CN103411939A - Tea geometrid odorant binding protein-based plant attractant screening method - Google Patents

Abstract

The invention discloses a tea geometrid odorant binding protein-based plant attractant screening method and belongs to the technical field of bioengineering. The tea geometrid odorant binding protein-based plant attractant screening method comprises the following processes of collecting tea geometrid antenna total RNA, acquiring total length of a tea geometrid odorant binding protein by RT-PCR, constructing a prokaryotic expression vector of the tea geometrid odorant binding protein, inducing the expression of the recombinant tea geometrid odorant binding protein by IPTG, carrying out the purification of the recombinant tea geometrid odorant binding protein by nickel sepharose gel affinity column, acquiring a binding reaction spectrum of the recombinant tea geometrid odorant binding protein and tea leaf smell volatiles by a competitive fluorescent combination method, and determining that the tea leaf smell volatile reducing relative fluorescence intensity of 1-NPN to less than 50% and having the binding constant of 13-45 micromoles per liter is the plant attractant of tea geometrid. The tea geometrid odorant binding protein-based plant attractant screening method provides a novel means for screening and designing a formula of a plant tea geometrid smell information attractant.

Description

A kind of screening technique of the botanical attractant based on the tea geometrid OBP

Technical field

The invention belongs to technical field of bioengineering, be specifically related to a kind of screening technique of the botanical attractant based on the tea geometrid OBP.

Background technology

Tea geometrid Ectropis obliquaProut, belong to Lepidoptera, and Geometridae is a kind of great Pests of Tea-Plants that tea province is respectively produced in the Yangtze river basin that is distributed widely in, and due to its happiness food tea leaf, and generation is many, and breeding is fast, and while seriously occurring, blade is all eaten up, and causes the Tea Industry underproduction even to have no harvest.For a long time, control tea geometrid and mainly rely on chemical pesticide control, very easily cause the Pesticide Residue in commodity tealeaves, the normal development of serious threat China tea trade industry, Given this, be necessary the non-harmful tea geometrid biological prevention of development of new outside chemical pesticide.Wherein, utilize its Olfactory behavior and design a kind of important means that its attractant becomes the current tea geometrid comprehensive regulation, and take, identify that by the chemical apparatuses analysis means tea geometrid is to the technology of tea leaf volatile matter as main representative, because the tea leaf volatile constituent is very complicated, so the very difficult design and development effectively of the method goes out to be applicable to the sense of smell attractant formula of tea geometrid.

Summary of the invention

For the problem that prior art exists, the object of the invention is to design the technical scheme of the screening technique that a kind of botanical attractant based on the tea geometrid OBP is provided.

The screening technique of described a kind of botanical attractant based on the tea geometrid OBP is characterized in that comprising following processing step:

1) collect the total RNA of tea geometrid feeler;

2) by RT-PCR, obtain the total length of tea geometrid OBP;

3) build the prokaryotic expression carrier of tea geometrid OBP;

4) by IPTG, induce tea geometrid restructuring smell binding protein expression, and by nickel Ago-Gel affinity column, it is carried out to purifying;

5) by the competitiveness fluorescent associated methods, obtain the association reaction spectrum of tea geometrid restructuring OBP and tea leaf smell volatile matter, the relative intensity of fluorescence of 1-NPN is reduced to below 50%, and binding constant is the botanical attractant that the volatile matter of the tea leaf of 13～45 μ mol/L is defined as tea geometrid.

The screening technique of described a kind of botanical attractant based on the tea geometrid OBP, it is characterized in that in described step 4), IPTG induces the inductive condition of tea geometrid restructuring smell binding protein expression to be: IPTG concentration is 1mmol/L, temperature is 30 ℃, inducing rotating speed is 200rpm, and induction time is 4h.

The screening technique of described a kind of botanical attractant based on the tea geometrid OBP is characterized in that competitiveness fluorescent associated methods in described step 5) obtains the step that tea geometrid restructuring OBP and the association reaction of tea leaf smell volatile matter compose as follows:

1) fluorescence aglucon 1-NPN and the protein-bonded combination of tea geometrid restructuring smell

Under the 282nm excitation wavelength, the 1-NPN to successively adding 1mmol/L in the tea geometrid of 1.5 μ mol/L restructuring OBP, fully mix;

2) aglucon combination

Choosing tea leaf smell volatile matter and 1-NPN is at war with in conjunction with tea geometrid restructuring OBP, the relative intensity of fluorescence of 1-NPN is reduced to below 50%, and binding constant is the botanical attractant that the volatile matter of the tea leaf of 13～45 μ mol/L is defined as tea geometrid.

