CN103245647B - Citrus fruit fly odorant binding protein-based attractant screening method - Google Patents

Abstract

The invention discloses a citrus fruit fly odorant binding protein-based attractant screening method belonging to the technical field of bioengineering. The citrus fruit fly odorant binding protein-based attractant screening method comprises the following steps of: collecting the total RNA (Ribonucleic Acid) of antennae of a citrus fruit fly; obtaining the overall length of a citrus fruit fly odorant binding protein through RT-PCR (Reverse Transcription-Polymerase Chain Reaction); constructing a prokaryotic expression vector of the citrus fruit fly odorant binding protein; inducing the expression of a citrus fruit fly recombinant odorant binding protein through IPTG (isopropyl-beta-d-thiogalactoside), and purifying the citrus fruit fly recombinant odorant binding protein through a nickel sepharose gel affinity column; obtaining a conjugation reaction spectrum of the citrus fruit fly recombinant odorant binding protein and a host fruit odor volatile matter through a competitive fluorescence combing method, wherein a dissociation constant (KD) is lower than below 10 mu mol/L host fruit smell; and the IC 50 value of fluorescence competition is less than 30 mu mol/L, determining a host fruit smell attractant suitable for the citrus fruit fly. The invention provides a new strategy for screening and designing a citrus fruit fly odorant host fruit smell odor information attractant formula.

Description

Based on the attractant screening technique of citrus fruit fly OBP

Technical field

The invention belongs to technical field of bioengineering, be specifically related to the attractant screening technique based on citrus fruit fly OBP.

Background technology

Citrus fruit fly bactrocera dorsalis, have another name called orient fruit fly (Orient fruit fly), belong to Diptera (Diptera), Tephritidae (Tephritidae), Bactrocera bactrocera Macquart, be the important quarantine pest of a class.This worm host range is wide, it is reported 46 section 250 various fruits of can causing harm, and causes harm seriously, cause huge economic loss to China's fruit industry to banana, guava, mango and oranges and tangerines etc.Current production is mainly taked chemical prevention control its harm, but chemical pesticide not only easily causes fruit and environmental pollution, and and be difficult to prove effective because of its mode that causes harm special (mainly with Adult worms producting eggs under fruit rind, larva is dived and occupies fruit flesh and take food, arillate physical shielding).Due to plant-feed insect to the host plant smell that it is had a liking for have significantly select tendency, and citrus fruit fly also has significantly hobby property for multiple host fruit, as the important fruit host that the fruit such as guava, grape, sweet orange, banana are all citrus fruit flies, and citrus fruit fly also can both produce significant sense of smell to them and lures reaction, therefore we according to the main host plants fruit volatile matter screening of citrus fruit fly and can obtain its Olfactory Attractants.But because the volatile constituent kind of various fruit is complicated, how to filter out the bottleneck that suitable attractant formula becomes the especially female worm attractant of restriction citrus fruit fly Olfactory Attractants.

Summary of the invention

For prior art Problems existing, the object of the invention is to design the technical scheme of the attractant screening technique provided based on citrus fruit fly OBP.

The described attractant screening technique based on citrus fruit fly OBP, is characterized in that comprising following processing step:

1) citrus fruit fly feeler total serum IgE is collected;

2) total length of citrus fruit fly OBP is obtained by RT-PCR;

3) prokaryotic expression carrier of citrus fruit fly OBP is built;

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

5) composed by the association reaction of competitiveness fluorescent associated methods acquisition citrus fruit fly restructuring OBP and host fruit smell volatile matter, dissociation constant k _dbelow the sense of smell of 10 μm of ol/L host fruits, the IC of fluorescence competition ₅₀when value is less than 30 μm of ol/L, be defined as the host fruit Olfactory Attractants of applicable citrus fruit fly.

The described attractant screening technique based on citrus fruit fly OBP, it is characterized in that the inductive condition of IPTG induction citrus fruit fly restructuring smell binding protein expression in described step 4) is: IPTG concentration is 0.8 ~ 1.2mmol/L, temperature is 28 ~ 32 DEG C, induction rotating speed is 150 ~ 250rpm, and induction time is 4 ~ 6h.

