Application of the Xanthomonas oryzae hexose phosphomutase-encoding gene PXO_03174 in regulating plant disease susceptibility

Abstract

The present invention belongs to the technical field of plant genetic engineering and relates to a Xanthomonas oryzae ( Xanthomonas oryzae ) Application of hexose phosphate mutase encoding gene in regulating the susceptibility of cruciferous plant Arabidopsis thaliana, the present invention constructs a recombinant plasmid and combines Xanthomonas oryzae Xanthomonas oryzae pv. rice ( Xoo )PXO99 A Hexose phosphate mutase encoding gene PXO_03174 As a target gene, it was introduced into the cruciferous plant Arabidopsis thaliana, and it was found that plants overexpressing this gene were resistant to the pathogenic bacteria Pseudomonas syringae. Pseudomonas syringae pv. tomato ( Psst ) DC3000, showing significantly enhanced susceptibility. The findings of this invention can be used to develop more sensitive bacterial disease detection methods, thereby improving the accuracy of bacterial disease detection. They can also be used to prepare pathogen-sensitive plants, identify toxic genes of plant pathogens such as Pseudomonas syringae, and study the mechanisms of plant infection. They can also be used to study plant resistance to Pseudomonas syringae and develop antibacterial agents.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a gene encoding hexose phosphate mutase of Xanthomonas oryzae. Background Art

[0002] Xanthomonas are a group of Gram-negative bacteria belonging to the kingdom Bacteria, phylum Primonae, group Gamma-Prionae, class Fermentobacteria, orders Xanthomonadales, and family Xanthomonadaceae. They are obligate aerobic, chemoorganic plant pathogens that infect over 400 staple and cash crops, such as rice, wheat, citrus, and tomatoes, severely impacting agricultural production. Xanthomonas have complex pathogenic mechanisms, utilizing a variety of plant nutrients to enhance their pathogenicity.

[0003] Xanthomonas oryzae is a plant pathogen that specifically causes rice diseases. Among them, rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a major bacterial disease in rice production worldwide, severely impacting rice yield and quality. PXO_03174 encodes the gene for the hexose phosphomutase XanA in Xoo. XanA, a carbon source metabolizing enzyme, is involved in carbohydrate metabolism in bacteria. Furthermore, studies have shown that XanA is a key pathogenicity factor in Xoo. Mutational studies of the Xan A encoding gene, PXO_03174, revealed that knockout mutants of the PXO_03174 gene exhibited significantly reduced pathogenicity in rice compared to wild-type strains, nearly eliminating it. This suggests that XanA is essential for the pathogenicity of Xoo. In addition, XanA is also involved in regulating life activities such as the formation of Xoo biofilm, synthesis of extracellular polysaccharides, energy metabolism, flagellar movement and cell wall synthesis, which are essential for pathogenic bacteria to adapt to the external environment and attach and survive on the surface of plant hosts.

[0004] Microbial genomes, including bacterial genomes, contain a rich reservoir of gene sequences. By introducing specific microbial genes into plants, they can confer novel phenotypic traits. Their application in plants has gradually become a key development direction in modern agricultural synthetic biology and agricultural biotechnology. The most successful example to date is the Bacillus thuringiensis (Bt) gene, which confers resistance to lepidopteran pests. As an important model plant, Arabidopsis thaliana is widely used to study the functions of exogenous genes in plants due to its short growth cycle, high fruit set rate, and mature transformation research system. Furthermore, the interaction system between Pseudomonas syringae Pst DC3000 and its host plant, Arabidopsis thaliana, has also become a popular tool for studying the regulation of plant disease resistance and susceptibility by exogenous genes. Summary of the Invention

[0005] PXO_03174 is a key carbon source metabolism gene in Xanthomonas oryzae pv.oryzae (Xoo), a pathogenic blight pathogen of rice. This gene encodes the hexose phosphomutase XanA, which plays a crucial role in the pathogenicity of plant pathogens. However, the function and role of the pathogenic bacterial hexose phosphomutase XanA in plants have not yet been fully understood, requiring further research and development.

