Application of tobacco NbCu / Zn-SOD-1 in prevention and treatment of plant viruses

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of tobacco NbCu / Zn-SOD-1 in prevention and treatment of plant viruses. The invention aims to solve the technical problem of finding more SOD (superoxide dismutase) and providing a new choice for preventing and treating plant viruses. According to the technical scheme, the invention relates to application of tobacco NbCu / Zn-SOD-1 in prevention and treatment of plant viruses. The amino acid sequence of the NbCu / Zn-SOD-1 is as shown in SEQ ID No.1. The invention also relates to application of the tobacco NbCu / Zn-SOD-1 in prevention and treatment of plant viruses. According to the invention, NbCu / Zn-SOD-1 is cloned from nicotiana benthamiana, the NbCu / Zn-SOD-1 is subjected to structural characterization and functional research, and the result shows that the NbCu / Zn-SOD-1 can effectively inhibit TVMV and PVY.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of tobacco NbCu / Zn-SOD-1 in preventing and controlling plant viruses. Background Art

[0002] Reactive oxygen species (ROS) are produced in both non-stressed and stressed cells and are by-products of oxygen metabolism, including superoxide anion (O2 - ), singlet oxygen ( 1 O2), hydroxyl radical (·OH), and hydrogen peroxide (H2O2). Plants have a perfect ROS defense system, including limiting the formation of ROS and a scavenging system. Under non-stress conditions, the formation and removal of O2 are balanced. However, when ROS formation increases under stress conditions, the defense system may be destroyed. Inside the cell, superoxide dismutases (SODs) constitute the first line of defense against ROS. SOD is an important antioxidant enzyme that can catalyze the dismutation reaction of superoxide anion, converting it into H2O2 and O2. When plants are subjected to abiotic stresses such as drought, high temperature, low temperature, and salinity, ROS are produced in cells, causing oxidative stress. At this time, the SOD activity in plants will increase significantly to enhance the scavenging ability of O2 - and resist oxidative damage.

[0003] The charged O2 - molecule cannot pass through the phospholipid membrane. Therefore, the presence of SODs is crucial for the removal of O2 - in the region where O2 - free radicals are formed. According to the metal cofactors utilized by the enzyme, SODs are divided into three groups: iron SOD (FeSOD), manganese SOD (MnSOD), and copper-zinc SOD (Cu / ZnSOD), and these SODs are localized in different regions of the cell. FeSODs are localized in chloroplasts, MnSODs are localized in mitochondria and peroxisomes, and Cu / ZnSODs are localized in chloroplasts and the cytoplasm and may also be located in the extracellular space. Comparison of the deduced amino acid sequences of the three different types of SODs shows that MnSODs and FeSODs are more ancient types of SODs, and these enzymes most likely originated from the same ancestral enzyme, while Cu / ZnSODs have no sequence similarity with MnSODs and FeSODs and may have evolved separately in eukaryotes.

[0004] Generally, the well-developed defense system in organisms will limit the formation or scavenging of ROS. If the excessive ROS generated by stress cannot be effectively scavenged, a series of biochemical reactions will be triggered, leading to DNA damage, protein inactivation, and biomembrane disruption, and in severe cases, even cell dysfunction and apoptosis.

[0005] In Arabidopsis thaliana, a series of oxidative stresses occur in 7 SODs, including 3 FeSODs (FSD1, FSD2, and FSD3), 3 Cu / ZnSODs (denoted as CSD1, CSD2, and CSD3 respectively), and 1 MnSOD (MSD1) at the mRNA and protein levels. It has been reported that the mRNA level of FSD2 increases under ultraviolet irradiation and strong light, but the FSD2 mRNA does not respond to ozone exposure. It was found that FSD1 is controlled by the biological clock at the mRNA level.

[0006] A report on two pea cultivars cvs Progress and cvs Nugget, which are insensitive and sensitive to SO2 respectively, disclosed the effect of SO2 on the activity of Cu / ZnSOD enzyme. After treating these plants with SO2, the activities of cytoplasmic and chloroplast SODs both increased in cv. Progress, while in cv. Nugget, the activities of both enzymes decreased. In cv. Nugget after treatment, the expression level of chloroplast Cu / ZnSOD mRNA decreased, while in cv. Progress, after an initial decrease, the number of transcripts recovered.

