Protein PtsuOBP16 combined with multiple cinnamomum camphora volatile compounds, cinnamomum camphora tooth elephant attractant and application thereof
阅读说明：本技术 一种与多种香樟挥发性化合物结合的蛋白PtsuOBP16、香樟齿喙象引诱剂及其应用 (Protein PtsuOBP16 combined with multiple cinnamomum camphora volatile compounds, cinnamomum camphora tooth elephant attractant and application thereof ) 是由 耿薏舒 陈聪 郝德君 王焱 张岳峰 樊斌琦 韩阳阳 于晓航 于 2021-07-22 设计创作，主要内容包括：本发明提供了一种与多种香樟挥发性化合物结合的蛋白PtsuOBP16、香樟齿喙象引诱剂及其应用,属于分子生物学技术领域。源于香樟齿喙象成虫气味结合蛋白PtsuOBP16如SEQ ID NO：1所示,其编码基因如SEQ ID NO.2所示。所述蛋白对香樟齿喙象寄主植物香樟中罗勒烯、芳樟醇与α水芹烯等具有较强的结合能力,同时对右旋龙脑以及樟脑具有较弱的结合能力,为解析香樟齿喙象为害香樟的嗅觉分子机制奠定分子基础,同时为开发以昆虫嗅觉关键基因为靶点的害虫监测防控技术提供参考依据；所述蛋白能够特异性结合罗勒烯、芳樟醇与α水芹烯等香樟挥发性化合物,可用于制备引诱剂,为监测和防控香樟齿喙象提供了新思路。(The invention provides a protein PtsuOBP16 combined with a plurality of camphor volatile compounds, a camphor tooth elephant attractant and application thereof, belonging to the technical field of molecular biology. The adult odorant binding protein PtsuOBP16 derived from cinnamomum camphora's tooth proboscis as shown in SEQ ID NO:1 and the coding gene is shown as SEQ ID NO. 2. The protein has strong binding capacity to ocimene, linalool, alpha phellandrene and the like in a cinnamomum camphora tooth proboscis host plant cinnamomum camphora, has weak binding capacity to d-borneol and camphor, lays a molecular foundation for analyzing an olfactory molecular mechanism of the cinnamomum camphora tooth proboscis as an insect pest, and provides a reference basis for developing a pest monitoring prevention and control technology taking an insect olfactory key gene as a target spot; the protein can be specifically combined with the camphor volatile compounds such as ocimene, linalool, alpha-phellandrene and the like, can be used for preparing an attractant, and provides a new idea for monitoring and controlling the camphor tooth beak elephant.)
1. A protein PtsuOBP16 combined with a plurality of camphor volatile compounds is characterized in that the amino acid sequence of PtsuOBP16 is shown as SEQ ID NO. 1.
2. Use of PtsuOBP16 according to claim 1 for identifying and/or binding camphor volatile compounds.
3. The use according to claim 2, wherein the camphor volatile compounds comprise one or more of the following compounds: linalool, ocimene, d-borneol, alpha-phellandrene and camphor.
4. The use according to claim 3, wherein PtsuOBP11 has a dissociation constant Ki < 10 μ M from ocimene, linalool or alpha phellandrene.
The dissociation constant of PtsuOBP11 and d-borneol or camphor is 10 mu M < Ki < 20 mu M.
5. The encoding gene of the protein PtsuOBP16 combined with the plurality of cinnamomum camphora volatile compounds according to claim 1, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2.
6. A recombinant vector for expressing a foreign gene, wherein the foreign gene comprises the coding gene of claim 5.
7. Use of the coding gene of claim 5 or the recombinant vector of claim 6 for the recombinant expression of PtsuOBP16 associated with a plurality of cinnamomum camphora volatile compounds.
