Method and sensor for detecting sex pheromone based on insect sex pheromone receptor

Abstract

The invention provides a method and a sensor for detecting sex pheromones based on insect sex pheromone receptors, and belongs to the field of pest population monitoring and biosensors. According to the method, on the basis of specific recognition of a spodoptera frugiperda sex pheromone receptor SfruOR13 / Orco on sex pheromone Z-9-tetradecyl acetate, ion flow on the two sides of a membrane is generated after the receptor is activated, target sex pheromone is detected through current change, and the sensor and the device are provided on the basis of the method. According to the sensor and the device, the insect sex pheromone receptor protein is used as a sensitive element, and the design of automatic sampling and low-speed steady flow is adopted, so that the flexible biological sensitive element can be effectively protected, and a standardized and repeatable automatic environment is provided for biological sensing detection. The method is efficient, simple and high in operability, the device is simple in structure and convenient to operate, and detection of the insect sex pheromone can be achieved without expensive large experimental instruments and complex experimental operation steps.

Description

Technical Field

[0001] This invention relates to the fields of pest population monitoring and biosensors, and in particular to a method and sensor for detecting sex pheromones based on insect sex pheromone receptors. Background Technology

[0002] In the field of agricultural pest control, precise monitoring, early warning and forecasting, and real-time tracking are key means of effective pest control. Especially for invasive pests, migratory pests, and boring pests, early detection and control can effectively improve control efficiency and significantly reduce control costs and difficulty. Among them, the fall armyworm (Spodopterafrugiperda) is a migratory pest under global early warning by the Food and Agriculture Organization of the United Nations, native to tropical and subtropical regions of the Americas. This pest has a wide diet and damages many important food crops such as corn, rice, and wheat. Currently, pest population monitoring methods include radar monitoring, light monitoring, pheromone trap monitoring, and field surveys, but these cannot simultaneously meet the needs of precise monitoring and convenient, efficient, and intelligent field management.

[0003] Insects possess sophisticated and efficient gas molecule detection systems. Male moths can specifically identify and track females of the same species, a process dependent on the specific recognition of sex pheromones by pheromone receptors (PRs). Insect sex pheromone receptors belong to the ordorant receptor (OR) family, and their seven transmembrane domains localize them to the cell membrane, exhibiting high specificity. Insect sex pheromones are composed of several volatile compounds, and the composition and proportions of sex pheromones vary among different insect species, demonstrating interspecies specificity. Based on the specificity and sensitivity of sex pheromones and their receptors, efficient, convenient, and interspecies-specific biosensors can be developed to detect population changes in pests.

[0004] Existing research has shown that by designing and synthesizing specific peptides based on the ligand binding sites of sex pheromone receptors and coupling them to sensors, the monitoring of sex pheromones in pests can be achieved, opening up new directions for sex pheromone receptor sensing research. Field environments are complex, with cross-occurrence of closely related pests, whose sex pheromone components are highly similar, necessitating improvements in the specificity of sex pheromone sensors. Using the full-length protein of the sex pheromone receptor as the sensitive material helps preserve its narrow tuning spectrum. On the other hand, bio-nanopore sensors offer advantages such as single-molecule level, direct, and rapid detection of ligand molecules, and have been successfully applied in single-molecule sequencing and biosensing. Insect olfactory receptors are tetrameric ion channel proteins composed of ligand-specific odorant receptors (Odorant receptor x, ORx) and odorant receptor co-receptors (Orco), possessing the potential to serve as bio-nanopores. However, research on the structure of insect sex pheromone receptors and their downstream signaling pathways is still incomplete, and the following problems remain: (1) whether sex pheromone receptors are also heterotetrameric ion channel proteins; (2) whether the activation and signal transduction of sex pheromone receptors depend on intracellular G proteins. The unresolved problems in insect sex pheromone receptors and their signaling pathways limit the application of these receptors in the field of bio-nanopore sensors, resulting in research on novel sensors based on insect sex pheromone receptors still being in the early exploratory stage. Summary of the Invention

[0005] The purpose of this invention is to provide a method and sensor for detecting sex pheromones based on insect sex pheromone receptors, which can realize the direct detection of insect sex pheromones.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for detecting sex pheromones based on insect sex pheromone receptors. The method comprises: generating an electric current by specifically recognizing the sex pheromone through the insect sex pheromone receptor, and detecting the sex pheromone by the change in the current; wherein the insect sex pheromone receptor is the fall armyworm sex pheromone receptor SfruOR13 / Orco, and the sex pheromone is Z-9-tetradecanoic acid ester.

