Application of tobacco NbASL gene in improvement of plant virus resistance

Abstract

The invention discloses application of a tobacco NbASL gene in improvement of plant virus resistance, and belongs to the technical field of molecular biology. The nucleotide sequence of the NbASL gene is as shown in SEQ ID NO: 1. The NbASL gene is silenced in nicotiana benthamiana, the sensitivity of the plant to potato Y virus (PVY) and pepper vein mottle virus (ChiVMV) is obviously enhanced, and the virus accumulation amount is obviously increased. On the contrary, overexpression of the NbASL gene can significantly inhibit infection of PVY and reduce accumulation of virus RNA. And the expression of the NbASL gene in the nicotiana benthamiana can be induced to be up-regulated by PVY or ChiVMV infection. Therefore, the NbASL gene is a positive regulatory factor in antiviral reaction of plants. The invention provides important gene resources and theoretical basis for cultivating broad-spectrum and durable antiviral new crop varieties by using the NbASL gene, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of the tobacco NbASL gene in enhancing plant antiviral resistance. Background Technology

[0002] Tobacco is an important global economic crop; however, viral diseases are a key factor restricting its yield and quality during cultivation. Potato virus Y (PVY), a typical member of the Pottyvirus genus, is one of the most serious viral diseases affecting tobacco, potatoes, and other solanaceous crops. PVY is mainly transmitted by aphids in a non-persistent manner, causing symptoms such as mosaic patterns, vein necrosis, and stunted growth, resulting in significant economic losses. In recent years, another member of the Pottyvirus genus—Chilli veinal mottle virus (ChiVMV)—has also become increasingly serious in solanaceous crops, causing leaf mottling, wrinkling, and fruit deformities, further exacerbating the pressure on disease control in production.

[0003] Currently, there is a lack of highly efficient and specific chemical agents for the control of plant viruses such as PVY and ChiVMV. Existing control strategies mainly rely on breeding and utilizing resistant varieties. However, crops themselves possess limited natural antiviral gene resources, and virus populations are constantly evolving, often overcoming crop resistance and rendering resistant varieties ineffective. Furthermore, while agricultural management measures (such as controlling vectors and removing diseased plants) can mitigate damage to some extent, their effectiveness is unstable and costs are high. Therefore, discovering novel antiviral genes within plants and elucidating their mechanisms of action is crucial for developing new virus control strategies and breeding broad-spectrum, durable resistant crop varieties.

[0004] Plants have developed a complex immune system to combat pathogen infection over a long period of evolution. Key enzymes and their mediated metabolic pathways play crucial roles in this defense response. Therefore, there is an urgent need to discover new key genes that can effectively participate in plant antiviral responses and to develop methods for their application in antiviral breeding of economic crops such as rice, tobacco, and tomato, in order to overcome the shortcomings of existing control technologies. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide the application of the tobacco NbASL gene in improving plant antiviral resistance.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of a tobacco NbASL gene in enhancing plant virus resistance, wherein the amino acid sequence encoded by the tobacco NbASL gene is shown in SEQ ID NO:2.

[0007] Based on the above scheme, the nucleic acid sequence of the tobacco NbASL gene is shown in SEQ ID NO:1.

[0008] Based on the above scheme, the virus is a virus belonging to the Potato Virus Y genus.

[0009] Based on the above scheme, the virus of the Potato Virus Y genus is Potato Virus Y or Pepper Vein Mottle Virus.

[0010] Application of products containing the tobacco NbASL gene in enhancing plant virus resistance, wherein the amino acid sequence encoded by the tobacco NbASL gene is shown in SEQ ID NO:2.

[0011] Based on the above scheme, the nucleic acid sequence of the tobacco NbASL gene is shown in SEQ ID NO:1.

[0012] Based on the above scheme, the product is an expression vector containing the NbASL gene, an expression cassette, or a host cell containing the expression vector.