The described attractant screening technique based on citrus fruit fly OBP, it is characterized in that the inductive condition of IPTG induction citrus fruit fly restructuring smell binding protein expression in described step 4) is: IPTG concentration is 1.0mmol/L, temperature is 30 DEG C, induction rotating speed is 200rpm, and induction time is 5h.

The described attractant screening technique based on citrus fruit fly OBP, is characterized in that the processing step that the association reaction that in described step 5), competitiveness fluorescent associated methods obtains citrus fruit fly restructuring OBP and host fruit smell volatile matter is composed comprises:

1) Fluorescent ligands 1-NPN and citrus fruit fly are recombinated the protein-bonded combination of smell

Under 282nm excitation wavelength, in the citrus fruit fly restructuring OBP of 1 μm of ol/L, successively add the 1-NPN of 1mmol/L, fully mix;

2) aglucon combines and EAG checking

Choosing citrus fruit fly host fruit key odor volatile matter and 1-NPN is at war with in conjunction with citrus fruit fly restructuring OBP, if the competition of citrus fruit fly host fruit key odor volatile matter, 1-NPN and BdorOBP relative fluorescence is to less than 50%, and dissociation constant k _dbelow the sense of smell of 10 μm of ol/L host fruits, the IC of fluorescence competition ₅₀when value is less than 30 μm of ol/L or fluorescent value competition to less than 30% time, be then defined as the host fruit Olfactory Attractants of applicable citrus fruit fly.

The present invention is from the physiological pattern of citrus fruit fly sense of smell, its OBP full-length gene has been cloned by Protocols in Molecular Biology, and the recombinant protein of this gene of coding is obtained by prokaryotic expression technology, the affinity of the host fruit sense of smell volatile matter that it is originated from different tea tree has been inquired into finally by biological chemistry combination technology.The present invention is that the screening of citrus fruit fly host fruit smell information attractant formula and design provide new strategy.

Accompanying drawing explanation

Fig. 1 is the pcr amplification figure of BdorOBP2 gene;

Fig. 2 is bacterium colony PCR qualification figure;

Fig. 3 is the separation and purification figure of restructuring citrus fruit fly smell protein B dorOBP;

Fig. 4 is the fluorescence binding curve figure of restructuring BdorOBP albumen and Fluorescent ligands 1-NPN;

Fig. 5 is the competition binding figure of candidate's aglucon and Fluorescent ligands 1-NPN and BdorOBP albumen of recombinating;

Fig. 6 is that the female male worm of citrus fruit fly reacts fluorescence in conjunction with proof diagram the EAG of different fruit volatile matter.

In Fig. 3: 1: the BL21(DE3 of the pET32a-BdorOBP do not induced) thalline; 2: through the pET32a-BdorOBP thalline of IPTG induction; The precipitation of 3:pET32a-BdorOBP after ultrasonication; The supernatant of 4:pET32a-BdorOBP after ultrasonication; 5: the pET32a-BdorOBP albumen after purifying.

Embodiment

The present invention is further illustrated below in conjunction with embodiment.

Embodiment 1: the gene clone of collecting citrus fruit fly feeler total serum IgE and OBP OBP

The clone of 1 citrus fruit fly OBP BdorOBP2

The extraction of 1.1 citrus fruit fly total serum IgE

Adopt Trizol reagent to extract the total serum IgE of citrus fruit fly, concrete operation step is as follows:

1) take out from-80 DEG C the citrus fruit fly sample preserved, put into the mortar with Liquid nitrogen precooler, add liquid nitrogen to grind fast, this step operates on ice, after pulverize, take 100 mg tissue samples to load in 1.5 mL centrifuge tubes, then add 1 ml Trizol reagent, eddy mixer mixes;

2) sample is left standstill 5 min in room temperature, nucleic acid-protein compound is separated completely;

3) 4 DEG C, 12 000 × g, centrifugal 5 min;

4) to be transferred to by supernatant in new centrifuge tube (being sure not to draw precipitation), the chloroform then adding 200 μ L turns upside down mixing 15 s, and room temperature places 15 min;

5) 4 DEG C, 12 000 × g, centrifugal 15 min;