[0008] Furthermore, the fall armyworm sex pheromone receptor SfruOR13 / Orco is a ligand-gated ion channel protein, and the fall armyworm sex pheromone receptor SfruOR13 / Orco contains two different subunits, namely the insect-specific sex pheromone receptor SfruOR13 and the insect odor receptor co-receptor Orco.

[0009] In a second aspect, the present invention provides a sensor for detecting sex pheromones prepared based on the method described in the first aspect. The sensor includes two chambers containing an electrolyte solution, the two chambers being separated by a separator with micropores. Each chamber integrates an electrode for detecting current changes caused by activation of insect sex pheromone receptors. A phospholipid bilayer containing the fall armyworm sex pheromone receptor SfruOR13 / Orco is constructed at the micropores.

[0010] Thirdly, the present invention provides an apparatus for detecting sex pheromones based on the sensor described in the second aspect, the apparatus comprising: a sample introduction system for delivering a sample to be tested to a detection area; a detection system comprising a sensor containing a phospholipid bilayer of the fall armyworm sex pheromone receptor SfruOR13 / Orco for receiving and analyzing the sex pheromone; and a sample discharge system for discharging the tested sample.

[0011] Fourthly, the present invention provides an application of the above-mentioned sensor and device in detecting insect sex pheromones, wherein the insect sex pheromone is Z-9-tetradecanoic acid ester.

[0012] The beneficial effects of this invention are: This invention provides a method for detecting sex pheromones based on insect sex pheromone receptors and a biosensor and device that can identify insect sex pheromones. This invention utilizes a ligand-gated ion channel protein, specifically the fall armyworm sex pheromone receptor SfruOR13 / Orco, and prepares a sensor and device based on the specific recognition of sex pheromones by this receptor.

[0013] The sensor and device prepared by this invention have three main advantages: (1) Using the insect pheromone receptor SfruOR13 / Orco with ligand-gated ion channel activity as the sensitive element, the direct detection and signal conversion of the target pheromone molecule can be achieved by utilizing the specific recognition of sex pheromones by the insect pheromone receptor; (2) The detection process can be automated and standardized through integrated design and low-speed steady flow control, effectively protecting the biological sensitive element; (3) It does not rely on large-scale experimental instruments, the operation steps are simple, and it has the potential to detect the dynamic changes of field pest populations. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1The interaction diagram of the specific pheromone receptor SfruOR13 and the co-receptor Orco provided in an embodiment of the present invention.

[0016] Figure 2 An immunoblot image for identifying the expression results of the sex pheromone receptor SfruOR13 / Orco according to an embodiment of the present invention;

[0017] Figure 3 An SDS-PAGE image of the purification results of the sex pheromone receptor SfruOR13 / Orco provided in an embodiment of the present invention;

[0018] Figure 4 This is a schematic diagram of an apparatus for detecting the binding process of insect pheromone receptors and pheromones according to an embodiment of the present invention. The apparatus includes a detection chamber-1, silver / silver chloride electrode I-2, silver / silver chloride electrode II-3, a fixing clip-4, a single micron-pore membrane-5, a phospholipid bilayer-6, an insect sex pheromone receptor protein-7, a sample inlet I-8, a sample inlet II-9, a sample inlet III-10, a sample inlet IV-11, a three-way valve-12, a sample inlet peristaltic pump I-13, a sample inlet peristaltic pump II-14, a sample outlet peristaltic pump I-15, a sample outlet peristaltic pump II-16, and a waste liquid tank-17.

[0019] Figure 5 This is a schematic diagram illustrating the coupling mechanism of a sex pheromone receptor and a lipid bilayer on a single microporous membrane, according to an embodiment of the present invention.

[0020] Figure 6 A current signal diagram of Z9-14:OAc detected by a nanopore sensing device based on SfruOR13 / Orco according to an embodiment of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0023] Example 1: Testing the subunit composition of the sex pheromone receptor of the fall armyworm.

