Use of a pea aphid protein in feeding deterrents

Abstract

The present invention relates to the use of a pea aphid protein having an amino acid sequence as shown in SEQ ID No. 7 and / or SEQ ID No. 8 for antifeeding.

Description

Technical Field

[0001] This invention relates to the field of biological control. Background Technology

[0002] Aphids are a global agricultural and forestry pest that causes serious damage by directly feeding on plants and spreading plant viruses.

[0003] Aphids reproduce rapidly, have short generations, and are highly resistant to pesticides, making them difficult to control. The use of traditional chemical pesticides causes serious environmental pollution problems. Therefore, there is an urgent need to develop environmentally friendly plant-derived pesticides for control. Summary of the Invention

[0004] One aspect of the present invention provides the application of pea aphid protein in feeding deterrence, wherein the amino acid sequence of the pea aphid protein is shown in SEQ ID No. 7 and / or SEQ ID No. 8.

[0005] In one specific embodiment, the nucleic acid encoding the pea aphid protein with the amino acid sequence shown in SEQ ID No. 7 is shown in SEQ ID No. 3; the nucleic acid encoding the pea aphid protein with the amino acid sequence shown in SEQ ID No. 8 is shown in SEQ ID No. 6.

[0006] In one specific embodiment, the antifeedant compound recognized by the pea aphid protein is polydialdehyde. That is, when the pea aphid protein is functioning normally, the pea aphid will refuse to eat food sources that produce polydialdehyde.

[0007] The beneficial effects of this invention are:

[0008] This invention is the first to discover that the pea aphid protein with an amino acid sequence such as SEQ ID No. 7 or SEQ ID No. 8 can mediate the pea aphid's antifeeding behavior, especially the antifeeding behavior to food sources containing the compound polydialdehyde. This indicates that the protein belongs to the pea aphid antifeeding receptor protein. The identification of this antifeeding receptor target provides an important theoretical basis for the application of screening polydialdehyde antifeeding analogues. Attached Figure Description

[0009] Figure 1 The graph shows the feeding rejection index of aphids under different concentrations of polydialdehyde treatment within 24 hours.

[0010] Figure 2Figure 1 shows the response of Xenopus oocytes expressing the ApTR gene to polydialdehyde (PDE) stimulation. Figure 2 shows the response intensity of ApTR (A) expressed in Xenopus oocytes to different concentrations of PDE stimulation. Figure 3 shows the response intensity of ApTR (B) expressed in Xenopus oocytes to different concentrations of PDE stimulation. Figure 4 shows the comparative analysis of the differences in the responses of ApTR (A) and ApTR (B) of pea aphids to different concentration gradients of PDE.

[0011] Figure 3 The graph shows the feeding rejection index of pea aphids after RNAi of the ApTR gene. Compared with the blank control group and the negative control group, the same letter in a and b indicates no significant difference at the α=0.05 level, while different letters indicate significant differences at the α=0.05 level. Detailed Implementation

[0012] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0013] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0014] PCR kit (Takara, Japan);

[0015] RNA extraction reagent Trizol (Invitrogen, USA);

[0016] Revert Aid First Strand cDNA Synthesis Kit (Transgen, China);

[0017] SYBR PRemix Ex Taq™ Real-Time PCR Kit (Takara, Japan);

[0018] Agarose gel extraction kit (TransGold, China);

[0019] Antibiotics ampicillin, IPTG, and X-gal (Sigma, USA);

[0020] Cloning vector pEASY-BluntT (Transgen, China);

[0021] Expression vector pT7Ts (Invitrogen, USA);

[0022] Competent Trans1-T1 cells (Full-Gold, China);

[0023] Plasmid miniprep kit (Tiangen, China);

[0024] cRNA Synthesis Kit (mMESSAGEmMACHINE T7 kit) (Ambion, USA);

[0025] dsRNA Synthesis Kit (Ambion, USA);

[0026] Water-saturated phenols (Biotech, China);

