Application of Arabidopsis thaliana uridine diphosphate glucosyltransferase genes AtUGT74F1 and AtUGT74F2 in resistance to potyvirus

Abstract

The application belongs to the field of plant biotechnology, and particularly relates to an Arabidopsis thaliana uridine diphosphate glucosyltransferase gene AtUGT74F1 and AtUGT74F2 application in resisting turnip mosaic virus. The application discloses a recombinant expression vector containing AtUGT74F1 and AtUGT74F2 is constructed to be overexpressed in Arabidopsis thaliana, and it is found that the related gene can enhance the ability of Arabidopsis thaliana to resist turnip mosaic virus, and the accumulation amount of the virus is significantly reduced compared with a wild type, while the accumulation amount of the virus is greatly increased when the related gene is knocked out. The related finding of the application can be used for preparation of a transgenic plant resisting turnip mosaic virus, and can also be used for identification and screening of a high-virus-resistance plant variety, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology. Specifically, this invention relates to the application of Arabidopsis thaliana uridine diphosphate glucosyltransferase genes AtUGT74F1 and AtUGT74F2 in resistance to turnip mosaic virus. Background Technology

[0002] Turnip mosaic virus (TuMV) belongs to the genus Pottyvirus (Potyvirus) of the family Pottyviridae and is one of the most prevalent plant viruses worldwide. TuMV has a very wide host range, systematically infecting cruciferous vegetables, rapeseed (Brassica napus), and other crops, causing mosaic and wrinkled leaves, stunted growth, and deformed flower stalks, severely reducing crop yield and quality and posing a significant threat to agricultural production safety. Currently, control methods for TuMV mainly rely on chemical control and agricultural cultivation measures, lacking efficient, safe, and sustainable resistance gene resources. Therefore, discovering endogenous antiviral genes in plants and cultivating resistant varieties are the fundamental ways to control this virus.

[0003] In the plant immune regulatory network, salicylic acid (SA) serves as a core defense signaling molecule, and the precise regulation of its endogenous content and activity directly affects the plant's resistance to pathogenic microorganisms. Glycosylation modification of SA is a key metabolic step in maintaining its homeostasis, primarily catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT; a member of the GT family of glycosyltransferases). Plant UGT uses activated uridine diphosphate sugar (UDP-sugar) as a glycosyl donor, catalyzing the formation of O-glycosides, N-glycosides, S-glycosides, and C-glycosides, and widely participating in physiological processes such as plant hormone modification, secondary metabolite transformation, and responses to biotic and abiotic stresses.

[0004] Previous studies have shown that members of the UGT family play complex and diverse regulatory roles in plant-pathogen interactions. For example, Arabidopsis UGT74F1 positively regulates the plant's basal resistance to *Pseudomonas syringae*, with mutations leading to decreased SA accumulation and suppressed expression of downstream defense genes. Its isoenzyme UGT74F2 negatively regulates free SA content by catalyzing the synthesis of salicylic acid-2-O-β-D-glucoside (SAG), synergistically activating the jasmonic acid (JA) / abscisic acid (ABA) signaling pathway, thus reducing resistance to *Pseudomonas syringae*. The peach (*Prunus persica*) homolog PpUGT74F2 enhances susceptibility to fungal diseases by inhibiting methyl salicylate (MeSA) accumulation. Furthermore, the tea plant (Camellia sinensis) CsUGT95B11 can promote quercetin glucoside synthesis, enhancing the plant's resistance to the gray-winged moth (Spodoptera littoralis). These studies confirm that members of the UGT family can perform diverse and even antagonistic biological functions in plant immune pathways by precisely regulating substrate glycosylation levels.

[0005] At present, research on the disease resistance of the plant UGT gene family mainly focuses on bacteria, fungi and herbivorous insects. Systematic research on the effects of this gene family on plant viruses is still relatively scarce, and the role of the plant UGT gene family in resisting plant viruses needs further research. Summary of the Invention

[0006] In response to the shortcomings of existing technologies, this invention has conducted in-depth research and found that Arabidopsis plants overexpressing AtUGT74F1 and AtUGT74F2 exhibit significant resistance to TuMV infection, providing a basis for the development of antiviral crops and also providing important evidence for elucidating the mechanism by which UGT participates in the plant virus infection process.

