Tibetan fimbriatus odor binding protein gene sequence and application thereof

Abstract

The application discloses a smell binding protein gene sequence of Tibet cluster horned beetle, including an OBP6 gene sequence and an OBP10 gene sequence, the OBP6 gene sequence is selected from SEQ ID No.1, and the OBP10 is selected from SEQ ID No.2. The application also provides applications of smell binding proteins coded by the OBP6 gene sequence and the OBP10 gene sequence in identifying smell compounds. The application of the smell binding protein coded by the OBP6 gene sequence in identifying the smell compound trans-2-hexenal. The application of the smell binding protein coded by the OBP10 gene sequence in identifying the smell compound geraniol. Through experiments, the application finds out that the Tibet cluster horned beetle combines the proteins coded by the OBP6 and OBP10 gene sequences with smell compounds to locate the attacked object (walnut), and accordingly, corresponding attractants can be developed to lure the Tibet cluster horned beetle to a capturing area.

Description

Technical Field

[0001] This invention relates to the gene sequence of odor-binding protein derived from the Tibetan tufted ... Background Technology

[0002] *C. cretifera thibetana*, belonging to the genus *Cacia* of the subfamily Laminae in the family Cerambycidae of the order Coleoptera, is a serious borer of economic trees such as walnut in Yunnan Province, my country. It is mainly distributed in Yunnan, Sichuan, Guangxi, and Tibet Autonomous Region. Adults emerge from boreholes in late April and migrate, laying eggs in May. Larvae begin boring into the trunk in late June, overwintering as larvae until they emerge as adults. Adults live inside the trunk until they migrate (Ji Baozhong et al., 2002). Adults carve grooves 1-2 cm apart into the bark of walnut trees in a "|" shape, extending to the phloem. Larvae primarily feed on the phloem before entering the xylem and bore into the wood, expelling large amounts of frass and sawdust from the boreholes (Lu Jifang et al., 2016). The damaged trees lose water due to the destruction of their nutrient organs, the bark of the damaged parts cracks, and even large burls form. Eventually, the trees gradually dry up and die, and are easily broken by the wind.

[0003] The Tibetan tufted tasseled longhorn beetle is primarily a larvae that bore into and damage host plants (Cai Xiaona and Huang Dazhuang, 2009). Currently, there are three main methods for controlling the Tibetan tufted tasseled longhorn beetle: (1) physical control methods such as manually pruning dead branches, manually killing adult beetles, and manually hooking larvae. However, the longhorn beetle reproduces and spreads rapidly, and physical control methods are only applicable to low-growing host plants and can only reduce the population density of the pest to a certain extent (Gao Guoping, 2011; Liang Kan, 2014); (2) chemical control, which involves spraying chemical insecticides on the damaged tree trunks during the hatching period of the longhorn beetle's eggs or the adult stage. However, the extensive use of insecticides can easily lead to the longhorn beetle developing resistance, and the residues of chemical pesticides cause serious environmental pollution (Liu Fenghua, 2014; Jiang Jing, 2012); (3) biological control, which utilizes the longhorn beetle's parasitic and predatory natural enemies for control. Parasitic natural enemies include the Sichuan scleroderma wasp, the Guan's scleroderma wasp, and the Dastarcus helophoroides parasitic beetle. Lu Xiping et al. (2011) used the parasitic Dastarcus helophoroides parasitic beetle to control the rusty-shouldered longhorn beetle Apriona swai nsoni; Han Mingli et al. used the great spotted woodpecker Dendrocopos major and the star-headed woodpecker Dendrocopos canicapillus to control the peach-necked longhorn beetle Aromia bungii, both achieving good control results (Huang Yonghuai et al., 2018; Han Mingli et al., 2019). Although biological control can achieve good control results, the breeding and protection of its natural enemies will undoubtedly increase the control costs for walnut growers, especially since seasonal changes will lead to bird migration, affecting the effectiveness of biological control. Therefore, it is necessary to develop a new, effective, environmentally friendly, and sustainable control method. Summary of the Invention

[0004] Therefore, this invention sequenced the gene sequence of the odor-binding protein of the Tibetan tufted-horned beetle, providing the amino acid sequence and gene sequence of the odor-binding protein of the Tibetan tufted-horned beetle that specifically binds to walnut odor compounds, wherein... The amino acid sequence of the odor-binding protein of the Tibetan tufted horned beetle includes the OBP6 and OBP10 amino acid sequences.

