Application of MpAcrP5 protein in affecting the transmission of turnip mosaic virus by green peach aphid

Abstract

The present invention relates to the use of the MpAcrP5 protein in influencing the transmission of Turnip Mosaic Virus by Myzus persicae. The present invention discovers that the MpAcrP5 protein of Myzus persicae can influence the transmission of Turnip Mosaic Virus by Myzus persicae. Reducing the expression of the MpAcrP5 protein in Myzus persicae can reduce the transmission of Turnip Mosaic Virus by Myzus persicae. Specifically, dsRNA of the MpAcrP5 gene can be used to interfere with the transcription of the MpAcrP5 gene in Myzus persicae, thereby reducing the transmission of Turnip Mosaic Virus by Myzus persicae, thereby preventing or alleviating plant diseases caused by Turnip Mosaic Virus.

Description

Technical Field

[0001] The present invention relates to the field of functional genes, and in particular to the application of functional genes as disease prevention agents. Background Art

[0002] Most plant viruses rely on vector insects for transmission, forming a complex three-way interaction relationship of "insect-borne virus-vector insect-host plant". Myzus persicae ) are important insect vectors of non-persistent viruses, and are known to transmit approximately 170 viruses, most of which belong to the genus Potyvirus ( Potyvirus Therefore, by blocking the virus transmission chain based on insect vectors, the difficulty of controlling viral damage can be significantly reduced, thereby effectively curbing the spread of viral damage. Summary of the Invention

[0003] The present invention provides the use of the MpAcrP5 protein having an amino acid sequence as shown in SEQ ID No. 2 in affecting the transmission of turnip mosaic virus by green peach aphids.

[0004] In one embodiment, the efficiency of peach aphids in transmitting Turnip mosaic virus to their host plants is reduced by reducing the expression of the MpAcrP5 protein in the peach aphid. When the expression of the MpAcrP5 protein in the peach aphid is reduced compared to the normal expression level of the protein in the peach aphid, the efficiency of the peach aphids in transmitting Turnip mosaic virus to their host plants is also reduced.

[0005] In one embodiment, the efficiency of peach aphids in transmitting Turnip mosaic virus to their host plants is reduced by interfering with the transcription of the MpAcrP5 gene in the peach aphid. When the transcription level of MpAcrP5 in the peach aphid is reduced compared to normal transcription levels of MpAcrP5 in the peach aphid, the efficiency of the peach aphids in transmitting Turnip mosaic virus to their host plants is also reduced.

[0006] In a specific embodiment, the nucleic acid sequence of the MpAcrP5 gene is shown as SEQ ID No. 1.

[0007] In one embodiment, dsRNA targeting the MpAcrP5 gene is used to interfere with the transcription of the MpAcrP5 gene in peach aphids to reduce the efficiency of peach aphids in transmitting Turnip Mosaic Virus to their host plants. Compared to normal transcription levels of MpAcrP5 in peach aphids, the dsRNA interference results in a decrease in the transcription level of MpAcrP5 in peach aphids, thereby reducing the efficiency of peach aphids in transmitting Turnip Mosaic Virus to their host plants.

[0008] In a specific embodiment, the dsRNA with a nucleic acid sequence such as that shown in SEQ ID No. 5 is used to interfere with the transcription of the MpAcrP5 gene in green peach aphid to reduce the efficiency of green peach aphid in transmitting turnip mosaic virus to the host plant.

[0009] In one embodiment, plant diseases caused by Turnip Mosaic Virus on host plants are prevented or alleviated by interfering with the transcription of the MpAcrP5 gene in peach aphids.

[0010] The present invention has the beneficial effects of discovering that the MpAcrP5 protein of peach aphid can affect the transmission of Turnip Mosaic Virus by peach aphids. Reducing the expression of the MpAcrP5 protein in peach aphids can reduce the transmission of Turnip Mosaic Virus by peach aphids. Specifically, dsRNA of the MpAcrP5 gene can be used to interfere with the transcription of the MpAcrP5 gene in peach aphids, thereby reducing the transmission of Turnip Mosaic Virus by peach aphids and achieving the purpose of preventing or alleviating plant diseases caused by Turnip Mosaic Virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The relative expression levels of the MpAcrP5 gene in Myzus persicae (Myzus persicae) 48 h after RNAi treatment are shown. ** indicates significant differences at the P < 0.001 level.

