CN109964935B - Application of terpene compounds in biological control of fall webworms - Google Patents

Abstract

An application of terpene compounds in biological control of fall webworm is disclosed. The invention discloses a new application of a terpene compound (3-carene) in biological control of fall webworm. Experiments prove that the 3-carene, the main component of monoterpene in the essential oil, has an evasion effect on the fall webworm, and meanwhile, the 3-carene can be combined with olfactory binding protein CcOBP2 to trigger olfactory reaction to attract a natural enemy insect, namely the fall webworm Chouioia. The research on the behavior ecology of the Chouioia cunea Yang and the natural enemy of the Chouioia cunea Yang on the hyphantria cunea will play an important role in the biological control of the hyphantria cunea Yang, and the application prospect is wide.

Description

Application of terpene compounds in biological control of fall webworms

The invention is subsidized by a national science fund (31702058) and an application basic plan project (18 JCYBJC 96300) in Tianjin City.

Technical Field

The invention relates to an application of terpene compounds in biological control of fall webworms, belonging to the technical field of plant protection and pest control.

Background

The fall moth is also called fall caterpillar, fall caterpillar and fall caterpillar, is a kind of worldwide quarantine pest with miscellaneous feeding, large breeding amount, strong adaptability, wide propagation path and serious harm, is listed as a first foreign invasive species by China and is listed as one of 6 engineering control pests by the national forestry Bureau. "national white moth epidemic area announcements in 2018 (No. 3 in 2018)" issued by the national forestry agency indicates that the national white moth epidemic area has spread to 11 provinces such as Beijing, Tianjin and the like and 573 county areas at present, wherein 15 Beijing, 16 Tianjin, 128 Hebei, 67 Liaoning, 1 Mongolian autonomous area, 16 Jilin, 50 Jiangsu, 61 Anhui, 137 Shandong, 71 Henan and 11 Hubei, and compared with 2017 years, 15 county areas are additionally added in the American white moth epidemic area. In order to prevent and control fall webworms, chemical insecticides are sprayed on a plurality of places by airplanes, so that the spreading tendency of the fall webworms is not effectively stopped, the drug resistance of diseases and pests is continuously enhanced, the environment is seriously polluted, and meanwhile, natural enemies are killed, and a vicious circle is formed.

Chouioia cunea Yang (a. cunea Yang)Chouioia cuneaYang) hymenoptera, family Apidae, are parasitic natural enemies in pupal stage of various lepidoptera insects such as hyphantria cunea, codling moth, willow moth, acacia ruled moth, and poplara. The natural enemy factor is used for inhibiting the generation of the fall webworm and plays an important role in controlling the harm of the fall webworm.

Insects mainly use an olfactory system to communicate with the external environment, find food, avoid enemies, seek puppet and mate, search hosts, select mating sites and the like, and olfactory recognition of insects is complex and requires participation of various protein molecules, including Odorant Binding Proteins (OBPs), Chemosensory proteins (CSPs), Odorant Receptors (ORs), Sensory Neuron Membrane Proteins (SNMPs) and the like. Wherein, the odor binding protein is a medium for the binding of external environmental chemical information and odor receptors in the antenna receptors. The odorant molecule enters the antenna sensor through the small hole on the epidermis of the antenna sensor on the antenna, is combined with the Odorant Binding Protein (OBP), is carried by the OBP to enter olfactory receptor cells, activates membrane-bound Odorant Receptor (OR), causes the opening and closing of the ion channel of the cell membrane to be converted into an electric signal to complete nerve conduction, and forms various behavioral instructions after the brain is processed and modified.

The odorant binding proteins are secreted by supporting cells inside the olfactory sensor, and are small, spherical, water-soluble acidic proteins with a molecular weight of about 14kDa, an isoelectric point between 4.0 and 5.2, and usually comprise 120-150 amino acids, which infiltrate into the sensor lymph fluid and are distributed around the axons of sensory neurons. It is currently generally accepted that insect OBPs have similar physiological functions: (1) as a cosolvent and a carrier, transporting fat-soluble odorant molecules across water-soluble lypoppy sensates; (2) selectively binding specific odor molecules as a peripheral filter; (3) as a stimulatory molecule to act in a specific manner on the odorant receptor protein to facilitate signal transduction; (4) scavenging, removing unwanted or toxic compounds around the odorant receptor; (5) OBPs may function to rapidly inactivate odorant molecules when they stimulate odorant receptors. However, the function of OBPs in the olfactory reaction of insects is still lack of specific molecular mechanism research.

