The identification method provided by the present invention has comprehensive evidence, and verifies whether the plant-derived compounds act on insect 5-hydroxytryptamine receptors from the dual perspectives of pharmacology and genetics, and the results are reliable.

[0025] 4. In summary, the method of the present invention has obvious advantages. The present invention can be used to screen out stable, efficient, non-toxic and green botanical insecticides, which has broad application prospects in pest control. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a dose curve of 5-hydroxytryptamine activating Drosophila 5-hydroxytryptamine receptor, where A is the dose of 5-hydroxytryptamine activating Drosophila 5-HT 1a Dose curve of 5-HT receptors, 5-HT 1a EC50, half maximal effect concentration 50 6.022 μM, B is 5-hydroxytryptamine activating Drosophila 5-HT 1b Dose curve of 5-HT receptors, 5-HT 1b EC50, half maximal effect concentration 50 is 55.99nM, C is 5-hydroxytryptamine activating Drosophila 5-HT 2a Dose curve of 5-HT receptors, 5-HT 2a EC50, half maximal effect concentration 50 is 208.8nM, D is 5-hydroxytryptamine activating Drosophila 5-HT 2bDose curve of 5-HT receptors, 5-HT 2b EC50, half maximal effect concentration 50 is 317.1nM, E is the dose curve of 5-HT activating Drosophila 5-HT7 receptor, and the half-maximal effect concentration value EC of 5-HT on 5-HT7 receptor 50 97.84nM;

[0027] Figure 2 The dose curve of 100 μM rutin inhibits the agonist activity of Drosophila 5-HT receptors, where:

[0028] A: 100 μM rutin inhibits the 5-HT 1a Dose curve of 5-HT receptor agonist activity, 5-HT 1a EC50, half maximal effect concentration 50 The effect of 5-HT on 5-HT was 6.022 μM. 1a EC50, half maximal effect concentration 50 A is 7.063μM, B is 100μM rutin inhibits Drosophila 5-HT 1b Dose curve of 5-HT receptor agonist activity, 5-HT 1b EC50, half maximal effect concentration 50 is 55.99 nM. After applying 100 μM gramine, 5-hydroxytryptamine has an inhibitory effect on 5-HT. 1b EC50, half maximal effect concentration 50 3.028μM, C 100μM rutin inhibits Drosophila 5-HT 2a Dose curve of 5-HT receptor agonist activity, 5-HT 2a EC50, half maximal effect concentration 50 208.8nM, after applying 100μM gramine, 5-hydroxytryptamine has an effect on 5-HT 2a EC50, half maximal effect concentration 50 9.366μM, D 100μM rutin inhibits Drosophila 5-HT 2b Dose curve of 5-HT receptor agonist activity, 5-HT 2b EC50, half maximal effect concentration 50 is 317.1nM. After applying 100μM gramine, 5-hydroxytryptamine has an effect on 5-HT 2b EC50, half maximal effect concentration 50 is 453.0nM, E is the dose curve of 100μM rutin to inhibit the agonist activity of Drosophila 5-HT7 receptor, and the half-maximal effect concentration value EC of 5-hydroxytryptamine on 5-HT7 receptor 50The half-maximal effect concentration of 5-HT on 5-HT7 receptor after applying 100 μM rutin is 97.84 nM. 50 240.7nM;

[0029] Figure 3 This is the death dose curve of 5-HT mutant fruit flies fed with rutin. 1118 The half-lethal concentration LC 50 3.9mM, mutant 5-HT 1a Gal4 The half-lethal concentration LC 50 5.1mM, 5-HT 1b Gal4 The half-lethal concentration LC 50 Greater than 10mM, 5-HT 2a Gal4 The half-lethal concentration LC 50 3.1mM, 5-HT 2b Gal4 The half-lethal concentration LC 50 3.8mM, 5-HT7 Gal4 The half-lethal concentration LC 50 2.8mM, 5-HT 1b ΔⅢ-Ⅴ The half-lethal concentration LC 50 The data were analyzed by nonlinear regression and compared with the additional sum of squares F test. Asterisks mark the significance of the F test between groups ( * P<0.05, ** P<0.01, *** P<0.001), 5-HT 1b Gal4 and 5-HT 1b ΔⅢ-Ⅴ LC 50 Significantly lower than wild type w 1118 . DETAILED DESCRIPTION

