Application of NDUFS1 protein or coding gene thereof in cultivation of antiviral plants

Abstract

The invention discloses application of NDUFS1 protein or a coding gene thereof in cultivation of antiviral plants. According to the invention, the NDUFS1 gene from potatoes is found to be capable of participating in infection of various viruses of potato Y viruses for the first time, and the silencing NDUFS1 can inhibit replication of the potato Y viruses and tobacco vein banding mosaic viruses; a gene editing technology is utilized, specific sgRNA is designed for nucleotide regions from the 221 site to the 242 site of a coding region of the potato StNDUFS1 gene (SEQ ID NO.3) for editing, a mutant potato plant with StNDUFS1 gene function deletion is obtained, and the plant has remarkable resistance to the potato Y virus. Therefore, the NDUFS1 protein can be used as a novel antiviral target, is used for cultivating plants resisting the potyvirus virus, and plays an important role in preventing and treating the potyvirus virus.

Description

Technical Field

[0001] This invention belongs to the field of genetic improvement of crop disease resistance. In particular, this invention relates to a new molecular target for breeding plants resistant to Potato Virus Y, the gene encoding the target, and a breeding method for creating virus-resistant plants by regulating the target. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Potatoes hold a vital strategic position in ensuring my country's food security; however, their production is severely threatened by viral diseases. There are more than 40 types of viruses that can infect potatoes, with the most serious being potato virus Y (PVY) and potato virus X (PVX). Tobacco vein mosaic virus (TVBMV) is a newly emerging virus on potatoes, and its harm is increasing year by year.

[0004] Planting disease-resistant varieties is the most economical and effective method for controlling crop viral diseases, but currently, resources of disease-resistant varieties are extremely scarce. With the development of modern bio-breeding technology, using gene editing technology to edit host susceptibility genes has become an effective way to cultivate virus-resistant plants, and screening new antiviral targets is of great significance for guiding the breeding of disease-resistant varieties.

[0005] Mitochondrial complex I, also known as NADH ubiquinone oxidoreductase, is the largest enzyme complex in the mitochondrial respiratory chain. It catalyzes the oxidation of NADH, transferring electrons to coenzyme Q10 and pumping protons to form a transmembrane electrochemical gradient, providing energy for ATP synthesis. NDUFS1, the largest subunit of mitochondrial complex I, plays a crucial role in maintaining the stability and enzyme activity of the complex. Deletion or mutation of NDUFS1 reduces the catalytic activity of mitochondrial complex I, disrupting NADH homeostasis. Infection with rice spur dwarf virus reduces the mRNA and protein levels of NDUFS1 in the vector insect brown planthopper, leading to decreased mitochondrial complex I activity, reduced ATP production, increased ROS accumulation, and induction of mitochondrial apoptosis, thereby promoting its own accumulation. Currently, the function of NDUFS1 in plant-virus interactions is unknown, and its application in the breeding of virus-resistant potato varieties remains unclear. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention is the first to discover that NDUFS1 is a target for breeding plants resistant to Potato Virus Y. Therefore, it provides an application of the NDUFS1 protein, its encoding gene, or its homologous gene in resistance to Potato Virus Y, and a method for preparing plants resistant to Potato Virus Y and its application.

[0007] The technical solution adopted in this invention is as follows: In a first aspect of the invention, there is provided the use of NDUFS1 protein as a target in screening or preparing products for improving plant resistance to Potato Virus Y; wherein the amino acid sequence of the NDUFS1 protein is as shown in SEQ ID NO.1 (StNDUFS1 amino acid sequence) and / or SEQ ID NO.2 (NbNDUFS1 amino acid sequence).

[0008] This invention is the first to discover that downregulating the expression or activity of this protein can significantly enhance plant resistance to Potato Virus Y. Therefore, this protein can serve as a key molecular target for screening or preparing products that enhance plant virus resistance, such as for designing gene silencing / editing tools.