8. An attractant for the beak elephant of cinnamomum camphora, wherein the active ingredient of the attractant comprises a volatile compound of cinnamomum camphora bound to the protein PtsuOBP16 of claim 1; the camphor volatile compounds comprise one or more of the following compounds: linalool, ocimene, d-borneol, alpha-phellandrene and camphor.
9. An attractant as set forth in claim 8 wherein when said camphorated volatile compounds include 2 species, it is comprised of two of camphor, ocimene and alpha phellandrene;
when the camphor volatile compounds include 3 species, it is composed of camphor, ocimene and alpha phellandrene.
10. Use of the attractant according to claim 8 or 9 for monitoring and/or controlling the rhynchophorus camphorata.
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a protein PtsuOBP16 combined with a plurality of camphor volatile compounds, a camphor tooth proboscis 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
In view of the above, the present invention aims to provide a pterocarpus camphoratus peck odor binding protein PtsuOBP16, wherein PtsuOBP16 can bind to a plurality of pterocarpus camphoratus peck volatile compounds.
The invention provides a cinnamomum camphora tooth beak weevil attractant and application thereof, and provides a new means for monitoring and controlling cinnamomum camphora tooth beak weevils.
The invention provides a protein PtsuOBP16 capable of being combined with a plurality of camphor volatile compounds, wherein the amino acid sequence of PtsuOBP16 is shown as SEQ ID NO. 1.
The invention provides application of the PtsuOBP16 in identification and/or combination of cinnamomum camphora volatile compounds.
Preferably, the camphor volatile compound comprises one or more of the following compounds: linalool, ocimene, d-borneol, alpha-phellandrene and camphor.
Preferably, the dissociation constant Ki of PtsuOBP11 with ocimene, linalool or alpha phellandrene is < 10 μ M;
the dissociation constant of PtsuOBP11 and d-borneol or camphor is 10 mu M < Ki < 20 mu M.
The invention provides a coding gene of the protein PtsuOBP16 combined with a plurality of camphor volatile compounds, 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 PtsuOBP16 combined with a plurality of cinnamomum camphora volatile compounds.
The invention provides a cinnamomum camphora tooth elephant attractant, which comprises key components of cinnamomum camphora volatile compounds combined with protein PtsuOBP 16; the camphor volatile compounds comprise one or more of the following compounds: linalool, ocimene, d-borneol, alpha-phellandrene and camphor.
Preferably, when the camphor volatile compound includes 2 species, it is composed of two of camphor, ocimene and alpha phellandrene;
when the camphor volatile compounds include 3 species, it is composed of camphor, ocimene and alpha phellandrene.
The invention provides application of the attractant in monitoring and/or controlling the cinnamomum camphora tooth proboscis.
The amino acid sequence of the protein PtsuOBP16 combined with a plurality of camphor volatile compounds provided by the invention is shown in SEQ ID NO. 1. The invention obtains an odor binding protein PtsuOBP16 from the camphor tooth elephant by cloning and prokaryotic expression methods. Fluorescence competitive binding experiments prove that the fluorescence values of PtsuOBP16 and 1-NPN have saturation effects along with the increase of the concentration of the 1-NPN, and the PtsuOBP16 has a single binding site and can be used for measuring fluorescence competitive binding force. Meanwhile, the active component identification of the host plant cinnamomum camphora volatile compound is reversely verified, and the result shows that PtsuOBP16 has strong binding force to ocimene (Ki is 8.14 mu M), linalool (Ki is 9.72 mu M) and alpha phellandrene (Ki is 8.90 mu M); meanwhile, PtsuOBP16 has weak binding force to camphor (Ki being 13.23 mu M) and dextroborneol (Ki being 13.21 mu M); (3) PtsuOBP16 can not bind 4 volatile compounds such as eucalyptol, 3-carene, trans-nerolidol and beta caryophyllene. The invention lays a molecular foundation for analyzing the olfactory molecular mechanism of the cinnamomum camphora, and provides a reference basis for developing a pest monitoring, prevention and control technology taking insect olfactory key genes as targets.
