Tomato leafminer common odor binding protein tabsgobp2 and application of coding gene thereof

Abstract

The application discloses a common odor binding protein of tomato leafminer and application of a coding gene thereof, and belongs to the technical field of molecular biology. The application clones a coding gene of the common odor binding protein of tomato leafminer from the tomato leafminer, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2, and the amino acid sequence of the TabsGOBP2 protein encoded by the coding gene is shown as SEQ ID NO. 1. Experimental results show that the TabsGOBP2 protein has an important role in specific identification of sex pheromones of the tomato leafminer, and can be used for identification of active components of odor compounds. The application lays a molecular foundation for analyzing the olfactory molecular mechanism of the tomato leafminer, and provides a target gene for screening of an olfactory behavior regulating agent. The application provides a new theoretical basis for development of a new type of green and environment-friendly pesticide based on behavior regulation and green prevention and control of the tomato leafminer.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to the application of TabsGOBP2, a common odor-binding protein of the tomato leafminer moth, and its encoding gene. Background Technology

[0002] Tomato leafminer ( Tuta absoluta The tomato leafminer, belonging to the family Gelechiidae in the order Lepidoptera, is a globally recognized quarantine pest native to Peru, South America. It has a wide host range, with approximately 39 species reported in 9 families, posing a particularly devastating threat to Solanaceae crops, including major economic crops such as tomatoes, potatoes, peppers, eggplants, tobacco, and goji berries. The tomato leafminer is characterized by its high concealment, rapid reproduction, short lifespan, and significant generation overlap. Improper control can lead to 80-100% yield reductions in tomatoes, seriously jeopardizing the health and safety of the tomato industry, making control a critical challenge. After hatching, the tomato leafminer larvae burrow into the host plant tissues to feed, making them difficult to detect in their early stages. Their excellent concealment makes it difficult for chemical pesticides to effectively reach the target area, resulting in poor control efficacy. Due to the rapid reproduction rate and significant generation overlap of the tomato leafminer, repeated application of pesticides within the same growing season leads to resistance to conventional chemical pesticides, further reducing the effectiveness of control measures. Meanwhile, chemical pesticides can also have adverse effects on non-target organisms such as pollinating insects and natural enemies, and cause problems such as environmental pollution and agricultural product safety. Therefore, it is urgent to develop and apply green control technologies.

[0003] The olfactory system of insects participates in a series of key life activities, including host localization, foraging, courtship and mating, oviposition selection, and predator avoidance. Odor-binding proteins (OBPs) are the "first line of defense" in the insect's olfactory recognition process. Located in the lymph of the antennal sensilla, they specifically bind and transport lipid-soluble hydrophobic odor molecules through the hydrophilic lymph to deliver them to olfactory receptors (ORs) to activate nerve signals. General odor-binding proteins (GOBPs) are an important subclass of the insect OBP family, particularly prominent in Lepidoptera. Unlike sex pheromone-binding proteins (PBPs) that specifically recognize sex pheromones, GOBPs mainly participate in sensing environmental odor molecules such as host plant volatiles, playing an irreplaceable role in host localization and feeding selection in herbivorous insects.

[0004] However, systematic research on the GOBP gene of the tomato leafminer is still in its early stages. To date, the expression characteristics of the TabsGOBP gene in the tomato leafminer, its binding properties with volatiles, and its specific function in olfactory recognition have not been systematically analyzed, and the development of novel control products based on TabsGOBP remains a blank. Summary of the Invention

[0005] The purpose of this invention is to provide the application of TabsGOBP2, a common odor-binding protein of the tomato leafminer, and its encoding gene, in order to solve the problems existing in the prior art. This invention clones the encoding gene of TabsGOBP2, a common odor-binding protein of the tomato leafminer, from the tomato leafminer. The TabsGOBP2 protein plays an important role in the specific recognition of sex pheromones in the tomato leafminer, providing a new theoretical basis for the development of novel green and environmentally friendly agents based on behavior regulation and the green control of the tomato leafminer.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an application of a common odor-binding protein from the tomato leafminer moth in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents; The common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.1 or the recombinant TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.3.

[0007] The present invention also provides an application of the gene encoding the common odor-binding protein of the tomato leafminer mentioned above in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents; When the common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; When the common odor-binding protein of the tomato leafminer is a recombinant TabsGOBP2 protein, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0008] The present invention also provides an application of a recombinant vector, wherein the recombinant vector comprises a gene with a nucleotide sequence as shown in SEQ ID NO. 4; The application is any one of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents.