[0007] In one aspect, the present invention discloses the application of Arabidopsis thaliana uridine diphosphate glucosyltransferase genes AtUGT74F1 and AtUGT74F2 in resistance to turnip mosaic virus, wherein the GenBank accession number of AtUGT74F1 is NM_129946 and the GenBank accession number of AtUGT74F2 is NM_129944.

[0008] In this invention, the amino acid sequences encoded by the AtUGT74F1 and AtUGT74F2 genes share 77% similarity, and both possess uridine diphosphate-dependent glycosyltransferase activity. Specifically, AtUGT74F1 catalyzes the conversion of salicylic acid (SA) to salicylate-2-O-β-D-glucoside (SAG); while AtUGT74F2 can simultaneously catalyze the conversion of SA to SAG and salicylate glucose ester (SGE). By regulating the dynamic balance between free and bound SA in the plant, these genes can mediate the plant's defense response against pathogens.

[0009] In some embodiments, the ability to resist turnip mosaic virus is improved by overexpressing the AtUGT74F1 and AtUGT74F2 genes in plants.

[0010] In some embodiments, the plant is selected from cruciferous vegetables, rapeseed, and Arabidopsis thaliana.

[0011] In some embodiments, a recombinant expression vector containing the AtUGT74F1 gene and / or the AtUGT74F2 gene is constructed, and then the recombinant expression vector is transduced into the plant for overexpression.

[0012] In some embodiments, Agrobacterium tumefaciens is transduced into plants for overexpression using a recombinant expression vector containing the AtUGT74F1 gene and / or the AtUGT74F2 gene.

[0013] In one embodiment, the expression vector is pBA-Flag-4×myc; and the Agrobacterium tumefaciens is Agrobacterium GV3101.

[0014] In one embodiment, the construction of the recombinant expression vector of the present invention includes: designing specific primers based on the full-length coding sequences of AtUGT74F1 and AtUGT74F2 recorded in GenBank, and introducing a Gateway recombinant cloning adapter sequence at the 5' end of the primers. Using Arabidopsis cDNA as a template, the target gene fragment is amplified by high-fidelity PCR. The target fragment is recombined into the pDONR207 entry vector using the GatewayBP reaction, and then the target fragment is directionally cloned into the plant expression vector pBA-Flag-4×myc driven by the cauliflower mosaic virus (CaMV) 35S promoter using the LR reaction, thereby constructing the recombinant expression vectors pBA-Flag-4×myc-AtUGT74F1 and pBA-Flag-4×myc-AtUGT74F2, respectively.

[0015] In one embodiment, obtaining the transgenic plants of the present invention includes: (1) transforming a recombinant expression vector containing the AtUGT74F1 gene and / or the AtUGT74F2 gene into Agrobacterium tumefaciens GV3101 competent cells by electroporation, and obtaining positive clones by antibiotic screening and colony PCR identification. The recombinant vector is then introduced into wild-type Arabidopsis thaliana Columbia-0 (Col-0) using the flower immersion method, and atUGT74F1 and AtUGT74F2 stably overexpressing transgenic lines are obtained by glufosinate resistance screening and Western blot detection.

[0016] (2) Gene knockout lines were constructed using a CRISPR / Cas9 dual-target guide RNA (gRNA) system. Specific prespacer sequences targeting AtUGT74F1 and AtUGT74F2 were designed using the CRISPOR online tool. The oligonucleotide fragments were inserted into the pHEE401E vector using Golden Gate cloning technology. After sequencing verification, the vector was transformed into Agrobacterium GV3101 and then into Col-0 via the flower immersion method. T1 generation seedlings were screened on 1 / 2 MS medium containing hygromycin B. The mutation sites were identified by PCR amplification and Sanger sequencing. Homozygous T2 generation knockout lines without Cas9 protein were obtained through continuous self-crossing and PCR screening.