[0030] The present invention discloses a method for identifying a target of a plant-derived compound acting on an insect 5-hydroxytryptamine receptor, comprising:

[0031] 1) Provide an insect 5-hydroxytryptamine receptor with functional expression in vitro, and determine the functional activity of the insect 5-hydroxytryptamine receptor in an in vitro expression system; the in vitro expression system includes: a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator, and polyoxyethylene polyoxypropylene ether; the method for determining the functional activity of the insect 5-hydroxytryptamine receptor is: in the in vitro expression system, an agonist 5-hydroxytryptamine is applied, which has an obvious calcium ion flow process within 180 seconds.

[0032] 2) Determine the agonistic effect of the plant-derived compound on the insect 5-hydroxytryptamine receptor of step 1). If the insect 5-hydroxytryptamine receptor can be directly activated by the plant-derived compound in reaction system A, it is determined to be the in vitro receptor of the plant-derived compound. The method for determining the agonistic effect of the plant-derived compound on the insect 5-hydroxytryptamine receptor of step 1) is as follows: applying equal concentrations of the plant-derived compound and 5-hydroxytryptamine, both of which have obvious calcium ion flow processes within 180 seconds. Reaction system A includes: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, Hank's balanced salt solution, a calcium ion indicator, and polyoxyethylene polyoxypropylene ether.

[0033] 3) Determine the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1). If the response of the insect 5-hydroxytryptamine receptor to 5-hydroxytryptamine in reaction system B is inhibited by the plant-derived compound to be tested, it is determined to be the in vitro receptor of the plant-derived compound to be tested. The method for determining the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1) is as follows: applying a fixed high concentration of the plant-derived compound to be tested while applying 5-hydroxytryptamine, which can significantly inhibit its response to 5-hydroxytryptamine within 180 seconds. Reaction system B includes: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator, and polyoxyethylene polyoxypropylene ether.

[0034] If the result of either step 2) or 3) is sufficient to determine the in vitro receptor of the plant-derived compound to be tested, the other step does not need to be performed.

[0035] 4) Through bioassay, a mutant insect strain resistant to the plant-derived compound to be tested is obtained, and its in vivo receptor is determined. Combined with the in vitro receptor determined in step 2) or 3), the target of the plant-derived compound to be tested is determined. The mutant insect strain is a strain in which the 5-hydroxytryptamine receptor function is completely lost. The target of the plant-derived compound to be tested refers to the receptor that is consistent with the in vitro receptor determined in step 2) or 3) and the in vivo receptor determined in step 4).

[0036] The insects are agricultural pests, horticultural pests, disease-carrying insects or parasites including the model organism Drosophila melanogaster. The plant-derived compounds to be tested are organic small molecules, inorganic small molecules, polysaccharides, peptides, proteins, nucleic acids or any one or more combinations thereof.

[0037] The specific implementation examples and the accompanying drawings are used to further illustrate the specific implementation methods of the present invention.

[0038] The invention discloses a method for identifying plant-derived compounds acting on insect 5-hydroxytryptamine receptors.

[0039] 1) Provide in vitro expression of functional insect (selected model organism Drosophila melanogaster) 5-HT receptors, including: Drosophila 5-HT 1a Receptor, Drosophila 5-HT 1b Receptor, Drosophila 5-HT 2a Receptor, Drosophila 5-HT 2b The functional activity of 5-HT receptors was determined in an in vitro expression system. 16 Co-expression (activation of 5-HT2 receptors will directly lead to an increase in calcium ion concentration, without the need to couple G proteins in the in vitro expression system) and applying different concentrations of 5-HT to verify receptor function.