[0006] Through extensive research, the present invention discovered that overexpression of the PXO_03174 gene in the cruciferous plant Arabidopsis thaliana weakens the plant's resistance to Pst DC3000, thereby enhancing its susceptibility to the disease. Therefore, the gene can be used to prepare plant materials susceptible to pathogenic bacteria, identify virulence genes in pathogenic bacteria that infect Arabidopsis thaliana, and study their pathogenicity. It can also be used to study Arabidopsis thaliana's resistance to pathogens such as Pst DC3000 and develop antibacterial chemicals. Therefore, the present invention provides the following technical solutions:

[0007] In one aspect of the present invention, the present invention provides an application of a Xanthomonas oryzae gene PXO_03174, wherein PXO_03174 is used to regulate the sensitivity of plants to diseases. The sequence of the gene PXO_03174 is shown in SEQ ID NO.1.

[0008] In the present invention, the regulation of plant sensitivity to disease is that the gene PXO_03174 is highly expressed in the plant relative to the wild type, which makes the plant susceptible to disease infection.

[0009] Preferably, the plant is a plant of the Poaceae family or the Cruciferae family.

[0010] Preferably, the plant is rice or Arabidopsis thaliana.

[0011] In an exemplary embodiment of the present invention, the disease is Xanthomonas oryzae pv.oryzae (Xoo) PXO99 A , or Pseudomonas syringae pv.tomato (Pst) DC3000.

[0012] In one embodiment, regulating the sensitivity of a plant to a disease is to increase the sensitivity of the plant to infection by the plant pathogenic bacterium Pseudomonas syringae by overexpressing the gene PXO_03174; preferably, the Pseudomonas syringae is Pseudomonas syringae Pst DC3000.

[0013] In one aspect, the present invention provides a method for preparing plants that are sensitive to infection by Pseudomonas syringae. The recombinant expression vector is constructed using the Xanthomonas gene PXO_03174 and introduced into the plant for overexpression.

[0014] Preferably, the vector is pCAMBIA2300. The recombinant expression vector is introduced into plants via Agrobacterium tumefaciens for overexpression.

[0015] In one aspect, the present invention provides a method for obtaining a Cruciferae plant material susceptible to pathogenic bacteria. The Xanthomonas gene PXO_03174 of the present invention is introduced as a target gene into the genome of a Cruciferae plant for overexpression, thereby cultivating a Cruciferae plant variety susceptible to pathogenic bacteria. Preferably, the Cruciferae plant is Arabidopsis thaliana.

[0016] The present invention has the beneficial effect of discovering for the first time that overexpression of the PXO_03174 (xanA) gene in plants affects their susceptibility to infection by Pseudomonas syringae. By constructing transgenic plants overexpressing the PXO_03174 (xanA) gene, pathogen-susceptible plant varieties can be obtained. These plants are applicable to functional studies of toxic genes in plant pathogens such as Pseudomonas syringae, as well as to research into the mechanisms of plant infection. They can also be used to study plant resistance to Pseudomonas syringae and develop antimicrobial chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the cloning map of the Xanthomonas oryzae gene PXO_03174;

[0018] Figure 2 Schematic diagram of the pCAMBIA2300-PXO_03174 recombinant vector structure;

[0019] Figure 3This is a WB identification image for screening PXO_03174 overexpression positive seedlings;

[0020] Figure 4 Figure 2 shows the disease phenotype of PXO_03174 transgenic plants and wild-type plants (CK) inoculated with Pst DC3000;

[0021] Figure 5 Figure 2 shows a quantitative analysis of the incidence of PXO_03174 transgenic plants and wild-type plants (CK) inoculated with Pst DC3000. The greater the dilution of the ground plant samples, the fewer successful infection colonies were found on the culture plates. Significant differences were observed between the two groups of samples at 10-fold, 100-fold, 1000-fold, and 10,000-fold dilutions. The number of successful infection colonies in the PXO_03174 transgenic plants (35S::31742# and 35S::317413#) was significantly higher than that in the non-transgenic plants, indicating that the PXO_03174 transgenic plants were more sensitive to infection by pathogenic bacteria and were susceptible to the disease. DETAILED DESCRIPTION

[0022] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] Example 1

[0024] 1. Materials

[0025] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0026] 2. Methods

[0027] 2.1 Rapid extraction of bacterial genomic DNA

[0028] (1) Sample processing

[0029] Gram-negative bacteria: Transfer 1 mL of overnight bacterial culture to a 1.5 mL centrifuge tube. Centrifuge at 8,000 rpm for 1 minute at room temperature. Discard the supernatant to collect the cells. Add 400 μL of Buffer Digestion and vortex to mix. Incubate at 65°C in a water bath for 1 hour until the cells are completely lysed.

