Application of PABP protein encoded by solanaceae plant in prevention and treatment of tobamovirus

Abstract

The present application relates to the technical field of biotechnology and crop disease prevention and treatment, and particularly relates to application of PABP protein coded by solanaceae in prevention and treatment of tobamovirus virus. The present application finds that PABP protein coded by solanaceae (taking NbPABP as an example) and its coding gene play an important role in pepper mild mottle virus (PMMoV) infection and proliferation, are a disease gene of PMMoV coded by a host plant, can be used for prevention and treatment of pepper mild mottle virus disease caused by PMMoV, and can be used for molecular breeding of PMMoV resistance / tolerance, etc. The present application provides valuable protein for plant antiviral infection, especially can improve the antiviral level of solanaceous fruit plants, ensures and promotes the production safety of solanaceous vegetables, and has wide application prospect in the field of plant antiviral breeding, etc.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and crop disease control technology, and in particular to the application of a PABP protein encoded by a Solanaceae plant in the control of Tobacco Mosaic Virus (TMN) viruses. Background Technology

[0002] Peppers and tomatoes are important solanaceous vegetable crops. In recent years, the incidence of pepper mild mottle virus (PMMoV) has shown a year-on-year increasing trend. PMMoV infection causes chlorotic mottling of leaves and stunted growth, posing a serious threat to the high and stable yields of peppers and other crops. Since there are currently no resistant / tolerant varieties and no effective control agents, it is urgent to identify disease-resistant genes and develop resistant materials to sustainably and more effectively control PMMoV and its damage, ensuring the production safety of peppers and other solanaceous vegetables.

[0003] As obligate intracellular parasites, plant viruses rely on and utilize host factors to complete life processes such as viral uncoating, genome replication, protein expression, viral particle assembly, and migration to neighboring cells during infection and proliferation. Therefore, identifying the host factors involved in plant virus infection and proliferation can help to better understand the mechanisms of viral infection and pathogenesis, thus providing new materials and theoretical basis for the prevention and control of viral diseases. Summary of the Invention

[0004] This invention provides the application of PABP protein encoded by Solanaceae plants in the control of Tobacco Mosaic Virus (PMMoV) viruses. This invention discovers that NbPABP protein and its encoding gene play an important role in the infection and proliferation of pepper mild mottle virus (PMMoV) of the Tobacco Mosaic Virus genus. It is a susceptible gene of PMMoV encoded by the host plant and can be used to control pepper mild mottle virus disease caused by PMMoV.

[0005] In a first aspect, the present invention provides an application of the NbPABP protein in any one or more of the following:

[0006] (1) Application in regulating the expression of Tobacco Mosaic Virus.

[0007] (2) Regulate the resistance of plants to diseases caused by Tobacco Mosaic Virus.

[0008] (3) Prevention and control of Tobacco Mosaic Virus (TMV) viral diseases.

[0009] (4) Regulate plant yield.

[0010] (5) Application in the improvement of plant germplasm resources.

[0011] (6) Application in the preparation of products for the prevention and control of Tobacco Mosaic Virus (TMV) virus diseases.

[0012] The amino acid sequence of the NbPABP protein includes any of the following:

[0013] (a) As shown in SEQ ID NO.1.

[0014] (b) A protein derived from SEQ ID NO.1 but retaining the function of the amino acid sequence shown in SEQ ID NO.1 by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1.

[0015] (c) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function as a protein.

[0016] (d) An amino acid sequence obtained by attaching a tag, restriction site and / or linking a peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (a)-(c).

[0017] Secondly, the present invention provides NbPABP The application of genes in one or more of the following:

[0018] (1) Application in regulating the expression of Tobacco Mosaic Virus.

[0019] (2) Regulate the resistance of plants to diseases caused by Tobacco Mosaic Virus.

[0020] (3) Prevention and control of Tobacco Mosaic Virus (TMV) viral diseases.

[0021] (4) Regulate plant yield.