The present invention is from the physiological pattern of tea geometrid sense of smell, by Protocols in Molecular Biology, cloned its OBP full-length gene, and by the prokaryotic expression technology, obtained the recombinant protein of this gene of encoding, finally by the biological chemistry combination technology, inquired into itself and the affinity of the plant volatiles in different tea trees source.The present invention provides new strategy for screening and the design of tea geometrid plant source smell information attractant formula.

The accompanying drawing explanation

Fig. 1 is the clone of tea geometrid EoblGOBP2 gene;

Fig. 2 is the optimization of restructuring tea geometrid OBP EoblGOBP2 abduction delivering time;

Fig. 3 is the separation and purification of restructuring tea geometrid OBP EoblGOBP2;

Fig. 4 is that candidate's smell information is combined with the competition of fluorescence aglucon 1-NPN and restructuring EoblGOBP2 albumen;

Fig. 5 is the antennae response current potential of candidate's smell attractant to tea geometrid.

Embodiment

Below in conjunction with embodiment, further illustrate the present invention.

Embodiment 1

1) collect the concrete steps of the total RNA of tea geometrid feeler;

Material: tea geometrid is tea place, Hangzhou Kill the larva, the laboratory artificial feeding to after adult for experiment.The larva raising condition is: 26 ± 1 ℃ of temperature, L:D=12:12, RH are 70%～80%.Utilize the TRIzol method to extract total RNA of adults of ectropis obliqua feeler, and utilize Super-Script II Reverse Transcriptase System (Takara) reverse transcription to obtain cDNA the first chain.

2) by RT-PCR, obtain the concrete steps of the total length of tea geometrid OBP;

1. the design of primers of tea geometrid EoblGOBP2.(the Genbank accession number is: ACN29681.1(12), utilize Primer Premier 5.0 Software for Design primers according to the gene order of the common OBP GOBP2 of tea geometrid in Genbank (EoblGOBP2).While for the later stage, expressing, conveniently genes of interest is cloned on expression vector to the restriction enzyme site (meaning with underscore) of design EcoR I and Hind III on forward and reverse primer.

Upstream primer: 5 '-GTGAATTCATGAAGTCTGTTCTGGTGGCGACGGT-3 ';

Downstream primer: 5 '-GGCAAGCTTGCTAATACTTCTCCATGACAGCTTC-3 '.

Primer is synthetic by Sangon Biotech (Shanghai) Co., Ltd..

2. the amplification of tea geometrid GOBP2 cDNA, Cloning and sequencing.Synthetic cDNA the first chain of the reverse transcriptase of take is template, and it carries out pcr amplification with primer pair.Through the PCR condition is optimized, finally determine the pcr amplification program of 6 genes: 94 ℃ of denaturation 3 min; 94 ℃ of 30 s, 62.1 ℃ of 30 s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 10 min.

The PCR product, through agarose gel electrophoresis the purifying of tapping rubber, is cloned in PMD 18-T vector carrier, after evaluation, delivers to Shanghai Sani Bioisystech Co., Ltd and check order, thereby obtain the genes of interest sequence, as shown in Figure 1.

3) build the concrete steps of the prokaryotic expression carrier of tea geometrid OBP;

With kit, extract cloned plasmids, warp EcoThe R I and HinAfter d III double digestion, enzyme is cut to product and carry out agarose gel electrophoresis and tap rubber purifying purpose fragment.Then the purpose fragment is connected with the linear pET-32a carrier of cutting processing through same enzyme, after according to common method, transforming Host Strains Trans 5 α, picking list bacterium colony, upgrading grain also carry out enzyme and cut evaluation.

4) by IPTG, induce tea geometrid restructuring smell binding protein expression, and by nickel Ago-Gel affinity column, it is carried out to the concrete steps of purifying;

1. the recombinate prokaryotic expression of tea geometrid GOBP2 albumen

The pET32a/GOBP2 Plasmid Transformation built is advanced to BL21 (DE3) competent cell, through flat board, be coated with, the monoclonal colony inoculation that picking obtains is in 1 mL LB nutrient culture media (containing Amp 100 μ g/mL), and 37 ℃ of 220 rpm shaken cultivation spent the night.After activation, (contain Amp 100 μ g/mL) by 1% (V/V) inoculum concentration, the 20 mL LB nutrient culture media of transferring, 37 ℃ of 220 rpm shaken cultivation is to OD600 to 0.6 left and right, get 1 mL bacterium liquid and make negative control, to original bacteria liquid, adding IPTG again is 1 mmol/L to final concentration, and 30 ℃ of 200 rpm continues to induce, and gets 1 mL bacterium liquid every 1h, be total to 5h, finally use the SDS-PAGE electrophoresis detection abduction delivering effect of different expression times, and determine that best induction time is 4 h, as shown in Figure 2.