[0017] In one aspect, the present invention discloses a method for screening crop varieties with high resistance to turnip mosaic virus. The method includes detecting the expression levels of functionally similar genes AtUGT74F1 and AtUGT74F2 in the crop variety. If the expression level is higher than that of the wild type, the crop variety has good resistance to turnip mosaic virus; otherwise, the resistance to turnip mosaic virus is low. Beneficial effects

[0018] Compared to the traditional strategy of using viral source genes or viral sequence fragments to cultivate resistant strains, the AtUGT74F1 and AtUGT74F2 used in this invention are endogenous genes of Arabidopsis thaliana. They enhance antiviral ability by regulating the glycosylation homeostasis of the plant's endogenous defense signaling molecule SA, and have outstanding advantages such as high biosafety, environmental friendliness, and no risk of virus escape.

[0019] This invention not only provides an important theoretical basis for elucidating the molecular mechanism by which UGT family genes participate in plant antiviral immunity, but also reserves high-quality gene resources for antiviral molecular breeding of cruciferous crops and other susceptible crops. Attached Figure Description

[0020] Figure 1This image shows the identification and phenotypic representation of the AtUGT74F1 and AtUGT74F2 overexpression lines. A shows the protein expression levels of AtUGT74F1 and AtUGT74F2 in the T2 generation overexpression lines as detected by Western blotting. B shows the transcriptional levels of AtUGT74F1 and AtUGT74F2 in the overexpression lines as detected by RT-qPCR. Arabidopsis thaliana Actin was used as an internal control gene. Error bars represent the standard deviations of the three biological replicates. Statistical significance analysis was performed using t-tests (*, p < 0.05, **, p < 0.01, ***, p < 0.001). C shows the growth phenotype of the AtUGT74F1 and AtUGT74F2 overexpression lines at the 4-6 rosette leaf stage. Scale bar = 5 cm.

[0021] Figure 2 This image shows the identification and phenotypic representation of the AtUGT74F1 and AtUGT74F2 gene-edited lines. A shows the sequencing validation results of the T2 generation CRISPR / Cas9 gene-edited plants. The red box indicates the NGG sequence as the prototype spacer. B shows the growth phenotype of the AtUGT74F1 and AtUGT74F2 gene-edited lines at the 4-6 rosette leaf stage. Scale bar = 5 cm.

[0022] Figure 3 The effects of AtUGT74F1 and AtUGT74F2 overexpression and knockout lines on TuMV infection compared to wild-type Arabidopsis. A shows the infection phenotype of AtUGT74F1 and AtUGT74F2 stably overexpressing and knockout lines 14 days after TuMV infection. Scale bar = 5 cm. B shows the TuMV RNA accumulation level of each line detected by RT-qPCR. Arabidopsis Actin was used as an internal control gene. Error bars represent the standard deviation of the three biological replicates. Statistical significance analysis was performed using t-tests (not significant, p > 0.05, *, p < 0.05, **, p < 0.01, ***, p < 0.001). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0024] Example 1: Creation of AtUGT74F1 and AtUGT74F2 overexpression lines

[0025] 1.1 Primer design and target gene amplification

[0026] The full-length coding sequences of AtUGT74F1 (GenBank: NM_129946) and AtUGT74F2 (GenBank: NM_129944) were retrieved from the NCBI database. Specific primers were designed, and a Gateway recombination adapter sequence was introduced into the 5' end of the primers. The primer sequences are as follows:

[0027]

[0028] Using Arabidopsis thaliana Col-0 cDNA as a template, PCR amplification was performed using PrimeSTAR® GXL Premix (2×). The 50 μL reaction mixture consisted of: 25 μL PrimeSTAR® GXL Premix (2×), 1.5 μL upstream primer (10 μM), 1.5 μL downstream primer (10 μM), 2 μL cDNA template, and 20 μL ddH2O. The PCR program was as follows: 98 °C pre-denaturation for 2 min; 98 °C denaturation for 15 s, 55 °C annealing for 20 s, and 68 °C extension for 30 s / kb, for a total of 32 cycles; final extension at 68 °C for 8 min; and storage at 12 °C.

