Cloning of tomato resistance gene slprepip2 and its application in small peptide slpip2

Abstract

The application provides a tomato anti-late blight gene SlprePIP2 and application of the gene and a small peptide SlPIP2, a nucleotide sequence of the tomato anti-late blight gene SlprePIP2 is shown as SEQ ID NO. 1, an amino acid sequence of a small peptide SlPIP2 precursor protein encoded by the tomato anti-late blight gene SlprePIP2 is shown as SEQ ID NO. 2, and the small peptide SlPIP2 is derived from a C-terminal end of SEQ ID NO. 2 and is shown as SEQ ID NO. 3. The application screens the SlprePIP2 gene which is significantly up-regulated in response to induction of a late blight bacterium, provides a cloning method of the gene, and constructs a PBI121-SlprePIP2 overexpression vector, and overexpression of the SlprePIP2 can improve the resistance of a plant to the late blight bacterium. In addition, the tomato plant is sprayed with the small peptide SlPIP2, the transcription level of a disease resistance protein PR is improved, the disease resistance of the plant is further enhanced, and this is helpful to mine a new type of plant disease resistance polypeptide, and provides a new idea for carrying out the work of preventing and treating the late blight.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the cloning of a tomato late blight resistance gene SlprePIP2 and its application with the small peptide SlPIP2. Background Technology

[0002] Tomatoes are one of the most widely grown and popular fruits and vegetables in the world, rich in nutrients and with a unique flavor. Late blight, caused by *Pseudomonas aeruginosa*, significantly impacts tomato yield and quality. Although chemical pesticides have some effect in controlling the occurrence and spread of late blight, the resulting environmental pollution and food safety issues have caused considerable trouble for agricultural production. Therefore, researching the interaction mechanism between tomato and late blight, and using molecular biology methods to improve tomato resistance to late blight, has become a superior research direction.

[0003] While the traits and flavor of tomatoes have improved during long-term domestication, continuous artificial selection has reduced their genetic diversity, leading to the near loss of many resistance genes. This has resulted in decreased resistance to pathogens in some cultivated varieties. Fortunately, these valuable resistance gene resources are still preserved in wild tomato strains. Therefore, the phylogenetic relationship between wild and cultivated tomatoes provides a new approach for identifying key regulatory factors, namely, introducing gene resources from wild species into cultivated tomatoes, potentially enhancing the latter's resistance. Previous studies have found significant differences in resistance to late blight between the wild currant tomato strain *Solanum pimpinellifolium* “L3708” (Sp3708) and the cultivated tomato variety *Solanum lycopersicum* “Zaofen No.2” (Zaofen2), with the former exhibiting high resistance while the latter is relatively susceptible. This suggests that identifying key genes from Sp3708 tomatoes and applying them to Zaofen2 may be an effective way to improve the disease resistance of cultivated tomatoes.

[0004] In nature, various adverse environmental factors such as pathogen infection and mechanical damage constantly threaten plant survival. Plants can often utilize exogenous or endogenous signaling molecules to recognize and effectively respond to these stresses. These signaling molecules include pathogen-associated molecular patterns (PAMPs) derived from pathogens and damage-associated molecular patterns (DAMPs) derived from the plant itself. To date, many DAMPs have been identified, and their functions in plant immunity have been partially understood. Plant DAMPs mainly consist of cytoplasmic proteins, peptides, nucleotides, and amino acids, which are released from damaged cells or secreted by intact cells that have experienced pathogen invasion.

[0005] In recent years, a novel class of small peptides, PIPs (PAMP-induced secreted peptides), has been discovered in plants. These peptides can act as DAMP molecules to regulate plant immunity. PIPs are widely present in most angiosperms, including some important crops such as potatoes. Previous studies have provided ample evidence to establish the crucial role of PIPs in plant resistance. For example, Arabidopsis PIPs are induced by flg22 and chitin. Studies have found that Arabidopsis overexpressing the PIP1 and PIP2 precursor genes exhibits enhanced defense against *F. oxysporum* f.sp. conglutinans strain 699 (Foc699). Exogenous application of mature PIP1 and PIP2 not only increases Arabidopsis resistance to Pst DC3000 but also induces the transcription of defense response genes such as FRK1, WRKY30, and PR1. Unlike the functions of the PIP1 and PIP2 precursor genes, overexpression of the PIP3 precursor gene led to increased susceptibility of Arabidopsis to Botrytis cinerea (B. cinerea) and Pseudomonas syringae (P. syringae). Overexpression of StPIP1 in potato reduced plant susceptibility to Potato virus Y. Exogenous spraying of synthetic StPIP1 onto potato leaves and stem nodes increased ROS levels in the plants, and consequently increased the expression of defense-related genes, thereby triggering an early defense response in potatoes.

