Application of tobacco NbCu / Zn-SOD-1 in preventing and controlling plant viruses

Abstract

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. 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. 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. The present invention cloned NbCu / Zn-SOD-1 from Nicotiana benthamiana, and carried out structural characterization and functional research on it. The results showed that the NbCu / Zn-SOD-1 could 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 restricting the formation of ROS and the removal 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 restrict the formation or removal of ROS. If the excessive ROS generated by stress cannot be effectively removed, a series of biochemical reactions will be triggered, leading to DNA damage, protein inactivation, and biofilm disruption, and in severe cases, even cell dysfunction and apoptosis.

[0005] In Arabidopsis thaliana, a series of oxidative stresses occurred 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 increase in FSD2 at the mRNA level under ultraviolet irradiation and strong light, but the FSD2 mRNA did not respond to ozone exposure, and it was found that FSD1 was controlled by the biological clock at the mRNA level.

[0006] A report on two pea varieties cvs Progress and cvs Nugget that are insensitive and sensitive to SO2 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 the treated cv. Nugget, 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 (from 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 treating 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:

[0013] MAAHTIFTTTTSTTNSLLFPVAAPNTNPSPSLHSSFHGVSLNLKSKTPQSLTLSAATAPKRLTVFAATKKAVAVLKGNSNVEGVVTLSQDDDGPTTVKVRITGLTPGLHGFHLHEFGDTTNGCMSTGPHFNPDGKTHGAPEDEIRHAGDLGNIVANADGVAEATIIDNQIPLTGPNSVIGRALVVHELEDDLGKGGHELSLTTGNAGGRLACGVLGLTPI*.

[0014] Furthermore, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown in SEQ ID No.2.

[0015] SEQ ID No.2 Nucleotide sequence of the coding gene of NbCu / Zn-SOD-1:

[0016] ATGGCCGCCCACACAATCTTCACTACCACCACCAGCACTACCAATTCTTTGTTATTCCCAGTCGCTGCCCCTAACACCAACCCCTCCCCTTCACTTCACTCTTCTTTCCACGGTGTTTCCCTCAATCTCAAGTCAAAGACTCCTCAATCTTTAACACTTTCTGCTGCCACTGCTCCTAAACGTCTCACTGTTTTTGCTGCTACTAAGAAAGCTGTTGCTGTCCTTAAGGGCAATTCCAATGTTGAGGGCGTTGTCACTCTCTCCCAAGATGATGATGGTCCAACCACTGTGAAAGTTCGCATAACTGGACTTACACCTGGACTTCATGGATTCCATTTGCACGAGTTCGGTGACACTACAAACGGGTGTATGTCTACAGGACCCCATTTCAATCCTGATGGCAAGACACATGGAGCTCCTGAAGATGAAATCCGTCATGCGGGTGACCTGGGAAACATAGTGGCCAATGCCGATGGTGTGGCTGAAGCAACAATTATAGATAATCAGATACCACTGACTGGTCCAAATTCAGTTATTGGTAGAGCACTTGTGGTTCACGAGCTTGAGGATGATCTTGGAAAGGGTGGCCATGAACTCAGCCTTACCACTGGGAATGCTGGTGGAAGATTGGCATGTGGAGTACTTGGTTTGACTCCAATATGA。

[0017] 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.

[0018] Among them, the plant is tobacco.

[0019] 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.

[0020] Furthermore, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0021] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0022] Among them, the plant is tobacco.

[0023] 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.

[0024] Furthermore, 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.

[0025] Furthermore, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0026] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0027] Among them, the plant is tobacco.

[0028] Particularly, the transformation is transient transformation or stable transformation.

[0029] 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.

[0030] Furthermore, 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.

[0031] Furthermore, the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown as SEQ ID No.2.

[0032] Specifically, the virus is tobacco vein mottling virus and potato virus Y.

[0033] Among them, the plant is tobacco.

[0034] Particularly, the transformation is transient transformation or stable transformation.

[0035] 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 the NbCu / Zn-SOD-1 could effectively inhibit TVMV and PVY viruses. Description of the Drawings

[0036] 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 the control. (B) Detection of the accumulation of TVMV virus by Western blot, using the TVMV coat protein (CP) as the antibody. (C) Gene density of the cDNA library. (D) Transcriptome sequencing (RNA-seq) analysis of the transcriptional expression of NbSOD homologs in Nicotiana benthamiana.

[0037] 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, with a Student's t-test of 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.

[0038] 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 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.

[0039] 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) Structural similarity matrix of NbCu / Zn-SOD-1. (D) Amino acid phylogenetic tree of NbCu / Zn-SOD-1.

[0040] 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 DH5α 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.

[0041] 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 coat protein (CP) of TVMV 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 coat protein (CP) of PVY 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

[0042] 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 effects of the present invention in conjunction with the accompanying drawings and preferred embodiments.

[0043] The vectors pDONR207, pEAQ-HT-DEST3 and Agrobacterium tumefaciens C58C1 used in the following examples were provided by Professor Juan Antonio García of the Spanish National Center for Biotechnology (CNB). The competent cells of Escherichia coli DH5α were purchased from Baori 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.

[0044] Table 1 Primer information

[0045] 。

[0046] Example 1 Expression of SOD homologs during the infection process of TVMV virus

[0047] 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.

[0048] 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, plants usually show dwarfing. Using a TVMV CP-specific antibody (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 the accumulation of TVMV CP protein 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.

