CN113563448B - Protein PtsuOBP39 combined with cinnamomum camphora volatile compound and insect pheromone, attractant and application thereof - Google Patents

Abstract

The invention provides a protein PtsuOBP39 combined with camphor volatile compounds and insect pheromone, an attractant and application thereof, and belongs to the technical field of molecular biology. The odor binding protein is PtsuOBP39 amino acid sequence shown as SEQ ID NO:1, and the coding gene is shown as SEQ ID NO:2, respectively. The protein and the gene can be used for reversely verifying the screening of key components of the insect pest cinnamomum camphora tooth beak attractant, lay a foundation for analyzing an olfactory molecular mechanism of the cinnamomum camphora tooth beak, and provide a reference basis for developing an insect pest prevention and control technology taking an insect pest olfactory key gene as a target. The 1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethyl bicyclo [4.1.0] heptane-3-alcohol and the cinnamomum camphora volatile compound which can be specifically combined with the protein can be used for preparing an attractant, and a new idea is provided for monitoring and controlling the cinnamomum camphora tooth beak condition.

Description

Protein PtsuOBP39 combined with cinnamomum camphora volatile compound and insect pheromone, attractant and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a protein PtsuOBP39 combined with camphor volatile compounds and insect pheromone, an attractant and application thereof.

Background

The toothed beak weevil of cinnamomum camphora (Pagiopthlees tsushimanus) is Coleptera (Coleptera), weevil (Curculionidae) and the toothed beak weevil (Pagiopthlees) and is a new recorded species of Chinese insect which damages cinnamomum camphora by boring. Adult insects mainly eat the epidermis of 1-2-year-old twigs, larvae eat cambium, and when the density of insect population is high, the growth vigor of the cinnamomum camphora is weakened or the whole cinnamomum camphora is withered. At present, the camphor tooth proboscis is only distributed in Shanghai city in China, 1 generation occurs in 1 year, and larvae and adults live through the winter.

Insects sense odor substances in the external environment through an olfactory system to regulate the behaviors of the insects, and methods for regulating the behaviors of the insects, which are developed based on an olfactory sensing mechanism of the insects, are applied to the control of agricultural and forestry pests. At present, a plurality of insect pheromones are used for monitoring and controlling pests, however, the process of screening the odor molecules with physiological activity to the pests needs to spend a great deal of manpower, material resources and financial resources, which seriously restricts the screening, popularization and application of the odor molecules with physiological activity. In view of the above, some researchers have proposed the concept of "Reverse Chemical Ecology", that is, screening a volatile Chemical substance having biological activity by using the binding force between an odorant molecule and an olfactory-related protein, rather than the conventional method of observing the insect behavioral reaction caused by a large amount of odorant molecules one by one, the workload of screening the odorant having biological activity can be significantly reduced, and the work efficiency can be significantly improved.

Odorous Binding Proteins (OBPs) are one of the major classes of insect olfactory-related Proteins, and are water-soluble acidic Proteins secreted by supporting cells within the insect olfactory sensor, infiltrating into the sensory lymph and distributed around the axons of sensory neurons. And is the first type of olfactory protein that an odorant molecule first contacts after entering an antenna sensor. The OBPs are necessary carrier proteins for insects to recognize external odorants, and the main functions of the OBPs are to bind and transport external odorant molecules to pass through hydrophobic lymph fluid of an antenna sensor to be released to olfactory receptors, so that chemical signals are converted into electric signals, therefore, the OBPs play a decisive role in the olfactory recognition process of insects, and the function of the OBPs is determined to be crucial to understanding the olfactory sensation system of insects.

Fluorescent competitive binding experiments (fluorescence competitive binding assays) are one of the main methods for studying the binding ability of insect OBPs to odor molecules, and have been widely applied to functional studies of insect OBPs, such as cockroaches (Leucophaea maderae), Holotrichia parallela (Holotrichia oblia), Antrodia camphorata (Anthragma achatina), Monochamus alternatus (Monochamus alternatus), Hyphantria cunea (Hypopharia cunea) and Holotrichia parallela (Holotrichia parallela).

At present, various insect OBPs have been cloned and researched, but the Cinnamomum camphora peck weevil adult odor binding protein OBPs have not been reported yet. The research on the adult odorant binding protein OBPs of the cinnamomum camphora tooth beak weevil not only lays a foundation for analyzing an olfactory molecular mechanism of the cinnamomum camphora tooth beak weevil as an insect pest, but also provides a reverse verification method for identifying key components of a host plant cinnamomum camphora volatile compound, and also provides a reference basis for further developing a pest monitoring, prevention and control technology taking an insect olfactory key gene as a target.

Disclosure of Invention

The invention aims to provide a cinnamomum camphora tooth peck odor binding protein PtsuOBP39 which can be combined with a cinnamomum camphora tooth peck host plant cinnamomum camphora volatile compound, can also be combined with insect pheromone, and has wider odor binding capacity.

The invention provides a protein PtsuOBP39 combined with a camphor volatile compound and insect pheromone, wherein the amino acid sequence of PtsuOBP39 is shown as SEQ ID NO. 1.

The invention provides application of the PtsuOBP39 in identification/combination of cinnamomum camphora volatile compounds and/or insect pheromone.