[0006] However, to date, there have been no studies on the interaction between SlprePIP2 and its mature peptide SlPIP2 in tomato and late blight pathogens. Summary of the Invention

[0007] To address the problems existing in the background art, the present invention provides a tomato late blight resistance gene SlprePIP2, its cloning method and application, as well as a tomato late blight resistance peptide SlPIP2 and its application.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] In a first aspect, the present invention provides a tomato late blight resistance gene SlprePIP2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a tomato late blight resistant peptide SlPIP2, wherein the peptide SlPIP2 is derived from the C-terminus of the peptide SlPIP2 precursor protein encoded by the gene SlprePIP2, the nucleotide sequence of the gene SlprePIP2 is shown in SEQ ID NO.1; the amino acid sequence of the peptide SlPIP2 precursor protein is shown in SEQ ID NO.2; and the amino acid sequence of the peptide SlPIP2 is shown in SEQ ID NO.3.

[0011] A third aspect of the present invention provides a method for cloning the above-mentioned tomato late blight resistance gene SlprePIP2, comprising the following steps:

[0012] Sa1. Using tomato cDNA as a template, PCR amplification was performed using specific primers; the specific primers are as follows:

[0013] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0014] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0015] Sa2. The obtained PCR product was ligated with the pMD-19T cloning vector to obtain the ligation product pMD-19T-SlprePIP2.

[0016] Sa3. Transform the ligation product pMD-19T-SlprePIP2 into Escherichia coli DH5α and spread it on LB solid medium containing ampicillin. Pick a single colony, shake it to extract the plasmid, perform plasmid PCR, and then send the plasmid for sequencing. If the sequencing result is the plasmid in SEQ ID NO.1, then it is correct.

[0017] A fourth aspect of the present invention provides an application of the above-mentioned tomato late blight resistance gene SlprePIP2, comprising the following steps:

[0018] Sb1. Using tomato cDNA as a template, PCR amplification was performed using specific primers; the specific primers are as follows:

[0019] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0020] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0021] Sb2. The obtained PCR product is ligated with the pMD-19T cloning vector to obtain the ligation product pMD-19T-SlprePIP2.

[0022] Sb3. The pMD-19T-SlprePIP2 plasmid, which was correctly sequenced, was digested with restriction endonucleases BamHI and SacI. The target fragment was recovered and ligated into the pBI121 expression vector with the GUS gene removed. The ligation product was transformed into E. coli DH5α and plated on LB solid medium containing kanamycin. After picking a single colony and shaking it, the plasmid was extracted. After plasmid PCR identification, the plasmid was sent for sequencing. The plasmid with correct sequencing was the recombinant expression vector pBI121-SlprePIP2.

[0023] Sb4. The recombinant expression vector was introduced into Agrobacterium GV3101 competent cells and plated on LB solid medium containing kanamycin, gentamicin and rifampin. Single colonies were picked for bacterial PCR verification to obtain positive engineered bacterial culture.

[0024] Sb5. Using five-leaf stage tomatoes as the treatment object, the prepared positive engineered bacterial solution was injected into the leaves of the five-leaf stage tomatoes to achieve overexpression of SlprePIP2.

[0025] In the preferred embodiment, the LB medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, and ultrapure water.

[0026] Specifically, the LB medium consists of 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, and ultrapure water to a final volume of 1L.

[0027] In a fifth aspect, the present invention provides an application of the above-mentioned tomato late blight resistant peptide SlPIP2, as described in any one of the following:

[0028] (1) Used for the prevention / treatment of tomato late blight;

[0029] (2) As a crop immune stimulant.

[0030] In the preferred embodiment, the application of the tomato late blight resistant peptide SlPIP2 is achieved by spraying the tomato plants with the tomato late blight resistant peptide SlPIP2.

