Use of abh i gene in preventing and treating bacterial wilt

Abstract

This invention discloses abhI The application of genes in the prevention and control of bacterial wilt. This invention involves... abhI Gene recombination and transformation yielded corresponding transformants, which demonstrated their ability to efficiently degrade methyl 3-hydroxymyristate, a quorum sensing signal molecule of Ralstonia solanacearum, thereby weakening the pathogen's virulence and achieving effective control of bacterial wilt. This invention provides a new strategy for the prevention and control of bacterial wilt in crops and the breeding of disease-resistant varieties, possessing significant scientific value and industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control and genetic engineering application technology, specifically involving abhI Application of genes in the prevention and control of bacterial wilt. Background Technology

[0002] Composed of Solanaceae Raulella complex ( Ralstonia solanacearum Bacterial wilt caused by the soil-borne pathogen RSSC is one of the ten most devastating bacterial plant diseases worldwide. This soil-borne pathogen can infect more than 450 species of plants from 54 families, including agricultural and economic crops such as tomatoes, peppers, peanuts, potatoes, ginger, and tobacco, as well as forest trees such as mulberry, eucalyptus, and casuarina. Due to its extremely high infectivity, it often causes severe economic losses in agriculture and forestry.

[0003] Currently, the main control strategies for Ralstonia solanacearum include chemical control, agricultural control, and biological control. For chemical control, treating seeds with a 10% trisodium phosphate solution or a 1% potassium permanganate solution can effectively prevent bacterial wilt. However, long-term or excessive use of chemical agents can easily lead to soil compaction, reduced fertility, and adverse effects on the ecological environment. Agricultural control is mainly achieved through breeding disease-resistant varieties. Although this method is widely used, the breeding cycle for disease-resistant varieties is long, and long-term large-scale planting of a single disease-resistant variety can easily lead to loss of resistance. Biological control utilizes biological resources to control pests and diseases, and is a green and sustainable control method. Among them, quorum quenching (QQ) technology weakens the pathogen's pathogenicity by interfering with the pathogen's quorum sensing system without inducing resistance evolution, showing broad application prospects.

[0004] Methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) are unique quorum sensing signaling molecules produced by Ralstonia solanacearum, making them ideal molecular targets for the control of bacterial wilt. Therefore, identifying genes that can degrade 3-OH PAME / MAME is of significant theoretical and practical value for developing QQ-based biocontrol strategies for bacterial wilt. Summary of the Invention

[0005] To address the aforementioned shortcomings of the prior art, this invention discloses... abhI Application of the gene in the control of bacterial wilt. By recombining and transforming the gene into a host bacterium, a recombinant engineered bacterium was obtained. Experiments showed that this strain could efficiently degrade the quorum sensing signal molecule of Ralstonia solanacearum—methyl 3-hydroxymyristate (3-OH MAME). This discovery provides a new strategy for the control of bacterial wilt and the breeding of new bacterial wilt-resistant varieties, and also lays the foundation for the application of innovative biological control methods based on quorum sensing quenching in crop protection and global food security.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention firstly... abhI The gene was cloned into the pUC19 plasmid, the recombinant plasmid pUC19-abhI was constructed, and it was transformed into Escherichia coli. Escherichia coli DH5 α Recombinant engineered bacteria were obtained from the strain. When this strain was applied to tomato plants infected with *Ralstonia solanacearum* (Syndrome aeruginosa NS25), it significantly enhanced the plants' resistance to bacterial wilt. Therefore, this invention provides... abhI The application of genes in the prevention and control of bacterial wilt, and their application in the preparation of drugs for the prevention and control of bacterial wilt.

[0007] The first objective of this invention is to provide abhI The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The second object of the present invention is to provide an expression abhI Recombinant gene expression vector. The expression vector may be pUC19 or other suitable expression vectors.

[0009] The third object of the present invention is to provide an expression abhI Genetically engineered bacteria. The host bacteria of the genetically engineered bacteria can be Escherichia coli, specifically DH5α. α strains.

[0010] A fourth objective of this invention is to provide a fungicide for controlling bacterial wilt of plants, comprising the expressed... abhI Genetically engineered bacteria and bacterial agents.