Preferably, the camphor volatile compound comprises one or more of the following compounds: camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene;

the dissociation constant of the PtsuOBP39 and camphor, ocimene, linalool, alpha phellandrene or beta caryophyllene is 10 mu M < Ki <20 mu M.

Preferably, the somatotropin comprises (1 α,3 α,4 α,6 α) -4,7, 7-trimethylbicyclo [4.1.0] heptan-3-ol;

the PtsuOBP39 has a dissociation constant of 1. mu.M < Ki < 2. mu.M with (1. alpha., 3. alpha., 4. alpha., 6. alpha.) -4,7, 7-trimethylbicyclo [4.1.0] heptan-3-ol.

The invention provides a coding gene of a protein PtsuOBP39 combined with a camphor volatile compound and insect pheromone, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

The invention provides a recombinant vector for expressing a foreign gene, wherein the foreign gene comprises the coding gene.

The invention provides application of the coding gene or the recombinant vector in recombinant expression of protein PtsuOBP39 combined with camphor volatile compounds and insect pheromone.

The invention provides a cinnamomum camphora tooth elephant attractant, which comprises the active ingredients of (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0] heptane-3-alcohol and/or one or more of the following compounds: camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene.

The invention provides application of the attractant in monitoring and/or controlling the cinnamomum camphora tooth proboscis.

The amino acid sequence of the protein PtSuOBP39 combined with the camphor volatile compound and the insect pheromone is shown as SEQ ID NO. 1, and the coding gene is shown as SEQ ID NO. 2. The protein and the gene provided by the invention can be used for reversely verifying the identification of the key components of the volatile information compound of the host plant cinnamomum camphora, also lay a molecular foundation for analyzing the olfactory molecular mechanism of the cinnamomum camphora with the odontoid rhynchophorus as the harmful cinnamomum camphora, and simultaneously provide a reference basis for developing a pest monitoring and controlling technology taking the insect olfactory key gene as a target spot. The invention also screens and obtains a plurality of volatile substances with strong binding force with the camphor tooth proboscis imago odor binding protein PtsuOBP39 through a fluorescence competitive binding experiment, which comprises the following steps: (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0] heptane-3-ol (imago insect pheromone) and camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene (host plant volatile matters) and the like can be used as candidate components for preparing an attractant for monitoring and controlling the camphor tooth beak image.

Drawings

FIG. 1 is a graph showing the relative expression amount of PtsuOBP39 gene in tissues of cinnamomum camphora kok-shaped male and female adults;

FIG. 2 is a diagram of the result of an SDS-PAGE analysis of PtsuOBP39 purification, note: in the figure, a lane M is Marker; 3 is the purified and collected target protein PtsuOBP 39;

FIG. 3 is a combination graph (a) and a Scatchard graph (b) of PtsuOBP39 and 1-NPN;

fig. 4 is a graph showing the fluorescent competitive binding of PtsuOBP39 with cinnamomum camphora volatile compound (a) and imago pheromone (b).

Detailed Description

The invention provides a protein PtSuOBP39 combined with a camphor volatile compound and insect pheromone, wherein the amino acid sequence of PtSuOBP39 is shown as SEQ ID NO:1 (MGKSAIFLCGICIYFLGVVQLTPVPYQHLSKEELHHIALECIEEVNIDRAVIEKVLKTQILPKENKKYKRYLECSYKKQGFLSPDGTQMLYNNLFQFLQRFYDRSELHALDQCKLIKAEDGGELCFQNLDCILNGLRTIENQKAIISNDIS). The PtsuOBP39 protein is coded by 151 amino acids, and the amino acid sequence is shown as the specification; comprises a signal peptide 21 aa; the molecular mass of the protein is 15.19 kDa; the isoelectric point was 6.07.

By verifying that the PtsuOBP39 has biological functions of being combined with the cinnamomum camphora volatile compounds and the insect pheromone, the invention provides application of the PtsuOBP39 in identifying/combining the cinnamomum camphora volatile compounds and/or the insect pheromone. The camphor volatile compounds comprise one or more of the following compounds: camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene.

In the present invention, the PtsuOBP39 function verification is performed by a fluorescent competitive binding assay. The fluorescence competitive binding experiment is used for pre-determining the applicability analysis of the fluorescent probe 1-NPN, and the result shows that the fluorescence values of PtsuOBP39 and 1-NPN have saturation effect along with the increase of the concentration of the 1-NPN, and simultaneously shows that PtsuOBP39 has a single binding site and can be used for fluorescence competitive bindingForce measurement, binding constant K of PtsuOBP39 and 1-NPN_1-NPN5.74 ± 0.48. By analyzing the volatile compounds of 9 host plants, namely cinnamomum camphora, the result shows that the dissociation constant of PtsuOBP39 and camphor, ocimene, linalool, alpha phellandrene or beta caryophyllene is 10 mu M < Ki <20 mu M. The imago body pheromone is as follows: (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0]Heptane-3-ol. The PtsuOBP39 and (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0]Dissociation constant of Heptane-3-ol 1. mu.M<Ki<2μM。

