A polypeptide for inducing plant to produce systemic acquired resistance and its application

Abstract

The present invention relates to a polypeptide for inducing plant to produce systemic acquired resistance and its application. The polypeptide sequence is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the plants include but are not limited to Arabidopsis, tobacco and rapeseed.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a polypeptide for inducing plant to produce systemic acquired resistance and application thereof. Background Art

[0002] 1. Plant SAR (Systemic Acquired Resistance)

[0003] Plant SAR (Systemic Acquired Resistance) refers to the ability of plants to establish a long-term, broad-spectrum disease resistance throughout the plant after local tissues are infected by pathogens. This state involves a series of physiological and biochemical changes, including the production and conduction of signal molecules. In plant immune responses, salicylic acid (SA) is one of the main signal molecules of SAR. Its content in the body increases sharply after plants are infected by pathogens, activating the plant's resistance response to pathogens.

[0004] The signal transduction of SAR involves multiple components, including the synthesis, perception, signal transduction and related signal transduction pathways of salicylic acid. During the signal transduction process, SA can induce the production of many disease resistance-related proteins, including pathogenesis-related proteins (PR proteins), thereby causing plants to produce SAR. The synthesis pathway of SA is generally divided into two types: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway. The ICS pathway is the main pathway for the synthesis of SA in plants.

[0005] In plant cells, SA activates downstream signaling elements by binding to specific proteins or receptors. Known SA-binding proteins include NPR1 (Nonexpressor of Pathogenesis-Related Genes 1), NPR3, and NPR4, which play a key regulatory role in the SA-mediated signal transduction process. NPR1 is a core transcriptional regulator in SA signal transduction. It exists in the form of oligomers in the cytoplasm. When the SA level rises, NPR1 undergoes conformational changes and enters the nucleus to activate the expression of PR genes.

[0006] In addition, SA is also involved in the response of plants to abiotic stress, but its specific biochemical mechanism still needs further study. In the study of plant disease resistance, SAR is an important research direction, which not only helps to understand the immune mechanism of plants, but also provides a theoretical basis for breeding new disease-resistant varieties and developing efficient immune inducers.

[0007] 2. OsCDR1 gene

[0008] The OsCDR1 gene in rice encodes an aspartic protease that plays an important role in plant disease resistance. Studies have shown that OsCDR1 functions in the apoplast of plant cell walls and is involved in plant disease resistance signaling. When OsCDR1 is ectopically expressed in rice and Arabidopsis, it can enhance plant resistance to bacterial and fungal pathogens. This enhanced resistance is associated with the induction of expression of pathogenesis-related genes and is dependent on the aspartic protease activity of the OsCDR1-encoded protein. In transgenic plants, OsCDR1 accumulates in the intercellular fluid and is able to induce systemic defense responses, indicating that the disease resistance function of OsCDR1 in Arabidopsis and rice is conserved (Prasad et al., 2009).

[0009] Prasad BD, Creissen G, Lamb C and Chattoo BB. 2009. Overexpression of rice (Oryza sativa L.) OsCDR1 leads to constitutive activation of defense responses in rice and Arabidopsis. Mol Plant Microbe Interact, 22: 1635-1644.

[0010] 3. OsLTP1.12 gene

[0011] OsLTP1.12 in rice is a nonspecific lipid transfer protein (nsLTP) that belongs to the lipid transfer protein family. This type of protein is widely present in plants and plays an important role in plant growth and development and response to environmental stress. OsLTP1.12 may be involved in the transport and deposition of lipids, which is essential for plant cuticle synthesis, embryonic development, and adaptation to various stress environments. For example, lipid transfer proteins may play a role in the plant's anti-pathogenic microbial response and enhance the plant's defense capabilities by participating in the synthesis and regulation of the cell wall.

[0012] In rice, the expression patterns and functions of OsLTP1 gene family members may be different, and they may play a role in different tissues and developmental stages. For example, some OsLTP genes may play a crucial role in seed development and seed quality formation. The study of these genes can provide important information for improving rice seed quality and enhancing its adaptability to environmental stress.

[0013] Specifically for OsLTP1.12, it may be expressed in specific tissues or developmental stages of rice and may be related to lipid metabolism and physiological and biochemical responses of plants.

[0014] However, the current information on the detailed functions and mechanisms of action of OsLTP1.12 may be relatively limited and requires further experimental studies to clarify.

[0015] Based on this, the present invention is proposed. Summary of the invention

[0016] The present invention firstly relates to a group of polypeptides capable of inducing systemic acquired resistance (SAR) in plants, wherein the plants include but are not limited to Arabidopsis thaliana, tobacco, and rapeseed; the polypeptide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3,

[0017] SEQ ID NO.1: KTLAGTIKSLNLGAAAGIPGK;

[0018] SEQ ID NO.2: KTLAGTIK;

[0019] SEQ ID NO.3: KTLAGTIKSLNLGAAAGIPGKCGVNVGFPISSLSTDCSKVS;

[0020] The present invention also relates to a plant antibacterial preparation, which contains: the polypeptide and necessary preparation auxiliary materials; the preparation auxiliary materials include but are not limited to: buffer, stabilizer, antioxidant; the plants include but are not limited to Arabidopsis, tobacco, rapeseed.

[0021] The present invention also relates to the use of the polypeptide in inducing SAR in plants, wherein the plants include but are not limited to Arabidopsis, tobacco, and rapeseed.

[0022] The beneficial effects of the present invention are:

[0023] 1. It was clarified that the small peptide produced by the hydrolysis of OSLTP1.12 by OsCDR1 can be used as a new SAR mobile signal. By verifying the long-distance transport of plant peptides, a new mechanism of plant peptides regulating plant disease resistance was discovered.

[0024] 2. A new strategy for inhibiting plant disease resistance was defined from the perspective of pathogens, providing a new strategy for plant defense and resistance to pathogen invasion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , differential proteins detected in WT apoplasts but not in Oscdr1 apoplasts after inoculation with PXO99.

[0026] Figure 2 , WB electrophoresis was used to identify the expression product of OsLTP1.12, 2A, exogenous cleavage results, lane 1: OsLTP1.12△SP-MBP 37℃0h, lane 2: OsLTP1.12△SP-MBP 37℃1h (the substrate will not self-degrade at 37 degrees), lane 3: OsLTP1.12△SP-MBP+OsCDR1 37℃1h; 2B, endogenous cleavage results, Ubi::OsCDR1-MBP and Ubi::OsCDR1 were co-expressed in tobacco leaves; 2C, protein structure diagram.