[0012] Figure 2 The figure shows the incidence of tobacco plants infected with TuMV-GFP virus transmitted by green peach aphids after 48 hours of RNAi targeting the MpAcrP5 gene of green peach aphids. *** indicates a highly significant difference at the P < 0.001 level.

[0013] Figure 3 Shown are EPG waveforms of peach aphids feeding on TuMV-GFP-infected tobacco plants 48 hours after disturbance. (a) Time of the first E1 wave; (b) Total E1 wave duration; (c) Number of E1 waves; (d) Average E1 wave duration; (e) Total E2 wave duration; (f) Number of E2 waves; (g) Average E2 wave duration; (h) Time of the first Pd wave; (i) Total Pd wave duration; (j) Number of Pd waves; (k) Average Pd wave duration; (l) Total C wave duration. Symbols above the bars indicate significant differences between the two groups (Mann-Whitney U test or Student's t test, n = 6, ns P>0.05; *P<0.05; **P<0.01). DETAILED DESCRIPTION

[0014] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0015] Unless otherwise specified, the strains, plasmids, and reagents in the examples of the present invention can be purchased through commercial channels.

[0016] Green peach aphid: Reared in an artificial climate chamber at 20±2 degrees Celsius, relative humidity 70+2%, and photoperiod L:D=16:8. Related article: Deshoux, M., Masson, V., Arafah, K., Voisin, S., Guschinskaya, N., vanMunster, M., Cayrol, B., Webster, CG, Rahbé, Y., Blanc, S., Bulet, P.,&Uzest, M. (2020). Cuticular Structure Proteomics in the Pea Aphid Acyrthosiphon pisum Reveals New Plant Virus Receptor Candidates at the Tip of Maxillary Stylets. Journal of proteome research, 19(3), 1319–1337.

[0017] Turnip mosaic virus is a TuMV-GFP strain. Related article: Ge, L., Jia, M., Shan, H., Gao, W., Jiang, L., Cui, H., Cheng, X., Uzest, M., Zhou, X., Wang, A., & Li, F. (2025). Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection. Nature communications, 16(1), 157.

[0018] Articles on dsRNA nanomaterials: Zheng, Y., Hu, Y., Yan, S., Zhou, H., Song, D., Yin, M., & Shen, J. (2019). A polymer / detergent formulation improves dsRNApenetration through the body wall and RNAi-induced mortality in the soybeanaphid Aphis glycines. Pest management science, 75(7), 1993–1999.

[0019] Agarose gel recovery kit was purchased from Quanshijin Company, China.

[0020] The antibiotics ampicillin, IPTG, and X-gal were purchased from Sigma, USA.

[0021] Water-saturated phenol was purchased from China Biotech Company.

[0022] Chloroform was purchased from China Biotech Company.

[0023] The sealing film was purchased from Parafilm Company, USA.

[0024] The cloning vector pEASY-Blunt was purchased from Quanshijin Company, China.

[0025] Competent cells Trans1-T1 were purchased from Quanshijin Company, China.

[0026] PrimeSTAR Max Premix PCR kit was purchased from Takara, Japan.

[0027] RNA extraction reagent Trizol was purchased from Invitrogen, USA.

[0028] The reverse transcription kit Revert Aid First Strand cDNA Synthesis Kit was purchased from Fermentas, USA.

[0029] The real-time fluorescence quantitative PCR kit SYBR PRemix Ex TaqTM was purchased from Takara Company, Japan.

[0030] Plasmid extraction kit was purchased from Tiangen Company, China.

[0031] The cRNA synthesis kit (mMESSAGE mMACHINE T7 kit) was purchased from Ambion, USA.

[0032] The dsRNA synthesis kit was purchased from Ambion, USA.

[0033] Example 1: Synthesis of dsRNA from the Myzus persicae MpAcrP5 gene

[0034] The nucleic acid sequence of the MpAcrP5 gene is shown in SEQ ID No. 1, and the amino acid sequence of the MpAcrP5 protein is shown in SEQ ID No. 2. Specific full-length primers used to amplify the MpAcrP5 gene are MpAcrP5-F (shown in SEQ ID No. 3) and MpAcrP5-R (shown in SEQ ID No. 4).

