CN107164364A - The method that screening anoplophora glabripennis lures composition and prepares anoplophora glabripennis attractant - Google Patents
The method that screening anoplophora glabripennis lures composition and prepares anoplophora glabripennis attractant Download PDFInfo
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Abstract
The method that screening anoplophora glabripennis lures composition and prepares anoplophora glabripennis attractant, the present invention relates to the method that screening efficiently lures composition and prepares anoplophora glabripennis attractant.The present invention proposes to solve host plant or plant-feed insect angle of the prior art in terms of single, it is difficult to filter out the shortcoming of efficient attractive activity composition.Step of the present invention is：Step one：The extraction and purifying of the total serum IgE of anoplophora glabripennis antennal sensilla；Step 2：Obtain anoplophora glabripennis odor-binding protein total length；Step 3：Build the prokaryotic expression carrier of anoplophora glabripennis odor-binding protein；Step 4：Anoplophora glabripennis recombinates the protein-bonded induction of smell and purifying；Step 5：Anoplophora glabripennis lures the screening of composition；Determined and field trapping experiments by GC EAG, indoor smell taxis, it is determined that optimal anoplophora glabripennis lures agent prescription.The present invention is used for forestry Forest Protection technical field.
Description
Technical field
The present invention relates to forestry Forest Protection technical field.
Background technology
Prior art confirms, along with chemistry, the maturation of ecological and molecular biotechnology and research method, insect Synthetic
Reason technology also there occurs qualitative leap, and this leap is embodied between comprehensive utilization insect and host plant, the chemistry of inter-species etc. leads to
News basis, so as to carry out the prophylactico-therapeutic measures research of insect from ecological, chemistry and biological technical field, i.e., using host insect,
Host plant, natural enemy etc., which can discharge specifically to have, to be attracted, refuses to keep away or do not attract what host insect was selected host plant
Volatile chemical realizes the purpose of pre- protection against insect.
Anoplophora glabripennis is a kind of stem borer kind omnivorous insect, but sucking action of the host plant to anoplophora glabripennis, danger
Evil degree has certain otherness, and different host tree species are transplanted in same place, there are only harm advantage host tree species, without
Endanger the situation of other seeds.If Acer negundo is as advantage host tree species, when with the tree kind such as elm, white birch, willow, willow same
When place is transplanted, all endanger Acer negundo very serious, and other seeds are not endangered or endangered but do not caused disaster.Research also demonstrates that,
Acer negundo volatile matter is to the attracting action of anoplophora glabripennis apparently higher than other seeds volatile matters.
From the point of view of existing investigative technique, the content of correlation anoplophora glabripennis attractant also compares many both at home and abroad, mainly from list
Pure anoplophora glabripennis sex pheromone composition proposes the preparation method of sex attractant and existing other botanical attractant applications
Into the preventing and treating of anoplophora glabripennis, host plant or plant-feed insect angle of the above method in terms of single propose, but
Insect is in actual living environment, and insect, to the M8003 line of host plant, is the volatile matter and elder brother discharged to host plant
A kind of concentrated expression of the aggregation pheromone constituents mixt of worm itself release.Meanwhile, waved as anoplophora glabripennis host tree species
Stimulating food and aggregation pheromone composition are more, are tested using conventional single Olfactory behavior, it is difficult to filter out efficient attractive activity
Composition.
The content of the invention
The invention aims to solve host plant or plant-feed insect angle of the prior art in terms of single
Propose, it is difficult to filter out the shortcoming of efficient attractive activity composition, and propose a kind of screening and efficiently lure composition and prepare light shoulder
The method of longicorn beetle attractant.
A kind of screening anoplophora glabripennis lures the method for composition to comprise the following steps：
Step one：Extract and purify the total serum IgE of anoplophora glabripennis antennal sensilla；
Step 2：Obtain anoplophora glabripennis odor-binding protein total length；
Step 3：Build the prokaryotic expression carrier of anoplophora glabripennis odor-binding protein；
Step 4：Anoplophora glabripennis recombinates the protein-bonded induction of smell and purifying；
Step 5：Anoplophora glabripennis lures the screening of composition.
A kind of detailed process for the method for preparing anoplophora glabripennis attractant is：
By 9 kinds of Acer negundo volatile matters and 8 kinds of anoplophora glabripennis aggregation pheromone volatile matters with volume ratio 2:After 1 mixing, according to
Volume ratio 1:2 are dissolved in atoleine, and anoplophora glabripennis attractant is made；
The cis- 3- hexenols of 9 kinds of Acer negundo volatile matters, 4- hexenes, 1- acetic acid esters, methyl palmitate, hexyl acetate, 3-
Hexene, 1- acetic acid esters, 4- hexenes, 1- butyrates, 4- hexenes, 1- valerates, 2- dipropyl-adipate ester, phthalic acid two
The mass percent of different monooctyl ester is followed successively by：2.78：34.70：5.06：2.07：0.38：12.36：6.38：5.74：0.79；
It is the 8 kinds of anoplophora glabripennis aggregation pheromone volatile matter 2- methyl-docosane, cis -9- tricosenes, suitable
Formula -9- pentacosa alkenes, cis -7- pentacosa alkenes, the alkene of cis -9- 27, the alkene of cis -7- 27,4- epoxides in heptan-butyraldehyde,
The volume ratio of 4- epoxides in heptan-n-butanol is：0.5：1：2：1：0.5：0.5：1:1.
