Identification method of bombyx mori sulfur kinin receptor

Abstract

The invention discloses an identification method of a bombyx mori sulfur kinin receptor, and relates to the technical field of sulfur kinin receptor identification. IP3 and Ca < 2 + > in cells are rapidly increased due to stimulation of the sulfur kinin, phosphorylation of ERK1 / 2 is remarkably enhanced, the process is sensitive to a G alpha q specific inhibitor, and food consumption and average weight of silkworms are obviously reduced when the silkworms are treated by the synthetic sulfur kinin; when the synthetic thiokinin is injected into the bombyx mori, the trehalose level in hemolymph is remarkably improved, and the effect is remarkably reduced through BNGR-A9dsRNA pretreatment; the sulfur kinin / BNGR-A9 signal channel is used as a key regulator for feeding behavior and hemolymph trehalose homeostasis in bombyx mori, and the effect of the sulfur kinin / BNGR-A9 signal channel in negative control of food intake and positive regulation of energy balance is highlighted.

Description

Technical Field

[0001] The present invention relates to the technical field of the identification of tachykinin receptors, and particularly relates to a method for identifying the tachykinin receptor of Bombyx mori. Background Art

[0002] The regulation of the feeding behavior of Bombyx mori has a crucial impact on its silk production and population health. However, the molecular mechanism of feeding regulation in this species is not yet fully understood. Tachykinin, an insect neuropeptide similar in function to gastrin / cholecystokinin in vertebrates, has been demonstrated to be involved in the regulation of feeding behavior in a variety of insects, including Schistocerca gregaria, Tribolium castaneum, and Dendroctonus armandi. In insects, tachykinin has been reported to enhance the release of major digestive enzymes in the intestine, increase the heart contraction frequency of Drosophila, and regulate various physiological processes such as odor preference, locomotion, and metabolism. However, the best-known function of tachykinin is to regulate feeding behavior as a satiety factor. In a variety of insect models, the injection of tachykinin has been demonstrated to significantly reduce food intake.

[0003] The tachykinin and its receptor of Bombyx mori and the possible mechanism involved in feeding regulation still need to be further explored. For this reason, we propose a method for identifying the tachykinin receptor of Bombyx mori. Summary of the Invention

[0004] The purpose of the present invention is: to solve the problems mentioned in the above background art, the present invention provides a method for identifying the tachykinin receptor of Bombyx mori.

[0005] The present invention specifically adopts the following technical solutions to achieve the above purpose:

[0006] A method for identifying the tachykinin receptor of Bombyx mori, comprising the following steps:

[0007] Step 1, Material preparation: Select Bombyx mori for feeding behavior determination and qRT-PCR analysis;

[0008] Step 2, Molecular cloning and plasmid construction: Obtain the entire coding region of BNGR-A9 by PCR and clone it into the pEGFP-N1 and pCMV-Flag vectors, and generate the Flag-BNGR-A9 construct for functional testing in functional testing;

[0009] Step 3, Cell culture and transfection: Culture HEK293 cells and BmN cells;

[0010] Step 4, CRE-driven cAMP luciferase assay: HEK293 and BmN cells were transfected with different Bombyx receptors, along with the corresponding CRE reporter plasmid 47. HEK293 cells and BmN cells were transfected with the CRE reporter plasmid 47. When the cell concentration reached 90 - 95%, they were treated with different concentrations, and after culturing for 4 hours, the cells were lysed and the luciferase activity was measured using a luciferase kit;

[0011] Step 5, Measurement of intracellular calcium: To monitor the changes in intracellular calcium, Fura-2AM was used as a calcium indicator. HEK293 cells stably expressing Flag-BNGR-A9 were exposed to different concentrations of Bombyx neuropeptides and measured using a fluorescence spectrometer. The calcium level was determined by the ratio of the fluorescence excitation value at 340 nm wavelength to the fluorescence excitation value at 380 nm wavelength;

[0012] Step 6, Internalization assay: HEK293 cells stably expressing BNGR-A9-EGFP were seeded onto coverslips and cultured overnight under normal growth conditions. The cells were starved for 1 hour with fresh serum-free DMEM and treated with 100 nM BmsSK or BmnsSK. After incubating for 30 minutes, the cells were rinsed three times with ice-cold PBS, then rinsed with cold water, treated with 3% paraformaldehyde for 10 minutes at room temperature, and photographed using a fluorescence microscope;