The invention provides a coding gene of the protein PtsuOBP39 combined with a camphor volatile compound and insect pheromone, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2 (ATGGGAAAAAGTGCAATATTTTTGTGTGGTATTTGTATCTACTTTTTGGGAGTAGTACAGCTTACACCTGTTCCATACCAACATTTATCAAAAGAAGAACTGCATCACATTGCTTTAGAATGTATAGAAGAAGTAAATATTGACAGAGCAGTTATAGAAAAAGTTCTTAAAACTCAGATACTTCCCAAAGAAAACAAAAAATACAAACGATATTTAGAATGCAGCTACAAAAAACAGGGATTTTTGTCACCAGATGGGACTCAAATGCTATATAACAACTTATTTCAGTTTTTGCAACGCTTTTATGATAGATCAGAATTGCATGCTTTAGATCAGTGCAAATTAATTAAAGCGGAAGATGGTGGAGAGTTGTGTTTTCAAAATTTGGACTGCATATTGAATGGGCTTAGGACGATAGAAAATCAAAAGGCCATTATCTCAAATGATATATCATAA). The coding gene is obtained by using cDNA of the cinnamomum camphora tooth elephant as a template and amplifying ORF of PtsuOBP39 gene by PCR. The primer for PCR amplification comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 (5'-ATGGGAAAAAGTGCAATATTTTTGT-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 (5'-TTATGATATATCATTTGAGATAATG-3'). The reaction system for PCR amplification is 750 ng/. mu.L cDNA template 1. mu.L, 2 XPrimerSTAR Max Premix 25. mu.L, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu.L, RNase Free ddH₂O was supplemented to 50. mu.L. The reaction program of the PCR amplification is 94 ℃ for 10 min; 10sec at 98 ℃, 30sec at 50 ℃ and 60sec at 72 ℃; 35 cycles; 2min at 72 ℃; keeping at 4 ℃.

The invention provides a recombinant vector for expressing a foreign gene, wherein the foreign gene comprises the coding gene. The backbone vector of the recombinant vector is not particularly limited in the present invention, and an expression vector well known in the art, such as TA/Blunt-Zero vector, may be used. The construction of the recombinant vector is preferably accomplished using the TA/Blunt-Zero Cloning Kit.

The invention provides application of the coding gene or the recombinant vector in recombinant expression of protein PtsuOBP39 combined with camphor volatile compounds and insect pheromone.

In the recombinant expression method of the cinnamomum camphora tooth beak-like odor binding protein PtsuOBP39, the recombinant vector constructed by the technical scheme is preferably introduced into a prokaryotic expression system, and the recombinant expression of the cinnamomum camphora tooth beak-like odor binding protein PtsuOBP39 is obtained through screening culture and induction.

The smell binding protein PtsuOBP39 based on the cinnamomum camphora tooth beak has specific binding capacity on the volatile compound of the host plant cinnamomum camphora and the sex pheromone of the adult cinnamomum camphora tooth beak, so that the cinnamomum camphora tooth beak attractant is provided. The active ingredients are preferably sequentially: (1 α,3 α,4 α,6 α) -4,7, 7-trimethylbicyclo [4.1.0] heptane-3-ol > ocimene > β caryophyllene > camphor > α phellandrene > linalool.

The PtsuOBP39 protein binding to cinnamomum camphora volatile compound and pheromone, attractant and use thereof provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

Cloning of PtsuOBP39 Gene

Raising of camphor tooth elephant

During feeding, the cinnamomum camphora teeth are separated from adult male and female insects by a gauze in the middle of a feeding box, and the adult male and female insects emerging in the same batch (35 days old, normally fed but not mated) are collected for sequencing transcriptome. The male and female adults are respectively provided with 3 independent biological repeats, and each repeat comprises 6 adults with the same sex. All samples were collected simultaneously and washed clean with 75% ethanol, immediately frozen rapidly in liquid nitrogen, stored at-80 ℃ or directly subjected to total RNA extraction.

(II) extracting RNA of cinnamomum camphora tooth elephant male and female adults

The method for extracting the total RNA of the cinnamomum camphora adult female and male in the beak like state by using a TRIzol (TaKaRa, Japan) method of Boehmeria biological engineering (Dalian) Co., Ltd.) comprises the following steps:

(1) placing each collected sample in a 1.5mL of an EP tube without RNase, adding 500. mu.L of TRIzol, and sufficiently grinding the sample with a grinding rod;

(2) after fully grinding, adding 500 mu LTRIzol to the EP tube again, and standing for 5min at room temperature;

(3) after centrifugation at 12000rpm for 10min at 4 ℃, the supernatant was transferred to a new RNase-free 1.5mL EP tube;

(4) adding 200 mu L of chloroform into a new EP tube, covering the centrifugal tube, violently shaking for 15sec, and standing for 3min at room temperature;

(5) centrifuging at 12000rpm at 4 deg.C for 15min, separating the mixture into three layers, and transferring the colorless water phase (RNA dissolved in water phase) to new RNase-free EP tube;

(6) adding isopropanol with the same volume as the colorless water phase obtained in the step (5), mixing uniformly, and standing at room temperature for 30min to fully precipitate RNA;

(7) centrifuging at 4 deg.C and 12000rpm for 10min, and removing supernatant;

(8) adding 1mL of 75% ethanol into the obtained RNA precipitate to wash the precipitate, centrifuging at 4 ℃ and 5000rpm for 5min, and carefully removing the supernatant;

(9) then repeating the step (8) three times;

(10) air drying at room temperature for 5-10 min, adding 20 μ L RNase Free ddH₂Dissolving the precipitate completely to obtain RNA solution, and storing at-80 deg.C;

(11) nanodrop 2000 is selected to detect the concentration and purity, OD, of RNA sample_260/280The value of (A) is between 1.8 and 2.1; the presence or absence of genomic residues or protein contamination and the integrity of the RNA bands were checked by electrophoresis in a 1.5% agarose gel.