[0027] Figure 3 , OsLTP1.12 expression product is hydrolyzed by OsCDR1; 3A, schematic diagram of the hydrolysis cleavage site; 3B, mass spectrometry analysis of the hydrolysis product.

[0028] Figure 4 , bacterial blight pathogenic bacteria can induce the expression of OsLTP1.12. Leaves of wild-type rice ZH11 were collected 0 hours, 2 hours, 6 hours and 8 hours after inoculation with PXO99, and the transcription level of OsLTP1.12 was detected by RT-qPCR.

[0029] Figure 5 , detection of OsLTP1.12 protein level in wild-type rice ZH11 leaves after inoculation with PXO99; 5A, samples were collected 0 hours and 6 hours after inoculation, and the protein level of OsLTP1.12 was detected using Anti-OsLTP1.12 antibody; 5B, detection results after sample dilution; 5C, the antibody was unable to detect negative control proteins OsLTP1.19 and OsLTP1.20.

[0030] Figure 6 , used the CRISPR / Cas9 system to edit the OsLTP1.12 gene to obtain the Osltp1.12 mutant.

[0031] Figure 7, growth results of un-infection leaves and infection leaves of rice wild type ZH11 (Wt) and Osltp1.12 mutant lines 14 days after inoculation with PXO99; 7A, phenotype of un-infection leaves 14 days after inoculation with PXO99; 7B, phenotype of infection leaves 14 days after inoculation with PXO99; 7C, statistics of lesion length (Local end is Un-infection, distal end is infection).

[0032] Figure 8 , rice wild type ZH11 (Wt), and different rice lines transformed with the same pUbiO plasmid (Ubi::OsLTP1.12 overexpression plasmid). The phenotypes of un-infected leaves and infected leaves 14 days after inoculation with PXO99; 8A, the phenotypes of un-infected leaves 14 days after inoculation with PXO99; 8B, the phenotypes of infected leaves 14 days after inoculation with PXO99.

[0033] Fig. 9 , rice wild type ZH11 (Wt), different rice lines transformed with the same pUbiO plasmid (Ubi::1.12SP-pep11.12 overexpression plasmid, overexpressing pep1 peptide), phenotypes of un-infected leaves and infected leaves 14 days after inoculation with PXO99; 9A, phenotypes of un-infected leaves 14 days after inoculation with PXO99; 9B, phenotypes of infected leaves 14 days after inoculation with PXO99; 9C, statistics of lesion length (Local end is Un-infection, distal end is infection); 9D, systemic acquired resistance levels of Wt and Ubi::Ubi::1.12SP-pep11.12 lines.

[0034] Fig.10 , the results of rice wild-type ZH11 and Oscdr1 mutant strains inoculated with PXO99 and pep1 polypeptide 48 hours after inoculation; 10A, PXO99-2d refers to the leaf phenotype of the leaves inoculated with PXO99 at the distal end two days after proximal inoculation; 10B, pep1-2d refers to the leaf phenotype of the leaves inoculated with pep1 synthesized in vitro at the distal end two days after proximal application; 10C, statistics of lesion length and the level of systemic acquired resistance of each strain; 10D, RT-qPCR was used to detect the transcription levels of three proteins, OsPR1a, OsPR15 and OsPR10, in different strains.

[0035] Fig.11Leaves of wild-type Arabidopsis thaliana, tobacco and rapeseed with or without exogenous application of pep1 polypeptide were collected 48 hours after the corresponding treatment, and the transcription levels of three SAR-related genes (AtPR1, AtPR2 and AtPR5) were detected by RT-qPCR. Control: leaves of the control group without exogenous application of pep1 polypeptide; pep1-treatment: leaves after exogenous application of pep1 polypeptide. DETAILED DESCRIPTION

[0036] 1. Extraction of total rice protein

[0037] Experimental Materials:

[0038] Fresh rice leaves, liquid nitrogen, mortar and pestle, protease inhibitors, high-speed refrigerated centrifuge, centrifuge tubes, micropipettes, protein quantification kit;

[0039] Total protein extraction buffer (formula as follows):

[0040] 50mM Tris-HCl (pH 7.5)

[0041] 150mM NaCl

[0042] 0.5% Triton X-100

[0043] 1mM EDTA

[0044] 1 mM PMSF (phenylmethylsulfonyl fluoride, protease inhibitor);

[0045] Experimental steps:

[0046] 1. Sample preparation:

[0047] Fresh rice leaves were collected and rinsed with distilled water to remove dust from the surface.

[0048] 2. Frozen samples:

[0049] Washed rice leaves were quickly frozen in liquid nitrogen to make them brittle.

[0050] 3. Grind the sample:

[0051] Transfer the frozen leaves to a mortar and add a small amount of liquid nitrogen.

[0052] Grind the leaves using a pestle until they become a fine powder.

[0053] 4. Extract protein:

[0054] Transfer the ground powder into a pre-chilled centrifuge tube.

[0055] Add total protein extraction buffer at a ratio of 200 μl to 0.1 g of powder.

[0056] Mix gently using a micropipette to ensure adequate contact between the powder and the buffer.

[0057] 5. Add protease inhibitors:

[0058] Add an appropriate amount of protease inhibitors to the centrifuge tube to prevent protein degradation.

[0059] 6. Centrifugation:

[0060] Place the centrifuge tube in a high-speed refrigerated centrifuge and set the centrifugation conditions to 4°C, 14,000 rpm, and centrifuge for 15 minutes.

[0061] 7. Collect the supernatant:

[0062] After centrifugation, carefully transfer the supernatant to a new centrifuge tube to avoid disturbing the pellet.

[0063] 8. Protein Quantification:

[0064] The protein concentration in the supernatant was determined using the BCA protein quantification kit according to the instructions.

[0065] 9. Preserve Protein:

[0066] The quantified protein samples were stored in a refrigerator at -80°C for subsequent experiments.

[0067] 2. BCA Quantification Method

[0068] The BCA protein concentration assay kit of Dingguo Company was used to detect the protein amount of different samples. The specific experimental steps are as follows:

[0069] 1. Preparation of protein standards:

[0070] Take an appropriate amount of 5 mg / ml protein standard (BSA) and dilute it with PBS to a final concentration of 0.5 mg / ml. For example, take 50 μl of 5 mg / ml protein standard and add 450 μl of PBS to prepare a 0.5 mg / ml protein standard.

[0071] 2. Preparation of BCA working solution:

[0072] According to the number of samples, prepare an appropriate amount of BCA working solution at a ratio of 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly. For example, 5 ml of BCA reagent A plus 100 μl of BCA reagent B, mix well, and prepare 5.1 ml of BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0073] 3. Protein concentration determination:

[0074] Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard into the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to a standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively.