[0009] In a second aspect of the invention, the application of the gene encoding the NDUFS1 protein in the breeding of plant varieties resistant to Potato Virus Y is provided. The amino acid sequence of the NDUFS1 protein is shown in SEQ ID NO.1 and / or SEQ ID NO.2; The encoding gene of the NDUFS1 protein is the nucleic acid molecule shown in SEQ ID NO.3 (StNDUFS1 nucleotide sequence) and / or SEQ ID NO.4 (NbNDUFS1 nucleotide sequence).

[0010] The application of this gene is reflected in the breeding of virus-resistant plant varieties. Specifically, it enhances plant resistance to Potato Virus Y by downregulating the expression or function of the gene encoding the NDUFS1 protein in plants. This provides a direct genetic target for disease resistance genetic improvement.

[0011] In a third aspect of the invention, a method for improving plant resistance to Potato Virus Y (PVY) viruses is provided, along with a method for preparing PVY-resistant potato plants and their application. The method includes the following steps: Downregulate the expression or activity of NDUFS1 protein in the target plant, wherein the amino acid sequence of the NDUFS1 protein is shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0012] In one or more embodiments of the present invention, the downregulation of the expression or activity of the NDUFS1 protein in the plant is achieved by silencing or knocking out the endogenous NDUFS1 gene in the plant.

[0013] Furthermore, the silencing is achieved through RNA interference technology; the knockout is achieved through gene editing technology. When gene editing technology is used, the target is designed to target the nucleotide region from position 221 to position 242 of the coding region of the StNDUFS1 gene shown in SEQ ID NO.3.

[0014] In a fourth aspect of the present invention, a method for preparing gene-edited plants resistant to Potato Virus Y is provided, the method comprising the following steps: Using gene editing technology, targeted mutations were made in specific regions of the NDUFS1 gene in the plant genome to obtain mutant plants with lost or reduced NDUFS1 protein function. The specific region is the nucleotide region from position 221 to position 242 of the coding region of the StNDUFS1 gene shown in SEQ ID NO.3.

[0015] In the above aspects of the present invention, the potato virus Y genus viruses include, but are not limited to, potato virus Y (Potatovirus Y). potato virus Y PVY) and / or tobacco vein mosaic virus (PVY) tobacco vein banding mosaic virus (TVBMV), the plants mentioned include, but are not limited to, potatoes and / or tobacco.

[0016] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Discovery of a novel antiviral target: the first time that a novel antiviral target has been elucidated NDUFS1 Its key role in the interaction between plant and potato virus Y provides a new direction for solving the problem of scarce crop germplasm resources for disease resistance breeding, establishing it as a direct target for disease resistance breeding.

[0017] (2) It provides a clear breeding path: It not only reveals the target, but also verifies the feasibility of obtaining resistance by manipulating the target in model plants and crops through technical paths such as gene silencing and gene editing, forming a complete technical chain from target discovery to variety creation.

[0018] (3) Broad-spectrum resistance potential: Experiments have shown that targeting NDUFS1 can not only inhibit the infection and replication of PVY, but is also effective against TVBMV of the same genus, indicating that this strategy may have broad-spectrum resistance to the potato virus Y genus and has broad application prospects.

[0019] (4) High technical operability: The gene silencing, gene editing and other technologies involved are all mature means in modern biological breeding, which are easy to promote and apply in different crops and accelerate the breeding process of disease-resistant varieties. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 Silence in Example 5.1 NDUFS1 Fluorescence images of leaves after PVY-GFP inoculation (left is control, right is silent plant).

[0022] Figure 2 Example 5.1 NDUFS1 Results of gene silencing efficiency testing.

[0023] Figure 3 Example 5.1 NDUFS1 The accumulation level of PVY CP in gene-silenced plants.

[0024] Figure 4 Example 5.1 NDUFS1 The accumulation level of PVY RNA in gene-silenced plants.