[0022] (5) Application in the improvement of plant germplasm resources.

[0023] (6) Application in the preparation of products for the prevention and control of Tobacco Mosaic Virus (TMV) virus diseases.

[0024] The NbPABP The nucleotide sequence of the gene is at least one of the following.

[0025] (a) As shown in SEQ ID NO.2.

[0026] (b) A nucleotide sequence that expresses the same functional protein by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.2.

[0027] (c) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.2 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS and washing the membrane with the solution.

[0028] (d) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein.

[0029] Preferably, the above-mentioned applications regulate plant resistance to Tobacco Mosaic Virus (NMPV) diseases, control crop yield, prevent and control NMPV diseases, or improve germplasm resources by adjusting the content or activity of NbPABP protein.

[0030] More preferably, the above-described application is achieved by regulating the content or activity of the NbPABP protein through genetic engineering technology.

[0031] More preferably, the above-mentioned application inhibits the replication and proliferation of tobacco mosaic virus by reducing the content or activity of NbPABP protein, thereby improving the plant's resistance to tobacco mosaic virus diseases, increasing crop yield, or improving germplasm resources.

[0032] Preferably, the above-mentioned application promotes the replication and proliferation of tobacco mosaic virus by increasing the content or activity of NbPABP protein, so as to construct a disease model of tobacco mosaic virus.

[0033] Thirdly, the present invention provides applications of inhibitors that reduce the content or activity of NbPABP protein in plants, including any of the following applications:

[0034] (1) Improve the plant’s resistance to Tobacco Mosaic Virus virus diseases.

[0035] (2) Increase plant yield.

[0036] (3) Prevention and control of Tobacco Mosaic Virus (TMV) viral diseases.

[0037] (4) Prepare pesticides, fertilizers or additives for the prevention and control of tobacco mosaic virus diseases.

[0038] The amino acid sequence of the NbPABP protein from the Solanaceae plant includes any of the following:

[0039] (a) As shown in SEQ ID NO.1.

[0040] (b) A protein derived from SEQ ID NO.1 but retaining the function of the amino acid sequence shown in SEQ ID NO.1 by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1.

[0041] (c) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function as a protein.

[0042] (d) An amino acid sequence obtained by attaching a tag, restriction site and / or linking a peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (a)-(c).

[0043] Preferably, the inhibitor can inhibit the expression or activity of NbPABP protein by inhibiting transcription, inhibiting transcriptional processing, or inhibiting post-transcriptional processing function. The inhibitor can be used as a pesticide, fertilizer, or fertilizer additive, and its types include, but are not limited to:

[0044] (1) Interfering RNAs such as siRNA or shRNA.

[0045] (2) Small molecule inhibitors.

[0046] (3) Enzyme inhibitors.

[0047] (4) Antibody drugs.

[0048] In practice, the content or activity of NbPABP protein can be regulated by controlling substances such as cDNA and RNA. For example, it can be regulated by RNA encoded by the nucleotide sequence shown in positions 996-1295 of SEQ ID NO.2 (silencing) NbPABP Gene).

[0049] In specific implementation processes, this includes, but is not limited to, using biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation to regulate the content or activity of NbPABP protein in plant cells.

[0050] Fourthly, the present invention provides a method for inhibiting the replication and proliferation of Tobacco Mosaic Virus (NMPV) viruses in plants, comprising: reducing the content or activity of NbPABP protein through genetic engineering techniques.

[0051] Preferably, the plant or crop described in any of the above applications, products or methods is a Solanaceae plant, preferably a plant of the genus *Nicotiana* or *Solanum*, and more preferably tobacco, chili pepper, tomato, potato, eggplant or wolfberry.

[0052] The tobacco mosaic virus genus viruses described in this invention include tobacco mosaic virus, tomato mosaic virus, tomato brown wrinkled fruit virus, cucumber green mottle mosaic virus, tobacco mild green mosaic virus, pepper mild mottle virus, etc., preferably pepper mild mottle virus.