[0030] (2) Add 200 μL of Buffer PB, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 min.

[0031] (3) Centrifuge at 10,000 rpm for 5 min at room temperature and transfer the supernatant (500-550 μL) to a new 1.5 mL centrifuge tube.

[0032] (4) Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0033] (5) Add 1 mL of 75% ethanol, rinse by inversion for 1-3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant.

[0034] (6) Repeat step (5) once.

[0035] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol is completely evaporated.

[0036] (8) Dissolve the obtained DNA in 50-100 μL TE Buffer. The extracted DNA can be used immediately for the next step or stored at -20°C.

[0037] 2.2 Construction of recombinant plasmid

[0038] The CDS sequence of the Xanthomonas gene PXO_03174 published in the database (as shown in SEQ ID NO.1, sequence source: gene number PXO_03174) was used to design primers using the homologous recombination method and perform PCR amplification reaction. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. Figure 1 As shown, the PCR product (amplified Xanthomonas gene PXO_03174 sequence) was detected by 0.1% agarose gel electrophoresis.

[0039] The cloned Xanthomonas gene PXO_03174 CDS sequence was inserted into Figure 2 The vector pCAMBIA2300 (the pCAMBIA 2300 vector is a Ca Mv35S promoter sequence inserted into the MCS of pCAMBIA 2300 by double digestion with EcoRI and SacI, and the promoter is used to promote the target sequence to achieve the purpose of overexpression) is shown to obtain a recombinant plasmid (pCAMBIA 2300-PXO_03174), which can then be transformed into Escherichia coli.

[0040] Table 3 PCR reaction system for target gene amplification

[0041]

[0042]

[0043] Table 4 PCR reaction program for target gene amplification

[0044]

[0045] 2.3 Extraction of small amounts of plasmid from E. coli

[0046] (1) Take a sterilized test tube and add 8 mL of LB medium and the corresponding antibiotic at a ratio of 1 / 1000. Select a single clone in the test tube and place the test tube in a shaker at 37°C, 200 rpm, and incubate overnight. Ensure sterile operation.

[0047] (2) Use a 2 mL centrifuge tube to collect the bacterial suspension, centrifuge 2-3 times, and discard the supernatant.

[0048] (3) Resuspend the cells in 200 μL of Solution I.

[0049] (4) Add 300 μL of Solution II. Note that Solution II must be prepared immediately before use. After adding Solution II, slowly invert the centrifuge tube 10 times to allow the bacteria to fully lyse. Let it stand for 2 minutes. After adding Solution II, the total time should not exceed 5 minutes.

[0050] (5) Add 300uL of Solution III. Slowly invert the centrifuge tube 10 times, then add 25mg / ml RNase A

[0051] Add 5uL, mix slowly by inverting up and down again, and let it stand at room temperature for more than 15 minutes.

[0052] (6) Centrifuge at 12,000 g for 15 min in a small centrifuge at room temperature. Use a pipette to transfer 700 μL of the supernatant to a new 2 mL centrifuge tube, taking care not to aspirate the white precipitate at the bottom of the tube. Discard the old centrifuge tube.

[0053] (7) Add 700uL of phenolform (Tris-phenol and chloroform 1:1), shake thoroughly and mix, let it stand for 3 minutes, and then centrifuge it at 8000g for 15 minutes in a small centrifuge at room temperature. At this time, the solution in the centrifuge tube is separated into layers, the upper layer is the water layer, and the lower layer is the organic layer of phenolform. Use a pipette to carefully draw 600uL of the water layer and transfer it to a new 1.5mL centrifuge tube, taking care not to draw the organic layer liquid. Discard the old centrifuge tube, add 0.7 times the volume of isopropanol (420uL), shake thoroughly and mix, and let it stand in a -20℃ medical refrigerator for more than 15 minutes.

