Method for marking positive single-stranded RNA virus replication site in plant living cell

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a method for marking a positive single-stranded RNA (Ribonucleic Acid) virus replication site in a plant living cell, which comprises the following steps: by taking pAB1677 plasmid as a template, amplifying dCas13BT3 by utilizing primers shown in SEQ ID NO.1 and SEQ ID NO.2, and connecting the dCas13BT3 into a fluorescent protein expression vector to obtain a recombinant vector; preparing gRNA of a target positive single-stranded RNA virus negative strand; and co-transferring the recombinant vector, the gRNA targeting the negative strand of the positive single-stranded RNA virus and the RNA of the positive single-stranded RNA virus into a plant, culturing, and observing the fluorescent protein expression position which is the replication site of the positive single-stranded RNA virus. According to the present invention, by designing the gRNA targeting the PnMV negative chain RNA and using the fusion of dCas13BT3 and the fluorescent protein, the fluorescence signal is accurately enriched in the virus replication complex, such that the background noise problem caused by the positive chain RNA targeting is effectively overcome;

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a method for marking the replication sites of positive single-stranded RNA viruses in living plant cells. Background Technology

[0002] In the fields of agricultural production and plant pathology research, plant viruses, especially positive single-stranded RNA viruses such as poinsettia mosaic virus, pose a particularly prominent threat. These viruses, with their rapid replication capabilities and wide host range, often lead to reduced crop yields and decreased quality, posing a persistent threat to food security and the agricultural economy. Therefore, a thorough understanding of their replication mechanisms—especially the replication sites within host cells, such as the assembly and dynamic changes of the replication complex—is a core prerequisite for developing effective control strategies.

[0003] However, current research techniques for plant virus replication sites have significant limitations, hindering in-depth exploration of the dynamic replication process of viruses. Among traditional detection methods, electron microscopy, while providing ultrastructural information on virus replication-related structures, relies on cell fixation and sectioning, capturing only static, transient states. Furthermore, it is complex, costly, and cannot reflect the real-time dynamics of the replication process. While immunofluorescence technology visualizes specific viral proteins through antibody labeling, it also requires cell or tissue fixation and permeabilization, which not only destroys cellular physiological activity but may also lead to results bias due to structural deformation during fixation, making continuous observation of viral replication within living cells difficult.

[0004] During replication of viruses like PnMV (positive ssRNA), the positive-sense viral RNA enters the cell and directly serves as mRNA for translation of viral replicase. The replicase uses the positive RNA as a template to synthesize the negative-sense RNA. Then, using the negative RNA as a template, it synthesizes a large amount of progeny positive RNA. The replication complex contains a double-stranded RNA intermediate and a newly formed RNA strand. Existing nucleic acid-targeting labeling methods mostly focus on the positive-sense viral RNA. However, positive-sense RNA not only participates in replication within the host cell but also performs multiple functions such as translation. Free positive-sense RNA easily creates background noise, severely interfering with the specific recognition of replication sites. This background interference makes it difficult for researchers to accurately distinguish the viral replication complex from free viral nucleic acid, greatly affecting the accuracy of replication mechanism analysis.

[0005] Against this backdrop, developing a non-invasive technique capable of real-time, specific labeling of positive single-stranded RNA virus replication sites within living plant cells has become an urgent need to overcome existing research bottlenecks. This technique must simultaneously overcome background noise interference and enable dynamic in vivo observation, thereby providing a research tool for elucidating the molecular mechanisms of viral replication and developing novel antiviral strategies. Summary of the Invention

[0006] The purpose of this invention is to provide a real-time imaging method for the replication site of positive single-stranded RNA viruses in living plant cells based on CRISPR-dCas13BT3. This invention achieves highly specific labeling by targeting the negative strand RNA of the virus, overcoming the limitation of traditional techniques that cannot be observed in vivo, and providing a non-invasive dynamic tracking tool for the study of the replication mechanism of positive single-stranded RNA viruses.