[0013] Step 7, Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR): Using a first-strand cDNA synthesis kit, it was synthesized in a 20 μl reaction using oligo(dT)18 primers and 1 μg of total RNA template. For qRT-PCR analysis of the bngr-a9 and sk genes, SYBR Ex Taq Premix was used. Melting curve analysis was further performed at the end of the PCR cycle to confirm the specificity of the primers, and relative quantification was carried out by the 2-ΔΔCT comparison method;

[0014] Step 8, In vitro dsRNA synthesis and injection: Double-stranded RNA was synthesized using the MEGAscript RNAi T7 Kit. The template for in vitro transcription was generated by PCR with primers containing the T7 polymerase promoter sequence. In each 20 μl reaction, 1 μg of the PCR product was added as a template. After transcription, the reaction mixture was purified by phenol / chloroform extraction and ethanol precipitation. The resulting dsRNA product was diluted with nuclease-free water to reach the required concentration;

[0015] Step 9, determination of feeding behavior: Inject 5 μg of dsRNA / BNGR-A9 or dsRNA / EGFP into the abdomen of larvae using a 10-μl microsyringe to knock out BNGR-A9. For silkworms treated and untreated with dsRNA, inject synthetic SK peptide (final concentration 10 nM) every day and rear them under standard conditions. Measure the larval body weight and food consumption every other day until pupation.

[0016] Step 10, determination of trehalose in hemolymph: Cool and anesthetize silkworm larvae on ice, then collect hemolymph from the abdominal incision using a micropipette. Add approximately 5 mg of phenylthiourea (final concentration 2.5% w / v) to the hemolymph in a 1.5-ml Ep tube to inhibit blood clotting, then centrifuge at 12,000 rpm for 10 minutes at 4°C to precipitate particles. Pretreat the hemolymph with trehalase, and subsequently, use a glucose oxidase-peroxidase kit to evaluate the sugar concentration in the hemolymph and determine trehalose.

[0017] Step 11, ERK phosphorylation and immunoblotting: Expose HEK293 cells with Flag-BNGR-A9 to 100 nM BmsSK for different durations, then lyse them using RIPA lysis buffer. In in vivo and in vitro experiments, treat the brains of 5th instar silkworm larvae with BmsSK (100 nM) and pretreat with a Gαq inhibitor for 30 minutes. After treatment, the samples are lysed, homogenized, and the protein concentration is determined using a BCA protein assay kit. When performing Western blot analysis, use an equal amount of protein lysate, and use rabbit monoclonal antibodies against total ERK and phosphorylated ERK provided by the protein primary antibody.

[0018] Step 12, data analysis: Use GraphPad Prism for data analysis. The results are expressed as mean ± SEM, and statistical significance is evaluated by Student's t-test or two-way ANOVA with multiple comparisons. A p-value less than 0.05 is considered significant. Generate the dose-response non-linear curve analysis, and use Adobe Photoshop to process the images to ensure consistent brightness and contrast settings under all conditions.

[0019] Furthermore, the silkworms selected in the material preparation are of the silkworm strain P50, and the larvae are cultured on fresh mulberry leaves at 25°C under standard conditions.

[0020] Furthermore, the molecular cloning and plasmid construction include CRE-luciferase, calcium mobilization, and ERK phosphorylation, while BNGR-A9-EGFP is used for receptor visualization. The BNGR-A9 gene is inserted into the pIZT / V5-His vector, and all constructs are sequenced by DNA.

[0021] Furthermore, in the cell culture and transfection, HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2. The bovine serum, the incubator temperature was 37°C, with 5% CO2. BmN cells were cultured in TC100 insect medium containing 10% fetal bovine serum in an incubator at 28°C.

[0022] Furthermore, in the measurement of intracellular calcium, cells were pretreated with U73122, FR900359, or KT5720 for 30 minutes before the start of detection. Experiments involving BmN cells expressing pIZT-BNGR-A9 were conducted at 28°C, and HBSS was replaced with HBM.

[0023] Furthermore, in the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the Ct value of the target gene was normalized to the geometric mean of the Ct values of the reference genes GAPDH and actin A3, and the experiments were performed in triplicate.

[0024] Furthermore, in the in vitro dsRNA synthesis and injection, the final volume was 2 - 5 μl.