[0027] Chloroform (Biotech, China);

[0028] PCR instrument (Bio-Rad, USA);

[0029] The ChemiDoc XR+ gel imaging system (Bio-Rad, USA);

[0030] NanoDrop ND-2000 Micro-Vitamin / Vis Spectrophotometer (Nanodrop, USA);

[0031] Cell incubator (SANYO, Japan);

[0032] 24-well cell culture plate (NEST, China);

[0033] Microinjection system (Nanoliter 2000, USA);

[0034] Voltage clamp recording system OC-725C oocyte clamp (Warner Instruments, USA);

[0035] Horizontal nucleic acid electrophoresis apparatus (June 1st, China);

[0036] -70℃ ultra-low temperature freezer (Haier, China).

[0037] Example 1

[0038] Polygonal aldehydes cause pea aphids to refuse to eat.

[0039] 1. Rearing and tissue collection of pea aphids

[0040] Pea aphids were reared in soil using broad beans by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The rearing temperature was 20±2℃, the relative humidity was 70%±5%, and the photoperiod was 16:8 (L:D).

[0041] 2. Experiment on the antifeeding behavior of pea aphids

[0042] The feeding preference of pea aphids for broad bean leaves treated with five concentrations of polydialdehyde (5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.1 mg / mL, and 0.05 mg / mL) was determined using a leaf disc apparatus. Broad bean leaves treated with 50 wt% ethanol aqueous solution served as a negative control.

[0043] The leaf disc apparatus was modified from a petri dish: Four 1.0 cm diameter holes were evenly punched into the bottom of a petri dish with a diameter less than 9 cm. Connecting the centers of these four holes sequentially formed a square, which was called the leaf cover plate. A 9 cm diameter petri dish was filled with 25 mL of 16 g / L agar substrate to maintain leaf humidity. Broad bean leaves were cut into 1.5 cm diameter circles using a perforator. Four circular leaves were placed on the agar substrate with their undersides facing up, connecting their centers sequentially to form a square. The leaf cover plate was then placed on top of the leaf-covered agar substrate, leaving the four circular leaves protruding from the four holes in the cover plate, forming a leaf disc. A 50 wt% ethanol aqueous solution was applied with a small brush to the two leaves forming a diagonal line. A polyvinyl aldehyde solution of the same concentration was applied with a small brush to the other two leaves forming a diagonal line to eliminate the influence of environmental factors such as orientation. 10 μL was applied to each leaf. After the solvent had completely dried, 20 five-day-old pea aphids were inoculated into each petri dish, and the dishes were covered to prevent the aphids from escaping. Each biological replicate consisted of 20 aphids, with a total of 6 replicates. The number of pea aphids feeding on leaves treated with polydialdehyde and those treated with 50 wt% ethanol aqueous solution was recorded and counted at 0.5, 1, 2, 4, 8, 20, and 24 h, and the antifeedant index was calculated. The antifeedant index (AI) was calculated as follows: (Number of feeding aphids on negative control leaves - Number of feeding aphids on leaves treated with polydialdehyde at a certain concentration) / (Number of feeding aphids on negative control leaves + Number of feeding aphids on leaves treated with polydialdehyde at a certain concentration). Results are shown below. Figure 1 .

[0044] Depend on Figure 1 It was found that polydialdehyde (PDA) exhibited antifeeding inhibition effects on pea aphids at relatively high concentrations of 5 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL. However, its antifeeding activity against pea aphids was not significant when the concentration was reduced to 0.05 mg / mL. Within the 24-hour observation period, the antifeeding ability (AI) of PDA against pea aphids remained relatively stable over time, with some fluctuations at earlier time points. The EC50 of the antifeeding effects at different concentrations was also discussed. 50 The value is calculated to obtain EC. 50 =0.467 mg / mL. This indicates that polydialdehyde causes significant feeding rejection behavior in pea aphids.