[0029] 1.2 Construction of recombinant expression vectors

[0030] After separation of PCR products by agarose gel electrophoresis, the target band was excised and purified by gel extraction. Using Gateway recombination cloning technology, the purified fragment was first subjected to a BP recombination reaction with the pDONR207 entry vector to construct an entry clone. Then, using the entry clone as a substrate, a LR recombination reaction was performed to directionally recombine the target fragment into the plant expression vector pBA-Flag-4×myc (driven by the CaMV 35S promoter), obtaining the recombinant expression vectors pBA-Flag-4×myc-AtUGT74F1 and pBA-Flag-4×myc-AtUGT74F2, respectively.

[0031] 1.3 Escherichia coli transformation and positive clone identification

[0032] Thaw 50 μL of *E. coli* DH5α competent cells (Weidi Bio, catalog number: DL1001) on ice. Add 5 μL of recombinant product, mix gently, and incubate on ice for 25–30 min. Heat shock at 42 ℃ for 45 s, then immediately incubate on ice for 3–5 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 37 ℃ with shaking at 200 rpm for 1 h. Spread 200 μL of the bacterial culture onto LB agar plates containing 50 μg / mL spectinomycin (Spec) and incubate at 37 ℃ for 12–16 h.

[0033] Pick 4–6 single colonies and inoculate them into 700 μL of LB liquid medium containing 50 μg / mL spectinomycin. Incubate at 37 ℃ and 200 rpm for 4–5 h until the bacterial culture becomes turbid. Perform PCR identification using the bacterial culture as a template. The 20 μL reaction system includes: 10 μL 2×Es Taq Master (Dye), 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 1.0 μL bacterial culture template, and 8.0 μL ddH2O. The PCR reaction program is as follows: 95 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, and 72 ℃ extension for 30 s / kb, for a total of 30 cycles; final extension at 72 ℃ for 8 min; store at 12 ℃.

[0034] PCR-positive colonies were inoculated into 5 mL of LB liquid medium containing 50 μg / mL spectinomycin and cultured at 37 ℃ with shaking at 200 rpm for 12–16 h. Plasmids were extracted using the EZNA® Plasmid Mini Kit I (Omega Bio-tek, catalog number: D6943) and verified by Sanger sequencing using universal vector primers or gene-specific primers. The correctly sequenced plasmids were named pBA-Flag-4×myc-AtUGT74F1 and pBA-Flag-4×myc-AtUGT74F2, respectively.

[0035] 1.4 Agrobacterium-mediated transformation

[0036] Take Agrobacterium GV3101 competent cells stored at -80 ℃ and thaw them on ice. Add 100 ng of the correctly sequenced recombinant plasmid to 100 μL of competent cells, mix well, and transfer to a pre-chilled electroporation cuvette. Perform electroporation transformation at 2.2 kV. After transformation, add 700 μL of LB liquid medium and incubate at 28 ℃ and 200 rpm for 30–40 min on a shaker. Spread the bacterial culture on LB agar plates containing 100 μg / mL spectinomycin and 100 μg / mL rifampin (Rif) and incubate at 28 ℃ until single colonies form.

[0037] 1.5 Transformation and Screening of Positive Plants by Flower Dipping Method

[0038] A single colony of segment 1.4 was picked and inoculated into 5 mL of double-antibiotic LB liquid medium and pre-cultured at 28 ℃ and 220 rpm with shaking. 200 μL of the pre-cultured bacterial solution was transferred to 200 mL of double-antibiotic LB medium and cultured under the same conditions for 12–14 h. The bacterial solution was aliquoted into 50 mL centrifuge tubes and centrifuged at 4 ℃ and 4000 rpm for 10 min to collect the cells. The cells were resuspended in sterile water and washed twice. The precipitate was finally resuspended in transgenic infiltration buffer (containing 5% sucrose and 0.05% Silwet L-77), and the bacterial solutions were combined and brought to a final volume of 100 mL.

[0039] Four-week-old, robust wild-type Arabidopsis thaliana (Col-0) plants were selected. Formed pods and open flower buds were removed, and the inflorescences were immersed in the bacterial solution for 4–6 seconds. This process was repeated after a certain interval. The transformed plants were then bagged to retain moisture and cultured in the dark for 24 hours, after which they were transferred to a greenhouse for normal cultivation under the following conditions: temperature 25 ± 2 ℃, relative humidity 60 ± 5%, and a photoperiod of 16 h light / 8 h darkness.