[0025] Furthermore, in the feeding behavior assay, 20 synchronized P50 larvae were used in each trial, and the silkworm larvae were fed with mulberry leaves three times a day after injection.

[0026] Furthermore, in the ERK phosphorylation and immunoblotting, the brain of the 5th instar silkworm larvae was treated for 1, 2, or 8 hours.

[0027] Furthermore, in the data analysis, all images and data provided were the results of at least three independent experiments.

[0028] The beneficial effects of the present invention are as follows:

[0029] 1. The stimulation of kinin in the present invention results in a rapid increase in intracellular IP3 and Ca 2+ and significantly enhances the phosphorylation of ERK1 / 2, and this process is sensitive to the Gαq-specific inhibitor. Treatment of silkworms with synthetic kinin was found to significantly decrease both food consumption and average body weight of silkworms.

[0030] 2. Injecting synthetic kinin into silkworms in the present invention significantly increases the trehalose level in the hemolymph, and this effect is significantly reduced by pretreatment with BNGR-A9 dsRNA.

[0031] 3. The kinin / BNGR-A9 signaling pathway in the present invention, as a key regulator of feeding behavior and hemolymph trehalose homeostasis in silkworms, highlights its role in the negative control of food intake and the positive regulation of energy balance.

[0032] 4. The present invention cloned and expressed the Bombyx mori SK receptor BNGR-A9, including a CRE-driven luciferase reporter system for cAMP measurement, a Fura-2AM-based calcium mobilization assay, and a receptor endocytosis assay, to determine BNGR-A9 as the specific receptor for Bombyx mori SKs. Further support from expression analysis and in vivo experiments indicates that the SK / BNGR-A9 signaling pathway plays a key role in regulating food intake and hemolymph trehalose levels in Bombyx mori. Detailed implementation manners

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0034] The present invention provides a method for identifying a Bombyx mori tachykinin receptor, including the following steps:

[0035] Step 1. Material preparation: Select Bombyx mori for feeding behavior determination and qRT-PCR analysis;

[0036] Step 2. Molecular cloning and plasmid construction: Obtain the entire coding region of BNGR-A9 by PCR and clone it into the pEGFP-N1 and pCMV-Flag vectors, and generate the Flag-BNGR-A9 construct for functional testing in functional tests;

[0037] Step 3. Cell culture and transfection: Culture HEK293 cells and BmN cells;

[0038] Step 4. CRE-driven cAMP luciferase assay: HEK293 and BmN cells were transfected with different Bombyx receptors, and the corresponding CRE reporter plasmid 47 was transfected into HEK293 cells and BmN cells. When the cell concentration reached 90-95%, they were treated with different concentrations, and after culturing for 4 hours, the cells were lysed and the luciferase activity was measured using a luciferase kit;

[0039] Step 5. Measurement of intracellular calcium: Monitor the change of intracellular calcium. Using Fura-2AM as a calcium indicator, HEK293 cells stably expressing Flag-BNGR-A9 were exposed to different concentrations of Bombyx mori neuropeptides and measured using a fluorescence spectrometer. The calcium level was determined by the ratio of the fluorescence excitation value at 340 nm wavelength to the fluorescence excitation value at 380 nm wavelength;

[0040] Step 6, internalization assay: HEK293 cells stably expressing BNGR-A9-EGFP were seeded onto coverslips and cultured overnight under normal growth conditions. The cells were starved for 1 hour with fresh serum-free DMEM and treated with 100 nM BmsSK or BmnsSK. After incubation for 30 minutes, the cells were rinsed three times with ice-cold PBS, then rinsed with cold water, fixed with 3% paraformaldehyde at room temperature for 10 minutes, and photographed using a fluorescence microscope.

[0041] Step 7, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR): Using a first-strand cDNA synthesis kit, cDNA was synthesized in a 20 μl reaction using oligo(dT)18 primers and 1 μg of total RNA template. qRT-PCR analysis of the bngr-a9 and sk genes was performed in a 20 μl reaction using SYBR Ex Taq Premix. Melting curve analysis was further performed at the end of the PCR cycle to confirm the specificity of the primers. Relative quantification was performed by the 2-ΔΔCT method.