6) supernatant of transferase 45 00 μ about L is in another new centrifuge tube, then adds the isopropyl alcohol of 200 μ L, turns upside down after fully mixing, leaves standstill 10 min at 15 DEG C;

7) 4 DEG C, 12 000 × g, centrifugal 10 min;

8) outwell supernatant, in centrifuge tube, then add the ethanol (preparation of DEPC water) that 1 ml concentration is 75 %, wash;

9) 4 DEG C, 8 000 × g, centrifugal 5 min;

10) outwell supernatant, first drying precipitated 5-10 min in atmosphere, then use DEPC water dissolution precipitation;

11) after RNA dissolves completely, the total serum IgE of extraction is stored in-80 DEG C of refrigerators or carries out reverse transcription and synthesize the first chain.

The synthesis of 1.2 first chain cDNA

Adopt the first chain cDNA of the Reverse Transcription box synthesis citrus fruit fly of TaKaRa company.Experimental technique carries out according to kit instructions, and specific experiment step is as follows:

Mixing, carries out of short duration centrifugal, PCR response procedures: 37 DEG C of 15 min, 85 DEG C of 5 s.Being tested to this stage has synthesized the first chain cDNA, it is saved backup under-20 DEG C of conditions.

1.3 gene clone

According to the citrus fruit fly BdorOBP total length (GenBank accession number: EU564816) logged in GenBank, design Auele Specific Primer:

Forward primer: 5 '-TAGAATTCATGCACTCCCGAAAGACTCTCCTGGG-3 ';

Reverse primer: 5 '-CCGAAGCTTGTTARATCAARAAATAATGCTTTGG-3 '.

In order to the needs of subsequent experimental operation, by genes of interest fragment subclone on other carriers, add in forward and reverse primer respectively when designing primer ecor I, hind III restriction enzyme site (representing with underscore).Primer is synthesized by Shanghai Sheng Gong company.

With citrus fruit fly cDNA for template, carry out PCR amplification BdorOBP2 gene by ExTaq enzyme.PCR reaction system following (20 μ L):

The reaction conditions of PCR is: 95 DEG C of 4 min denaturation, each circulation 94 DEG C of 30 s, 57 DEG C of 45 s, 72 DEG C of 45s, carries out 35 circulations, and then 72 DEG C extend 10 min, 16 DEG C of fover.The agarose gel electrophoresis that PCR terminates rear use 1 % carries out detection analysis to object band.

The rubber tapping of 1.4 PCR primer is reclaimed

Utilize Axygen DNA gel to reclaim kit to reclaim rubber tapping product.Concrete grammar is as follows:

1) under uviol lamp, cut object fragment, put into the centrifuge tube of 1.5 mL, first weigh, after then example converts in mass ratio, add 600 μ L Buffer DE-A, heating and melting (heating 5-7 min, notes mixing in heating process) under 75 DEG C of conditions;

2) then to 1) in centrifuge tube in add 250 μ L Buffer DE-B, fully load after mixing and prepare in pipe, centrifugal 1 min of 12 000 × g;

3) by the liquid after centrifugal in post, 500 μ L Buffer W1 are added, centrifugal 30 s of 12 000 × g;

4) outwell filtrate, add 700 μ L Buffer W2, centrifugal 30 s of 12 000 × g normal temperature;

5) outwell filtrate, add 700 μ L Buffer W2 and wash one time again, centrifugal 1 min of 12 000 × g;

6) 1.5 new ml centrifuge tubes are got, pillar is put into pipe, add 20 μ L ddH2O, after room temperature places 1 min, centrifugal 1 min of 12 000 × g, after abandoning pillar, the agarose gel electrophoresis with 1% detects and reclaims afterproduct purity, and for subsequent use with being kept at-20 DEG C after ultramicron UV spectrophotometer measuring concentration.