[0007] The antiviral activity of SODs against human viruses has been reported. In particular, the induction of Cu / Zn superoxide dismutase (SOD1) is related to the antioxidant and antiviral activities of acetylsalicylic acid in HCV-expressing cells.

[0008] In plant viruses, SODs are also involved in the virus infection process. During asymptomatic extreme resistance, the role of early accumulated ROS in inhibiting the replication of potato virus X (PVX) has been demonstrated. The possible biochemical mechanism of asymptomatic extreme virus resistance may be that the accumulation of ROS has a dual role in infected plants. Higher concentrations of ROS may promote the programmed cell death (PCD) of infected plant cells, and also cause the death or inhibition of invading pathogens such as viruses because of their high toxicity. On the other hand, in the cells near the infection site of healthy plants, low concentrations of ROS play an important role as key signaling compounds for activating host defense responses (including the induction of antioxidants).

[0009] For example, the early (6 to 10 hours after inoculation) accumulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent superoxide and reactive oxygen species has a certain role in the resistance to plant viruses such as tobacco mosaic virus (TMV) related to the hypersensitive response (HR) of plants. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to discover more SODs and provide a new option for preventing and controlling plant viruses.

[0011] The technical solution of the present invention is the application of tobacco NbCu / Zn-SOD-1 in preventing and controlling plant viruses, and the amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.1.

[0012] SEQ ID No.1 Amino acid sequence of NbCu / Zn-SOD-1: MAAHTIFTTTTSTTNSLLFPVAAPNTNPSPSLHSSFHGVSLNLKSKTPQSLTLSAATAPKRLTVFAATKKAVAVLKGNSNVEGVVTLSQDDDGPTTVKVRITGLTPGLHGFHLHEFGDTTNGCMSTGPHFNPDGKTHGAPEDEIRHAGDLGNIVANADGVAEATIIDNQIPLTGPNSVIGRALVVHELEDDLGKGGHELSLTTGNAGGRLACGVLGLTPI*.

[0013] Furthermore, the nucleotide sequence of the coding gene of the NbCu / Zn-SOD-1 is shown in SEQ ID No.2.

[0014] SEQ ID No.2 Nucleotide sequence of the coding gene of NbCu / Zn-SOD-1: ATGGCCGCCCACACAATCTTCACTACCACCACCAGCACTACCAATTCTTTGTTATTCCCAGTCGCTGCCCCTAACACCAACCCCTCCCCTTCACTTCACTCTTCTTTCCACGGTGTTTCCCTCAATCTCAAGTCAAAGACTCCTCAATCTTTAACACTTTCTGCTGCCACTGCTCCTAAACGTCTCACTGTTTTTGCTGCTACTAAGAAAGCTGTTGCTGTCCTTAAGGGCAATTCCAATGTTGAGGGCGTTGTCACTCTCTCCCAAGATGATGATGGTCCAACCACTGTGAAAGTTCGCATAACTGGACTTACACCTGGACTTCATGGATTCCATTTGCACGAGTTCGGTGACACTACAAACGGGTGTATGTCTACAGGACCCCATTTCAATCCTGATGGCAAGACACATGGAGCTCCTGAAGATGAAATCCGTCATGCGGGTGACCTGGGAAACATAGTGGCCAATGCCGATGGTGTGGCTGAAGCAACAATTATAGATAATCAGATACCACTGACTGGTCCAAATTCAGTTATTGGTAGAGCACTTGTGGTTCACGAGCTTGAGGATGATCTTGGAAAGGGTGGCCATGAACTCAGCCTTACCACTGGGAATGCTGGTGGAAGATTGGCATGTGGAGTACTTGGTTTGACTCCAATATGA。

[0015] Specifically, the virus is Tobacco vein mottling virus (TVMV) and Potato virus Y (PVY). The infectious clone of Potato virus Y is PVY-Ros1, which is constructed by inserting the cDNA encoding the Antirrhinum majus Rosea1 transcription factor into the PVY infectious clone within the Potato virus Y genome.

[0016] Among them, the plant is tobacco.

[0017] The present invention also provides the application of tobacco NbCu / Zn-SOD-1 in enhancing the antiviral ability of plants. The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.1.

[0018] Further, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0019] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0020] Among them, the plant is tobacco.

[0021] The present invention also provides a method for controlling plant viruses, which is achieved by increasing the expression level of NbCu / Zn-SOD-1 in plants. The amino acid sequence of NbCu / Zn-SOD-1 is shown as SEQ ID No.1.