[0013] A method for enhancing plant resistance to viruses, the method comprising introducing and expressing the aforementioned tobacco NbASL gene in a plant.

[0014] Based on the above scheme, the method includes transforming an expression vector containing the tobacco NbASL gene into plants.

[0015] Based on the above scheme, the method of transforming plant bodies is one of Agrobacterium-mediated transformation, gene gun transformation, electroporation transformation, PEG transformation, and liposome transformation.

[0016] Based on the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0017] Based on the above scheme, the plant is a plant of the Solanaceae family, and further, the plant is selected from tobacco, potato, tomato or pepper.

[0018] Advantages of the technical solution of this invention This invention reveals for the first time the crucial role of the tobacco NbASL gene in plant resistance to Potato Virus Y (especially PVY and ChiVMV), providing a novel and effective candidate gene for crop antiviral breeding. Furthermore, through loss-of-function (silencing) and gain-of-function (overexpression) experiments, the inhibitory effect of the NbASL gene on infection by these two important viruses, PVY and ChiVMV, was verified, indicating that this gene may possess broad-spectrum antiviral potential.

[0019] The NbASL gene and its coding sequence described in this invention can be directly used to improve the virus resistance of important Solanaceae crops such as tobacco, potato, tomato, and pepper through genetic engineering techniques (such as overexpression), and have important application prospects in virus-resistant crop breeding. Attached Figure Description

[0020] Figure 1 Silencing efficiency detection for pTRV:NbASL; Figure 2 Phenotypic diagrams of NbASL silent plants and control plants (top) and phenotypic diagrams of NbASL silent plants and control plants after inoculation with PVY-GFP (bottom). Figure 3 Phenotypic diagrams of pTRV:NbASL in the treatment group and pTRV00 in the negative control group on day 19 of silence; Figure 4 Changes in PVY and ChiVMV CP expression levels in tobacco after PVY or ChiVMV infection of NbASL-silenced plants; Figure 5 The changes in PVY mRNA expression after PVY infection of NbASL-overexpressing tobacco (A) and the fluorescence of leaves of NbASL-overexpressing plants on day 6 after inoculation with PVY-GFP (B).

[0021] In the above figures, * indicates a significant difference (P<0.05), and ** indicates an extremely significant difference (P<0.01). Detailed Implementation

[0022] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0024] In the following embodiments: The greenhouse cultivation conditions were: temperature 25℃, photoperiod of 16 h light / 8 h dark, and light intensity of 2000 lx. Plasmids pTRV2, pTRV1, Agrobacterium strain GV3101, potato virus Y, pepper vein mottle virus, plasmid pGWC, Escherichia coli competent cells DH5α, and PVY-WT plasmid were all obtained from the Department of Pathology Laboratory, College of Plant Protection, Henan Agricultural University.

[0025] Example 1 The tobacco NbASL gene has the nucleic acid sequence shown in SEQ ID NO:1 and the encoded amino acid sequence shown in SEQ ID NO:2.

