Tomato leaf miner odorant binding protein TabsPBP1.3 and application of coding gene of tomato leaf miner odorant binding protein TabsPBP1.3

Abstract

The invention discloses a tomato leaf miner odorant binding protein TabsPBP1.3 and application of a coding gene thereof, and belongs to the technical field of molecular biology. The PBP1.3 gene is cloned from an important invasive pest tomato leaf miner gene for the first time, the amino acid sequence of the tomato leaf miner odorant binding protein is shown as SEQ ID NO.1, and the coding gene of the odorant binding protein is shown as SEQ ID NO.2. The protein and the gene provided by the invention lay a molecular foundation for analyzing the olfactory molecular mechanism of the phyllocnistis citrella, and can be used for identifying active components of reverse verification odor compounds; and a potential target gene is provided for developing olfactory behavior regulators, such as screening of attractants or repellents.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a tomato leafminer odor-binding protein TabsPBP1.3 and an application of a coding gene thereof. Background Art

[0002] The tomato leafminer (Tuta absoluta), a member of the Gelechiidae family of the Lepidoptera order, is native to Peru in South America. After widespread global spread, it was first discovered in August 2017 on fresh-to-eat tomatoes grown in the Yili region of Xinjiang, my country. It has now broken out in several regions of Northwest, Southwest, North, and Central my country, with signs of spreading northward. Since invading Europe, Africa, and the Middle East, the tomato leafminer has posed a serious threat to greenhouse and field tomato production, resulting in an 80-100% reduction in yield. As the world's largest tomato producer, an outbreak of the tomato leafminer would cause significant losses to my country's tomato industry. Numerous studies have shown that tomatoes are the leafminer's optimal host, but the insect can also cause infestations on host plants such as eggplant, nightshade, and potato. Due to the hidden "stealth feeding" characteristics of tomato leafminer larvae, the use of chemical pesticides is not only difficult to kill them, but also has adverse effects on pollinating insects and natural enemies, and causes environmental pollution and agricultural product safety problems. Therefore, there is an urgent need to develop and apply green control technologies.

[0003] Olfaction plays a crucial role in insect host location, prey avoidance, oviposition, and mate recognition. Insects utilize a complex olfactory system to detect and analyze odor molecules in the environment, a prerequisite for survival and reproduction. External, fat-soluble odor molecules can enter the lymph fluid within insect olfactory receptors through micropores. They bind to odorant-binding proteins to form complexes, which are then transported to odorant receptors (ORs) or directly bind to ORs, triggering signal transduction. Specific recognition of odorants and odorant-binding proteins is crucial for peripheral signaling.

[0004] Insect odorant-binding proteins (OBPs) are a class of low-molecular-weight, acidic, soluble proteins. OBPs bind to odorant molecules (such as insect pheromones or plant volatiles) and transport compound ligands through the lymph fluid. This is the first step in insects' recognition of chemical cues. Clarifying the function of insect OBPs is crucial for understanding the insect olfactory system. Based on their function, they are primarily categorized as general odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs). PBPs primarily recognize sex pheromones, while GOBPs primarily recognize general odorant molecules. In some insects, they can also bind to sex pheromones, sometimes with greater affinity than PBPs.

[0005] While OBPs have been extensively studied and used to develop new pest control products, the key PBPs involved in the perception of sex pheromones in the tomato leafminer have yet to be reported. Studying PBPs in the tomato leafminer not only lays the foundation for understanding the olfactory molecular mechanisms of sex pheromone recognition in the tomato leafminer, but also provides important evidence for the development of behavioral modulators, such as sex pheromone analogs or antagonists, targeting PBPs in the tomato leafminer. Summary of the Invention

[0006] One object of the present invention is to provide the use of a tomato leafminer odor-binding protein TabsPBP1.3 and its encoding gene in screening reagents for regulating the behavior of tomato leafminers; secondly, the present invention provides a method for screening the tomato leafminer behavior-regulating reagent; in addition, the present invention also provides a recombinant protein of the tomato leafminer odor-binding protein TabsPBP1.3 and a preparation method thereof; the present invention also screened and found that terpinolene can be used as an active molecule for developing tomato leafminer behavior-regulating reagents.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In the first aspect of the present invention, the present invention provides a use of a tomato leafminer odorant binding protein TabsPBP1.3 and / or its encoding gene in at least one of the following:

[0009] a1) Application in the recognition and / or binding of odor molecules;

[0010] a2) Application in the preparation of products for screening agents for regulating the behavior of tomato leafminers.