[0042] Step 8, in vitro dsRNA synthesis and injection: Double-stranded RNA was synthesized using the MEGAscript RNAi T7 Kit. The template for in vitro transcription was generated by PCR using primers containing the T7 polymerase promoter sequence. In each 20 μl reaction, 1 μg of the PCR product was added as a template. After transcription, the reaction mixture was purified by phenol / chloroform extraction and ethanol precipitation. The resulting dsRNA product was diluted with nuclease-free water to the desired concentration.

[0043] Step 9, feeding behavior assay: 5 μg of dsRNA / BNGR-A9 or dsRNA / EGFP was injected into the abdomen of larvae using a 10 μl microsyringe to knock out BNGR-A9. The dsRNA-treated and untreated silkworms were injected with synthetic SK peptide (final concentration 10 nM) daily and reared under standard conditions. The larval weight and food consumption were measured every other day until pupation.

[0044] Step 10, determination of trehalose in hemolymph: Silkworm larvae were cooled and anesthetized on ice, and then hemolymph was collected from the abdominal incision using a micropipette. Approximately 5 mg of phenylthiourea (final concentration 2.5% w / v) was added to the hemolymph in a 1.5 ml Ep tube to inhibit coagulation, and then centrifuged at 12,000 rpm for 10 minutes at 4°C to precipitate particulate matter. The hemolymph was pretreated with trehalase, and subsequently, the sugar concentration in the hemolymph was evaluated using a glucose oxidase-peroxidase kit to determine trehalose.

[0045] Step 11, ERK phosphorylation and immunoblotting: HEK293 cells with Flag-BNGR-A9 were exposed to 100 nM BmsSK for different durations, and then lysed using RIPA lysis buffer. In in vivo and in vitro experiments, the brains of 5th instar silkworm larvae were treated with BmsSK (100 nM), and a Gαq inhibitor was pre-treated for 30 minutes. After treatment, the samples were lysed and homogenized, and the protein concentration was measured using a BCA protein assay kit. When performing Western blot analysis, an equal amount of protein lysate was used, and rabbit monoclonal antibodies against total ERK and phosphorylated ERK were provided by the protein primary antibody;

[0046] Step 12, Data analysis: GraphPad Prism was used for data analysis, and the results were expressed as mean ± SEM. Statistical significance was evaluated by Student's t-test or two-way analysis of variance with multiple comparisons. A p-value less than 0.05 was considered significant. It was generated by dose-response non-linear curve analysis, and Adobe Photoshop was used to process the images to ensure consistent brightness and contrast settings under all conditions.

[0047] In this example, preferably, the silkworms selected in the material preparation are of the silkworm strain P50, and the larvae are cultured on fresh mulberry leaves at 25 °C under standard conditions.

[0048] In this example, preferably, the molecular cloning and plasmid construction include CRE-luciferase, calcium mobilization, and ERK phosphorylation, while BNGR-A9-EGFP is used for receptor visualization. The BNGR-A9 gene was inserted into the pIZT / V5-His vector, and all constructs were sequenced by DNA sequencing.

[0049] In this example, preferably, in the cell culture and transfection, HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a humidified incubator at 37 °C with 5% CO2. The bovine serum, the incubator temperature was 37 °C, with 5% CO2. BmN cells were cultured in TC100 insect medium containing 10% fetal bovine serum in an incubator at 28 °C.

[0050] In this example, preferably, in the measurement of intracellular calcium, the cells were pre-treated with U73122, FR900359, or KT5720 for 30 minutes before the start of detection. Experiments involving BmN cells expressing pIZT-BNGR-A9 were carried out at 28 °C, and HBSS was replaced with HBM.

[0051] In this example, preferably, in the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the Ct value of the target gene was normalized to the geometric mean of the Ct values of the reference genes GAPDH and actin A3, and the experiment was performed in triplicate.

[0052] In this example, preferably, the final volume in the in vitro dsRNA synthesis and injection is 2 - 5 μl.

[0053] In this example, preferably, 20 synchronized P50 larvae are used in each trial for the feeding behavior assay, and the silkworm larvae are fed with mulberry leaves three times a day after injection.

[0054] In this example, preferably, the brain of the 5th instar silkworm larvae is processed for 1, 2, or 8 hours in the ERK phosphorylation and immunoblotting.

[0055] In this example, preferably, all the images and data provided in the data analysis are the results of at least three independent experiments.

[0056] The working principle and usage process of the present invention: When the device is used, it includes the following steps:

[0057] Step 1. Material preparation: When performing the feeding behavior assay and qRT-PCR analysis, the silkworm strain P50 is used. The larvae are cultured on fresh mulberry leaves at 25 °C under standard conditions.