[0035] Wash the homogenizer and dry it in an oven at 180 degrees Celsius for 3 hours. Wear a mask and disposable gloves during the entire operation and try to ensure that it is carried out under RNase-free conditions.

[0036] Extraction of total RNA from peach aphids: (1) Place the whole peach aphids in a 2 ml centrifuge tube, wash them 2 to 3 times with pre-cooled PBS buffer, aspirate the excess liquid with a pipette tip, and immediately freeze them in liquid nitrogen. Then place them on ice, add 500 μl of Trizol, grind them into a homogenate using an electric grinder, and then add 500 μl of Trizol and mix them upside down. Let them stand on ice for 15 minutes to fully lyse the sample; (2) Add 200 μl of chloroform, shake them thoroughly for 15 seconds, let them stand at room temperature for 15 minutes, centrifuge them at 12000 rpm and 4 degrees Celsius for 15 minutes, and separate them into layers. The lower layer is a pink organic phase, the middle layer is an impurity layer, and the upper layer is a colorless aqueous phase. 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) to obtain chloroform extract; (3) add isopropanol of equal volume to the chloroform extract, let it stand at -20 degrees Celsius for two hours to precipitate RNA, then centrifuge at 12000 rpm and 4 degrees Celsius for 15 minutes, discard the supernatant to obtain RNA precipitate; (4) add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water), vortex the precipitate until it is suspended, then centrifuge at 7500 rpm and 4 degrees Celsius for 10 minutes, discard the supernatant to remove impurities; then repeat this operation twice; (5) Invert the centrifuge tube on filter paper for about 15 minutes to dry the precipitate; (6) Add 30 microliters of RNase-free water to the dried precipitate and pipette thoroughly until the precipitate is dissolved to obtain RNA solution; (7) Take 1 microliter of RNA solution for gel electrophoresis to ensure that its bands are correct and bright, and then take 1 microliter of RNA solution for NANOdrop detection. The OD value is within the usable range (260 / 280: 1.8-2.2; 260 / 230: 1.5-2.4), and store at -80 degrees Celsius.

[0037] First-strand cDNA synthesis: The entire reverse transcription process was performed under RNase-free conditions as much as possible. The first-strand cDNA of green peach aphid was synthesized using the extracted RNA as a template according to the protocol of the Revert Aid First Strand cDNA Synthesis Kit.

[0038] Cloning of MpAcrP5: Using the first-strand cDNA of green peach aphid as a template and MpAcrP5-F and MpAcrP5-R as primers, PCR was performed with the PrimeSTAR Max Premix PCR kit to obtain the PCR product of MpAcrP5. The PCR product of MpAcrP5 was then ligated into the pEASY-Blunt vector, and the ligation product was transformed into the competent cells Trans1-T1. After PCR identification and sequencing analysis, the positive plasmid pEASY-Blunt-MpAcrP5 was obtained, and the positive strain was Trans1-T1 / pEASY-Blunt-MpAcrP5.

[0039] Preparation of dsRNA template for the MpAcrP5 gene and dsRNA synthesis: A dsRNA sequence was identified within the MpAcrP5 sequence of Myzus persicae (MYZ) as an RNAi target site (shown in SEQ ID No. 5). Forward and reverse primers were determined based on this fragment, and T7 promoters were added to both ends of the primers. The forward primer, dsAcrP5-F, is shown in SEQ ID No. 6, and the reverse primer, dsAcrP5-R, is shown in SEQ ID No. 7. Using the plasmid pEASY-Blunt-MpAcrP5 as a template and dsAcrP5-F and dsAcrP5-R as primers, PrimeSTAR DNA polymerase was used to amplify the resulting dsRNA template. This PCR product was extracted with phenol-chloroform to obtain a phenol-chloroform-extracted PCR product (OD values ​​as measured by NANOdrop: 260 / 280: 1.8-2.0; 260 / 230: 1.8-2.0). Using the phenol-chloroform-extracted PCR product as a template, reverse transcription and DNA and ssRNA removal were performed using the Revert Aid First Strand cDNA Synthesis Kit to generate a dsRNA product. Agarose gel electrophoresis revealed a single bright band, and NANOdrop analysis revealed OD values ​​of 260 / 280: 1.8-2.0 and 260 / 230: 1.8-2.0, respectively. Phenol-chloroform extraction of the dsRNA product yielded the phenol-chloroform-extracted MpAcrP5 gene, dsMpAcrP5. Agarose gel electrophoresis revealed a single bright band, and NANOdrop analysis revealed OD values ​​of 260 / 280: 1.8-2.0 and 260 / 230: 1.8-2.0, respectively.