[0011] In the present invention, the odor molecule is selected from sex pheromones and / or host volatiles. Preferably, the odor molecule is a sex pheromone.

[0012] The sex pheromone is selected from one or a combination of two or more of TDTA and TDDA.

[0013] The host is a plant of the Solanaceae family, including tomatoes, eggplants, peppers and wolfberries, and the host volatiles are selected from one or a combination of two or more of limonene, α-terpinene, terpinolene, methyl salicylate, trans-2-hexenal, 2-pentylfuran, 2-carene, 3-carene, nerolidol, myrcene, terpinene, α-phellandrene, ocimene, cis-3-hexen-1-ol, α-pinene, methyl jasmonate, etc.

[0014] In a specific embodiment of the present invention, the odor molecules include but are not limited to TDTA, TDDA, limonene, α-terpinene, terpinolene, methyl salicylate, trans-2-hexenal, 2-pentylfuran, and 2-carene.

[0015] In a preferred embodiment of the present invention, the odor molecule is TDTA and / or TDDA.

[0016] In the present invention, the behavior-modulating agent is selected from an attractant, trap, or repellent, wherein the active ingredient in the attractant, trap, or repellent is a sex pheromone, sex pheromone analog, or antagonist; the sex pheromone, sex pheromone analog, or antagonist is obtained by screening products prepared from the tomato leafminer odor-binding protein TabsPBP1.3 provided by the present invention. The behavior-modulating agent is used to detect, monitor, and / or control tomato leafminers, including male and female tomato leafminers.

[0017] The product for screening a behavior regulating agent for tomato leafminer contains an effective amount of the tomato leafminer odor-binding protein TabsPBP1.3.

[0018] The products include but are not limited to reagents, kits, test strips, membrane strips, biosensors or detection platforms.

[0019] The amino acid sequence of the tomato leafminer odor-binding protein TabsPBP1.3 is selected from any one of the following:

[0020] b1) the amino acid sequence shown in SEQ ID NO.1;

[0021] b2) a protein sequence that is at least 90%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1 and has the same activity;

[0022] b3) differs from the sequence shown in SEQ ID NO. 1 by no more than 5, 4, 3, 2 or 1 amino acids;

[0023] b4) A variant of SEQ ID NO. 1, wherein the difference between the variant and SEQ ID NO. 1 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

[0024] The SEQ ID NO.1 sequence is as follows:

[0025] MKMWKSVVLICVYLAIESRVDGSADIMKTISINFGKALNECKKEMELPDSIDTDFMNFWKEDYDVTNRFTGCAIMCLSTKLDLVSPDGTLHHGNAQDFAKKHGADETMAKKLVELLHGCEKSTPDDEDGCAKVLTVAKCFKAEIHKLNWAPNMDLIIAEVLAEEG

[0026] In one embodiment of the present invention, the nucleotide sequence encoding the tomato leafminer odorant binding protein TabsPBP1.3 is selected from any one of the following:

[0027] (c1) the nucleotide sequence shown in SEQ ID NO. 2;

[0028] (c2) Adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2 to form a mutant gene or allele that can encode a protein with the same function.

[0029] The SEQ ID NO.2 sequence is as follows:

[0030] ATGAAGATGTGGAAGTCGGTAGTGCTGATCTGTGTGTATTTGGCAATAGAATCAAGGGTAGATGGATCAGCCGATATCATGAAAACGATTTCAATCAATTTCGGGAAGGCGTTGAACGAGTGTAAGAAAGAGATGGAACTTCCCGATTCCATAGATACGGATTTTATGAACTTTTGGAAGGAGGACTATGATGTGACGAACCGCTTCACCGGTTGTGCCATCATGTGTCTAAGCACCAAGTTGGATTTG GTGTCTCCTGATGGAACTCTGCATCACGGCAATGCACAGGATTTTGCGAAGAAGCATGGCGCTGATGAAACCATGGCGAAAAAAACTGGTAGAACTCCTCCACGGCTGCGAAAAGTCGACTCCTGATGACGAAGATGGTTGCGCAAAAGTTCTGACAGTGGCTAAGTGCTTCAAAGCTGAGATTCACAAACTAAACTGGGTCCCTAATATGGATCTGATCATTGCTGAAGTCTTAGCTGAAGAAGGTTAA