[0007] The technical solution adopted in this invention is: This invention provides a method for labeling the replication site of positive single-stranded RNA viruses in living plant cells, comprising the following steps: Using pAB1677 plasmid as a template, dCas13BT3 was amplified using primers shown in SEQ ID NO.1 and SEQ ID NO.2. dCas13BT3 was then ligated into a fluorescent protein expression vector to obtain a recombinant vector. The base sequence of dCas13BT3 is shown in SEQ ID NO.3. Prepare gRNA targeting the negative strand of a positive single-stranded RNA virus, wherein the base sequence of the gRNA is shown in SEQ ID NO.4; The recombinant vector, gRNA, and RNA of the positive single-stranded RNA virus were co-transformed into plants, cultured, and the location of fluorescent protein expression was observed. The location of fluorescent protein expression is the replication site of the positive single-stranded RNA virus.

[0008] The positive single-stranded RNA virus is poinsettia leaf virus.

[0009] Preferably, the fluorescent protein expression vector is the peqFP611 vector.

[0010] Preferably, the ratio of the recombinant vector, the gRNA targeting the negative strand of the positive single-stranded RNA virus, and the RNA of the positive single-stranded RNA virus is 1 mol: 1 × 10⁻⁶. 6 g~5×10 6 g: 1×10 6 g~5×10 6 g.

[0011] Preferably, the process for preparing the gRNA targeting the negative strand of the positive single-stranded RNA virus is as follows: Synthesize the primers shown in SEQ ID NO.8 and SEQ ID NO.9; The primers were mixed and then annealed for extension to obtain a double-stranded DNA template. The double-stranded DNA template was transcribed in vitro to obtain the gRNA that targets the negative strand of the positive single-stranded RNA virus.

[0012] Preferably, the conditions for the annealing extension are: Denature at 95°C for 2 minutes, then extend at 72°C for 10 minutes.

[0013] Preferably, the process of linking dCas13BT3 into the fluorescent protein expression vector is as follows: The fluorescent protein expression vector was digested with enzymes to prepare a linearized fluorescent protein expression vector; dCas13BT3 was cloned into a fluorescent protein expression vector via homologous recombination.

[0014] Preferably, the restriction endonuclease used to digest the fluorescent protein expression vector is BamHI.

[0015] Preferably, the enzyme digestion condition is a reaction at 37°C for 2 hours.

[0016] Preferably, the homologous recombination is performed under the condition of incubation at 50°C for 15 minutes.

[0017] The present invention also provides a real-time imaging system for virus replication sites, comprising: The dCas13bt3 fluorescent protein fusion expression vector is driven by the 35S promoter. The gRNA targeting viral negative-strand RNA includes a spacer region specifically binding to the target viral negative strand, a T7 promoter, and a Cas13BT3 scaffold sequence; the gRNA works synergistically with dCas13BT3-RFP to specifically label the replication complex of a positive single-stranded RNA virus in living plant cells.

[0018] A third aspect of the present invention provides an application of the method in any of the following: Real-time dynamic tracking of the virus replication process within living plant cells.

[0019] Screening for compounds or genes that inhibit viral replication.

[0020] To elucidate the interaction mechanism between viral replication complexes and host factors.

[0021] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for labeling the replication site of positive single-stranded RNA viruses in living plant cells. The steps are as follows: using pAB1677 plasmid as a template, dCas13BT3 is amplified using primers shown in SEQ ID NO.1 and SEQ ID NO.2; dCas13BT3 is ligated into a fluorescent protein expression vector to obtain a recombinant vector; the base sequence of dCas13BT3 is shown in SEQ ID NO.3; gRNA targeting the negative strand of the positive single-stranded RNA virus is prepared, the base sequence of which is shown in SEQ ID NO.4; the recombinant vector, gRNA, and RNA of the positive single-stranded RNA virus are co-transformed into plants, cultured, and the location of fluorescent protein expression is observed. The location of fluorescent protein expression is the replication site of the positive single-stranded RNA virus. This invention achieves, for the first time, non-invasive real-time imaging of the replication site of positive single-stranded RNA viruses in living plant cells, overcoming the limitation of electron microscopy / immunofluorescence requiring sample fixation, and achieving a breakthrough in in vivo observation. Furthermore, through the core innovative design of targeting the negative strand RNA of the positive single-stranded RNA virus rather than the positive strand, background noise from free positive strand RNA is eliminated, achieving precise localization of the replication complex. Furthermore, the method described in this invention has strong universality and can be extended to other positive single-stranded viruses such as TMV, PVX, PnMV, PSTVd, etc. Attached Figure Description

[0022] Figure 1 The image shows the spectrum of the dCas13BT3-RFP recombinant vector prepared in this invention.