[0005] The gene sequence that encodes the amino acid sequence of the odor-binding protein of the Tibetan tufted horned beetle includes the OBP6 gene sequence and the OBP10 gene sequence, wherein the OBP6 gene sequence is selected from SEQ ID No. 1 and the OBP10 gene sequence is selected from SEQ ID No. 3.

[0006] This invention also provides the application of the OBP6 amino acid sequence and the OBP10 amino acid sequence in the identification of walnut odor compounds.

[0007] Furthermore, the application of the odor-binding protein compiled from the OBP6 gene sequence in recognizing the walnut odor compound trans-2-hexenal.

[0008] Furthermore, the application of the odor-binding protein compiled from the OBP10 gene sequence in recognizing the walnut odor compound geraniol.

[0009] The beneficial effects of this invention are: 1. This invention has screened out the odor-binding protein and its amino acid sequence of the Tibetan tufted-horned longhorn beetle that combines with walnut odor compounds through experiments, as well as the gene sequence that can encode the amino acid sequence. Based on this, corresponding attractants can be developed to lure the Tibetan tufted-horned longhorn beetle to the capture area.

[0010] 2. This invention elaborates in detail the sequencing process of the OBP6 and OBP10 gene sequences of the odor-binding proteins of the Tibetan tufted-horned longhorn beetle through experiments, as well as the screening and binding process of these genes with walnut odor compounds. It elucidates the olfactory recognition mechanism of the Tibetan tufted-horned longhorn beetle, enriches the chemical ecology theory of the Tibetan tufted-horned longhorn beetle, and provides new methods and approaches for the control of the Tibetan tufted-horned longhorn beetle and similar insects. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the construction of prokaryotic expression vectors for the OBP6 and OBP10 genes; Figure 2 This is a PCR electrophoresis image of the OBP6 and OBP10 genes. In the image, Lane 1: CcreOBP10 PCR band, Lane 2: CcreOBP6 PCR band. Figure 3 These are images showing positive PCR identification of OBP6 and OBP10 gene colonies; Figure 4 This is a graph showing the OBP6 gene sequencing results; Figure 5 This is a graph showing the OBP10 gene sequencing results; Figure 6 This is a graph showing the SDS-PAGE analysis results of purified OBP6 protein; Figure 7 This is a graph showing the SDS-PAGE analysis results of purified OBP10 protein; Figure 8 This is a curve showing the binding of OBP6 protein to 1-NPN; Figure 9 This is a curve showing the binding of OBP10 protein to 1-NPN; Figure 10This is a competitive binding curve of OBP6 with different odor molecules; Figure 11 This is a competitive binding curve of OBP10 with different odor molecules. Detailed Implementation

[0012] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present application and are not intended to limit its scope.

[0013] The test insect in this embodiment, adult Tibetan tufted-horned longhorn beetle, was collected in June 2018 from a walnut economic forest in Midu County, Dali Prefecture, Yunnan Province. Live adult Tibetan tufted-horned longhorn beetles were collected in the walnut forest and placed in an insect rearing cage before being brought back to the laboratory.

[0014] Test plants: Samples were collected in May 2018 from a walnut forest in Midu County, Dali Prefecture, Yunnan Province. Three walnut trees in the *Juglans sigillata* 'San-tai' forest that had been fed on by the Tibetan longhorn beetle for three days were randomly selected for sampling. Three healthy walnut trees of the same age were also selected as controls. Walnut branches 2 m above the ground were cut with scissors, and the bark and leaves were collected and placed in an icebox for laboratory processing. Each sample was tested in triplicate.

[0015] Example 1. Construction of the cDAN library of the Tibetan tufted-horned longhorn beetle 1.1 Total RNA was extracted from the antennae, head, male, abdomen, legs, and wings of male and female adult Tibetan longhorn beetles using the Trizol method. After the extracted total RNA samples passed the tests, eukaryotic mRNA was enriched using magnetic beads carrying Oligo(dT). Subsequently, fragmentation buffer was added to break the mRNA into short fragments. Using the mRNA as a template, one-stranded cDNA was synthesized using six-base random primers, followed by the synthesis of a second-stranded cDNA using DNA polymerase. After purification of the double-stranded cDNA using AMPure XP beads, end repair was performed, A-tails were added, and sequencing adapters were ligated. Fragment sizes were selected using AMPure XP beads. PCR amplification was performed, and the PCR amplification products were purified using AMPure XP beads to obtain the final library. After library construction, preliminary quantification was performed using Qubit 2.0, followed by dilution to 1.5 mg / μL and insertion size detection using an Agilent 2100. Once the insertion size met expectations, the effective concentration of the library was accurately quantified using Q-PCR to ensure library quality.