Drawings
FIG. 1 is a graph showing the relative expression amount of PtsuOBP16 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 PtsuOBP16 purification, note: in the figure, a lane M is Marker; 3 is the purified and collected target protein PtsuOBP 16;
FIG. 3 is a combination graph (a) and a Scatchard graph (b) of PtsuOBP16 and 1-NPN;
fig. 4 is a graph of the fluorescent competitive binding of PtsuOBP16 to camphor volatiles.
Detailed Description
The invention provides a protein PtsuOBP16 combined with a plurality of camphor volatile compounds, wherein the amino acid sequence of PtsuOBP16 is shown as SEQ ID NO:1 (MKHFIAVLLCALVASALARPEVNQDAVKESGRRIKEAHDKCQTDPATAIDEEALKNARKSGAPPAPPANSGPHSLCISKAVGWQNEDGSINKANIEEKAHAIFGEQSDIKNILDECVVAQENPEATAVHLFDCYRKHAPHPAGGPHPPPPHH). The PtsuOBP16 is encoded by 152 amino acids; contains a signal peptide formed by 18 aa; the molecular mass of the protein is 14.45 kDa; the isoelectric point was 5.97.
Based on the fact that PtsuOBP16 has the biological function of specifically binding to camphor tree volatile compounds, the invention provides application of PtsuOBP16 in identification and/or binding of camphor tree volatile compounds. The camphor volatile compounds comprise one or more of the following compounds: linalool, ocimene, d-borneol, alpha-phellandrene and camphor.
In the present invention, the PtsuOBP16 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 PtsuOBP16 and 1-NPN have saturation effect along with the increase of the concentration of the 1-NPN, and PtsuOBP16 has a single binding site and can be used for determining the fluorescence competitive binding force. Combination constant K of PtsuOBP16 and 1-NPN1-NPN5.47 ± 0.47. The competitive binding experiment of ligand odor molecules is carried out by taking 1-NPN as a fluorescent probe, and the result shows that PtsuOBP16 has strong binding force to ocimene (Ki is 8.14 mu M), linalool (Ki is 9.72 mu M) and alpha phellandrene (Ki is 8.90 mu M) by analyzing volatile compounds of 9 host plants namely cinnamomum camphora; meanwhile, PtsuOBP16 has weak binding force to camphor (Ki being 13.23 mu M) and dextroborneol (Ki being 13.21 mu M); (3) PtsuOBP16 can not bind 4 volatile compounds such as eucalyptol, 3-carene, trans-nerolidol and beta caryophyllene.
The invention provides a coding gene of the protein PtsuOBP16 combined with a plurality of camphor volatile compounds, and the nucleotide sequence of the coding gene is shown as SEQ ID NO.2 (ATGAAACACTTCATTGCTGTGCTTCTCTGCGCCCTTGTTGCATCTGCTCTGGCAAGACCCGAAGTTAACCAGGATGCAGTTAAAGAAAGCGGACGCAGAATCAAAGAAGCCCACGACAAATGCCAAACCGACCCTGCCACTGCTATAGACGAAGAGGCCCTCAAGAACGCCCGTAAATCAGGTGCTCCTCCAGCTCCTCCAGCCAACAGTGGACCTCACTCTCTTTGTATTTCCAAGGCTGTAGGATGGCAAAACGAAGATGGTTCAATCAATAAAGCCAACATTGAAGAAAAGGCTCACGCTATTTTCGGCGAGCAATCCGACATTAAAAATATCCTCGACGAATGTGTCGTAGCTCAGGAAAACCCCGAGGCAACCGCTGTCCACCTCTTCGACTGTTACAGAAAGCACGCTCCTCATCCCGCAGGCGGTCCCCACCCTCCTCCTCCTCACCATTAA).