2. the recombinate separation and purification of tea geometrid GOBP2 albumen

The EoblGOBP2 Escherichia coli bacteria liquid that 400mL is induced, the centrifugal 10min of 8000rpm, abandon supernatant, by microorganism collection in the 50mL centrifuge tube, add 10 mL lysis buffers and 100 μ L lysozymes, the rear 4 ℃ of cracking that fully suspend are spent the night, after next day, 5s/5s was interrupted ultrasonic 10min, 4 ℃ of 12000 centrifugal 10min of rpm, abandon supernatant, precipitation adds bacterial lysate, 4 ℃ of 12000 centrifugal 10min of rpm after room temperature cracking 1h, get supernatant to the good nickel NTA Ago-Gel post of balance, and with the solution of different imidazole concentrations, carry out gradient elution successively, amount to seven times, after collecting 7 pipe eluents, every pipe is got 10ul and is carried out the SDS-PAGE analysis, the position of the purity of testing goal albumen and place collection tube, as shown in Figure 3.

To determine that the eluent that contains pure EoblGOBP2 recombinant protein all shifts in bag filter, in 500mL contains that concentration is low and subtracts the PBS damping fluid (pH7.4) of urea, the urea initial concentration is 7mol/L, in 4 ℃ of dialysis 72h, during changed once fresh PBS damping fluid in every 8 hours.Then after the fusion of having dialysed being measured to concentration by the Bradford method, be transferred in 1.5 mL Eppendorf pipes, be stored in-80 ℃ of refrigerators standby.

5) pass through the association reaction spectrum that the competitiveness fluorescent associated methods obtains tea geometrid restructuring OBP and tea leaf smell volatile matter, screening obtains the concrete steps of the botanical attractant of applicable tea geometrid.

With methyl alcohol, 1-NPN fluorescence aglucon is made into to 1 mmol/L and is placed in 4 ℃ and keep in Dark Place, pipette the EoblGOBP2 of 1.5 μ mol/L in quartz colorimetric utensil, successively add the 1-NPN solution of 1mmol/L to carry out fluorescent scanning, add afterreaction 2min at every turn, under 282 wavelength, excite, record fluorescence emission spectrum, and the fluorescence intensity at maximum emission wavelength 328 nm places is according to Scatchard equation (1) linearization spectroscopic data:

(1)

Wherein [Dt] and [D] means respectively free 1-NPN concentration in total and system, and [Pt] is the concentration of EoblGOBP2 recombinant protein, and K is the binding constant of 1-NPN and EoblGOBP2, and n is binding site number.Can obtain the dissociation constant of 1-NPN and EoblGOBP2.

Then utilize competion experiment to study the dissociation constant of candidate's aglucon and tea geometrid EoblGOBP2 recombinant protein.In Competition binding assay, use 1-NPN as fluorescence report, different aglucons are mixed with 1 μ mol/l restructuring OBP.Chemical aglucon is dissolved in methanol solution, and being mixed with concentration is the 10mmol/L sample.Chemical aglucon is successively joined in 1-NPN and EoblGOBP2 mixed liquid of protein, add afterreaction time 2min at every turn, record the emission spectrum of fluorescence.According to equation (2) calculated candidate aglucon dissociation constant KD:

(2)

[IC wherein ₅₀] be that the concentration of competition aglucon can replace 50% 1-NPN time the, [1-NPN] they are not in conjunction with the concentration of 1-NPN, K _1-NPNDissociation constant for GOBP2 and 1-NPN.

Choose different being at war with property of tea leaf smell volatile matter fluorescence in conjunction with test, discovery accounts for the gaultherolin that tea plant volatiles content is higher, trans-2-hexenoic aldehyde, trans-2-decenal, benzaldehyde, alpha, beta-lonone and dibutyl phthalate all energy and tea geometrid EoblGOBP2 generation association reaction, concrete outcome such as following table and shown in Figure 4:

Table 1 candidate aglucon is combined with the competition of 1-NPN and restructuring EoblGOBP2 albumen

Embodiment 2

The efficiency assay of the botanical attractant that proof obtains by the present invention.

