Application of endogenous Bunyavirus element LsBuEVE and its dsRNA in blocking rice virus transmission in planthopper

Abstract

This invention discloses the application of an endogenous Bunyavirus element LsBuEVE and its dsRNA from the planthopper in blocking the transmission of rice viruses. The LsBuEVE genome contains the nucleotide sequence shown in SEQ ID NO.1, and its corresponding transcript contains a complete open reading frame, the nucleotide sequence of which is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3. Introducing the dsRNA targeting LsBuEVE into the planthopper effectively inhibits LsBuEVE expression, significantly reduces the transcriptional level of the RSV nucleoprotein gene, protein accumulation, and the RSV acquisition rate in the planthopper, thereby effectively blocking RSV replication and transmission within the planthopper. This invention reveals for the first time the important role of the endogenous viral element LsBuEVE in the transmission of plant viruses by vector insects and provides a precise targeted control strategy based on RNA interference. Compared with traditional chemical pesticides, this technology has advantages such as high specificity, environmental friendliness, and low likelihood of developing resistance, and has broad application prospects and commercial value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biological control, specifically to the application of the endogenous Bunyavirus element LsBuEVE and its dsRNA in blocking the transmission of rice viruses in the planthopper. Background Technology

[0002] The brown planthopper (Laodelphax striatellus) is a highly destructive vector insect in my country's agricultural production. It is not only a key vector for Rice Black-streaked Dwarf Virus (RBSDV) but also a core vector for Bunyaviruses such as Rice Stripe Virus (RSV). The virus feeds on the vector insect through its piercing and sucking action, undergoing key stages within the planthopper including virus acquisition, cyclical replication, and release via salivary glands, ultimately causing large-scale crop diseases. Currently, field control still heavily relies on chemical pesticides. Long-term pesticide use has led to a surge in pesticide resistance in vector insects, disrupted the ecological balance of farmland, and resulted in significant problems such as pesticide residues in agricultural products. Furthermore, the limited genetic background and narrow resistance spectrum of resistant crop varieties make it difficult to effectively address the control pressure posed by the migration and spread of vectors.

[0003] Endogenous viral elements (EVEs) are genetic remnants formed after ancient viral sequences integrate into the host genome, and are considered "molecular fossils" recording the history of virus-host interactions. Recent studies have confirmed that some functionalized EVEs can participate in biological processes such as host antiviral immunity and developmental regulation. However, significant gaps remain in the functional research of Bunyavirus-derived Endogenous Viral Elements (BuEVEs) in vector insect genomes. It is currently unclear whether these EVEs participate in regulating the infection and proliferation of contemporary plant Bunyaviruses (such as RSV) in vectors, and the underlying epigenetic regulatory mechanisms and cross-species conservation lack systematic analysis.

[0004] In existing technologies, regulatory targets for the planthopper-Buyavirus interaction are mostly limited to classical immune pathway genes (such as the Toll and IMD pathways), and no antiviral mechanism centered on endogenous Bunyavirus elements has been discovered. Furthermore, there is a lack of targeted control technologies developed using these specific EVEs, resulting in limited and imprecise control measures. RNA interference (RNAi) technology, which specifically degrades target mRNA using small double-stranded RNA, has significant advantages in high specificity and efficiency and is currently widely used in agricultural pest control. Therefore, there is an urgent need to combine RNAi technology to target and discover key BuEVE functional elements in the planthopper and develop green and efficient control solutions based on novel molecular mechanisms. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an application of the endogenous Bunyavirus element LsBuEVE (Laodelphax striatellus Bunyavirus-derived Endogenous Viral Element) and its dsRNA (double-stranded RNA) in blocking the transmission of rice viruses. By discovering a novel endogenous Bunyavirus element LsBuEVE from the planthopper as a novel molecular target, and combining it with RNAi technology to further target and interfere with the expression of this functional element in the planthopper, the replication and transmission of rice viruses in vector insects can be blocked, providing an effective molecular tool and a novel technical approach for the green control of rice viral diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides an endogenous Bunyavirus element LsBuEVE for the planthopper, the genome of which comprises a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence having at least 90% homology with the SEQ ID NO.1.

[0008] Preferably, the transcript corresponding to LsBuEVE contains a complete open reading frame, and the amino acid sequence encoded by the open reading frame is shown in SEQ ID NO.3.

[0009] Preferably, the nucleotide sequence of the open reading frame is as shown in SEQ ID NO.2, or a nucleotide sequence having at least 90% homology with the one shown in SEQ ID NO.2 and encoding an amino acid as shown in SEQ ID NO.3.

[0010] A second aspect of the present invention provides a dsRNA that targets and silences the aforementioned LsBuEVE, wherein the target sequence of the dsRNA is located within the nucleotide sequence region shown in SEQ ID NO.2.