[0022] Further, the way to increase the expression level of NbCu / Zn-SOD-1 in plants is: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plants.

[0023] Further, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0024] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0025] Among them, the plant is tobacco.

[0026] Particularly, the transformation is transient transformation or stable transformation.

[0027] The present invention also provides a method for improving the antiviral ability of plants, which is achieved by increasing the expression level of NbCu / Zn-SOD-1 in plants. The amino acid sequence of NbCu / Zn-SOD-1 is shown as SEQ ID No.1.

[0028] Further, the way to increase the expression level of NbCu / Zn-SOD-1 in plants is: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plants.

[0029] Further, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0030] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0031] Among them, the plant is tobacco.

[0032] Particularly, the transformation is transient transformation or stable transformation.

[0033] Advantages of the present invention: In the present invention, differential expression of SOD homologs (3 NbCu / Zn-SODs, 4 NbFe-SODs, and 2 NbMn-SODs) in Nicotiana benthamiana was discovered during the infection process of Tobacco vein mottling virus. NbCu / Zn-SOD-1 was cloned from Nicotiana benthamiana, its structure was characterized, and the effects of NbCu / Zn-SOD-1 on the infection of TVMV and PVY-Ros1 viruses were studied. The results showed that this NbCu / Zn-SOD-1 could effectively inhibit TVMV and PVY viruses. Description of the Drawings

[0034] Figure 1 Expression of SOD homologs during the infection process of TVMV virus. (A) Inoculation of Tobacco vein mottling virus (TVMV), with healthy Nicotiana benthamiana as a control. (B) Detection of the accumulation amount of TVMV virus by western blot, using the TVMV coat protein (CP) as an antibody. (C) Gene density of the cDNA library. (D) Transcriptome sequencing (RNA-seq) analysis of the transcriptional expression of NbSOD homologs in Nicotiana benthamiana.

[0035] Figure 2 Identification of NbCu / Zn-SOD in Nicotiana benthamiana infected with TVMV. (A) Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the relative expression of SOD homologs in Nicotiana benthamiana after TVMV infection. * represents p < 0.05, and "ns" represents p > 0.5, and the student t-test is 0.5. (B) Structure and multiple sequence alignment of the coding region and non-coding region (CDS-UTR) of SOD homologs in Nicotiana benthamiana. The yellow rectangle represents the CDS, the black line represents the exon, and the green rectangle represents the non-coding region.

[0036] Figure 3 Multiple sequence alignment of SOD homologs. (A) Multiple sequence alignment of Cu / Zn-SOD homologs in Nicotiana benthamiana. Dashes between amino acid letters indicate blanks introduced for optimal alignment. The background color of the letters represents the high variability (white) to low variability (red) at certain sites in the sequence, and the blue line represents the shared conserved sequence. (B) Multiple sequence alignment of Fe-SOD homologs, with the green line representing the common conserved sequence. (C) Multiple sequence alignment of Mn-SOD homologs, with the purple line representing the common conserved sequence.

[0037] Figure 4, Structural modeling and analysis of NbCu / Zn-SOD-1. (A) Secondary structure of NbCu / Zn-SOD-1. (B) Tertiary structure of NbCu / Zn-SOD-1, colored by predicted confidence score. (C) Structure similarity matrix of NbCu / Zn-SOD-1. (D) Amino acid phylogenetic tree of NbCu / Zn-SOD-1.

[0038] Figure 5 , Construction of NbCu / Zn-SOD subcellular vector and observation of subcellular localization. (A) The target fragment of NbCu / Zn-SOD-1 was amplified by polymerase chain reaction (PCR) using primers 1300-NbCu / Zn-SOD-F / 1300-NbCu / Zn-SOD-R and electrophoresed in agarose gel. (B) The 1300-GFP-EV vector fragment was digested with BamH I / Sal I. (C) The 1300-NbCu / Zn-SOD-GFP fragment was amplified in Escherichia coli DH5a by PCR. (D) The 1300-NbCu / Zn-SOD-GFP was digested with BamH I / Spe I. The marker fragment sizes are shown on the right. (E) GFP fluorescence was observed at an excitation wavelength of 488 nm. The 1300-GFP-EV empty vector was used as a control.