[0023] Figure 2 Image of protoplasts prepared in this invention Figure 3 The image shows the fluorescence expression of the dCas13BT3-RFP recombinant vector prepared in this invention in tobacco protoplasts.

[0024] Figure 4 This diagram illustrates how the CRISPR-dCas13BT3 system is used in this invention to mark the replication sites of the PnMV virus. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0026] The inventive concept of this invention is as follows: The existing technology has the following drawbacks when labeling the replication sites of positive single-stranded RNA viruses (PnMV): (1) Live observation is not possible; (2) Background noise interference is large; (3) Operation is complicated and costly.

[0027] To address the above deficiencies, this invention proposes a CRISPR-dCas13BT3-mediated method for marking the replication site of the plant in vivo positive single-stranded RNA virus PnMV. This method specifically solves the problems existing in the prior art, as detailed below: (1) The problem that existing detection technologies cannot achieve real-time observation of live cells: Traditional virus replication site detection technologies such as electron microscopy and immunofluorescence rely on cell or tissue fixation and processing, which can only obtain static information and cannot reflect the dynamic process of virus replication. This invention, through a visualization system constructed based on dCas13BT3-RFP and gRNA targeting PnMV, has for the first time achieved real-time observation of the virus PnMV replication site in plant live cells, breaking through the limitations of immobilization technology.

[0028] (2) Background noise interference exists in positive-sense RNA targeting labeling: Existing nucleic acid targeting labeling methods mostly target viral positive-sense RNA, which has multiple functions such as translation in host cells. Free positive-sense RNA is prone to background noise, making it impossible to accurately identify replication sites. This invention designs gRNA that targets viral negative-sense RNA, taking advantage of the characteristic that negative-sense RNA is mainly present in the replication complex, so that the fluorescence signal is accurately enriched in the replication complex, effectively overcoming background noise interference and improving the specificity of labeling.

[0029] (3) Problems of complex operation and insufficient accuracy of existing technologies: Traditional technologies such as electron microscopy are complex and costly, and immunofluorescence may lead to deviations in results due to the fixation process. The technical solution of the present invention is relatively simple. Through specific targeting and fluorescent labeling, it can more accurately locate the viral replication site, reduce errors caused by technical operation, and provide more reliable results for research.

[0030] The resolution of these technical problems laid an important foundation for in-depth research on the replication mechanism of the PnMV virus and promoted technological progress in related fields.

[0031] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0032] The list of abbreviations for this invention is shown in Table 1.

[0033] Table 1. List of abbreviations for this invention 1. The experimental materials involved in this invention.

[0034] (1) Strains and vectors.

[0035] Escherichia coli strain JM109, used for the amplification and preservation of all plasmids.

[0036] Vector plasmids: pAB1677, REPAIR.t3, Addgene#103849, containing the catalytically inactivated Cas13BT3 coding sequence; peqFP611 vector, containing the RFP fluorescent tag and CaMV 35S promoter.

[0037] (2) Enzymes and reagents.

[0038] Cloning reagents: In-Fusion HD cloning kit, purchased from Takara Bio, JP; restriction endonuclease BamHI; Taq DNA polymerase, purchased from Thermo Fisher Scientific, USA.

[0039] Nucleic acid purification reagents: QIAquick PCR purification kit (Qiagen); GeneJET plasmid mini-prep kit (Thermo Fisher Scientific, USA); TRIzol reagent (Invitrogen); PureLink™ RNA mini kit (Promega).

[0040] Transcription reagent: RiboMAX™ Large Scale RNA Production System-T7, purchased from Promega.

[0041] Reagents related to plant protoplasts: Cellulase R10, with an enzyme activity of 10000 U / g, purchased from Yakult, JP; Isolase R10, with an enzyme activity of 3000 U / g, purchased from Yakult, JP; PEG 4000; Mannitol, KCl, CaCl2, MES and other chemical reagents were all of analytical grade.

[0042] (3) Plant materials.

[0043] Ben's tobacco ( N.benthamiana Plants: Cultured in an artificial climate chamber at 22°C with a photoperiod of 16 hours light / 8 hours dark. Fully expanded leaves from 4-week-old plants were selected for protoplast isolation and virus inoculation.

[0044] (4) Virus materials Poinsettia leaf virus: It was propagated in Nicotiana benthamiana by mechanical friction inoculation. Systemically infected leaves were collected 4 weeks after inoculation, and total RNA was extracted as RNA template for PnMV detection.