(III) cloning of PtsuOBP39 Gene

Analyzing ORFs of the PtSUOBP39 gene according to Open Reading Frame (ORFs) predicted websites (http:// www.ncbi.nlm.nih.gov/gorf. html), and designing specific primers of the PtSUOBP39 gene by using Primer Premier 5.0 software to carry out PCR amplification on ORF of the PtSUOBP39 gene;

the specific primers are as follows:

F：5′-ATGGGAAAAAGTGCAATATTTTTGT-3′(SEQ ID NO:3)；

R：5′-TTATGATATATCATTTGAGATAATG-3′(SEQ ID NO:4)。

the reaction system is shown in Table 1.

TABLE 1 PCR amplification reaction System

cDNA template	1μL(750ng/μL)
		PrimerSTAR Max Premix(2×)	25μL
Forward primer (10. mu.M)	1μL
		Reverse primer (10. mu.M)	1μL
RNase Free ddH2O	Make up to 50 μ L

After the mixture is flicked and mixed, the reaction solution is collected to the bottom of the tube by low-speed short-time centrifugation, and the reaction procedure is shown in table 2.

TABLE 2 PCR amplification reaction procedure

(4) After the reaction, agarose gel (2%) electrophoresis was performed to determine whether the size of the PCR product band was the same as that of the target gene.

(5) And (5) cutting and recovering the target strip by using a glue recovery kit. The method comprises the following steps:

firstly, placing agarose gel in an ultraviolet environment, cutting a part containing a target fragment, and minimizing the number of the fragments;

transferring the rubber block into an EP tube with the volume of 2mL, adding 400 mu L of Binding Solution, setting a metal bath at 60 ℃, and heating the rubber block in the EP tube until the rubber block is completely dissolved;

thirdly, the mixed solution is moved into an adsorption tube with a filter membrane, stands for 2min at room temperature, and is centrifuged for 1min at 6000 rpm;

fourthly, repeating the third step to remove residual liquid in the collecting pipe;

fifthly, adding 500 mu L WA into the adsorption tube, centrifuging at 12000rpm for 1min, and removing residual liquid in the collection tube;

sixthly, adding 500 mu L of Wash Solution into the adsorption tube, centrifuging for 1min at 12000rpm, and removing residual liquid in the collection tube;

seventhly, repeating the step sixthly, and removing residual liquid in the collecting pipe;

then centrifuging at 12000rpm for 2min, transferring the adsorption tube to a new 1.5mL EP tube, and standing at room temperature for 10 min;

ninthly, dropping 20 mu L of RNase Free ddH on the filter membrane in the middle of the adsorption tube₂O, standing for 3min at room temperature, and centrifuging at 12000rpm for 2min to obtain a product recovered from the glue;

purity and concentration of recovered product were checked by agarose gel electrophoresis (2%) and Nano Drop 2000.

(6) The product after gel cutting recovery and purification was ligated to the vector TA/Blunt-Zero, the ligation reaction system is shown in Table 3.

TABLE 3 ligation reaction System

Glue recovery product	1～4μL
		5×TA/Blunt-Zero Cloning Mix	1μL
RNaseFreeddH2O	Make up to 5. mu.L

(7) Introducing the recombinant plasmid obtained in the step (6) into a competent cell DH5 alpha, and carrying out clone screening and purification by the following specific steps:

putting a competent cell DH5 alpha in an ice-water mixture for thawing;

when the mixture is completely thawed, subpackaging the mixture into 50 mu L of competent cells in each tube, respectively adding 5 mu L of the recombinant plasmid obtained in the step (6), flicking the tube bottom to uniformly mix the recombinant plasmid, and standing the mixture in an ice water mixture for 30 min;

taking out the centrifugal tube after the standing is finished, thermally shocking the centrifugal tube for 45sec at 42 ℃, and quickly taking out the centrifugal tube and transferring the centrifugal tube to an ice-water mixture for standing for 2 min;

fourthly, 900 mu L of liquid LB culture medium is added into the mixed liquid after the reaction of the third step, and the mixed liquid is placed in a shaking table at 37 ℃ and 200rpm for shaking culture for 1h to recover competent cells;

fifthly, after shaking for 1h, centrifuging at 2500rpm for 3min, taking out 900 mu L of supernatant by using a pipettor, removing, after re-suspending the thalli, uniformly coating on an LB plate containing ampicillin resistance;

sixthly, transferring the flat plate into an incubator at 37 ℃ for several minutes until the bacterial liquid is dried, inverting the flat plate and culturing overnight;

seventhly, respectively selecting 3 monoclones, transferring the monoclones into 1mL of LB liquid culture medium, shaking for 4 hours on a shaking table, streaking the bacterial liquid on an LB flat plate for culture, then respectively selecting 6 monoclones, and shaking for 4 hours by the same method;

subjecting the purified monoclonal bacteria liquid to PCR verification by using an M13 primer, finally selecting 6 positive clones, sequencing to obtain a sequence and verifying the accuracy of the sequence.

The ORF sequence of the gene of PtsuOBP39 obtained by sequencing is shown as SEQ ID NO:2, the expression protein is shown as SEQ ID NO:1 is shown.