[0026] SEQ ID NO:1 (5'→3') ATGAGTGACTCTTCTGCTCAATACATCCACATGGTGCAGCACTTGATAGAAGAGTGTATAATTTTCAAAATGAACAGAGAAGAATGCATGGAAGCACTCTCCAAACATGCAAATATCCAGCCTATTATCACTTCCACGGTGTGGAAGGAGTTGG AGAAGGAGAACAAAGAATTCTTTGAGGCATACAATAAAACGAGAGAGGAAAAATCATCACCAATATTATCAGCAGAATCACAATTGGAGACGACAAGACGAAGAATCCATAATATGATGTTGGATTCGTCTTCTAAAGATTCCAATGAAAAGTGA SEQ ID NO:2 MSDSSAQYIHMVQHLIEECIIFKMNREECMEALSKHANIQPIITSTVWKELEKENKEFFEAYNKTREEKSSPILSAESQLETTRRRIHNMMLDSSSKDSNEK Example 2 The cloning method for the tobacco NbASL gene involves the following steps: (1) Total RNA was extracted from Nicotiana benthamiana using the TRIzol method. The specific method is as follows: Take healthy Nicotiana benthamiana seedlings, cut fresh leaves and grind them into a fine powder in a mortar. After grinding, take about 100 mg of leaf powder into a 1.5 mL centrifuge tube, add 1 mL of RNAiso Plus (Takara) and mix well. Incubate on ice for 5 min, centrifuge at 12000 g for 10 min at 4 ℃ to allow the leaf tissue to fully precipitate. Transfer the supernatant and add 200 μL of chloroform and mix well. Incubate on ice for 5 min, centrifuge at 12000 g for 15 min, take 500 μL of the supernatant and add an equal volume of isopropanol, mix well, incubate on ice for 10 min, centrifuge at 12000 g for 10 min, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, incubate for 5 min, centrifuge at 7500 g for 5 min, discard the supernatant, and add 100 μL of RNase-free... The total RNA from tobacco was obtained by dissolving and precipitating it in water, and its concentration was determined. The precipitate was then stored at -20 °C.

[0027] (2) Using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara), cDNA was obtained by reverse transcription of total RNA from tobacco according to the kit manual; (3) Using the extracted Nicotiana benthamiana cDNA as a template, the fragment was amplified using specific primers NbASL-GWCF / R and PrimeSTAR enzyme (Takara). The reaction program was as follows: 98℃ for 3 min; 98℃ for 15 s, 55℃ for 20 s, 72℃ for 20 s, 35 cycles; 72℃ for 5 min; and stored at 4℃. The fragment was recovered by agarose gel electrophoresis using a gel recovery kit (Shanghai Sangon Biotech).

[0028] The sequence of the specific primer NbASL-GWCF / R is as follows: NbASL-GWCF: 5'-CTTTGACTTTAGGTCCGGCCGGATGAGTGACTCTTCTGCT-3' (SEQ ID NO: 3); NbASL-GWCR: 5'-GGGTCTAGAGACTTTAGGTCCTGCTTTTCATTGGAATCTTT-3' (SEQ ID NO: 4).

[0029] Example 3 Construction of a tobacco NbASL gene silencing vector (1) Using Nicotiana benthamiana cDNA as a template, the NbASL silent fragment was amplified by PCR using the NbASL-TRV2F / R primer pair; NbASL-TRV2F: 5'-CCGGAATTCCATACAATAAAACGAG-3' (SEQ ID NO: 5); NbASL-TRV2R: 5'-CCGCTCGAGGAATCCAACATCATAT-3' (SEQ ID NO: 6); (2) Linearize the vector plasmid pTRV2 using restriction endonuclease EcoRI and XhoI (BioLabs), and ligate the amplified target fragment to the vector using T4 DNA ligase (Takara); thus obtaining the tobacco NbASL gene silencing vector (pTRV2:NbASL).

[0030] Example 4 The effect of silencing the NbASL gene on viral infection (1) pTRV1 and pTRV2:NbASL were transformed into Agrobacterium strain GV3101, with Agrobacterium strain GV3101 transformed with empty vector pTRV2 as a control. The specific steps are as follows: Agrobacterium competent strain GV3101 was added to the target plasmid, placed on ice for 5 min, treated with liquid nitrogen for 5 min, bathed in water at 37℃ for 5 min, and then placed on ice for 5 min. After adding 800 μL of antibiotic-free LB liquid medium, it was cultured in a constant temperature shaker at 28℃ for 2-3 h, centrifuged at 5000 rpm for 2 min, and after discarding part of the supernatant, the precipitate was mixed by suction and spread on solid medium containing kanamycin (Kan) and rifampin (Rif), and placed in an incubator at 28℃ for 2-3 days until colonies grew.