[0040] Comparative Example 1: dsRNA synthesis of GFP gene

[0041] The nucleic acid sequence of the GFP gene is shown in SEQ ID No. 8. A dsRNA sequence was identified within the GFP gene sequence as an RNAi target site. Forward and reverse primers were determined based on this fragment, and T7 promoters were added to both ends of the primers. The forward primer GFP-F-T7 is shown in SEQ ID No. 9, and the reverse primer GFP-R-T7 is shown in SEQ ID No. 10.

[0042] A synthetic GFP RNAi target site nucleic acid sequence was used as a template for PCR amplification using primers GFP-F-T7 and GFP-R-T7. This PCR product was extracted with phenol-chloroform to obtain a phenol-chloroform-extracted PCR product (OD values ​​as measured by NANOdrop: 260 / 280: 1.8-2.0; 260 / 230: 1.8-2.0). Using this phenol-chloroform-extracted PCR product as a template, reverse transcription and DNA and ssRNA removal were performed using the Revert Aid First Strand cDNA Synthesis Kit to generate a dsRNA product. A single bright band was detected by agarose gel electrophoresis and NANOdrop analysis, with OD values ​​as 260 / 280: 1.8-2.0; 260 / 230: 1.8-2.0. Next, the dsRNA product was subjected to phenol-chloroform extraction to obtain the GFP gene dsRNA product dsGFP after phenol-chloroform extraction. The result of agarose gel electrophoresis was a single bright band, and the OD value of NANOdrop detection was: 260 / 280: 1.8-2.0; 260 / 230: 1.8-2.0.

[0043] Example 2: Myzus persicae dsRNA interference

[0044] The preparation process of dsMpAcrP5-nanomaterial complex is as follows: a. Mixing of nanomaterial and dsRNA. The MpAcrP5 gene dsRNA was diluted to 1 μg / μl and mixed with liquid A (nanocarrier) at a volume ratio of 1:1, i.e., 2 μl of dsRNA + 2 μl of liquid A. After mixing, the mixture was vortexed 1 to 2 times and allowed to stand at room temperature for 15 minutes to obtain a dsMpAcrP5-nanocarrier liquid. The volume of the dsMpAcrP5-nanocarrier liquid was recorded as 100%, and 10% of liquid B (surfactant), i.e., 0.4 μl of liquid B, was added to the dsMpAcrP5-nanocarrier liquid and mixed evenly to obtain a dsMpAcrP5-nanocomplex, which was used to penetrate into the body of peach aphid through the body wall and exert its effect.

[0045] The dsGFP-nanomaterial complex was prepared in the same manner as a control.

[0046] Third-instar peach aphid nymphs were gently picked with a soft brush for injection. Using a microinjector, 18.9 nanoliters of the dsMpAcrP5-nanocomplex was dripped onto the aphid's stylet using a microinjector. The dsMpAcrP5-nanocomplex mixture completely penetrated the insects within approximately 1 minute. Aphids were then randomly assigned to individual healthy tobacco plants of similar growth. Each cage was housed in a single insectary, controlled at 25 ± 2°C and 70 ± 2% humidity, under a 16 L:8 D photoperiod. The aphids were reared for 48 hours to obtain aphids in which MpAcrP5 gene transcription had been disrupted by dsRNA. A dsGFP-nanocomplex was used as a control. At least 200 aphids were dripped per biological replicate, with 50 aphids placed on each tobacco plant, for a total of four tobacco plants. Three biological replicates were performed for each treatment.