Bioinformatic analysis of the (tetra) PtsuOBP39 protein

The ExPasy software shows that the PtsuOBP39 protein is encoded by 151 amino acids, and the amino acid sequence is shown in the specification; comprises a signal peptide 21 aa; the molecular mass of the protein is 15.19 kDa; the isoelectric point was 6.07.

Example 2

Relative expression level of PtsuOBP39 gene in cinnamomum camphora rostrum adult tissue

Raising Cinnamomum camphora (I) toothed rhynchophylla and collecting samples

The wild collection and breeding of the camphor tooth proboscis were the same as in example 1. During breeding, the male and female imagoes are separated by a gauze in the middle of a breeding box, and the same batch of eclosion male and female imagoes (35 days old, normally fed but not mated) are collected for dissection. The male and female adults are respectively provided with 3 independent biological repeats, and each repeat comprises 30 adults with the same sex. All samples were collected simultaneously and washed clean with 75% ethanol, immediately dissected on ice to obtain tissues such as tentacles, heads (without tentacles), breasts, abdominal ends, wings and feet, and rapidly frozen in liquid nitrogen, and immediately stored at-80 ℃ or directly subjected to total RNA extraction.

(II) Total RNA extraction of Each sample

The method and procedure were as in example 1.

(III) qRT-PCR primer design and amplification efficiency calculation thereof

Based on the ORF of the sequence-verified PtsuOBP39 gene, qRT-PCR primers for the PtsuOBP39 gene were designed using the in-line program (https:// www.ncbi.nlm.nih.gov/tools/primer-blast /). The qRT-PCR amplification primers were as follows:

F：5′-TGTCACCAGATGGGACTCAA-3′(SEQ ID NO:5)；

R：5′-GCCCATTCAATATGCAGTCCA-3′(SEQ ID NO:6)。

the cDNA template concentration of the sample is diluted to 500 ng/. mu.L, and then diluted by 10 times for 4 times, finally cDNA templates with 5 concentrations, 500 ng/. mu.L and 500X 10 respectively, are obtained^-1(50ng/μL)、500×10^-2(5ng/μL)、500×10^-3(0.5 ng/. mu.L) and 500X 10^-4(0.05 ng/. mu.L). The reaction system for qRT-PCR is shown in Table 4.

TABLE 4 reaction System for qRT-PCR

Hieff qPCR SYBR Green Master Mix	10μL
		Template cDNA	2μL
Primer (F/R)	1+1μL
		RNase Free ddH2O	6μL

The reaction solution was collected to the bottom of the tube by brief centrifugation at low speed, and the reaction procedure of qRT-PCR is shown in Table 5.

TABLE 5 reaction procedure for qRT-PCR

Each treatment comprises 3 biological repetitions and 3 technical repetitions, respectively, amplifying the standard curve to obtain the correlation coefficient R of the standard curve equation²And slope, calculating the amplification efficiency of each primer according to a formula.

Amplification efficiency E ═ 10^{[－1/slope]－1)} X 100 formula I.

After the reaction, the dissolution curve of the PtsuOBP39 gene qRT-PCR primer is shown to be a single peak, which indicates that the primer has specificity and no primer dimer exists. In addition, amplification characteristics of the PtsuOBP39 gene qRT-PCR primers are shown in table 6 below.

TABLE 6 amplification characteristics of the PtsuOBP39 Gene qRT-PCR primers

Name of Gene	Read length (bp)	Slope	Efficiency (%)	Coefficient of correlation R2	Linear regression equation
						PtsuOBP39	162	－3.2255	104.188	0.9939	y＝－3.2255x+36.713

The amplification efficiency of the primers was 104.188%, which ranged from 80% to 120%, indicating that the amplification was good for the primer specificity. The PtsuOBP39 gene has the highest relative expression in antennal tissues of male and female adults and is obviously higher than other tissues; the relative expression level in the abdomen of male and female adults is the second; there was no significant difference in the relative expression levels between the same tissues of the male and female adults (see FIG. 1).

Example 3

Prokaryotic expression and purification of PtsuOBP39

(I) prokaryotic expression vector construction based on homologous recombination principle

(1) Removing a signal peptide sequence (SEQ ID NO: 9: ATGGGAAAAAGTGCAATATTTTTGTGTGGTATTTGTATCTACTTTTTGGGAGTAGTACAGCTT) of the PtsuOBP39 gene, and designing target fragment primers containing homologous sequences at two ends of a linearized vector by using a primer Design software CE Design (http:// www.vazyme.com, Novozao network) according to a sequence without the signal peptide and a sequence of a prokaryotic expression vector pET28a (+); the primers for constructing the PtsuOBP39 prokaryotic expression vector are as follows:

F：5′-cgcggatccgaattcgagctcACACCTGTTCCATACCAACATTTATC-3′(SEQ ID NO:7)；

R：5′-tggtggtgctcgagtgcggccgcTTATGATATATCATTTGAGATAATGGCC-3′(SEQ ID NO:8)。

(2) the expression vector pET28a (+) was double digested with two restriction enzymes to linearize pET28a (+) and the PCR reaction is shown in Table 7.