[0031] (2) Pick the above Agrobacterium colonies, incubate overnight at 28℃ and 200 r / min, and use resuspension (10 mmol·L⁻¹) -1 MES, 10 mmol·L -1 MgCl2, 200 μmol·L -1 Acetyleugenol (AS) was resuspended to OD600 = 0.4. A bacterial suspension containing pTRV1 plasmid and a bacterial suspension containing pTRV2 plasmid were mixed at a 1:1 volume ratio, allowed to stand for 2 h, and then injected into 4-leaf-stage Nicotiana benthamiana, designated as the pTRV:00 group. A bacterial suspension containing pTRV1 plasmid and a bacterial suspension containing pTRV2:NbASL recombinant plasmid were mixed at a 1:1 volume ratio; allowed to stand for 2 h, and then injected into 4-leaf-stage Nicotiana benthamiana, designated as the pTRV:NbASL group.

[0032] The relative expression levels of the NbASL gene were analyzed using QuantStudio™ 3 (thermofisher) and ChamQ Universal SYBR qPCR MasterMix (Vazyme). NbASL-YF / R was used as the primer for quantitative detection of NbASL, with Nicotiana benthamiana 18S selected as an internal control. 2 -ΔΔCt The method quantitatively calculates the relative changes in gene transcription levels.

[0033] Nb18sRNA-YF: 5'-GGTGGAGCGATTTGTCTGGT-3' (SEQ ID NO: 7); Nb18sRNA-YR: 5'-CAGGCTGAGGTCTCGTTCGT-3' (SEQ ID NO: 8); NbASL-YF: 5'-TTCACGGAAAACGCAACCAAAA-3' (SEQ ID NO: 9); NbASL-YR: 5'-TTAGCTGCGCCATACAGCCCTC-3' (SEQ ID NO: 10); The results are as follows Figure 1 As shown, 14 days after NbASL was injected and silenced, the expression level of NbASL in pTRV:NbASL plants was only 18.9% of that in the control, indicating that NbASL silencing was successful.

[0034] Using pTRV:00 as a control, after silencing for 19 days, no significant phenotypic difference was found between the silenced plants (pTRV: NbASL) and the control plants (pTRV:00). Figure 2 As shown in the top row, this indicates that silencing NbASL does not affect the growth of Nicotiana benthamiana.

[0035] 200 mg of preserved infectious clone PVY-GFP leaves and ChiVMV leaves were ground in a mortar, and 2 mL of 10×PBS (Servicebio) buffer and 18 mL of deionized water were added to form a homogenate. The supernatant was then used to inoculate the 2nd and 3rd leaf positions of the pTRV:00 and pTRV:NbASL plants. On day 5 after inoculation, RNA was extracted from the systemic leaves, reverse transcribed, and then quantitatively analyzed using real-time quantitative PCR to determine the infection status of PVY and ChiVMV after silencing NbASL. The primers for PVY quantification were PVY-YF4 and PVY-YR4, and the primer for ChiVMV quantification was ChiVMV-CPF / R.

[0036] PVY-YF4: 5'-GGGTTGCCTGTTCATCTTAGTGT-3' (SEQ ID NO: 11); PVY-YR4: 5'-CATACGCAGTGTTGGCTTCTTG-3' (SEQ ID NO: 12); ChiVMV-CPF: 5'-ATGATGTCACAGATGAAC-3' (SEQ ID NO: 13); ChiVMV-CPR: 5'-GTCTAAATGATGTGTTTGG-3' (SEQ ID NO: 14); The results are as follows Figure 2 As shown in the bottom row, when NbASL was silenced for 14 days and then inoculated with PVY-GFP, the fluorescence of the inoculated leaves in the silenced plants was significantly stronger than that in the control plants on day 5. Real-time quantitative PCR (qPCR) Figure 3 The results showed that the contents of PVY and ChiVMV CP in the leaves of NbASL-silenced plants were significantly higher than those in the control group. PVY was 13.7 times that of the control group, and ChiVMV CP was 2.02 times that of the control group. This indicates that silencing NbASL promotes PVY-GFP and ChiVMV infection. Therefore, NbASL may act as a positive regulator to inhibit viral infection.