[0045] Example 2

[0046] Two-electrode voltage clamp function verification experiment of ApTR gene

[0047] 1. Extraction of total RNA from pea aphids

[0048] The homogenizer was washed and placed in an oven at 180°C for 3 hours. Masks and disposable gloves were worn throughout the entire process to ensure the procedure was performed under RNase-free conditions. The entire RNA extraction process is shown below:

[0049] (1) Quickly add whole adult pea aphids stored at -70℃ into a glass homogenizer pre-cooled with liquid nitrogen, immediately add 1mL of Trizol reagent to the homogenizer, and grind the tissue thoroughly; use an RNase-free pipette tip to transfer the ground tissue solution into an RNase-free 1.5mL centrifuge tube and temporarily place it on ice.

[0050] (2) Centrifuge at 12000g for 15 min at 4℃, and transfer the supernatant to a new RNase-free 1.5mL centrifuge tube;

[0051] (3) After placing the supernatant at room temperature for 5 minutes, add 0.2 mL of chloroform to the centrifuge tube, shake vigorously for 15 seconds, and then let it stand at room temperature for 2-3 minutes.

[0052] (4) Centrifuge at 12000g for 15 min at 4℃. After centrifugation, the mixture will separate into layers. The lower layer is a red organic phase and the upper layer is a colorless aqueous phase.

[0053] (5) Use an RNase-free pipette tip to transfer the upper aqueous phase to a new RNase-free 1.5 mL centrifuge tube. Be careful not to aspirate the middle layer to avoid protein or DNA contamination.

[0054] (6) Add 0.5 mL of isopropanol, mix well, and let stand at room temperature for 10 min to precipitate RNA;

[0055] (7) After centrifuging at 12000g for 10 minutes at 4℃, the observed precipitate is RNA. Discard the supernatant (absorb as much as possible), add 1 mL of 75% ethanol (prepared with DEPC water), gently shake the centrifuge tube to suspend the precipitate, and wash the precipitate.

[0056] (8) After centrifuging at 7500g for 10 minutes at 4℃, discard the supernatant (absorb as much as possible), and dry the precipitate in a clean bench for 5-10 minutes;

[0057] (9) Add 10-20 μL of RNase-free water (depending on the amount of RNA), gently tap and centrifuge to fully dissolve the precipitate;

[0058] (10) After incubation at 55-60℃, take 1μL of sample and dilute it to 5μL. 2.5μL of the sample is used for electrophoresis detection, and the concentration of the other 2.5μL is detected by Nanodrop instrument. The remaining sample is stored in a -70℃ freezer or used for cDNA synthesis.

[0059] 2. Synthesis of first-strand cDNA

[0060] The entire reverse transcription process should be carried out under conditions free from RNase contamination, following the operating procedures of the Revert Aid FirstStrand cDNA Synthesis Kit, as follows:

[0061] (1) Add the following to a PCR tube without RNase in sequence: 2 μg RNA (calculate the volume of RNA to be added based on the concentration of RNA), 1 μL 10×Reaction Buffer with MgCl2, 1 μL DNase I, and make up the volume to 10 μL with RNA-free water; vortex gently to mix and then centrifuge.

[0062] (2) Incubate at 37℃ for 30 min, then add 1 μL of 50 mM EDTA to the system, mix well and centrifuge, incubate at 65℃ for 10 min to remove DNANase I (the temperature should be controlled by a PCR instrument as much as possible throughout the process).

[0063] (3) Add 1 μL of Oligo(dT) 18 After mixing and centrifuging, incubate at 65°C for 5 minutes, then immediately place on ice;

[0064] (4) Add the following reagents in sequence: 4 μL of 5×Reaction buffer, 2 μL of dNTP Mix (10 mM), 1 μL of Revert Aid Reverse Transcriptase and 1 μL of Ribolock RNase Inhibitor, mix well and centrifuge.

[0065] (5) Incubate at 42℃ for 60 min, then incubate at 70℃ for 5 min;

[0066] After synthesis, store in a refrigerator at -20℃ or -70℃. Dilute several times as needed before use.

