Application of tobacco NtMATE34 gene and protein in regulation of bacterial wilt resistance

Abstract

The application discloses application of a tobacco NtMATE34 gene and protein in regulation of a bacterial wilt resistance. The tobacco NtMATE34 gene is induced to express by inoculation of a bacterial wilt pathogen Ralstonia solanacearum, and participates in a tobacco bacterial wilt stress response. After artificial inoculation of the bacterial wilt, compared with K326, the tobacco plant with overexpression of the NtMATE34 gene shows strong bacterial wilt resistance. The tobacco NtMATE34 gene plays an important role in regulation of the tobacco bacterial wilt resistance, and can be applied to gene function research and biological breeding for improvement of the tobacco bacterial wilt resistance.

Description

Technical Field

[0001] This invention belongs to the field of tobacco genetic engineering technology, specifically relating to the application of the tobacco NtMATE34 gene and protein, which are related to plant resistance to bacterial wilt, in regulating plant resistance to bacterial wilt. Background Technology

[0002] Bacterial wilt of tobacco is a soil-borne bacterial disease caused by *Ralstonia solanancearum*. It occurs during the seedling and field stages of tobacco, severely impacting growth and development and causing significant economic losses to tobacco production. Control methods for tobacco bacterial wilt include chemical, agricultural, and biological control, but these methods are currently ineffective. Discovering and utilizing bacterial wilt resistance genes in tobacco and other crops is a fundamental approach to controlling the disease. Isolating bacterial wilt resistance-related genes from tobacco using genetic engineering techniques and applying them to molecular breeding of resistant tobacco is of significant practical importance for ensuring both tobacco quality and yield.

[0003] The multidrug and toxic compound extrusion (MATE) protein family is an important member of the multidrug extrusion transporter, primarily involved in the transport of organic compounds such as organic acids, plant hormones, and secondary metabolites like flavonoids. Different MATE subfamilies perform different functions, including transporting and accumulating flavonoids and alkaloids, expelling endogenous or exogenous compounds from plants, regulating disease resistance, and responding to abiotic stresses. The MATE family exhibits diverse functions in plants. However, the biological functions of tobacco MATE family genes are currently poorly reported.

[0004] This invention is the first to discover a MATE-type transporter protein associated with resistance to bacterial wilt in tobacco, namely the transporter protein encoded by the tobacco NtMATE34 gene, providing a new pathway for tobacco resistance to bacterial wilt. Summary of the Invention

[0005] This invention utilizes a recombinant overexpression vector constructed from the NtMATE34 gene, which is associated with resistance to bacterial wilt in tobacco, to transform tobacco. This increases the expression level of the NtMATE34 gene, thereby enhancing the resistance of tobacco to bacterial wilt and providing intermediate breeding materials for improving tobacco resistance to bacterial wilt and for breeding varieties with resistance to bacterial wilt.

[0006] The primary objective of this invention is to provide an application of the tobacco NtMATE34 gene, which is associated with resistance to bacterial wilt in tobacco, in regulating resistance to bacterial wilt. The CDS sequence of the tobacco NtMATE34 gene is shown in SEQ ID NO.1.

[0007] Furthermore, the gene sequence also includes gene sequences with similar functions and a similarity of not less than 95%; the similar functions include expressing proteins that resist bacterial wilt.

[0008] Furthermore, the specific target of the antibacterial disease is tobacco.

[0009] The second objective of this invention is to provide an application of tobacco NtMATE34 protein in regulating resistance to bacterial wilt, the protein sequence of which is shown in SEQ ID NO.2.

[0010] Furthermore, the protein sequence also includes protein sequences with similar functions, having a similarity of not less than 95%; the similar functions include resistance to bacterial wilt.

[0011] Furthermore, the specific target of the antibacterial disease is tobacco.

[0012] A third objective of this invention is to provide the application of the tobacco NtMATE34 gene or tobacco NtMATE34 protein in improving resistance to bacterial wilt.

[0013] Furthermore, it is used to improve the resistance of tobacco to bacterial wilt.

[0014] Furthermore, by overexpressing the tobacco NtMATE34 gene, tobacco transformants with enhanced resistance to bacterial wilt were obtained.

[0015] Furthermore, the vector for overexpressing the tobacco NtMATE34 gene is pCHF3.

[0016] The recombinant overexpression vector pCHF3 is a binary Agrobacterium vector.