[0040] 2) The above receptors were co-expressed with G protein Gα16 in HEK293T cells (activation of 5-HT2 receptors will directly lead to an increase in calcium ion concentration without coupling with G protein), different concentrations of the plant-derived compounds to be tested were applied to reaction system A, and the calcium ion flow of 5-hydroxytryptamine receptors within 180° was measured to determine the in vitro receptors of the plant-derived compounds to be tested;

[0041] 3) In HEK293T cells, the above receptors were combined with G protein Gα 16 Co-expression (activation of 5-HT2 receptors will directly lead to an increase in calcium ion concentration without coupling with G proteins), add different concentrations of endogenous agonist 5-hydroxytryptamine and 100μM of the plant-derived compound to be tested in reaction system B, measure the inhibition of the endogenous response of the plant-derived compound to be tested on the 5-hydroxytryptamine receptor within 180 seconds, and determine the in vitro receptor of the plant-derived compound to be tested;

[0042] 4) Through bioassay, a mutant insect strain that is resistant to the plant-derived compound to be tested is obtained, and its in vivo receptor is determined at the genetic level. Combined with the in vitro receptor determined at the pharmacological level in step 2) or 3), the target of the plant-derived compound to be tested is finally determined.

[0043] Implementation Cases:

[0044] Expression of functional insect 5-HT receptors in vitro

[0045] (1) Construct an expression plasmid containing the Drosophila melanogaster 5-HT receptor.

[0046] Plasmid information is shown in Table 1:

[0047]

[0048] (2) HEK293T cells were used to transiently express the 5-HT receptor of Drosophila. The cells were cultured on a 60 mm culture dish and placed in an incubator at 37°C and 5% CO2. When the cell density was 70%, the 5-HT receptor plasmid was transfected with the G protein plasmid Gα using the liposome transfection reagent X-tremeGENE™ HP DNA. 16 co-transfected into HEK293T cells. 2a / b The exception is the expression plasmid, which is G q Endogenously coupled to the protein, it can be directly activated to increase intracellular calcium levels. Gα 16 G i / G s Receptor-coupled conversion to phospholipase C pathway.

[0049] Detection of insect 5-HT receptor functional activity

[0050] (1) Configuration of an in vitro expression system, including candidate insect 5-HT receptors with physiological functions expressed by an in vitro eukaryotic system, 5-HT, Hank's balanced salt solution, calcium ion indicator and polyoxyethylene polyoxypropylene ether. The specific steps of component configuration are as follows:

[0051] a. Prepare Hank's balanced salt solution, which includes: 152 mM NaCl, 5.4 mM KCl, 0.8 mM CaCl2, 1.8 mM MgCl2, 5.5 mM glucose and 10 mM HEPES (pH = 7.4).

[0052] b. Prepare calcium ion indicator: dissolve the substance in Hank's buffer so that the prepared solution contains a final concentration of 2 μmol / L calcium ion indicator Fura 2-AM and 0.05% polyoxyethylene polyoxypropylene ether.

[0053] c. Configuration of 5-HT: In a 96-well plate, dilute 5-HT to a 5-fold reaction concentration to form a 5-HT reaction concentration gradient, such as (0.05 nM, 0.5 nM, 5 nM, 50 nM, 500 nM, 5 μM, 50 μM, 500 μM).

[0054] (2) Detection of the functional activity of insect 5-hydroxytryptamine receptors. The transfected cells were cultured in a 96-well plate, washed three times with Hank's balanced salt solution, and incubated for 60 minutes with 50 μL of calcium ion indicator. After incubation, the cells were washed three times with Hank's balanced salt solution, 200 μL of buffer solution was added to each well, and the cells were incubated in an incubator for 15 minutes. The corresponding concentration of 5-hydroxytryptamine was added to each well, and the calcium ion level within 180 seconds was detected using the 340 / 380 nm excitation wave channel and the 510 nm emission wave channel. The half-maximal effect concentration (EC) was calculated by fitting the dose-response curve. 50 The value is the concentration that can cause 50% of the maximum effect of insect serotonin receptors.