TABLE 7 reaction System for PCR

10×QuickCut Buffer	5μL
		Restriction enzyme I	1μL
Vector pET28a(+)	<1μg
		RNase Free ddH2O	Up to 50μL

(3) After flicking and mixing uniformly, carrying out low-speed short-time centrifugation, collecting the reaction solution to the bottom of the tube, and incubating for 15min at 37 ℃ in a PCR instrument;

(4) after the end, 1 mu L of restriction enzyme II is added, and the mixture is incubated for 15min in a PCR instrument at 37 ℃;

(5) selecting a 1.5% agarose gel electrophoresis detection product, cutting the gel of a target band, and recovering and purifying;

(6) selecting high-fidelity enzyme and target fragment primers containing homologous sequences to perform PCR amplification, product gel cutting recovery and purification, vector connection and sequencing by taking the cloned plasmid as a template;

(7) and (3) recombination reaction: the recombinant reaction system was prepared on ice as shown in Table 8.

TABLE 8 recombination reaction System

5×CE Ⅱ Buffer	2μL
		Linearized fragments	80ng
Linearized vector	100ng
		Exnase Ⅱ	1μL
RNase Free ddH2O	Up to 10μL

After flicking and uniformly mixing, carrying out low-speed short-time centrifugation to collect reaction liquid to the bottom of the tube, and incubating for 30min at 37 ℃ in a PCR instrument;

(8) and (3) transformation of a recombinant product: selecting a DH5 alpha cloning recombinant plasmid PtSuOBP39/pET28a (+), selecting M13F/R to verify positive cloning, and finally sequencing and verifying;

(9) extracting and transforming positive clone plasmids:

taking 10mL of overnight shake-cultured bacterial liquid respectively, centrifuging for 1min at 12000rpm, and removing a culture medium;

adding 500 mu L of Solution I to fully resuspend the thalli, then adding 500 mu L of Solution II, quickly reversing, fully mixing to obtain a transparent egg white shape, finally adding 700 mu L of Solution III, fully mixing, standing at room temperature for 5min, and centrifuging at 12000rpm for 10 min;

thirdly, respectively adding the supernatant into a special adsorption tube for plasmid extraction (an adsorption column is subjected to balance treatment) for several times, centrifuging at 12000rpm for 1min, removing the waste liquid in the collection tube, and repeating the steps until the waste liquid is completely removed;

fourthly, adding 1500 mu L Solution W into the tube, centrifuging for 1min at 12000rpm, and removing waste liquid in the collecting tube;

adding 600 mu L of Wash Solution into the tube, centrifuging at 12000rpm for 1min, removing the waste liquid in the collecting tube, and repeating the steps until the waste liquid is completely removed;

sixthly, centrifuging for 2min at 12000rpm, transferring the adsorption tube to a new 1.5mL EP tube, and airing for 10min at room temperature;

seventhly, RNase Free ddH is dripped into the middle of the membrane in the adsorption tube₂O100 mu L, standing for 2min at room temperature, and centrifuging at 12000rpm for 2min to obtain purified plasmid;

detecting the purity and concentration of the plasmid by agarose gel (1.5%) electrophoresis and Nano Drop 2000;

ninthly, transferring the positive clone plasmid into an expression carrier BL21(DE3), prolonging the heat shock time to 90sec, taking the positive bacterial plaque for shake culture, taking the monoclonal after streak purification for shake culture again for 5h, and after PCR detection of bacterial liquid, sending to sequencing verification and reserving the strain for later use.

(II) inducing expression of recombinant plasmid

Inducing expression by the recombinant plasmid: after activation of the retained PtsuOBP39 glycerol bacteria, the ratio of 1: 100 into 1L liquid LB medium (containing Kana);

selecting isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.6mM, and inducing at the temperature of 20 ℃;

other same conditions and steps are as follows: culturing at 150rpm until OD value is 0.6-0.8, adding IPTG, and performing induced culture for 12 hr. The cells were centrifuged at 12000rpm at 4 ℃ for 15min, and the cells were collected, 100mL of buffer (50mM PB, 300mM NaCl, 20mM imidazole, pH 7.4) was added to resuspend the cells, and PMSF, a protease inhibitor, was added to a final concentration of 1mM, and the resuspension was sonicated in an ice bath (sonication cycle: 3sec, 5sec off) and centrifuged at 12000rpm for 30min, and the supernatants were collected. Respectively taking 20 mu L of the supernatant, respectively adding 5 mu L of Loadingbuffer, uniformly mixing, carrying out water bath at 100 ℃ for 10min, carrying out short-time centrifugation, collecting to the bottom of the tube, and detecting the protein expression condition by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE, 100V, 120min) in combination with a Coomassie brilliant blue staining and decolorizing method.

(III) purification of Key PtsuOBP39 recombinant protein

Filtering the supernatant after ultrasonic crushing with a filter membrane with the aperture of 0.2 mu m;

② the heavy suspension balances the nickel ion resin gravity column (1mL Ni +/column) for standby;

thirdly, the supernatant fluid filtered by the filter membrane passes through the gravity column (2 times) successively;

fourthly, eluting the hybrid protein in the supernatant of the target protein by using 6mL of multiplied by 4Wash buffer (50mM PB, 300mM NaCl, 80mM imidazole, pH 7.4);

fifthly, eluting and collecting target protein by 300 uL multiplied by 5 elusion buffer (300mM NaCl, 300mM imidazole, 50mM PB, 10% glycerol, 1mM PMSF, pH 7.4);

sixthly, the collected target proteins are respectively put into dialysis bags, and each target protein is dialyzed for 16 hours at the temperature of 4 ℃ by respectively using dialysate I (200mM NaCl, 200mM imidazole, 40mM PB, and the pH value is 7.4); dialyzing each protein of interest against dialysate II (100mM NaCl, 100mM imidazole, 20mM PB, pH 7.4) under the same conditions for a further 16 h; finally transferring the mixture into pure water for dialysis for 16 hours;

seventhly, freezing and dialyzing the target protein at the temperature of minus 80 ℃ for 4 hours, and then transferring the target protein to a freeze dryer for freeze drying until the protein is completely dried into powder;