[0039] Figure 6 , Effect of overexpression of NbCu / Zn-SOD-1 on plant virus accumulation. (A) Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression level of NbCu / Zn-SOD-1 (mean ± SD). (B) Western blot was used to detect the accumulation of TVMV virus. The TVMV coat protein (CP) was used as an antibody, and the ribulose bisphosphate carboxylase (Rubisco) protein band was used as an internal reference (loading control). (C) Quantification value of the protein band (mean ± standard deviation). CTRL: control, * represents p < 0.05, and the student t-test is 0.5. (D) Western blot was used to detect the accumulation of PVY-Ros1 virus. The PVY coat protein (CP) was used as an antibody, and the ribulose bisphosphate carboxylase (Rubisco) protein band was used as an internal reference (loading control). (E) Quantification value of the protein band (mean ± standard deviation). CTRL: control, * represents p < 0.05, and the student t-test is 0.5. Detailed implementation manners

[0040] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following describes in detail the specific implementation manners, structures, features, and their effects of the present invention with reference to the accompanying drawings and preferred embodiments.

[0041] In the following examples, the vectors pDONR207, pEAQ-HT-DEST3, and Agrobacterium tumefaciens C58C1 were provided by Professor Juan Antonio García of the Spanish National Center for Biotechnology (CNB). Competent Escherichia coli DH5α cells were purchased from Baorui Biotechnology (Beijing) Co., Ltd. The infectious clone of TVMV was provided by Professor Zhao Mingmin (Zhao M, García B, Gallo A, et al. Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones[J]. Phytopathol Res, 2020, 2:36.). The primers used in the following examples are shown in Table 1.

[0042] Table 1 Primer Information 。

[0043] Example 1 Expression of SOD Homologs during TVMV Virus Infection The seeds of Nicotiana benthamiana plants were sown in soil and cultured in a greenhouse at 22 - 26°C with a 16 / 8 hour light / dark cycle. When the N. benthamiana plants grew to 4 - 5 leaves, they were inoculated with TVMV virus by injection. The symptoms of N. benthamiana plants were observed on the 9th day after inoculation.

[0044] Compared with the healthy control, the leaves of the plants inoculated with TVMV showed obvious shrinkage and downward curling of the edges ( Figure 1 A). In severe TVMV infections, the plants usually showed dwarfing. Using a TVMV CP-specific antibody (Aiboteck ABclonal Technology), the virus accumulation in the samples on the 9th day was detected by Western blotting. The results showed that TVMV CP was not detected in the healthy control group, but TVMV CP protein accumulation was detected in both biological replicates of the plants inoculated with TVMV in N. benthamiana leaves ( Figure 1 B). Therefore, this confirmed the successful infection of TVMV in N. benthamiana.

[0045] To verify whether SOD homologs are regulated by TVMV infection, transcriptome sequencing was performed on Nicotiana benthamiana plants 9 days after TVMV infection. The treatment group was healthy Nicotiana benthamiana plants injected with the TVMV infectious clone, and the healthy Nicotiana benthamiana plants were used as the control. Total RNA was extracted using TRIzol reagent (manufacturer LAB, catalog number R1000) according to the manufacturer's instructions. Transcriptome sequencing and data analysis were carried out with reference to the literature (Zhang Jidan, Sun Zhenqi, Yue Jianying, et al. Transcriptome study on the infection of Nicotiana benthamiana by Tobacco vein mottling virus [J]. Acta Laser Biology Sinica, 2023, 32(06): 525 - 536.). The transcriptome consisted of four cDNA libraries, including two biological replicates and two sets of treatments. The NbSODs genes were screened from the transcriptome sequencing data.

[0046] The box plot results showed high repeatability between the TVMV-infected group and the healthy control group ( Figure 1 C), demonstrating the reliability of the data and providing a basis for further analysis. The results showed that the expression of 17 transcripts encoding SOD homologs was different in the TVMV-infected samples compared with the healthy control plants (Table 2, Figure 1 D).

[0047] Table 2 Differential expression of SOD homologs after TVMV infection of Nicotiana benthamiana .

[0048] Example 2 Identification of NbCu / Zn-SOD-1 in Nicotiana benthamiana Total RNA was extracted from plant leaves for cDNA synthesis reactions; during reverse transcription, about 1 μg of total RNA was converted into cDNA using the SYBR Green PremixPro Taq HS qPCR kit according to the manufacturer's regulations. Real-time fluorescence quantitative PCR reactions were performed using gene-specific primers on a QuantStudio™ 3 and 5 real-time PCR system MAN0010407 (Thermo Fisher Scientific) instrument. The expression was normalized using NbUBI as an internal reference and calculated by the ΔΔCT method.