Beneficial effects of the present invention are：
The present invention is on the basis of existing research, and selection screens glabripenis from comprehensive taxis behavior and molecular biology method
Efficient attractive activity composition in the volatile matter and anoplophora glabripennis aggregation pheromone of longicorn advantage host tree species Acer negundo, by luring
Experiment is caught to establish the optimum formula of attractant.It is Acer negundo volatile matter to have attracting action most strong formula to anoplophora glabripennis
Mixture：Aggregation pheromone mixture=2:1.
Feeler odorant receptor protein screening anoplophora glabripennis advantage host tree is utilized it is an object of the invention to provide one kind
Plant Acer negundo volatile matter and the efficient of aggregation pheromone lures compositions, method and lure composition and aggregation to believe using efficient plant source
The method that the plain composition of breath prepares anoplophora glabripennis attractant.Its innovative point is to utilize the anoplophora glabripennis with attracting action excellent
Volatile constituent is pressed in the Acer negundo volatile matter of gesture host tree species special ratios and the aggregation pheromone of anoplophora glabripennis special ratios
Different proportion is mixed, filter out the special ratios with efficient attracting action efficiently lure composition lure agent composition.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of feeler total serum IgE 0.01；
Fig. 2 is target protein pET-32a (+)-Agla-OBP1 electrophoretograms；
Fig. 3 is target protein pET-32a (+)-Agla-OBP2 electrophoretograms；
Fig. 4 is target protein pET-32a (+)-Agla-OBP1 electrophoretograms after purification；
Fig. 5 is target protein pET-32a (+)-Agla-OBP2 electrophoretograms after purification；
Fig. 6 is that different ratio lures agent prescription to the EAG response diagrams of anoplophora glabripennis；
Fig. 7 lures agent prescription to anoplophora glabripennis smell taxis figure for different ratio；
Fig. 8 is that anoplophora glabripennis prevention effect is traped in field.
Embodiment
Embodiment one：A kind of screening anoplophora glabripennis lures the method for composition to comprise the following steps：
Step one：Extract and purify the total serum IgE of anoplophora glabripennis antennal sensilla；
Step 2：Obtain anoplophora glabripennis odor-binding protein total length；
Step 3：Build the prokaryotic expression carrier of anoplophora glabripennis odor-binding protein；
Step 4：Anoplophora glabripennis recombinates the protein-bonded induction of smell and purifying；
Step 5：Anoplophora glabripennis lures the screening and identification of composition.
Embodiment two：Present embodiment from unlike embodiment one：Glabripenis in the step one
The detailed process of extraction and the purifying of the total serum IgE of longicorn antennal sensilla is：
Anoplophora glabripennis feeler total serum IgE is extracted using Trizol reagents, non-enzyme dna removing-A (DNA-Be-Gone- are utilized
A the DNA remained in total serum IgE) is removed, and total serum IgE integrality is detected using 0.01 agarose gel electrophoresis；Utilize SMART IV
Oligonucleotide and CDS III/3 ' PCR primer (primer) reverse transcription, 42 DEG C are bred 1h, generate the chains of cDNA first.
Other steps and parameter are identical with embodiment one.
Embodiment three：Present embodiment from unlike embodiment one or two：Obtained in the step 2
The detailed process for taking anoplophora glabripennis odor-binding protein total length is：
Agla-OBP1, Agla-OBP2 design of primers,
Agla-OBP1 primer sequences：
Forward：5'-ATGAAACTTTTTGTATTCGTCC-3'
Reverse：5'-TTAGGGCAAGAAATAATTCGAG-3'
Agla-OBP2 primer sequences：
Forward：5'-ATGTCGCTCAGAATCGTTATCG-3'
Reverse：5'-CTATACCAGGAACCAGTTCTCC-3'
Anoplophora glabripennis odor-binding protein Agla-OBP1 and Agla-OBP2 cDNA amplification, clone and sequencing.With anti-
It is template to transcribe the first chains of enzymatic synthesis cDNA, and with primer pair, it enters performing PCR amplification；Reaction system：Pfu 0.5 μ l, Pfu
Buffer+Mg2+(SO4 2-) 5.0 μ l, dNTP 4.0 μ l, cDNA 1.0 μ l, Agla-OBP1-forward 1.0 μ l, Agla-
OBP1-reverse 1.0 μ l, ddH2O 37.5μl；PCR amplification programs：95℃3min；95℃30s、62.1℃30s、72℃
40s, 35 circulations；72℃10min；By 4 DEG C of preservations of PCR primer, through agarose gel electrophoresis and purifying of tapping rubber, PMD is cloned in
In 18-T vector carriers, identify and send biotech firm to be sequenced, so as to obtain objective gene sequence.