[0031] In the present invention, the process of screening a tomato leafminer behavior regulating agent includes: adding a candidate odor molecule to a mixed system containing the tomato leafminer odor-binding protein TabsPBP1.3 and 1-NPN; allowing the candidate odor molecule to compete with 1-NPN for binding to the tomato leafminer odor-binding protein TabsPBP1.3, calculating the dissociation constant of the candidate odor molecule, and using an odor molecule with a dissociation constant Ki < 20 μmol / L as a tomato leafminer behavior regulating agent.

[0032] In the second aspect of the present invention, the present invention provides a method for screening a behavior regulating agent for tomato leafminer, the screening method comprising: adding a candidate odor molecule to a mixed system containing the tomato leafminer odor binding protein TabsPBP1.3 and 1-NPN; allowing the candidate odor molecule to compete with 1-NPN for binding to the tomato leafminer odor binding protein TabsPBP1.3, calculating the dissociation constant of the candidate odor molecule, and using the odor molecule with a dissociation constant Ki < 20 μmol / L as a behavior regulating agent for tomato leafminer.

[0033] In a specific embodiment of the present invention, the method for screening a behavior regulating agent for tomato leafminer comprises the following steps:

[0034] S1) Add a 2 μmol / L Tris-HCl solution of the tomato leafminer odorant binding protein TabsPBP1.3 to a 2 ml cuvette, mix well, let stand, and record the maximum fluorescence value;

[0035] S2) Add 4 μl of 1 mmol / L 1-NPN to the cuvette to achieve a final concentration of 2 μmol / L, mix well, let stand, and record the maximum fluorescence peak;

[0036] S3) Gradually add 4 μl of the candidate odorant at a concentration of 1 mmol / L to the cuvette to a final concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 μmol / L, mix well, let stand, and record the peak fluorescence intensity;

[0037] S4) Drawing a competition binding curve, calculating the dissociation constant, and using an odor molecule with a dissociation constant Ki < 20 μmol / L as a behavior regulating agent for tomato leafminer.

[0038] In a third aspect of the present invention, the present invention provides a recombinant protein of TabsPBP1.3 of a tomato leafminer, wherein the amino acid sequence of the recombinant protein is selected from any one of the following:

[0039] d1) the amino acid sequence shown in SEQ ID NO. 3;

[0040] d2) a protein sequence that is at least 90%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 3 and has the same activity;

[0041] d3) differs from the sequence shown in SEQ ID NO. 3 by no more than 5, 4, 3, 2 or 1 amino acids;

[0042] d4) A variant of SEQ ID NO. 3, wherein the difference between the variant and SEQ ID NO. 3 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

[0043] The SEQ ID NO.3 sequence is as follows:

[0044] SADIMKTISINFGKALNECKKEMELPDSIDTDFMNFWKEDYDVTNRFTGCAIMCLSTKLDLVSPDGTLHHGNAQDFAKKHGADETMAKKLVELLHGCEKSTPDDEDGCAKVLTVAKCFKAEIHKLNWAPNMDLIIAEVLAEEG

[0045] In the fourth aspect of the present invention, the present invention provides a method for preparing a recombinant protein of the tomato leafminer TabsPBP1.3, the method comprising: subcloning the gene fragment shown in SEQ ID NO.4 into the pET-28a vector to obtain a recombinant plasmid; transfecting the recombinant plasmid into Escherichia coli and selecting positive colonies; inducing expression with IPTG; and affinity purification with a Ni column to obtain the recombinant protein.

[0046] In a fifth aspect, the present invention provides an organism comprising a recombinant vector or an engineered bacterium, wherein the recombinant vector is a plasmid containing the gene segment set forth in SEQ ID NO. 4, and the engineered bacterium is a cell containing the gene segment set forth in SEQ ID NO. 4 or the recombinant vector. In a preferred embodiment of the present invention, the cell is Escherichia coli.