[0075] Add appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20μl, add standard diluent to make it up to 20μl. Please remember to record the sample volume.

[0076] Add 200 μl of BCA working solution to each well and place at 37°C for 20-30 minutes. Alternatively, place at room temperature for 2 hours or at 60°C for 30 minutes. When the BCA method is used to determine protein concentration, the color will deepen over time, and the color development reaction will accelerate as the temperature rises.

[0077] The absorbance at A562 or other wavelengths between 540-595 nm was measured using a microplate reader.

[0078] 4. Calculate sample protein concentration based on the standard curve:

[0079] The protein concentration of the samples was calculated based on the standard curve and the sample volume used.

[0080] 3. The vacuum extraction method of rice apoplast protein mainly includes the following steps:

[0081] 1) Sample preparation: Take fresh rice leaves or roots, weigh them and put them into small tubes.

[0082] 2) Pre-cooled washing: Rinse the sample twice with pre-cooled ddH2O, 15 minutes each time.

[0083] 3) Vacuum infiltration: Place the small tube containing the sample into a centrifuge tube, add pre-cooled extraction solution, and then place the bottle into a vacuum pump to evacuate to 35kPa for 5-6 minutes.

[0084] 4) Restore air pressure: Slowly restore the air pressure to normal atmospheric pressure level, remove the small tube, and place it at room temperature for 5 minutes to allow excess extract to flow out.

[0085] 5) Centrifugal collection: Place the small tube back into a suitable centrifuge tube, centrifuge at 1000 g for 10 minutes at 4°C, repeat the above steps, and combine the extracts from the two centrifugations.

[0086] 6) Filtration and concentration: The collected extract was filtered through a 0.22 μm filter membrane and concentrated to about 500 μL.

[0087] IV. 293T Cell Culture

[0088] 1. Cell recovery:

[0089] Take out the cryovials from liquid nitrogen or -80℃ freezer and immerse them directly in 37℃ warm water to thaw quickly.

[0090] After melting, open the lid in the clean bench, aspirate the cell suspension with a pipette tip, add it to a 15ml centrifuge tube containing 3-6mL complete culture medium, and flick to mix.

[0091] Centrifuge and remove the supernatant, then add 1-3 mL of complete culture medium to resuspend the cells.

[0092] 2. Cell culture:

[0093] Transfer the resuspended cell suspension to a new culture flask and add new complete culture medium to 8-10 mL / flask.

[0094] 293T cells have weak adhesion ability and can be cultured in Corning CellBIND flasks.

[0095] The culture conditions were DMEM+10% FBS+1% P / S, air, 95%; carbon dioxide (CO2), 5%.

[0096] 3. Cell passaging:

[0097] The recommended ratio for the first subculture is 1:2. Discard the old culture medium, add 0.25% trypsin to the culture flask, and digest at 37°C for 2-3 minutes.

[0098] After most of the cells have become round and fallen off, add complete medium to terminate digestion, gently blow the cells, and aspirate them into a centrifuge tube after they have completely fallen off.

[0099] Centrifuge (1000 rpm, 5 min), discard the supernatant, and add 1-3 mL of complete culture medium to resuspend.

[0100] The resuspended cell suspension was transferred to a new culture flask and supplemented with new complete culture medium.

[0101] 4. Cell cryopreservation:

[0102] Complete cell culture medium 92% + DMSO 8% was used as the freezing medium formula.

[0103] The cell suspension is dispensed into cryopreservation tubes at a certain density, and the cell name, culture generation, cryopreservation date, batch number and other information are marked.

[0104] After placing the cryovials in a programmed freezing box (cooling at 1°C per minute), place them directly in a -80°C refrigerator overnight and transfer them to a liquid nitrogen tank for long-term storage the next day.

[0105] 5. 293T cell transfection

[0106] 1. Cell culture:

[0107] 293T cells were seeded into 6-well plates, and 2 mL of DMEM medium containing 10% FBS was added to each well.

[0108] The cells were cultured until the day of transfection when the cell confluence reached 80%.

[0109] 2. Preparation of transfection reagents:

[0110] Lipofectamine 2000 was selected as the transfection reagent.

[0111] Prepare plasmid DNA and adjust the concentration to 1 μg / μL.

[0112] 3. Preparation of the complex:

[0113] 0.5 μg of plasmid DNA was transfected into each well.

[0114] In a sterile centrifuge tube, add 50 μL of serum-free DMEM and then add 0.5 μg of plasmid DNA.

[0115] Take another sterile centrifuge tube and add 1.5 μL Lipofectamine 2000 and 50 μL serum-free DMEM.

[0116] Mix gently and incubate at room temperature for 5 minutes.

[0117] Add the Lipofectamine 2000 solution to the tube containing DNA, mix gently and incubate at room temperature for 20 minutes to form a complex.

[0118] 4. Transfection operation:

[0119] Discard the culture medium in the 6-well plate and gently rinse the cells once with 1 mL of serum-free DMEM.

[0120] Add 100 μL of serum-free DMEM containing DNA-Lipofectamine 2000 complex to each well of cells.

[0121] Gently shake the plate to evenly distribute the complex.

[0122] 5. Cultivation and testing:

[0123] The culture plate was returned to a 37°C, 5% CO2 incubator for 6 hours.

[0124] After 6 hours, the complexes were discarded and 2 mL of DMEM medium containing 10% FBS was added to each well.

[0125] The cells were cultured for 48 hours after transfection, and the cells or culture supernatant were collected for subsequent experiments.

[0126] 6. Western blot

[0127] 1) Clean the electrophoresis tank, transfer clip, gasket, and filter paper used in subsequent experiments in advance, and soak the gasket and filter paper in protein transfer buffer;

[0128] 2) Prepare a 0.45 μm PVDF membrane of appropriate size in advance and soak it in methanol in advance;

[0129] 3) Assemble the transfer device: assemble the transfer clamp according to the positive and negative order (gasket, filter paper, gel after electrophoresis, PVDF membrane, filter paper, gasket), install the transfer clamp into the electrophoresis tank according to the positive and negative poles, fill the sandwich with ice cubes, and fill the electrophoresis tank with protein transfer buffer;

[0130] 4) Turn on the power and transfer the membrane at 4°C (200 mA, constant current electrophoresis for 120 minutes);

[0131] 5) Wash the transferred membrane in 1×TTBS solution three times, 5 minutes each time;

[0132] 6) Place the membrane in a 1×TTBS solution containing 5% skimmed milk powder, place it on a shaker at 60 rpm, and block it at room temperature for 2 hours;

[0133] 7) Wash three times in 1×TTBS solution, 5 minutes each time;

[0134] 8) Add primary antibodies at appropriate concentrations and incubate according to the optimal incubation conditions for different proteins;

[0135] 9) Wash three times in 1×TTBS solution, 5 minutes each time;

[0136] 10) Add appropriate concentration of secondary antibody that matches the primary antibody and incubate according to the optimal incubation conditions for different proteins;

[0137] 11) Wash three times in 1×TTBS solution, 5 minutes each time;

[0138] 12) Add color developing solution to develop color.