[0053] The tobacco mosaic virus disease described in this invention refers to viral diseases caused by viruses of the tobacco mosaic virus genus that occur in plants such as peppers, tomatoes, and tobacco, preferably viral diseases caused by PMMoV infection that occur in plants such as peppers and tobacco.

[0054] The above NbPABP The gene originates from tobacco Bunsen, given that tobacco Bunsen NbPABP The PABP gene is highly conserved in Solanaceae economic crops such as chili peppers, therefore this invention can also be used in dicotyledonous host plants of Solanaceae such as chili peppers.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] This invention reveals that the NbPABP protein and its encoding gene play a crucial role in PMMoV infection and proliferation, and are a susceptibility gene for PMMoV encoded by the host plant. NbPABP In the silence experiment, NbPABP With a 60% downregulation of expression levels, the accumulation of PMMoV genomic RNA was downregulated by 70%, and the accumulation of PMMoV CP protein was downregulated by 60%, indicating that silencing Buprofen... NbPABP The gene can significantly reduce the levels of PMMoV genomic RNA and CP protein, thus inhibiting PMMoV infection.

[0057] The above findings can be used to control mild mottle virus disease of peppers caused by PMMoV, and can be used in molecular breeding for resistance / tolerance to PMMoV. This invention can provide a basis for studying the role of plant proteins in virus infection, for antiviral breeding, and other fields, improve the research level of antiviral protection in solanaceous vegetables, and ensure and promote the production safety of solanaceous vegetables. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 The silence in Embodiment 2 of the present invention NbPABPThe following is a schematic diagram of the results of inoculated leaves of *Nicotiana sambac*. The image was taken under UV light 5 days after PMMoV-GFP inoculation; the dots represent PMMoV-GFP observed under UV light, and their quantity indicates the amount of PMMoV-GFP accumulated in the leaf. TRV: mCherry This is the control group, indicating that the person was not silent. NbPABP Bunsen tobacco leaves; TRV: NbPABP The experimental group represents silence. NbPABP Bunsen tobacco leaves.

[0060] Figure 2 The silence in Embodiment 2 of the present invention NbPABP The following is a statistical analysis of the fluorescence spot counts on inoculated leaves of *Nicotiana bungeana*. The fluorescence spots on the inoculated leaves were counted and analyzed using Graphpad Prism software. (The last sentence appears to be incomplete and possibly refers to a different topic.) t -test performs statistical analysis of significant differences, **** indicates p <0.0001.

[0061] Figure 3 The silence in Embodiment 2 of the present invention NbPABP Afterwards, the leaves of the tobacco plant were inoculated. NbPABP The results of gene expression levels were plotted using RT-qPCR. NbPABP The relative expression levels of genes were analyzed using Graphpad Prism software. Student's... t -test performs statistical analysis of significant differences, ** indicates p <0.01.

[0062] Figure 4 This is a graph showing the accumulation of PMMoV genomic RNA in inoculated leaves of *Nicotiana benthamiana* after silencing NbPABP in Example 2 of this invention. The relative expression levels of PMMoV genomic RNA were detected using RT-qPCR and analyzed using Graphpad Prism software. (Student's...) t -test performs statistical analysis of significant differences; * indicates p <0.05.

[0063] Figure 5 The silence in Embodiment 2 of the present invention NbPABP The following image shows the results of PMMoV CP accumulation in inoculated leaves of Nicotiana Bunsenii. PMMoV CP accumulation levels were detected using Western blotting, with Actin as a protein loading control. Coomassie Brilliant Blue (CBB) staining indicated the total protein content. PMMoV CP was 17 kDa, and Actin was 43 kDa. Quantitative values ​​were calculated using ImageJ software. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0065] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer.

[0066] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0067] The biological materials used in the following examples are as follows:

[0068] Bunsen tobacco: See Goodin MM, Zaitlin D, Naidu RA, Lommel SA. 2008. Nicotiana benthamiana : its history and future as a model for plant-pathogeninteractions. Molecular Plant-Microbe Interactions 21:1015-1026.