[0025] Figure 5 Silence in Example 5.1 NDUFS1 Fluorescence photographs of leaves inoculated with PVY-GFPrep.

[0026] Figure 6 Example 5.1 NDUFS1 Accumulation level of PVY-GFPrep in gene-silenced plants.

[0027] Figure 7 Example 5.1 NDUFS1 The level of accumulation of negative strand RNA in the genome of gene-silenced plants PVYrep.

[0028] Figure 8 Silence in Example 5.2 NDUFS1 Fluorescent photographs of leaves after inoculation with the TVBMV-GFP system.

[0029] Figure 9 Example 5.2 NDUFS1 Accumulation level of TVBMV CP in gene-silenced plants.

[0030] Figure 10 Silence in Example 5.2 NDUFS1 Fluorescence images of leaves after inoculation with TVBMV-GFPrep.

[0031] Figure 11 Example 5.2 NDUFS1 Accumulation level of TVBMV-GFPrep in gene-silenced plants.

[0032] Figure 12 Example 5.2 NDUFS1 The level of accumulation of negative strand RNA in the genome of gene-silenced plants TVBMVrep.

[0033] Figure 13 Edited in Example 5.3 NDUFS1 The fluorescence and CP accumulation levels of potato plants inoculated with PVY-GFP. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0036] As mentioned earlier, there is currently limited research on the function of the NDUFS1 protein in the process of plant virus infection.

[0037] In view of this, the present invention has conducted an in-depth study on the function of the StNDUFS1 protein derived from potato. The amino acid sequence of the StNDUFS1 protein is shown in SEQ ID NO.1; the amino acid sequence of the NbNDUFS1 protein is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the StNDUFS1 protein is shown in SEQ ID NO.3; and the nucleotide sequence of the gene encoding the NbNDUFS1 protein is shown in SEQ ID NO.4.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1: Vector Construction 1.1 Viral Infectivity Cloning Modification

[0040] Based on the existing TVBMV and PVY-GZ isolate sequences in the laboratory, specific primers were designed to amplify the full-length sequences of TVBMV-GFP and PVY-GFP and then ligated into the binary vector pCB301 to obtain pCB301-TVBMV-GFP and pCB301-PVY-GFP (the construction method can be found in the published literature Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain).

[0041] Based on this, to facilitate subsequent experimental observation, the 35S-mCherry-HDEL expression cassette was ligated into the pCB301-TVBMV-GFP and pCB301-PVY-GFP vectors. Nos later.

[0042] Mutations were introduced into the P3N-PIPO of TVBMV and PVY (e.g., introducing an early stop codon into the P3N-PIPO coding sequence of TVBMV and PVY to prematurely terminate their translation), resulting in pCB301-TVBMV-GFP replicons (pCB301-TVBMV-GFPrep) and pCB301-PVY-GFP replicons (pCB301-PVY-GFPrep) that cannot migrate between cells but can replicate normally (for the construction principle and method, please refer to the published literature Potato virus Y viral protein 6K1 inhibits the interaction between defense proteins during virus infection).

[0043] 1.2 Construction of virus-induced gene silencing vectors

[0044] For silence NbNDUFS1 Genes, Utilization rTaq DNA polymerase amplification of *Nycium benzoate* cDNA Should A specific fragment of the gene was extracted and ligated into the TRV silencing vector pTRV2 to obtain the recombinant plasmid pTRV2-NbNDUFS1. The empty vector pTRV2 served as a control.

[0045] 1.3 Construction of gene editing vectors

[0046] To knock out the StNDUFS1 gene in potatoes, a gene editing vector based on the CRISPR / Cas9 system was constructed.