1.5 coupled reaction

Product is reclaimed in the rubber tapping of genes of interest fragment be connected on pMD 18-T Vector carrier, be converted into Trans 5 α competent cell.Linked system is as follows:

。

Embodiment 2: citrus fruit fly OBP BdorOBP2 expression vector establishment

2.1PCR amplification BdorOBP2 gene

Extract the total serum IgE of citrus fruit fly, as template after cDNA first chain that reverse transcription obtains, PCR reaction is carried out with primer before and after the citrus fruit fly BdorOBP2 of design and synthesis, PCR result is through agarose gel electrophoresis, as shown in fig. 1, obtain the object fragment of about 447bp, consistent with expection fragment length.

The qualification of 2.2 bacterium liquid PCR

After BdorOBP2 gene PCR increases and to be connected with pMD 18-T Vector after the object fragments gel that obtains reclaims purifying and to transform, after blue hickie and the qualification of ammonia benzyl, the clone bacterium of the white chosen carries out the qualification of positive colony, and as shown in Figure 2, object stripe size meets the requirements PCR the result.

2.3 are converted into competent cell Trans 5 α

Utilize bacterium liquid PCR to verify to recombinant plasmid pMD18/BdorOBP2, then send into the Shanghai Sani company that checks order and check order.If order-checking is correct, lower step by pMD18/BdorOBP2 plasmid and expression vector pET32a (+) plasmid all through EcoRI and Hind III restriction enzymes double zyme cutting, then object fragment and pET32a (+) carrier are with mole ratio 3:1, and under the effect of T4 ligase, 4 DEG C of connections are spent the night and built pET32/BdorOBP2.Product after connection first transforms and enters Escherichia coli Trans5 α, to choose after positive colony bacterium colony after PCR qualification, the positive colony bacterium containing pET32/BdorOBP2 choosing qualification expands to be cultivated, and extract plasmid, the plasmid of extraction through double digestion digestion verification and serve extra large Sani check order company order-checking determine further.Linked system and double digestion system as follows:

1) double digestion system (20 μ L):

2) linked system (10 μ L):

3) conversion of product is connected

A. get-70 DEG C of frozen competent cells (100 μ L), after thawed on ice, be distributed into two pipes, often pipe 50 μ L;

B. add 5 μ L and connect product, piping and druming evenly gently, ice bath 30 min;

C. bacterium liquid is put into 42 DEG C of water-bath heat shock 90 s, rapid dislocation places 5 min on ice;

D. the LB nutrient culture media (without ammonia benzyl) of 700 ul is added, 200 rpm on 37 DEG C of constant-temperature tables, incubation 1h;

E. by bacterium liquid 3000 rpm/min, centrifugal 3 min, sucking-off supernatant 500 μ L;

F. remaining 200 μ L draw 100 μ L and are coated with flat board, after bacterium liquid is absorbed by flat board, are inverted overnight incubation in 37 DEG C;

G. the white list bacterium colony on picking flat board, puts into containing 1 mL LB(containing ammonia benzyl) 1.5 mL centrifuge tubes, 37 DEG C, 220 rpm, incubated overnight 10-12 h;

H. whether it is positive colony to utilize bacterium colony PCR method to identify;

I. be accredited as the bacterium liquid of positive colony, expansion can be carried out and cultivate and order-checking.

2.4 are transformed into competent cell BL21(DE3)

The pET32-ABdorOBP2 vector successfully constructed is entered the competence BL21(DE3 for prokaryotic expression) in bacterium.Picking list bacterium colony is sent into the Shanghai Sani company of checking order again and is checked order after expanding and cultivating, if the result that order-checking obtains is correct, this bacterium liquid can be stored in-20 DEG C of refrigerators.

Embodiment 3: the abduction delivering of citrus fruit fly OBP BdorOBP2

Single bacterium colony after checking 37 DEG C of shaken cultivation in the LB nutrient culture media of 3 mL containing 100 μ g/mL ammonia benzyl mycins are spent the night, next day is with 1%(V/V) inoculum concentration be inoculated in 20 mL LB nutrient culture media to expand and be cultured to OD600 about 0.5, adding IPTG to final concentration is that 1 mmol/L carries out 30 DEG C of 200rpm and starts abduction delivering.Take out 1 mL bacterium liquid every 1 h, coinduction 5 h, with the bacterium liquid of not inducing for negative control, after induction terminates, by the bacterium liquid of collection per hour centrifugal 10 min precipitations under 8000rpm, and use ddH ₂the centrifugal 10min of 5000rpm after 0 difference Eddy diffusion, collect thalline, add 1 × SDS sample buffer suspension bacterial sediment of 150 μ l, and water proof boils 10min and makes the abundant cracking of thalline at 100 DEG C, sample in each pipe is carried out carrying out in electrophoresis, determining whether thalline has expression in the SDS-PAGE glue of the concentrated glue of 5% and the separation gel of 12% afterwards.