[0069] Vectors pJM23, pJG1024, and pLoxP-Cre: See Yin, Y., Hua M., Zhao K., WanQ., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen, J. 2022. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456-2463.

[0070] For vectors pTRV1 and pTRV2, see: Liu, Y., Schiff M and Dinesh-Kumar SP.2002.Virus-induced gene silencing in tomato.The Plant journal 32:777-786.

[0071] Pepper mild mottle virus (PMMoV) See: Yin, Y., Hua M., Zhao K., Wan Q., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen, J. 2022. Construction of chimeric viruses based on pepper mildmottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456-2463.

[0072] The NbPABP protein sequence in the following examples is shown in SEQ ID No. 1. The gene encoding the NbPABP protein is named... NbPABP The gene, with its sequence shown in SEQ ID No. 2.

[0073] Example 1: Construction of the recombinant vector pTRV2-NbPABP

[0074] 1. Extraction of raw tobacco ( Nicotiana benthamiana The total RNA was reverse transcribed into cDNA.

[0075] 2. Using the cDNA obtained in step 1 as a template, use pTRV2:: NbPABP -F (SEQ ID No. 3) and pTRV2:: NbPABP The primer pair consisting of -R (SEQ ID No. 4) was used for PCR amplification to obtain... NbPABP PCR amplification product of the gene fragment containing the nucleotide sequence of positions 996-1295 in the coding region of (SEQ ID No. 2); recovery NbPABP The PCR product of the gene fragment shown in positions 996-1295 of the coding region.

[0076] 3. Using restriction endonucleases XbaI and KpnI The pTRV2 vector was digested with enzymes, and the vector backbone was recovered.

[0077] 4. Connect the recovered product from step 2 with the vector backbone from step 3 to obtain the recombinant vector pTRV2-NbPABP.

[0078] 6. Based on the sequencing results, the structure of the recombinant vector pTRV2-NbPABP is described as follows: In the vector pTRV2... XbaI and KpnI A double-stranded DNA molecule, as shown in SEQ ID No. 11, was inserted between the restriction enzyme sites.

[0079] Example 2 NbPABP Gene silencing experiments

[0080] 1. Extraction of PMMoV-GFP crude extract

[0081] (1) Streak the Agrobacterium strain containing pJM23, pJG1024 and pLoxP-Cre plasmids stored at -80℃ on LB resistance plates (containing 50 μg / ml Kan and 100 μg / ml Rif) and incubate at 28℃ for about 48 h; inoculate single colonies on LB resistance plates into 3~4 ml of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) and incubate at 28℃ and 180 rpm for 14~18 h.

[0082] (2) Inoculate 100 μL of bacterial culture into 10 mL of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and culture at 28℃ and 180 rpm for 10-12 h to obtain bacterial cultures of Agrobacterium pJM23, pJG1024 and pLoxP-Cre.

[0083] (3) Collect the bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, suspend the precipitate in 3 mL of soaking buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 µM acetylsyl syringone), and determine the concentration of the bacterial suspension using a spectrophotometer.

[0084] (4) Add 3 mL of Agrobacterium pJM23 bacterial suspension (OD) 600nm 3 mL of Agrobacterium pJG1024 bacterial suspension (concentration of 0.5%) 600nm The concentration was 0.5 g / mL, and 3 mL of Agrobacterium pLoxP-Cre bacterial suspension (OD200) was used. 600nm Mix the samples (at a concentration of 0.25%) and let them stand at room temperature for 2-3 hours to obtain a mixture. Then, use a needleless syringe (1 mL) to inject the mixture into the lower epidermis of the tobacco leaves and incubate at 22-24℃ for 6 days.

[0085] (5) Take the leaves of this tobacco plant, add 0.1 M phosphate buffer and grind them, centrifuge at 4500 g for 5 min, and collect the virus supernatant containing the crude extract of PMMoV-GFP.