[0011] Preferably, the dsRNA is synthesized by in vitro transcription using the positive strand primer shown in SEQ ID NO.4 and the antisense strand primer shown in SEQ ID NO.5.

[0012] A third aspect of the present invention provides a biological agent for blocking the spread of rice viruses, comprising the above-described dsRNA and an agriculturally acceptable carrier or excipient.

[0013] The fourth aspect of the present invention provides the use of the above-described LsBuEVE, the above-described dsRNA, or the above-described biological agent in the preparation of a product for blocking the spread of rice virus.

[0014] Preferably, the product blocks the transmission of rice viruses by inhibiting the expression of LsBuEVE in planthoppers, thereby reducing the acquisition rate of rice viruses by planthoppers or inhibiting the replication and accumulation of rice viruses in planthoppers.

[0015] Preferably, the rice virus is rice stripe virus.

[0016] The fifth aspect of the present invention provides a method for blocking the spread of rice viruses, comprising injecting and / or feeding the above-mentioned dsRNA or the above-mentioned biological agent into the rice planthopper, or applying the above-mentioned biological agent to the rice plant or cultivation environment.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention is the first to identify and isolate a novel endogenous Bunyavirus element, LsBuEVE, derived from the capsid protein gene of Fushun phasmavirus 2, from the genome of the planthopper. dsRNA targeting LsBuEVE was designed and synthesized using RNA interference technology. After the dsRNA was introduced into the planthopper, LsBuEVE expression was significantly inhibited; simultaneously, the transcriptional level and protein accumulation of the RSV nucleoprotein gene both decreased significantly, and the RSV acquisition rate in the planthopper was also significantly reduced. This indicates that LsBuEVE plays a crucial proviral role in RSV transmission, and targeting LsBuEVE can effectively block RSV replication and transmission within the vector insect, thus providing an effective technical pathway and a novel molecular intervention target for blocking planthopper-mediated rice virus transmission.

[0019] (2) The dsRNA technology developed in this invention based on endogenous viral elements unique to the genome of the planthopper has significant advantages over existing control methods that rely on chemical pesticides, such as high specificity, environmental friendliness, and low likelihood of developing resistance. It meets the strategic requirements of green agricultural production and sustainable development and has broad application prospects and commercial value. At the same time, the endogenous elements are relatively conserved in different geographical populations and have good targeting universality. Attached Figure Description

[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0021] Figure 1 The diagram shows the sequence identification and transcriptional expression analysis of the endogenous Bunyavirus element LsBuEVE in the planthopper in Example 1. In this diagram, A is the genomic location and sequence alignment of LsBuEVE on chromosome 2 of the planthopper, and B is the transcript structure of LsBuEVE and its predicted open reading frame (ORF).

[0022] Figure 2 The image shows the RT-qPCR detection of the effect of dsLsBuEVE silencing on RSV accumulation in Example 2. In the image, dsGFP represents the mixed introduction group of GFP dsRNA and crude RSV extract (control group), and dsLsBuEVE represents the mixed introduction group of LsBuEVE dsRNA and crude RSV extract (experimental group).

[0023] Figure 3 This is a Western blotting image showing the effect of dsLsBuEVE silencing on RSV accumulation in Example 2. In this image, dsGFP represents the dsRNA-introduced group of GFP (control group), and dsLsBuEVE represents the dsRNA-introduced group of LsBuEVE (experimental group).

[0024] Figure 4 This is a statistical analysis chart showing the effect of silencing LsBuEVE on the virus acquisition rate of planthoppers in Example 3. In the chart, dsGFP represents the mixed introduction group of GFP dsRNA and crude RSV extract (control group), and dsLsBuEVE represents the mixed introduction group of LsBuEVE dsRNA and crude RSV extract (experimental group).

[0025] Figure 5This is a yeast two-hybrid verification diagram of the interaction between LsBuEVE and LsSAMS in Example 4. In this diagram, BD-LsBuEVE+AD-LsSAMS is the experimental group, BD-LsSAMS+AD-T7 and BD-LsBuEVE+AD-T7 are both negative self-activation control groups, and AD-T7-T + BD-53 is the positive control group.

[0026] Figure 6 This is an RT-qPCR detection diagram of the effect of silencing LsSAMS on the transcriptional levels of LsSAMS and RSV-NP in planthoppers in Example 4. In this diagram, dsGFP represents the mixed introduction group of GFP dsRNA and crude RSV extract (control group), and dsLsSAMS represents the mixed introduction group of LsSAMS dsRNA and crude RSV extract (experimental group).