Terpene compound 3-carene (C)₁₀H₁₆) The structural formula (II) is as follows:

physical and chemical properties: boiling point 168-169 ℃ 705mm Hg, density 0.857g/mLat 25 ℃, refractive index n 20/D1.474 and flash point 115 ℃ F, and mainly play a role: the 3-carene exists in various essential oils such as turpentine, pepper oil and angelica rotundifolia oil, the specific action is unknown, and the 3-carene has a toxic effect on the mountain pine beetles and can inhibit the beetles from spawning in a literature report, and the 3-carene can play a role in the interaction of plants, phytophagous insects and natural enemies as a plant volatile compound.

Disclosure of Invention

The invention overcomes the technical defects of chemical pesticides for preventing and controlling the fall webworms, and provides a biological control strategy of the fall webworms based on the olfactory mechanism of natural enemy insects. The invention aims to solve the behavioral ecological problem of natural enemy insects in the biological control of fall webworms.

In order to achieve the purpose, the invention discloses the following technical contents:

an application of a terpene compound 3-carene in preventing and treating American white moth organisms mainly refers to: evasion effect of 3-carene on american white moth. Wherein the evasion effect of 3-carene on the fall webworm refers to that: the white moth larvae tend to be far away from the 3-carene volatilization region; the attraction of 3-carene to Chouioia cunea of the white moth means that Chouioia cunea tends to move towards the 3-carene volatilization region. The invention discloses a terpene compound 3-carene which can cause an antenna potential reaction of Chouioia cunea Yang. The 3-carene can be combined with Chouioia cunea odor binding protein CcOBP 2. The experimental results show that a low concentration (0.05 mM) of 3-carene can cause an obvious antennary potential reaction (about 6.5 times), the CcOBP2 protein is expressed and purified in vitro, the binding condition of the protein and the 3-carene is detected by a fluorescence competitive binding experiment, and the fact that as the concentration of the 3-carene in a system is gradually increased, the protein is more and more bound to the 3-carene, the binding capacity of the protein and the probe is weaker and weaker, the fluorescence intensity of the substrate is reduced, the IC50 (the concentration of the 3-carene when 50% competitive binding effect is achieved) is 17.01 mu M, the Ki (the concentration of the 3-carene when 50% competitive binding effect is detected and is calculated by using Michaelis-Menten dynamics) is 14.07 mu M, and the 3-carene is obviously bound to the CcOBP2 is shown.

The invention mainly considers the olfaction mechanism of the host recognition of Chouioia cunea Yang of the white moth, and mainly solves the action mechanism of the compound on the Chouioia cunea Yang of the white moth, and the invention has the difficulty that how the 3-carene is combined with the Chouian cunea Yang odor binding protein CcOBP2 is explained, and the innovation point of the invention is that a terpene compound (3-carene) is found to have the attraction effect on the Chouian Chouioia cunea of the white moth.

Drawings

(1) FIG. 1 is a graph of the evasive effect of 3-carene on the fall webworm;

(2) FIG. 2 is a graph of the attraction of 3-carene to Chouioia cunea Yang;

(3) FIG. 3 is an antennal potential response of Chouioia cunea to 3-carene;

(4) FIG. 4 shows the result of purification of the olfactory binding protein CcOBP2 of Chouioia cunea Yang;

(5) fig. 5 is a fluorescent binding experiment showing that 3-carene binds specifically to CcOBP 2.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available. Of which 3-carene was purchased from Protechs GmbH (cat. No. 423988).

Example 1:

evasion effect of 3-carene on American white moth

As shown in fig. 1, a Y-type olfactometer experiment was used to detect the behavioral effects of 3-carene on the larvae of fall webworms, and the specific method included the following steps:

and (3) collecting fall webworm larvae in the field, and feeding the fall webworm larvae to 5 instars by using fresh poplar leaves.

The 3-carene was prepared at five concentrations of 100mM, 50mM, 5mM, 0.5mM and 0.05mmol/L, respectively, using liquid paraffin as a solvent.