[0047] The SEQ ID NO.4 sequence is as follows:

[0048] TCTGCTGACATCATGAAGACCATCTCCATCAACTTCGGTAAAGCTCTGAACGAATGCAAGAAAGAAATGGAACTGCCGGACAGCATCGACACTGACTTCATGAACTTCTGGAAAGAAGACTACGACGTGACTAACCGTTTCACTGGTTGCGCAATCATGTGCCTGTCTACCAAACTGGATCTGGTATCCCCAGATGGTACTCTGCACCACGGTA ACGCGCAAGACTTCGCAAAGAAACACGGTGCAGACGAAACCATGCCTAAGAAACTGGTCGAACTGCTGCATGGTTGCGAGAAATCTACTCCGGATGACGAAGACGGTTGTGCGAAAGTGCTGACCGTTGCTAAATGCTTCAAAGCTGAAATCCACAAACTGAACTGGGCACCAAACATGGACCTGATCATCGCGGAAGTACTGGCGGAAGAAGGT

[0049] In a sixth aspect of the present invention, the present invention provides a use of the tomato leafminer TabsPBP1.3 recombinant protein according to the third aspect of the present invention or the organism according to the fifth aspect in at least one of the following:

[0050] e1) Application in the recognition and / or binding of odor molecules;

[0051] e2) Application in the preparation of products for screening agents for regulating the behavior of tomato leafminers.

[0052] The definitions of odor molecules and behavior regulation agents are as shown in the first aspect of the present invention.

[0053] This invention, for the first time, cloned the tomato leafminer odorant binding protein gene PBP1.3 from the genes of the important invasive pest, the tomato leafminer. This PBP plays an important role in the specific recognition of the tomato leafminer's sex pheromone. The protein and gene provided by this invention lay the molecular foundation for understanding the molecular mechanism of olfaction in the tomato leafminer and can be used to reverse-validate the identification of active components of odor compounds. It also provides a potential target gene for the development of olfactory behavior modulators (attractants / repellents). This invention provides a new theoretical basis for the development of new, green and environmentally friendly agents based on behavioral regulation for the green control of tomato leafminer.

[0054] During the screening of potential active compounds for use as agents for regulating the behavior of tomato leafminers using the prepared recombinant protein TabsPBP1.3, the present invention unexpectedly discovered that terpinolene binds well to the odor-binding protein. Therefore, terpinolene can be used as an attractant in the development of agents for regulating the behavior of tomato leafminers, thereby trapping both sexes of adult insects. In a seventh aspect, the present invention provides the use of terpinolene in the preparation of agents for regulating the behavior of tomato leafminers.

[0055] The behavior regulation agent is an attractant or trap, the active ingredient in the behavior regulation agent is terpinolene, the tomato leafminer includes male and female tomato leafminers, and the behavior regulation agent can realize the detection, monitoring and / or prevention of both sexual adults of tomato leafminers. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Analysis of the expression characteristics of the odor-binding protein TabsPBP1.3 in different tissues of male and female adults of the tomato leafminer; Note: FA: female antennae; MA: male antennae; FH: female head; MH: male head; FL: female legs; ML: male legs; Data in the figure are mean ± SD; different letters indicate significant differences among samples (P < 0.05) by one-way ANOVA and Tukey's test.

[0057] Figure 2 SDS-PAGE analysis of the prokaryotic expression conditions of the recombinant protein of the tomato leafminer TabsPBP1.3. Note: M, marker; 1, supernatant of 16℃ induction; 2, precipitate of 16℃ induction; 3, supernatant of 30℃ induction; 4, precipitate of 30℃ induction.

[0058] Figure 3 SDS-PAGE analysis results of the purified recombinant protein of the tomato leafminer TabsPBP1.3. Note: M, marker; 1, TabsPBP1.3 protein.

[0059] Figure 4 Binding curve and Scatchard plot of TabsPBP1.3 recombinant protein and fluorescent probe 1-NPN.

[0060] Figure 5 Competition binding curves of TabsPBP1.3 recombinant protein with sex pheromone components and different plant volatiles. DETAILED DESCRIPTION

[0061] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0062] Example 1 Cloning of the TabsPBP1.3 gene of the tomato leafminer

[0063] (1) TabsPBP1.3 gene amplification

[0064] S1) Sampling: After sexing during the pupal stage, the insects were placed in separate cages for eclosion. Tissues including antennae, heads (without antennae), and legs were collected from unmated adult tomato leafminers within three days of eclosion. These tissues were placed in 1.5 mL centrifuge tubes, frozen in liquid nitrogen, and stored at -80°C for later use or direct RNA extraction. Three independent biological replicates were performed for each tissue from both male and female adults, with tissues collected from 200 adults in each replicate.