[0139] VII. Amplification and recovery of target fragments

[0140] This patent amplifies OsCDR1 ( OsCDR1 (constitutive disease resistance 1) encodes an aspartic acid protease that participates in the plant's defense response to pathogens. The expression of OsCDR1 is activated after treatment with disease resistance signaling molecules such as benzothiadiazole and salicylic acid. Overexpression of OsCDR1 enhanced resistance to bacterial and fungal diseases and was associated with the induced expression of pathogenesis-related genes. This suggests that the function of OsCDR1 is conserved in rice and Arabidopsis and is able to induce systemic defense responses.), OsCDR1R97A (OsCDR1R97A is a mutant of OsCDR1, in which a specific amino acid residue (Arg97) Change. Studies have found that pathogens The induced methylglyoxal (MG) inhibits the protease activity of OsCDR1 by modifying the Arg97 residue. This suggests that OsCDR1R97A plays a key role in the pathogen infection process. Different disease resistance characteristics may be present in ) and the CDS fragment of OsLTP1.12△SP (△SP: without signal peptide) ( CDS Coding Sequence fragments refer to DNA sequences that correspond one to one with protein sequences. These sequences are The CDS does not contain any non-coding sequences, such as introns, etc. It is a key part that encodes a specific protein ); and OsLTP1.12, pep1, pOsCDR1::OsCDR1, and pOsCDR1::OsCDR1R97A fragments used for transgenic plant construction. Using rice ZH11 ("ZH11" refers to a rice variety, the full name of which is "Zhonghua No. 11", bred by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Variety source: "Zhonghua No. 11" is a rice variety bred by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Main characteristics: the variety has good disease resistance and lodging resistance, and strong adaptability) cDNA or genome as a template, use specific primers (10μM) at both ends of the corresponding fragment, and use Vazyme's Phanta DNA polymerase for amplification. Use Vazyme's FastPure @ The target fragment was recovered by using the Gel DNA Exraction Mini Kit.

[0141] 8. Extraction, linearization and extraction of vector plasmid

[0142] This experiment uses Plasmid Mini Kit (Vazyme) was used to extract the vector plasmid.

[0143] In this experiment, the extracted plasmid was linearized using restriction endonucleases with corresponding enzyme cutting sites (Thermo Scientific Fast Digest). The enzyme cutting system (200 μl) was as follows:

[0144] Plasmid DNA: Add an appropriate volume based on the purification status and concentration of your plasmid DNA. For purified plasmid DNA, usually add 10 μl (about 5 μg).

[0145] FastDigest restriction endonuclease: Select the corresponding FastDigest restriction endonuclease according to your target restriction site and add 1μl.

[0146] FastDigest Green Buffer or Colorless Buffer: Add 2 μl of 10X FastDigest Green Buffer or Colorless Buffer.

[0147] Water, nuclease-free: make up to a total volume of 200 μl. For plasmid DNA, add 15 μl of water.

[0148] Total volume: Ensure that the final reaction system reaches 200 μl.

[0149] Incubation conditions: Incubate the reaction at the recommended digestion temperature for 5-15 minutes. FastDigest enzymes work at 37°C.

[0150] Using Vazyme Plasmid Mini Kit was used to extract the linearized vector plasmid.

[0151] IX. Recombination of linearized vector and target fragment and transformation of recombinant product into competent cells

[0152] use Ultra One Step Cloning Kit (Vazyme) uses a one-step recombination method to reconstitute the linearized vector and the target fragment. The recombination system (10 μl) is as follows:

[0153] Linearized vector: Based on the size of your vector, use the formula [0.02 × number of base pairs of cloning vector] ng (0.03pmol) to calculate the amount of vector required. For example, for a 5kb vector, the optimal amount is 100ng.

[0154] Target fragment: For the insert fragment, use the formula [0.04 × number of base pairs of the insert fragment] ng (0.06 pmol) to calculate the amount of the required fragment. For example, for a 2 kb insert fragment, the optimal amount is 80 ng.

[0155] 2×ClonExpress Mix: 5μl. This is the premix provided in the kit, which contains the recombinant enzyme and other necessary components.

[0156] ddH2O: make up to a total volume of 10 μl.

[0157] The specific steps of the recombination experiment are as follows:

[0158] 1) Take out the DH5α competent cell stored in -80℃ refrigerator, thaw it on ice, add the above-mentioned recombination system, mix gently, and let it stand on ice for 30 minutes;

[0159] 2) Transfer the above system to a 42°C water bath and incubate for 30-60 seconds, then immediately remove and place on ice for 2-5 minutes;

[0160] 3) On a clean bench, add 500 μl of antibiotic-free LB liquid culture medium to the above system, place in a 37°C shaker, 100-150 rpm, and culture for 30-60 minutes;

[0161] 4) Spread the bacterial solution onto solid LB medium of the corresponding resistance and invert it in a 37°C incubator for overnight culture (more than 12 hours).

[0162] 10. Identification of positive colonies

[0163] In the above experimental steps, many single colonies will grow on the plates cultured overnight. In order to identify which single colonies are positive colonies with correct recombination, we use universal identification primers (10μM) on both sides of the multi-cloning site of the corresponding vector to amplify and identify. PCR SuperMix (TransGen Biotech), positive colony identification PCR system is as follows:

[0164] Template DNA: Use a single colony as template and take 1 μl of the colony suspension.

[0165] Upstream primer (10 μM): 1 μl, used to amplify the regions on both sides of the vector multiple cloning site.

[0166] Downstream primer (10 μM): 1 μl, paired with the upstream primer, used to amplify the regions on both sides of the vector multiple cloning site.

[0167] PCR SuperMix: 5 μl, containing DNA Polymerase, dNTPs and optimized reaction buffer at a concentration of 2×.

[0168] ddH2O: make up to a total volume of 10 μl.

[0169] PCR amplification conditions: pre-denaturation at 95°C for 5 minutes; followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds; finally extension at 72°C for 5 minutes; maintained at 4°C after completion.