[0016] 2. Identification of odor-binding protein genes After the cDAN library passed verification, Illumina HiSeq sequencing was performed. Raw reads were obtained after sequencing. Reads containing adapters and low-quality reads were removed, and then the raw reads were filtered to obtain high-quality clean reads. Trinity was used to assemble and filter the clean reads to obtain transcript sequences, and the longest transcript was selected as the unigenes. A total of 222,946 transcripts were obtained after assembly and splicing, with an average length of 807 bp and an N50 length of 1584 bp, resulting in 89,897 unigenes. The average length and N50 length of the unigenes were 1036 bp and 1626 bp, respectively.

[0017] The OBP gene was identified from the transcriptome of *Euonymus alatus* (Tibetan longhorn beetle) using BLAST homology search and alignment. OBP gene sequences from *Euonymus rubrum*, *Euonymus glabripennis*, *Euonymus alatus*, *Euonymus chinensis*, and *Euonymus sibirica* were downloaded from the NCBI (National Center for Biotechnology Information) Protein database and from publicly available literature accessions as target sequences. BLAST homology searches were performed using TBtools software to identify candidate OBP genes. Finally, a Protein BLAST homology search was conducted in the NCBI database to confirm the OBP gene in *Euonymus alatus*.

[0018] Based on the sequencing results, candidate genes were cloned and expressed, and candidate proteins were purified. Through fluorescence competition assays, two genes that can recognize trans-2-hexenal and geraniol were screened from 31 OBP genes. The sequence of the first gene is SEQ ID No.1, and the sequence of the second gene is SEQ ID No.3.

[0019] 3. PCR amplification and sequencing of OBP6 and OBP10 gene sequences 3.1 Primer Design Two OBP genes (CcreOBP6 and CcreOBP10) were selected, and the N-terminal signal peptide was removed. Characteristic primers were designed, and based on the prokaryotic expression vector diagram, such as... Figure 1 As shown in Table 1, NdeI and XhoI restriction sites (underlined) were designed and added to the primers.

[0020] Table 1 Primers for cloning the CcreOBP6 and CcreOBP10 genes 3.2 PCR amplification and sequencing Using cDNA from the antennae of adult Tibetan tufted-horned longhorn beetle as a template, PCR amplification was performed according to the kit instructions. The amplification volume was 50 μL. The PCR reaction system is shown in Table 2.

[0021] Table 2 PCR reaction system PCR reaction procedure: The PCR products were detected by electrophoresis on a 1% agarose gel.

[0022] Using antennal cDNA from adult Tibetan tufted-horned longhorn beetle as a template, PCR amplification of the CcreOBP6 and CcreOBP10 genes was performed. The results are as follows: Figure 2 As shown in the figure, PCR yielded 386 bp and 395 bp fragments, which are consistent with the size of the target gene fragment.

[0023] 3.3 Construction of expression vector: The PCR product and expression vector were double-digested using two restriction endonucleases, NdeI and XhoI. The digestion system is shown in Table 3.

[0024] Table 3 Enzyme digestion systems After mixing the above enzyme digestion system, the mixture was placed in a 37 ℃ water bath for 15 min, followed by gel digestion and recovery. The target fragment recovered from the gel was then ligated to the pABe vector using T4 DNA Ligase in a 20 μl ligation reaction mixture at 37 ℃ for 5 min. See Table 4.

[0025] Table 4 Connection System The ligation product was transformed into BL21(DE3) competent cells, and the specific steps are as follows: (1) Add the ligation product from the previous step to 50 μL of BL21(DE3) competent cells and place on ice for 30 min.

[0026] (2) Heat shock accurately in a 42 ℃ water bath for 90 seconds, and then quickly place it on ice to cool for 3-5 minutes.

[0027] (3) Add 1 mL of LB liquid culture medium to the tube, mix well, and then culture at 37 °C and 160 rpm for 1 h to allow the bacteria to return to normal growth.

[0028] (4) Centrifuge the above bacterial culture, remove 800 μL of supernatant, and then mix the remaining culture medium by aspiration and spread it on LK screening plates.