[0055] The dose curve of 5-HT activating Drosophila 5-HT receptors is shown in the figure below. Figure 1 As shown, Figure 1 AE in Drosophila 5-HT 1a , 5-HT 1b , 5-HT 2a , 5-HT 2b and the dose curve of 5-HT7 receptor. 5-HT can activate the 5-HT receptor of Drosophila, indicating that the Drosophila 5-HT receptor expressed in vitro functions normally.

[0056] Inhibition experiment of lycopene on insect 5-hydroxytryptamine receptors

[0057] (1) Reaction system B is prepared, including gramine, candidate insect 5-hydroxytryptamine receptor with physiological function expressed by in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, calcium ion indicator and polyoxyethylene polyoxypropylene ether. The specific steps of component preparation are as follows:

[0058] a. Prepare Hank's balanced salt solution, which includes: 152 mM NaCl, 5.4 mM KCl, 0.8 mM CaCl2, 1.8 mM MgCl2, 5.5 mM glucose and 10 mM HEPES (pH = 7.4).

[0059] b. Prepare calcium ion indicator: dissolve the substance in Hank's buffer so that the prepared solution contains calcium ion indicator Fura 2-AM with a final concentration of 2 μmol / L and 0.05% polyoxyethylene polyoxypropylene ether.

[0060] c. 5-HT configuration: In a 96-well plate, dilute 5-HT to a 10-fold reaction concentration to form a 5-HT reaction concentration gradient, such as (0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM).

[0061] d. Preparation of gramine: Prepare 1 mM gramine at 10 times the actual detection concentration and mix it with the prepared 5-hydroxytryptamine at various concentrations.

[0062] (2) Detection of the inhibition of insect 5-HT receptor response to 5-HT by gramineous lycopersicum. The transfected cells were cultured in a 96-well plate, washed three times with Hank's balanced salt solution, and incubated for 60 minutes with 50 μL of calcium ion indicator. After incubation, the cells were washed three times with Hank's balanced salt solution, 200 μL of buffer solution was added to each well, and the cells were incubated in an incubator for 15 minutes. A mixture of 5-HT and a fixed concentration of gramineous lycopersicum was added to each well, and the calcium ion level within 180 seconds was detected using the 340 / 380 nm excitation wave channel and the 510 nm emission wave channel. The half-maximal effect concentration (EC) was calculated by fitting the dose-response curve. 50 The value is the concentration that can cause 50% of the maximum effect of insect serotonin receptors.

[0063] Luzhulin inhibits the agonist activity of 5-HT on various 5-HT receptors in Drosophila Figure 2 As shown, Figure 2 AE in the experiment was 100 μM rutin to inhibit the 5-HT 1a , 5-HT 1b , 5-HT 2a , 5-HT 2b and 5-HT7 receptor agonist activity. 1b and 5-HT 2a After applying 100 μM rutin, its ability to respond to the endogenous agonist 5-HT agonist activity was significantly reduced, indicating that in vitro rutin can antagonize 5-HT 1b and 5-HT 2a Receptors affect its activity and determine 5-HT 1b and 5-HT 2a The receptor is the in vitro receptor for gramine.

[0064] Bioassay of gramine against Drosophila melanogaster

[0065] (1) Prepare fruit flies for testing, including mutant 5-HT 1a Gal4 , 5-HT 1b Gal4 , 5-HT 2a Gal4 , 5-HT 2b Gal4 , 5-HT7 Gal4 , 5-HT 1b ΔⅢ-Ⅴ , wild type w 1118, select 3-5 day old female fruit flies, 10 per fruit fly tube, 3 tubes per group.

[0066] (2) Preparation of sucrose water for feeding. Dissolve sucrose in deionized water to prepare a 5% sucrose aqueous solution. Dilute 1M gramine with sucrose water to the reaction concentration, such as (0mM, 1mM, 2.5mM, 5mM, 7.5mM, 10mM), and add it to the absorbent cotton;

[0067] (3) Feeding sucrose water. The prepared female fruit flies of each strain were transferred to the fruit fly tube containing sucrose water and fed for 3 days. The number of deaths was then counted and the half-lethal concentration (LC) was calculated by fitting the dose-response curve. 50 value.