The coding gene is obtained by using cDNA of the cinnamomum camphora tooth elephant as a template and amplifying ORF of PtsuOBP16 gene by PCR. The primer for PCR amplification comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 (5'-ATGAAACACTTCATTGCTGTGCTTC-3') and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 (5'-TTAATGGTGAGGAGGAGGAGGGTGG-3'). The reaction system for PCR amplification is 750 ng/. mu.L cDNA template 1. mu.L, 2 XPrimer STARMax 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 51 ℃ 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 cinnamomum camphora tooth beak elephant odor binding protein PtsuOBP 16.
In the recombinant expression method of the cinnamomum camphora tooth beak-like odor binding protein PtsuOBP16, 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 PtsuOBP16 is obtained through screening culture and induction.
The PtsuOBP16 protein based on the cinnamomum camphora tooth elephant has the specific binding capacity on the sex information compound of a host cinnamomum camphora, so the invention provides the cinnamomum camphora tooth elephant attractant, which comprises key components of the attractant, including ligand odor molecules capable of binding with PtsuOBP 16; the ligand odor molecules include linalool, ocimene, d-borneol, alpha phellandrene and camphor. When the camphor volatile compounds include 2 species, it is preferably composed of two of camphor, ocimene and alpha phellandrene; when the camphor volatile compounds include 3 species, it is composed of camphor, ocimene and alpha phellandrene. The attractant also comprises other auxiliary materials acceptable in the field, such as a solvent, a lure and the like. The preparation method of the attractant is not particularly limited, and the attractant can be prepared by adopting the types well known in the field.
The invention provides application of the attractant in monitoring and controlling the cinnamomum camphora tooth proboscis. The attractant is also preferably matched with other attracting devices or reagents for monitoring and controlling the cinnamomum camphora tooth beak image.
The following examples are provided to illustrate PtsuOBP16, the cinnamomum camphora kom attractant and the use thereof in combination with various cinnamomum camphora volatile compounds, but they should not be construed as limiting the scope of the present invention.
Example 1
Cloning of PtsuOBP16 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 12000 rpm 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 12000 rpm 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 12000 rpm 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 sample260/280The 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 PtsuOBP16 Gene
Analyzing ORFs of the PtSUOBP16 gene according to Open Reading Frame (ORFs) predicted websites (http:// www.ncbi.nlm.nih.gov/gorf. html), and designing specific primers of the PtSUOBP16 gene by using Primer Premier 5.0 software to amplify the ORF of the PtSUOBP16 gene;
the cloning primers were as follows:
F：5′-ATGAAACACTTCATTGCTGTGCTTC-3′(SEQ ID NO:3)；
R：5′-TTAATGGTGAGGAGGAGGAGGGTGG-3′(SEQ ID NO:4)。
the reaction system is shown in Table 1.
TABLE 1 reaction System for specific primer amplification of PtsuOBP16 Gene
cDNA template
1μL(750ng/μL)
PrimerSTAR Max Premix(2×)
25μL
Primer1(10μM)
1μL
Primer2(10μM)
1μL
RNase Free ddH2O
up to 50μL
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 reaction procedure for specific primer amplification of PtsuOBP16 Gene
(4) After the amplification reaction is finished, agarose gel (2%) electrophoresis is used for detecting whether the size of the band of the PCR product is 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, and cutting a part containing a target fragment as little as possible;
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 microliter WA into the adsorption tube, centrifuging for 1min at 12000 rpm, and removing residual liquid in the collection tube;
sixthly, adding 500 mu L of Wash Solution into the adsorption tube, centrifuging for 1min at 12000 rpm, and removing residual liquid in the collection tube;
seventhly, repeating the step sixthly, and removing residual liquid in the collecting pipe;
then centrifuging at 12000 rpm 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 for 2min at 12000 rpm to obtain a product recovered from the glue;
the purity and concentration of the recovered product was checked by agarose gel (2%) electrophoresis 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 reaction System for ligation reaction
Glue recovery product
1～4μL
5×TA/Blunt-Zero Cloning Mix
1μL
RNase Free ddH2O
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 cells are completely thawed, subpackaging the cells 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 on a shaking table to be shaken for 1h at 37 ℃ and 200rpm so as to recover competent cells;
fifthly, after 1h of shake culture, centrifuging for 3min at 2500rpm, taking out 900 microliter of supernatant by using a pipettor, removing the supernatant, and after the thalli are resuspended, uniformly coating the thalli 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 accurate ORF sequence of the PtsuOBP16 gene 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) PtsuOBP16 protein
The ExPasy software indicates that the PtsuOBP16 protein is encoded by 152 amino acids; the N end contains a signal peptide 18aa consisting of 18 aa; the molecular mass of the protein is 14.45 kDa; the isoelectric point was 5.97.