[0054] (8) Centrifuge at 12000g for 15 min in a small centrifuge at room temperature and discard the supernatant.

[0055] (9) Add 1 mL of 75% ethanol and centrifuge at 12,000 g for 5 min at room temperature. Discard the supernatant. Pipette away any remaining liquid at the bottom of the tube and dry in an oven.

[0056] (10) Add 50uL ddH2O to fully dissolve and store in a -20℃ medical refrigerator for later use.

[0057] 2.4 Agrobacterium infection of Arabidopsis thaliana

[0058] 2.4.1 Transformation of Agrobacterium Competent Cells

[0059] (1) Take the competent Agrobacterium stored at -80℃ and place it in the room temperature or in the palm of your hand for a while until it partially melts. When it is in an ice-water mixture, insert it into ice.

[0060] (2) Add 0.01-1 μg of plasmid DNA per 100 μL competent medium, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0061] (3) Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking for 2 to 3 hours.

[0062] (4) After centrifugation at 6000 rpm for 1 min, collect the bacteria and take about 100 μL of supernatant. Gently pipette and smear the resuspended bacteria on LB plates containing Kana, Gent, and Rif antibiotics. Place the plates upside down in a 28°C incubator and culture for 2-3 days.

[0063] Reagent formula:

[0064] LB medium preparation:

[0065] Components Required per 1L Typetone 10g Yeastextract 5g NaCl 5g

[0066] Use ddH2O to make up to 1 L. If solid culture medium is required, 15 g / L agar can be added and sterilized by high temperature and high pressure at 121°C for 20 min.

[0067] 2.4.2 Genetic transformation of Arabidopsis thaliana

[0068] In genetic experiments using Arabidopsis thaliana, it's often necessary to knock out or overexpress a gene, or to restore a gene in a mutant context to observe the phenotype. These transgenic materials are typically obtained using Agrobacterium infection. The principle is that Agrobacterium integrates sequences from the T-DNA region of the vector into the host cell's DNA. Typically, the target of infection is the flowers of the Arabidopsis plant. To be more precise, the target is the germ cells of the plant, not the flowers. The rationale is that the DNA of these infected cells is altered, and the resulting plants become transgenic.

[0069] Infection experiment steps:

[0070] (1) Add 20 mL of LB liquid medium to a sterilized 50 mL centrifuge tube and add Kana, Gent, and Rif antibiotics. Pick a single clone from the plate activated in 3.2.3.1 and place it in a 50 mL centrifuge tube. Shake gently in a 28°C constant temperature test tube shaker.

[0071] (2) Transfer the bacterial suspension from the small shaker to 500 mL of LB liquid medium and add Kana, Gent, and Rif antibiotics. Use a double-layer constant temperature shaker to adjust the temperature to 28°C and 200 rpm to culture overnight.

[0072] (3) Centrifuge at 4000 rpm for 15 min at 16°C in a floor-standing high-speed low-temperature centrifuge and discard the supernatant to enrich the cells.

[0073] (4) Resuspend Agrobacterium with infection solution and adjust the concentration of Agrobacterium to OD600 value of 0.8-1.5 with infection solution.

[0074] (5) The Agrobacterium infection solution was transferred to a 250 mL glass beaker, and Silwet-77 (at a ratio of 1 / 2000) and 100 mM acetosyringone (at a ratio of 1 / 1000) were added.

[0075] (6) Remove the excess fruit pods of the Arabidopsis thaliana to be transformed, leaving only the flower buds.

[0076] (7) Immerse the Arabidopsis inflorescence in the Agrobacterium infection solution for about 20 seconds.

[0077] (8) Place the transformed Arabidopsis thaliana upside down on a black tray that has been moistened in advance. Cover with a black plastic bag to protect it from light. After 16-24 hours, stand the Arabidopsis thaliana upright. To achieve higher transformation efficiency, you can re-infect the Arabidopsis thaliana one week later, depending on the condition of the Arabidopsis thaliana.