Other steps and parameter are identical with embodiment one or two.
Embodiment four：Unlike one of present embodiment and embodiment one to three：The step 3
It is middle build anoplophora glabripennis odor-binding protein the detailed process of prokaryotic expression carrier be：
Anoplophora glabripennis odor-binding protein Agla-OBP1 and Agla-OBP2 belong to the sub- eggs of odor-binding protein Minus C
White family, containing 6 conservative Cys, forms 3 pairs of disulfide bond.Utilize Prokaryotic expression vector construction expressing fusion protein system side
Method, is transformed pET-32a (+) carrier, removes the Thrombin restriction enzyme sites that carrier is carried；Design carries Xha I and Xho
The primer of two restriction enzyme sites of I, one section of base containing Thrombin is removed, clone again using bridge-type PCR method
One section of carrier sequence without Thrombin.Then it will expand from XbaI-XhoI purpose fragments (being free of Thrombin) bridging PCR
Increase；CDNA fragments to amplification use XbaI and XhoI to carry out digestion to pET-32a (+) and cDNA fragments, identical with process afterwards
PET-32a (+) carrier of digestion processing is attached, after the α of conversion Host Strains Trans 5, and picking single bacterium colony, upgrading grain are simultaneously carried out
Digestion is identified.
Other steps and parameter are identical with one of embodiment one to three.
Embodiment five：Unlike one of present embodiment and embodiment one to four：The glabripenis
The protein-bonded induction of longicorn restructuring smell and the detailed process purified are：
Induce anoplophora glabripennis to recombinate smell binding protein expression by IPTG, and pass through nickel Ago-Gel affinity chromatography
Post is purified to it；
The prokaryotic expression of destination protein：By PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla- built
OBP2 plasmids are transformed into BL21 (DE3) competent cell, and picking monoclonal or glycerol stock are seeded to LBs of the 5ml containing corresponding antibiotic
In nutrient solution, 37 DEG C, 230R/min concussion and cultivates are stayed overnight；1ml overnight cultures are taken to access 50ml containing appropriate corresponding antibiosis rope
In LB nutrient solutions, 37 DEG C, shaking table 230r/min is cultivated, and takes culture 20mL to be forwarded to the LB that 1L contains appropriate corresponding antibiotic after 3h
In nutrient solution, 37 DEG C, shaking table 230r/min is cultivated to OD600 0.6-0.8；In 16 DEG C of the inductive condition filtered out, IPTG concentration
For 0.5mM, overnight induction, induced expression target protein；Cell is collected with 6000r/min centrifugations 15min in 4 DEG C, wet thallus is protected
It is stored in -80 DEG C of purifying continued below；
Target protein PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 purifying：400mL is induced
Good PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 thalline are resuspended in lysate Buffer A,
Buffer A are by pH7.0,40mM Tris, 300mM NaCI, 5%Glycerol composition, according to bacterium solution:Buffer A=2L：
50ml ratio dissolving thalline；PSMF (100mM) is added, 50ml phage solutions add 50 μ l PSMF, MgC12(1M), 5Oml
Solution adds 50 μ l MgCI2, and DNAse (24mg/ml), height crushes bacterium after 10 μ l DNAse of 1L bacterium addition are mixed, and pressure is
1200bar, until bacterium solution in drops outflow can (3-4 all over), then at 4 DEG C, 6000r/min, 1h take supernatant to having balanced
Nickel NTA agarose Gel columns, and successively with different imidazole gradients (0mM, 10mM, 50mM, 100mM, 250mM, 500mM (100%
Buffer B) solution eluted, seven times altogether, collect after 7 pipe eluents, often manage and take 10ul to carry out SDS-PAGE point
Analysis, the purity of testing goal albumen；
Collect the eluent containing pure PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 recombinant proteins
Afterwards, transfer them in bag filter, in 500ml urea containing descending concentrations (initial concentration 7mol/L) PBS (pH7.4)
In, dialyse 72h at 4 DEG C, and changes a PBS every 8h.
Other steps and parameter are identical with one of embodiment one to four.
Embodiment six：Unlike one of present embodiment and embodiment one to five：The step 5
The detailed process of the screening of middle anoplophora glabripennis attractant composition is：
Anoplophora glabripennis restructuring smell albumen and Acer negundo volatile matter and glabripenis are obtained using competitiveness fluorescent associated methods
The association reaction spectrum of longicorn aggregation pheromone volatile matter, filters out the Acer negundo volatile matter for efficiently luring anoplophora glabripennis and aggregation letter
The plain composition of breath, 8 kinds of volatile matters of 9 kinds of Acer negundo volatile matters and anoplophora glabripennis aggregation pheromone can be with anoplophora glabripennis PET-32a
(+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 produces association reaction.