[0065] S2) RNA extraction: RNA was extracted using TransZol Up Enhanced RNA Extraction Reagent (Tiangen). The specific steps were as described in the kit instructions. The concentration and purity of the RNA sample were detected using Nanodrop 2000. The OD260 / 280 value was between 1.8 and 2.1. RNA was detected by conventional agarose gel electrophoresis to detect intact samples. Using the above RNA as a template, the reverse transcription kit was used. cDNA was obtained by reverse transcription amplification using IIIRT SuperMix for qPCR.

[0066] S3) TabsPBP1.3 gene cloning: The open reading frames (ORFs) of TabsPBP1.3 were predicted, and specific primers were designed for the gene. The cDNA was used as a template and amplified using Qingke 2×T8 High-Fidelity MasterMix.

[0067] The specific primers designed for the TabsPBP1.3 gene of the present invention are as follows:

[0068] Upstream primer / F 5′-ATGAAGATGTGGAAGTCGGTAGT-3′ SEQ ID NO.5 Downstream primer / R 5′-TTAACCTTCTTCAGCTAAGACTTCA-3′ SEQ ID NO.6

[0069] The amplification system is:

[0070] Components Volume (μL) 2×T8 High-Fidelity Master Mix 20 10μM Primer F 2 10μM Primer R 2 Template (cDNA) 2 water 14 Total 40

[0071] The amplification procedure is:

[0072]

[0073] Take 2 μl of the PCR product obtained by amplification according to the above method and perform agarose electrophoresis detection to compare whether the product band obtained by electrophoresis detection is the same size as the target gene.

[0074] (2) PCR product gel recovery

[0075] Add 250 μL of Buffer BL to the adsorption column and centrifuge at 12,000 g for 1 min to activate the silica membrane. After separating the target bands of the PCR products by electrophoresis on a 2% agarose gel, excise the desired DNA band with a clean razor blade under 365 nm long-wave UV light and place it into a 2 mL centrifuge tube. Add 500 μL of Buffer GL; incubate at 65°C in a water bath for 4-6 minutes, mixing by inversion every 2-3 minutes, until the gel is completely melted and the solution turns pale yellow. Transfer the solution to an EC adsorption column and centrifuge at 12,000 g for 1 minute. Discard the waste solution and return the EC adsorption column to an empty collection tube. Add 700 μL of Buffer W2 to the adsorption column and centrifuge at 12,000 g for 1 minute. Discard the waste solution (repeat once). Return the adsorption column to the empty collection tube and centrifuge at 12,000 g for 2 minutes. Remove the adsorption column and 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 minutes, and then centrifuge at 12,000 g for 2 minutes. Recover the purified PCR product by electrophoresis to estimate its approximate concentration. Recover the product for cloning and ligation.

[0076] (3) TA cloning ligation transformation

[0077] The product recovered and purified after gel excision is connected to the carrier. The connection system is as follows:

[0078] Components Volume (μL) PCR products 3 5x pClone007Versatile Simple Vector mix 4 <![CDATA[ddH2O]]> 3 Total volume 10

[0079] After the ligation system is mixed, ligation is carried out at room temperature for 5 minutes. The ligation product is directly added to 100μL of competent medium melted in an ice bath, pipetted once with a pipette tip, and allowed to stand on ice for 25 minutes. Heat shock in a 42℃ water bath for 30-45 seconds, quickly transfer to an ice bath, and let it stand for 2 minutes. Add 500μL of LB medium without antibiotics to the centrifuge tube, mix well, and recover at 37℃, 200rpm for 45 minutes. Pipette 200ul of the recovery solution and evenly spread it on the LB medium plate containing Amp antibiotics, and place the plate in a 37℃ incubator for overnight incubation. Pick up a single white colony with a pipette tip and add it to 20μL of sterile water. After pipetting and mixing, take 2μL as a template and perform bacterial P using 2×T5 super PCRMix (Colony) reagent. The amplified product is identified by electrophoresis, and the positive clone is determined according to the fragment size and Sanger sequencing is performed. The exact sequence of the TabsPBP1.3 gene obtained by sequencing is shown in SEQ ID NO.2, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.1.