[0027] Figure 7 This is a Western blotting image showing the effect of silencing LsBuEVE on LsSAMS protein expression in the planthopper in Example 5. In this image, dsGFP represents the mixed introduction group of GFP dsRNA and crude RSV extract (control group), and dsLsSAMS represents the mixed introduction group of LsSAMS dsRNA and crude RSV extract (experimental group). Detailed Implementation

[0028] To make the objectives and technical solutions of this invention clearer and more complete, the invention will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the technical solutions of this invention, are all within the scope of protection of this invention.

[0029] Unless otherwise specified, all reagents and materials involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0030] Example 1: Identification and Transcriptional Expression Analysis of the LsBuEVE Sequence of Endogenous Bunyavirus Element from the Grey Planthopper

[0031] 1. Identification of the LsBuEVE sequence of endogenous Bunyavirus element in the grey planthopper

[0032] To systematically identify endogenous viral elements of the Bunyavirus order in the genome of the planthopper, this embodiment uses the amino acid sequences of identified Bunyavirus structural proteins as query sequences and performs tBLASTn homology comparisons with the whole genome data of the planthopper. To ensure the reliability of the screening results, the following strict criteria are established: First, only those with an expected value (E-value) lower than 1×10⁻⁶ are retained. -9 The matching results are used to exclude random matches with low confidence; secondly, sequences with a length of less than 300 bp are removed to ensure that the candidate sequences have minimum coding potential and structural integrity.

[0033] To further verify that the candidate sequences were indeed of viral origin, the candidate EVEs obtained from the initial screening were searched using BLASTx in the NCBI Non-Redundant Protein Database (Nr), and viral amino acid sequences that were highly homologous to them were selected and downloaded for subsequent analysis.

[0034] Through the above process, this embodiment successfully identified an endogenous viral element derived from the capsid protein (CP) of Fushun phasmavirus 2 (a baculovirus isolated from Fushun, belonging to the family Phasmaviridae), named LsBuEVE. It is located at positions 56,766,738-56,768,729 on chromosome 2 of the planthopper, and shares 89% amino acid sequence similarity with the CP protein. Figure 1 (A in the sequence). The original LsBuEVE integration fragment corresponding to this genomic locus is 1992 bp in length, and the specific genomic sequence is shown in SEQ ID NO.1.

[0035] 2. Transcriptional expression analysis of LsBuEVE

[0036] To clarify the in vivo transcriptional activity and protein-coding potential of the obtained LsBuEVE, this embodiment further performed homology alignment of the LsBuEVE genome sequence (SEQ ID NO.1) with the *Agrostis spp.* transcriptome database using BLASTn, and used the NCBI ORF Finder tool to predict open reading frames (ORFs) of the matched transcript sequences. The transcriptome alignment results showed a high degree of match between the LsBuEVE genome sequence and the endogenous transcript sequences in *Agrostis spp.*, confirming that the LsBuEVE segment integrated into the *Agrostis spp.* genome can be recognized and effectively transcribed by the host transcription system, forming corresponding transcripts in vivo. Open reading frame analysis showed that the transcript corresponding to this LsBuEVE contains a complete 2124 bp ORF, and the region corresponding to this ORF has no intron insertions. Figure 1(B in the original text). The nucleotide sequence of the ORF corresponding to the obtained LsBuEVE is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3. It should be noted that, in this embodiment, although the genome sequence of the LsBuEVE element itself is only 1992 bp, the transcript sequence it forms is longer than 1992 bp, indicating that it also contains a part of the host genome sequence during transcription.

[0037] The genome sequence of LsBuEVE (SEQ ID NO.1):

[0038]

[0039] The nucleotide sequence of the ORF corresponding to LsBuEVE (SEQ ID NO.2):

[0040]

[0041] The amino acid sequence encoded by the ORF corresponding to LsBuEVE (SEQ ID NO.3):

[0042] MADKQTGPAQGPDPNAIVPAQQVQTAAITLDKRAQLAKAVVEKSPENLANHINETNFEVRAMSPDEFMDKHSSTTFDLQGLFNDFKEVCPDFEEDFGQSSKARFCIGFCSDIISTVGPESRKLKKDSRDKSWCFVFGEGEQAKYVYIATFKNDETKVNYARKDRSVLCLTMKQASL ISVEILSRLVTVIQRDGKSKDKIVLTPLAGAIFSRNDMATMAHLMGKSLSEGINAVNRSCQSGSQYFPIGKGSSEIAVVAAITATRNMKSKSTMESIITKTLKQYLNNDHPFDRELLKALSIFANCGLPDGITADTLISDYESAKKQTDVADLRNLYKTYYQTINKPGNQGGGGPYG GNPPGGNPPGNQGGAPPKGRKPPPGFPPINKGPASKPGLPTTTTLPSILKQGQSTRKRKSEGPAMPSMLEMRTAIEALERADPVDMERNIPKLAALQGPSIVIPDTDVDVPAPKSPRHEVGSRQQKQNVFADAHHAPDEDIFEQDNDNEGDHHDHEERHIVGETTAHSTQPGEEASS EGQSDNISSASFDIRSLLTISTEDSDDYNHKQKKAPLAYDKKLLKRTDRADRYEARRDVRRNSYSASHHPEVLKFIAQMENIHDLSTFNPGTKLQTMISMYMLGFKPGGMQYLREQVCMGNPNISKPVRVFLSLSRRDCEMMYEQGCTDSITFPKHDEIEAASSSGNQAQSPGSDTSE