[0049] RT-qPCR analysis was performed on 9 SOD homologs, including 3 NbCu / Zn-SODs, 4 NbFe-SOD2s, and 2 NbMn-SODs. The primer sequence numbers used were SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25, SEQ ID No.26. RT-qPCR system: TB Green Premix Ex Taq II (Tli RNaseH Plus) 2×, 10 μL; forward primer (10 μM), 0.8 μL; reverse primer (10 μM), 0.8 μL; ROX Reference Dye II (50×), 2 μL; cDNA template, 2 μL; ddH2O was made up to 20 μL. Program: 95°C, 10 min; 95°C, 15 s; 60°C, 1 min. The results showed that after infection with TVMV, the expression of 3 SODs (NbCu / Zn-SOD-1, NbCu / Zn-SOD-2, NbCu / Zn-SOD-3) was up-regulated, and the expression of 6 SODs (NbFe-SOD2-1, NbFe-SOD2-2, NbFe-SOD2-3, NbFe-SOD2-4, NbMn-SOD-1, and NbMn-SOD-2) was down-regulated. The expression of the NbCu / Zn-SOD-1 gene was significantly up-regulated, and the expression of the NbFe-SOD2-3 gene was significantly down-regulated ( Figure 2 A).

[0050] The physicochemical properties of NbCu / Zn-SOD-1 were analyzed using ExPaSy-Protparam. As shown in Table 3, the amino acid composition of the SOD homologs was between 101 and 305 aa, and the relative molecular weight was 10.3 - 34.9 kD. Among them, NbFe-SOD2-3, NbMn-SOD-1, and NbMn-SOD-2 were basic proteins with an isoelectric point greater than 7, and the remaining 6 were acidic proteins with an isoelectric point less than 7. Hydrophilicity analysis showed that the GRAVY values of the SOD proteins were all negative, belonging to hydrophilic proteins. Subcellular localization prediction of NbCu / Zn-SOD-1 was performed using Plantm-PLoc. The results showed that 2 NbMn-SODs were localized to mitochondria, and the remaining SODs were localized to chloroplasts.

[0051] Table 3 Characteristics and Subcellular Localization of Nicotiana benthamiana SOD Homologs TBtools analyzed the CDS-UTR structure and multiple sequence alignment of Nicotiana benthamiana SOD homologs. The length of the Nicotiana benthamiana SOD locus was 2,658 - 9,691 bp. Three NbCu / Zn-SOD homologs contained 3 - 9 CDS regions, and four NbFe-SOD2 homologs each contained 8 - 9 CDS regions. The NbMn-SOD homolog contained 6 CDS regions ( Figure 2 B).

[0052] The amino acid sequences of Nicotiana benthamiana SOD homologs were aligned using the multiple sequence alignment method with ESPript 3.x and MEGA 11. The conserved domain of NbCu / Zn-SOD-1 was predicted using Scan Prosite. The results showed that the conserved sequences "GFHLHEfGDtT" and "GFHVHAlGDtT" were present in the Cu / Zn-SOD amino acid sequence of Nicotiana benthamiana ( Figure 3 A). A conserved sequence "DvWEHAYY" was distributed on the amino acid sequence of Fe-SOD ( Figure 3 B). A conserved sequence "DvWEHAYY" was distributed in the Mn-SOD amino acid sequence, which was the same as the conserved sequence distributed in the Fe-SOD amino acid sequence ( Figure 3 C).

[0053] Example 3 Subcellular Localization of NbCu / Zn-SOD-1 in Nicotiana benthamiana The expression of 9 SODs screened by transcriptome sequencing in Nicotiana benthamiana was analyzed by qPCR. Among them, the expression of the NbCu / Zn-SO-1 gene was significantly up-regulated and the expression level was relatively high, so it was selected as the research object.