(iii) RNase Free ddH₂Dissolving the dried target protein by using O;

ninthly, detecting the purified target protein solution by SDS-PAGE (100V, 120min), and storing at-80 ℃ for later use.

The purification result of PtsuOBP39 is shown in fig. 2, and PtsuOBP39 obtained by purification and collection can be used for carrying out functional verification of the odor binding protein.

Example 4

Functional verification of PtsuOBP39

The fluorescent competitive binding experiment of the target protein comprises the following steps:

preparing a protein solution and a ligand odor molecule solution: the lyophilized protein powder of PtsuOBP39 was dissolved in 50mM Tris-HCl buffer (pH 7.4), and the concentration of the protein was measured with a NanoDrop 1000 nucleic acid protein quantifier. Chromatographic grade methanol is selected to respectively dissolve fluorescent probe N-phenyl-1-naphthylamine (N-phenyl-l-naphthylamine, 1-NPN), host plant camphor volatile compounds (camphor, ocimene, d-borneol, linalool, eucalyptol, 3-carene, trans-nerolidol, alpha phellandrene and beta caryophyllene) and adult insect body pheromones (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0] heptane-3-ol and other ligand odor molecules, 10mM mother liquor is prepared and stored at-20 ℃ for later use, and the mother liquor is diluted into 1mM working solution by the methanol during experiments.

(II) suitability determination of the fluorescent probe 1-NPN: the excitation wavelength of the fluorescent probe 1-NPN is 337nm, a proper amount of 20mM Tris-HCl (pH 7.4) buffer solution (the total volume of the reaction system is 250 muL) is respectively added into micropores of a 96 black microplate, then the fluorescent probe 1-NPN is respectively added to enable the final concentration to be 2 muM, a scanning method (Spectrum) is selected to carry out scanning measurement under the condition of the wavelength range of Em 400-500 nm, the fluorescence emission peak exists at the wavelength of 460nm, the addition amount is calculated according to the concentration of the target protein PtSuOBP39, the final concentration of the target protein PtSuOBP39 is 2 muM, the target protein is fully combined with the 1-NPN after reaction for 2min at room temperature, and whether the fluorescence emission peak of the reaction system has obvious blue shift and the fluorescence intensity is obviously increased or not is detected under the same experimental conditions. Otherwise, other kinds of probes are selected for retesting. Subsequently, 1-NPN was added to 2 μ M of the target protein solution in series to give final concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μ M, respectively, and the reaction was carried out at room temperature for 2min, and fluorescence values were recorded, respectively, and 3 technical repetitions were carried out for the target protein PtSuOBP39, respectively. The experimental parameter conditions are as follows: the exciting light (Ex) is 337nm, and the absorbing light (Em) is a value corresponding to the maximum fluorescence emission peak after blue shift of the combined spectrum of 1-NPN and PtsuOBP 39. The fluorescence values from PtsuOBP39 were plotted against the concentration of fluorescent probe 1-NPN and the binding constants were calculated. Meanwhile, a Scatchard method linearization curve is selected, the abscissa is the concentration of the combined ligand 1-NPN, and the ordinate is the ratio of the concentration of the combined ligand 1-NPN to the concentration of the free ligand 1-NPN. Therefore, whether the fluorescent probe 1-NPN is suitable for the fluorescent competitive binding experiment of PtSUOBP39 or not is detected. If the combination of the PtSUOBP39 and the fluorescent probe 1-NPN has a saturation effect and a remarkable Scatchard linearization relationship, a single combination site exists between the target protein PtSUOBP39 and the fluorescent probe 1-NPN and the influence of an allosteric effect does not exist, so that the fluorescent probe 1-NPN is suitable for a subsequent fluorescent competitive combination experiment.

(III) competitive binding experiment of ligand odor molecules: an appropriate amount of 20mM Tris-HCl (pH 7.4) buffer (250 μ L total reaction volume) was added to each well of a 96 black microplate, the amount of addition was calculated based on the concentration of the target protein PtsuOBP39 so that the final concentration of PtsuOBP39 was 2 μ M, 1-NPN was added so that the final concentration was 2 μ M, the reaction was carried out at room temperature for 2min, and the fluorescence value (initial fluorescence value) was measured and recorded by the end point method (Endpoint) under excitation light at 337nm and the maximum emission peak wavelength. Subsequently, the host cinnamomum camphora volatile compound was added to the reaction system at final concentrations of 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 12. mu.M, 16. mu.M and 20. mu.M or the imago pheromone was added at final concentrations of 0.2. mu.M, 0.4. mu.M, 0.6. mu.M, 0.8. mu.M, 1.2. mu.M, 1.6. mu.M, 2.0. mu.M, 3.0. mu.M and 4.0. mu.M, and the mixture was reacted at room temperature for 2min, and fluorescence values were measured and recorded by an end-point method (Endpoint) under excitation light of 337nm and a maximum emission peak wavelength.