[0080] (4) Bioinformatics analysis of TabsPBP1.3 protein

[0081] The protein signal peptide was predicted on the website SignaIP 5.0 server. The analysis results showed that the protein consists of a total of 165 amino acids, has no transmembrane region, has a signal peptide, and the signal peptide sequence is: MKMWKSVVLICVYLAIESRVDG; the protein molecular mass is 16.8kDa.

[0082] Example 2 Analysis of expression characteristics of TabsPBP1.3 gene in different tissues of male and female adults of the tomato leafminer

[0083] The collection of tomato leafminer samples was the same as in Example 1.

[0084] Using PrimeScript TM Reverse transcription was performed using the RT reagent kit with gDNA Eraser (Perfect Real Time). RNA samples extracted from the antennae, heads, and legs of male and female tomato leafminers were used as templates. gDNA was removed and then reverse transcribed into cDNA. Refer to the manufacturer's instructions for the specific steps to obtain cDNA. The reverse-transcribed cDNA was diluted 10-fold and used as a template. qRT-PCR primers were designed for the gene using Primer 3.0. The primers are as follows:

[0085] Upstream primer / qF 5′-ATGGAACTCTGCATCACGGC-3′ SEQ ID NO.7 Downstream primer / qR 5′-AGCCACTGTCAGAACTTTTGCG-3′ SEQ ID NO.8

[0086] The reaction system for qRT-PCR is:

[0087] Components volume TB Green Premix Ex Taq II(Tli RNaseH Plus)(2X) 5μL RCR Forward Primer (10μM) 0.4μL RCR Reverse Primer (10μM) 0.4μL RNase-free Water 3.2 μL cDNA 1 μL Total 10 μL

[0088] The qRT-PCR reaction procedure was as follows: 95°C pre-denaturation for 30 seconds; 95°C for 5 seconds, 60°C for 30 seconds, for 40 cycles. After the reaction, the PCR product was subjected to a melting curve analysis: 95°C for 5 seconds, 60°C for 1 minute, and 95°C. RNA was replaced with enzyme-free water for reverse transcription, and the resulting sample served as a negative control. Three biological replicates and three technical replicates were performed. After the reaction, the Ct values ​​of the target gene and the internal reference gene were collected and analyzed using 2 -△△ The expression levels of different treatments were determined by Ct relative quantitative method.

[0089] The results are as follows Figure 1 As shown, the expression of TabsPBP1.3 gene in the antennae of tomato leafminer was significantly higher than that in other body parts, and there was no significant difference in the relative expression levels in the antennae of male and female adults.

[0090] Example 3 Prokaryotic expression and purification of the recombinant protein of the tomato leafminer TabsPBP1.3

[0091] (1) Transformation of TabsPBP1.3 recombinant vector into Escherichia coli Shuffle

[0092] The signal peptide of the TabsPBP1.3 gene was removed and codon-optimized before gene synthesis (the optimized sequence is shown in SEQ ID NO. 4). The target fragment was subcloned into the pET-28a vector and fused with a His tag to express the target protein in an E. coli expression system (E. coli Shuffle). Expression and purification tests were performed. The constructed plasmid information is as follows: (pET-28a + NcoI + target gene + XhoI + 6*His included in the vector)

[0093] 1.1) Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 minutes;

[0094] 1.2) Heat shock at 42°C for 90 seconds, immediately place on ice for 5 minutes, and add 600 μL LB medium;

[0095] 1.3) Shake at 220 rpm for 1 h at 37°C. After centrifugation, spread the entire volume onto an LB plate containing 50 μg / mL Kan+ and incubate inverted at 37°C overnight.

[0096] (2) Expression and identification of recombinant bacterial fusion protein induced by IPTG

[0097] The expression conditions are as follows: culture E. coli at 37℃ to an OD value of 0.5-0.6, induce at 30℃ for 15-18h; IPTG concentration is 0.2mM. After testing, it was found that the expression in inclusion bodies ( Figure 2 , lane 4).