[0043] Example 2: dsRNA of LsBuEVE from the planthopper inhibits RSV accumulation in planthoppers.

[0044] To investigate whether the endogenous viral element LsBuEVE identified above functions in the process of planthopper resisting rice stripe virus (RSV) infection, this embodiment uses RNAi technology to specifically knock down the transcript of LsBuEVE in planthopper and observe its effect on RSV virus accumulation.

[0045] 1. Synthesis and purification of dsRNA (dsLsBuEVE) from the planthopper LsBuEVE

[0046] (1) Synthesis of dsLsBuEVE

[0047] Using gene-specific primers linking the T7 promoter sequence and the nucleotide sequence shown in SEQ ID NO.2 as a template, the target interference fragment of LsBuEVE was amplified by PCR, with a GFP gene fragment used as a control. In a preferred embodiment, the target interference fragment is located within the ORF region of the corresponding transcript of LsBuEVE. The specific amplification process is a conventional technique in the art and will not be described in detail here. Subsequently, according to the instructions of the Yisheng Bio T7 High Yield RNASynthesis Kit, the PCR amplification product was used as template DNA to transcribe and synthesize the corresponding double-stranded RNA. The specific reaction system is shown in Table 1, and the dsLsBuEVE primers and dsGFP primers with the T7 promoter sequence are shown in Table 2.

[0048] Table 1 Reaction system for dsRNA synthesis

[0049]

[0050] Table 2. Primers for dsLsBuEVE and dsGFP with T7 promoter sequences

[0051]

[0052] (2) Purification of dsLsBuEVE

[0053] Specifically, the purification reaction conditions are as follows: After gently mixing the above mixture, incubate at 37°C for 12-16 hours. Then, add 1 μL of DNase I and incubate at 37°C for 15 min to digest the residual DNA template. After the reaction is complete, centrifuge the system at 12,000 rpm for 2 min at 4°C and collect the supernatant. Finally, take 1 μL of the product and verify the dsRNA synthesis efficiency by agarose gel electrophoresis. Store the LsBuEVE dsRNA at -80°C for later use.

[0054] 2. RT-qPCR detection of dsLsBuEVE silencing and RSV accumulation

[0055] To analyze whether LsBuEVE affects RSV replication, this embodiment involves injecting a mixture of dsLsBuEVE and crude RSV extract into a non-toxic planthopper. A dsGFP+RSV crude extract serves as a negative control. The transcriptional levels of LsBuEVE and RSV nucleoprotein (RSV-NP) genes were detected using quantitative real-time PCR (RT-qPCR). The specific implementation process is as follows:

[0056] (1) Microinjection

[0057] a. Preparation of microinjection needles: Glass capillaries were drawn into microinjection needles using a microelectrode needle puller (PC-100) at 65°C. The prepared injection needles were then neatly placed in a culture dish with double-sided tape for later use.

[0058] b. Preparation of the agarose injection platform: Pour a 2% agarose solution dissolved in water into a petri dish. Before solidification, place a glass capillary tube parallel to the liquid surface to form a shallow groove. After the agarose has completely solidified, remove the capillary tube to obtain the grooved agarose gel platform. Before injection, remove the agarose gel platform from the 4°C freezer and pre-cool it on ice.

[0059] c. Sample preparation and injection system assembly: Prepare the injection mixture for the experimental group, with dsLsBuEVE concentration of 3-5 μg / μL and RSV crude extract diluted at a ratio of 1:2, and mix the two liquids at a 1:1 volume ratio. Use a 10 μL pipette to inject the sample into the microinjection needle. Then, attach the injection needle to the fixing connector of the microinjection instrument, and under a microscope, use fine tweezers to break off the needle tip, creating a moderately angled opening. The size of the needle tip opening should be such that it can easily penetrate the body wall of the planthopper with minimal solution leakage.