[0047] The stylets of peach aphids after dsRNA interference were collected and quickly frozen in liquid nitrogen. The same procedures as in Example 1 were then used for RNA extraction and first-strand cDNA synthesis. The cDNA was diluted with RNase-free water at a ratio of 1:5 and used as a template. MperActin and MperEF1α were used as internal reference genes. Real-time fluorescence quantitative PCR was used to analyze the expression of the MpAcrP5 gene in peach aphids after interference. The specific amplification steps were referred to the instructions of the SYBR PRemix Ex TaqTM Real-time Fluorescence Quantitative PCR Kit of TakaRa Company. The relative expression of the target gene was measured using 2 -ΔΔCT The calculation formula is: 2 -ΔΔCT =2 -[(MpAcrP5 CT- MperActin CT)+(MpAcrP5 CT- MperEF1α CT)] / 2 .

[0048] The sequence of the upstream primer qPCRMpAcrP5-F for MpAcrP5 real-time fluorescence quantitative PCR is shown in SEQ ID No. 11, and the sequence of the downstream primer qPCRMpAcrP5-R is shown in SEQ ID No. 12.

[0049] The sequence of the upstream primer MperActin-F for MperActin real-time fluorescence quantitative PCR is shown in SEQ ID No. 13, and the sequence of the downstream primer MperActin-R is shown in SEQ ID No. 14.

[0050] The sequence of the upstream primer MperEF1α-F for MperEF1α real-time fluorescence quantitative PCR is shown in SEQ ID No. 15, and the sequence of the downstream primer MperEF1α-R is shown in SEQ ID No. 16.

[0051] The amplification program was set as follows: 94°C pre-denaturation for 30 s; 40 cycles of 94°C denaturation for 5 s; and 60°C annealing for 34 s. Each biological replicate consisted of 200 aphid stylets, three biological replicates per treatment, and three mechanical PCR replicates per biological replicate. Figure 1 . Figure 1 The results showed that at 48 h, the expression of MpAcrP5 gene was significantly downregulated in the dsMpAcrP5-treated group (n = 3; Student's t test, ns P >0.05,*** P <0.001).

[0052] Example 3: Transmission of Myzus persicae by dsRNA interference

[0053] Virus inoculation: (1) Place 1 to 2 leaves of Nicotiana benthamiana infected with Turnip mosaic virus (TuMV-GFP) in a mortar, add 5 mL of infiltration buffer (MgCl 10 mmol / L, 2-morpholineethanesulfonic acid 10 mmol / L, acetosyringone 100 μmol / L), and grind thoroughly to obtain leaf grinding liquid. (2) Spray corundum (200 to 400 mesh) evenly on the surface of the healthy Nicotiana benthamiana leaves to be inoculated, and mark the leaves to be inoculated with a marker. (3) Dip the leaf grinding liquid with a grinding stick or finger and evenly apply it to the surface of the leaves to be inoculated. When applying, gently apply it in one direction. (4) Rinse the inoculated leaves with ddH2O and culture the inoculated Nicotiana benthamiana in a low light intensity environment in the greenhouse. (5) After 5 days, the disease occurs and the infected Nicotiana benthamiana is obtained for subsequent virus transmission experiments.

[0054] Symptoms of turnip mosaic virus: systemic mosaic, wrinkling, necrosis and leaf drop; infected plants initially show mosaic, visible veins and wrinkling, and later show deformities, dwarfing and delayed growth period.

[0055] Treatment of Myzus persicae: The Myzus persicae dsMpAcrP5 interference procedure was the same as in Example 2. At least 200 aphids were inoculated per replicate, with three replicates per treatment. Aphids were randomly assigned to individual healthy tobacco plants of similar growth, with approximately 50 aphids placed on each plant, for a total of four plants. Each aphid was housed in a separate insect cage, and all cages were placed in the same insectary. The aphids were reared for 48 hours at 25±2°C, 70±2% humidity, and a photoperiod of 16 L:8 D. The dsMpAcrP5-interferenced aphids were obtained. A dsGFP-nanomaterial was used as a control.

[0056] To promote feeding, the dsRNA-interferenced peach aphids were starved for 1 hour to obtain starvation-+dsRNA-interferenced peach aphids. The specific procedure was as follows: approximately 120 peach aphids of uniform size and high activity were selected, placed in a disposable petri dish, sealed with parafilm, and starved for 1 hour.