[0058] Step 2. Molecular cloning and plasmid construction: The entire coding region of BNGR-A9 is obtained by PCR and cloned into the pEGFP-N1 and pCMV-Flag vectors. The Flag-BNGR-A9 construct is generated for functional tests in functional tests, including CRE-luciferase, calcium mobilization, and ERK phosphorylation, while BNGR-A9-EGFP is used for receptor visualization. In addition, for expression in the BmN cell system, the BNGR-A9 gene is inserted into the pIZT / V5-His vector, and all constructs are sequenced by DNA.

[0059] Step 3. Cell culture and transfection: HEK293 cells are cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a humidified incubator at 37 °C with 5% CO2, fetal bovine serum, the incubator temperature is 37 °C, with 5% CO2. BmN cells are cultured in TC100 insect medium (containing 10% fetal bovine serum) in an incubator at 28 °C.

[0060] Step 4, CRE-driven cAMP luciferase assay: HEK293 and BmN cells were transfected with different Bombyx receptors. Among them, HEK293 cells were transfected with Flag-BNGR-A9, -A24, -A32 or A33, and the corresponding CRE reporter plasmid 47. HEK293 cells and BmN cells were transfected with the CRE reporter plasmid 47. When the cell concentration reached 90 - 95%, they were treated with different concentrations. After culturing for 4 hours, the cells were lysed and the luciferase activity was measured using a luciferase kit.

[0061] Step 5, Measurement of intracellular calcium: To monitor the changes in intracellular calcium, Fura-2AM was used as a calcium indicator. HEK293 cells stably expressing Flag-BNGR-A9 were exposed to different concentrations of Bombyx neuropeptides and measured using a fluorescence spectrometer. The calcium level was determined by the ratio of the fluorescence excitation value at 340 nm wavelength to the fluorescence excitation value at 380 nm wavelength. When necessary, the cells were pretreated with U73122, FR900359 or KT5720 for 30 minutes before starting the detection. Experiments involving BmN cells expressing pIZT-BNGR-A9 were carried out at 28 °C, and HBSS was replaced with HBM (HBM: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM Na2HPO4, 5 mM NaHCO3, 10 mM glucose, and 20 mM HEPES-NaOH, CaCl2 (1 mM), pH 6.2).

[0062] Step 6, Internalization assay: HEK293 cells stably expressing BNGR-A9-EGFP were seeded onto coverslips and cultured overnight under normal growth conditions. The cells were starved for 1 hour with fresh serum-free DMEM and treated with 100 nM BmsSK or BmnsSK. After incubating for 30 minutes, the cells were rinsed three times with ice-cold PBS, then rinsed with cold water, and treated with 3% paraformaldehyde at room temperature for 10 minutes, and photographed using a fluorescence microscope.

[0063] Step 7, Quantitative reverse transcription polymerase chain reaction (qRT-PCR): Using the PrimeScript First Strand cDNA Synthesis Kit produced by Takara Bio Inc., Japan, it was synthesized in a 20 μl reaction using oligo(dT)18 primer and 1 μg total RNA template. For the qRT-PCR analysis of bngr-a9 and sk genes, SYBR Ex Taq Premix was used for the qRT-PCR analysis of bngr-a9 and sk genes. At the end of the PCR cycle, melting curve analysis was further performed to confirm the specificity of the primers. Relative quantification was performed by the 2-ΔΔCT comparison method. Specifically, the Ct value of the target gene was normalized to the geometric mean of the Ct values of the reference genes GAPDH and actin A3. The experiment was performed in triplicate.

[0064] Step 8, In vitro dsRNA synthesis and injection: Double-stranded RNA was synthesized using the MEGAscript RNAi T7 Kit. The template for in vitro transcription was generated by PCR with primers containing the T7 polymerase promoter sequence. In each 20 μl reaction, 1 μg of the PCR product was added as a template. After transcription, the reaction mixture was purified by phenol / chloroform extraction and ethanol precipitation. The resulting dsRNA product was diluted with nuclease-free water to reach the desired concentration, and the final volume was 2 - 5 μl.