1) for the examination insect.The mature larva of tea geometrid picks up from the population into artificial feeding in this laboratory.The raising condition is: temperature (26 ± 1) ℃, relative humidity (75 ± 5) %, photoperiod L:D=12:12(light 12 h, dark 12 h).After it is pupated, sprouts wings, collect the adult in sexal maturity period and test, hungry 3 h before experiment.

2) experimental apparatus and reagent.Experimental apparatus adopts tentaculum electric potential instrument (Dutch Syntech company); Various tea leaf volatile matter standard items, all purchased from the lark prestige, are dissolved in whiteruss by every kind of compound respectively, and are made into 0.1(v/v) solution carries out EAG mensuration.(2 cm * 0.5 cm) drips 10 uL testing sample solutions on the filter paper bar in advance, makes solvent evaporates one after the meeting, then filter paper is filled in the Pasteur pipe, with masking foil, mouth of pipe two ends sealed, and prevents the sample volatilization.Separately get 10 uL whiteruss+filter paper as blank.

3) experimental technique.At first with scalpel, the tea geometrid feeler is cut from base portion, then carefully the two ends of tea geometrid feeler are sticked respectively on the metal electrode of EAG with conducting resinl.During test, Pasteur Guan Yiduan being inserted to the snorkel outside diameter is in the aperture of 2 mm, and the snorkel mouth of pipe is vertically vertical with feeler, and with feeler at a distance of about 1 cm.Step on pedal with stimulation time 0.2 s, stimulating air-flow is 100 mL/min, between twice stimulation, makes feeler recover 2 min.Before working sample, first carry out contrast CK(whiteruss 1 time) and criterion referenced stimulate, specimen then, each testing sample repetition 6 times.The EAG reacting value calculates with reference to following formula:

In formula, Sr is the relative value of tea geometrid to this kind compd E AG reaction, Sc is the EAG reacting value of this kind compound, CKm is the mean value of the twice EAG reaction of contrast whiteruss before and after this kind of mensuration compound, and Rm is for measuring the mean value of the front criterion referenced EAG reaction of this kind compound.

4) data are processed and are analyzed

Utilize SPSS 16.0 softwares to process and analyze the experimental data obtained.Result shows that the tea geometrid feeler is followed successively by for the reaction size of tea plant volatiles: trans-2-hexenoic aldehyde > benzaldehyde > acetophenone > gaultherolin, and the dissociation constant of above-mentioned volatile matter and tea geometrid EoblGOBP2 albumen is 13～45 μ mol/L, therefore determine dissociation constant in this scope the volatile matter of tea leaf be the botanical attractant composition of tea geometrid, as shown in Figure 5.

Claims (3)

1. screening technique based on the botanical attractant of tea geometrid OBP is characterized in that comprising following processing step:

1) collect the total RNA of tea geometrid feeler;

2) by RT-PCR, obtain the total length of tea geometrid OBP;

3) build the prokaryotic expression carrier of tea geometrid OBP;

4) by IPTG, induce tea geometrid restructuring smell binding protein expression, and by nickel Ago-Gel affinity column, it is carried out to purifying;

5) by the competitiveness fluorescent associated methods, obtain the association reaction spectrum of tea geometrid restructuring OBP and tea leaf smell volatile matter, the relative intensity of fluorescence of 1-NPN is reduced to below 50%, and binding constant is the botanical attractant that the volatile matter of the tea leaf of 13～45 μ mol/L is defined as tea geometrid.

2. the screening technique of a kind of botanical attractant based on the tea geometrid OBP as claimed in claim 1, it is characterized in that in described step 4), IPTG induces the inductive condition of tea geometrid restructuring smell binding protein expression to be: IPTG concentration is 1mmol/L, temperature is 30 ℃, inducing rotating speed is 200rpm, and induction time is 4h.

3. the screening technique of a kind of botanical attractant based on the tea geometrid OBP as claimed in claim 1 is characterized in that competitiveness fluorescent associated methods in described step 5) obtains the step that tea geometrid restructuring OBP and the association reaction of tea leaf smell volatile matter compose as follows:

1) fluorescence aglucon 1-NPN and the protein-bonded combination of tea geometrid restructuring smell

Under the 282nm excitation wavelength, the 1-NPN to successively adding 1mmol/L in the tea geometrid of 1.5 μ mol/L restructuring OBP, fully mix;

2) aglucon combination

Choosing tea leaf smell volatile matter and 1-NPN is at war with in conjunction with tea geometrid restructuring OBP, the relative intensity of fluorescence of 1-NPN is reduced to below 50%, and binding constant is the botanical attractant that the volatile matter of the tea leaf of 13～45 μ mol/L is defined as tea geometrid.

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