[0009] The present invention also provides the use of recombinant bacteria comprising the above-described recombinant vector in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents.

[0010] Furthermore, the tomato leafminer behavior regulation agent is an attractant, trap, or repellent.

[0011] Furthermore, the odor molecules are sex pheromones or host plant volatiles.

[0012] Furthermore, the sex pheromone is (3E,8Z,11Z)-tetradecanetriene acetate or (3E,8Z)-tetradecanediene acetate.

[0013] This invention also provides a method for screening reagents to regulate the behavior of tomato leafminer, comprising the following steps: The common odor binding protein of the tomato leafminer moth, the fluorescent probe and the odor molecule to be tested were mixed, the dissociation constant of the odor molecule to be tested was detected, and the odor molecule to be tested with a dissociation constant of less than 20 μmol / L was retained as the behavior regulation reagent of the tomato leafminer moth. The common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.1 or the recombinant TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.3.

[0014] Furthermore, the tomato leafminer behavior regulation agent is an attractant, trap, or repellent.

[0015] Furthermore, the fluorescent probe is N-phenyl-1-naphthylamine.

[0016] The present invention discloses the following technical effects: This invention cloned the encoding gene of a common odor-binding protein from the tomato leafminer moth, with its nucleotide sequence shown in SEQ ID NO.2, encoding the TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.1. Experimental results show that the TabsGOBP2 protein plays an important role in the specific recognition of sex pheromones in the tomato leafminer moth and can be used for the identification of active components of odor compounds. This invention lays the molecular foundation for elucidating the olfactory molecular mechanism of the tomato leafminer moth and provides target genes for the screening of olfactory behavior regulators. This invention provides new theoretical basis for the development of novel green and environmentally friendly pesticides based on behavior regulation and for the green control of the tomato leafminer moth. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in 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.

[0018] Figure 1 This study analyzed the expression characteristics of the odor-binding protein TabsGOBP2 in different tissues of male and female tomato leafminer moths. FA represents the antennae of female moths; MA represents the antennae of male moths; FH represents the head of female moths; MH represents the head of male moths; FL represents the legs of female moths; and ML represents the legs of male moths. Data in the figure are mean ± standard error. Different letters indicate significant differences between samples according to Tukey's test in one-way ANOVA (P < 0.05). Figure 2 SDS-PAGE analysis results of prokaryotic expression conditions of recombinant Tabs GOBP2 protein from the tomato leafminer; where M is the marker; 1 is the supernatant induced at 16℃; 2 is the precipitate induced at 16℃; 3 is the supernatant induced at 30℃; 4 is the precipitate induced at 30℃. Figure 3 SDS-PAGE analysis results of purified TabsGOBP2 recombinant protein from the tomato leafminer; where M is the marker and 1 is the TabsGOBP2 recombinant protein; Figure 4 The binding curve of the recombinant protein TabsGOBP2 from the tomato leafminer moth to the fluorescent probe 1-NPN; Figure 5Competitive binding curves of the TabsGOBP2 recombinant protein of the tomato leafminer moth with sex pheromone components and different plant volatiles. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1: Cloning of the TabsGOBP2 gene in the tomato leafminer moth 1. TabsGOBP2 gene amplification 1.1 Sampling After separating males and females during the pupal stage, the pupae of the tomato leafminer were placed in separate cages according to sex and awaited emergence. Tissues from unmated adult tomato leafminers within 3 days of emergence were collected, including antennae, head (without antennae), and legs. These tissues were placed in 1.5 mL centrifuge tubes, frozen in liquid nitrogen, and stored at -80°C for later use or for direct RNA extraction. Three independent biological replicates were established for each tissue from both male and female adults, with each replicate consisting of tissues from 200 adults.

[0025] 1.2 RNA extraction RNA extraction was performed using TransZol Up enhanced RNA extraction reagent (Tiangen), following the kit instructions. The concentration and purity of the RNA sample were assessed using NanoDrop 2000, with OD260 / 280 values ​​between 1.8 and 2.1. RNA integrity was assessed using standard agarose gel electrophoresis. Using the RNA as a template, cDNA was obtained through reverse transcription amplification using the SynScript® III RTSuperMix for qPCR kit.