Example 2
Relative expression level of PtsuOBP16 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 PtSUOBP16 gene, qRT-PCR primers for the PtSUOBP16 gene were designed using an in-line program (https:// www.ncbi.nlm.nih.gov/tools/primer-blast /).
The cloning primers were as follows:
F：5′-TCATTGCTGTGCTTCTCTGC-3′(SEQ ID NO：5)；
R：5′-GTCTATAGCAGTGGCAGGGT-3′(SEQ ID NO：6)。
the cDNA template concentration of the sample was diluted to 500ng/. mu.L, successively diluting 10 times, diluting 4 times to obtain 5 concentrations of cDNA templates, 500 ng/. mu.L and 500 × 10-1(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 scheme 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, respectively calculating the amplification efficiency of each primer according to formula I.
Amplification efficiency E ═ 10[－1/slope]－1) X 100 formula I.
The dissolution curve of the qRT-PCR primer for PtsuOBP16 gene was shown to be unimodal after the reaction, indicating that the primer is specific and no primer dimer is present. In addition, amplification characteristics of the PtsuOBP16 gene qRT-PCR primers are shown in table 6 below.
TABLE 6 amplification characteristics of the PtsuOBP16 Gene qRT-PCR primers
Name of Gene
Read length (bp)
Slope
Efficiency (%)
Coefficient of correlation R2
Linear regression equation
PtsuOBP16
140
－2.9680
117.234
0.9902
y＝－2.968x+37.955
The amplification efficiency of the primers was 117.234%, which ranged from 80% to 120%, indicating that the amplification was good for the primer specificity. The PtsuOBP16 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 wings of male and female adults is less; the relative expression level in the wing tissues of female adults was significantly higher than that of male adults, while the relative expression level in the feet of male adults was significantly higher than that of female adults (fig. 1).
Example 3
Prokaryotic expression and purification of PtsuOBP16
(I) prokaryotic expression vector construction based on homologous recombination principle
(1) The signal peptide sequence (SEQ ID NO:9: ATGAAACACTTCATTGCTGTGCTTCTCTGCGCCCTTGTTGCATCTGCTCTGGCA) of PtsuOBP16 was removed, and a primer Design software CE Design (SEQ ID NO:9: ATGAAACACTTCATTGCTGTGCTTCTCTGCGCCCTTGTTGCATCTGCTCTGGCA) was used based on the sequence not containing the signal peptide and the sequence of the prokaryotic expression vector pET28a (+) (CE Design)http://www.vazyme.comNuo kean official network) to design target fragment primers containing homologous sequences at both ends of the linearized vector; the primers for constructing the PtsuOBP16 prokaryotic expression vector are as follows:
F：5′-cgcggatccgaattcgagctcAGACCCGAAGTTAACCAGGATG-3′(SEQ ID NO:7)；
r: 5'-tggtggtgctcgagtgcggccgcTTAATGGTGAGGAGGAGGAGGG-3' (SEQ ID NO: 7). Wherein the lower case letters are the cleavage sites.