[0078] Reagent formula:

[0079] Preparation of Agrobacterium infection solution:

[0080] Components Required per 1L sucrose 50g MS powder 2.2g MES 0.5g SilwetL-77 500μL

[0081] Dissolve the reagent in 1 L ddH2O and adjust the pH of the infection solution to 5.7 with 1 M KOH solution.

[0082] 2.5 Arabidopsis cultivation and growth conditions

[0083] 2.5.1 Soil culture of Arabidopsis thaliana

[0084] The nutrient soil used in this study is Danish Pinstop peat nutrient soil (fiber length 0-10mm). Before planting, soak the required square plastic flower pots and black trays in 10% 84 disinfectant for more than 20 minutes to remove insect eggs, then rinse with tap water, and prepare clean transparent plastic covers. First, take an appropriate amount of nutrient soil and rub it into fine powder, and add an appropriate amount of nutrient solution. After thoroughly stirring, put the nutrient soil into the square plastic flower pot, and place the plastic flower pot on the black tray. Then add 500mL of nutrient solution to each black tray to allow the nutrient soil to fully absorb water. Cover with a transparent plastic cover to prevent debris from falling into the soil.

[0085] Select the seeds to be sown according to the needs of the experiment, and the sowing density is generally 5 or 9 seeds per flower pot. Use a toothpick moistened with water to pick up a single seed and place it on the soil surface. After sowing, spray an appropriate amount of tap water on the soil surface to allow the seeds to grow close to the soil. Cover with a transparent plastic cover and place in a 4°C refrigerator for 3 days to break the seed dormancy. In this study, long-day light was used for the phenotypic observation of Arabidopsis, identification of genotypes, and acquisition of transgenic materials. Culture conditions: culture temperature 22°C, 16h light, 8h dark, light intensity 150μmol m -2 s -1 , humidity is 65%.

[0086] 2.5.2 Soilless Culture of Arabidopsis

[0087] For screening of transgenic Arabidopsis positive seedlings, all materials were grown on 1 / 2MS solid medium. The specific steps are as follows:

[0088] (1) Place a small amount of seeds to be washed into a 2.0 mL EP tube (about 100 μL), add 1 mL of sterile ddH2O and shake to soak.

[0089] (2) Add 1 mL of sterile ddH2O to wash the seeds, invert and mix for 3 minutes. After the seeds naturally settle to the bottom of the tube, remove the waste liquid with a pipette. Repeat this step 3 times. Then add 1 mL of 75% ethanol solution and invert and mix for about 6 minutes. After the seeds naturally settle to the bottom of the tube, remove the waste 75% ethanol solution with a pipette.

[0090] (3) Wash with sterile water 6-7 times to remove residual 75% ethanol. Repeat once.

[0091] (4) Add 1 mL of 10% 84 solution and mix thoroughly by inversion for 6 min. After the seeds naturally settle to the bottom of the tube, use a pipette to remove the waste 10% 84 solution in a clean bench and quickly wash with sterile ddH2O for more than 8 times.

[0092] (5) Spread the washed seeds evenly on a 1 / 2 MS solid culture medium plate, or use a pipette to draw sterile seeds and evenly spot them on a 1 / 2 MS solid culture medium plate, and add an appropriate volume of antibiotics to the culture medium.

[0093] (6) Seal 1 / 2 of the MS solid culture medium plate with breathable medical tape.

[0094] (7) After 3 days of treatment at 4°C in the dark, place the 1 / 2 MS solid culture medium plate flat or upright in a growth chamber for observation.

[0095] Reagent formula:

[0096] 1 / 2MS medium was prepared as follows:

[0097] Components Required per 1L MS solid powder 2.2g sucrose 5g MES 0.5g agar powder 8g

[0098] Add approximately 800 mL of ddH2O, adjust the pH to 5.8 with 1 M KOH, and then dilute to 1 L. Add 8 g of agar and place in a high-temperature, high-pressure steam sterilizer at 121°C for 20 minutes. Once the culture medium has cooled to a comfortable temperature, add the appropriate reagents, such as antibiotics and hormones, as needed for the experiment. Plate the culture medium while it is still cool.