[0067] 3. Cloning of the ApTR gene in the pea aphid

[0068] Using the synthesized cDNA as a template, the ApTR(A) gene was amplified (SEQ ID No. 3) using primers ApTR(A)-F (SEQ ID No. 1) and ApTR(A)-R (SEQ ID No. 2); using the synthesized cDNA as a template, the ApTR(B) gene was amplified (SEQ ID No. 6) using primers ApTR(B)-F (SEQ ID No. 4) and ApTR(B)-R (SEQ ID No. 5). The amino acid sequence of the protein encoded by the ApTR(A) gene is shown in SEQ ID No. 7; the amino acid sequence of the protein encoded by the ApTR(B) gene is shown in SEQ ID No. 8.

[0069] The PCR products of the ApTR(A) and ApTR(B) genes were electrophoresed on 1% agarose gels. The gel containing the desired DNA fragment was excised under long-wave UV light and placed in a 1.5 mL centrifuge tube. The fragments were then recovered using a full-gold agarose gel extraction kit (following the instructions provided with the kit). The recovered fragments were ligated into the full-gold pEASY-BluntT cloning vector, following the instructions for use of the vector. This yielded the positive recombinant vectors pEASY-BluntT-ApTR(A) and pEASY-BluntT-ApTR(B). The ligation system was used to transform Trans1-T1 fully gold competent cells (transformation steps were performed according to the instructions accompanying the competent cells). After overnight culture, 16 white single clones were selected for PCR verification. Positive clones were cultured overnight in liquid LB medium containing 100 μg / mL ampicillin with shaking. After preservation, the clones were sent for sequencing, and positive strains Trans1-T1 / pEASY-BluntT-ApTR(A) and Trans1-T1 / pEASY-BluntT-ApTR(B) were screened.

[0070] 4. Construction of ApTR gene expression vector and synthesis of cRNA

[0071] Using pEASY-BluntT-ApTR(A) as a template, the ApTR(A) gene was amplified using primers ApTR(A)-F-SpeI (SEQ ID No. 9) and ApTR-R-Xho1 (SEQ ID No. 10); using pEASY-BluntT-ApTR(B) as a template, the ApTR(B) gene was amplified using primers ApTR(B)-F-SpeI (SEQ ID No. 11) and ApTR-R-Xho1 (SEQ ID No. 12).

[0072] The PCR products of the ApTR(A) and ApTR(B) genes were subjected to electrophoresis on 1% agarose gels. A gel containing the desired target DNA fragment was excised under long-wave UV light and placed in a 1.5 mL centrifuge tube. The fragments were then recovered using a Full Gold agarose gel extraction kit (procedure steps were followed according to the kit's instructions). The recovered fragments and the expression vector pT7Ts were double-digested with enzymes from the primers. The digested fragments of the two genes were then ligated to the digested backbone fragment of the expression vector pT7Ts using T4 ligase, yielding the positive recombinant vectors pT7Ts-ApTR(A) and pT7Ts-ApTR(B). The ligation system was transformed into Full Gold Trans1-T1 competent cells, cultured overnight at 37°C, and eight clones were selected for PCR verification. Positive clones were cultured overnight in liquid LB medium containing ampicillin (containing 100 μg / mL of antibiotic Amp). After preservation the next day, the clones were sent for sequencing and screening. Positive strains Trans1-T1 / pT7Ts-ApTR(A) and Trans1-T1 / pT7Ts-ApTR(B) were screened.

[0073] After successful sequencing, the recombinant plasmid was extracted, linearized by single enzyme digestion, and cRNA was synthesized. The experimental steps were as follows: pT7Ts-ApTR(A) and Trans1-T1 / pT7Ts-ApTR(B) were digested with SmaI. The digestion system consisted of 45 μL of recombinant plasmid, 8 μL of 10×FastDigest Buffer, and 4 μL of SmaI, with water added to make up to 80 μL. The mixture was incubated at 30°C for 3 hours. Then, 1 μL of the digestion product was taken for electrophoresis to check for complete cleavage. After confirming complete cleavage, phenol-chloroform extraction was performed. Finally, 7 μL of LEPC water was added to dissolve the precipitate, and 0.5 μL was taken for electrophoresis. Ideally, the result should be a single, very bright band.