[0170] The corresponding PCR amplification program is as follows:

[0171] 1) 95°C, 5 minutes

[0172] 2) 95°C, 30 seconds

[0173] 3) 58℃~62℃ (select according to different fragment characteristics), 30 seconds

[0174] 4) 72°C, 1 minute / 1kb (select according to the target fragment length)

[0175] Repeat steps 2, 3, and 4 for 38 cycles;

[0176] 5) 72°C, 10 minutes

[0177] 6) 16°C, 10 minutes

[0178] The obtained PCR products were then subjected to electrophoresis detection, and single colonies that could amplify fragments consistent with the predicted band size were marked. They were re-transferred to liquid LB culture medium with corresponding resistance, cultured in a shaking incubator at 37°C overnight, and the plasmids were extracted by referring to the experimental steps in 2.2.4.3. The plasmids were sent to the company for sequencing, and the plasmids whose sequencing results completely corresponded to the expected fragment sequence were saved for subsequent experiments.

[0179] 11. Preservation of strains

[0180] The recombinant strains identified as positive bacteria are preserved as follows:

[0181] 1) Prepare sterilized 1.5 ml eppendorf tubes, 80% glycerol, and pipette tips in advance;

[0182] 2) Pipette 750 μl of the bacterial solution corresponding to the correctly sequenced plasmid into a 1.5 ml EP tube, add 250 μl of 80% glycerol, and mix well with a pipette tip;

[0183] 3) Mark the corresponding bacterial strains, seal the EP tube with sealing film, and store the preserved bacteria in a -80℃ refrigerator for long-term storage.

[0184] 12. Mass spectrometry detection

[0185] 1) Sample preparation: The system used for mass spectrometry detection was concentrated to about 100 μg / ml. Since the mass spectrometry system of this paper was hydrolyzed by the rice apoplast protein extracted in this paper or the proteolytic enzyme expressed by eukaryotes, conventional trypsin treatment was not required. The system was directly desalted and freeze-dried.

[0186] 2) HPLC: The dried samples were reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000g for 10 minutes, and the supernatant was injected; the samples were separated by Thermo UltiMate 3000UHPLC. The samples were first enriched and desalted in a trap column, and then connected in series with a self-assembled C18 column (75μm inner diameter, 3μm column particle size, 25μm column length), and separated at a flow rate of 300nL / min through the following effective gradient: 0-5 minutes, 5% mobile phase B (98% ACN, 0.1% FA); 5-45 minutes, mobile phase B linearly increased from 5% to 25%; 45-50 minutes, mobile phase B increased from 25% to 35%; 50-52 minutes, mobile phase B increased from 35% to 80%; 52-54 minutes, 80% mobile phase B; 54-60 minutes, 5% mobile phase B. The end of the nanoliter liquid phase separation is directly connected to the mass spectrometer.

[0187] 3) Detection: The system after liquid phase separation was ionized by nanoESI source and then entered into the tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA) for DDA (Data Dependent Acquisition) mode detection. Main parameter settings: ion source voltage was set to 1.9 kV; primary mass spectrometry scanning range was 350-1,500 m / z; resolution was set to 60,000; secondary mass spectrometry starting m / z was fixed to 100; resolution was 15,000. The screening conditions for the parent ions of the secondary fragmentation were: charge 2+ to 6+, peak intensity exceeding 10,000, and the parent ions ranked in the top 30. The ion fragmentation mode was HCD, and the fragment ions were detected in the Orbitrap. The dynamic exclusion time was set to 30 s. AGC was set to: primary 3E6, secondary 1E5.

[0188] 13. Transformation steps of genetically modified rice:

[0189] 1. Construction of transgenic vector

[0190] Ensure that the transgenic vector (plasmid) has been successfully constructed and contains the target gene and the selectable marker gene.

[0191] 2. Transformation of Agrobacterium EHA105

[0192] Transformation of Agrobacterium EHA105 using the calcium ion method:

[0193] EHA105 colonies were picked from the glycerol tube, inoculated into LB medium containing the corresponding antibiotics, and cultured at 37°C with shaking overnight.

[0194] On the next day, 1.5 mL of overnight culture solution was added to new LB medium containing antibiotics, and cultured at 37°C with shaking until OD600 reached 0.6-0.8.

[0195] The cells were collected and centrifuged at 12000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in ice-cold sterile water.

[0196] Take 100-200 μL of resuspended bacteria, add it to a pre-cooled 0.2 mL centrifuge tube, and add 1-5 μL of plasmid DNA (about 50-100 ng).

[0197] Add 250 μL of pre-cooled 2×YT medium and 20 μL of 2.5 M CaCl2, mix gently and place on ice for 20-30 minutes.

[0198] Heat shock at 42°C for 90 seconds, then quickly transfer to ice and cool for 10 minutes.

[0199] Add 800 μL of 2×YT medium without antibiotics and culture at 37°C with shaking for 2-3 hours.

[0200] Spread onto LB plates containing corresponding antibiotics and culture at 37°C overnight.

[0201] 3. Screening of transformants

[0202] Single colonies were selected for PCR verification and sequence analysis to confirm whether the transformation was successful.

[0203] 4. Agrobacterium-mediated rice transformation

[0204] The successfully transformed Agrobacterium was cultured to OD600=0.8 and used to infect rice callus or embryo.

[0205] The callus or embryos were co-cultured with Agrobacterium for 2-3 days.

[0206] The transformed callus or embryo containing the resistance gene is selected for further culture and screening.

[0207] 5. Regeneration and Screening

[0208] The transformed callus was transferred to regeneration medium to induce regeneration of plants.

[0209] The regenerated plants were transferred to screening medium and the stably transformed plants were selected.

[0210] 6. Identification of positive colonies

[0211] The positive colonies were identified using the method described in Example 2.

[0212] 7. Transformation of wild-type rice callus

[0213] The callus tissue of wild-type rice transformed with positive Agrobacterium was selected and cultured at 20° C. in the dark for three days.

[0214] 8. Screening Culture

[0215] The callus was transferred to a screening medium containing the corresponding resistance and cultured at 28°C in the dark for 14 to 21 days.

[0216] 9. Differentiation Culture

[0217] The callus grown on the screening medium was transferred to the differentiation medium and cultured under light conditions of 28°C for several days until visible seedlings were differentiated.

[0218] 10. Rooting culture

[0219] The differentiated seedlings are inserted into test tubes containing rooting medium and cultured at 28°C for several days under light conditions until new roots grow.

[0220] 11. Short-term storage

[0221] When the seedlings have grown relatively completely (the leaves and root system are relatively intact), add ddH2O to the test tubes and store them in light at 28°C for a short period of time (not too long, generally not more than 14 days).