The experiment was carried out using liquid paraffin as a blank control. Sucking 1 μ L of the drug with a micropipette, uniformly dropping the drug on a 2cm × 0.5cm filter paper strip, placing the drug in a sample tube, sucking 1 μ L of liquid paraffin with another micropipette, uniformly dropping the liquid paraffin on a 2cm × 0.5cm filter paper strip, and placing the liquid paraffin in another sample tube. The experiment was started after opening the device switch.

During the experiment, the test insects are led in from the pipe orifice of the main arm of the Y-shaped pipe, are placed at a position 2-3cm away from the bifurcation, move against the airflow, observe the selection made at the bifurcation of the Y-shaped pipe, and enter one arm within 5min, so that the reagent on the side can be considered to have the attraction effect on the test insects, and otherwise, the test insects have the avoidance effect. For each concentration, three experiments were performed with 17 different worm records selected. And cleaning up the residual reagent after the reagent experiment of each concentration is finished so as to prevent the reagent of the next concentration from being polluted. The white moth larvae have strong activity under the condition of light source, can be carried out in the daytime, and do not need to specially control the light source.

Example 2:

attraction of 3-carene to Chouioia cunea Yang

As shown in fig. 2, the influence of 3-carene on the ethology of Chouioia cunea Yang on the white moth was detected by using a Y-type olfactometer experiment, and the specific method comprises the following steps:

the Chouioia cunea Yang which is cultured in the laboratory and substitutes the tussah pupa as the host.

The 3-carene was prepared at five concentrations of 100mM, 50mM, 5mM, 0.5mM and 0.05mmol/L, respectively, using liquid paraffin as a solvent.

The experiment was carried out using liquid paraffin as a blank control. Sucking 1 μ L of the drug with a micropipette, uniformly dropping the drug on a 2cm × 0.5cm filter paper strip, placing the drug in a sample tube, sucking 1 μ L of liquid paraffin with another micropipette, uniformly dropping the liquid paraffin on a 2cm × 0.5cm filter paper strip, and placing the liquid paraffin in another sample tube. The experiment was started after opening the device switch.

During the experiment, the test insects are led in from the pipe orifice of the main arm of the Y-shaped pipe, are placed at a position 2-3cm away from the bifurcation, move against the airflow, observe the selection made at the bifurcation of the Y-shaped pipe, and enter one arm within 5min, so that the reagent on the side can be considered to have the attraction effect on the test insects, and otherwise, the test insects have the avoidance effect. For each concentration, three experiments were performed with 17 different worm records selected. And cleaning up the residual reagent after the reagent experiment of each concentration is finished so as to prevent the reagent of the next concentration from being polluted. The Chouioia cunea Yang has very strong phototropism, therefore need carry out the shading when carrying out the experiment and handle, otherwise can influence the accuracy of data greatly.

Example 3:

antennal potential reaction of Chouioia cunea to 3-carene

As shown in fig. 3, the specific method for detecting the antennal potential reaction of Chouioia cunea to 3-carene by using the EAG experiment comprises the following steps:

the 3-carene was prepared at five concentrations of 100mM, 50mM, 5mM, 0.5mM and 0.05mmol/L, respectively, using liquid paraffin as a solvent.

Treating the wasps: dipping the pupated vespids into finger-shaped test tubes by using a writing brush, taking one vespid and placing under a dissecting mirror, completely cutting off one tentacle by using a dissecting blade, and then cutting off the trunk of the vespids 1/3 and the tip of the other tentacle.

Fixing the wasps: and (3) coating proper conductive glue on the tentacles and the abdomens of the wasps, and respectively fixing the tentacles and the abdomens of the wasps treated by the conductive glue on two joints of the electrode, wherein the breasts of the wasps are not coated with the conductive glue and the two joints of the electrode are not touched.

Adjusting the instrument: the prepared odor tube of the electroantennal potentiometer is properly angularly adjusted to be about 1cm away from the bee. Preparing a 0.5cm × 0.5cm filter paper strip, dipping a proper amount of the solution to be detected, and placing the filter paper strip into a sample tube, wherein the tail end of the sample tube is connected with a gas supply device.