[0054] To verify the subcellular localization of NbCu / Zn-SOD-1 in plant cells, the NbCu / Zn-SOD-1 sequence was cloned into the subcellular localization recombinant vector 1300-GFP-EV (the vector was kindly provided by Academician He Zuhua of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences). The target fragment (663 bp) of NbCu / Zn-SOD-1 was amplified from healthy Nicotiana benthamiana using the primers with the sequence numbers SEQ ID No.7 and SEQ ID No.8 ( Figure 5 A). The vector 1300-GFP-EV was digested with BamH I / Sal I to obtain a 10,497 bp fragment ( Figure 5B). Then, NbCu / Zn-SOD-1 was ligated with the 1300-GFP-EV vector to obtain an expression vector named 1300-NbCu / Zn-SOD-1-GFP. The recombinant plasmid 1300-NbCu / Zn-SOD-1-GFP was transformed into Escherichia coli DH5α, and a 663-bp fragment was verified by PCR ( Figure 5 C), and fragments of 10485 bp and 672 bp were obtained using BamH I / Spe I enzymes ( Figure 5 D). The plasmid 1300-NbCu / Zn-SOD-1-GFP was transformed into Agrobacterium tumefaciens C58C1 and injected into Nicotiana benthamiana. Fluorescence was observed at 3 dpi. GFP fluorescence was detected in the cytoplasm, indicating that NbCu / Zn-SOD-1 was localized in the cytoplasm ( Figure 5 E).

[0055] Example 4 Characterization of NbCu / Zn-SOD-1 The secondary structure of NbCu / Zn-SOD-1 was analyzed using NovoPro. Sixteen α-helix structures and three β-sheet structures were identified from the structure of NbCu / Zn-SOD-1 ( Figure 4 A). The three-dimensional structure of NbCu / Zn-SOD-1 predicted by Swiss-Model showed a conserved core with a high prediction confidence, flanked by less conserved termini with variable numbers of disordered residues ( Figure 4B). To confirm the structural correlation of the cloned NbCu / Zn-SOD-1 homologous gene with known Cu / Zn-SOD genes in other plants, for example, tomato superoxide dismutase Cu-Zn (NP 001234769.2), Solanum pennellii superoxide dismutase Cu-Zn (XP 015058855.1), potato superoxide dismutase Cu-Zn (XP 015164932.1), pepper superoxide dismutase Cu-Zn (XP 016547975.1), Lycium barbarum superoxide dismutase Cu-Zn (XP 060169665.1), Petunia hybrida superoxide dismutase Cu-Zn (CAA32534.1), Nicotiana benthamiana superoxide dismutase Cu-Zn (NbCu / Zn-SOD-1), Nicotiana tabacum superoxide dismutase Cu-Zn (XP 016486719.1), Populus alba superoxide dismutase Cu-Zn (BAF80585.1), Spinacia oleracea superoxide dismutase Cu-Zn (NP 001413319.1). Structural comparison was carried out using the reference structure obtained by X-ray crystallography or high-resolution modeling, and phylogenetic tree alignment analysis was performed using MEGA11. The results of the structural similarity matrix and phylogenetic tree showed that NbCu / Zn-SOD-1 was very similar to Cu / Zn-SOD in Nicotiana tabacum ( Figure 4 C and 4D).

[0056] Example 5 Effect of NbCu / Zn-SOD-1 on Plant Virus Infection To detect the effect of NbCu / Zn-SOD-1 on plant virus infection, the NbCu / Zn-SOD-1 (663 bp) fragment of Nicotiana benthamiana was amplified using SEQ ID No.3 and SEQ ID No.4 and verified by sequencing. The target fragment of NbCu / Zn-SOD-1 was amplified using SEQ ID No.5 and SEQ ID No.6 primers, and the NbCu / Zn-SOD-1 fragment was cloned into pEAQ-HT-DEST3 (purchased from Invitrogen) through the Gateway recombination system to obtain pEAQ-NbCu / Zn-SOD-1. The plasmid pEAQ-NbCu / Zn-SOD-1 was transformed into Agrobacterium tumefaciens C58C1 and injected into the leaves of Nicotiana benthamiana. RT-qPCR was performed using SEQ ID No.9 and SEQ ID No.10 primers to detect the expression level of NbCu / Zn-SOD-1. The results showed that the expression of NbCu / Zn-SOD-1 increased significantly compared with the healthy control group ( Figure 6 A). It was proved that the transient expression of NbCu / Zn-SOD-1 had a certain function.