[0098] (3) Ni column affinity purification

[0099] 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 (20mM Tris-HCl, 0mM imidazole, 0.5M NaCl, pH 8.0). The target protein was eluted using Ni-NTA Elution Buffer containing 50mM, 100mM, and 500mM imidazole. The flow-through was collected and analyzed by SDS-PAGE.

[0100] (4) Dilution and renaturation

[0101] The collected protein solution was concentrated and slowly added dropwise to 1*PBS at a ratio of 1:10, allowed to stand at 4°C overnight, and then concentrated by ultrafiltration; and then purified again using Ni column affinity purification.

[0102] (5) Dialysis concentration

[0103] The collected protein solution was dialyzed into PBS at a ratio of 1:30 overnight. The dialysate was replaced the next day and the dialysis was continued for 8 hours. The results were analyzed by SDS-PAGE. Figure 3 As shown, the TabsPBP1.3 recombinant protein was purified and collected for functional verification of the odorant binding protein.

[0104] Example 4 Functional verification of the recombinant protein of the tomato leafminer TabsPBP1.3

[0105] This experiment was conducted on an F-7000 fluorescence spectrometer using a 3.5 mL (10 mm × 10 mm × 35 mm) four-way glass cuvette. The experimental parameters were: Emission scanning mode, Fluorescence data mode, excitation slit 5.0 nm, emission slit 5.0 nm, excitation wavelength (EX WL) 337 nm, and emission wavelength (EM) range 380–450 nm.

[0106] (1) Protein-fluorescent probe binding experiment

[0107] The ligand and probe 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 the cuvette to a final concentration of 2 μmol / L of the recombinant protein. The total volume of the solution was 2 mL. The mixture was allowed to stand for 2 minutes. The fluorescence intensity was measured by the instrument and the maximum fluorescence value was recorded. 4 μl of 1-NPN with a concentration of 1 mmol / L was gradually added to the cuvette to a final concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 μmol / L. The mixture was allowed to stand for 2 minutes. The peak fluorescence intensity was recorded. The experiment was repeated three times. The fluorescence value of TabsPBP1.3 was fitted with the concentration of fluorescent probe 1-NPN. The bound 1-NPN concentration was used as the horizontal axis and the bound 1-NPN concentration / free 1-NPN concentration was used as the vertical axis. The Scatchard plot was drawn by linear regression and the binding constant (K) was calculated using the Scatchard equation. 1-NPN ), namely K d .

[0108] The results showed that the binding of TabsPBP1.3 recombinant protein to 1-NPN had a saturation effect and a significant Scatchard linear relationship ( Figure 4 ), K d It is 2.41 μM, indicating that there is a single binding site between the TabsPBP1.3 recombinant protein and the fluorescent probe and there is no allosteric effect. 1-NPN is suitable for subsequent fluorescence competitive binding experiments.

[0109] (2) Fluorescence competition binding experiment between protein and odor ligand

[0110] Add the recombinant protein prepared in Example 3 and Tris-HCl to a cuvette to a final concentration of 2 μmol / L. The total volume of the solution is 2 ml. Mix well and let stand for 2 minutes. Place the solution in an instrument to test the fluorescence intensity and record the maximum fluorescence value. Then add 4 μl of 1-NPN at a concentration of 1 mmol / L to the cuvette to a final concentration of 2 μmol / L. Mix well and let stand for 2 minutes. Record the maximum fluorescence peak. Then gradually add 4 μl of odor ligand at a concentration of 1 mmol / L to the cuvette to a final concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 μmol / L. As the ligand is continuously added, the fluorescence value decreases. After each addition of the odor ligand, mix well and let stand for 2 minutes before placing the solution in an instrument to test and record its peak fluorescence intensity. Set up 3 replicates for each odor ligand. The data were processed using Graphpad Prism 8.0.2, and the competition binding curves were drawn using Binding Saturation in nonlinear correlation regression and One Site Total.

[0111] Calculation of ligand odor molecule IC 50 The value (the concentration of the ligand when the fluorescence value drops to half of the initial fluorescence value of the protein and probe) and the dissociation constant Ki. Calculation formula Ki = [IC 50 ] / (1+[1-NPN] / K [1-NPN] ), [1-NPN] is the concentration of free 1-NPN when the 1-NPN concentration is 2 μmol / L; K [1-NPN] is the binding constant between the recombinant protein and the probe.