Air blowing experiment of vespids: after baseline stabilization, stimulation was given at intervals of at least 20s per stimulation to ensure complete recovery of the antennary receptors. Each drug was tested sequentially from low to high concentrations, each concentration was blown in three more times, and the measurements were first performed with liquid paraffin as a control before starting the concentration of a single sample. After 5 concentrations of the same drug were completed, the sample tube was thoroughly washed with alcohol and air-dried naturally to eliminate residual odor.

Recording of data: the liquid paraffin control value and the three EAG values of different concentrations of each drug are recorded for each time, and finally the data are collated and processed.

And (3) processing data: (1) EAG reaction relative value: the EAG value of 2-fold stimulus was divided by the sum of the previous and subsequent 2 liquid paraffin control measurements. (2) The calculated EAG response relative values were in three groups, and the mean and standard deviation were determined and plotted. (3) Data were processed using the duncan new complex pole difference method in SPSS17 software and analyzed for significant differences.

Example 4:

the olfactory binding protein CcOBP2 of Chouioia cunea Yang is expressed and purified

As shown in fig. 4, the olfactory binding protein CcOBP2 of Chouioia cunea Yang was expressed and purified in vitro, and the specific method comprises the following steps:

extracting Chouioia cunea Yang RNA: (1) selecting female bees which emerge on the same day and have stronger vitality for experiment, placing about 60 female bees into a 1.5mL EP tube treated by DEPC water, and adding liquid nitrogen for grinding. After the grinding, 600. mu.l Trizol was added, and the tissue and Trizol were thoroughly combined by repeated pipetting and allowed to stand at room temperature for 5 min. (2) Add 200. mu.l chloroform to each tube, cover tightly, shake vigorously up and down for 30-50s, incubate for 3min at room temperature. (3) Centrifuge at 13000rpm for 15min at 4 ℃. After being taken out, the sample is divided into 3 layers, the upper layer is a colorless aqueous phase, the middle layer is a protein layer, and the lower layer is organic phase chloroform. The extracted RNA was mainly present in the upper aqueous phase and carefully aspirated with a pipette and placed in a new EP tube. (4) 400 mul of isopropanol is added into the obtained upper water phase, after being mixed evenly, the mixture is placed at minus 20 ℃ for 25min, and then centrifuged at 13000rpm for 10min at 4 ℃. (5) Discarding the supernatant, adding 600 μ l 75% ethanol into each tube, mixing by vortex, centrifuging at 7500rpm for 5min at 4 deg.C, and discarding the supernatant. (6) Drying at room temperature for 3min, taking care to avoid that too long drying time affects the solubility of the extracted RNA. (7) The RNA was dissolved in 20. mu.l of an RNA-dissolving solution, and the RNA solubility was increased by heating in a water bath at 55 ℃ for 10 min. (8) The RNA extraction effect was checked by 1% agarose gel electrophoresis, and the concentration of extracted RNA was determined by NanoDrop 2000.

Reverse transcription of cDNA: first Strand cDNA was synthesized with reference to the TransScript First-Strand cDNA Synthesis SuperMix (AT301) kit.

pET-28a-CcOBP2 expression plasmid construction: the method comprises the steps of performing antennal transcriptome sequencing on Chouioia cunea Yang according to the Chouioia cunea Yang to obtain a CcOBP2 gene sequence, designing a gene specific primer, performing PCR amplification by using ExTaq Premix with cDNA as a template, verifying by adopting 1% agarose Gel electrophoresis, and recovering target fragments by using a Gel Extraction Kit recovery Kit (purchased in the Kangji century). The plasmid was digested with BamHI/HindIII restriction enzymes and ligated to the expression vector pET-28 a. The ligation product was added to 50. mu.l of Trans1-T1 competent cells (purchased from holo-type gold Biotechnology Co., Ltd.) for transformation, and plasmids were extracted and subjected to enzyme digestion to verify the correctness, followed by sequencing verification by Jinzhi Biotechnology Co., Ltd.