[0024] Sex pheromone receptors are membrane proteins on insect olfactory neurons that sense specific sex pheromone molecules and are key molecules for olfactory neurons to recognize extracellular sex pheromone compounds. Previous studies have shown that insect-specific sex pheromone receptors and odorant receptor co-receptors are co-expressed in the same cell, but the mechanisms by which they participate in sex pheromone recognition and signal transduction remain unclear. To clarify whether sex pheromone receptors are heteromeric proteins composed of specific subunits and co-receptor subunits, this example uses the fall armyworm-specific sex pheromone receptor SfruOR13 and the odorant receptor co-receptor Orco as examples, employing co-immunoprecipitation (Co-IP) to detect the structural relationship between SfruOR13 and Orco. Details are as follows:

[0025] The complete coding region nucleic acid sequences of the fall armyworm specific receptor SfruOR13 and co-receptor Orco were retrieved from the NCBI database, with sequence numbers ON310519.1 and ON693217.1, respectively. The gene sequences of the specific receptor SfruOR13 and co-receptor Orco were ligated into the plasmid pIZT / V5-His, respectively. A DNA sequence encoding a 3×Flag tag was added to the 5' end of the SfruOR13 gene, and a DNA sequence encoding a 3×HA tag was added to the 5' end of the Orco gene.

[0026] After sequencing verification, the recombinant plasmid was transfected into Sf9 cells using the transfection reagent Cellfectin II (Gibco, catalog number 10362-100), and three treatments were set up:

[0027] (1) Transfected with recombinant plasmid pIZT / V5-HA-Orco;

[0028] (2) Transfected with recombinant plasmid pIZT / V5-Flag-SfruOR13;

[0029] (3) Co-transfect recombinant plasmids pIZT / V5-HA-Orco and pIZT / V5-HA-Orco.

[0030] Forty-eight hours after transfection, cells were collected. 100 μL of lysis buffer containing protease inhibitors (0.8 µM aprotinin, 2 µg / mL leupeptin, 2 µM pepstatin, and 1 mM PMSF) was mixed with the cells, and the mixture was gently pipetted to ensure adequate contact between the lysis buffer and cells. Lysis was performed on ice for 10 min. After complete lysis, the cells were centrifuged at 14,000 × g at 4°C for 5 min, and the supernatant was collected as the protein sample for Western blotting. 8 μL of anti-Flag magnetic bead suspension was added to each protein sample, and the mixture was incubated overnight at 4°C on a rotary mixer. After incubation, the cells were separated on a magnetic rack for 10 seconds, and the supernatant was removed. 0.5 mL of lysis buffer containing protease inhibitors was added, and the magnetic beads were gently resuspended by pipetting. The cells were separated on a magnetic rack for 10 seconds, and the supernatant was removed. The cells were washed three times with lysis buffer containing protease inhibitors. Add 100 μL of 3× Flag peptide elution buffer to each sample group, mix well, and incubate at 4°C for 2 hours on a rotary mixer. After incubation, separate on a magnetic rack for 10 seconds, and transfer the supernatant to a new centrifuge tube. This contains the eluted Flag-tagged protein and its complex, which is used for Western blotting detection. The results are as follows: Figure 1 As shown, the results of co-transfection with recombinant plasmids pIZT / V5-HA-Orco and pIZT / V5-HA-Orco indicate that SfruOR13 binds to Orco in spatial structure.

[0031] Example 2: Preparation of sex pheromone receptors for fall armyworm.

[0032] This embodiment utilizes a baculovirus-insect cell expression system to express and purify sex pheromone receptors. The gene sequences of the specific sex pheromone receptor SfruOR13 and the co-receptor Orco were ligated into the plasmid pFastBac1, respectively. DNA sequences encoding Flag and His tags were ligated to the 5' end of the SfruOR13 gene, and DNA sequences encoding Flag, Twin-Strep, and GFP tags were ligated to the 5' end of the Orco gene. A TEV protease restriction site was added between the tags and the target protein. These two recombinant plasmids were transformed into DH10Bac competent cells, and recombinant baculoviruses were obtained through blue-white screening. The recombinant baculovirus DNA was extracted. Using Cellfectin II transfection reagent, the obtained SfruOR13 recombinant baculoviruses and Orco recombinant baculovirus DNA were transfected into Sf9 cells. Four days after transfection, the cells were separated from the culture supernatant by centrifugation. The culture supernatant contained recombinant baculovirus and was temporarily stored at 4°C in the dark. Lysis buffer was added to the cell pellet, and the mixture was centrifuged at 14,000 × g at 4°C for 5 minutes. The pellet was then separated from the supernatant and used to detect the expression of SfruOR13 and Orco. The results of Western blotting are shown below. Figure 2 As shown.