[0031] Beneficial effects of the present invention

[0032] 1. This invention constructs a recombinant expression vector for SlprePIP2. Overexpression of SlprePIP2 using this invention allows for the regulation of tomato late blight resistance genes. Specifically, the transient overexpression of SlprePIP2 in plants obtained by this method shows higher SlprePIP2 expression levels than in unexpressed plants, resulting in superior resistance to late blight. This invention enhances tomato resistance to late blight through overexpression of the SlprePIP2 gene, which is of great significance for breeding late blight-resistant tomato varieties.

[0033] 2. Since the gene encoding the SlprePIP2 precursor peptide can enhance the resistance of tomatoes to late blight, further analysis was conducted to determine whether the mature SlPIP2 peptide at its C-terminus also promotes resistance to late blight in tomatoes. This invention also provides a tomato late blight-resistant peptide, SlPIP2, which, when sprayed onto tomato plants, can enhance tomato immunity and increase the resistance of tomato plants to late blight pathogens. This contributes to the protection of tomato resistance and the development of the tomato industry.

[0034] 3. SlPIP2 can enhance the transcriptional accumulation of the disease-resistance protein PR in plants, and PR proteins can directly affect plant disease resistance. It can serve as a widely useful crop immunostimulant for tomatoes against various pathogens. Therefore, our research will provide new insights for crop protection and pesticide development. Furthermore, these advances may provide a reference point for crop disease resistance breeding and lay a theoretical foundation for effective and broad-spectrum disease management in modern agriculture.

[0035] 4. An overexpression plasmid for SlprePIP2 was constructed using the PBI121 vector, with a transient expression of the empty PBI121 vector serving as a control. Both the SlprePIP2 overexpression plasmid and the empty PBI121 vector were transiently expressed in tomato leaves. After 3 days, the transcriptional activity of SlprePIP2 was 5.3 times that of the transiently expressed empty PBI121 vector (EV) in tomato leaves. Then, an in vitro leaf inoculation experiment was conducted. Five days after infection of the transient expression site with *Phytophthora infestans*, compared to the EV group, the SlprePIP2 overexpression group showed lower lesion severity and smaller lesion diameter. Attached Figure Description

[0036] Figure 1 The expression level of the SlprePIP2 gene at different time points of infection with Late Pesticides;

[0037] Figure 2To enhance disease resistance by overexpressing the SlprePIP2 gene;

[0038] Figure 3 Changes in disease resistance phenotype and PR gene transcription level after spraying tomatoes with SlPIP2 peptide. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] This invention provides a cloning of the tomato late blight resistance gene SlprePIP2 and the application of its mature peptide SlPIP2.

[0041] A tomato late blight resistance gene, SlprePIP2, has the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence of its encoded precursor protein is shown in SEQ ID NO.2. The small peptide SlPIP2 is derived from the C-terminus of the amino acid sequence SEQ ID NO.2 as shown in SEQ ID NO.3.

[0042] A method for cloning the tomato late blight resistance gene SlprePIP2 includes the following steps:

[0043] Sa1. Using tomato cDNA as a template, PCR amplification was performed using specific primers; the heterogeneous primers are as follows:

[0044] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0045] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0046] Sa2. The obtained PCR product was ligated with the pMD-19T cloning vector to obtain the ligation product pMD-19T-SlprePIP2.

[0047] Sa3. Transform the ligation product pMD-19T-SlprePIP2 into Escherichia coli DH5α and spread it on LB solid medium containing ampicillin. Pick a single colony, shake the culture, extract the plasmid, perform plasmid PCR, and then send the plasmid for sequencing. If the sequencing result is the plasmid (pMD-19T-SlprePIP2 plasmid) in SEQ ID NO.1, then it is correct.

[0048] The application of the tomato late blight resistance gene SlprePIP2 includes the following steps:

[0049] S1. Construct the recombinant expression vector of SlprePIP2;

[0050] S2. The recombinant expression vector was introduced into Agrobacterium GV3101 competent cells and plated on LB solid medium containing kanamycin, streptomycin and rifampin. Single colonies were picked for bacterial PCR verification to obtain positive engineered bacterial culture.

[0051] S3. Take five-leaf stage tomatoes as the treatment object, and inject the prepared positive engineered bacterial solution (Agrobacterium transient infection solution) into the leaves of seven-leaf stage tomatoes to achieve overexpression of SlprePIP2.