[0047] sgRNA design and synthesis: Specific sgRNAs were designed targeting the potato StNDUFS1 gene sequence (SEQ ID NO.3). Nucleotides 221 to 242 were designed as specific sgRNA target sites, specifically: 5'-GTACCCGGTCAAAATCCCTA-3' (SEQ ID NO.5), and cloned into the sgRNA expression backbone; Vector assembly: The above sgRNA expression cassette was inserted into the plant binary expression vector pNK2 containing the Cas9 protein-coding gene and hygromycin resistance selection marker using Golden Gate assembly technology; Obtaining recombinant plasmids: The recombinant plasmid that was correctly sequenced was named pNK2-StNDUFS1-sgRNA and used for subsequent genetic transformation.

[0048] Example 2: Virus inoculation, obtaining gene-silenced plants, and transient protein expression

[0049] Common methods for viral inoculation include Agrobacterium tumefaciens infiltration inoculation and friction inoculation. In the Agrobacterium tumefaciens infiltration inoculation experiment, infectious viral clones (pCB301-TVBMV-GFP, pCB301-PVY-GFP), viral replicons (pCB301-TVBMV-GFPrep, pCB301-PVY-GFPrep), and gene silencing vectors (pTRV2-NbNDUFS1 and its empty vector control pTRV2) were transformed into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method. Single colonies were picked and placed in LB liquid containing 50 μg / mL kanamycin and 100 μg / mL rifampin, and cultured overnight in a horizontal shaker at 28°C. The overnight culture was centrifuged at 6000 rpm for 3 min to enrich the bacterial cells, and then resuspended in resuspension buffer (10 mM MES [pH 5.6], 10 mM MgCl2, and 200 uM AS) and incubated at 28°C for 3 h. For invasive clones, adjust the bacterial concentration to OD. 600 =0.5, inoculate tobacco of appropriate age; for viral replicons, adjust the bacterial concentration to OD0.5. 600 =0.2, appropriate age for tobacco vaccination.

[0050] In the friction inoculation experiment, diseased leaves were collected and ground with phosphate buffer at a ratio of 1:1 (w / v) to obtain a crude virus extract. The extract was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was collected for later use. A small amount of carborundum was sprinkled on the upper part of the plant to be grafted to create micro-wounds. The leaves of the plant to be grafted were then rubbed in one direction to unfold the leaves.

[0051] For transient expression experiments, each plasmid was transformed into Agrobacterium GV3101 strain and cultured according to the method described above. In the immunoprecipitation and transient expression experiments, the final concentration of single bacteria was adjusted to OD0.05. 600 =0.35 (immunoprecipitation assay, luciferase assay, and colocalization assay) or OD 600 =0.2 (bimolecular fluorescence complementation experiment), mix with the RNA silencing repressor pBin-P19 bacterial solution and inoculate into plants of appropriate age.

[0052] Example 3: Detection of gene silencing efficiency and viral RNA accumulation level

[0053] Total RNA was extracted from plants according to the instructions for use with TransZol (catalog number ET101-01). Primer 5 software was used to analyze the viral load. CP qPCR primers were designed based on gene or plant gene sequences. In experiments detecting viral accumulation or gene expression levels, the extracted total RNA was used to remove the plant genome using gDNA removal enzyme. Using 1-2 µg of total RNA as a template, reverse transcription was performed using 5×HiScript II qRT Super Mix II. The cDNA was then used as a template and detected using 2×ChamQ SYBR qPCR MasterMix and specific primers. In experiments detecting viral replication levels, taking PVY replication levels as an example, the extracted total RNA was used to remove the plant genome using gDNA removal enzyme. Using 1-2 µg of total RNA as a template, reverse transcription was performed using 5×HiScript II RT select Super Mix with qPVY-CP-F+EF1α-R or qPVY-CP-R+EF1α-R primers. The obtained cDNA was then used as a template and detected using 2×ChamQ SYBR qPCR Master Mix and CP-specific primers.