(IV) data analysis: selecting GraThe phPad Prism 7.0 is used for fitting and plotting the fluorescence value of PtsuOBP39 combined with the maximum emission peak of the fluorescent probe 1-NPN and the concentration of the fluorescent probe 1-NPN, the curve is linearized (Y ═ bound/free, X ═ bound) by using a Scatchard method, a regression equation of the curve is obtained, and then the binding constant (binding constant, K) is calculated by using the Scatchard equation_1-NPN). Calculation of IC for various ligand odor molecules₅₀The concentration of ligand odor molecules at which the fluorescence value drops to half of the initial fluorescence value of PtsuOBP39 bound to fluorescent probe 1-NPN. Finally, calculating Dissociation constants (Ki) of various ligand odor molecules by using a formula II;

Ki＝[IC₅₀]/(1+[1-NPN]/K_1-NPN) Formula II

In the formula, IC₅₀(Halfmaximal inhibition center) is the concentration of the ligand odorant molecule at which the fluorescence intensity is reduced by half; [1-NPN]The concentration of unbound fluorescent probe was 1-NPN.

The results are as follows:

(1) combination characteristics of PtsuOBP39 and 1-NPN

As can be seen from FIG. 3, there is a significant linear relationship between the PtsuOBP39 odor binding protein and the fluorescent probe 1-NPN, and R of the regression equation²The result shows that the fluorescence values of PtsuOBP39 and 1-NPN have saturation effect with the increase of the concentration of the 1-NPN, and the PtsuOBP39 has a single binding site and can be used for measuring the fluorescence competitive binding force. Furthermore, the combination constant K of PtsuOBP39 and 1-NPN_1-NPN＝5.74±0.48。

(2) Binding characteristics of PtsuOBP39 and host cinnamomum camphora volatile compound odorant ligand

The binding force judgment standard of plant volatile matters is as follows: ki is less than 10 mu M, which indicates strong binding force; the binding force is weak when the Ki is more than 10 mu M and less than 20 mu M; ki > 20. mu.M indicates no binding.

The binding affinity of PtsuOBP39 to camphor volatile compounds is shown in table 9.

TABLE 9 binding affinities of PtsuOBP39 to Cinnamomum camphora volatile Compounds

Note: r.f. (Relative fluorescence) indicates the Relative percentage fluorescence at maximum ligand concentration.

As shown in fig. 4 and table 9: among the 9 host cinnamomum camphora volatile information compounds, (1) PtsuOBP39 has weak binding force to camphor (Ki ═ 14.71 μ M), ocimene (Ki ═ 11.29 μ M), linalool (Ki ═ 19.19), α phellandrene (Ki ═ 18.14 μ M), and β caryophyllene (Ki ═ 14.13 μ M); (2) PtsuOBP39 does not bind 4 volatiles such as d-borneol, eucalyptol, 3-carene and trans-nerolidol.

(3) Binding characteristics of PtsuOBP39 to adult insect pheromone odor ligands

The judgment standard of the binding force of pheromone is as follows: ki <1 μ M indicates strong binding force; 1 μ M < Ki <2 μ M indicates moderate binding; 2 μ M < Ki <4 μ M indicates weak binding; ki > 4. mu.M indicates no binding.

PtsuOBP39 showed moderate binding to adult insect pheromone (1 α,3 α,4 α,6 α) -4,7, 7-trimethylbicyclo [4.1.0] heptan-3-ol (Ki ═ 1.11 μ M).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and optimization can be made without departing from the principle of the present invention, and these modifications and optimization should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of forestry

Shanghai Forestry Station

<120> protein PtsuOBP39 combined with cinnamomum camphora volatile compound and insect pheromone, attractant and application thereof

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Arg Ala Val Ile Glu Lys Val Leu Lys Thr Gln Ile Leu Pro Lys Glu

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Claims (4)

1. Use of PtsuOBP39 for the identification/binding of camphor volatile compounds and/or insect pheromones, said camphor volatile compounds consisting of one or several of the following compounds: camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene;

   the amino acid sequence of the PtsuOBP39 is shown as SEQ ID NO. 1;

   the polypide pheromone is (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethyl bicyclo [4.1.0] heptane-3-alcohol.
2. 2. The use of claim 1, wherein the PtsuOBP39 has a dissociation constant of 10 μ Μ < Ki <20 μ Μ with camphor, ocimene, linalool, alpha phellandrene, or beta caryophyllene.
3. 3. The use according to claim 1,

   the PtsuOBP39 has a dissociation constant of 1. mu.M < Ki < 2. mu.M with (1. alpha., 3. alpha., 4. alpha., 6. alpha.) -4,7, 7-trimethylbicyclo [4.1.0] heptan-3-ol.
4. 4. The application of the cinnamomum camphora tooth beak attractant in monitoring and/or controlling the cinnamomum camphora tooth beak is characterized in that the active ingredient of the cinnamomum camphora tooth beak attractant consists of (1 alpha, 3 alpha, 4 alpha, 6 alpha) -4,7, 7-trimethylbicyclo [4.1.0] heptane-3-alcohol and/or one or more of the following compounds: camphor, ocimene, linalool, alpha phellandrene and beta caryophyllene.

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