[0029] (5) Invert the petri dish and incubate at 37 ℃ for 16-24 h.

[0030] (6) Select approximately 10 clones and place them in 600 mL of LK liquid medium. Place the culture on a shaker (200 r / min, 3-4 h) and perform positive PCR identification. The PCR system follows the same procedure as above. Run the PCR products on a gel. Finally, send the positive clones to a sequencing company for sequencing. The identification results are as follows: Figure 3 As shown, the positive clone was sent to the company for sequencing, and the sequencing results are as follows. Figure 4 and 5 As shown in the figure, the sequencing results are consistent with the sequences of the target genes CcreOBP6 and CcreOBP10.

[0031] 4. Expression and purification of OBP6 and CcreOBP10 proteins 4.1 Induced expression of protein Positive BL21 cells were inoculated into 5 mL of LB liquid medium, and 5 μL of kanamycin was added. The culture was carried out at 37 ℃ and 200 rpm in a full-temperature shaking incubator until the logarithmic growth phase (OD600 = 0.6-0.8). Then, 5 μL of IPTG (1000 mM) was added to a final concentration of 1 mM (the negative control was without IPTG inducer), and the culture was induced at 37 ℃ and 200 rpm in a full-temperature shaking incubator for 4 h.

[0032] 4.2 Protein purification (1) The bacterial cells were resuspended in a lysis buffer and then sonicated.

[0033] (2) For samples where the protein is present in the supernatant, centrifuge at 12000 g for 5 min at 4 ℃ and collect the supernatant. For samples where the protein is present in inclusion bodies, collect the precipitate by low-temperature centrifugation, dissolve the precipitate in denaturing buffer, and then centrifuge to collect the supernatant.

[0034] (3) The supernatant obtained above is used for protein purification via Ni affinity chromatography. The purified protein is then dialyzed, or the refolded protein is dialyzed. The steps are as follows: ① Wash with 5 column volumes of deionized water to remove air and 20% ethanol; ②5-10 column volume buffer equilibration column, buffer: 0.02 M PB, 0.5 M NaCl, 10 mM imidazole; ③ Flow the sample through the nickel column at a rate of 0.5 mL / min; ④ Use the above buffer to balance the columns; ⑤ Elute with 50 mM imidazole, 300 mM imidazole, and 500 mM imidazole respectively; ⑥ Perform SDS-PAGE gel analysis on the eluted samples to determine if the target protein is present.

[0035] Prokaryotic expression vectors of the CcreOBP6 and CcreOBP1 genes were induced with IPTG and then purified to obtain CcreOBP6 and CcreOBP10 proteins using Ni affinity chromatography. SDS-PAGE analysis of the purified proteins is shown below. Figure 6 7. As can be seen from the figure, the molecular weights of CcreOBP6 and CcreOBP10 proteins are approximately 14.7 kDa and 13.0 kDa, respectively, and the protein bands are consistent with the size of the target gene proteins.

[0036] 5. Fluorescence competition binding experiment 5.1 Extraction of volatile substances from walnut bark and leaves Volatile substances from walnut bark and leaves were extracted using a simultaneous distillation-extraction apparatus (Zesangzi et al., 2011). 100 g each of fresh, healthy bark and leaves (after consumption) were chopped and placed separately in 2000 mL round-bottom flasks. 400 mL of distilled water was added, and distillation was carried out for 4 h. Simultaneous extraction was then performed using 20 mL of chromatographically pure n-hexane. Dry anhydrous sodium sulfate was then added to the n-hexane extract for dehydration, and the extract was concentrated using N2 purging.

[0037] 5.2 Gas Chromatography-MS (GC-MS) Testing Chromatographic column: HP-5MS flexible quartz capillary column (30 m × 0.25 mm × 0.25 μm), injection volume 2.0 μL, splitless injection. Temperature program: column temperature 60 ℃, hold for 2 min, ramp to 100 ℃ at 4 ℃ / min, hold for 5 min, ramp to 260 ℃ at 10 ℃ / min, hold for 5 min. Ionization method: EI source, ion source temperature 230 ℃; electron energy 70 eV; interface temperature 280 ℃; mass scan range 40–500 amu. Identification of volatile components: peak times and mass spectra were matched with standard compounds and the NISTD2 mass spectrometry library. The relative contents of various volatile compounds were determined by area normalization (Du Jiawei, 2001).