[0026] 1.3 Cloning of the TabsGOBP2 gene Predict the open reading frames (ORFs) of TabsGOBP2, design gene-specific primers, and amplify the cDNA using the 2×T8 High-Fidelity Master Mix.

[0027] The specific primers designed for the TabsGOBP2 gene in this invention are as follows: Upstream primer F: 5′-ATGTTTCGCTGGGTTGTTTTTG -3′, SEQ ID NO.5; Downstream primer R: 5′-CTAATAGTGCTCCATGACAGCCTC-3′, SEQ ID NO.6.

[0028] The amplification system is shown in Table 1, and the amplification procedure is shown in Table 2.

[0029] Table 1 Amplification System Table 2 Amplification Procedure Take 2 μL of the PCR product obtained by the above method and perform agarose gel electrophoresis to detect whether the product band obtained by electrophoresis is the same size as the target gene.

[0030] 2. Gel recovery of PCR products Add 250 μL of Buffer BL to the adsorption column, centrifuge at 12,000 g for 1 min to activate the silica membrane; after separating the target band of the PCR product by 2% agarose gel electrophoresis, cut off the DNA band to be recovered with a clean blade under a 365 nm long-wave ultraviolet lamp and put it into a 2 mL centrifuge tube. Add 500 μL of Buffer GL; incubate at 65°C for 4-6 min, inverting and mixing every 2-3 min until the gel is completely melted and the solution turns pale yellow. Transfer the solution to an adsorption column EC, centrifuge at 12,000 g for 1 min, discard the waste liquid, and return the adsorption column EC to an empty collection tube. Add 700 μL of Buffer W2 to the adsorption column, centrifuge at 12,000 g for 1 min, discard the waste liquid (repeat once); return the adsorption column to an empty collection tube, centrifuge at 12,000 g for 2 min, remove the adsorption column, place it in a clean 1.5 mL centrifuge tube, add 40 μL of Eluent to the center of the adsorption membrane, incubate at 20-25°C for 2 min, and centrifuge at 12,000 g for 2 min. Estimate the approximate concentration of the recovered purified PCR product by electrophoresis, and use the recovered product for the next step of cloning and ligation.

[0031] 3. TA clone ligation conversion The product, after gel extraction, recovery, and purification, was ligated with a carrier, and the ligation system is shown in Table 3.

[0032] Table 3 Connection System After mixing the ligation system, ligate at room temperature for 5 min. Add the ligation product directly to 100 μL of competent cells thawed on ice, pipette once, and incubate on ice for 25 min. Heat shock at 42℃ for 30-45 s, then quickly transfer to ice and incubate for 2 min. Add 500 μL of antibiotic-free LB medium to a centrifuge tube, mix well, and incubate at 37℃, 200 rpm for 45 min. Spread 200 μL of the recovery solution evenly onto LB agar plates containing Amp antibiotic, and incubate overnight at 37℃. Pick a single white colony with a pipette tip and add it to 20 μL of sterile water, mix well, and use 2 μL as a template. Perform colony PCR using 2×T5 superPCR Mix (Colony) reagent. Identify the amplified products by electrophoresis, determine positive clones based on fragment size, and perform Sanger sequencing. The accurate sequence of the TabsGOBP2 gene was obtained by sequencing, as shown in SEQ ID NO.2, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.1.

[0033] SEQ ID NO.1: MFRWVVFGMILGITRVKSDAEVMSHVTAHFGKALEECREESGLTTDVMDEFKHFWREDFEVVHRELGCAIICMSNKFSLLKEDTRIHHMNMGDYIKNFPNGEILAQKMVDLIHNCEKFYEDITDDCSRVVKVSACFKKDAKKEGIAPEVAMIEAVMEHY.

[0034] SEQ ID NO.2: ATGTTTCGCTGGGTTGTTTTTGGGATGATCCTGGGTATCACAAGGGTTAAAAGTGATGCAGAAGTGATGAGCCATGTTACTGCTCATTTTGGTAAGGCCTTGGAAGAATGTCGAGAAGAGTCGGGATTGACCACCGATGTGATGGACGAGTTTAAGCATTTCTGGCGTGAGGACTTCGAAGTGGTCCACCGGGAGCTCGGCTGCGCCATCATCTGTATGTCCAATAAGTTCTCCCTATTG AAGGAGGACACTCGAATCCATCACATGAACATGGGCGATTATATCAAGAACTTCCCTAATGGTGAGATCCTGGCCCAAAAGATGGTGGACCTGATCCACAACTGTGAGAAGTTCTACGAAGACATCACAGATGACTGCAGCCGTGTTGGTGAAGGTTTCTGCCTGCTTCAAGAAGGACGCCAAGAAAGAGGGCATAGCCCCCGAGGTCGCTATGATCGAGGCTGTCATGGAGCACTATTAG.