[0060] d. Anesthesia and fixation of planthoppers: Third-instar planthoppers were anesthetized with CO2 for 1 minute, and then lightly covered with their hands for about 20 seconds to maintain the anesthesia. The anesthetized planthoppers were then neatly arranged in a pre-cooled agarose gel tray in a dorsoventral position (lying flat) using a fine brush.

[0061] e. Microinjection parameter settings: Set the basic parameters of the microinjector as follows: injection pressure 1000 Pa, injection time 0.1 seconds, and compensation pressure 10 Pa. In actual operation, these parameters can be adjusted appropriately according to the size of the insect and the diameter of the needle tip.

[0062] f. Microinjection procedure: The injection site is selected on the lateral epidermis between the first and second pairs of legs on the thorax of the planthopper. The injection needle should be inserted at a small angle, taking care to control the insertion depth to avoid damaging internal tissues. Each planthopper is injected once, with an injection volume of approximately 40 nL. The planthoppers are then fed to healthy rice seedlings.

[0063] Using the same method, a mixture of dsGFP and crude RSV extract (with the same concentration and ratio as the experimental group) was introduced into the planthopper as a negative control group. It should be noted that the above parameters are exemplary parameters for this embodiment, and those skilled in the art can make adaptive adjustments according to actual needs.

[0064] 2. Real-time quantitative PCR (RT-qPCR) detection

[0065] On day 6 post-injection (6 dpi), samples of planthoppers from each group were collected, and total RNA was extracted and reverse transcribed into cDNA. Real-time quantitative PCR (RT-qPCR) was used to determine the transcription levels of LsBuEVE and RSV-NP, respectively. Specifically, the Hieff® qPCR SYBR Green PCR Master Mix kit from Yisheng Biotechnology was used, and RT-qPCR analysis was performed according to its instructions. Primers capable of amplifying 150-250 bp specific fragments were used, and the cDNA template was diluted 10-20 times before use. The primer sequences used are shown in Table 4, and the specific reaction system is shown in Table 3.

[0066] Table 3 RT-qPCR reaction system

[0067]

[0068] Table 4. RT-qPCR primers for LsBuEVE, RSV-NP, and internal reference genes.

[0069]

[0070] The real-time quantitative PCR instrument used was a Roche LightCycler® 480 II. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 60℃ annealing for 30 s; 70℃ extension for 30 s; for a total of 40 cycles. The melting curve program was run according to the instrument settings. 2 -ΔΔCT Methods: Quantitative data were analyzed, and the Student's t-test was used to test the significance of differences. P < 0.05 (*) indicated a significant difference, while P < 0.01 (**) and P < 0.001 (***) indicated extremely significant differences.

[0071] The results are as follows Figure 2As shown, dsLsBuEVE can effectively silence LsBuEVE expression; and in the planthopper where LsBuEVE expression is significantly suppressed, the transcriptional level of RSV-NP also shows a highly significant decreasing trend. This indicates that the absence of LsBuEVE severely hinders the transcriptional replication of RSV virus in the planthopper.

[0072] 3. Western blotting detection of RSV accumulation using dsLsBuEVE

[0073] To further verify the effect of dsLsBuEVE on RSV replication at the protein level, this embodiment introduced dsLsBuEVE into infected planthoppers, using dsGFP as a negative control, and detected RSV-NP protein levels by Western blotting. The specific implementation process is as follows:

[0074] (1) Poisoned planthopper population: The poisoned planthopper population was maintained and fed by this laboratory. The virus carrying rate was tested regularly and kept above 90%.

[0075] (2) Microinjection: dsLsBuEVE (experimental group) or dsGFP (control group) were injected into the two groups of planthoppers respectively. The injection concentration was 3-5 μg / μL and the volume was about 40 nL. The microinjection method was the same as described above.

[0076] (3) Western blotting analysis: On the 3rd day after injection, samples of gray planthoppers from each group were collected and Western blotting was performed according to the following steps.

[0077] a. Protein sample preparation: Add each group of planthoppers to 1×Lysis Buffer containing protease inhibitors and grind thoroughly. Centrifuge the lysate at 4℃, 12,000 rpm for 10 min, collect the supernatant, and mix it with 5×Loading Buffer at an appropriate volume ratio. Boil the mixed sample in a 95℃ metal bath for 10 min, then briefly centrifuge before use.

[0078] b. SDS-PAGE electrophoresis: Select an appropriate separating gel concentration (8%–15%) based on the molecular weight of the target protein. The gel thickness (1.0 mm or 1.5 mm) and the number of comb wells (10 or 15 wells) should be determined according to specific experimental requirements. Load 10 μL of sample into each well. The electrophoresis program is set as follows: stacking gel stage at 80 V constant voltage for 30 min, separating gel stage at 100 V constant voltage for 60 min.