Get the bacterium liquid after 50 mL inductions, under 8000 rpm, centrifugal 10 min collect thalline, after abandoning supernatant, with bacteria lysis damping fluid (the 50 mmol/L Tris-HCl of the lysozyme of 5 mL, 2 mmol/L EDTA, 100 mmol/L NaCl, 0.5 % TritonX-100, 1 mg/mL lysozyme) resuspension fluids is placed on 4 DEG C of abundant cracking and spends the night, cell ultrasonic degradation instrument is used next day to make the abundant cracking of thalline, after centrifugal 10 min of thalline after cracking 12 000 rpm at 4 DEG C, supernatant is transferred in new centrifuge tube for subsequent use, inclusion body lysate (the 50 mmol/L Tris-HCl of 3 mL are added in precipitation, pH 8.0, 1 mmol/L EDTA, 100 mmol/L NaCl, 8 mol/L urea) carry out 4 DEG C of abundant cracking and spend the night, solution after cracking next day centrifugal supernatant of 12 000 rpm at 4 DEG C takes out.Supernatant after inclusion body cracking cleer and peaceful in bacterial lysate cracking is respectively taken out after 1 × SDS sample buffer that 20 μ L add 20 μ L boils 3 min in boiling water, electrophoresis in the SDS-PAGE albuminous degeneration glue of the concentrated glue of 5 % and the separation gel of 12 %, to observe the expression-form of BdorOBP2 recombinant protein.

Table 1 SDS-PAGE electrophoresis concentrates glue and separation gel composition

Interpretation of result: for obtaining more BdorOBP, we were optimized expression time, through SDS-PAGE, analyze electrophoretogram with Bandscan software, result shows, when IPTG concentration is 1mmol/L, temperature 30 DEG C, when induction rotating speed is 200rpm, the inducing amount of 4h is maximum.And specific protein band is at about 36kD, consistent with expection.

Embodiment 4: the purifying of citrus fruit fly OBP BdorOBP2

4.1 protein purification

1) Escherichia coli of 500 mL are induced after determining BdorOBP2 expression of recombinant proteins form, collect the bacterial precipitation containing destination protein, sex change is carried out to inclusion body: in inclusion body solution, add 5 mL inclusion body lysate (50 mmol/L Tris-HCl, pH 8.0,1 mmol/L EDTA, 100 mmol/L NaCl, 8 mol/L urea), piping and druming dissolving 1 h, gets supernatant after 10 000 rpm are centrifugal.

2) with purifying destination protein containing nickel NTA agarose affinity chromatography post, with damping fluid I (0.5 mol/L NaH2PO4, the 0.5 mol/L Na2HPO4 of 5 times of column volumes, NaCl, urea) balance nickel NTA agarose column, pillar on the protein solution that 3.1.3 is obtained, coutroi velocity is at 2 mL/min

3) then wash with the damping fluid I of 5 times of column volumes,

4) use different gradient imidazoles (10,20,50,100,200,300,400 mol/L) to carry out wash-out respectively, collect the eluent under variable concentrations respectively, finally use the purity of SDS-PAGE electrophoresis detection albumen.Various gradient imidazole elution formula is as follows:

The formula rate of table 2 eluent

4.2 dialysis desalinations

1) process of bag filter

A. get 1 mL 0.5 mol/L EDTA, then add 499 mL deionized waters and be made into 1 mmol/L EDTA solution;

B. in above-mentioned solution, add 10 g NaHCO3, fully stir and evenly mix;

C. bag filter is cut into suitable size, then puts in the solution configured, boil 10 min;

D. dislysate dialysis is added after distilled water wash clean again.

2) dialyse

Prepare the PBS solution (pH 7.4 of 500 mL 0.01 mol/L, containing 7 mol/L urea), the eluent determined in 3.1.3 containing pure BdorOBP2 recombinant protein is all shifted in bag filter, put into PBS solution, 4 DEG C are successively reduced dialysate concentration, carry out dialysis renaturation 3 d, this one-phase, every day changes fresh PBS damping fluid once sooner or later respectively, finally the albumen Bradford method of having dialysed is measured the concentration of albumen, with 1.5 ml centrifuge tube packing ,-20 DEG C of Refrigerator stores are for subsequent use.