[0040] After the transformed plants have set fruit, T1 generation seeds are harvested. Seeds are vernalized at 4 ℃ for 2–3 days and then sown in the cultivation substrate and placed in a greenhouse for cultivation. Four–5 days after germination, a 100 mg / L glufosinate solution is sprayed for three consecutive days to screen for resistance. Healthy, green plants are retained and transplanted individually. Transplanted plants are then kept in a greenhouse at 25 ± 2 ℃, relative humidity 60 ± 5%, and a photoperiod of 16 h light / 8 h darkness. After 10–15 days of growth, 100–200 mg leaf samples are collected for Western blotting to identify the overexpression level of the target gene.

[0041] Example 2: Creation of AtUGT74F1 and AtUGT74F2 gene knockout lines

[0042] Knockout lines of AtUGT74F1 and AtUGT74F2 were constructed using the CRISPR / Cas9 dual-target gRNA system. Dual guide RNA sequences targeting the exon regions of the two genes were designed using the online tool CRISPOR (http: / / crispor.tefor.net / ): AtUGT74F1 5'-3': ATCTCCGATGGCTATGACC (RNA1 SEQ ID NO: 5); TCCATGACCTCGACCTTCA (RNA2 SEQ ID NO: 6); AtUGT74F2 5'-3': CAGTGCCGTACCCAACGCA (RNA1 SEQ ID NO: 7); TGGGCACTTGACGTTGCTA (RNA2 SEQ ID NO: 8) . Annealed oligonucleotide double-stranded fragments were inserted into the pHEE401E vector using Golden Gate cloning technology. After Sanger sequencing confirmed the recombinant vector sequence was correct, it was transformed into Agrobacterium GV3101.

[0043] The recombinant CRISPR / Cas9 vector was transformed into Col-0 using the flower immersion method, with transformation and culture conditions identical to those in Example 1. T1 generation resistant seedlings were obtained by screening on 1 / 2 MS solid medium containing 25 μg / mL hygromycin B. Genomic DNA was extracted from the T1 generation plants and amplified by PCR using target-specific primers. The amplified products were then identified by Sanger sequencing to determine the mutation sites. T2 generation plants were obtained through continuous self-pollination, and homozygous mutant lines lacking the Cas9 protein-coding sequence were further screened using PCR for subsequent virus inoculation experiments.

[0044] Example 3: Molecular biological detection of transgenic plants

[0045] 3.1 Western blot analysis of overexpression lines

[0046] (1) Extraction of total plant protein: Leaf samples were quantitatively obtained using a perforator, flash-frozen in liquid nitrogen, and then thoroughly pulverized in a grinder. Protein lysis buffer (containing protease inhibitors) was added, vortexed, and lysed on ice for 30 min. The samples were centrifuged at 4 ℃ and 12000 rpm for 15 min, and the supernatant was collected. 5×SDS loading buffer was added and mixed, and the samples were denatured at 95 ℃ in a metal bath for 5 min, cooled on ice, and briefly centrifuged. The resulting samples could be directly used for electrophoresis.

[0047] (2) Polyacrylamide gel electrophoresis: Separating gel and stacking gel were prepared using the Yaxin 12.5% ​​color rapid gel kit. After gel assembly, electrophoresis buffer was added, and protein samples were loaded sequentially. Electrophoresis was performed at a constant voltage of 100 V. Electrophoresis was stopped when the bromophenol blue indicator migrated to the bottom of the gel.

[0048] (3) Western blot analysis: PVDF membranes activated and equilibrated with methanol were transferred using a rapid transfer apparatus. The transferred membranes were blocked with 5% skim milk powder at room temperature for 0.5–1 h, and then incubated sequentially with primary antibody (anti-Flag-tagged antibody, 1:5000 dilution) and secondary antibody (HRP-labeled goat anti-mouse IgG, 1:10000 dilution). After each incubation, the membranes were thoroughly washed with TBST solution. ECL chemiluminescence was used for color development and imaging. Simultaneously, the gels were stained with Coomassie brilliant blue and destained to verify the consistency of sample loading amounts.