[0049] 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 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 of tobacco vein mottling virus infecting Nicotiana benthamiana [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.

[0050] 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 TVMV-infected samples compared with healthy control plants (Table 2, Figure 1 D).

[0051] Table 2 Differential expression of SOD homologs after TVMV infection of Nicotiana benthamiana

[0052] .

[0053] Example 2 Identification of NbCu / Zn-SOD-1 in Nicotiana benthamiana

[0054] Total RNA was extracted from plant leaves for cDNA synthesis reaction; during reverse transcription, the SYBR Green PremixPro Taq HS qPCR kit was used to convert approximately 1 μg of total RNA into cDNA 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 the internal reference and calculated by the ΔΔCT method.

[0055] 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).

[0056] The physicochemical properties of NbCu / Zn-SOD-1 were analyzed using ExPaSy-Protparam. As shown in Table 3, the amino acid composition of SOD homologs was between 101 - 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 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.

[0057] Table 3 Characteristics and Subcellular Localization of Nicotiana benthamiana SOD Homologs

[0058]

[0059] TBtools analyzed the CDS-UTR structure and multiple sequence alignment of Nicotiana benthamiana SOD homologs. The length of the Nicotiana benthamiana SOD locus is 2658 - 9691 bp. Three NbCu / Zn-SOD homologs contain 3 - 9 CDS regions, and four NbFe-SOD2 homologs contain 8 - 9 CDS regions respectively. The NbMn-SOD homolog contains 6 CDS regions ( Figure 2 B).

[0060] The amino acid sequences of Nicotiana benthamiana SOD homologs were aligned by multiple sequence alignment methods using ESPript3.x and MEGA11. The conserved domain of NbCu / Zn-SOD-1 was predicted using Scan Prosite. The results showed that the conserved sequences "GFHLHEfGDtT" and "GFHVHAlGDtT" existed 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 amino acid sequence of Mn-SOD, which was the same as the conserved sequence distributed in the amino acid sequence of Fe-SOD ( Figure 3 C).

[0061] Example 3 Subcellular Localization of NbCu / Zn-SOD-1 in Nicotiana benthamiana

[0062] 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.

[0063] 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 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 10497 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).

[0064] Example 4 Characterization of NbCu / Zn-SOD-1

[0065] 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).

[0066] Example 5 Effect of NbCu / Zn-SOD-1 on Plant Virus Infection

[0067] 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 the primers SEQ ID No.5 and SEQ ID No.6, 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. The expression level of NbCu / Zn-SOD-1 was detected by RT-qPCR using the primers SEQ ID No.9 and SEQ ID No.10. The results showed that the expression of NbCu / Zn-SOD-1 was significantly increased 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.

[0068] Agrobacterium tumefaciens carrying pEAQ-NbCu / Zn-SOD-1, pEAQ-HT-DEST3, and the infectious clone of TVMV (pLX-TVMV) was cultured to an OD600 of 1.0 respectively. A mixture of pEAQ-NbCu / Zn-SOD-1 / TVMV at a volume ratio of V:V = 1:1 was prepared respectively. Co-injection of pEAQ-HT-DEST3 with each virus (TVMV and PVY-Ros1) at a volume ratio of 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 (a 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 as described 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.).

[0069] 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 and ground into a powder, then added to 2 volumes of 5% SDS solution and mixed well until homogeneous. It was heated in a boiling water bath at 95 °C for 5 min, centrifuged 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, heated again in a boiling water bath at 95 °C for 5 min, and placed on ice for 2 min. Centrifuged at 4 °C and 12,000 rpm for 10 min. The clear upper protein solution obtained was collected for Western blot analysis.

[0070] 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.

[0071] The results showed that the virus accumulation of TVMV and PVY-Ros1 in the transiently expressed NbCu / Zn-SOD-1 in plants was significantly reduced compared with the control group (P value < 0.05) ( Figure 6 B, Figure 6 C, Figure 6 D, Figure 6 E). It indicated that NbCu / Zn-SOD-1 had an obvious inhibitory effect on virus infection.

[0072] 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 the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments with equivalent changes 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 modification, equivalent change and modification 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, and the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown in SEQ ID No.2; the virus is tobacco vein mottling virus and potato virus Y; the plant is tobacco.

2. Application of tobacco NbCu / Zn-SOD-1 in enhancing plant antiviral ability, characterized in that: The amino acid sequence of NbCu / Zn-SOD-1 is shown in SEQ ID No.1, and the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown in SEQ ID No.2; the virus is tobacco vein mottling virus and potato virus Y; the plant is tobacco.

3. A method for preventing and controlling plant viruses, characterized in that: It 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; the method for increasing the expression level of NbCu / Zn-SOD-1 in plants is: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plant; the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown in SEQ ID No.2; the virus is tobacco vein mottling virus and potato virus Y; the plant is tobacco.

4. The method according to claim 3, characterized in that: The transformation is transient transformation or stable transformation.

5. A method for improving the antiviral ability of plants, characterized in that: It 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; the method for increasing the expression level of NbCu / Zn-SOD-1 in plants is: constructing a vector expressing NbCu / Zn-SOD-1 and transforming the plant; the nucleotide sequence of the coding gene of NbCu / Zn-SOD-1 is shown in SEQ ID No.2; the virus is tobacco vein mottling virus and potato virus Y; the plant is tobacco.

6. The method according to claim 5, wherein: The transformation is transient transformation or stable transformation.

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