[0222] 14. CRISPR / Cas9 gene editing

[0223] 1) The genomes of successfully transformed CRISPR / Cas9 gene-edited T0 rice and wild-type rice were extracted using the CTAB method;

[0224] 2) Design specific identification primers near the gene editing target site, use the genome of the corresponding material as a template, perform PCR program, perform PCR amplification, and send the PCR product to the company for sequencing;

[0225] 3) Compare the sequencing results of the gene-edited T0 generation rice with those of the wild-type rice, and summarize and record the specific gene editing conditions of each strain;

[0226] 4) The strains with editing around the target site and a single peak in sequencing results are homozygous T0 generation strains. The T1 generation seeds obtained by self-pollination of the homozygous T0 generation strains are numbered according to the gene editing situation and collected individually;

[0227] 5) The strains with editing around the target site and double peaks in sequencing results are heterozygous T0 generation strains. The T1 generation seeds obtained by self-pollination of the heterozygous T0 generation strains are screened with hygromycin, and the screened seeds are continued to be used in subsequent experiments.

[0228] CTAB method rice genome:

[0229] 1) Sampling: Take leaves of the strain whose genome needs to be identified, quickly freeze them in liquid nitrogen, and then transfer them to a -20°C refrigerator for storage;

[0230] 2) Wash the grinding rod and mortar, dry them, and grind the sample leaves;

[0231] 3) Add 500 μl of CTAB solution to each fully ground sample, mix by inverting, and place in a 65°C water bath for 30 minutes to fully lyse the cells;

[0232] 4) Move the sample to room temperature, add 500 μl of chloroform, mix by inversion, and centrifuge at 12,000 g for 10 minutes at room temperature;

[0233] 5) Take a new EP tube, add 80 μl of anhydrous ethanol in advance, take about 400 μl of the supernatant in step 4 and add it, mix it upside down, and place it in a -20℃ refrigerator overnight;

[0234] 6) Take out the overnight sample and centrifuge at 4°C, 12000g for 10 minutes;

[0235] 7) Pour out the supernatant, place the EP tube upside down on absorbent paper laid in advance, dry it, and add 30μl ddH2O (if storage is required, the prepared genomic samples should be stored at -20℃).

[0236] Example 1: Identification of OsLTP1.12 by mass spectrometry and WB

[0237] 1. Differential gene expression of rice Oscdr1 mutant after inoculation with PXO99

[0238] Since OsCDR1 is located in the apoplast of rice, we extracted apoplast proteins of WT (wild type, the rice wild type ZH11 was used in this experiment) and Oscdr1 (rice Oscdr1 mutant, Oscdr1 deficient) two days after inoculation with PXO99 (the pathogen of bacterial leaf blight PXO99 is a Gram-negative bacterium Xanthomonas oryzae pv. oryzae, Xoo) and performed mass spectrometry detection. The differential proteins detected in the WT apoplast after inoculation with PXO99 but not in the Oscdr1 apoplast are shown in Figure 1 The results showed that the lack of OsCDR1 protein resulted in 44 fewer proteins in the apoplast than the normal WT type.

[0239] The rice apoplast is the area between the plant cell wall and the plant cell plasma membrane, including the cell wall, intercellular space and tracheal system. It is one of the important channels for the transport of water and nutrients in the plant body, and is also involved in multiple processes such as material storage and transformation, nutrient accumulation, signal transduction, and plant-microorganism interaction. In the process of plant-microorganism interaction, ascorbic acid in the apoplast is an important component of the redox system. Its dynamic balance is precisely regulated by ascorbic acid oxidase, thereby maintaining the level of reactive oxygen in the apoplast, which plays an important role in controlling plant immunity. As the first battlefield for the interaction between host plants and pathogens, the level of reactive oxygen in the apoplast space is strictly controlled by the plant redox system. Therefore, the apoplast plays an important role in plant growth and development and in sensing external environmental stimuli.

[0240] 2. Effect of OsCDR1 on differentially expressed proteins

[0241] We used mammalian 293T eukaryotic cells to express OsCDR1 protein in vitro (OsCDR1 is an important gene in rice, encoding an aspartic acid protease associated with disease resistance signaling, Genbank accession number: GenBank: AY372184,

[0242] The gene sequence of OsCDR1 protein is shown in SEQ ID NO.4:

[0243] SEQ ID NO.4:

[0244]

[0245] Mammalian cells 293T expressing eukaryotic OsCDR1 protein were incubated with apoplast proteins from inoculated PXO99 at 37°C for 1 hour before mass spectrometry detection.

[0246] In this experiment, the synthesized OSCDR1 protein was incubated with the apoplasts with 44 protein differences after inoculation. The OSCDR1 protein reacted with some of these 44 proteins. In the results, we searched for proteins detected in the WT apoplasts but not in the Oscdr1 apoplasts ( Figure 1 , red and annotated proteins); among these proteins, OsLTP1.12 ( Figure 1 , Q2RBD1) protein marked in blue.

[0247] OsLTP1.12 (Q2RBD1) is a non-specific lipid transfer protein (LTPs). Many chemicals that induce systemic acquired responses in plants require non-specific lipid transfer proteins. Therefore, subsequent work will start with this protein. Gene name: OsLTP1.12, LOC number: LOC_Os11g02400, Os number: Os11g0116000.

[0248] 3. Isolate OsLTP1.12 by western-blot to verify that OsLTP1.12 is a substrate of OsCDR1

[0249] (1) Exogenous (in vitro) cleavage experiment:

[0250] When OsLTP1.12△SP-MBP was treated at 37℃ for 1h, the protein did not undergo hydrolysis compared with that without treatment, indicating that the protein would not self-degrade at 37℃;

[0251] Subsequently, OsLTP1.12△SP-MBP and OsCDR1 were incubated together at 37°C for 1 h. The results showed that the protein was enzymatically hydrolyzed and a 43 kD band appeared after cleavage ( Figure 2 );

[0252] (△SP: signal peptide removed), signal peptide is a short peptide located at the N-terminus of a protein, responsible for carrying protein secretion information, and is ubiquitous in all prokaryotes and eukaryotes. Their main function is to guide newly synthesized proteins to the secretory pathway, ensuring that proteins can be correctly transferred across the membrane to specific parts of the cell, such as the endoplasmic reticulum, Golgi apparatus, nucleus, mitochondria, etc.

[0253] MBP is a natural protein found in Escherichia coli, whose encoding gene is malE, which is responsible for the transport and metabolism of maltose. In genetic engineering, MBP can be used as a fusion tag to help expression and purification by improving the solubility and stability of the target protein.