[0017] When constructing the recombinant expression vector, the CDS base sequence of the NtMATE34 gene was inserted after the cauliflower mosaic virus (CAMV) 35S promoter.

[0018] The transformed tobacco was prepared by infecting tobacco callus tissue with Agrobacterium containing a recombinant overexpression vector, and then using Agrobacterium-mediated transformation to integrate the cauliflower mosaic virus 35S promoter and the tobacco NtMATE34 gene into the tobacco genome. Within the tobacco plant, overexpression of the NtMATE34 gene enhances resistance to bacterial wilt.

[0019] This invention marks the first successful cloning, expression, and functional analysis of the NtMATE34 gene in tobacco. The analysis results indicate that this gene is associated with resistance to bacterial wilt in tobacco. Furthermore, overexpression of the NtMATE34 gene in tobacco significantly enhances its resistance to bacterial wilt, thus providing materials for improving tobacco resistance to bacterial wilt and for breeding varieties with resistance to this disease. Attached Figure Description

[0020] Figure 1 Analysis of NtMATE34 gene expression pattern after tobacco inoculation with bacterial wilt;

[0021] Figure 1 MOCK in the middle represents the control group;

[0022] Figure 2 Analysis of NtMATE34 expression level in tobacco plants overexpressing the NtMATE34 gene;

[0023] Figure 3 Evaluation of bacterial wilt resistance in tobacco plants overexpressing the NtMATE34 gene and the K326 control;

[0024] Figure 4 The proliferation of bacterial wilt pathogens in the roots of tobacco plants overexpressing the NtMATE34 gene and the K326 control. Detailed Implementation

[0025] The present application will be further explained below with reference to the embodiments, without limiting the present invention; before introducing the specific embodiments, the basic situation of some biological materials, experimental reagents, experimental instruments and other aspects involved in the following embodiments will be briefly introduced as follows.

[0026] Biological materials: Tobacco materials, specifically the cultivated tobacco (Nicotiana tabacum) variety K326, are preserved in our laboratory. The vector plasmid pCHF3 used for overexpressing tobacco genes is also preserved in our laboratory; the vector construction method can be found in the reference (https: / / www.mdpi.com / 2073-4409 / 8 / 1 / 50).

[0027] Experimental Reagents: The reagents and kits used in this experiment are as follows: restriction endonucleases were purchased from NEB (Beijing) Co., Ltd.; the RNA extraction TRIzol kit was purchased from Kangwei Century Biotechnology Co., Ltd.; high-fidelity DNA amplification enzyme, reverse transcription kit, and real-time fluorescence kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; the DNA gel recovery kit MiniBEST Agarose Gel DNA Extraction Kit and the plasmid DNA small-scale purification kit MiniBEST Plasmid purification Kit were purchased from Invitrogen; and antibiotics such as kanamycin and rifampin were purchased from Shanghai Sangon Biotech Co., Ltd.

[0028] Example 1: This example describes the process of obtaining the NtMATE34 gene.

[0029] 1. Total RNA extraction

[0030] RNA was extracted using the TRIzol reagent extraction method from Kangwei Century Company. The specific steps are as follows: After harvesting 4-week-old cultivated tobacco K326 under normal growth conditions, the tissue was rapidly ground into powder in liquid nitrogen. 1 ml of TRIzon Reagent (cwbiotech) was added to every 30-50 mg of tissue, and the mixture was thoroughly mixed. After incubating at room temperature for 5 min, 200 μl of chloroform was added, and the mixture was vigorously shaken for 15 seconds and incubated at room temperature for 2 min. The mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was carefully collected and transferred to another centrifuge tube, along with an equal volume of 70% ethanol. The entire mixture was transferred to an adsorption column, centrifuged at 12,000 rpm for 20 seconds, and the waste liquid in the collection tube was discarded. 700 μl of Buffer RW1 was added, and the mixture was centrifuged at 12,000 rpm for 20 seconds, and the waste liquid in the collection tube was discarded. 500 μl of Buffer RW2 was added, and the mixture was centrifuged at 12,000 rpm for 20 seconds, and the waste liquid in the collection tube was discarded. After 2 minutes of air centrifugation, discard the waste liquid in the collection tube, incubate at room temperature for 5 minutes, add 30 μl of water, incubate at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, collect the RNA solution, and store the RNA at -70℃ to prevent degradation. The extracted RNA was then treated with DNase (Fermentas).