[0074] Based on the correct detection, cRNA was synthesized using mMESSAGE MmachineT7. The experimental steps are as follows:

[0075] (1) Add the following reagents to a new RNase-free 1.5 mL centrifuge tube to make the system 20 μL: 10 μL of T7 2×NTP / CAP, 2 μL of T7 10×Reaction Buffer, 6 μL of linearized plasmid template and 2 μL of Enzyme mix; mix thoroughly and incubate at 37 °C for 2 h.

[0076] (2) Add 30 μL of RNA-free water and 30 μL of lithium chloride solution to the system respectively; mix thoroughly and let stand overnight at 4°C;

[0077] (3) Centrifuge at 4℃ and 12000 rpm for 30 min;

[0078] (4) Discard the supernatant, add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 min, discard the ethanol, and air dry the precipitate;

[0079] (5) Add 8 μL of DEPC water to dissolve the precipitate, take 1 μL and dilute it five times. Use half for electrophoresis detection and half for content determination.

[0080] 5. cRNA injection, expression, and two-electrode voltage-clamp recording

[0081] The synthesized ApTR gene cRNA was diluted to 2 μg / μL and injected into healthy, mature Xenopus oocytes at a dose of 50 nL. The injected cells were then cultured in an 18°C ​​incubator. After 2-3 days of culture, the oocyte responses to the compound stimulation were recorded using a dual-electrode voltage clamp on an OC-725 Coocyte clamp. Specific data acquisition and analysis were performed using a Digidata 1440A instrument and pCLAMP 10.2 software.

[0082] Preparation of oocyte culture medium, washing solution, and perfusion buffer: Sterilize distilled water and filtration flasks beforehand. Weigh out NaCl: 56.1g, KCl: 1.5g, MgCl2·6H2O: 10.2g, and HEPES: 11.9g. Make up to 1L with sterile water (10×Ringer). Adjust the pH to 7.6 with NaOH. Filter and set aside. Prepare stock solutions of tetracycline, streptomycin, gentamicin, and sodium pyruvate with sterile water at concentrations of 50mg / mL, 100mg / mL, 10mg / mL, and 275mg / mL, respectively. Prepare a 100×CaCl2 stock solution with CaCl2·2H2O. For Xenopus oocyte washing solution, make up to 1L with 100mL of 10×Ringer solution and 1mL of gentamicin stock solution, then filter and set aside. Xenopus oocyte culture medium was prepared by mixing 100 mL of 10× Ringer, 10 mL of 100× CaCl2, 50 mL of horse serum, 1 mL of tetracycline stock solution, 1 mL of streptomycin stock solution, and 2 mL of sodium pyruvate stock solution with sterile water to a final volume of 1 L, followed by filtration. The perfusion buffer (1× Ringer) consisted of essentially the same components as the washing buffer, except that 10 mL of 100× CaCl2 stock solution was added per liter of the perfusion solution. The oocyte responses to different temperatures were plotted and analyzed using GraphPad Prism 5.0.