[0045] 2. The solution formulation involved in this invention.

[0046] (1) Enzymatic hydrolysate: containing 1.5% cellulase R10, 0.4% dissociative enzyme R10, 0.4M mannitol, 20mM MKCl, 10mM CaCl2, 20mM MES, and 0.1% BSA.

[0047] (2) W5 solution: 154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES, pH 5.7.

[0048] (3) MMG buffer: 4mM MES pH5.7, 0.4M mannitol, 15mM MgCl2.

[0049] (4) PEG solution: 40% PEG 4000, 0.2M mannitol, 100mM CaCl2.

[0050] (5) WI buffer: 0.5M mannitol, 20mM KCl, 4mM MES pH5.7.

[0051] Example 1 A method for labeling the replication site of positive single-stranded RNA viruses in living plant cells is as follows: 1. Construction of core vector: Using pAB1677 plasmid as template, dCas13BT3 was amplified using primers shown in SEQ ID NO.1 and SEQ ID NO.2. dCas13BT3 was then ligated into a fluorescent protein expression vector to obtain the recombinant vector pdCas13BT3-RFP.

[0052] (1) Amplification of the target fragment: Using pAB1677 plasmid as a template, the catalytically inactivated Cas13BT3 coding sequence dCas13BT3 was amplified by PCR using specific primers F and R, ensuring that the amplified fragment contained homologous arm sequences complementary to the BamHI restriction sites of the peqFP611 vector at both ends. The base sequences of specific primers F and R are shown in SEQ ID NO.1 and SEQ ID NO.2; the base sequence of dCas13BT3 is shown in SEQ ID NO.3.

[0053] SEQ ID NO.1: 5'-GGACTCTAGAGGATCATGGCCCAGGTGTCCAAGCAGACC-3'.

[0054] SEQ ID NO. 2: 5'-TTCATCCCGGGGATCCTACCGCCTGACCCTCCC-3'.

[0055] SEQ ID NO.3:

[0056] (2) Vector digestion: The fluorescent protein expression vector peqFP611 was digested with the restriction endonuclease BamHI. After reacting at 37°C for 2 h, the linearized peqFP611 vector fragment was recovered by agarose gel electrophoresis.

[0057] (3) Recombination ligation: Using the In-Fusion HD cloning kit, the dCas13BT3 fragment was ligated with the linearized peqFP611 vector and incubated at 50°C for 15 min to insert dCas13BT3 into the BamHI site to construct a recombinant vector whose expression is regulated by the CaMV35S promoter.

[0058] (4) Plasmid verification: The ligation product was transformed into E. coli JM109 competent cells. Single colonies were picked and cultured, and plasmids were extracted using the GeneJET plasmid miniprep kit. The correctness of the recombinant plasmid construction was verified by PCR identification and sequencing. The positive recombinant vector was named pdCas13BT3-RFP. The diagram is shown below. Figure 1 .

[0059] (5) Verification of fluorescent expression of recombinant vector.

[0060] 1) Preparation of protoplasts.

[0061] Take leaves from 6-leaf-aged *Tobacco Benedict*, cut them into 1mm wide strips with a fresh blade, and place them in pre-warmed enzymatic hydrolysate. The hydrolysate needs to be preheated at 55°C for 10 min to inactivate DNase, and then filtered through a 0.45μm syringe filter for sterilization. Mix the leaf strips with the hydrolysate, incubate under vacuum in the dark for 30 min, and then incubate at room temperature for 3 h for enzymatic hydrolysis. Filter the hydrolysate through a 75μm nylon mesh to remove residue, collect the filtrate in a centrifuge tube, centrifuge at 100g for 2 min, and discard the supernatant. Resuspend the precipitate in W5 solution, repeat centrifugation and washing once, and finally adjust the protoplast concentration to 2×10⁻⁶ with W5 solution. 5 Cells / mL were prepared for use. Protoplast samples were placed in a dedicated confocal culture dish and observed using a Leica SP8 confocal laser scanning microscope. Chlorophyll autofluorescence was detected at a wavelength of 633 nm. The protoplasts prepared in this invention are spherical with visible chloroplasts inside, indicating that the prepared protoplasts are intact and undamaged. Results are shown below. Figure 2 .

[0062] 2) Transformation of recombinant vectors.