Other steps and parameter are identical with one of embodiment one to five.
Embodiment seven：Unlike one of present embodiment and embodiment one to six：9 kinds of Acer negundos
Volatile matter is specially：
Cis- 3- hexenols (3-Hexen-1-o1, (Z)), 4- hexenes, 1- acetic acid esters (4-Hexen-1-o1, acetate),
Methyl palmitate (Methyl palmitate), hexyl acetate (acetic acid, hexyl ester), 3- hexenes, 1- second
Acid esters ((Z) -3-Hexen-1-ol acetate), 4- hexenes, 1- butyrates (Butanoic acid, 4-hexen-1-yl
Ester), 4- hexenes, 1- valerates (Pentanoic acid, 4-hexen-1-yl ester), 2- dipropyl-adipate ester
(Hexanedioic acid, bis (2-methylpropyl) ester), diisooctyl phthalate (Diisooctyl
phthalate)。
Other steps and parameter are identical with one of embodiment one to six.
Embodiment eight：Unlike one of present embodiment and embodiment one to seven：8 kinds of volatilizations
Thing stimulating food is specially：
It is 2- methyl-docosane (2-Methyldocosane), cis -9- tricosenes ((Z) -9-Tricosene), suitable
Formula -9- pentacosa alkenes ((Z) -9-Pentacosene), cis -7- pentacosa alkenes ((Z) -7-Pentacosene), cis -9- two
17 alkene ((Z) -9-Heptacosene), the alkene of cis -7- 27 ((Z) -7-Heptacosene), 4- epoxides in heptan-butyraldehyde (4-
(Heptyloxy)-butana), 4- epoxides in heptan-n-butanol (4- (Heptyloxy)-butan-1-ol).
Other steps and parameter are identical with one of embodiment one to seven.
Embodiment nine：A kind of detailed process for the method for preparing anoplophora glabripennis attractant is：
Relative intensity of fluorescence is reduced to less than 50% by selection, and binding constant is in 13-45umol/L Acer negundo volatile matter
And anoplophora glabripennis aggregation pheromone composition is used as the composition of attractant；Acer negundo volatile constituent and aggregation by different proportion
The mixing of pheromone component, is determined and field trapping experiments by GC-EAG, indoor smell taxis, it is determined that optimal attractant is matched somebody with somebody
Side.
9 kinds of Acer negundo volatile content ratios setting Acer negundo volatile matter mixture, 8 kinds of anoplophora glabripennis according to filtering out gather
Collect pheromones volatile content ratio setting aggregation pheromone mixture；With Acer negundo volatile matter mixture：Aggregation pheromone is mixed
Thing=1：5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5：1 after 1 mixing:2 are dissolved in atoleine, form different proportion and match somebody with somebody
Side, is that, as (CK) is compareed, filtered out using GC-EAG has attracting action is most strong to match somebody with somebody to anoplophora glabripennis using atoleine
Sugar cube maple volatile matter mixture：Aggregation pheromone mixture=2:1.Indoor taxis is determined：Using Y-shaped smell sensing apparatus with atoleine
As control (CK), attractant Acer negundo volatile matter mixture is determined：Aggregation pheromone mixture=1：5、1:4、1:3、1:2、1:
1、2:1、3:1、4:1、5：1 after 1 mixing:2 are dissolved in smell taxis of the atoleine to anoplophora glabripennis, as a result show, to light shoulder
There is longicorn beetle attracting action most strong formula to be Acer negundo volatile matter mixture：Aggregation pheromone mixture=2:1.
Beneficial effects of the present invention are verified using following examples
Embodiment one：
Screening (efficient) anoplophora glabripennis attractant composition and prepare anoplophora glabripennis attractant method include following step
Suddenly：
1) extraction and purifying of anoplophora glabripennis antennal sensilla total serum IgE：
1. adopted for examination Anoplophora Glabripennis Adults in Harbin City's Acer negundo greening-tree, during mid-July to August, now adopt existing
With, utilize Trizol reagents extract anoplophora glabripennis feeler total serum IgE.
2. the DNA remained in total serum IgE is removed using non-enzyme dna removing-A (DNA-Be-Gone-A), and utilizes 0.01 agar
Its integrality of sugared detected through gel electrophoresis, as shown in Figure 1.
3. the PCR primer reverse transcriptions of SMART IV oligonucleotide and CDS III/3 ' are utilized, 42 DEG C are bred 1h,
Generate the chains of cDNA first.
2) specific steps of anoplophora glabripennis odor-binding protein are obtained using RT-PCR.
1. the design of primers of anoplophora glabripennis odor-binding protein Agla-OBP1, Agla-OBP2.According to light in Genbank
The gene order of shoulder longicorn beetle odor-binding protein Agla-OBP1, Agla-OBP2 (2010, Wang Wei), utilizes software Oligo7.0
Carry out design of primers.