As the separation and purification figure that Fig. 3 is restructuring citrus fruit fly smell protein B dorOBP, in figure: 1: the BL21(DE3 of the pET32a-BdorOBP do not induced) thalline; 2: through the pET32a-BdorOBP thalline of IPTG induction; The precipitation of 3:pET32a-BdorOBP after ultrasonication; The supernatant of 4:pET32a-BdorOBP after ultrasonication; 5: the pET32a-BdorOBP albumen after purifying.

Embodiment 5: citrus fruit fly restructuring OBP and host fruit smell volatile matter fluorescence competitive assay

5.1 materials and methods

5.1.1 reagent and instrument

5.1.1.1 experiment reagent

The standard model of testing fluorescence probe N-phenyl-1-naphthylamine (N-phenyl-1-naphthylamine, 1-NPN) used and various scented volatile materials is all purchased from lark prestige company, and purity is all at 97 more than %.Testing protein is the BdorOBP2 recombinant protein that above-mentioned purifying is good, and final concentration of protein is 1 μm of ol/L.

5.1.1.2 experimental apparatus

RF-5301PC type fluorospectrophotometer (Japanese Shimadzu Corporation)

5.1.2 the preparation of experiment reagent

Fluorescent ligands 1-NPN is made into 10 mmol/L mother liquors and is placed in 4 DEG C and keep in Dark Place.Each standard model is all dissolved in HPLC level methyl alcohol, is mixed with the solution 4 DEG C preservation of 10 mmol/L.

5.1.3BdorOBP2 the mensuration of binding ability

First the binding curve of BdorOBP2 and 1-NPN is measured.Pipette 1 μm of ol/ BdorOBP2 protein solution in quartz colorimetric utensil, then after progressively adding 1-NPN solution reaction 2 min of 1 mmol/L of 3 μ L, excite under 282 nm wavelength, fluorescent value when record fluorescence emission spectrum and maximum emission wavelength 328 nm place, carries out linearization (1) according to Scatchard equation to spectroscopic data:

（1）

Wherein [Dt] represents 1-NPN concentration total in solution, and [D] represents 1-NPN concentration free in solution, and [Pt] is the concentration of BdorOBP2 fusion, kfor the binding constant of 1-NPN and BdorOBP2, n is binding site number.

Then use 1-NPN as fluorescence probe, utilize competion experiment to study the dissociation constant of fruit volatile matter and citrus fruit fly BdorOBP2 recombinant protein.Successively will join in 1-NPN and BdorOBP2 mixed liquid of protein for examination volatile substance, and add rear reaction time 2 min at every turn, then record fluorescent value.The dissociation constant of volatile matter to be measured is calculated according to formula (2) k _d:

（2）

Wherein [IC ₅₀] be the concentration when Percentage bound of 1-NPN and albumen can be down to 50 % by volatile matter, [1-NPN] is 1-NPN concentration unconjugated in solution, k _1-NPNfor the dissociation constant of albumen/1-NPN compound.

5.1.4 fluorescent spectroscopy

(1) combination of Fluorescent ligands 1-NPN and recombinant protein

Under 282nm excitation wavelength, the 1-NPN of 1mmol/L is successively added in the restructuring BdorOBP protein solution of 1 μm of ol/L, carry out fluorescent scanning after abundant mixing, the fitting of a polynomial related coefficient of fluorescence spectrum reaches 0.9953, and matching better (Fig. 4); After Scatchard equation (formula 1) linearization spectroscopic data, fitting correlation coefficient is 0.9927, and matching better (Fig. 5).The dissociation constant K of 1-NPN and BdorOBP albumen can be tried to achieve according to formula 1 _1-NPNbe 3.03 μm of ol/L.