[0065] Step 9, Feeding behavior assay: Synchronized 5th instar day 1 larvae were selected for the feeding behavior assay. The silkworms were starved for 12 hours to unify the starting level. To knock out BNGR-A9, 5 μg of dsRNA / BNGR-A9 or dsRNA / EGFP was injected into the larval abdomen using a 10 μl microsyringe. For both dsRNA-treated and untreated silkworms, synthetic SK peptide (final concentration 10 nM) was injected daily. Twenty synchronized P50 larvae were used in each trial. After injection, the silkworm larvae were fed sufficient mulberry leaves three times a day and reared under standard conditions. The larval weight and food consumption were measured every other day until pupation.

[0066] Step 10, Determination of trehalose in hemolymph: The silkworm larvae were cooled and anesthetized on ice, and then hemolymph was collected from the abdominal incision using a micropipette. Approximately 5 mg of phenylthiourea (final concentration 2.5% w / v) was added to the hemolymph in a 1.5 ml Ep tube to inhibit clotting, and then centrifuged at 12,000 rpm for 10 minutes at 4°C to precipitate any particulates. To determine trehalose, the hemolymph was pretreated with trehalase. Subsequently, the sugar concentration in the hemolymph was evaluated using a glucose oxidase-peroxidase kit.

[0067] Step 11, ERK phosphorylation and immunoblotting: HEK293 cells with Flag-BNGR-A9 were exposed to 100 nM BmsSK for different durations and then lysed using RIPA lysis buffer. In in vivo and in vitro experiments, the brains of 5th instar silkworm larvae were treated with BmsSK (100 nM) for 1, 2, or 8 hours and pretreated with a Gαq inhibitor for 30 minutes. After treatment, the samples were lysed, homogenized, and the protein concentration was determined using a BCA protein assay kit. When performing Western blot analysis, an equal amount of protein lysate was used, and the total ERK and phosphorylated ERK rabbit monoclonal antibodies were provided by the protein primary antibody.

[0068] Step 12, Data analysis: Data analysis was performed using GraphPad Prism, and the results are expressed as mean ± SEM. Statistical significance was evaluated by Student's t-test or two-way ANOVA with multiple comparisons. A p-value less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). It was generated by dose-response non-linear curve analysis, and images were processed using Adobe Photoshop to ensure consistent brightness and contrast settings under all conditions. All images and data provided are the results of at least three independent experiments.