[0063] Resuspend the protoplasts in MMG buffer, maintaining a concentration of 2 × 10⁻⁶. 5cells / mL. Take 50 μL of protoplast suspension, add 2 pmol pdCas13BT3-RFP and mix gently. Then add 55 μL of freshly prepared PEG solution and incubate at room temperature for 10 min. Add 220 μL of W5 solution to terminate the reaction, centrifuge at 100g for 2 min, discard the supernatant, and then culture. The culture process is as follows:

[0064] The precipitate was resuspended in WI buffer and incubated at room temperature in the dark for 48 hours. After 48 hours of incubation, protoplast samples were placed in a confocal microscopy dish to observe the RFP fluorescence signal. Observation was performed using a Leica SP8 confocal laser scanning microscope. Excitation wavelength settings: RFP signal 561 nm; chlorophyll autofluorescence detection wavelength 633 nm. Figure 3 As shown, a red fluorescent signal was found in the protoplast, and the signal was randomly distributed in the protoplast, indicating that the dCas13BT3-RFP was successfully constructed.

[0065] 2. Preparation of gRNA targeting the negative strand of a positive single-stranded RNA virus.

[0066] (1) Target sequence design.

[0067] A target-specific spacer sequence was designed targeting the conserved region of the negative-strand RNA of the viral PnMV. This spacer sequence was then fused with the T7 promoter and the Cas13BT3 backbone sequence to form a complete gRNA sequence. The gRNA sequence targeting the PnMV negative strand is shown in SEQ ID NO.4.

[0068] SEQ ID NO.4: 5'-TAATACGACTCACTATAGGGAACATGGTGTCCTCTTGGTGTTTTAGTACTCTGG-3'.

[0069] Structural description of the gRNA sequence targeting the negative strand of PnMV: The T7 promoter, located at positions 1 to 20, is shown in SEQ ID NO. 5 and serves to provide a start signal for subsequent in vitro transcription. The PnMV negative strand targeting region, located at positions 21 to 40, is shown in SEQ ID NO. 6. This region is complementary to the conserved region of the PnMV negative strand RNA and can specifically recognize and bind to the target sequence, ensuring the targeting accuracy of the gRNA. The Cas13BT3 scaffold region, located at positions 41 to 54, is shown in SEQ ID NO. 7. This region can bind to the dCas13BT3 protein to form a functional complex, ensuring that the gRNA can carry the dCas13BT3-fluorescent protein fusion to the replication site of the PnMV negative strand RNA.

[0070] SEQ ID NO. 5: TAATACGACTCACTATAGGG.

[0071] SEQ ID NO. 6: AACATGGTGTCCTCTGGTG.

[0072] SEQ ID NO.7: TTTTAGTACTCTGG.

[0073] (2) DNA template preparation.

[0074] The base sequence of the forward primer for synthesizing gRNA targeting the negative strand of PnMV is shown in SEQ ID NO.8, and the base sequence of the reverse primer is shown in SEQ ID NO.9.

[0075] SEQ ID NO.8: 5'-TAATACGACTCACTATAGGGAACATGGTGTCCTCTTGGTGT-3'.

[0076] SEQ ID NO.9: 5'-CCAGAGTACTAAAAACACCAAGAGGACACCATGTT-3'.

[0077] The forward and reverse primers were mixed in equal amounts and annealed and extended using Taq DNA polymerase in the PCR reaction system: denaturation at 95°C for 2 min, extension at 72°C for 10 min, to form a double-stranded DNA template.

[0078] (3) In vitro transcription of gRNA.

[0079] Double-stranded DNA template was purified using the QIAquick PCR purification kit. Using this template, in vitro transcription was performed using the RiboMAX™ T7 RNA synthesis system. The transcript was extracted with phenol:chloroform, purified by ethanol precipitation, quantified using NanoDrop 2000, and the concentration was adjusted to 2 μg / μL. The transcript was then stored at -80°C for later use.

[0080] 3. The recombinant vector, the gRNA targeting the negative strand of the positive single-stranded RNA virus, and the RNA of the positive single-stranded RNA virus are co-transformed into plant cells, cultured, and the location of fluorescent protein expression is observed. The location of fluorescent protein expression is the replication site of the positive single-stranded RNA virus.