[0031] 2. Reverse transcription reaction: The reverse transcription step was performed using the Novizan R323 kit. The specific steps are as follows: Take 1 μg of K326 total RNA for reverse transcription, add 4 μl of 4×gDNA wiper Mix, and add deionized water to make up to 16 μl; incubate at 42℃ for 2 min, then add 4 μl of 5...

[0032] ×HiScript III qRT SuperMix, final reaction volume 20 μl; reaction at 37℃ for 15 min, then terminated by heating at 85℃ for 5 s. The obtained cDNA was stored at -20℃.

[0033] 3. Identification and Cloning of the NtMATE34 Gene: Analysis of transcriptome data before and after tobacco bacterial wilt inoculation revealed a tobacco gene, NtMATE34, whose expression was significantly upregulated after inoculation. The tobacco NtMATE34 gene has the accession number Nta13g14960.1 in the Tobacco Genome Database (http: / / lifenglab.hzau.edu.cn / Nicomics / ). This gene is annotated to encode a transporter protein, the specific function of which is unknown.

[0034] PCR amplification was performed using the In-fisuon method from Clontech. A pair of primers were artificially synthesized, and 20bp vector sequences were added to their 5' and 3' ends, respectively. The vector sequences were F: 5′-AGAACACGGGGGACGAGCTC-3′, as shown in SEQ ID NO.3; R: 5′-GATCCCCGGGTACCGAGCTC-3′, as shown in SEQ ID NO.4.

[0035] The upstream and downstream primers are as follows:

[0036] NtMATE34-F: 5′-ATGAGTCGCAACACAGAGCCC 3′, as shown in SEQ ID NO.5; NtMATE34-R: 5′-TTAGTCAGTTGCAGCTGCTCCTC3′, as shown in SEQ ID NO.6;

[0037] Using cDNA from whole tobacco seedlings of the K326 variety as a template, and NtMATE34-F and NtMATE34-R as primers, PCR amplification was performed using a high-fidelity 2×Phanta Max Master Mix (Dye Plus Vazyme); the 50 μl reaction system was designed as follows:

[0038]

[0039] The reaction procedure was as follows: 95℃ for 3 min pre-denaturation; 95℃ for 15 s; 56℃ for 15 s; 72℃ for 1 min; 35 cycles, with a final extension at 72℃ for 5 min. After the reaction, the PCR results were detected by electrophoresis. Following electrophoresis, the gel was excised and recovered under UV light using a gel extraction kit (TAKARA). The recovered amplified product was sequenced, identifying the NtMATE34 gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0040] Example 2: Analysis of NtMATE34 gene expression pattern after tobacco inoculation with bacterial wilt

[0041] Tobacco cultivar K326 was treated with bacterial wilt inoculation. Root damage treatment was performed before inoculation with the bacterial wilt pathogen. The specific method for root damage treatment was as follows: when the tobacco seedlings grew to the size of three leaves and one bud in indoor cultivation, they were transplanted into 9.0 cm diameter seedling pots, and then inoculated with bacterial wilt when they reached the size of four leaves and one bud. Before treating the tobacco plants with the preparation and bacterial wilt pathogen, root damage pretreatment was performed by cutting off one-third of the tobacco roots with sharp scissors before re-transplanting into flower pots. The specific inoculation method and process for bacterial wilt was as follows: the bacterial wilt strain CQPS-1, preserved in a -80℃ ultra-low temperature freezer, was taken out, NB medium was prepared, and the bacterial wilt pathogen was inoculated in a clean bench and cultured overnight at 28℃ / 220 rpm. Root drenching inoculation was used, with 10.0 mL of approximately 10 ml of the pathogen inoculated around the base of each tobacco seedling. 8 A bacterial suspension of CFU / mL was prepared. The ambient temperature was adjusted to 30°C and the humidity to 80% for incubation. Disease control was then conducted, and the proliferation of *Ralstonia solanacearum* in tobacco roots was assessed at 0, 2, 4, and 6 days. RNA was extracted from the roots of diseased tobacco plants using the method described in Example 1, and RNA was also extracted from the roots of uninoculated tobacco plants as a control. The extracted RNA was reverse transcribed to obtain cDNA, and real-time quantitative PCR was performed using the cDNA as a template (the reverse transcription steps were the same as in Example 1) with specific primers for NtMATE34. The primer sequences are as follows:

[0042] Real-time quantitative primer F: CAATTCTCCTTTTGGCTGGTCAAAC, as shown in SEQ ID NO.7.