(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
Make up to 50 μ L
(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×CEⅡBuffer
2μL
Linearized fragments
80ng
Linearized vector
100ng
ExnaseⅡ
1μL
RNase Free ddH2O
Make up to 10 μ L
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 30min at 37 ℃ in a PCR instrument;
(8) and (3) transformation of a recombinant product: selecting DH5 alpha clone recombinant plasmid PtsuOBP16/pET28a (+) and selecting M13F/R to verify positive clone, and finally sequencing and verifying;
(9) extracting and transforming positive clone plasmids:
firstly, respectively taking 10mL of overnight shake-cultured bacterial liquid, centrifuging at 12000 rpm for 1min, and removing a culture medium;
② respectively adding 500 mu L Solution I to fully resuspend the thalli, then adding 500 mu L Solution II, quickly reversing and fully mixing to be transparent egg white, finally adding 700 mu L Solution III to fully mix, standing for 5min at room temperature, and centrifuging for 10min at 12000 rpm;
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 for 1min at 12000 rpm, 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 12000 rpm, and removing waste liquid in the collecting tube;
adding 600 mu L of Wash Solution into the tube, centrifuging at 12000 rpm 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 12000 rpm, 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 at room temperature for 2min, and centrifuging at 12000 rpm 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 vector BL21(DE3), prolonging the heat shock time to 90sec, selecting the positive bacterial plaque for shake culture, taking the single clone for shake culture again for 5h after streak purification, and sending to sequencing verification and reserving the strain for later use after PCR detection of bacterial liquid.
(II) inducing expression of recombinant plasmid
Inducing expression by the recombinant plasmid: after activation of the retained PtsuOBP16 glycerol bacteria, the ratio of 1: 100 into 1L of 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 12000 rpm 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, PMSF was added to a final concentration of 1mM, the suspension was sonicated under ice bath (sonication cycle: 3sec, stop 5sec), and centrifuged at 12000 rpm for 30min, and the supernatants were collected. Respectively taking 20 mu L of the supernatant, respectively adding 5 mu L of Loading buffer, 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) combined with a Coomassie brilliant blue method dyeing and decoloring method.
(III) purification of Key PtsuOBP16 recombinant protein
Filtering the supernatant after ultrasonic crushing with a filter membrane with the aperture of 0.2 mu m;
② heavy suspension balancing 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, respectively eluting and collecting target protein by 300 mu L multiplied by 5Elution buffer (300mM NaCl, 300mM imidazole, 50mM PB, 10% glycerol, 1mM protease inhibitor 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 and dialyzing 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 (SDS-PAGE, 100V, 120min) detecting the purified target protein solution, and storing at-80 ℃ for later use.
The purification result of PtsuOBP16 is shown in fig. 2, and PtsuOBP16 obtained by purification and collection can be used for carrying out functional verification of the odor binding protein.
Example 4
Functional verification of PtsuOBP16
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 PtsuOBP16 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. A chromatographic grade methanol is selected to dissolve a fluorescent probe N-phenyl-1-naphthylamine (N-phenyl-l-naphthylamine, 1-NPN) and host plant camphor volatile matters (camphor, ocimene, d-borneol, linalool, eucalyptol, 3-carene, trans-nerolidol, alpha phellandrene and beta caryophyllene) and other ligand odor molecules to prepare a 10mM mother solution, the mother solution is stored at the temperature of minus 20 ℃ for later use, and the mother solution is diluted into a 1mM working solution by using methanol during experiments.