[0038] 5.3 Fluorescent probe binding experiment Prepare a 50 mM Tris-HCl (pH=7.4) buffer solution. Use N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe and chromatographic grade methanol as the solvent. Dissolve the fluorescent probe 1-NPN and 20 odor standards in methanol to prepare a 1 mM solution. Store the prepared solution at -20 ℃ for later use. Add samples using a 96 Micro Well™ microplate (Nunclon™). Set the excitation wavelength of the multi-functional microplate reader (VARIOSKAN FLASH) to 337 nm and the scanning emission wavelength range to 370–550 nm. Set both the excitation and emission slits to 5 nm.

[0039] To determine the binding constants of CcreOBP6 and CcreOBP10 proteins to the fluorescent probe 1-NPN, CcreOBP6 and CcreOBP10 protein solutions were added to microplates, along with Tris-HCl (pH=7.4) buffer, to bring the final concentrations of CcreOBP6 and CcreOBP10 proteins to 2 μM. 1-NPN concentrations of 2, 4, 6, 8, 12, 16, and 20 μM were added sequentially, and the fluorescence intensity at the highest emission wavelength (Em=410 nm) was recorded after each addition. The experiment was repeated three times.

[0040] The binding affinity of CcreOBP6 and CcreOBP10 proteins to 20 odor standards was determined. CcreOBP6 and CcreOBP10 protein solutions, along with 1-NPN, were added to 96-well Micro Well™ plates. Tris-HCl (pH=7.4) buffer was then added to bring the final concentration of both protein solutions and 1-NPN to 2 μM. After the protein solutions and the fluorescent probe 1-NPN were fully bound, the fluorescence intensity (Em=410 nm) was measured and recorded. Odor standards at concentrations of 2, 4, 6, 8, 12, 16, and 20 μM were then added sequentially, and the changes in fluorescence intensity were recorded. The experiment was repeated three times. The dissociation constant between the protein and the odor standard was calculated using the formula: Ki=[IC50] / (1+[1-NPN] / K1-NPN). Where IC 50 represents the concentration of the odor standard when the fluorescence intensity value is reduced by half, [1-NPN] is the concentration of unbound 1-NPN, and K 1-NPN is the binding constant of the protein and the 1-NPN complex.

[0041] Table 5-5 Odor Standards for Fluorescence Competitive Binding Experiment 4.4 Analysis of the binding characteristics of OBP6 and OBP10 proteins with different odors This experiment selected 20 volatile compounds released from walnut, the host plant of the Tibetan tufted-horned beetle, and their odor-binding proteins for fluorescent competitive binding experiments. These included 9 terpenes, 5 alcohols, 3 ketones, and 3 aldehydes. The results showed that the odor-binding proteins CcreOBP6 and CcreOBP10 from the Tibetan tufted-horned beetle exhibited excellent binding ability to the fluorescent probe 1-NPN. Figure 5-8 9. The dissociation constants of CcreOBP6 and CcreOBP10 are 7.09 and 6.18 μmol / L, respectively. CcreOBP6 does not bind to four odor standards: caryophyllene oxide, 1,6-cyclodecadiene, n-hexanol, and phytol. Figure 5-10 It bound to all 16 other odor standards. The only odor standards with relative fluorescence values ​​below 50% were terpineol (IC50 value of 19.71, Ki value of 15.64 μmol / L) and trans-2-hexenal (IC50 value of 16.25, Ki value of 12.90 μmol / L). The odor standard with the strongest binding ability was trans-2-hexenal, and the odor standard with the weakest binding ability was eucalyptol, with a relative fluorescence value decrease of only 10.84%.