2. Agla-OBP1 primer sequences:
Forward：5'-ATGAAACTTTTTGTATTCGTCC-3'.
Reverse：5'-TTAGGGCAAGAAATAATTCGAG-3'.
3. Agla-OBP2 primer sequences：
Forward：5'-ATGTCGCTCAGAATCGTTATCG-3'.
Reverse：5'-CTATACCAGGAACCAGTTCTCC-3'.
4. anoplophora glabripennis odor-binding protein Agla-OBP1 and Agla-OBP2cDNA amplification, clone and sequencing.With
Reverse transcriptase synthesis cDNA first chains are template, and with primer pair, it enters performing PCR amplification.Reaction system：Pfu 0.5μl,Pfu
Buffer+Mg2+(SO4 2-)5.0μl,dNTP 4.0μl,cDNA 1.0μl,Agla-OBP1-forward 1.0μl,Agla-
OBP1-reverse 1.0μl,ddH2O 37.5μl.PCR amplification programs：95℃3min；95℃30s、62.1℃30s、72℃
40s, 35 circulations；72℃10min.
5. by 4 DEG C of preservations of PCR primer, through agarose gel electrophoresis and purifying of tapping rubber, PMD 18-T vector loads are cloned in
In vivo, identify and send biotech firm to be sequenced, so as to obtain objective gene sequence, as shown in Figure 1.
3) structure of anoplophora glabripennis odor-binding protein prokaryotic expression carrier.
1. solubility expression of the heterologous protein of disulfide bond in Escherichia coli：Anoplophora glabripennis odor-binding protein
Agla-OBP1 and Agla-OBP2 belong to the sub- protein families of odor-binding protein Minus C, containing 6 conservative Cys, form 3
To disulfide bond.Using Prokaryotic expression vector construction expressing fusion protein systems approach, pET-32a (+) carrier is transformed, gone
The Thrombin restriction enzyme sites carried except carrier.
2. primer of the design with Xha I and Xho two restriction enzyme sites of I, will be contained using bridge-type PCR method
Thrombin one section of base is removed, and one section of carrier sequence for being free of Thrombin is cloned again.Then will be from XbaI-XhoI
Purpose fragment (being free of Thrombin) bridging PCR amplifications；CDNA fragments to amplification use XbaI and XhoI to pET-32a (+)
Digestion is carried out with cDNA fragments, pET-32a (+) carrier of digestion processing identical with process is attached afterwards, converts Host Strains
After the α of Trans 5, picking single bacterium colony, upgrading grain simultaneously carry out digestion identification.
4) induce anoplophora glabripennis to recombinate smell binding protein expression by IPTG, and pass through the affine layer of nickel Ago-Gel
Analysis post is purified to it.
1. the prokaryotic expression of destination protein：By PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla- built
OBP2 plasmids are transformed into BL21 (DE3) competent cell, and picking monoclonal or glycerol stock are seeded to LBs of the 5ml containing corresponding antibiotic
In nutrient solution, 37 DEG C, 230R/min concussion and cultivates are stayed overnight.1ml overnight cultures are taken to access 50ml containing appropriate corresponding antibiosis rope
In LB nutrient solutions, 37 DEG C, shaking table 230r/min is cultivated, and takes culture 20mL to be forwarded to the LB that 1L contains appropriate corresponding antibiotic after 3h
In nutrient solution, 37 DEG C, shaking table 230r/min is cultivated to OD600 0.6-0.8.In 16 DEG C of the optimal inductive condition filtered out, IPTG
Concentration is 0.5mM, overnight induction, induced expression target protein, as shown in Figures 2 and 3.15min is centrifuged with 6000r/min in 4 DEG C
Cell is collected, wet thallus is stored in -80 DEG C of purifying continued below.
2. target protein PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 purifying：400mL is lured
PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 thalline led are resuspended in lysate Buffer A,
Buffer A are by pH7.0,40mM Tris, 300mM NaCI, 5%Glycerol composition, according to bacterium solution:Buffer A=2L：
50ml ratio dissolving thalline.PSMF (100mM) is added, 50ml phage solutions add 50 μ l PSMF, MgC12(1M),5Oml
Solution adds 50 μ l MgCI2, and DNAse (24mg/ml), height crushes bacterium after 10 μ l DNAse of 1L bacterium addition are mixed, and pressure is
1200bar, until bacterium solution in drops outflow can (3-4 all over), then at 4 DEG C, 16000r/min, 1h take supernatant to balancing
Nickel NTA agarose Gel columns, and eluted successively with the solution of different imidazole gradients, seven times altogether, collect 7 pipe eluents
Afterwards, often manage and take 10ul to carry out SDS-PAGE analyses, the purity of testing goal albumen, wherein 1-7 are collecting pipe position, such as Fig. 4
With shown in Fig. 5.