[0079] c. Transfer: After electrophoresis, remove the stacking gel and excess separating gel, and equilibrate the target gel region in pure water. Activate the PVDF membrane, pre-cut to the same size as the gel, in methanol for 10 seconds. Assemble in the following order: transfer mesh—PVDF membrane—protein gel—transfer mesh, ensuring no air bubbles between layers. Place the transfer clamp in an automated transfer apparatus (GenScript) to complete the transfer.

[0080] d. Sealing: After the transfer is completed, immerse the PVDF membrane in a 5% skim milk powder solution and seal it on a shaker at room temperature for 2 hours.

[0081] e. Primary antibody incubation: Add the target protein RSV-NP primary antibody to the blocking buffer at a ratio of 1:5000, and incubate for 2 hours on a shaker at room temperature, or overnight on a shaker at 4°C. Actin protein is used as an internal control.

[0082] f. Washing the membrane: After the primary antibody incubation is complete, wash the membrane three times with 1×TBST buffer on a shaker at room temperature for 10 min each time.

[0083] g. Secondary antibody incubation: Add the corresponding species of secondary antibody (rabbit or mouse) to the freshly prepared blocking solution at a ratio of 1:10000, and incubate on a shaker at room temperature for 1 hour.

[0084] h. Washing the membrane: After the secondary antibody incubation is complete, wash the membrane three times with 1×TBST buffer on a shaker at room temperature for 10 min each time.

[0085] i. Chemiluminescence color development: Mix HRP chemiluminescence substrate solution A and solution B in a 1:1 ratio and store in the dark. Add the mixed working solution evenly to the target band region of the PVDF membrane, react in the dark for 3 minutes, and then detect the chemiluminescence signal using a protein imaging system.

[0086] The results are as follows Figure 3 As shown, silencing LsBuEVE in infected planthoppers resulted in a significant decrease in RSV-NP protein accumulation compared to the control group. This result corroborates the findings of RT-qPCR, further demonstrating that LsBuEVE plays a crucial role in regulating RSV viral protein synthesis.

[0087] Example 3: Silencing LsBuEVE inhibits RSV acquisition rate in planthoppers.

[0088] To investigate whether LsBuEVE affects the ability of planthoppers to acquire rice viruses, this example further statistically analyzes the virus acquisition rate of planthoppers. Specifically, according to the dosage and method described in Example 2, second-instar virus-free planthoppers were injected with a mixture of dsLsBuEVE and crude RSV extract (experimental group), while the control group was introduced with the same dose of a mixture of dsGFP and crude RSV extract. They were then fed to healthy rice seedlings for 6 days.

[0089] After the virus feeding was completed, individual samples were taken from each planthopper. Total RNA was extracted, and the RSV virus carriage status of each planthopper was detected by reverse transcription and RT-PCR to calculate the virus acquisition rate for each group. The RSV-NP RT-PCR primers used in the experiment are shown in Table 5.

[0090] The results are as follows Figure 4 As shown, silencing LsBuEVE significantly reduced the virus acquisition rate in planthoppers compared to the control group. This result indicates that introducing LsBuEVE dsRNA effectively reduces the ability of planthoppers to acquire RSV virus.

[0091] Table 5 Primers for RT-PCR detection of RSV-NP

[0092]

[0093] Example 4: Interaction identification and functional study of LsBuEVE with S-adenosylmethionine synthase (SAMS) of the planthopper

[0094] To elucidate the molecular mechanism by which LsBuEVE regulates RSV replication, this embodiment screened and identified the host proteins of the planthopper that interact with it using yeast two-hybrid technology, and verified and explored the function of this interaction. The specific implementation process is as follows:

[0095] 1. Screening and identification of LsBuEVE interacting host proteins

[0096] To systematically screen for planthopper proteins that interact with LsBuEVE, its complete open reading frame (ORF) sequence was first cloned into the yeast two-hybrid vector pGBKT7 to construct the bait plasmid (BD-LsBuEVE). Simultaneously, a planthopper cDNA library was constructed and cloned into the prey vector pGADT7.

[0097] The bait yeast strain AH109 (carrying BD-LsBuEVE) was subjected to large-scale hybridization transformation with a cDNA library from the planthopper. Transformants were plated on auxotrophic medium SD / -Leu / -Trp / -His (triple-deficient medium) for rigorous selection; yeast can only grow in this medium when the bait protein interacts with the prey protein and activates the reporter gene. Prey bombmids were extracted from the growing positive clones and sequenced. The obtained sequences were BLASTx aligned to the NCBI non-redundant protein database (Nr), identifying a protein interacting with LsBuEVE, which is the planthopper's S-adenosylmethionine synthetase (SAMS), named LsSAMS in this embodiment. The nucleotide sequence of the coding region of the LsSAMS gene is shown in SEQ ID NO.16, and its encoded amino acid sequence is shown in SEQ ID NO.17.