[0043] The real-time quantitative primer R: TGAGCTTGTAAGAATTTCTGAATTGG, as shown in SEQ ID NO.8,

[0044] The results showed that the expression of the NtMATE34 gene was significantly upregulated 6 days after inoculation with bacterial wilt. Figure 1 (), approximately 7.6 times that of the control.

[0045] Example 3

[0046] Using the NtMATE34 gene obtained in Example 1, the inventors further constructed the overexpression vector pCHF3-NtMATE34 for transformation. The relevant process is briefly described below.

[0047] First, the NtMATE34 gene obtained in Example 1 was ligated with the pCHF3 plasmid digested with Sac I. Following the Clontech In-fusion seamless ligation instructions, a 10 μL ligation reaction system was established as follows: 2 μL 5x in-fusion; 4 μL pCHF3 (Sac I digested); 4 μL NtMATE34 gene amplification product; incubated at 50°C for 15 min on ice for the next transformation step. The ligation product was transformed into competent E. coli cells using a heat shock method. The specific procedure was as follows: Under aseptic conditions, 2 μL of the ligation product was added to competent cells, gently mixed, and incubated on ice for 30 min; heat-shocked at 42°C for 90 s, and the centrifuge tube was quickly transferred to ice for 2-3 min; 800 μL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C with gentle shaking for approximately 1 h; 200 μL of the culture solution was spread onto LB solid medium containing 100 μg / ml spectinomycin and incubated upside down at 37°C for 12-16 h.

[0048] White bacterial colonies were picked from the culture medium and inoculated into LB liquid medium containing 100 μg / ml spectinomycin, and cultured with shaking for 12-16 h. PCR verification was performed using self-primers and vector primers. The primer sequences are as follows:

[0049] Self-primer: CTTAATAGCTATTACAGCAAT, as shown in SEQ ID NO.9;

[0050] Vector primers: GTGTGTGCGCAATGAAACTG, as shown in SEQ ID NO.10;

[0051] Positive clones with correct results are further sent to the company for sequencing to ensure that the recombinant plasmid is constructed correctly.

[0052] Example 4

[0053] Using the heat shock method, the pCHF3-NtMATE34 vector prepared in Example 3 was transformed into Agrobacterium GV3101 (purchased from Beijing TransGen Biotech Co., Ltd.). Single colonies were selected for PCR verification to confirm that the expression vector pCHF3-NtMATE34 was successfully transformed into Agrobacterium.

[0054] Transform tobacco plants:

[0055] Transgenic tobacco plants were obtained using Agrobacterium-mediated leaf disc transformation. The specific steps are as follows:

[0056] 1. Aseptic seedling culture: Take an appropriate amount of K326 seeds, surface disinfect with 75% alcohol for 30 seconds, rinse three times with sterile water, then soak in 15% hydrogen peroxide for 8 minutes, rinse three times with sterile water, and then soak in sterile water for 24 hours. Sow the disinfected seeds on MS culture dishes. After the seedlings have grown three small leaves, transfer them to tissue culture bottles containing MS for culture. Culture in an artificial climate chamber for about 45 days, and select healthy leaves for Agrobacterium infection.

[0057] 2. Agrobacterium infection: Take the correctly identified and preserved Agrobacterium culture, and after complete thawing, transfer 500 μL to 50 mL of YEP liquid medium with the appropriate antibiotic resistance. Incubate at 28℃ / 220 rpm until the OD600 reaches 0.6. Take 50 mL of the culture, centrifuge at 4000 rpm for 10 minutes, and discard the supernatant. Collect the bacterial cells and resuspend them until the OD600 reaches 0.6. Then add AS to a final concentration of 20 mg / L for infection. Cut off the leaf edges of sterile seedlings and cut the leaves into 1 cm sections along the main vein. 2 The leaf discs were immersed in Agrobacterium infection solution for 5 minutes.