(2) aglucon Binding experiment

Select 10 kinds of aglucons (3,4-dimethylbenzaldehyde, benzaldehyde, trans-2-hexenoic aldehyde, butyl butyrate, alpha, beta-lonone, isoamyl acetate, phenylacetaldehyde, dibutyl phthalate, isoamyl butyrate and capraldehyde) carry out fluorescence Binding experiment respectively, these 10 kinds of aglucons all can with 1-NPN competition binding BdorOBP, and binding ability is all better, can by the competition of 1-NPN and BdorOBP relative fluorescence to less than 50% (Fig. 5).The relative fluorescence of 1-NPN and relative fluorescence BdorOBP is competed to 50% the strongest aglucon be 3,4-dimethylbenzaldehyde, binding constant reaches 2.07 μm of ol/L.

The competition binding of table 3 candidate aglucon and 1-NPN and BdorOBP albumen of recombinating

As shown in Fig. 5 and table 3, multiple tea leaf volatile matter is as trans-2-hexenoic aldehyde, benzaldehyde, 3 to utilize the present invention to find, 4-dimethylbenzaldehyde, dibutyl phthalate etc. can produce stronger affinity with citrus fruit fly OBP, find that two kinds of aglucon phenylacetaldehydes and alpha, beta-lonone also can produce stronger affinity with this albumen in addition, show to utilize above-mentioned odoring substance to screen and be directed to the botanical attractant formula that the especially female fly sense of smell of citrus fruit fly is lured.

Embodiment 6: the efficiency assay being screened the host fruit Olfactory Attractants obtained by the present invention

6.1 materials and methods

6.1.1 for examination insect

Citrus fruit fly is the population of artificial feeding in this laboratory.Rearing conditions is: temperature (26 ± 1) DEG C, and relative humidity (75 ± 5) %, photoperiod L:D=12:12(light 12 h, dark 12 h).After pupating until it, sprouting wings, the female worm of collecting sexal maturity period is tested, hungry 3 h before experiment.

6.1.2 experimental apparatus and reagent

6.1.2.1 experimental apparatus

Tentaculum electric potential instrument is produced by Dutch Syntech company, obtains controller IDAC-2, stimulates gas flow controller (Syntech CS-55), inching operation instrument (Syntech MN-151) and Syntech software processing system four part to form by intelligent data.

6.1.2.2 reagent

Various standard items, all purchased from lark prestige.Conducting resinl, scalpel, blade, masking foil, qualitative filter paper.

6.1.3 research method

6.1.3.1 the preparation of reagent and stimulated samples

Respectively often kind of compound being dissolved in whiteruss, and being made into 0.1(v/v) solution carries out EAG mensuration.During dose response test, select compound representative in fluorescence Binding experiment, with solvent in contrast.

6.1.3.2 tentaculum electric potential measures

1) first with scalpel, citrus fruit fly feeler is cut from base portion, then carefully the two ends conducting resinl of citrus fruit fly feeler is sticked respectively on the metal electrode of EAG.At this moment observe the baseline situation of change on display screen, after waiting until that baseline steadily, show that feeler is stablized, and reaction is good, now just can starts experiment.

2) on filter paper bar, (2 cm × 0.5 cm) drips 10 uL testing sample solutions, makes solvent volatilize one after the meeting, then is filled in by filter paper in Pasteur pipe, with masking foil, mouth of pipe two ends are sealed, prevent sample from volatilizing.Separately get 10 uL whiteruss+filter paper as blank.During test, snorkel outside diameter being inserted in Pasteur pipe one end is in the aperture of 2 mm, and the snorkel mouth of pipe is longitudinally vertical with feeler, and with feeler at a distance of about 1 cm.Put hitter's mark simultaneously and step on pedal, stimulation time 0.2 s, stimulating air-flow to be 100 mL/min, twice stimulation time is separated by 2 min, to ensure that the sensory function of Antennal Sensilla is recovered completely.Random mensuration testing sample, due to the prolongation along with the time, the physiologically active of feeler also can and then reduce, in order to avoid this kind of situation causes adverse influence to experiment, using 0.01(v/v) leaf-alcohol (solvent is whiteruss) carry out standardization correction as the irritant reaction of standard reference stimuli to each test sample.Because find that the reaction of citrus fruit fly to leaf-alcohol exists stable peak value by preliminary experiment, select leaf-alcohol standard reference the most.1 contrast CK(whiteruss is first carried out before working sample) and standard reference stimuli, then 5 samples are tested, carry out contrast and standard reference stimuli again, so move in circles, each feeler about tests 5 samples, each testing sample repeats 6 times, and EAG reacting value is with reference to the method calculating of paying big grade [101] dawn, and formula is as follows.