[0057] Agrobacterium tumefaciens carrying pEAQ-NbCu / Zn-SOD-1, pEAQ-HT-DEST3, and pLX-TVMV (the infectious clone of TVMV) was cultured to OD 600 = 1.0 respectively. A pEAQ-NbCu / Zn-SOD-1 / TVMV mixture with V:V = 1:1 was prepared respectively. Co-injection of pEAQ-HT-DEST3 with each virus (TVMV and PVY-Ros1) at V:V = 1:1 was used as a control. Symptoms were observed 9 days later. Agrobacterium tumefaciens carrying pEAQ-NbCu / Zn-SOD-1 was injected into Nicotiana benthamiana. Three days later, PVY-Ros1 (the cDNA encoding the Antirrhinum majus Rosea1 transcription factor was inserted into the PVY infectious clone in the potato virus Y genome to construct the PVY-Ros1 virus) was inoculated according to the description by Fabio et al. (Pasin, F., Shan, H., García, B., Müller, M., SanLeón, D., Ludman, M., Fresno, D. H., Fátyol, K., Munné-Bosch, S., Rodrigo, G., & García, J. A. (2020). Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus. Plant communications, 1(5), 100099.).

[0058] Nine days after virus injection, systemic leaves were collected for western blot analysis. Approximately 0.2 g of diseased leaf tissue was frozen in liquid nitrogen, ground into a powder, and added to 2 volumes of 5% SDS solution, and mixed well until homogeneous. Incubate in a boiling water bath at 95 °C for 5 min, centrifuge at 4 °C and 12,000 rpm for 10 min. 2× loading buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate, 0.01% bromophenol blue, 250 mM dithiothreitol) was added to the supernatant at a ratio of 1:1, and heated again in a boiling water bath at 95 °C for 5 min, then placed on ice for 2 min. Centrifuge at 4 °C and 12,000 rpm for 10 min. The clear upper protein solution obtained was collected for Western blot analysis.

[0059] The accumulation of TVMV virus was detected using the antibody TVMV CP, and the accumulation of PVY-Ros1 virus was detected using the antibody PVY CP. Horseradish peroxidase-conjugated goat anti-rabbit IgG (ab205718, Abcam) antibody was used as the secondary antibody. Protein signals were detected by chemiluminescence.

[0060] The results showed that the virus accumulation levels of TVMV and PVY-Ros1 in the transiently expressed NbCu / Zn-SOD-1 in plants were significantly reduced compared with the control group (P value < 0.05) ( Figure 6 B, Figure 6 C, Figure 6 D, Figure 6 E). This indicates that NbCu / Zn-SOD-1 has an obvious inhibitory effect on virus infection.

[0061] As described above, it is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent variations using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent variations, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Application of tobacco NbCu / Zn-SOD-1 in preventing and controlling plant viruses, characterized in that: The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that: Has at least one of the following characteristics: a. The nucleotide sequence of the gene encoding NbCu / Zn-SOD-1 is shown in SEQ ID No.2; b. The viruses are tobacco vein mottle virus and potato virus Y; c. The plant is tobacco.

3. Application of tobacco NbCu / Zn-SOD-1 in improving plant antiviral ability, characterized by: The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.

1.

4. The application according to claim 3, characterized in that: Has at least one of the following characteristics: d. The nucleotide sequence of the gene encoding NbCu / Zn-SOD-1 is shown in SEQ ID No. 2; e. The viruses are tobacco vein mottle virus and potato virus Y; f. The plant is tobacco.

5. A method for preventing and controlling plant viruses, characterized in that: This is achieved by increasing the expression level of NbCu / Zn-SOD-1 in plants. The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.

1.

6. The method according to claim 5, characterized in that: Has at least one of the following characteristics: g. The method of increasing the expression of NbCu / Zn-SOD-1 in plants is as follows: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plants; h. The nucleotide sequence of the gene encoding NbCu / Zn-SOD-1 is shown in SEQ ID No.2; i. The viruses are tobacco vein mottle virus and potato virus Y; j. The plant is tobacco.

7. The method according to claim 6, characterized in that: The transformation is either a transient transformation or a stable transformation.

8. A method for improving plant antiviral ability, characterized in that: This is achieved by increasing the expression level of NbCu / Zn-SOD-1 in plants. The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.

1.

9. The method according to claim 8, characterized in that: Has at least one of the following characteristics: k. The method of increasing the expression of NbCu / Zn-SOD-1 in plants is as follows: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plants; 1. The nucleotide sequence of the gene encoding NbCu / Zn-SOD-1 is shown in SEQ ID No.2; m. The viruses are tobacco vein mottle virus and potato virus Y; n. The plant is tobacco.

10. The method according to claim 9, characterized in that: The transformation is either a transient transformation or a stable transformation.

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