[0112] The experiment tested the affinity of the recombinant TabsPBP1.3 protein for the seven odor molecules (two sex pheromone components and five host plant volatiles) shown in the table below. The results showed that the recombinant protein TabsPBP1.3 had strong binding affinity to both female insect sex pheromone components (Ki < 10 μmol / L; TDTA: Ki = 7.15 ± 0.52 μmol / L; TDDA: Ki = 7.95 ± 0.99 μmol / L; Figure 5 ), the recombinant protein also has a moderate binding affinity to the plant volatile terpinolene (Ki = 17.12 ± 0.17 μmol / L < 20 μmol / L). Therefore, terpinolene can be used as a new attractant to trap both sexes of adult insects.

[0113]

[0114]

[0115] TDTA: (3E,8Z.11Z)-tetradecatrien-1-ylacetate

[0116] TDDA: (E3,Z8)-tetradecadien-1-yl acetate

[0117] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a tomato leafminer odor-binding protein TabsPBP1.3 and / or its encoding gene in at least one of the following: a1) Application in the recognition and / or binding of odor molecules; a2) Use in the preparation of a product for screening a tomato leafminer behavior regulating agent; The amino acid sequence of the tomato leafminer odor-binding protein TabsPBP1.3 is selected from any one of the following: b1) the amino acid sequence shown in SEQ ID NO.1; b2) a protein sequence that is at least 90%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1 and has the same activity; b3) other amino acid sequences that differ from the amino acid sequence of SEQ ID NO. 1 by no more than 5, 4, 3, 2 or 1 amino acids; b4) A variant of SEQ ID NO. 1, wherein the difference between the variant and SEQ ID NO. 1 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the tomato leafminer odor-binding protein TabsPBP1.3 is selected from any one of the following: (c1) the nucleotide sequence shown in SEQ ID NO. 2; (c2) Adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2 to form a mutant gene or allele that can encode a protein with the same function.

3. The use according to claim 1, characterized in that The odor molecules are selected from sex pheromones and / or host volatiles.

4. The use according to claim 1, characterized in that The behavior regulating agent is selected from attractants, traps or repellents; the product for screening the behavior regulating agent of tomato leafminer contains an effective amount of tomato leafminer odor binding protein TabsPBP1.

3.

5. A method for screening a behavior-regulating agent for tomato leafminer, the method comprising: A candidate odor molecule is added to a mixed system containing the tomato leafminer odor-binding protein TabsPBP1.3 described in claim 1 and 1-NPN; the candidate odor molecule is allowed to compete with 1-NPN for binding to the tomato leafminer odor-binding protein TabsPBP1.3, the dissociation constant of the candidate odor molecule is calculated, and the odor molecule with a dissociation constant Ki less than 20 μmol / L is used as a tomato leafminer behavior regulation agent.

6. A recombinant protein of TabsPBP1.3, wherein the amino acid sequence of the recombinant protein is selected from any one of the following: d1) the amino acid sequence shown in SEQ ID NO. 3; d2) a protein sequence that is at least 90%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 3 and has the same activity; d3) differs from the sequence shown in SEQ ID NO. 3 by no more than 5, 4, 3, 2 or 1 amino acids; d4) A variant of SEQ ID NO. 3, wherein the difference between the variant and SEQ ID NO. 3 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

7. A method for preparing the recombinant protein of TabsPBP1.3 of tomato leafminer according to claim 6, comprising: The gene fragment shown in SEQ ID NO.4 was subcloned into the pET-28a vector to obtain a recombinant plasmid; the recombinant plasmid was transfected into Escherichia coli, and positive colonies were selected; IPTG was induced for expression; and Ni column affinity purification was performed to obtain the recombinant protein.

8. An organism comprising a recombinant vector or an engineered bacterium, characterized in that: The recombinant vector is a plasmid containing the gene fragment shown in SEQ ID NO.4, and the engineered bacteria are cells containing the gene fragment shown in SEQ ID NO.4 or the recombinant vector.

9. Use of the TabsPBP1.3 recombinant protein of claim 6 or the organism of claim 8 in at least one of the following: e1) Application in the recognition and / or binding of odor molecules; e2) Application in the preparation of products for screening agents for regulating the behavior of tomato leafminers.

10. Application of terpinolene in the preparation of behavioral regulation reagents for tomato leafminer.

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