[0052] The method for constructing the recombinant expression vector encoding the SlprePIP2 gene includes digesting the correctly sequenced pMD-19T-SlprePIP2 plasmid with restriction endonucleases BamHI and SacI, recovering the target fragment, ligating the target fragment with the pBI121 expression vector (with the GUS gene removed), transforming the ligation product into *E. coli* DH5α and plating it on LB agar containing kanamycin, picking single colonies, shaking the culture, extracting the plasmid, performing plasmid PCR identification, and sequencing the plasmid. The plasmid with correct sequencing is the recombinant expression vector pBI121-SlprePIP2. The pMD-19T-SlprePIP2 plasmid is derived from a cloning method, i.e., its preparation process is the same as steps Sa1-Sa2 described above.

[0053] A small peptide for resisting late blight in tomatoes, SlPIP2, has the amino acid sequence shown in SEQ ID NO.3.

[0054] This invention also provides the application of the tomato late blight-resistant peptide SlPIP2 to the resistance of tomatoes to late blight. The tomato plants were sprayed with the tomato peptide SlPIP2, followed by spraying with a late blight spore suspension. The plant lesions were observed, and the SlPIP2-induced response produced effective tomato immunity.

[0055] The aforementioned tomato late blight resistant peptide SlPIP2 is used as a crop immune stimulant.

[0056] Example 1

[0057] Obtaining the SlprePIP2 gene

[0058] First, genes responding to late blight induction were screened using an existing transcriptome library of *P. infestans* P12103 infected tomato. Peptides encoding fewer than 150 amino acids were selected, and the gene encoding the small peptide SpPIP of interest was found. Using real-time quantitative PCR, the gene SlprePIP2, which responds to late blight induction, was further screened, and its mature peptide was named SlPIP2.

[0059] 1. Cultivation of biological materials and inoculation of tomatoes with Late Blight pathogen

[0060] Seeds of Sp3708 tomatoes were soaked and germinated at 28℃, then sown in soil. Tomato plants at the 4-5 leaf stage were used as experimental materials after 16 hours of light and 8 hours of darkness at 28±2℃.

[0061] Late blight pathogens were inoculated onto oat solid medium and cultured at 20°C for 3–4 weeks. 10 mL of sterile water was added to the medium covered with mycelia. Mycelia were gently scraped off from the surface and placed at 4°C to promote zoospore release. The filtrate was collected using three layers of sterile gauze, and the number of spores was counted using a hemocytometer. The suspension concentration was adjusted to 1 × 10⁻⁶ spores per mL with sterile water. 6 One spore.

[0062] Tomatoes were treated with late blight pathogen using a spray inoculation method. A suspension of pathogen spores was evenly sprayed onto the surface of each tomato leaf and stem. After inoculation, the plants were incubated in the dark at 90%–100% humidity and 20±1℃ for 24 hours, followed by 16 hours of light and 8 hours of dark incubation. Tomato leaves were collected at 0, 3, 6, 12, 24, 48, 72, and 96 hours after infection and rapidly frozen in liquid nitrogen for later use.

[0063] 2. Total RNA extraction and reverse transcription

[0064] Total RNA was extracted from all tomato leaves using RNAiso Plus. Transcriptome sequencing was performed on total RNA from both uninoculated and late blight-inoculated tomatoes of good quality. The specific steps are as follows:

[0065] 1) Extraction of total RNA from tomato leaves

[0066] ① The mortar and pestle are baked in an oven. Liquid nitrogen is poured into the mortar before grinding the sample and it is fully pre-cooled. All pipette tips and centrifuge tubes must be treated with RNase-free agents during extraction.

[0067] ② Take an appropriate amount of tomato leaves and grind them thoroughly into powder in a mortar. Add the powder to 1 mL of pre-cooled Trizol, shake to mix immediately, and let stand for 5–10 minutes.

[0068] ③ Add 250 μL of chloroform solution to the centrifuge tube, invert to mix, let stand for 5 min, and centrifuge at 4℃ and 12000 rpm for 10 min;

[0069] ④ Transfer the colorless supernatant from centrifugation to a new centrifuge tube and add an equal volume of pre-cooled isopropanol solution. Mix thoroughly and let stand for 10 minutes, then centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0070] ⑤ Discard the supernatant, retain the precipitate, add 1 mL of 75% ethanol to carefully resuspend and wash the precipitate, centrifuge at 4°C and 12000 rpm for 5 min; then repeat this step again.