3. the elution containing pure PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 recombinant proteins is collected
After liquid, transfer them in bag filter, in 500ml low PBSs for subtracting urea (initial concentration 7mol/L) containing concentration
(pH7.4) in, dialyse 72h at 4 DEG C, and changes a PBS every 8h.Then using Bradford methods to dialysing
Fusion protein determine concentration, be transferred in 1.5ml Eppendorf microcentrifugal tubes, be stored in -80 DEG C of refrigerators standby.
5) anoplophora glabripennis restructuring smell albumen and Acer negundo volatile matter and light shoulder are obtained using competitiveness fluorescent associated methods
The association reaction spectrum of longicorn beetle aggregation pheromone volatile matter, filters out Acer negundo volatile matter and the aggregation for efficiently luring anoplophora glabripennis
Pheromone component.
1. Fluorescent ligands 1-NPN is dissolved in methanol, is configured to be kept in dark place under the conditions of 1mmol/L solution, 4 DEG C, takes
1.5 μm of ol/L PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 are placed in the fluorescence cuvette of 1cm four-ways
In, the 1-NPN solution that 1mmol/L is gradually added into cuvette carries out fluorescent scanning, 2min is reacted after adding every time, 282
Excited under wavelength, the fluorescence intensity at record fluorescence emission spectrum, and maximum emission wavelength 328nm is according to Scatchard equations
(1) spectroscopic data is linearized：
Wherein DtRepresent 1-NPN concentration that is total and dissociating in system respectively with D, Pt is PET-32a (+)-Agla-OBP1
With the concentration of pET-32a (+)-Agla-OBP2 recombinant proteins, K is 1-NPN and PET-32a (+)-Agla-OBP1 and pET-32a
(+)-Agla-OBP2 binding constant, n is binding site number.It can obtain 1-NPN and Agla-OBP1/Agla-OBP2 dissociation
Constant.
Wherein IC50Concentration during 50% 1-NPN can be replaced for competition aglucon, 1-NPN is uncombined 1-NPN concentration,
K1- NPN is PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 and 1-NPN dissociation constant.
2. choose different sugar maple Leaf odor volatile matter and being at war with property of anoplophora glabripennis aggregation pheromone volatile matter is glimmering
Light binding tests, to the 26 kinds of Acer negundo volatile matters identified using GCSM, are filtered out 9 kinds of Acer negundo volatile matters and anoplophora glabripennis are poly-
Collecting 8 kinds of volatile matters of pheromones can be with anoplophora glabripennis PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 production
Raw association reaction, concrete outcome such as 1, table 2.
The Acer negundo volatile matter aglucon of table 1 and 1-NPN and restructuring PET-32a (+)-Agla-OBP1, pET-32a (+)-Agla-
The competition binding of OBP2 (A1/A2) albumen
The anoplophora glabripennis aggregation pheromone aglucon of table 2 and 1-NPN and restructuring PET-32a (+)-Agla-OBP1, pET-32a
The competition binding of (+)-Agla-OBP2 (A1/A2) albumen
6) anoplophora glabripennis lures the proposition of agent prescription：
1. GC-EAG screens optimum formula：According to the above-mentioned efficient 9 kinds of Acer negundo volatile contents ratio setting filtered out
Acer negundo volatile matter mixture, 8 kinds of anoplophora glabripennis aggregation pheromone volatile content ratio setting aggregation pheromone mixtures；And
Afterwards with Acer negundo volatile matter mixture：Aggregation pheromone mixture=1：5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5：1 mixes
1 after conjunction:2 are dissolved in atoleine, form different proportion formula, using atoleine as control (CK), are screened using GC-EAG
Go out there is anoplophora glabripennis attracting action most strong formula Acer negundo volatile matter mixture：Aggregation pheromone mixture=2:1, such as
Fig. 6.
2. indoor taxis is determined：By the use of Y-shaped smell sensing apparatus using atoleine as control (CK), the volatilization of attractant Acer negundo is determined
Thing mixture：Aggregation pheromone mixture=1：5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5：1 after 1 mixing:2 are dissolved in liquid
As a result body paraffin show to the smell taxis of anoplophora glabripennis, and it is sugar to have attracting action most strong formula to anoplophora glabripennis
Maple volatile matter mixture：Aggregation pheromone mixture=2:1, as shown in Figure 7.
7) field trapping experiments：Using it is above-mentioned it is resulting lure agent prescription, using utility model patent
Trapper and method for entrapping in CN201620147068.7, trapping glabripenis in field are carried out in late July, 2016 in early September
Longicorn, investigates Anoplophora Glabripennis Adults quantity on check plot (CK) and trapping area (YB) 30 plants of sugar maple trees, check plot amounts to respectively
11, and trapping area amounts to 189, average individual plant insect population number reduces 5.93, illustrates that the attractant that the present invention is obtained is matched somebody with somebody
Side is obvious to the prevention effect of anoplophora glabripennis, as shown in Figure 8.With it is existing it is simple using sex pheromone and plant volatile as
Anoplophora glabripennis attractant compares, with obvious attractant effect.