[0035] 4. Bioinformatics analysis of TabsGOBP2 protein The signal peptide of the protein was predicted using the SignalP 6.0 server. The analysis results showed that the protein consists of 159 amino acids and has no transmembrane region; the protein molecular weight is 18.28 kDa and it has a signal peptide with the sequence: MFRWVVFGMILGITRVKS (SEQ ID NO.9).

[0036] Example 2: Expression characteristics of the TabsGOBP2 gene in different tissues of male and female tomato leafminer moths. Tomato leafminer samples were collected in the same manner as in Example 1.

[0037] Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time). RNA samples extracted from the antennae, head, and legs of male and female tomato leafminer moths were used as templates. gDNA was first removed, and then reverse transcribed into cDNA, following the manufacturer's instructions. The resulting cDNA was diluted 10-fold and used as a template. qRT-PCR primers were designed using Primer 3.0 targeting the gene. The primers are as follows: Upstream primer qF: 5′-GTGATGCAGAAGTGATGAGCCA-3′, SEQ ID NO.7; Downstream primer qR: 5′-TCGAAGTCCTCACGCCAGAA-3′, SEQ ID NO.8.

[0038] The reaction system for qRT-PCR is shown in Table 4.

[0039] Table 4 qRT-PCR reaction system The qRT-PCR reaction program was as follows: 95℃ pre-denaturation for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles. After the reaction, the PCR products were subjected to melting curve determination: 95℃ for 5 sec, 60℃ for 1 min, 95℃. Enzyme-free water was used instead of RNA for reverse transcription, and the resulting sample served as a negative control. Three biological replicates and three technical replicates were performed.

[0040] After the reaction, the Ct values ​​of the target gene and the internal reference gene were collected, and the results were analyzed using 2... -△△Ct The expression levels of different treatments were determined by relative quantification.

[0041] The results are as follows Figure 1 As shown, the expression level of the TabsGOBP2 gene in the antennae of the tomato leafminer moth is significantly higher than that in other body parts. The expression level in the antennae of the male moth is significantly higher than that in the female moth, and is twice that in the antennae of the female moth.

[0042] Example 3: Prokaryotic expression and purification of recombinant TabsGOBP2 protein from Tomato Leafminer 1. The TabsGOBP2 recombinant vector was transformed into E. coli Shuffle bacteria. The TabsGOBP2 gene was modified by removing the signal peptide and codon optimization before gene synthesis (the optimized sequence is shown in SEQ ID NO.4, encoding a protein with an amino acid sequence shown in SEQ ID NO.3). The target fragment was subcloned into the pET-28a vector and fused with a His tag for expression in the E. coli expression system. E.Coli The target protein was expressed using a shuffle plasmid, and expression and purification tests were performed. The constructed plasmid information is as follows: (pET-28a+NcoI+target gene+XhoI+vector-included 6×His).

[0043] SEQ ID NO.3: DAEVMSHVTAHFGKALEECREESGLTTDVMDEFKHFWREDFEVVHRELGCAIICMSNKFSLLKEDTRIHHMNMGDYIKNFPNGEILAQKMVDLIHNCEKFYEDITDDCSRVVKVSACFKKDAKKEGIAPEVAMIEAVMEHY.

[0044] SEQ ID NO.4: GATGCAGAAGTAATGTCTCACGTTACTGCGCACTTCGGTAAAGCTCTGGAAGAATGCCGTGAAGAATCCGGTCTGACCACTGATGTAATGGACGAATTCAAACACTTCTGGCGTGAAGATTTCGAAGTTGTACATCGTGAACTGGGTTGCGCGATCATCTGCATGTCCAACAAATTCAGCTTGCTGAAAGAAGACACTCGTATCCACCACAT GAACATGGGTGACTACATCAAGAACTTCCCGAACGGCGAAATCTTGGCTCAGAAGATGGTTGACCTGATCCACAACTGCGAGAAATTCTACGAAGATATCACTGACGACTGTTCTCGTGTGGTTAAAGTGTCTGCGTGCTTCAAGAAAGACGCTAAGAAAGAAGGCATCGCTCCGGAAGTTGCGATGATCGAAGCGGTTATGGAACACTAC.