[0069] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying a thiokinin receptor of silkworm, characterized in that: The steps include: Step 1. Material preparation: Select silkworms for feeding behavior measurement and qRT-PCR analysis; Step 2, molecular cloning and plasmid construction: The entire coding region of BNGR-A9 was obtained by PCR and cloned into pEGFP-N1 and pCMV-Flag vectors, and the Flag-BNGR-A9 construct was generated for functional testing; Step 3, cell culture and transfection: culturing HEK293 cells and BmN cells; Step 4, CRE-driven cAMP luciferase assay: HEK293 and BmN cells were transfected with different Bombyx receptors and the corresponding CRE reporter plasmid 47. HEK293 cells and BmN cells were transfected with CRE reporter plasmid 47. When the cell concentration reached 90-95%, they were treated with different concentrations. After 4 hours of culture, the cells were lysed and the luciferase activity was measured using a luciferase kit; Step 5, measurement of intracellular calcium: To monitor the changes in intracellular calcium, Fura-2AM was used as a calcium indicator, and HEK293 cells stably expressing Flag-BNGR-A9 were exposed to different concentrations of Bombyx mori neuropeptide and measured using a fluorescence spectrometer. The calcium level was determined by the ratio of the fluorescence excitation value at a wavelength of 340 nm to the fluorescence excitation value at a wavelength of 380 nm. Step 6, internalization assay: HEK293 cells stably expressing BNGR-A9-EGFP were seeded on coverslips and cultured overnight under normal growth conditions, starved with fresh serum-free DMEM for 1 h, and treated with 100 nM BmsSK or BmnsSK. After incubation for 30 min, the cells were rinsed three times with ice-cold PBS, then rinsed with cold water, treated with 3% paraformaldehyde at room temperature for 10 min, and photographed with a fluorescence microscope; Step 7, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR): synthesize with a first-strand cDNA synthesis kit using oligo (dT) 18 primer and 1 μg of total RNA template in a 20 μl reaction, perform qRT-PCR analysis of bngr-a9 and sk genes using SYBR Ex Taq Premix, further perform melting curve analysis at the end of the PCR cycle to confirm the specificity of the primers, and perform relative quantification by the 2-ΔΔCT comparison method; Step 8. In vitro dsRNA synthesis and injection: Double-stranded RNA was synthesized using MEGAscriptRNAiT7Kit. The template for in vitro transcription was generated by PCR using primers containing the T7 polymerase promoter sequence. In each 20 μl reaction, 1 μg of PCR product was added as a template. After transcription, the reaction mixture was purified by phenol / chloroform extraction and ethanol precipitation. The obtained dsRNA product was diluted with nuclease-free water to achieve the desired concentration. Step 9, feeding behavior assay: 5 μg dsRNA / BNGR-A9 or dsRNA / EGFP was injected into the abdomen of larvae using a 10 μl microsyringe. BNGR-A9 knockout, dsRNA-treated and untreated silkworms were injected with synthetic SK peptide (final concentration of 10 nM) every day and raised under standard conditions. Larval body weight and food consumption were measured every other day until pupation; Step 10, determination of trihalogenose in hemolymph: placing silkworm larvae on ice to cool and anesthetize, then collecting hemolymph from the abdominal incision with a micropipette, adding about 5 mg of phenylthiourea (final concentration of 2.5% w / v) to the hemolymph in a 1.5 ml Eppendorf tube to inhibit coagulation, then centrifuging at 12,000 rpm for 10 minutes at 4°C to precipitate particles, pre-treating the hemolymph with trihalogenase, and then evaluating the sugar concentration in the hemolymph using a glucose oxidase-peroxidase kit to determine the trihalogenose; Step 11, ERK phosphorylation and immunoblotting: HEK293 cells carrying Flag-BNGR-A9 were exposed to 100 nM BmsSK for different time periods and then lysed using RIPA enzymatic buffer. In in vitro and in vivo experiments, brains of 5th instar silkworm larvae were treated with BmsSK (100 nM) and pretreated with Gαq inhibitor for 30 minutes. After treatment, samples were lysed and homogenized, and protein concentrations were determined using a BCA protein kit. When performing Western blot analysis, equal amounts of protein lysate were used, and total ERK and phosphorylated ERK rabbit monoclonal antibodies were provided by protein primary antibodies; Step 12. Data analysis: GraphPad Prism was used for data analysis. The results were expressed as mean ± SEM. Statistical significance was assessed by Student's t-test or two-way analysis of variance with multiple comparisons. A p value less than 0.05 was considered significant. The data were generated by dose-response nonlinear curve analysis. Adobe Photoshop was used to process images, ensuring consistent brightness and contrast settings under all conditions.

2. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: The silkworms used in the material preparation were selected from the silkworm strain P50, and the larvae were cultured on fresh mulberry leaves at 25°C under standard conditions.

3. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: The molecular cloning and plasmid construction included CRE-luciferase, calcium mobilization and ERK phosphorylation, while BNGR-A9-EGFP was used for receptor visualization. The BNGR-A9 gene was inserted into the pIZT / V5-His vector, and all constructs were sequenced by DNA.

4. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: In the cell culture and transfection, HEK293 cells were cultured in Dulbecco's modified glucose medium containing 10% fetal bovine serum in a humidity incubator at 37°C and 5% CO2, and BmN cells were cultured in TC100 insect culture medium containing 10% fetal bovine serum in an incubator at 28°C.

5. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: In the measurement of intracellular calcium, cells were pretreated with U73122, FR900359 or KT5720 for 30 minutes before the start of the assay. Experiments involving BmN cells expressing pIZT-BNGR-A9 were performed at 28°C, and HBSS was replaced with HBM.

6. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: In the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the Ct value of the target gene was normalized to the geometric mean of the Ct values ​​of the reference genes GAPDH and actin A3, and the experiment was performed in triplicate.

7. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: The final volume in the in vitro dsRNA synthesis and injection was 2-5 μl.

8. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: Twenty synchronized P50 larvae were used in each test in the feeding behavior assay, and the silkworm larvae were fed mulberry leaves three times a day after injection.

9. The method for identifying a silkworm thiokinin receptor according to claim 1, characterized in that: Brains from 5th instar silkworm larvae were processed for 1, 2, or 8 hours for the ERK phosphorylation and immunoblotting.

10. The method for identifying the silkworm thiokinin receptor according to claim 1, characterized in that: All images and data presented in the data analysis are the results of at least three independent experiments.