[0037] Example 5 Construction of a tobacco NbASL gene overexpression vector (1) Using Nicotiana benthamiana cDNA as a template, the fragment was amplified using specific primers NbASL-GWCF / R and PrimeSTAR enzyme (Takara). The reaction program was as follows: 98℃ for 3 min; 98℃ for 15 s, 55℃ for 20 s, 72℃ for 20 s, 35 cycles; 72℃ for 5 min; stored at 4℃. The fragment was recovered by agarose gel electrophoresis and using a gel recovery kit (Shanghai Sangon Biotech).

[0038] (2) The target fragment was recombined into the entry vector plasmid pGWC, which was digested with restriction endonucleases PpumI and EagI (BioLabs), using the Clon Express II One Step Cloning Kit (Vazyme) and transformed into competent E. coli DH5α cells. After incubation at 37℃ for 12-16 h, single colonies were picked for colony PCR identification. Positive clones were picked and cultured in a culture medium, and plasmids were extracted using a plasmid extraction kit (Shanghai Sangon Biotech). The plasmids were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The target fragment was recombined from the correctly sequenced vector pGWC into the fluorescent expression vector PGWC-GFP using the LRClonase™ II enzyme mix (Invitrogen) to construct the fluorescent expression vector GFP-NbASL.

[0039] (3) Using plasmid GFP-NbASL as a template, the fragment was amplified with primers PVY-S-EGFPF and PVY-S-R1 and then recovered by gel extraction. The PCR product was then amplified with primers PVY-S-EGFPF and PVY-S-R2 and the gel was cut and recovered. The PVY-WT plasmid was digested with restriction endonuclease SacII, and the fragment was recombined with the vector to obtain the recombinant vector PVY / GFP-NbASL, which was used to simultaneously express PVY virus, NbASL gene and GFP fluorescent protein in Nicotiana benthamiana.

[0040] PVY-S-EGFPF: 5'-ATGATTCAGTTCTCTTCCGCGGAAATGGTGAGCAAGGGCGAG-3' (SEQ IDNO: 15); PVY-S-R1: 5'-AGCCTGGTGATGAACCTCGTATAGAGACTTTAGGTCCTG-3' (SEQ ID NO: 16); PVY-S-R2: 5'-CCTTCCAAAAGCTTTCCGCGGAAGAGTTAGCCTGGTGATGAACCTCGTA-3' (SEQ ID NO: 17).

[0041] An infectious clone PVY-GFP was constructed as a control. Using plasmid pGWC-GFP as a template, the fragment was amplified with primers PVY-S-EGFPF and PVY-S-R1 and recovered via gel extraction. The PCR product was then amplified again with primers PVY-S-EGFPF and PVY-S-R2 and recovered via gel extraction. The PVY-WT plasmid was digested with the restriction endonuclease SacII, and the fragment was recombined with a vector to obtain the recombinant vector PVY-GFP, which was used to simultaneously express PVY virus and GFP fluorescent protein in Nicotiana benthamiana.

[0042] Example 6 The effect of NbASL gene overexpression on viral infection (1) The recombinant vector PVY / GFP-NbASL was transformed into Agrobacterium strain GV3101. The specific steps are as follows: Agrobacterium competent strain GV3101 was added to the correctly sequenced plasmid, placed on ice for 5 min, treated with liquid nitrogen for 5 min, bathed in water at 37℃ for 5 min, and then placed on ice for 5 min. After adding 800 μL of antibiotic-free LB liquid medium, it was cultured in a constant temperature shaker at 28℃ for 2-3 h, centrifuged at 5000 rpm for 2 min, and after discarding part of the supernatant, the precipitate was mixed by suction and spread on solid medium containing kanamycin (Kan) and rifampin (Rif), and placed in an incubator at 28℃ for 2-3 days until colonies grew.