[0083] Polygonal dialdehyde was prepared into a 10-fold solution using dimethyl sulfoxide (DMSO). -4The stock solution was prepared with 1× Ringer (9.6 mM NaCl, 0.2 mM KCl, 0.5 mM MgCl2·6H2O, 0.5 mM HEPES, 0.6 mM CaCl2, pH 7.6) to a final concentration of 5×10⁻⁶. -5 2×10 -5 10 -5 5×10 -6 2×10 -6 10 -6 The electrophysiological responses of Xenopus oocytes expressing ApTR gene cRNA to the above concentrations of polydialdehyde were recorded using an OC-725 Coocyte clamp dual-electrode voltage clamp recording system. Figure 2 Oocytes expressing both ApTR(A) and ApTR(B) were activated by polydialdehyde, with type B showing a significantly stronger response than type A, and the response value increasing with increasing concentration. The results were fitted using GraphPad Prism 5.0 software. Figure 2 The reaction values ​​versus dosage curves are shown, and the EC50 values ​​of polyvinylpyrrolidone activation of ApTR(A) and ApTR(B) are calculated. 50 The values ​​are 5.37 × 10 -5 and 4.26×10 -5 mol / L. Data are expressed as mean ± SEM (n ≥ 6).

[0084] Example 4

[0085] RNAi validation of in vivo function

[0086] Preparation of 1st and 3rd instar pea aphids

[0087] The pea aphid was selected as the third instar nymph. The collection method for the third instar nymphs is as follows: Collect newly hatched first instar nymphs and place them on fresh broad bean seedlings. After about 4 days, all the first instar nymphs will grow into third instar nymphs. When transferring the pea aphid nymphs, gently pick them up with a soft brush to avoid causing unnecessary mechanical damage to the nymphs.

[0088] 2. Preparation of dsRNA template and synthesis of ApTR gene

[0089] A dsRNA sequence, i.e., the RNAi target site, was identified on the shared sequence of ApTR(A) and ApTR(B) in the pea aphid. Forward and reverse primers were designed based on this fragment, and a T7 promoter was added to the front end of the primers. Using the plasmid pEASY-BluntT-ApTR(A) (since it amplifies the shared sequence of ApTR(A) and ApTR(B), pEASY-BluntT-ApTR1(B) can also be used) as a template, the ApTR gene fragment (SEQ ID No. 15) was amplified using the primers ApTR-F-T7 (SEQ ID No. 13) and ApTR-R-T7 (SEQ ID No. 14).

[0090] The PCR products were subjected to electrophoresis on a 1% agarose gel. If the obtained band length matched the target fragment length and the result showed a single bright band, the remaining PCR products were extracted and purified using phenol-chloroform. The precipitate was then dissolved in 5 μL of RNase-free water. 1 μL of the solution was dissolved in 4 μL of RNase-free H₂O, and the concentration and OD value were measured using a concentration measuring instrument. The product uniformity was assessed by gel electrophoresis. If the detected band was a single bright band and its OD value was:

[0091] 260 / 280: 1.8-2.0

[0092] 260 / 230: 1.8-2.0

[0093] This indicates that the PCR product is of good quality and can be used as a template for the next step of dsRNA synthesis. The dsRNA of the ApTR gene was synthesized using a dsRNA synthesis kit.

[0094] The reaction system for synthesizing dsRNA was (400 μL): 80 μL 5× buffer, 8 μL each of dNTPs, 8-15 μg DNA template, 12 μL T7 enzyme, and DEPC water to a final volume of 400 μL. After thoroughly mixing all components, the mixture was incubated in a metal bath at 37°C for 4 hours for synthesis. The synthesized dsRNA product was purified by phenol-chloroform extraction, and the precipitate was finally dissolved in 50 μL of RNase-free water.

[0095] Take 1 μL of dissolved dsRNA product and dilute it in 4 μL of DEPC H2O. Use gel electrophoresis to check the product's uniformity.

[0096] The concentration and OD value were then measured using a concentration measuring instrument. If the measured band is a single, bright band, and its OD value is:

[0097] 260 / 280: 1.8-2.0

[0098] 260 / 230: 1.8-2.0

[0099] This proves that the dsRNA is of good quality and can be used for subsequent pea aphid RNAi bioassay experiments.