[0054] Example 4: Detection of Virus Accumulation

[0055] Leaves of the plant to be tested were collected and placed in a pre-cooled mortar. Liquid nitrogen was added and the mixture was rapidly ground into powder. The sample and protein extraction solution (25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 10% glycerol, 0.15% Nonidet P-40 and 1×protease inhibitor cocktail) were thoroughly mixed by shaking at a ratio of 1:1 to 1:3. After incubation on ice for 10 min, the mixture was centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new centrifuge tube, 5×SDS was added in the appropriate ratio, the mixture was boiled in water for 10 min, placed on ice for 5 min, and stored at -20 °C.

[0056] The protein gel was prepared by adjusting the separation gel concentration according to the target protein size, and the sample to be tested was subjected to polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a nitrocellulose membrane using a wet transfer method. After the transfer, the membrane was blocked with 0.5% skim milk powder (10 mL of TBST buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20 per 0.5 g of skim milk powder) at room temperature for 1 h or at 4°C overnight. The corresponding antibody was added according to the antibody titer, and after incubation at room temperature for 1 h, the membrane was washed with 1×TBST for 10 min / wash three times. The secondary antibody was added according to the primary antibody source, and after incubation at room temperature for 1 h, the membrane was washed with 1×TBST for 10 min / wash twice, and then washed with 1×TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20) for 10 min / wash once. After washing the membrane, ECL western blotting substrate was added, and protein signals were detected using a chemiluminescence detector.

[0057] Example 5: Verification of NDUFS1 gene function 5.1 Silencing NDUFS1 inhibits PVY replication

[0058] To clarify the role of the NDUFS1 gene in PVY infection, the NDUFS1 gene in Nicotiana benthamiana was silenced using the TRV system according to the method described in Example 2. After 12 days of cultivation, the silenced plants showed normal growth and development. Figure 1 Gene silencing efficiency testing (using the same method as in Example 3) showed that the silencing efficiency of NbNDUFS1 was 26.4% ( Figure 2 ).

[0059] Subsequently, following the friction inoculation method of Example 2, PVY-GFP was inoculated onto the aforementioned silent plants and control plants. On the 5th day after inoculation, observation under ultraviolet light revealed that, compared to the control plants, the GFP fluorescence in the systemic leaves of the NDUFS1 silent plants was weaker (…). Figure 1 Virus accumulation detection (method as in Example 4) showed that its CP accumulation level was approximately 81% of the control. Figure 3 Viral RNA accumulation level detection (method as in Example 3) showed that the PVY RNA accumulation level was approximately 12% of the control. Figure 4 The above results indicate that silencing NDUFS1 inhibits PVY infection.

[0060] To further clarify the role of NDUFS1 in PVY replication, PVY replicons (PVY-GFPrep) were inoculated into leaves of the NDUFS1-silenced Nicotiana benthamiana system using the Agrobacterium tumefaciens infiltration method described in Example 2. The results showed that, compared to the control, the GFP fluorescence in the inoculated leaf areas of the NDUFS1-silenced plant system was weaker. Figure 5 ), the CP accumulation was approximately 60% of the control ( Figure 6 Viral replication level detection (using the same method as in Example 3) showed that the accumulation levels of PVY genomic RNA and negative strand RNA in NDUFS1 silenced plants were approximately 40% of those in control plants. Figure 7 The results in summary indicate that silencing NDUFS1 inhibits PVY replication.

[0061] 5.2 Silencing NDUFS1 inhibits TVBMV replication

[0062] Tobacco vein mosaic virus ( tobacco vein banding mosaic virus TVBMV is another representative virus of the Potato Virus Y genus. To verify the broad-spectrum function of NDUFS1, TVBMV-GFP was inoculated into the leaves of the NDUFS1-silenced Nicotiana benthamiana system according to the method in Example 2. On the 5th day after inoculation, observation under ultraviolet light showed that, compared with the control plants, the GFP fluorescence in the systemic leaves of the NDUFS1-silenced plants was weaker (…). Figure 8 The accumulation level of CP decreased significantly. Figure 9 ).