[0058] 3. Co-culture and Subculture: Infected leaves were removed, dried with filter paper to remove Agrobacterium, and laid flat on the co-culture medium with the leaf surface facing down. The leaves were then placed in a climate chamber and cultured in the dark for 3 days. S1 Subculture: Leaves cultured for 3 days were transferred to S1 differentiation medium with the leaf surface facing up. After about one week of dark culture in a climate chamber, they were transferred to light and cultured until buds of about 0.5 cm appeared at the leaf margins. S2 Subculture: Buds from S1 were transferred to S2 differentiation medium. Leaves that had not yet developed buds were removed. The plants were cultured under light for 2 weeks until the buds developed into seedlings. S3 Subculture: Seedlings from S2 were transferred to S3 differentiation medium and cultured under light for 2 weeks. Rooting Culture: Swollen parts and yellowing leaves at the base of the seedlings were removed. The seedlings were transferred to tissue culture flasks containing rooting medium and cultured under light for 2 weeks.

[0059] 4. Obtaining genetically modified tobacco: When the seedlings have developed about 8 roots, each about 3cm long, open the cap of the culture bottle to harden them off. After 3 days, transfer the seedlings to flowerpots filled with sterile soil and cover them with plastic film to keep them warm. Remove the plastic film after one week to allow them to grow rapidly under natural conditions.

[0060] 5. Positive Identification of Transgenic Tobacco: Total RNA was extracted from the transgenic tobacco plants obtained in Example 1, and cDNA was obtained by reverse transcription. Real-time quantitative PCR was performed using the specific primers for the NtMATE34 gene from Example 2. The results showed that, compared with the control (the control refers to plants not transformed by Agrobacterium), the expression level of the NtMATE34 gene in the OE2 and OE3 transgenic tobacco plants was significantly increased, by 111.3 times and 62.9 times, respectively, compared with the control. Figure 2 ).

[0061] Bacterial wilt resistance identification

[0062] The positive plants OE2 and OE3 of the above-mentioned tobacco transgenic pCHF3-NtMATE34 were transferred to a greenhouse for cultivation, self-pollination, and transgenic seeds were collected. Meanwhile, tobacco K326 transgenic with the empty pCHF3 vector was used as a control (K326) for subsequent bacterial wilt resistance identification.

[0063] When the plants reached 45 days of growth, the bacterial wilt inoculation and investigation methods described in Example 2 were used to treat NtMATE34 gene-overexpressing tobacco plants and pCHF3 empty vector-transformed tobacco control plants with bacterial wilt, respectively. The disease incidence was observed 6 days later. Figure 3 As shown, compared with the control, tobacco plants overexpressing the NtMATE34 gene exhibited significant resistance to bacterial wilt. Furthermore, regarding the proliferation of Bacterium bacterial wilt in the tobacco roots, the proliferation rate of Bacterium bacterial wilt in the roots of tobacco plants overexpressing the NtMATE34 gene was significantly slower than that in the control. Figure 4 In summary, overexpression of the NtMATE34 gene in tobacco significantly enhances resistance to tobacco bacterial wilt.