The present invention can also have other various embodiments, in the case of without departing substantially from spirit of the invention and its essence, this area
Technical staff works as can make various corresponding changes and deformation according to the present invention, but these corresponding changes and deformation should all belong to
The protection domain of appended claims of the invention.
Claims (9)
1. a kind of screen the method that anoplophora glabripennis lures composition, it is characterised in that：The screening anoplophora glabripennis lures composition
Method concretely comprise the following steps：
Step one：Extract and purify the total serum IgE of anoplophora glabripennis antennal sensilla；
Step 2：Obtain anoplophora glabripennis odor-binding protein total length；
Step 3：Build the prokaryotic expression carrier of anoplophora glabripennis odor-binding protein；
Step 4：Anoplophora glabripennis recombinates the protein-bonded induction of smell and purifying；
Step 5：Anoplophora glabripennis lures the screening of composition.
2. the method that a kind of screening anoplophora glabripennis according to claim 1 lures composition, it is characterised in that：The step
The detailed process of the total serum IgE of extraction and purifying anoplophora glabripennis antennal sensilla is in one：
Anoplophora glabripennis feeler total serum IgE is extracted using Trizol reagents, removes what is remained in total serum IgE using non-enzyme dna removing-A
DNA, and utilize the integrality of 0.01 agarose gel electrophoresis detection total serum IgE；Using SMART IV oligonucleotide and
The PCR primer reverse transcriptions of CDS III/3 ', 42 DEG C are bred 1h, generate the chains of cDNA first.
3. the method that a kind of screening anoplophora glabripennis according to claim 2 lures composition, it is characterised in that：The step
The detailed process of acquisition anoplophora glabripennis odor-binding protein total length is in two：
Agla-OBP1, Agla-OBP2 design of primers,
Agla-OBP1 primer sequences：
Forward：5'-ATGAAACTTTTTGTATTCGTCC-3'
Reverse：5'-TTAGGGCAAGAAATAATTCGAG-3'
Agla-OBP2 primer sequences：
Forward：5'-ATGTCGCTCAGAATCGTTATCG-3'
Reverse：5'-CTATACCAGGAACCAGTTCTCC-3'
The chains of cDNA first are synthesized as template using reverse transcriptase, it enters performing PCR amplification with primer pair；Reaction system：The μ l of Pfu 0.5,
Pfu Buffer+Mg2+(SO4 2-) 5.0 μ l, dNTP 4.0 μ l, cDNA1.0 μ l, Agla-OBP1-forward 1.0 μ l, Agla-
OBP1-reverse 1.0 μ l, ddH2O 37.5μl；PCR amplification programs：95℃3min；95℃30s、62.1℃30s、72℃
40s, 35 circulations；72℃10min；By 4 DEG C of preservations of PCR primer, through agarose gel electrophoresis and purifying of tapping rubber, PMD is cloned in
In 18-T vector carriers, identify and be sequenced, obtain objective gene sequence.
4. the method that a kind of screening anoplophora glabripennis according to claim 3 lures composition, it is characterised in that：The step
The detailed process of the prokaryotic expression carrier of structure anoplophora glabripennis odor-binding protein is in three：
Using Prokaryotic expression vector construction expressing fusion protein systems approach, pET-32a (+) carrier is transformed, removes and carries
The Thrombin restriction enzyme sites that body is carried；Primer of the design with Xha I and Xho two restriction enzyme sites of I, utilizes bridge-type PCR's
Method removes one section of base containing Thrombin, and one section of carrier sequence for being free of Thrombin is cloned again；Will be from
XbaI-XhoI purpose fragment bridgings PCR is expanded；CDNA fragments to amplification use XbaI and XhoI to pET-32a (+) and cDNA
Fragment carries out digestion, and pET-32a (+) carrier of digestion processing identical with process is attached, after the α of conversion Host Strains Trans 5,
Picking single bacterium colony, upgrading grain simultaneously carry out digestion identification.