[0081] This invention utilizes the CRISPR-dCas13bt3 system to observe the replication sites of the PnMV virus. The specific process is as follows: (1) Preparation of transfection system: Resuspend protoplasts in MMG buffer and maintain a concentration of 2×10⁻⁶. 5cells / mL. Take 50 μL of protoplast suspension, add 2 pmol dCas13BT3-RFP recombinant vector, 2 μg / μL 1 μL PnMV RNA, 1 μL 2 μg / μL in vitro transcribed gRNA targeting negative strand RNA, mix gently, and use the treatment without gRNA as a blank control.

[0082] (2) PEG-mediated transfection: Add 55 μL of freshly prepared PEG solution to the above mixture and incubate at room temperature for 10 min. Terminate the reaction by adding 220 μL of W5 solution, centrifuge at 100 g for 2 min, discard the supernatant, and culture. The culture process is as follows:

[0083] The precipitate was resuspended in WI buffer and incubated at room temperature in the dark for 48 hours. After 48 hours of incubation, protoplast samples were placed in a confocal microscopy dish to observe the distribution of RFP fluorescence signals and record the fluorescence enrichment at viral replication sites. Observation was performed using a Leica SP8 confocal laser scanning microscope. Excitation wavelength settings: RFP signal 561 nm; chlorophyll autofluorescence detection wavelength 633 nm.

[0084] Comparative Example 1 A method for labeling the replication site of positive-stranded single-stranded RNA viruses in plant living cells. This comparative example uses gRNA designed from the conserved region of the positive-stranded RNA of PnMV virus to label the replication site, as detailed below: (1) The relevant information of gRNA designed targeting the conserved region of the positive-sense RNA of PnMV virus is as follows: The complete gRNA base sequence is shown in SEQ ID NO.10.

[0085] SEQ ID NO.10: 5'-TAATACGACTCACTATAGGGTTCGTACCGTGAAACAGTGGTTTTTAGTACTCTGG-3'.

[0086] Structural Description: The T7 promoter, located at positions 1 to 20, has a sequence identical to SEQ ID NO.5. Its function is to provide a start signal for subsequent in vitro transcription, ensuring that gRNA is effectively synthesized in the in vitro transcription system.

[0087] The PnMV positive strand targeting region, from 21st to 40th nt, has the base sequence shown in SEQ ID NO. 11. This region is complementary to the conserved region of the PnMV positive strand RNA, enabling it to specifically recognize and bind to the positive strand target sequence, ensuring the targeting specificity of the gRNA to the positive strand RNA. SEQ ID NO. 11: TTCGTACCGTGAAACAGTGG.

[0088] The Cas13BT3 scaffold region, located at positions 41 to 54, has a sequence identical to SEQ ID NO.7. It can bind to the dCas13BT3 protein to form a functional complex, guiding the dCas13BT3-fluorescent protein fusion to precisely locate the replication or translation site of the PnMV positive-sense RNA.

[0089] (2) DNA template preparation.

[0090] The base sequence of the forward primer for synthesizing the gRNA targeting the positive strand of PnMV is shown in SEQ ID NO.12, and the base sequence of the reverse primer is shown in SEQ ID NO.13.

[0091] SEQ ID NO.12: 5'-TAATACGACTCACTATAGGGTTCGTACCGTGAAACAGTGG-3'.

[0092] SEQ ID NO.13: 5'-CCAGAGTACTAAAAACCACTGTTTCACGGTACGAAC-3'.

[0093] In this comparative example, gRNA was designed using a conserved region of the positive-sense RNA of the PnMV virus, and all other conditions were exactly the same as in Example 1.

[0094] The results of Example 1 and Comparative Example 1 were analyzed, and the results are shown in the figure. Figure 4 In the blank control without gRNA, the red fluorescence signal of pdCas13BT3-RFP was randomly distributed, indicating that without gRNA guidance, pdCas13BT3-RFP is randomly distributed within the cell. After adding gRNA targeting the positive strand of PnMV RNA, the red fluorescence signal of pdCas13BT3-RFP was distributed on chloroplasts and in the interchloroplast space. Since positive strand RNA has multiple functions in the host cell, including replication, translation, and regulation of host cell functions, the chloroplast and interchloroplast space may be sites of replication, translation, and regulation of host cell functions, making it impossible to accurately identify the replication site. However, after adding gRNA targeting the negative strand of PnMV RNA, the red fluorescence signal of pdCas13BT3-RFP was all localized to the chloroplast membrane. The negative strand RNA is mainly present in the replication complex, indicating that the replication site of PnMV is on the chloroplast membrane.