[0049] Using wild-type Arabidopsis thaliana Col-0 as a negative control, Western blot validation was performed on the transgenic T1 lines initially obtained through Basta screening using a specific antibody against the Myc tag. The results showed that significant accumulation of Flag-4×myc-AtUGT74F1 and Flag-4×myc-AtUGT74F2 were detected in AtUGT74F1 OE#3, #6, #8 and AtUGT74F2 OE#2, #5, #7, indicating overexpression (see results). Figure 1 (A). AtUGT74F1 OE#3, #6 and AtUGT74F2 OE#5, #7 lines were selected. RT-qPCR was used to analyze the overexpression levels of AtUGT74F1 and AtUGT74F2. Compared to Col-0, the overexpression levels were 1000-1500-fold and 8-12-fold, respectively (see results). Figure 1 (B). Under normal growth conditions, all four overexpression lines exhibited significantly larger rosette leaf growth phenotypes (see results). Figure 1 (C)

[0050] 3.2 PCR amplification and sequencing identification of knockout lines

[0051] (1) Genomic DNA extraction: A suitable amount of Arabidopsis thaliana leaves were placed in a centrifuge tube containing sterile steel beads, flash-frozen in liquid nitrogen, and then pulverized into powder using a grinder. DNA extraction buffer (50 mL system formula: 10 mL 1 M Tris-Cl (pH 7.5), 0.75 g NaCl, 0.36 g EDTA, 2.5 mL 10% SDS) was added, and the mixture was vortexed thoroughly and then lysed on ice. The mixture was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected. An equal volume of isopropanol was added, the mixture was allowed to stand, and the DNA precipitate was collected by centrifugation again. The precipitate was washed with 75% ethanol and then dried thoroughly. The DNA precipitate was dissolved in preheated elution buffer, and the nucleic acid concentration was determined. The precipitate was then sealed and stored at -40 °C.

[0052] (2) PCR amplification and sequencing: The following primers were used to amplify the target site region:

[0053]

[0054] The PCR reaction system and procedure were the same as those in Section 1.3 of Example 1. The amplified products were sequenced by Sanger sequencing, and the mutation type was determined by sequence alignment analysis.

[0055] Based on the Col-0 genome sequence, sequence alignment results showed that the AtUGT74F1 candidate knockout line had a 313 bp deletion in the target region, and was named AtUGT74F1. ko AtUGT74F2 yielded two edited strains: one with a 293 bp insertion and a 303 bp deletion at the target site, and the other with a large 304 bp deletion at the target site. These were named AtUGT74F2, respectively. ko -1、AtUGT74F2 ko -2 (See results) Figure 2 (A). Under normal growth conditions, these knockout lines showed no significant growth differences compared to the wild type (see results). Figure 2 (B)

[0056] Example 4: Resistance analysis of overexpression and knockout lines to TuMV

[0057] 4.1 Preparation of the source of the toxin

[0058] Agrobacterium containing TuMV infectious clones were activated and cultured on LB agar plates containing the corresponding antibiotics for 12 h. Single colonies were then inoculated into LB liquid medium containing the corresponding antibiotics and cultured at 28 ℃ and 220 rpm with shaking for 12–16 h. After collecting the bacterial cells, they were repeatedly washed with transient transfection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsylphenone), resuspended, and the OD of the bacterial culture was adjusted. 600 To 0.1. Using a needleless syringe, the bacterial solution was injected from the underside of the leaves into 4-5 week old *Nicotiana benthamiana* plants. The injected plants were first cultured overnight in darkness, then transferred to normal light conditions for 2-3 days. 7-10 days after inoculation, systemic leaves of infected *Nicotiana benthamiana* plants were used as the source of the virus for subsequent *Arabidopsis thaliana* TuMV friction inoculation experiments.

[0059] 4.2 Mechanical friction inoculation

[0060] Leaves infected with TuMV-GFP in the *Nicotiana benthamiana* system were placed in a pre-cooled mortar and ground thoroughly with a small amount of quartz sand at a ratio of 0.2 g of leaf to 10 mL of 1×PBS buffer. After settling, the supernatant was used as the virus inoculation solution. Healthy *Arabidopsis* plants with uniform growth (wild-type Col-0, overexpression lines, and knockout lines) were selected. Quartz sand was gently sprinkled on the surface of two leaves at the same leaf position. The leaf surface was then gently wiped 5 times in the same direction with a finger or cotton ball dipped in the virus solution to complete mechanical inoculation. After the inoculation solution remained for 1-2 minutes, it was gently rinsed with sterile water. The plants were cultured overnight in the dark, and then placed in a greenhouse at a temperature of 25±2 ℃, a relative humidity of 60±5%, and a photoperiod of 16 h light / 8 h darkness for normal culture.