[0057] Transmission of the virus by peach aphids: The peach aphids that were starved and interfered with by dsRNA were taken out of the culture dish and placed on the top leaves of the infected Nicotiana benthamiana for feeding. The feeding time was 15 minutes. After the feeding, the peach aphids were transferred to the second to third top leaves of the non-toxic Nicotiana benthamiana. There were 10 peach aphids per plant, and 1 plant was repeated for each. All the peach aphids were removed 2 days after the transmission of the virus. On the 7th day after the transmission of the virus, the Nicotiana benthamiana was observed under a portable ultraviolet light (because the TuMV virus carries a GFP tag, the amount of virus accumulation can be judged by the intensity of fluorescence under ultraviolet light. Strong green fluorescence indicates a large amount of virus accumulation) to determine the location of the diseased spots. Then, a laser confocal microscope (CLSM) was used to detect the fluorescence signal of the diseased leaves. Combined with the symptoms of the disease, it was determined whether they were infected with the virus and the incidence rate was calculated, where the incidence rate = (number of diseased plants / total number of plants) × 100%. See the results. Figure 2 .

[0058] According to the statistical data, after dsGFP interfered with peach aphid feeding, the tobacco morbidity rate was 81.08%. In contrast, after dsMpAcrP5 interfered with peach aphid feeding, the tobacco morbidity rate was significantly reduced (dsGFP treatment, n=37; ds MpAcrP5 treatment, n=29; Student's t test, *** P <0.001), only 51.72%, indicating that the efficiency of myzus persicae transmission decreased with downregulation of MpAcrP5 gene expression. Therefore, MpAcrP5 gene expression is positively correlated with plant disease incidence.

[0059] Example 4: Electrophysiological analysis of green peach aphids interfered with by dsRNA

[0060] Myzus persicae (Peach Aphids) were starved for 1 hour after 48 hours of exposure to dsMpAcrP5. Their feeding behavior on infected Nicotiana benthamiana plants was monitored using the Electrical Penetration Graph (EPG). dsGFP was used as a control. The specific steps for EPG monitoring are as follows.

[0061] (1) Preparation of insect electrodes: The required materials include copper nails, copper wire, silver glue, gold wire, and tin. First, tightly wrap the copper wire around the copper nail 3 to 4 times and use a tin soldering gun to evenly solder and secure it. Next, take a 5 to 6 cm section of gold wire, wind it into a very thin small loop at one end, and apply silver glue to the small loop. Using the viscosity of the silver glue, fix the gold wire to the unsoldered end of the copper wire, and the preparation of the insect electrode is complete.

[0062] (2) Preparation of test plants and plant electrodes: Before the test, eight Nicotiana benthamiana plants with a one-month growth cycle were prepared to maintain consistent growth. During the test, an appropriate amount of water was added to the soil to ensure the moisture content of the plant electrodes. Dry soil may cause a chaotic waveform signal, affecting the test results. The plant electrodes are copper rods approximately 10 cm in length. When used, they are inserted into moist soil near the plant roots.

[0063] (3) Connection between peach aphids and the insect electrode: Select a starved peach aphid and place it under a microscope for observation. In step (1), one end of the gold wire was coiled into a small loop and fixed to the unsoldered end of the copper wire. A small amount of silver glue was used to form a small ball at the other end of the gold wire. The ball was then precisely adhered to the middle of the peach aphid's pronotum. Let it stand for 1 minute to allow the silver glue to fully solidify. This ensures that the normal activities of the peach aphid are not affected. The insect electrode is then suspended. The connection between the electrode and the peach aphid is checked for stability during the experiment.

[0064] (4) Setting up the EPG system: Insert the copper nail into the EPG probe hole and insert the plant electrode into the moist soil near the roots of the Nicotiana benthamiana plant. After starting the controller, run the Stylet+d software on the computer and set the experiment name and duration. At the same time, ensure that all equipment is properly connected to facilitate accurate data collection and analysis.

[0065] (5) Insect placement: First, adjust the probe height to place the peach aphid on the leaves of N. benthamiana. Then, slightly adjust the probe to a position where the aphid can move freely. Close the Faraday cage to isolate external noise and reduce interference factors. Then, click the "Start" button in the software to officially start the monitoring process and record the feeding behavior of the peach aphid for 8 hours. Each treatment was replicated 8 times, with 6 peach aphids in each replicate.