[0042] Depend on Figure 8-11 It can be seen that, except for hexanal and phytol, CcreOBP10 has the ability to bind with all 18 other odor standards. The odor standards with relative fluorescence values ​​decreasing to below 50% include myrcene, 1-caryophyllene, terpineol, and trans-2-hexenal. Among them, CcreOBP10 has the strongest binding ability with geraniol, with an IC50 value of 16.54 and a Ki value of 12.73 μmol / L. Myrcene is the second strongest, with an IC50 value of 17.91 and a Ki value of 13.81 μmol / L. The binding ability with diisobutyl phthalate is the weakest, with a relative fluorescence value decreasing by only 6.4%. sequence list <110> Southwest Forestry University <120> The odor-binding protein gene of the Tibetan tufted horned beetle and its application <130> 20210426 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 369 <212> DNA <213> Tibetan cretifera thibetana (Tibetan tufted-horned longhorn beetle) <400> 1 gcgttatcag agaacaact aaatgcaacg aaaaaacttg tacgaacac atgttttaat 60 aaagctaac cagaaattga acagatagat gcaatgcaga aaggactt cagtggtgac 120 aaaaatgctc agtgttacct gtactgcata ctaaatacgt acaaattgtt atctaaagac 180 aatgcctttg attgggaagg gggtgtcaag gcacttgctg caaacgctcc accaagtgta 240 gctgatccgg gcattataag cataaaaaac tgtaagatg cagtaaaaac cccttcggac 300 aaatgtatgg ctgccacgga atatcaaa tgcatatacg aggacaatcc atcgaattac 360 ttctttcca 369 <210> 2 <211> 142 <212> PRT <213> (Thibetan cretifer hunting) <400> 2 Met Arg Phe Cys Leu Phe Leu Leu Cys Leu Tyr Cys Val Thr His Tyr 1 5 10 15 Val Tyr Thr Ala Leu Serves On Glu Lys Gln Leu Asn On Thr Lys Lys 20 25 30 Val Arg Asn Thr Cys Leu Asn Lys Ala Lys Pro Glu Ile Glu Gln Ile 35 40 45 Asp Ala Met Gln Lys Gly Asp Phe Ser Gly Asp Lys Asn Ala Gln Cys 50 55 60 Tyr Leu Tyr Cys Ile Leu Asn Thr Tyr Lys Leu Leu Ser Lys Asp Asn 65 70 75 80 Ala Phe Asp Trp Glu Gly Gly Val Lys Ala Leu Ala Ala Asn Ala Pro 85 90 95 Pro Ser Val Ala Asp Pro Gly Ile Ile Ser Ile Lys Asn Cys Lys Asp 100 105 110 Ala Val Lys Thr Pro Ser Asp Lys Cys Met Ala Ala Thr Glu Ile Ser 115 120 125 Lys Cys Ile Tyr Glu Asp Asn Pro Ser Asn Tyr Phe Phe Pro 130 135 140 <210> 3 <211> 345 <212> DNA <213> Tibet Cluster-horned Elephant Longhorn Beetle (Cacia cretifera thibetana) <400> 3 ctaagtgagg aaatgcagga attagcaaac atgcttcata atacttgttt ggaagaaaca 60 gggacattag aagattatat agaaaaagcc aggggaggag actttactga tgacgaaaaa 120 tttaaatgct atattatgtg cattatggcg caaatggcat gtatagacga agacggtgtt 180 atagacgtag aagcaacgat agcagttatt ccagaagaat atcaagact ggcggcacca 240 atcataagga aatgtgatac gcaaaggga tccacctt gtgagaatgc tggctgaca 300 cataaatgtt attaatga aaactccaag gcttacttct tagtt 345 <210> 4 <211> 136 <212> PRT <213> (Thibetan cretifer hunting) <400> 4 Met Val Asn Lys Leu Ser Ala Val Cys Ala Leu Leu Leu Phe Val Phe 1 5 10 15 Pro Phe Val Arg Gly Leu Ser Glu Glu Met Gln Glu Leu Ala Asn Met 20 25 30 From His Asn Thr Cys to Glu Glu Thr Gly Thr to Glu Asp Tyr Ile 35 40 45 Glu Lys Ala Arg Gly Gly Asp Phe Thr Asp Asp Glu Lys Phe Lys Cys 50 55 60 Tyr With Cys And Met Ala Gln Met Ala Cys With Asp Glu Asp Gly 65 70 75 80 Val Island Asp Val Glu Ala Thr Island Ala Val Island Pro Glu Glu Tyr Gln 85 90 95 Glu Leu Ala Ala Pro Ile Ile Arg Lys Cys Asp Thr Gln Lys Gly Ser 100 105 110 Thr Pro Cys Glu Asn Ala Trp Leu Thr His Lys Cys Tyr Tyr Asn Glu 115 120 125 Asn Ser Lys Ala Tyr Phe Leu Val 130 135

Claims

1. A gene that encodes the odor-binding protein OBP6 from the Tibetan tufted-horned longhorn beetle, characterized by: The sequence of the gene is selected from SEQ ID No. 1.

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