[0043] (2) Pick the above Agrobacterium colonies, incubate overnight at 28℃ and 200 r / min, and use resuspension (10 mmol·L⁻¹) -1 MES, 10 mmol·L -1 MgCl2, 200 μmol·L -1 Acetyleugenone) resuspended to OD 600 =0.4, after 2 hours of stillness, inject F. benthamiana, wait 1-2 weeks for the disease to develop, and then cut off the diseased leaves to preserve the source of the toxin.

[0044] (3) Good quality *Nicotiana benthamiana* plants were selected and divided into two groups, inoculated with an overexpressing infectious clone (PVY / GFP-NbASL) and PVY-GFP, respectively. On day 4, samples of inoculated leaves were taken, and the PVY genomic RNA content was detected by real-time quantitative PCR. The results showed that the viral RNA content in the PVY / GFP-NbASL inoculated group was significantly lower than that in the control group PVY-GFP (…). Figure 4 The expression level of PVY-GFP was reduced by 68% compared to the control group. On day 6 post-inoculation, the fluorescence of PVY-GFP was observed under a handheld UV lamp, revealing that the fluorescence intensity of the control group was significantly stronger than that of the overexpression group. Figure 4 (B) indicates that overexpression of NbASL can inhibit PVY virus infection.

[0045] Example 7 Effect of viral infection on NbASL gene expression in Nicotiana benthamiana Six-leaf-stage Nicotiana benthamiana was inoculated with PVY-GFP and ChiVMV, with PBS inoculation serving as a control. RNA was extracted from inoculated leaves 1.5 days after inoculation, and the expression level of NbASL was detected after reverse transcription.

[0046] The results showed that 1.5 days after PVY infection, NbASL expression was significantly higher than that in the PBS control, being 6.62 times higher than that in the control group. Figure 5 In the control group (CheVMV), 1.5 days after ChiVMV infection, the NbASL expression level was 2.37 times that of the control group. Figure 5 (B). The above results indicate that PVY-GFP and ChiVMV infection of *N. benthamiana* induces NbASL expression, and NbASL is associated with the antiviral performance of *N. benthamiana*.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of a tobacco NbASL gene in enhancing plant virus resistance, characterized in that, The amino acid sequence encoded by the tobacco NbASL gene is shown in SEQ ID NO:

2.

2. The application of the tobacco NbASL gene according to claim 1 in enhancing plant virus resistance, characterized in that, The nucleic acid sequence of the tobacco NbASL gene is shown in SEQ ID NO:

1.

3. The application of the tobacco NbASL gene according to claim 1 or 2 in enhancing plant virus resistance, characterized in that, The virus in question belongs to the Potato Virus Y genus.

4. The application of the tobacco NbASL gene according to claim 3 in improving plant virus resistance, characterized in that, The virus in question is either Potato Virus Y or Pepper Vein Mottle Virus.

5. The application of products containing the tobacco NbASL gene in enhancing plant virus resistance, characterized in that... The amino acid sequence encoded by the tobacco NbASL gene is shown in SEQ ID NO:

2.

6. The application of the product containing the tobacco NbASL gene according to claim 5 in improving plant virus resistance, characterized in that, The nucleic acid sequence of the tobacco NbASL gene is shown in SEQ ID NO:

1.

7. The application of the product containing the tobacco NbASL gene according to claim 5 in improving plant virus resistance, characterized in that, The product is an expression vector, expression cassette, or host cell containing the NbASL gene.

8. A method for improving plant resistance to viruses, characterized in that, The method includes introducing and expressing the tobacco NbASL gene of claim 1 in a plant.

9. The method for improving plant antiviral resistance according to claim 8, characterized in that, The method includes transforming an expression vector containing the tobacco NbASL gene into a plant.

10. The method for improving plant virus resistance according to claim 8 or 9, characterized in that, The plant in question belongs to the Solanaceae family.

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