[0064] Sequence Listing SEQ ID NO: 1

[0065] ATGAGTCGCAACACAGAGCCCCTTCTTGATAATGATCAGCTGGACGGAGAAGAAAAGCAGGCGGTGAGT

[0066] AGACGTGGCAATAACGACGACGTTGGATTTGTTAAGGAATTTGGTTGGGAGTCGAAGAGGCTATGGGAG

[0067] CTGGCTGGTCCTGCCATTTTCACTACCGTATGTCAGTATTCACTTGGTGCACTCACTCAGACTTTTGCC

[0068] GGTCAAATTGGGGAGCTTGAACTGGCTGCTGTCTCTATTGAAAACTCTGTTATTACCGGCCTTGCTTTT

[0069] GGTGTCATGCTGGGGATGGGAAGTGCATTGGAGACATTATGCGGGCAAGCATTTGGTGCAGGACATCTG

[0070] AGGATGCTGGGAATATACATGCAAAGATCTTGGGTTATTTTGCTCACCACGGCTTGTATTTTGGTTCCC

[0071] GTTTATGTCTTTTCTCCTCCAATTCTCCTTTTGGCTGGTCAAACCACTCGCATCTCAAATGCTGCTGGA

[0072] AAATTTGCTTTGTGGATGATACCACAATTGTTTGCTTATGCAATGAACTTTCCAATTCAGAAATTCTTA

[0073] CAAGCTCAGAGGAAAGTGTTAGTGATGGCATGGATATCTGCTGGTGTTCTTGTGCTACATGTCCTTTTT

[0074] AGCTGGTTATTCATACTGAAACTTAAATGGGGATTAATTGGAGCAGCTGTCACTCTGAATATCTCGTGG

[0075] TGGCTCATTGTAATTCTACAGCTTCTCTATATCTTTGTCACCAAATCTGATGGAGCTTGGACTGGTTTC

[0076] TCATCCTTGGCATTTCAAGACTTGTTTGGATTTGTCAAGCTATCTTTGGCCTCTGCTGTCATGTTATGC

[0077] TTGGAGTTCTGGTATTTGATGATACTGATCATAATCACAGGTCTTCTGCCTAACCCTTTGGTGCCAGTT

[0078] GATGCTATGTCTATATGCATGAACATTAATGGATGGCAGGCCATGATTTCTTTAGGATTTAATGCTGCC

[0079] ATCAGTGTGAGAATATCAAATGAACTTGGAGCTGGTAATGCACGACTTGCAAAATTTTCAGTGATTGTA

[0080] GTCTCCTTAACATCAACTGTCATAGGAGTAGCTTGCATGATTGTCGTGCTAGCAACAAGAGAATATTTT

[0081] CCCTACCTTTTCACTAACAGTGAAGCAGTTGCCAATGAGACAAAGAAACTGGCAATGTTGCTTGCCTGG

[0082] ACAGTTCTATTAAACAGCCTTCAGCCTGTCTTATCCGGTGTTGCTGTTGGAGCTGGATGGCAGGCACTT

[0083] GTGGCATATTAACATTGCCTGTTACTATATTGTTGGTTTGCCGGCTGGTGTACTTTTGGGCTTCAAA

[0084] TTTGATTTTGGAGCTATGGGAATCTGGGGTGGGATGATTGGAGGCATTTGTTTGCAGACCATTATCTTA

[0085] ATAGCTATTACAGCAATGACAAACTGGGAGAAAGAGGCAAGTCTAGCTGCAAGTCGTGTAAAGCAATGG

[0086] GGAGGAGCAGCTGCAACTGACTAA

[0087] SEQ ID NO:2

[0088] MSRNTEPLLDNDQLDGEEKQAVSRRGNNDDVGFVKEFGWESKRLWELAGPAIFTTVCQYSLGALTQTFA

[0089] GQIGELELAAVSIENSVITGLAFGVMLGMGSALETLCGQAFGAGHLRMLGIYMQRSWVILLTTACILVP

[0090] VYVFSPPILLLAGQTTRISNAAGKFALWMIPQLFAYAMNFPIQKFLQAQRKVLVMAWISAGVLVLHVLF

[0091] SWLFILKLKWGLIGAAVTLNISWWLIVILQLLYIFVTKSDGAWTGFSSLAFQDLFGFVKLSLASAVMLC

[0092] LEFWYLMILIIITGLLPNPLVPVDAMSICMNINGWQAMISLGFNAAISVRISNELGAGNARLAKFSVIV

[0093] VSLTSTVIGVACMIVVLATREYFPYLFTNSEAVANETKKLAMLLAWTVLLNSLQPVLSGVAVGAGWQAL

[0094] VAYINIACYYIVGLPAGVLLGFKFDFGAMGIWGGMIGGICLQTIILIAITAMTNWEKEASLAASRVKQW

[0095] GGAAATD

Claims

1. An application of the tobacco NtMATE34 gene in regulating bacterial wilt resistance, characterized in that, The gene CDS sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The gene sequence also includes gene sequences with similar functions, having a similarity of not less than 95%; the similar functions include expressing proteins that resist bacterial wilt.

3. The application according to claim 2, characterized in that, The specific target for combating bacterial wilt is tobacco.

4. The application of tobacco NtMATE34 protein in regulating bacterial wilt resistance, characterized in that, The protein sequence is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, The protein sequences also include protein sequences with similar functions, having a similarity of not less than 95%; the similar functions include resistance to bacterial wilt.

6. The application according to claim 5, characterized in that, The specific target for combating bacterial wilt is tobacco.

7. The use of the tobacco NtMATE34 gene according to any one of claims 1-3, or the tobacco NtMATE34 protein according to any one of claims 4-6, in improving resistance to bacterial wilt.

8. The application according to claim 7, characterized in that, Used to improve tobacco's resistance to bacterial wilt.

9. The application according to claim 8, characterized in that, By overexpressing the tobacco NtMATE34 gene, tobacco transformants with enhanced resistance to bacterial wilt were obtained.

10. The application according to claim 9, characterized in that, The vector for overexpressing the tobacco NtMATE34 gene is pCHF3.

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