（3）

In formula, Sr is the relative value that citrus fruit fly reacts this kind of compd E AG, Sc is the EAG reacting value of this kind of compound, CKm is the mean value of the contrast whiteruss twice EAG reaction measured before and after this kind of compound, and Rm is the mean value of the standard before measuring this kind of compound with reference to EAG reaction.

6.1.4 data process&analysis

SPSS 16.0 software is utilized to process obtained experimental data and analyze.The relative value of EAG reaction adopts Duncan method to calculate its conspicuousness situation, and female, male worm then adopts t to check (P<0.05) to the variance analysis of same test substances.

Table 4 citrus fruit fly reacts the EAG of fruit volatile matter

Trans-2-hexenoic aldehyde, benzaldehyde, 3 can be obtained from Fig. 6 and table 4,4-dimethylbenzaldehyde etc. all can make the female worm of citrus fruit fly produce the sense of smell stronger compared with male worm and lure reaction, and corresponding fluorescence in conjunction with dissociation constant also very little (less show that OBP is stronger in conjunction with smell aglucon ability).

Claims (2)

1., based on the attractant screening technique of citrus fruit fly OBP, it is characterized in that comprising following processing step:

1) citrus fruit fly feeler total serum IgE is collected;

2) total length of citrus fruit fly OBP is obtained by RT-PCR;

3) prokaryotic expression carrier of citrus fruit fly OBP is built;

4) citrus fruit fly restructuring smell binding protein expression is induced by IPTG, and by nickel Ago-Gel affinity column, purifying is carried out to it, the inductive condition of described IPTG induction citrus fruit fly restructuring smell binding protein expression is: IPTG concentration is 0.8 ~ 1.2mmol/L, temperature is 28 ~ 32 DEG C, induction rotating speed is 150 ~ 250rpm, and induction time is 4 ~ 6h;

5) composed by the association reaction of competitiveness fluorescent associated methods acquisition citrus fruit fly restructuring OBP and host fruit smell volatile matter, be defined as the host fruit host fruit sense of smell Olfactory Attractants of applicable citrus fruit fly, concrete technology step comprises:

A) Fluorescent ligands 1-NPN and citrus fruit fly are recombinated the protein-bonded combination of smell

Under 282nm excitation wavelength, in the citrus fruit fly restructuring OBP of 1 μm of ol/L, successively add the 1-NPN of 1mmol/L, fully mix;

B) aglucon combines and EAG checking

Choosing citrus fruit fly host fruit key odor volatile matter and 1-NPN is at war with in conjunction with citrus fruit fly restructuring OBP, if the competition of citrus fruit fly host fruit key odor volatile matter, 1-NPN and BdorOBP relative fluorescence is to less than 50%, and dissociation constant KD is lower than below the sense of smell of 10 μm of ol/L host fruits, when the IC50 value of fluorescence competition is less than 30 μm of ol/L or fluorescent value compete to less than 30% time, be then defined as the host fruit host fruit sense of smell Olfactory Attractants of applicable citrus fruit fly.

2. as claimed in claim 1 based on the attractant screening technique of citrus fruit fly OBP, it is characterized in that the inductive condition of IPTG induction citrus fruit fly restructuring smell binding protein expression in described step 4) is: IPTG concentration is 1.0mmol/L, temperature is 30 DEG C, induction rotating speed is 200rpm, and induction time is 5h.