[0095] This invention provides a visualization method for targeting negative-strand RNA viruses based on dCas13BT3-GFP, achieving the following objectives: Real-time observation in vivo: Breaking through the limitations of existing technologies that rely on cell fixation, this technology enables real-time observation of the replication site of virus PnMV within living plant cells, providing a possibility for studying the dynamic process of virus PnMV replication.

[0096] Improving label specificity: By designing gRNAs that target the negative-sense RNA of viral PnMV, fluorescent signals are precisely enriched in the replication complex, overcoming the background noise problem caused by positive-sense targeting. This allows for precise differentiation between the viral replication complex and free viral nucleic acid, improving the accuracy of replication site identification. This is because negative-sense RNA exists only within the replication complex and does not participate in translation or packaging, making it a specific marker of the replication site. Positive-sense RNA is distributed throughout the cell, and targeting the positive strand may lead to background noise. This also avoids interference with the viral life cycle.

[0097] Provides non-invasive research tools: This method does not require destructive treatment of cells, providing a non-invasive tool for studying the viral PnMV replication mechanism, avoiding result bias caused by cell structure damage or processing, and more realistically reflecting the replication of the virus in the host cell.

[0098] Advancing the research on viral replication mechanisms: This research provides researchers with an efficient, precise, and non-invasive tool that helps elucidate the molecular mechanisms of viral PnMV replication, such as the formation of the replication complex and the interaction mechanisms between the virus and host cells. These research findings will lay a solid foundation for the development of novel antiviral drugs and prevention and control strategies, and have significant scientific and applied value.

[0099] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0100] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for labeling the replication site of a single-stranded RNA virus within living plant cells, characterized in that, The steps are as follows: Using pAB1677 plasmid as a template, dCas13BT3 was amplified using primers shown in SEQ ID NO.1 and SEQ ID NO.

2. dCas13BT3 was then ligated into a fluorescent protein expression vector to obtain a recombinant vector. The base sequence of dCas13BT3 is shown in SEQ ID NO.

3. Prepare gRNA targeting the negative strand of a positive single-stranded RNA virus, wherein the base sequence of the gRNA targeting the negative strand of the positive single-stranded RNA virus is shown in SEQ ID NO.4; The recombinant vector, gRNA targeting the negative strand of the positive single-stranded RNA virus, and RNA of the positive single-stranded RNA virus were co-transformed into plants, cultured, and the location of fluorescent protein expression was observed. The location of fluorescent protein expression is the replication site of the positive single-stranded RNA virus.

2. The method as described in claim 1, characterized in that, The positive single-stranded RNA virus is poinsettia leaf virus.

3. The method as described in claim 1, characterized in that, The fluorescent protein expression vector is the peqFP611 vector.

4. The method as described in claim 1, characterized in that, The ratio of the recombinant vector, gRNA targeting the negative strand of the positive single-stranded RNA virus, and RNA of the positive single-stranded RNA virus is 1 mol: 1 × 10⁻⁶. 6 g~5×10 6 g: 1×10 6 g~5×10 6 g.

5. The method as described in claim 1, characterized in that, The process for preparing the gRNA targeting the negative strand of the positive single-stranded RNA virus is as follows: Synthesize the primers shown in SEQ ID NO.8 and SEQ ID NO.9; The primers were mixed and then annealed for extension to obtain a double-stranded DNA template. The double-stranded DNA template was transcribed in vitro to obtain the gRNA that targets the negative strand of the positive single-stranded RNA virus.

6. The method as described in claim 5, characterized in that, The conditions for the annealing extension are: Denature at 95°C for 2 minutes, then extend at 72°C for 10 minutes.

7. The method as described in claim 1, characterized in that, The process of linking dCas13BT3 into the fluorescent protein expression vector is as follows: The fluorescent protein expression vector was digested with enzymes to prepare a linearized fluorescent protein expression vector; dCas13BT3 was cloned into a fluorescent protein expression vector via homologous recombination.

8. The method as described in claim 7, characterized in that, The restriction endonuclease used to digest the fluorescent protein expression vector is BamHI.

9. The method as described in claim 7, characterized in that, The enzyme digestion conditions were 37°C for 2 hours.

10. The method as described in claim 7, characterized in that, The conditions for homologous recombination were incubation at 50°C for 15 minutes.

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