[0071] ⑥ After discarding the supernatant, open the lid and let it stand until the ethanol evaporates. Then add 20 μL of RNase-Free ddH2O to dissolve the precipitate.

[0072] 2) Reverse transcription of total RNA

[0073] The extracted tomato total RNA was reverse transcribed into cDNA. Specifically, following the reverse transcription system instructions of Takara (Code No. RR036A), reagents were added to a 200 μL RNase-free PCR tube. The tube was then placed in a PCR instrument and reacted at 37°C for 15 min, then at 85°C for 5 s to obtain tomato cDNA. Finally, the sample was stored at -20°C for subsequent RT-qPCR experiments (real-time polymerase chain reaction).

[0074] 3. Obtaining the SlprePIP2 gene

[0075] like Figure 1 As shown, the transcriptional level of the gene encoding the PIP polypeptide precursor protein was detected using real-time quantitative PCR. A gene with significantly upregulated transcriptional level after infection with Late Blight was obtained. The PIP polypeptide sequences encoded in Arabidopsis thaliana and potato were compared and named SlprePIP2, and its mature peptide was named SlPIP2.

[0076] The above real-time quantitative PCR reaction system is as follows:

[0077]

[0078] Example 2

[0079] Application of the late blight resistance gene SlprePIP2

[0080] To analyze the role of SlprePIP2 in the interaction mechanism between tomato and late blight pathogens, an overexpression plasmid of SlprePIP2 was constructed using the PBI121 vector and transiently expressed in tomato leaves. After 3 days, the transcriptional activity of SlprePIP2 was almost 5.3 times that of the transiently expressed empty PBI121 vector (EV) in tomato leaves. Then, an in vitro leaf inoculation experiment was conducted. Five days after infection with late blight pathogens at the transient expression site, compared with the EV group, the SlprePIP2 overexpression group showed lower lesion severity and smaller lesion diameter.

[0081] 1. Cloning of the SlprePIP2 gene

[0082] 1) Using tomato cDNA as a template, PCR amplification was performed using specific primers.

[0083] The specific primers used for cloning are as follows:

[0084] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0085] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0086] The reaction conditions are as follows:

[0087]

[0088] 2) Recovery and purification of PCR amplification products

[0089] After detecting the PCR products by 1% agarose gel electrophoresis, the PCR products that meet the target fragment size are recovered using a gel extraction kit (purchased from Takara).

[0090] 3) Ligation of the target fragment with the cloning vector

[0091] The target fragment obtained above was ligated with the cloning vector pMD-19T (purchased from Takara), and the reaction system is as follows:

[0092]

[0093] The ligation product pMD-19T-SlprePIP2 was obtained by ligation at 16℃ for 8 hours.

[0094] 4) Transformation of the ligation product into E. coli

[0095] (1) Add 10 μL of the ligation product to E. coli DH5α competent cells, mix by pipetting, and then incubate in an ice water bath for 30 min.

[0096] (2) Immediately transfer the mixture after the ice-water bath to a 42°C water bath, and after 90 seconds, put it in an ice-water bath for 2 minutes.

[0097] (3) Add 1 mL of fresh antibiotic-free LB medium to the above mixture and incubate at 180 rpm for 1 h in a constant temperature shaker at 37°C.

[0098] (4) Centrifuge the above bacterial solution at 12000 r / min, discard the supernatant, leave a small amount of bacterial solution, resuspend it by pipetting, spread it on LB plates containing 50 μg / L ampicillin, and incubate overnight in a constant temperature incubator at 37℃.

[0099] (5) Pick a single white colony and inoculate it into LB liquid medium containing 50 μg / L ampicillin. Incubate overnight at 37°C with shaking at 180 rpm.

[0100] 5) Extraction of pMD-19T-SlprePIP2 plasmid

[0101] Following the instructions of the plasmid miniprep kit (purchased from TIANGEN), the pMD-19T-SlprePIP2 plasmid was extracted from the bacterial culture. A 5 μL sample of the plasmid was then analyzed by 1% agarose gel electrophoresis.