[0066] (6) Waveform calibration and monitoring: 5 minutes before monitoring begins, evaluate the waveform output voltage and its stability. Perform waveform calibration using the control box. During monitoring, when unusual waveforms appear, observe the behavior of the peach aphids and record them in detail. If the waveform peak rises sharply or drops sharply, the output voltage may exceed the detection range. In this case, timely adjustments must be made using the control box to ensure the accuracy and reliability of the monitoring data.

[0067] (7) The DC-EPG system (Giga-8d, EPG Systems, Tisch, USA) was used to record the feeding behavior of peach aphids for 8 h. There were seven waveform types, including E1 wave (secretion of water-soluble saliva), E2 wave (passive feeding on the phloem), C wave (path wave), Pd wave (probing wave), Np wave (non-probing wave), G wave (active feeding on the xylem), and F wave (mechanical puncture obstruction).

[0068] (8) Data collection and processing: After the monitoring time is over, the data are saved and the waveform is processed using Stylet+a software (Wageningen University & Research (WUR), Netherlands), and the specific frequency and output voltage of the waveform are recorded.

[0069] See the results Figure 3 Compared with the dsGFP control group, silencing the MpAcrP5 gene significantly affected the feeding pattern of peach aphids ( Figure 3 Specific results are as follows: During the water-soluble saliva secretion stage, the first appearance of the E1 wave was significantly delayed (Mann-Whitney U Test, P<0.05), and the total duration (Mann-Whitney U Test, P<0.01) and average duration (Mann-Whitney U Test, P<0.01) of the E1 wave were significantly shortened. During the passive phloem feeding stage, the total duration (Mann-Whitney U Test, P<0.05) and average duration (Mann-Whitney U Test, P<0.01) of the E2 wave were significantly shortened. During the exploratory behavior stage, the total duration of the C wave was significantly prolonged (Student's t test, F=0.8143, P<0.01). However, there were no significant differences between the two groups in terms of the total number of E1 waves (Student's t test, F=0.031, P=0.362), the total number of E2 waves (Student's t test, F=0.047, P=0.701), the time to the first appearance of the Pd wave (probing wave) (Mann-Whitney U Test, P=0.109), the total time of the Pd wave (Student's t test, F=1.39, P=0.150), the number of Pd waves (Student's t test, F=2.434, P=0.105), and the average time of the Pd wave (Student's t test, F=1.761, P=0.454). This indicates that silencing MpAcrP5 prolongs the time it takes for the peach aphid's stylet to reach the phloem for feeding, and also makes it difficult for the peach aphid to secrete saliva to inhibit the plant's immune defense response, affecting the peach aphid's probing and feeding, that is, the successful feeding time is significantly shortened, thereby reducing the peach aphid's acquisition or transmission of the virus.

Claims

1. Use of the MpAcrP5 protein having the amino acid sequence shown in SEQ ID No. 2 in preventing green peach aphids from transmitting turnip mosaic virus.

2. The use according to claim 1, characterized in that By reducing the expression level of MpAcrP5 protein in peach aphids, the efficiency of peach aphids in transmitting turnip mosaic virus to host plants can be reduced.

3. The use according to claim 1, characterized in that By interfering with the transcription of the MpAcrP5 gene in the peach aphid, the efficiency of the peach aphid in transmitting turnip mosaic virus to the host plant is reduced.

4. The use according to claim 3, characterized in that The nucleic acid sequence of the MpAcrP5 gene is shown in SEQ ID No.

1.

5. The use according to claim 3, characterized in that The dsRNA of the MpAcrP5 gene was used to interfere with the transcription of the MpAcrP5 gene in the peach aphid, thereby reducing the efficiency of the peach aphid in transmitting turnip mosaic virus to the host plant.

6. The use according to claim 3, characterized in that The dsRNA with the nucleic acid sequence shown in SEQ ID No. 5 is used to interfere with the transcription of the MpAcrP5 gene in the green peach aphid, thereby reducing the efficiency of the green peach aphid in transmitting turnip mosaic virus to the host plant.

7. The use according to any one of claims 2 to 6, characterized in that Plant diseases caused by turnip mosaic virus on host plants can be prevented or alleviated by interfering with the transcription of the MpAcrP5 gene in peach aphids.

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