[0068] Results Figure 3 The death dose curve of 5-HT mutant Drosophila fed with rutin indicated that 5-HT 1b The loss of the receptor or the incompleteness of its functional domain leads to its resistance to rutin, which determines the 5-HT 1b The receptor is its in vivo receptor. Based on the results of in vitro receptor identification, 5-HT 1b Receptors are the targets of gramine.

[0069] Although the present invention has been disclosed according to specific implementation cases of the present invention, it should be understood that the present invention has additional modifications, and the present invention application is intended to cover generally follow the principles of the present invention, including public technologies or commonly used technical means that do not belong to the scope of the present invention but belong to the field to which the present invention belongs, which can be applied to any variation, use or change of the essential features described above and listed in the scope of the appended claims.

[0070] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to the above embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for identifying a target of a plant-derived compound that acts on an insect 5-hydroxytryptamine receptor, characterized in that: include: 1) Provide functional insect 5-HT receptors expressed in vitro and measure the functional activity of insect 5-HT receptors in the in vitro expression system; 2) Determine the agonistic effect of the plant-derived compound on the insect 5-hydroxytryptamine receptor of step 1). If the insect 5-hydroxytryptamine receptor can be directly activated by the plant-derived compound in reaction system A, it is determined to be the in vitro receptor of the plant-derived compound; 3) Determine the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1). If the response of the insect 5-hydroxytryptamine receptor to 5-hydroxytryptamine in reaction system B is inhibited by the plant-derived compound to be tested, then it is determined to be the in vitro receptor of the plant-derived compound to be tested; 4) Through bioassay, a mutant insect strain resistant to the plant-derived compound to be tested is obtained, and its in vivo receptor is determined. Combined with the in vitro receptor determined in step 2) or 3), the target of the plant-derived compound to be tested is determined.

2. The method according to claim 1, characterized in that: The in vitro expression system of step 1) comprises: a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

3. The method according to claim 1 or 2, characterized in that: The method for determining the functional activity of insect 5-hydroxytryptamine receptors in step 1) is as follows: in an in vitro expression system, an agonist 5-hydroxytryptamine is applied, which has an obvious calcium ion flow process within 180 seconds.

4. The method according to claim 3, characterized in that: The method for determining the agonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1) in step 2) is as follows: applying equal concentrations of the plant-derived compound to be tested and 5-hydroxytryptamine, both of which have obvious calcium ion flow processes within 180 seconds, and the reaction system A comprises: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

5. The method according to claim 4, characterized in that: The method for determining the antagonistic effect of the plant-derived compound to be tested on the insect 5-hydroxytryptamine receptor of step 1) in step 3) is: applying a fixed high concentration of the plant-derived compound to be tested while applying 5-hydroxytryptamine, which can significantly inhibit its response to 5-hydroxytryptamine within 180 seconds, and the reaction system B includes: the plant-derived compound to be tested, a candidate insect 5-hydroxytryptamine receptor with physiological function expressed by an in vitro eukaryotic system, 5-hydroxytryptamine, Hank's balanced salt solution, a calcium ion indicator and polyoxyethylene polyoxypropylene ether.

6. The method according to claim 1 or 2 or 4 or 5, characterized in that: If the result of either step 2) or 3) is sufficient to determine the in vitro receptor of the plant-derived compound to be tested, the other step does not need to be performed.

7. The method according to claim 6, characterized in that: The mutant insect strain in step 4) is a strain that completely loses the function of 5-hydroxytryptamine receptors.

8. The method according to claim 7, characterized in that: The target of the plant-derived compound to be tested in step 4) refers to the receptor that is consistent with the in vitro receptor determined in step 2) or 3) and the in vivo receptor determined in step 4).

9. The method according to claim 1 or 2 or 4 or 5 or 7 or 8, characterized in that: The insects are agricultural pests, horticultural pests, disease-carrying insects or parasites including the model organism Drosophila melanogaster.

10. The method according to claim 9, characterized in that: The plant-derived compound to be tested is an organic small molecule, an inorganic small molecule, a polysaccharide, a peptide, a protein, a nucleic acid or any one or more combinations thereof.

Images