[0045] Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min; Heat shock at 42℃ for 90 seconds, then immediately place on ice for 5 minutes, and add 600 μL of LB culture medium; Shake at 37℃ and 220 r / min for 1 h, then centrifuge and spread the entire mixture onto a substrate containing 50 μg / mL Kans. + LB plates were incubated overnight at 37°C inverted.

[0046] 2. Identification of IPTG-induced expression of recombinant bacterial fusion protein The expression conditions were as follows: *E. coli* cultured at 37℃ until the OD value reached 0.5-0.6, induced at 16 / 30℃ for 15-18 h; IPTG concentration 0.2 mM. After testing, expression was observed in inclusion bodies. Figure 2 Lanes 2 and 4 in the middle of the swim).

[0047] 3. Ni column affinity purification Purification was performed using a gravity column. The sample solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (20 mM Tris-HCl, 0 mM imidazole, 0.5 M NaCl, pH 8.0). The target protein was eluted with Ni-NTA Elution-Buffer containing 50 mM, 100 mM, and 500 mM imidazole, and the eluent was collected. The results were analyzed by SDS-PAGE.

[0048] 4. Dilution and refolding The collected protein solution was concentrated and then slowly added dropwise to 1×PBS at a volume ratio of 1:10. The solution was incubated overnight at 4°C and then concentrated by ultrafiltration. Finally, it was purified again using Ni column affinity.

[0049] 5. Dialysis Concentration The collected protein solution was dialyzed into PBS at a 1:30 volume ratio overnight. The dialysate was changed the next day, and dialyzing was continued for 8 hours. The results were then analyzed by SDS-PAGE. The purification results are as follows: Figure 3 As shown, the recombinant TabsGOBP2 protein was purified and collected for functional validation of odor-binding proteins.

[0050] Example 4 Functional verification of the TabsGOBP2 recombinant protein from the tomato leafminer moth The experiments in this embodiment were conducted on an F-7000 fluorescence spectrometer, using a 3.5 mL (10 mm × 10 mm × 35 mm) four-channel glass cuvette. Experimental parameters were set as follows: scanning mode (Emission), data mode (Fluorescence), excitation slit 5.0 nm, emission slit 5.0 nm, excitation wavelength (EX WL): 337 nm, emission wavelength (EM) range 380-450 nm.

[0051] 1. Protein-fluorescent probe binding experiment The ligands and probes used in the experiment were diluted with chromatographic grade methanol to a final concentration of 1 mmol / L. The recombinant protein prepared in Example 3 and 0.05 mol / L Tris-HCl (pH=7.4) were added to a cuvette to achieve a final concentration of 2 μmol / L for the recombinant protein and a total solution volume of 2 mL. After mixing and standing for 2 min, the solution was placed in an instrument to test the fluorescence intensity and record the maximum fluorescence value. Then, 4 μL of the fluorescent probe N-phenyl-1-naphthylamine (1-NPN) at a concentration of 1 mmol / L was gradually added to the cuvette, resulting in final concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 μmol / L. After mixing and standing for 2 min, the solution was placed in an instrument to test the fluorescence intensity and record the peak value. This experiment was repeated three times. Based on the correlation between the fluorescence value of TabsGOBP2 and the concentration of the fluorescent probe 1-NPN, a Scatchard plot was constructed using linear regression with the bound 1-NPN concentration on the x-axis and the ratio of bound 1-NPN concentration to free 1-NPN concentration on the y-axis. The binding constant (K) was then calculated using the Scatchard equation. 1-NPN ), that is, K d .

[0052] The results are as follows Figure 4 As shown, the binding of the TabsGOBP2 recombinant protein to 1-NPN exhibits a saturation effect and a significant Scatchard linearization relationship, K d The value was 3.944 μM, indicating that there is a single binding site between the TabsGOBP2 recombinant protein and the fluorescent probe and there is no allosteric effect. 1-NPN is suitable for subsequent fluorescence competitive binding experiments.