[0011] The fifth object of the present invention is to provide abhI Application of genes in the prevention and control of bacterial wilt in plants.

[0012] Preferably, the bacterial wilt is a plant disease caused by *Laurella multocida*, a member of the Solanaceae family.

[0013] Preferably, the plants are tomatoes, eggplants, peppers, potatoes, tobacco, ginger, and casuarina.

[0014] Preferably, the method for controlling bacterial wilt in plants involves degrading methyl 3-hydroxymyristic acid (3-OH MAME), a quorum sensing signal molecule produced by *Raylordia solani*. Experiments have shown that... abhI Transformants obtained after gene recombination expression can effectively degrade the signaling molecule, weaken the pathogenicity of the pathogen, and thus achieve effective control of bacterial wilt. abhIGene-based control of bacterial wilt involves inhibiting the pathogenicity of *Rauvolfia solanaceae* by degrading quorum sensing signaling molecules produced by *Rauvolfia solanaceae*.

[0015] The sixth objective of this invention is to provide a method for preventing and controlling bacterial wilt of plants, by applying the genetically engineered bacteria or the bacterial agent to plants, or constructing an expression... abhI Genetically modified plants.

[0016] Preferably, the application method is root drenching or injection.

[0017] This invention also relates to abhI The application of the gene in the construction of transgenic plants resistant to bacterial wilt: by recombinating and transforming the gene into Agrobacterium and achieving functional expression, a new gene is provided for the genetic control of bacterial wilt.

[0018] The present invention has the following beneficial effects: This invention will abhI Gene recombination and transformation yielded transformants that not only possess the ability to degrade ester substrates (such as tributyrate), but also degrade the quorum sensing signal molecule 3-OH MAME of Ralstonia solanacearum through hydrolysis, thereby significantly enhancing resistance to bacterial wilt. This invention provides a new gene resource and application strategy for the biological control of bacterial wilt, and has significant scientific and industrial application value. Attached Figure Description

[0019] Figure 1 To construct the obtained DH5α(pUC19- abhI PCR verification results of recombinant transformants; Note: Lane M is the 2K DNA marker, lanes 1-3 are the amplification bands of the empty vector DH5α (pUC19) strain, lanes 4-6 are the amplification bands of DH5α (pUC19- abhI Amplification bands of the strain.

[0020] Figure 2 For DH5α(pUC19- abhI The esterification of tricresyl glycerol by the invertor.

[0021] Figure 3 For DH5α(pUC19- abhI The degradation rate of 3-OH MAME by the transformant.

[0022] Figure 4 For DH5α(pUC19- abhI The biocontrol effect of the transformant on tomato seedlings inoculated with Ralstonia solanacearum NS25. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1: Construction of Transformants I. Extraction of pUC19 plasmid DNA E. coli containing pUC19 plasmid E. coli DH5 α The bacterial strain was streaked onto LB solid medium containing 50 μg / mL ampicillin (Amp) and incubated at 37°C for 12 hours. Single colonies with good growth were picked and inoculated into 10 mL of LB liquid medium containing 50 μg / mL Amp, and cultured at 37°C with shaking at 200 rpm until the bacteria reached the logarithmic growth phase. pUC19 plasmid DNA was extracted using a plasmid miniprep kit and... Hind III and Bam HI restriction endonuclease was used to digest the DNA fragments at 37°C for 30 min. The digestion products were separated by 1% agarose gel electrophoresis, and the target DNA fragments were recovered using a universal DNA purification and recovery kit.

[0025] two, abhI Amplification of the target gene fragment Tohoni Pseudomonas ( Pseudomonas tohonis Using Q4-3 (Genome Accession Number: CP115820.1) genomic DNA as a template, the primer pairs shown in Table 1 were used ( abhI -F: 5'-gaccatgattacgccaagcttATGAAAAAACTGCTT- 3'; abhI -R: 5'-gagctcggtacccggggatccTCACTGCGAATCGAG-3'), and the target gene was processed according to the PCR reaction system in Table 1. abhI PCR amplification of the fragment. PCR reaction conditions were: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 33 cycles; final extension at 72℃ for 5 minutes. The amplified product was purified using a DNA purification kit. abhI Gene fragment. Amplified. abhI The nucleotide sequence of the gene is shown in SEQ ID NO.1 (924 bp).