[0063] To further analyze the effect of silencing NDUFS1 on TVBMV replication, TVBMV replicons (TVBMV-GFPrep) were inoculated into the leaves of the silencing system. Four days after inoculation, compared with the control, the GFP fluorescence in the inoculated leaf area of ​​the NDUFS1-silenced plant system was weaker. Figure 10 ), the CP accumulation was approximately 40% of the control ( Figure 11 The accumulation levels of TVBMV genomic RNA and negative-strand RNA in NDUFS1 silenced plants were approximately 20% of those in control plants. Figure 12 The results in summary indicate that silencing NDUFS1 inhibits TVBMV replication.

[0064] 5.3 Knocking out the NDUFS1 gene enhances the resistance of potato plants to potato virus Y.

[0065] The gene-editing vector pNK2-StNDUFS1-sgRNA constructed in Example 1.3 was transformed into stem explants / stalks of the potato tetraploid cultivar 'Desiree' using Agrobacterium-mediated transformation. Resistant callus tissue was obtained after screening on a hygromycin-containing medium and induced to regenerate into complete plants. Genomic DNA was extracted from the regenerated plants, and PCR amplification and sequencing were performed on the genomic region containing the StNDUFS1 gene sgRNA target sequence (SEQ ID NO. 5). Through PCR amplification and sequencing analysis, edited plants with effective frameshift mutations in all four or most (e.g., three) of the StNDUFS1 alleles were screened for subsequent resistance identification. These plants can be considered loss-of-function mutants at the tetraploid level.

[0066] Following the friction inoculation method of Example 2, PVY sap carrying GFP was rubbed onto the above-identified qualified gene-edited plants and wild-type control plants. On day 12 post-inoculation, obvious fluorescence was observed in the leaves of the wild-type control system, and CP accumulation was high. However, no GFP fluorescence was observed in the leaves of the edited plant system, and no virus accumulation was detected. This indicates that effectively editing multiple StNDUFS1 alleles in tetraploid potatoes using CRISPR / Cas9 technology improved their resistance to PVY. Figure 13 ).

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an NDUFS1 protein as a target in screening or preparing products for improving plant resistance to Potato Virus Y; wherein, The amino acid sequence of the NDUFS1 protein is shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The viruses of the Potato Virus Y genus include Potato Virus Y and / or Tobacco Vein Mosaic Virus.

3. Application of the NDUFS1 protein encoding gene in breeding plant varieties resistant to Potato Virus Y; The amino acid sequence of the NDUFS1 protein is shown in SEQ ID NO.1 and / or SEQ ID NO.2; The gene encoding the NDUFS1 protein is a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3 and / or SEQ ID NO.

4.

4. The application as described in claim 3, characterized in that, The application is to enhance plant resistance to Potato Virus Y by downregulating the expression or function of the gene encoding the NDUFS1 protein in plants.

5. A method for improving plant resistance to Potato Virus Y, characterized in that, The method includes downregulating the expression or activity of NDUFS1 protein in the target plant; the amino acid sequence of the NDUFS1 protein is shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

6. The method as described in claim 5, characterized in that, The downregulation of NDUFS1 protein expression or activity in plants is achieved by silencing or knocking out the endogenous NDUFS1 gene in the plant.

7. The method as described in claim 6, characterized in that, The silencing is achieved through RNA interference technology; the knockout is achieved through gene editing technology.

8. The method as described in claim 7, characterized in that, When gene editing technology is used, the target is the nucleotide region from position 221 to position 242 of the coding region of the StNDUFS1 gene shown in SEQ ID NO.

3.

9. A method for preparing gene-edited plants resistant to Potato Virus Y, characterized in that, The method includes the following steps: Using gene editing technology, targeted mutations were made in specific regions of the NDUFS1 gene in the plant genome to obtain mutant plants with lost or reduced NDUFS1 protein function. The specific region is the nucleotide region from position 221 to position 242 of the coding region of the StNDUFS1 gene shown in SEQ ID NO.

3.

10. The preparation method according to claim 9, characterized in that, The plant in question is a potato.

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