[0033] The results showed that both SfruOR13 and Orco were expressed and localized to the cell membrane. Recombinant baculovirus was used to infect Sf9 cells at an MOI of 0.1 for viral amplification, with amplification for 1-2 generations until the viral titer was approximately 1 × 10⁻⁶. 8 pfu / mL. Recombinant baculovirus SfruOR13 and Orco were co-infected with Sf9 cells at MOIs of 7.5 and 2.5, respectively. Cells were collected after 4 days for subsequent protein purification.

[0034] Add dissolution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% LMNG) to the expression cell pellet and incubate at 4°C for 2 hours. Centrifuge at 20,000 × g for 1 hour at 4°C and collect the supernatant. Perform Flag affinity purification, Twin-Strep affinity purification, TEV restriction enzyme digestion to remove the tag, and Superose 6 Increase molecular sieve purification sequentially to obtain the target pheromone receptor protein. The detection results are shown below. Figure 3 As shown, the SEC E2 sample is the purified SfruOR13 and Orco complex.

[0035] Example 3: Constructing a biosensor-based device based on the insect sex pheromone receptor SfruOR13 / Orco.

[0036] The device includes a sample introduction system, a detection system, and a sample dispensing system, such as Figure 4 As shown, it consists of the following parts: detection chamber 1, silver / silver chloride electrode I 2, silver / silver chloride electrode II 3, fixing clip 4, single micron-pore membrane 5, phospholipid bilayer 6, insect sex pheromone receptor 7, sample inlet I 8, sample inlet II 9, sample inlet III 10, sample inlet IV 11, three-way valve 12, sample inlet peristaltic pump I 13, sample inlet peristaltic pump II 14, sample outlet peristaltic pump I 15, sample outlet peristaltic pump II 16, waste liquid tank 17. The biosensor consists of a detection chamber 1, a clamping clip 4, a single microporous membrane 5, a phospholipid bilayer 6, an insect sex pheromone receptor 7, a silver / silver chloride electrode I 2, and a silver / silver chloride electrode II 3. The detection chamber 1 is divided into two chambers by the single microporous membrane 5. The clamping clip 4 is used to fix the single microporous membrane 5. The silver / silver chloride electrode I 2 and the silver / silver chloride electrode II 3 are connected to the detector and used to collect changes in the current signal. A schematic diagram of the coupling mode between the sex pheromone receptor and the lipid bilayer on the single microporous membrane is shown below. Figure 5 As shown.

[0037] A 5 mM Hepes / KOH electrolyte solution (containing 0.8 mM CaCl2, 96 mM NaCl, 2 mM KCl, and 5 mM MgCl2, pH 7.6) was added to injection tank I 8. The three-way valve 12 was rotated to align with injection tank I 8, and the peristaltic pump I 13 was turned on at a perfusion rate of 1 μL / s. After a perfusion time of 50 s, the peristaltic pump I 13 was turned off. A phospholipid bilayer was prepared at the pores of the single microporous membrane 5 using a brush method. The phospholipid used was 20 mg / mL DOPC:DOPE (DOPC to DOPE mass ratio 3:1). The sex pheromone receptor SfruOR13 / Orco complex protein prepared in Example 2 was diluted with 5 mM Hepes / KOH electrolyte solution to prepare a protein dilution buffer with a final concentration of 2 ng / μL. The protein dilution buffer was added to injection tank II 9, and the three-way valve 12 was rotated to align with injection tank II 9. The peristaltic pump I 13 was turned on at a perfusion rate of 1 μL / s, and the pump was turned off after 50 s of perfusion. Incubation was performed for 10-15 minutes to allow the sex pheromone receptor to insert into the lipid bilayer membrane. The coupling mechanism between the sex pheromone receptor and the lipid bilayer on the single microporous membrane 5 is detailed in [link to documentation]. Figure 5 Set a voltage of +50 mV, and record the current signal as a function of time under a sampling frequency of 5 kHz and a low-pass filter of 1 kHz.