[0086] 2. Utilizing TRV VIGS to silence smog NbPABP Gene

[0087] (1) The recombinant plasmid pTRV2-NbPABP obtained in Example 1 was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium TRV2-NbPABP, and stored in a -80℃ freezer.

[0088] (2) Agrobacterium strains carrying pTRV1, pTRV2-mCherry and pTRV2-NbPABP plasmids stored at -80℃ were streaked on LB resistance plates (containing 50 μg / ml Kan and 100 μg / ml Rif) and cultured at 28℃ for about 48 h; single colonies on LB resistance plates were inoculated into 3~4 ml of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) and cultured at 28℃ and 180 rpm for 14~18 h.

[0089] (3) Inoculate 100 μL of bacterial culture into 10 mL of LB liquid resistance medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and culture at 28℃ and 180 rpm for 10~12 h to obtain Agrobacterium culture carrying pTRV1, pTRV2-mCherry and pTRV2-NbPABP plasmids.

[0090] (4) Collect the bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, suspend the precipitate in 3 mL of soaking buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 µM acetylsyl syringone), and determine the concentration of the bacterial suspension using a spectrophotometer.

[0091] (5) Add 3 mL of Agrobacterium tumefaciens culture (OD) carrying the pTRV1 plasmid. 600nm A bacterial suspension of 1.0 g of recombinant Agrobacterium pTRV2-NbPABP plasmid (OD200) was mixed with 3 mL of the suspension. 600nm The concentration was 1.0) and 3 mL of pTRV2-mCherry agrobacterium bacterial suspension (OD). 600nm Mix the ingredients (1.0 concentration) and let stand at room temperature for 2-3 hours to obtain a mixture. Then, use a needleless syringe (1 mL) to inject the mixture into the lower epidermis of the 3rd-4th true leaves of Nicotiana benthamiana and incubate at 22-24℃ for 6 days.

[0092] 3. PMMoV-GFP challenge inoculation of Nicotiana benthamiana plants

[0093] (1) Take the silence from step 2 (5) to complete the process. NbPABP The original smoke or silence mCherry The *Nicotiana spp.* (as a control) were cultured in alternating light and dark conditions (16 h / 20℃ light, 8 h / 18℃ darkness) for 6 days. A small amount of carborundum was sprinkled on the 5th-6th true leaves of *Nicotiana spp.* and the PMMoV-GFP crude extract obtained in step 1 was rubbed inoculated (inoculation amount: 50 μL, concentration: 0.3 g / mL). After that, the inoculated *Nicotiana spp.* plants were cultured in alternating light and dark conditions (16 h / 20℃ light, 8 h / 18℃ darkness).

[0094] 4. Detect silence NbPABP PMMoV replication and proliferation levels in Nicotiana bungeana plants

[0095] The disease incidence in *Nicotiana bungeana* leaves after PMMoV inoculation was continuously observed. On the 5th day after PMMoV inoculation, inoculated leaves were taken and the PMMoV-GFP fluorescence spots were observed under ultraviolet light. The results are shown in […]. Figure 1 Fluorescence images were analyzed using Graphpad Prism software, and Student's... t -The test was used to perform statistical analysis of significant differences, and the results are shown in [the table]. Figure 2 Total RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, quantitative real-time PCR was performed using the Beijing Adley Biotechnology fluorescent PCR kit. Actin Genes used as internal reference genes for RT-qPCR identification NbPABP The relative expression levels of the gene and the content of PMMoV genomic RNA are shown in the following results. Figure 3 , Figure 4 Simultaneously, total protein was extracted from tobacco leaves. Western blot analysis was performed using TMV CP antiserum as the primary antibody, HRP-labeled goat anti-rabbit antibody as the secondary antibody, and Actin expression level as an internal control. The accumulation of PMMoV CP protein was detected by Western blotting, and quantification was performed using ImageJ software. Results are shown below. Figure 5 .