[0100] 3. Preparation of dsRNA template and synthesis of GFP gene

[0101] The difference between this method and the preparation and synthesis of dsRNA templates for the ApTR gene lies in the following: a sequence from the GFP gene (GenBank accession number: U76561) is determined for dsRNA synthesis. Using a laboratory-preserved Cloning vector pEGFP plasmid as a template, the GFP gene fragment (SEQ ID No. 18) is amplified using GFP-F-T7 (SEQ ID No. 16) and GFP-R-T7 (SEQ ID No. 17) primers. This amplified fragment is then used as the template for dsRNA synthesis. The synthesis of GFP dsRNA (dsGFP) is performed using the same method as the synthesis of dsRNA (dsApTR) for the ApTR gene.

[0102] 4. Hybridization of nanomaterials with dsRNA

[0103] The synthesized dsApTR was diluted to 1 μg / μL and then mixed with a certain proportion of nanomaterials to obtain the dsApTR-nanomaterial complex, which was used for the next step of body wall penetration into the pronotum of the soybean aphid. The preparation process of the dsApTR-nanomaterial complex is described in Yang Zheng, Yiseng Hu, Shuo Yan,*Hang Zhou, Dunlun Song, Meizhen Yin and Jie Shen*. A polymer / detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybeanaphid Aphis glycines. Pest Management Science. 2019; DOI:10.1002 / ps.5313 and Yang Zheng, Shusen You, Chendong Ji, Meizhen Yin, Wantai Yang, and Jie Shen; Adv. Mater. 2016, 28, 1375–1380. Details are as follows:

[0104] a. Mix 4 μL dsApTR + 1 μL nanocarrier at room temperature, dilute with water until the solution is clear and free of precipitate, vortex 1 to 2 times, and let stand at room temperature for 15 min to obtain dsApTR-nanocarrier dilution.

[0105] b. With the total volume of the dsApTR-nanocarrier diluent as 100%, add 10% of the volume of surfactant to the dsApTR-nanocarrier diluent, mix well, and obtain the dsApTR-nanomaterial composite.

[0106] The dsGFP-nanomaterial complex was prepared in the same manner as a control.

[0107] 5. RNAi function assay of target genes

[0108] A nano-mixture of dsApTR was administered via microinjection at a rate of 100 nmol / dose to the pronotum of third-instar pea aphids. The dsApTR nano-mixture was completely absorbed into the aphids within 1-2 minutes. A nano-mixture of dsGFP was used as a negative control, and water was used as a blank control. At least 80 pea aphids were treated per treatment and then transferred to single broad bean seedlings of similar growth for rearing. Each seedling was placed in a single rearing cage, and the cages were kept in the same rearing chamber. After 48 hours, the effect of polygalactocyanate on the pea aphids' feeding rejection was assessed using a leaf disc device, following the same method as in Example 1 (where the concentration of polygalactocyanate used was 1 mg / mL). The feeding rejection index of the pea aphids in different treatment groups was calculated, and the results are shown below. Figure 3 .

[0109] Figure 3 The results showed that the antifeeding index of pea aphids in the dsApTR nanomixture group was significantly lower than that in the dsGFP nanomixture and water control groups, indicating that the sensitivity of pea aphids to recognizing polydialdehyde decreased after interfering with the ApTR gene (mean±SEM, n=3; Tukey's HSD tests, α=0.05). RNAi technology confirmed that the pea aphid ApTR gene is an important target gene for recognizing the antifeeding compound polydialdehyde, providing an important theoretical basis for the application of aphid antifeedants.

Claims

1. Use of a pea aphid protein in the identification of an antifeedant compound, the amino acid sequence of the pea aphid protein being as set forth in SEQ ID No. 7 or as set forth in SEQ ID No. 8, the antifeedant compound identified by the pea aphid protein being leguminous aglycone, which induces antifeeding behavior in pea aphids.

2. Use according to claim 1, characterized in that, The nucleic acid encoding the pea aphid protein having the amino acid sequence as set forth in SEQ ID No. 7 is as set forth in SEQ ID No. 3; the nucleic acid encoding the pea aphid protein having the amino acid sequence as set forth in SEQ ID No. 8 is as set forth in SEQ ID No. 6.

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