[0099] 2.6 Resistance screening of transgenic Arabidopsis plants:

[0100] The specific experimental steps for screening positive transgenic Arabidopsis seedlings are described in 2.5.2. The following points are particularly noted.

[0101] (1) Resistance addition to 1 / 2MS solid culture medium. Usually, we use hygromycin resistance and kanamycin resistance plasmids to transform plants to obtain transgenic materials. Therefore, when we use resistance screening to screen positive transgenic plant seedlings, we need to add antibiotics to the 1 / 2MS solid culture medium. The concentration of hygromycin stock solution is 50mg / ml, and it is added at a ratio of 1 / 2000. The concentration of kanamycin stock solution is 50mg / ml, and it is added at a ratio of 1 / 1000.

[0102] (2) When screening for hygromycin-resistant transgenic plants, first wash and sterilize the seeds, evenly spread them on a hygromycin-resistant 1 / 2MS solid culture medium plate, treat them in the dark at 4°C for 3 days, then place them in the light for 8 hours, and then grow them in the dark at 22°C for 3-5 days. Then observe the hypocotyl of the seeds. If there are plants with obvious hypocotyl elongation, they are positive seedlings. Then place the plate in the light for cultivation. After the positive seedlings turn green, they can be transferred to the soil.

[0103] (3) When screening for kanamycin-resistant transgenic plants, first wash and sterilize the seeds, evenly spread them on a kanamycin-resistant 1 / 2MS solid culture medium plate, treat them in the dark at 4°C for 3 days, and then culture them under light. Plants that can turn green and grow normally on the kanamycin-resistant 1 / 2MS solid culture medium plate are positive seedlings and can be transferred to the soil.

[0104] (4) After the positive seedlings are transferred to the soil, they need to be wrapped with plastic wrap for the first two days to prevent moisture loss. They should also be placed under low light to recover for 2-3 days. After that, they can grow normally.

[0105] 3 Experimental verification

[0106] 3.1 Extraction of plant total protein

[0107] (1) Take about 100 mg of fresh plant material and immediately place it in liquid nitrogen.

[0108] (2) Use a grinding shaker to fully shake the plant material for 90 seconds at 30 Hz.

[0109] (3) Carefully open the lid and add 100 μL of Extraction Buffer and 20 μL of 5× Protein Loading Buffer.

[0110] (4) After thorough mixing, centrifuge at 4°C, boil in a 99°C metal bath for 5-10 minutes, and store at 4°C or -20°C.

[0111] Reagent formula:

[0112] Extraction Buffer preparation:

[0113]

[0114]

[0115] 3.2 Western blotting

[0116] (1) After completing SDS-PAGE electrophoresis according to 3.2.12, cut the target protein according to its molecular weight and the position indicated by the protein marker.

[0117] (2) Cut the appropriate PDVF membrane according to the size of the gel and soak the membrane in methanol for 30 seconds.

[0118] (3) Peel off the protein gel from the front and back plates.

[0119] (4) Assemble the transfer sandwich. The order from negative electrode to positive electrode is: sponge / filter paper / protein glue / membrane / filter paper / sponge. After each layer is placed, remove the air bubbles.

[0120] (5) Place the transfer tank in an ice bath, adjust the voltage to 100 V, and transfer the membrane for about 2 hours.

[0121] (6) After transfer, add 20 mL of blocking buffer and block at room temperature for 1-2 hours.

[0122] (7) Rinse the hybridization membrane twice quickly with 1×TBST Buffer to remove residual blocking buffer.

[0123] (8) Dilute the primary antibody according to the antibody instructions, add 6 mL of the diluted primary antibody, and incubate slowly at 4°C overnight.

[0124] (9) Recover the primary antibody and wash the membrane four times with 1× TBST buffer, each time for 8 min.

[0125] (10) Dilute the secondary antibody according to the antibody instructions, add 6 mL of the appropriate secondary antibody, and incubate slowly at room temperature for 1-2 hours; remove the secondary antibody, recover the primary antibody with a pipette, add 1× TBST Buffer, wash the membrane 4 times, each time for 8 minutes; add 1 mL of ThermoSuperSignalTMWestPico PLUS luminescent substrate, mix and incubate for 1 minute.