[0098] The coding region nucleotide sequence of the LsSAMS gene (SEQ ID NO.16):

[0099]

[0100] The amino acid sequence encoded by the LsSAMS gene (SEQ ID NO.17):

[0101] MPETSHIINGFTNGHMEPETPEGESFLFTSESVGEGHPDKMCDQISDAILDAHLRQDPDAKVACETVTKTGMVLLCGEITSKANVDYQKVVRDTIQHIGY DDSSKGFDYRTCNVLTAVDQQSPHIADGVHKNKEEGDIGAGDQGLMFGYATDETEECMPLTVVLAHKLNQKVADLRRRSGEFWWARPDTKTQVTCEYCFRRG SCVPQRVHTVVVSVQHSEKISLEDLRAAVTEKVVKEVIPAKYLDSKTIIHINPCGAFVLGGPQCDAGLTGRKIIVDTYGGWGAHGGGAFSGKDFTKVDRSA AYAARWVAKSLVKNGYCKRCLVQVSYAIGVAEPISITLFDYGTSTKSQKELLAIVRKNFDLRPGMIVRDLNLKNPIYQKTSTYGHFGRDIFPWEQPKKLVE

[0102] 2. Yeast two-hybrid one-to-one verification of the interaction between LsBuEVE and LsSAMS

[0103] To further confirm the above screening results, the following one-to-one yeast two-hybrid verification experiment was conducted.

[0104] (1) Vector construction: The complete ORF of the LsSAMS gene to be identified was cloned into the prey vector pGADT7 to construct the AD-LsSAMS plasmid.

[0105] (2) Preparation of competent yeast cells: Yeast strain AH109 was streaked onto a YPDA plate for activation. Single colonies were then picked and cultured overnight in liquid YPDA medium at 30°C with shaking. The cells were collected, washed with ddH2O, and resuspended in 1 M LiAc. Simultaneously, salmon sperm carrier DNA was subjected to two pre-denaturation treatments at 95°C for 5 min each. The LiAc-treated cells were collected by centrifugation and the following solutions were added sequentially: 200 μL 1 M LiAc, 690 μL 60% PEG3350, 100 μL 1 M DTT, and 10 μL 10 mg / mL denatured carrier DNA. After thorough vortexing, the mixture was dispensed into 1.5 mL EP tubes, 200 μL per tube, which were the competent yeast cells.

[0106] (3) Co-transformation: Add 5 μL each of co-transformation plasmids BD-LsBuEVE and AD-LsSAMS to 200 μL of competent cells and vortex to mix. Three control groups were set up at the same time. BD-LsBuEVE + AD-T7 (pGADT7 empty vector) and BD-LsSAMS + AD-T7 were used as negative self-activation controls; AD-T7-T (expressing SV40 large T antigen) + BD-53 (expressing p53 protein) were used as positive controls.

[0107] (4) Transformation and screening: The above mixture was heat-shocked in a 42℃ water bath for 30 minutes, with gentle turning and mixing every 10 minutes. After heat shock, the EP tubes were immediately transferred to ice and allowed to stand for 10-15 minutes. The transformed mixture was then evenly spread on SD / -Leu / -Trp (dual deficiency) deficient medium plates and incubated at 30℃ for 3-5 days to screen for cotransformants.

[0108] (5) Mutual verification: Positive clones were picked from the two-deficient plates, resuspended, and serially diluted 10-fold (original solution, 10... -1 10 -2 10 -3 The samples were spotted onto SD / -Leu / -Trp (two-deficient) and SD / -Leu / -Trp / -His (three-deficient) malformation medium plates and incubated upside down at 30°C for 3 days.

[0109] The results are as follows Figure 5 As shown, yeast co-transformed with BD-LsBuEVE and AD-LsSAMS grew normally at all dilutions on double-depleted plates. On strictly triple-depleted plates, this group of yeasts also grew normally, with colony growth decreasing with increasing dilution, indicating a dose-dependent effect. All negative control groups did not grow on triple-depleted plates, while the positive control groups grew normally at all dilutions on triple-depleted plates. These results confirm the specific interaction between LsBuEVE and LsSAMS in yeast.

[0110] 3. The impact of silencing LsSAMS on RSV replication

[0111] To investigate the effect of the interacting protein LsSAMS on RSV replication, this embodiment silences LsSAMS using RNA interference technology. Specifically, following the aforementioned dsRNA synthesis and microinjection procedures, a specific dsRNA (dsLsSAMS) was designed and synthesized targeting the coding region sequence of the LsSAMS gene (SEQ ID NO.16). The primer sequences are shown in SEQ ID NO.18 and SEQ ID NO.19 in Table 6.