5. the method that a kind of screening anoplophora glabripennis according to claim 4 lures composition, it is characterised in that：The step
The detailed process of the protein-bonded induction of anoplophora glabripennis restructuring smell and purifying is in four：
Induce anoplophora glabripennis to recombinate smell binding protein expression by IPTG, and pass through nickel Ago-Gel affinity column pair
It is purified；
The prokaryotic expression of destination protein：By pET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 matter built
Grain is transformed into BL21 (DE3) competent cell, and picking monoclonal or glycerol stock are seeded to LB nutrient solutions of the 5ml containing corresponding antibiotic
In, 37 DEG C, 230R/min concussion and cultivates are stayed overnight；1ml overnight cultures are taken to access LB cultures of the 50ml containing appropriate corresponding antibiosis rope
In liquid, 37 DEG C, shaking table 230r/min is cultivated, and takes culture 20mL to be forwarded to the LB nutrient solutions that 1L contains appropriate corresponding antibiotic after 3h
In, 37 DEG C, shaking table 230r/min is cultivated to OD600 in 0.6-0.8；In 16 DEG C of the inductive condition filtered out, IPTG concentration is
0.5mM, overnight induction, induced expression target protein；Cell is collected with 6000r/min centrifugations 15min in 4 DEG C, wet thallus is preserved
In the purifying of -80 DEG C of continuation；
Target protein pET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 purifying：400mL has been induced
PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 thalline are resuspended in lysate Buffer A, Buffer A
By pH7.0,40mM Tris, 300mM NaCI, 5%Glycerol composition, according to bacterium solution:Buffer A=2L：50ml ratio
Dissolve thalline；PSMF (100mM) is added, 50ml phage solutions add 50 μ l PSMF, MgC12, 50 μ l of 5Oml solution addition
MgCI2, and DNAse, 1L bacterium add 10 μ l DNAse mix after height crush bacterium, pressure is 1200bar, until bacterium solution be in drops
Outflow, then at 4 DEG C, 6000r/min, 1h take supernatant to the nickel NTA agarose Gel columns balanced, and successively with without imidazoles
100%Buffer B and imidazole gradient be 10mM, 50mM, 100mM, 250mM, 500mM 100%Buffer B are washed
It is de-, seven times altogether, collect after 7 pipe eluents, often manage and take 10ul to carry out SDS-PAGE analyses, the purity of testing goal albumen；
Collect after the eluent containing pure PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 recombinant proteins, will
It is transferred in bag filter, in the PBS of 500ml urea containing descending concentrations, and dialyse 72h at 4 DEG C, and every 8h more
A PBS is changed, the initial concentration of the descending concentrations urea is 7mol/L.
6. a kind of method for screening anoplophora glabripennis attractant composition according to claim 5, it is characterised in that：The step
The detailed process of the screening of anoplophora glabripennis attractant composition is in rapid five：
Anoplophora glabripennis restructuring smell albumen and Acer negundo volatile matter and anoplophora glabripennis are obtained using competitiveness fluorescent associated methods
The association reaction spectrum of aggregation pheromone volatile matter, filters out 9 kinds of Acer negundo volatile matters for luring anoplophora glabripennis and 8 kinds of glabripenis day
Ox aggregation pheromone volatile matter, 9 kinds of Acer negundo volatile matters and 8 kinds of anoplophora glabripennis aggregation pheromone volatile matters can be with glabripenis day
Ox PET-32a (+)-Agla-OBP1 and pET-32a (+)-Agla-OBP2 produces association reaction.
7. the method that a kind of screening anoplophora glabripennis according to claim 6 lures composition, it is characterised in that：Described 9 kinds
Acer negundo volatile matter is specially：
Cis- 3- hexenols, 4- hexenes, 1- acetic acid esters, methyl palmitate, hexyl acetate, 3- hexenes, 1- acetic acid esters, 4- hexenes,
1- butyrates, 4- hexenes, 1- valerates, 2- dipropyl-adipate ester, diisooctyl phthalate.
8. the method that a kind of screening anoplophora glabripennis according to claim 7 lures composition, it is characterised in that：Described 8 kinds
Anoplophora glabripennis aggregation pheromone volatile matter is specially：
2- methyl-docosane, cis -9- tricosenes, cis -9- pentacosa alkenes, cis -7- pentacosa alkenes, cis -9- two
17 alkene, the alkene of cis -7- 27,4- epoxides in heptan-butyraldehyde, 4- epoxides in heptan-n-butanol.
9. a kind of method for preparing anoplophora glabripennis attractant, it is characterised in that：The side for preparing anoplophora glabripennis attractant
The detailed process of method is：
By 9 kinds of Acer negundo volatile matters and 8 kinds of anoplophora glabripennis aggregation pheromone volatile matters with volume ratio 2:After 1 mixing, according to volume
Than 1:2 are dissolved in atoleine, and anoplophora glabripennis attractant is made；
The cis- 3- hexenols of 9 kinds of Acer negundo volatile matters, 4- hexenes, 1- acetic acid esters, methyl palmitate, hexyl acetate, 3- oneself
Alkene, 1- acetic acid esters, 4- hexenes, 1- butyrates, 4- hexenes, 1- valerates, 2- dipropyl-adipate ester, phthalic acid two are different
The mass percent of monooctyl ester is followed successively by：2.78：34.70：5.06：2.07：0.38：12.36：6.38：5.74：0.79；
The 8 kinds of anoplophora glabripennis aggregation pheromone volatile matter 2- methyl-docosane, cis -9- tricosenes, cis -9-
Pentacosa alkene, cis -7- pentacosa alkenes, the alkene of cis -9- 27, the alkene of cis -7- 27,4- epoxides in heptan-butyraldehyde, 4- oxygen in heptan
The volume ratio of base-n-butanol is：0.5：1：2：1：0.5：0.5：1:1.
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