[0061] 4.3 RT-qPCR detection of viral RNA accumulation levels

[0062] Total RNA was extracted from Arabidopsis thaliana inoculated leaves using the Akerui SteadyPure Universal RNA Extraction Kit. Reverse transcription was performed using the Akerui Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver. 2 to generate the first strand of cDNA. Real-time quantitative PCR was performed using the Akerui SYBR Green Premix Taq HS qPCR Kit (UNG Plus) to detect the relative expression level of the TuMV coat protein (CP) gene. Arabidopsis thaliana AtActin2 was used as an internal control gene.

[0063] The primer sequences are as follows:

[0064]

[0065] Each sample was tested in triplicate, and the relative expression level of the TuMV CP gene was calculated using the 2^(-ΔΔCt) method.

[0066] 4.4 Results Analysis

[0067] Fourteen to twenty-one days after friction inoculation, the disease symptoms of each strain were systematically observed and recorded, including leaf mosaic, degree of leaf wrinkling, plant dwarfing, and flower stalk malformation. Simultaneously, aboveground leaf samples were collected, and RNA was extracted for RT-qPCR detection.

[0068] The results showed that, compared with the wild-type Col-0, the AtUGT74F1 and AtUGT74F2 overexpression lines exhibited significantly reduced symptoms, a smaller viral infection range, and effectively alleviated flower bud malformation and dwarfing phenotypes. The viral RNA accumulation in leaves was also significantly reduced. The AtUGT74F1 knockout lines showed a similar disease severity to Col-0, suggesting that endogenous AtUGT74F1 is not essential for basic plant antiviral defense in this infection system, and its function may be compensated for by other redundant genes. The AtUGT74F2 knockout lines, however, showed a more severe disease severity than Col-0, with a significantly increased viral RNA accumulation. Figure 3 (A and B in the original text). The above results confirm that AtUGT74F1 and AtUGT74F2 can positively confer resistance to TuMV infection in Arabidopsis thaliana. The above results confirm that AtUGT74F1 and AtUGT74F2 can positively regulate resistance to TuMV infection in Arabidopsis thaliana.

[0069] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. Arabidopsis thaliana uridine diphosphate glucosyltransferase gene AtUGT74F1 and AtUGT74F2 Its application in combating turnip mosaic virus is characterized by... The AtUGT74F1 GenBank login number is NM_129946; AtUGT74F2 The GenBank login ID is NM_129944.

2. The application according to claim 1, characterized in that, Will AtUGT74F1 Genes and / or AtUGT74F2 The gene is overexpressed in the plant to enhance its resistance to turnip mosaic virus.

3. The application according to claim 2, characterized in that, The plants were selected from cruciferous vegetables, rapeseed, and Arabidopsis thaliana.

4. The application according to claim 2, characterized in that, By constructing AtUGT74F1 Genes and / or AtUGT74F2 Gene recombinant expression vectors are then transduced into plants for overexpression.

5. The application according to claim 4, characterized in that, Through containing AtUGT74F1 Genes and / or AtUGT74F2 Agrobacterium tumefaciens, a gene recombination expression vector, is used to transduce and overexpress the gene in plants.

6. The application according to claim 5, characterized in that, The expression vector is pBA-Flag-4×myc; the Agrobacterium tumefaciens is Agrobacterium GV3101.

7. A method for screening crop varieties highly resistant to turnip mosaic virus, characterized in that, The method includes detecting in crop varieties AtUGT74F1 , AtUGT74F2 The expression level of functionally similar genes is considered. If the expression level is higher than that of wild-type genes, the crop variety has good resistance to turnip mosaic virus. Conversely, if the expression level is lower, the resistance to turnip mosaic virus is lower.

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