(II) determination of suitability of 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 PtSuOBP16, the final concentration of the target protein PtSuOBP16 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. mu.M of the target protein solution in series to give final concentrations of 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, 12. mu.M, 14. mu.M, 16. mu.M, 18. mu.M and 20. mu.M, respectively, and the reaction was carried out at room temperature for 2min, and the fluorescence values were recorded, respectively, and 3 technical repetitions were carried out for the target protein PtSuOBP16, 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 16. The fluorescence values from PtsuOBP16 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 PtSUOBP16 or not is detected. If the combination of the PtSUOBP16 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 PtSUOBP16 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 experiments of ligand odorant 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 PtsuOBP16 so that the final concentration of PtsuOBP16 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, host Cinnamomum camphora volatiles were added to the reaction system in a final concentration of 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 12. mu.M, 16. mu.M and 20. mu.M, respectively, and reacted at room temperature for 2min, respectively, and fluorescence values were measured and recorded by the end point method (Endpoint) under the conditions of 337nm excitation light and maximum emission peak wavelength.
(IV) data analysis: the fluorescence value of PtsuOBP16 combined with the maximum emission peak of the fluorescent probe 1-NPN is plotted by matching the concentration of the fluorescent probe 1-NPN with GraphPad Prism 7.0, the curve is linearized by the Scatchard method (Y ═ bound/free, X ═ bound) to obtain a regression equation of the curve, and the binding constant (K binding constant) is calculated by the Scatchard equation1-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 PtsuOBP16 bound to fluorescent probe 1-NPN. Finally, calculating Dissociation constants (Ki) of various ligand odor molecules by using a formula I;
formula Ki ═ IC50]/(1+[1-NPN]/K1-NPN) Formula II
In the formula, IC50(Halfmaximal inhibition center) is the concentration of ligand odorant molecules at which the fluorescence intensity drops by half; [1-NPN]The concentration of unbound fluorescent probe was 1-NPN.
The results are as follows:
(1) combination characteristics of PtsuOBP16 and 1-NPN
As can be seen from FIG. 3, there is a significant linear relationship between the PtsuOBP16 odor binding protein and the fluorescent probe 1-NPN, and R of the regression equation2The result shows that the fluorescence values of PtsuOBP16 and 1-NPN have saturation effect with the increase of the concentration of the 1-NPN, and the PtsuOBP16 has a single binding site and can be used for measuring the fluorescence competitive binding force. Furthermore, the combination constant K of PtsuOBP16 and 1-NPN1-NPN＝5.47±0.47。
(2) Binding characteristics of PtsuOBP16 to odorant ligands
The standard for judging the binding force of plant volatile compounds 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 PtsuOBP16 to camphor volatiles is shown in table 9.
TABLE 9 PtsuOBP16 and 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 compounds, (1) PtsuOBP16 p-ocimene (Ki ═ 8.14 μ M), linalool (Ki ═ 9.72 μ M), and α phellandrene; (2) PtsuOBP16 has weak binding force to camphor (Ki 13.23 μ M) and d-borneol (Ki 13.21 μ M); (3) PtsuOBP16 can not bind 4 volatile compounds such as eucalyptol, 3-carene, trans-nerolidol and beta caryophyllene.
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 decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Nanjing university of forestry
Shanghai Forestry Station
<120> protein PtsuOBP16 combined with multiple cinnamomum camphora volatile compounds, cinnamomum camphora tooth elephant attractant and application thereof
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Ile Asp Glu Glu Ala Leu Lys Asn Ala Arg Lys Ser Gly Ala Pro Pro
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Leu Asp Glu Cys Val Val Ala Gln Glu Asn Pro Glu Ala Thr Ala Val
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tgccaaaccg accctgccac tgctatagac gaagaggccc tcaagaacgc ccgtaaatca 180
ggtgctcctc cagctcctcc agccaacagt ggacctcact ctctttgtat ttccaaggct 240
gtaggatggc aaaacgaaga tggttcaatc aataaagcca acattgaaga aaaggctcac 300
gctattttcg gcgagcaatc cgacattaaa aatatcctcg acgaatgtgt cgtagctcag 360
gaaaaccccg aggcaaccgc tgtccacctc ttcgactgtt acagaaagca cgctcctcat 420
cccgcaggcg gtccccaccc tcctcctcct caccattaa 459
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