[0026] Table 1 PCR reaction system III. DH5α(pUC19- abhI Transformer construction According to the ClonExpress II One Step Cloning Kit instructions, the above abhI The gene fragment was ligated with the target DNA fragment of the enzyme-digested pUC19 plasmid. Subsequently, the product was added to 100 μL of *E. coli* DH5α. α Competent cells were incubated on ice for 30 minutes, then heat-shocked in a 42°C water bath for 45 seconds, followed immediately by ice-cooling for 5 minutes. 600 μL of LB liquid medium was then added, and the cells were cultured at 37°C with shaking at 200 rpm for 1 hour. After centrifugation at 4000 rpm for 5 minutes, most of the supernatant was discarded, retaining approximately 100 μL of bacterial culture and the precipitated cells. The cells were gently resuspended and spread onto LB solid medium containing 50 μg / mL Amp, and cultured at 37°C for 15 hours. Finally, transformants were screened and cultured using primer pairs M13-F (5'-GTAAAACGACGGCCAG-3') and M13-R (5'-CAGGAAACAGCTATGAC-3') to obtain transformants containing the recombinant plasmid pUC19- abhI The transformant was named DH5α(pUC19- abhI ), transformant amplification bands as follows Figure 1 As shown.

[0027] Example 2: Determination of the esterification ability of the transformants DH5α(pUC19-) were selected from Example 1. abhI Single colonies of DH5α (pUC19) were inoculated into LB liquid medium containing 50 mg / L Amp and cultured with shaking at 28°C and 200 rpm until the bacterial concentration reached OD500. 600 = 1.2 is reserved for later use.

[0028] Pour 40 mL of LB medium containing 1% (v / v) tribonitrile into a 12×12 cm square petri dish to prepare a plate. Punch holes using a 5 mm diameter punch. Add 20 μL of the bacterial culture to each hole and incubate the plate upright at 28°C for 4 days. After incubation, wash away the bacterial cells and photograph the plate. Measure the diameter of the clear degradation zone using ImageJ 1.52a software. Repeat the experiment three times.

[0029] The results are as follows Figure 2 As shown, DH5α(pUC19- abhI Both strains DH5α and DH5α(pUC19) were able to degrade glycerol tartrate to some extent. Compared with the control strain DH5α(pUC19), DH5α(pUC19- abhI The transformants exhibited stronger esterification ability.

[0030] Example 3DH5α(pUC19- abhI Determination of the degradation effect of transformants on 3-OH MAME Take the DH5α(pUC19-) obtained in Example 1 abhI Single colonies of strains DH5α and DH5α(pUC19) were inoculated into LB liquid medium containing 50 mg / L Amp and cultured with shaking at 28°C and 200 rpm until the bacterial culture reached OD. 600 = 1.2. Subsequently, the culture was inoculated into MSM medium containing 0.5 μM 3-OH MAME and cultured with shaking at 28°C and 200 rpm for 24 hours. The residual concentration of 3-OH MAME in the culture medium was determined by LC-MS.

[0031] LC MS analysis conditions are as follows: Instrument model: Thermo Fisher Scientific Q Exactive Focus liquid chromatography-mass spectrometry system; Chromatographic column: Waters ACQUITY UPLC HSS T3 Column (SKU: 186003539, 100Å, 1.8 μm, 2.1×100 mm). Flow rate: 0.3 mL / min; Column temperature: 40℃; Mobile phase: methanol / water (80:20, v / v); Characteristic ion: [M+Na] + Molecular weight: 281.20, elution time: 3.0-4.0 minutes, injection volume: 10 μL.

[0032] The results are as follows Figure 3 As shown, in the quantitative degradation experiment using 3-OH MAME as a substrate, compared with the negative control DH5α (pUC19), the expression abhI The transformant DH5α(pUC19- abhI The degradation rate of the substrate 3-OH MAME reached 88.80% within 24 hours.