[0126] (11) Exposure using a chemiluminescence analyzer Bio-Rad ChemiDoc™ Touch Imaging System, the results are as follows Figure 3 shown.

[0127] Reagent formula:

[0128] Membrane transfer buffer:

[0129] Components Each 1L requires Glycine 2.9g Tris-base 5.8g

[0130] Add approximately 600 mL of ddH2O to a beaker and stir thoroughly to dissolve. Add 200 mL of methanol, then add ddH2O to bring the volume up to 1 L. Pre-cool in a -20°C medical freezer.

[0131] 1×TBST buffer:

[0132] Components Each 1L requires NaCl 8.8g 1MTris-HCl (pH=8.0) 20mL

[0133] Add approximately 800 mL of ddH2O to a beaker and stir thoroughly to dissolve. Add 0.5 mL of Tween 20 and mix thoroughly. Then add ddH2O to bring the volume up to 1 L.

[0134] Blocking buffer (50 mL): Weigh 2.5 g of skim milk and dissolve it in 50 mL of 1× TBST. Stir thoroughly to dissolve and use immediately.

[0135] 3.3 Indoor identification experiment of disease resistance of transgenic plants

[0136] 4 to 5-week-old transgenic positive seedlings (PXO_03174) and wild type (CK) were selected and inoculated with the pathogen Pseudomonas syringae Pst DC3000 for plant disease resistance identification and analysis. 7 transgenic positive seedlings and 7 wild type plants were inoculated with 3 leaves from each plant. Observation and recording were performed every day to record the growth of the leaves of the inoculated bacteria (transgenic plants and wild type plants were placed in the same black pot). When obvious differences appeared, the inoculated leaves were cut and two pieces were taken for crushing. Figure 4 The leaves were washed with 75% ethanol solution for 30 seconds, then with sterile water for 30 seconds. The residual sterile water on the surface was removed with absorbent paper and placed in 2.0 mL centrifuge tubes. Two small steel balls were placed in each tube. After adding 200 μL of sterile water, the tubes were shaken and broken. The tubes were centrifuged at 6000 r / min for 2 minutes. 10 μL of supernatant was taken with a pipette for gradient dilution. The dilution multiples were 10 and 10. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , the bacterial solutions of different concentrations were sequentially spotted on the rifampicin-resistant LB solid medium, and cultured in a 30℃ constant temperature incubator for 48 hours. The number of colonies on the medium was observed and statistical results were obtained, such as Figure 5 shown.

[0137] Experimental conclusion: Compared with the wild type, transgenic Arabidopsis thaliana overexpressing the Xanthomonas gene PXO_03174 showed a weakened disease resistance phenotype, indicating that plants overexpressing the PXO_03174 gene are more sensitive to infection by pathogenic bacteria. They can subsequently be used to prepare plant materials susceptible to pathogenic bacteria, identify virulence genes of pathogenic bacteria infecting Arabidopsis thaliana, and study the function of infection and pathogenicity. They can also be used to study the disease resistance of Arabidopsis thaliana to pathogenic bacteria such as Pst DC3000 and develop antibacterial chemicals.

[0138] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. An application of a gene encoding hexose phosphomutase from Xanthomonas oryzae, characterized in that: Xanthomonas oryzae Xanthomonas oryzae pv. oryzae ( Xoo )PXO99 A Hexose phosphate mutase encoding gene PXO_03174 For regulating the susceptibility of a plant to a disease, the gene PXO_03174 The sequence of SEQ ID NO.1 is shown, the plant is Arabidopsis thaliana, and the overexpression in Arabidopsis thaliana PXO_03174 Genes are susceptible to infection with the disease, the disease is Pseudomonas syringae Pst Caused by DC3000 infection.

2. A method for Pseudomonas syringae Pst The method for preparing plants susceptible to DC3000 infection is characterized in that: Using Xanthomonas oryzae genes PXO_03174 A recombinant expression vector is constructed and introduced into a plant for overexpression, wherein the plant is Arabidopsis thaliana.

3. The preparation method according to claim 2, characterized in that The vector is pCAMBIA2300.

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