[0112] Subsequently, dsLsSAMS and crude RSV extract were mixed and injected into second-instar non-toxic planthoppers (experimental group), while dsGFP+RSV crude extract was used as a negative control group. The planthoppers were then reared on healthy rice plants. Samples were collected on day 6 post-injection (6 dpi), and the transcriptional levels of LsSAMS and RSV-NP genes were detected by RT-qPCR. The quantitative primers for LsSAMS are shown in SEQ ID NO.20 and SEQ ID NO.21 in Table 6.

[0113] Table 6 Primers for dsRNA synthesis and RT-qPCR quantification of LsSAMS

[0114]

[0115] The results are as follows Figure 6 As shown, compared with the control group, the transcriptional levels of both LsSAMS and RSV-NP were significantly reduced in the experimental group. This result indicates that the expression of the interfering interaction protein LsSAMS can inhibit RSV replication, and its phenotype is consistent with that of silenced LsBuEVE.

[0116] Example 5: Effect of silencing LsBuEVE on LsSAMS expression levels

[0117] To further investigate the regulatory relationship between LsBuEVE and LsSAMS, in this embodiment, dsLsBuEVE (experimental group) or dsGFP (control group) was mixed with crude RSV extract and injected into non-toxic planthoppers, using the same method as before.

[0118] Samples were collected on day 6 post-injection (6 dpi), and the expression level of LsSAMS protein in the planthopper was detected by Western blotting.

[0119] The results are as follows Figure 7 As shown, compared with the control group, the protein expression levels of both LsBuEVE and LsSAMS were significantly decreased in the LsBuEVE silencing experimental group. This result demonstrates that LsBuEVE can positively regulate the expression of its interacting protein LsSAMS, thereby participating in promoting RSV replication.

[0120] In summary, this invention provides the first identification of a transcriptionally active endogenous Bunyavirus element, LsBuEVE, in the rice planthopper and confirms it as a key host factor for the replication and transmission of rice stripe virus (RSV) within the vector insect. Specific silencing of LsBuEVE using RNAi technology significantly inhibits virus acquisition and accumulation in the planthopper. Mechanistic studies show that LsBuEVE synergistically promotes RSV proliferation by directly interacting with and upregulating the expression of host S-adenosylmethionine synthase (LsSAMS). This invention provides a novel mechanism for elucidating the regulation of virus transmission by endogenous viral elements and offers a new molecular target for the green control of rice viral diseases.

Claims

1. An endogenous Bunyavirus element LsBuEVE for the gray planthopper, characterized in that, The genome of the LsBuEVE contains a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence that has at least 90% homology with the sequence shown in SEQ ID NO.

1.

2. The LsBuEVE according to claim 1, characterized in that, The transcript corresponding to LsBuEVE contains a complete open reading frame, and the amino acid sequence encoded by the open reading frame is shown in SEQ ID NO.

3.

3. The LsBuEVE according to claim 2, characterized in that, The nucleotide sequence of the open reading frame is as shown in SEQ ID NO.2, or a nucleotide sequence having at least 90% homology with SEQ ID NO.2 and encoding an amino acid as shown in SEQ ID NO.

3.

4. A method for targeting and silencing the dsRNA of LsBuEVE according to any one of claims 1-3, characterized in that, The target sequence of the dsRNA is located within the nucleotide sequence region shown in SEQ ID NO.

2.

5. The dsRNA according to claim 4, characterized in that, The dsRNA was synthesized in vitro via transcription using the positive-sense primer shown in SEQ ID NO.4 and the antisense primer shown in SEQ ID NO.

5.

6. A biological agent for blocking the transmission of rice viruses, characterized in that, It contains the dsRNA as described in claim 4 or 5, and an agriculturally acceptable vector or adjuvant.

7. The use of LsBuEVE according to any one of claims 1-3, dsRNA according to claim 4 or 5, or the biological agent according to claim 6 in the preparation of products for blocking the spread of rice viruses.

8. The application according to claim 7, characterized in that, The product blocks the transmission of rice viruses, including by inhibiting the expression of LsBuEVE in planthoppers, thereby reducing the acquisition rate of rice viruses by planthoppers or inhibiting the replication and accumulation of rice viruses in planthoppers.

9. The application according to claim 8, characterized in that, The rice virus in question is rice stripe virus.

10. A method for blocking the transmission of rice viruses, characterized in that, This includes injecting and / or feeding the planthopper with the dsRNA of claim 4 or 5, or the biological agent of claim 6, or applying the biological agent of claim 6 to rice plants or the cultivation environment.

Images