[0096] Used for identification NbPABP The primer pairs for the gene are as follows:

[0097] NbPABP-qRT-F: 5'-GCCACAGGGTGGTGTGAATA-3' (SEQ ID No. 5)

[0098] NbPABP-qRT-R: 5'-TCAGCACTTCCAATGCCCTT-3' (SEQ ID No. 6)

[0099] Used for identification Actin The primer pairs for the gene are as follows:

[0100] NbActin-qRT-F: 5'-TGCATTCTCCGTCTTGACT-3' (SEQ ID No. 7)

[0101] NbActin-qRT-R: 5'-TGCAGTCTCGAGTTCCTGTT-3' (SEQ ID No. 8)

[0102] The primer pairs used to identify PMMoV CP are as follows:

[0103] PMMoV CP-qRT-F: 5'-TGGAAGACTATTCCGACCGC-3' (SEQ ID No. 9)

[0104] PMMoV CP-qRT-R: 5'-CCGTGCCACGAACTAACTCA-3' (SEQ ID No. 10)

[0105] The obtained data is used in 2 -ΔΔCT The analysis is performed using the following steps:

[0106] First, for the experimental group samples (test) and control group samples (calibrator), the C of the reference gene (ref) was used. T C of the target gene is normalized. T value:

[0107] ΔC T (test) = C T (target, test) - C T (ref, test)

[0108] ΔC T (calibrator) = C T (target, calibrator) - C T (ref, calibrator)

[0109] Secondly, using the ΔC of the control group sample T ΔC of the normalized experimental group sample T value:

[0110] ΔΔC T = ΔC T (test) - ΔC T (calibrator)

[0111] Finally, calculate the expression level ratio: 2 -ΔΔCT = The ratio of the amount of expression.

[0112] Results Analysis: Figure 1 ,Figure 2 The results showed that on day 5 after PMMoV vaccination, compared with the control group (TRV-mCherry), the silencing effect was significantly reduced. NbPABP The experimental group (TRV-NbPABP) showed fewer green fluorescent spots on leaves infected with Nicotiana bungeana.

[0113] Depend on Figure 3 It can be seen that, compared with the control group, silence... NbPABP The genetically derived tobacco plant, NbPABP Expression levels were downregulated by approximately 60%; from Figure 4 It can be seen that, compared with the control group, the accumulation level of PMMoV genomic RNA was downregulated by about 70%.

[0114] Depend on Figure 5 It can be seen that, compared with the control group, silence... NbPABP In the Nicotiana Bunsenifolia plant, the accumulation of PMMoV CP protein was downregulated by about 60%.

[0115] The above research results indicate that silencing smog... NbPABP The gene significantly reduced PMMoV genomic RNA and CP protein levels, inhibiting PMMoV infection. These results indicate that the NbPABP protein and its encoding gene play an important role in PMMoV replication and proliferation, and are host susceptibility genes that promote PMMoV infection. They can be used to control mild mottle virus disease of pepper caused by PMMoV, and for molecular breeding of PMMoV-resistant varieties.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of inhibitors that reduce the content or activity of NbPABP protein in tobacco in the preparation of pesticides for controlling pepper mild mottle virus, characterized in that, The amino acid sequence of the NbPABP protein includes any of the following: (a) As shown in SEQ ID NO.1; (b) A protein derived from SEQ ID NO.1 and retaining the function of the amino acid sequence shown in SEQ ID NO.1 by substitution, deletion and / or addition of one or more amino acid residues; (c) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function as a protein; The inhibitor is interfering RNA.

2. The application according to claim 1, characterized in that, The inhibitor can suppress the expression or activity of NbPABP protein by inhibiting its post-transcriptional processing function, and the inhibitor is a pesticide.

3. A method for preventing and controlling pepper mild mottle virus, characterized in that, include: Reduce the content or activity of NbPABP protein through genetic engineering techniques.

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