[0038] Example 4: Detection of ligand pair Z9-14:OAc by a biosensor based on insect sex pheromone receptor SfruOR13 / Orco.

[0039] The fall armyworm sex pheromone receptor SfruOR13 / Orco can tune the sex pheromone (Z)-9-tetradecanoic acid ester (Z9-14:OAc). This embodiment utilizes the insect pheromone receptor-based device described in Example 3 to detect the response of the sex pheromone receptor SfruOR13 / Orco to the sex pheromone Z9-14:OAc in a non-cellular environment.

[0040] Dilute DMSO with 5 mM Hepes / KOH electrolyte solution to prepare a 0.1% (v / v) DMSO dilution. Add the DMSO dilution to injection tanks III 10 and IV 11. Rotate the three-way valve 12 to align with injection tank III 10. Turn on peristaltic pumps I 13 and II 14 at a perfusion rate of 0.5 μL / s. After a perfusion time of 10 s, turn off peristaltic pumps I 13 and II 14. The output current fluctuates around 0 pA. Figure 6 As shown. Rotate the three-way valve 12 to align with the sample inlet 18, and turn on the sample inlet peristaltic pumps 13, 14, 15, and 16. The perfusion rate is 1 μL / s. After a perfusion time of 300 s, turn off the sample inlet peristaltic pumps 13, 14, 15, and 16.

[0041] The Z9-14:OAc to be detected was dissolved in DMSO to prepare a 100 mM ligand stock solution. The Z9-14:OAc stock solution was diluted with 5 mM Hpeps / KOH electrolyte solution to prepare a 0.1 mM Z9-14:OAc dilution. This dilution was added to injection tanks III 10 and IV 11. The three-way valve 12 was rotated to align with injection tank III 10. Peristaltic pumps I 13 and II 14 were turned on at a perfusion rate of 0.5 μL / s. After a perfusion time of 10 s, peristaltic pumps I 13 and II 14 were turned off. A change in the current signal was detected, such as... Figure 6 As shown.

[0042] The results indicate that the fall armyworm sex pheromone receptor SfruOR13 / Orco is a ligand-gated ion channel protein. Upon recognition of the ligand, it opens the ion channel, forming a pathway across the membrane and generating an electric current. Therefore, using the sex pheromone receptor as a sensing element, the binding process between the sex pheromone receptor and the sex pheromone can be detected by changes in current. This invention provides an efficient and reliable method, biosensor, and device for detecting insect sex pheromones, aiming to meet the practical application needs in this field. The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to this invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this invention should be included within the scope of the claims of this invention.

Claims

1. A method for detecting sex pheromones based on insect sex pheromone receptors, characterized in that, The method involves generating an electric current through the specific recognition of sex pheromones by insect sex pheromone receptors, and detecting sex pheromones by changes in this current. The insect sex pheromone receptor is the fall armyworm sex pheromone receptor SfruOR13 / Orco, and the sex pheromone is... Z -9-Tetradecanoic acid ester.

2. The method according to claim 1, characterized in that, The fall armyworm sex pheromone receptor SfruOR13 / Orco is a ligand-gated ion channel protein.

3. The method according to claim 2, characterized in that, The fall armyworm sex pheromone receptor SfruOR13 / Orco contains two different subunits: the insect-specific sex pheromone receptor SfruOR13 and the insect odor receptor co-receptor Orco.

4. A sensor for detecting sex pheromones that implements the method of any one of claims 1-3, characterized in that, The sensor includes two chambers containing an electrolyte solution, which are separated by a microporous separator. Each chamber integrates an electrode for detecting current changes caused by activation of insect sex pheromone receptors. A phospholipid bilayer containing the fall armyworm sex pheromone receptor SfruOR13 / Orco is constructed at the micropores.

5. A device for detecting sex pheromones, characterized in that, The device includes: The sample delivery system is used to transport the sample to be tested to the detection area; A detection system comprising a sensor for detecting pheromones as described in claim 4, for receiving and analyzing the pheromones; The sample dispensing system is used to dispense the tested samples.

6. The application of the sensor for detecting sex pheromones according to claim 4 or the device according to claim 5 in detecting insect sex pheromones.

7. The application according to claim 6, characterized in that, The insect sex pheromone mentioned is Z -9-Tetradecanoic acid ester.

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