[0033] Example 4DH5α(pUC19- abhI Biocontrol efficacy determination of transformants mixed with Ralstonia solanacearum NS25 The DH5α(pUC19- obtained in Example 1 abhIStrains DH5α and DH5α (pUC19) were activated by streaking on LB agar containing 50 mg / L Amp, while Ralstonia solanacearum NS25 was activated by streaking on TTC agar. Single colonies were picked and inoculated into the corresponding liquid media and cultured with shaking to obtain seed culture. The next day, the seed culture was transferred to 200 mL of the corresponding fresh liquid media at a ratio of 1:100 and cultured with shaking at 28°C and 200 rpm until the bacterial growth rate reached OD. 600 It reached 1.2.

[0034] Tomato seedlings were inoculated using the root-damage drenching method. The roots of the tomato seedlings were lightly pruned with sterile scissors. The following treatments were established: Ralstonia solanacearum NS25 bacterial solution + LB liquid medium (positive control group), LB liquid medium + TTC liquid medium (negative control group), Ralstonia solanacearum NS25 bacterial solution + DH5α (pUC19) bacterial solution, and Ralstonia solanacearum NS25 bacterial solution + DH5α (pUC19- abhI ) bacterial suspension, DH5α (pUC19- abhI The bacterial culture solution and TTC liquid medium were mixed at a 1:1 volume ratio in each treatment combination. Each tomato seedling was watered with 20 mL of the mixture, and the plant growth was observed and recorded regularly. When all tomato seedlings in the control group had died, all plants in all groups were photographed and recorded. Each treatment was replicated in triplicate, and the entire experiment was repeated twice at different time points.

[0035] Experimental results showed that the group treated with Ralstonia solanacearum NS25 bacterial suspension + LB liquid medium exhibited severe wilting symptoms in tomato plants within 7 days after inoculation; while the group treated with Ralstonia solanacearum NS25 bacterial suspension + DH5α (pUC19- abhI The disease severity of tomato plants in the bacterial suspension group was significantly lower than that in the Ralstonia solanacearum NS25 bacterial suspension + LB liquid medium group and the Ralstonia solanacearum NS25 bacterial suspension + DH5α (pUC19) bacterial suspension group, indicating that DH5α (pUC19- abhI It has a certain control effect on bacterial wilt of tomato caused by Ralstonia solanacearum. The LB liquid medium + TTC liquid medium group and the DH5α (pUC19-) group showed similar effects. abhI No plantlets in the bacterial culture + TTC liquid medium group showed mortality, indicating that the recombinant transformant DH5α(pUC19-) was effective. abhI It itself is not pathogenic to tomato plants. Figure 4 ).

Claims

1. abhI Genes, characterized by, The nucleotide sequence is shown in SEQ ID NO.

1.

2. A representation of the invention described in claim 1 abhI Recombinant gene expression vectors.

3. A representation of the invention described in claim 1 abhI Genetically engineered bacteria.

4. A fungicide for controlling bacterial wilt of plants, comprising the expression described in claim 3. abhI Genetically engineered bacteria and bacterial agents.

5. The claim 1 abhI Application of genes in the prevention and control of bacterial wilt in plants.

6. The application according to claim 5, characterized in that, The bacterial wilt mentioned above is a plant disease caused by Raulella ovalis, a member of the Solanaceae family.

7. The application according to claim 5, characterized in that, The plants mentioned are tomatoes, eggplants, peppers, potatoes, tobacco, ginger, and casuarina.

8. The application according to claim 5, characterized in that, The method described for controlling bacterial wilt of plants involves degrading methyl 3-hydroxymyristate, a quorum sensing signal molecule produced by *Raylorhizium anisopliae*.

9. A method for preventing and controlling bacterial wilt in plants, characterized in that, Apply the genetically engineered bacteria of claim 3 or the bacterial agent of claim 4 to plants, or construct a system expressing the bacterial agent of claim 1. abhI Genetically modified plants.

10. The method according to claim 9, characterized in that, The application method is either root drenching or injection.

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