[0053] 2. Fluorescent competitive binding assay between protein and odorant ligand The recombinant protein prepared in Example 3 and Tris-HCl were added to a cuvette to achieve a final protein concentration of 2 μmol / L and a total solution volume of 2 mL. The mixture was stirred, allowed to stand for 2 min, and then placed in an instrument to test the fluorescence intensity, recording the maximum fluorescence value. Next, 4 μL of 1-NPN at a concentration of 1 mmol / L was added to the cuvette to achieve a final 1-NPN concentration of 2 μmol / L. The mixture was stirred, allowed to stand for 2 min, and then the maximum fluorescence peak was recorded. Then, 4 μL of odorant ligand at a concentration of 1 mmol / L was gradually added to the cuvette, resulting in final concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 μmol / L. With each addition of odorant ligand, the fluorescence value decreased. After each addition of odorant ligand, the mixture was stirred, allowed to stand for 2 min, and then placed in an instrument to test, recording the peak fluorescence intensity. Three replicates were performed for each odorant ligand. The obtained data was processed using Graphpad Prism 8.0.2, and the Binding Saturation function in nonlinear correlation regression was used to plot the competing combination curves by selecting One site Total.

[0054] Calculate the IC of ligand odor molecules 50 The fluorescence value (the concentration of the ligand when the fluorescence value drops to half of the initial fluorescence value of protein and probe binding) and the dissociation constant Ki. The calculation formula is as follows: Ki = [IC 50 ] / (1 + [1-NPN] / K [1-NPN] ); Wherein, [1-NPN] is the concentration of free 1-NPN at a 1-NPN concentration of 2 μmol / L; K [1-NPN] The binding constant between the recombinant protein and the probe is denoted as .

[0055] The affinity of the seven odor molecules (two sex pheromone components and five host plant volatiles) shown in Table 5 to the TabsGOBP2 recombinant protein was tested. Of the two sex pheromone components, TDTA is (3E,8Z,11Z)-tetradecatrien-1-yl acetate; and TDDA is (3E,8Z)-tetradecadien-1-yl acetate.

[0056] The test results are shown in Table 5 and Figure 5As shown, recombinant TabsGOBP2 has a strong affinity for the sex pheromone component TDTA (Ki = 7.69 μM) and a moderate affinity for the minor sex pheromone component TDDA (Ki = 13.27 μM). However, it did not show any affinity for the five plant volatiles tested, including limonene, α-terpinene, terpinene oil, methyl salicylate, and trans-2-hexenal (Ki > 40 μM).

[0057] Table 5. Affinity test results of 7 odor molecules The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a common odor-binding protein of the tomato leafminer moth in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents; The common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.1 or the recombinant TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.

3.

2. The use of the gene encoding the common odor-binding protein of the tomato leafminer as described in claim 1 in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents; When the common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; When the common odor-binding protein of the tomato leafminer is a recombinant TabsGOBP2 protein, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

3. An application of a recombinant vector, characterized in that, The recombinant vector contains a gene with a nucleotide sequence as shown in SEQ ID NO.4; The application is any one of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents.

4. The use of a recombinant bacterium comprising the recombinant vector of claim 3 in any of the following: (1) Applications in recognizing and / or binding odor molecules; (2) Application in the preparation of products that recognize and / or bind odor molecules; (3) Application in screening reagents for regulating the behavior of tomato leafminer; (4) Application in the preparation of products for screening tomato leafminer behavior regulation reagents.

5. The application as described in any one of claims 1-4, characterized in that, The tomato leafminer behavior regulation agent is an attractant, trap, or repellent.

6. The application as described in any one of claims 1-4, characterized in that, The odor molecules are sex pheromones or host plant volatiles.

7. The application as described in claim 6, characterized in that, The sex pheromone is (3E,8Z,11Z)-tetradecanetriene acetate or (3E,8Z)-tetradecanediene acetate.

8. A method for screening reagents to regulate the behavior of tomato leafminer, characterized in that, Includes the following steps: The common odor binding protein of the tomato leafminer moth, the fluorescent probe and the odor molecule to be tested were mixed, the dissociation constant of the odor molecule to be tested was detected, and the odor molecule to be tested with a dissociation constant of less than 20 μmol / L was retained as the behavior regulation reagent of the tomato leafminer moth. The common odor-binding protein of the tomato leafminer is the full-length TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.1 or the recombinant TabsGOBP2 protein with the amino acid sequence shown in SEQ ID NO.

3.

9. The screening method as described in claim 8, characterized in that, The tomato leafminer behavior regulation agent is an attractant, trap, or repellent.

10. The screening method as described in claim 8, characterized in that, The fluorescent probe is N-phenyl-1-naphthylamine.

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