[0254] WB test results Figure 2 The results showed that when OsCDR1 was co-expressed, OsLTP1.12△SP-MBP underwent similar endogenous cleavage.

[0255] Figure 2 In the WB electrophoresis strip, the size of each element is:

[0256] OsLTP1.12ΔSP-MBP is 52.04 kD (43 + 11.44 2.4 = 52.04), MBP tag is 43 kD, OSLTP1.12 is 11.44 kD, signal peptide is 2.4 kD, and OsLTP1.12ΔSP is 8.64 kDa;

[0257] OsCDR1 cuts off about 5.15kD, so the band produced after cutting below is about 46.89kD

[0258] OSLTP1.12, 11.44 kD, is hydrolyzed by aspartic protease (OsCDR1) to produce a polypeptide fragment pep1 as shown in SEQ ID NO.3:

[0259] SEQ ID NO. 3: KTLAGTIKSLNLGAAAGIPGKCGVNVGFPISSLSTDCSKVS.

[0260] (2) OsLTP1.12△SP-MBP is expressed alone in tobacco cells, or OsLTP1.12△SP-MBP and OsCDR1 are co-expressed in tobacco cells, and the tobacco transformation technology is used by Agrobacterium tumefaciens. The basic steps are as follows:

[0261] 1) Preparation stage:

[0262] Tobacco (eg, Nicotiana benthamiana) is grown under conditions of 14 hours light / 10 hours dark, 25°C, and 70% relative humidity for about 4 to 5 weeks.

[0263] 2) Transformation and cultivation of Agrobacterium:

[0264] The vector containing the target gene was transferred into Agrobacterium, Agrobacterium strain: GV3101.

[0265] Select a single clone of Agrobacterium containing the target vector and culture it in LB medium containing the corresponding antibiotics.

[0266] The Agrobacterium bacterial suspension was transferred to LB medium containing antibiotics and acetosyringone for expansion culture until the Agrobacterium grew to the logarithmic phase (OD600=0.5-0.6).

[0267] 3) Treatment of Agrobacterium:

[0268] Agrobacterium cells were collected by centrifugation and suspended in an inoculum containing MgCl2, MES and acetosyringone to OD600 = 1.0.

[0269] Let stand at room temperature for 2 to 3 hours to activate the infection ability of Agrobacterium.

[0270] 4) Transformation of tobacco leaves:

[0271] Use a 1mL needle to gently make a small hole on the back of the tobacco leaf, and inject the Agrobacterium solution into the leaf using a needle-free syringe.

[0272] The leaves were ground into powder, resuspended in lysis buffer (containing Tris-HCl, glycerol, PVP, NaCl, Triton X-100 and protease inhibitors), and incubated on ice for 1 hour.

[0273] After centrifugation at 14,000 rpm for 10 minutes, the supernatant was taken for Western blot analysis to detect the expression of specific proteins.

[0274] Example 2: Identification of the cleavage site of OsLTP1.12 by OsCDR1

[0275] According to the results of Example 1, we synthesized pep1 containing 45 amino acids at the C-terminus of OsLTP1.12 in vitro (SEQ ID NO.3: KTLAGTIKSLNLGAAAGIPGKCGVNVGFPISLSTDCSKVS). After comparing with the peptide database, we focused on the site near 78L (aspartate hydrolase prefers to cut L), and found that:

[0276] Aspartate hydrolases tend to cleave peptide bonds with aromatic amino acids (such as phenylalanine F, tryptophan W and tyrosine Y) or leucine L at the amino or carboxyl termini (Bhakat, 2021). OsCDR1 is an aspartate hydrolase (Bhakat S. 2021. Pepsin-like aspartic proteases (PAPs) as model systems for combining biomolecular simulation with biophysical experiments. RSC Adv, 11: 11026-11047.)

[0277] pep1 containing the C-terminal 45 amino acids of OsLTP1.12 was synthesized in vitro. After incubation of pep1 and OsCDR1 proteins at 37°C for 30 minutes, mass spectrometry was performed and two peptides were detected:

[0278] Cleavage product polypeptide 1: TLAGTIKSLNLGAAAGIPGK (SEQ ID NO. 1);

[0279] The abundance of the two peptide fragments of cleavage product peptide 2: TLAGTIK (SEQ ID NO. 2) was the highest.

[0280] Therefore, OsLTP1.12( Figure 1 The Q2RBD1 protein (marked in blue in the middle) is cut between position 78 L and position 79 K ( Figure 3 )

[0281] Example 3: OsCDR1 cleavage of substrate is induced by bacterial infection

[0282] It is known that OsCDR1 expression is not regulated. In order to verify whether the reaction of OsCDR1 cleavage substrate OsLTP1.12 (Q2RBD1) identified in Example 1 can be induced by pathogens, the following experiment was designed:

[0283] 1. Determination of OsLTP1.12 gene expression under bacterial infection conditions

[0284] (1) Use a pipette to absorb sterilized ddH2O to rinse the cultured PXO99 and dilute the collected bacterial solution to OD 600 = 0.5. Leaf cutting method (scissors dipped in diluted bacterial solution and cut the leaves 2 cm below the leaf tip) inoculated with Bacillus subtilis PXO99;

[0285] (2) Detect the changes in the transcription level of OsLTP1.12 after inoculation with PXO99;

[0286] The results are as follows Figure 4 It was found that the transcription level of OsLTP1.12 gradually increased after inoculation. Six hours after inoculation with PXO99, its expression was nearly 8 times that before inoculation.

[0287] 2. The antibody against OsLTP1.12 was prepared to quantitatively detect the changes in the amount of OsLTP1.12 protein under bacterial infection. The results are as follows Figure 5 As shown, 6 hours after infection, the protein level of OsLTP1.12 was significantly increased and could still be detected after 20-fold dilution; while as negative controls, there was no significant difference in the gene expression of OsLTP1.19 and OsLTP1.20 after inoculation.

[0288] Example 4: Relationship between OsLTP1.12 and pep1 polypeptides and plant SAR

[0289] 1. After OsLTP1.12 mutation, the SAR level of the plant was significantly reduced;

[0290] The results of Example 3 showed that OsLTP1.12 could be induced to express by bacterial infection. Therefore, three mutants of OsLTP1.12 #4, #10, #28 ( Figure 6 , a single A base was deleted in Osltp1.12#4, a single A base was inserted in Osltp1.12#10, and the ATC base was deleted in Osltp1.12#28);.