CcOBP2 protein expression purification: taking 10 mu l of recombinant plasmid with correct sequencing, adding 100 mu l of Transetta (DE3) expression bacteria for transformation, selecting a single clone for amplification culture, performing shake culture at 37 ℃ until the OD value is 0.8, taking 600 mu l of shaken bacterial liquid as a control, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM into the residual bacterial liquid for induction expression. Centrifuging the induced bacteria liquid at 8000rpm for 2min, adding 30ml PBS into each 50ml centrifuge tube to resuspend the bacteria, centrifuging again at 8000rpm for 2min, weighing the centrifuged bacteria precipitate, adding 5ml Buffer B and lysozyme with working concentration of 1mg/ml into each g precipitate to resuspend, cracking on ice for 30min, slowly swirling the liquid every few minutes to avoid foaming, and paying attention to low temperature during operation. And (3) putting the bacteria liquid obtained by cracking into 50ml centrifuge tubes, wherein the liquid content of each centrifuge tube is not more than 25ml, fixing the centrifuge tubes in an ice-water mixture, carrying out ultrasonic crushing, wherein the crushing power is 60%, stopping for 5s after 5s, and repeating for 3-5 times to fully crush the thalli, wherein the thalli are relatively clear, and the ice-water mixture is timely replaced during the ultrasonic crushing to prevent protein denaturation. And subpackaging the crushed liquid into 2ml EP tubes, centrifuging at 13000rpm for 30min at 4 ℃ by a centrifuge, respectively sampling the supernatant and the precipitate, detecting whether the target protein exists in the supernatant in a form of an inclusion body by SDS-PAGE electrophoresis, starting purification and renaturation after success, and storing the separated supernatant in an ice box at 4 ℃. Purified CcOBP2 protein was quantified using the Thermo Scientific Pierce BCA protein quantification kit.

Example 5:

detection of specific binding of 3-carene and CcOBP2 by fluorescence binding assay

As shown in fig. 5, the specific binding of 3-carene to CcOBP2 was detected using a fluorescence binding assay, the specific method comprising the steps of:

1-NPN was chosen as the fluorescent probe for this experiment. To 100. mu.l of the system was added 2. mu.M final protein (protein samples diluted with 50mM Tris-HCl pH 7.4) and 1-NPN (chromatographic grade methanol) at different concentrations, respectively, with 0, 5, 10, 15, 20, 25, 30, 35, 40. mu.M final concentration for each set of probes, and finally the system was supplemented with 50mM Tris-HCl pH 7.4. 5 groups of parallel experiments are designed, the above systems are prepared and mixed uniformly, and are sequentially added into a 96-hole fluorescence microplate reader plate, and the experimental conditions of the microplate reader are as follows: 337nm wavelength excitation, and recording the emission spectrum in the wavelength range of 380-500 nm. Whether the activity of the protein can be subjected to the subsequent experiment is judged according to the binding condition of the target protein and the probe.

Mu.l of the system was added with 2. mu.M of the target protein, 2. mu.M of probe 1-NPN, and sequentially with 0, 5, 10, 15, 20, 25, 30. mu.M of 3-carene (diluted with chromatographic grade methanol), and finally the system was made up with 50mM Tris-HCl pH 7.4. The procedure set up on the microplate reader was the same as before, and 5 sets of parallel experiments were still designed. The data obtained are plotted and the binding of the odorant molecule to the protein is recorded as a function of the change in fluorescence intensity of the substrate.

The fluorescence competition combination experiment detects the combination of the odor binding protein and various odor molecules, and the essence of the fluorescence competition combination experiment is to measure the change of the fluorescence value of a substrate. The protein substrate should increase the fluorescence intensity with the increasing concentration of the added fluorescent probe; if the odor molecules are added in an increasing amount when the concentration of the protein substrate and the fluorescent probes is fixed, once the protein and the odor molecules are combined, the odor molecules occupy the positions of the fluorescent probes, so that the overall fluorescence value of the substrate is reduced.

It was found that as the concentration of 3-carene was increased, the 3-carene displaced the binding of the probe (1-NPN) to the protein CcOBP2, resulting in a gradual decrease in the fluorescence intensity of the substrate, with an IC50 (concentration of 3-carene at 50% competitive binding) of 17.01 μ M and a Ki (concentration of 3-carene at 50% competitive binding, as calculated using Michaelis-Menten kinetics) of 14.07 μ M. The strong binding capacity of 3-carene and CcOBP2 is shown.

It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the scope and spirit of the invention, and it is intended that all such changes and modifications as fall within the true spirit and scope of the invention be interpreted in accordance with the principles of the invention. And the invention is not limited to the example embodiments set forth in the description.

Claims (1)

1. Application of terpene compound 3-carene in repelling American white moth is provided.

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