[0291] The other three strains #11, #12, and #23 were different rice strains transformed with the same pUbiO plasmid (all were Ubi::OsLTP1.12 overexpression strains) to overexpress OsLTP1.12;

[0292] To verify whether the mutated protein will cause changes in the SAR level of plants, the experimental design is as follows:

[0293] (1) The second fully expanded leaf on 60-day-old rice tillers was inoculated with PXO99;

[0294] (2) Two days later, the third fully expanded leaf on the tiller (this leaf was defined as the infection leaf) was inoculated with PXO99 and its resistance was analyzed;

[0295] (3) As a control, we treated the second fully expanded leaf on the tiller of another plant in the same way, but without the pathogen, and then analyzed the resistance of the third fully expanded leaf on the tiller (this leaf was defined as the un-infection leaf) to the bacterial blight pathogen PXO99.

[0296] The experimental steps are as follows:

[0297] (1) pep1 is an exogenously synthesized peptide in this study. We took 1 mg of pep1 powder, dissolved it in 5 mL of sterilized ddH2O, and applied pep1 exogenously to the second fully expanded leaf grown on the tiller of 60-day-old rice by the leaf cutting method (scissors were dipped in the peptide solution and the leaves were cut 2 cm below the leaf tip);

[0298] (2) Two days later, the third fully expanded leaf on the tiller was inoculated with Bacterial Blight PXO99 using the leaf cutting method;

[0299] (3) After 14 days, the length of the lesions was observed and measured.

[0300] The experimental results are as follows Figure 7 , Figure 8 ,The results showed that simply overexpressing Osltp 1.12 (the amount of OsCDR1 was the same) could not directly increase the SAR level of rice;

[0301] In order to further verify the function of pep1 (pep1 is the pep1 polypeptide hydrolyzed from OsLTP1.12, with the sequence of SEQ ID NO.3), we constructed an Ubi::1.12SP-LTP1.12 overexpression strain that overexpresses pep1 with the OsLTP1.12 signal peptide sequence in wild-type rice.

[0302] Since pep1 in wild-type rice is produced by OsCDR1 hydrolyzing OsLTP1.12 after inoculation, wild-type rice does not directly express 1.12SP-pep1. Therefore, we used three strains (Ubi::1.12SP-LTP1.12#2, #11, #20) with high expression levels of pep1 (OsLTP1.12 peptide) for subsequent experiments.

[0303] The experimental results are as follows Fig. 9 The results showed that overexpression of pep1 polypeptide (SEQ ID NO.3) can directly increase the SAR level of rice.

[0304] The pUbiO plasmid is a plant expression vector that uses the rice Ubiquitin promoter (often referred to as UBI promoter) to drive the expression of foreign genes. The Ubiquitin promoter is a strong promoter that enables efficient and stable gene expression in plants. The pUbiO plasmid is commonly used in plant genetic transformation and functional gene research, allowing researchers to place target genes under the control of the UBI promoter to ensure that the genes are effectively expressed in plants.

[0305] The main features of pUbiO plasmids include:

[0306] 1. Strong promoter: Use UBI promoter, which has broad expression activity in plants and can drive efficient gene expression in a variety of plant tissues.

[0307] 2. Plant expression: Expression vectors designed specifically for plant systems are suitable for a variety of plant species, including monocots and dicots.

[0308] 3. Selectable marker: Plasmids usually contain antibiotic resistance genes, such as kanamycin resistance gene, which is used to select transformed cells.

[0309] 4. Multiple cloning sites: provide multiple restriction enzyme cutting sites to facilitate researchers to insert target genes.

[0310] 5. Reporter gene: Sometimes pUbiO plasmids are used in combination with reporter genes (such as GUS or GFP) to facilitate monitoring and verification of gene expression.

[0311] The specific construction and application of pUbiO plasmid may vary according to different research purposes and needs, but its core purpose is to utilize the powerful driving force of the UBI promoter to achieve efficient expression of foreign genes in plants.

[0312] The above results indicate that the OsLTP1.12 polypeptide needs to be hydrolyzed into the pep1 peptide fragment in order to have the activity of increasing the SAR level.

[0313] 2. Testing the activity of pep1 peptide in Oscdr1 loss-of-function mutant strains

[0314] #8, #21 and #26 are Oscdr1 loss-of-function mutant strains

[0315] #16, #37, and #41 are Oscdr1 loss-of-function mutant strains, but they contain the plasmid Ubi::1.12SP-pep1 and can express pep1

[0316] The experimental results are as follows Fig.10 As shown, it is shown that the OsLTP1.12 polypeptide needs to be hydrolyzed into the pep1 peptide fragment before it has the activity of increasing the SAR level.

[0317] Example 5: Exogenous addition of chemically synthesized pep1 polypeptide fragments can enhance the expression of plant disease resistance genes.

[0318] It has been reported that SAR mobile signal molecules are conserved (it has been reported that the leaf exudates of cucumber leaves after inoculation can directly induce the SAR response of Arabidopsis), so we applied exogenously synthesized pep1 peptides to the leaves of Arabidopsis, tobacco and rapeseed.

[0319] Specifically, 1 mg of pep1 polypeptide powder was dissolved in 5 mL of sterilized ddH2O, and the pep1 polypeptide was exogenously applied to the second fully expanded leaf grown on the 60-day-old rice tiller by the leaf cutting method (scissors were dipped in the polypeptide solution and the leaves were cut 2 cm below the leaf tip). Two days later, the third fully expanded leaf grown on the tiller was inoculated with bacterial blight PXO99 by the leaf cutting method, and 14 days later, the length of the lesions was observed and measured.

[0320] The results are as follows Fig.11 , indicating that exogenous pep1 polypeptide can also induce the expression of disease resistance-related genes in the corresponding plants.

[0321] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the technical content of the present invention, and are not used to limit the protection scope of the present invention.

Claims

1. A group of polypeptides capable of inducing systemic acquired resistance (SAR) in plants, wherein the plants include but are not limited to Arabidopsis, tobacco, and rapeseed; the polypeptide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3, SEQ ID NO.1: KTLAGTIKSLNLGAAAGIPGK; SEQ ID NO.2: KTLAGTIK; SEQ ID NO. 3: KTLAGTIKSLNLGAAAGIPGKCGVNVGFPISSLSTDCSKVS.

2. A plant antibacterial preparation, comprising: the polypeptide according to claim 1 and necessary preparation excipients; the preparation excipients include but are not limited to: buffers, stabilizers, antioxidants; the plants include but are not limited to Arabidopsis, tobacco, and rapeseed.

3. Use of the polypeptide according to claim 1 in inducing SAR in plants, wherein the plants include but are not limited to Arabidopsis, tobacco, and rapeseed.

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