[0102] 6) Plasmid PCR detection

[0103] Using the extracted pMD-19T-SlprePIP2 plasmid as a template, PCR was performed using SlprePIP2-FP and SlprePIP2-RP primers. The plasmid was then sequenced, and the plasmid that matched SEQ ID NO.1 was the correct plasmid for linking pMD-19T (pMD-19T-SlprePIP2 plasmid).

[0104] 2. Construction of the SlprePIP2 recombinant expression vector

[0105] 1) Enzyme digestion of pMD-19T-SlprePIP2 plasmid

[0106] The pMD-19T-SlprePIP2 plasmid was double-digested with restriction endonucleases BamHI and SacI (purchased from Takara), and the target fragment was recovered. The digestion reaction system and method are as follows:

[0107]

[0108] The enzyme was digested at 37℃ for 2 hours, and the digestion products were detected by 1% agarose gel electrophoresis.

[0109] 2) Double digestion of pBI121 plasmid

[0110] The pBI121 plasmid was double-digested with restriction endonucleases BamHI and SacI to remove the GUS gene, and the pBI121 fragment (target fragment) was recovered. The reaction system is as follows:

[0111]

[0112] The enzyme was digested at 37℃ for 2 hours, and the digestion products were detected by 1% agarose gel electrophoresis.

[0113] 3) Target fragment ligated to pBI121 vector

[0114] The target fragment was ligated to the pBI121 vector (with the GUS gene removed) using T4 DNA ligase (purchased from Takara). The reaction system is as follows:

[0115]

[0116] The cells were ligated at 16℃ for 4 hours. The ligation product was then transformed into E. coli DH5α competent cells (same transformation process as for the SlprePIP2 gene clone). After transformation, the cells were plated on LB solid medium containing kanamycin. Single colonies were picked and shaken to extract the plasmid. After PCR identification, the plasmid was sent for sequencing. The plasmid with correct sequencing results was the recombinant expression vector pBI121-SlprePIP2.

[0117] 3. Application of SlprePIP2

[0118] 1) Preparation of engineered Agrobacterium tumefaciens pBI121-SlprePIP2

[0119] The correctly sequenced recombinant plasmid pBI121-SlprePIP2 was introduced into Agrobacterium strain GV3101 competent cells using a freeze-thaw method, and then gently mixed. The cells were frozen in liquid nitrogen for 2 min, incubated at 37°C for 5 min, and then 1 mL of LB liquid medium was added. The cells were incubated at 28°C and 180 rpm for 2–3 h. After centrifugation, 800 μL was aspirated, and the remaining bacterial culture was mixed thoroughly. The culture was then spread using a spreader onto LB solid medium containing 100 mg / L streptomycin, 100 mg / L rifampin, and 50 mg / L kanamycin. After absorption, the culture was transferred to a 28°C incubator and inverted for 18–24 h.

[0120] 2) Identification of positive clones

[0121] Single colonies were picked from the plates and inoculated into LB broth containing antibiotics, and incubated at 28°C and 180 rpm for 12–16 h. PCR verification was performed using specific primers. An equal volume of glycerol was added to the positive clone culture for preservation.

[0122] 3) Establishment of a transient infection system and related detection

[0123] (1) Preparation of Agrobacterium transient infection solution

[0124] ① Agrobacterium tumefaciens activated bacterial suspension containing transient expression pBI121 empty vector (EV group) and pBI121-SlprePIP2 was inoculated into 5 mL of LB liquid medium containing 50 mg / L Gent, 100 mg / L Rif and 50 mg / L Kana, and cultured at 28℃ with shaking at 180 rpm for 12-16 h.

[0125] ② Add 50 mg / L Gent, 100 mg / L Rif, 50 mg / L Kana, 10 mmol / L MES (morpholinoethanesulfonic acid monohydrate), 20 μmol / L AS (acetylsyleugenone), and 2 mmol / L MgSO4 to LB medium, and adjust the pH to 5.6. Add 1 mL of the activated bacterial solution to this liquid medium, and culture under the same conditions as above.

[0126] ③ Collect bacterial cells by centrifugation at 4℃ and 4000 rpm for 10 min. Resuspend the bacterial cells in MMA solution (containing 10 mmol / L MgCl2, 10 mmol / L LMEs, 20 μmol / L AS, pH 5.6) and measure OD using a microplate reader. 600 Adjust to 1.0, 28℃, shake at 180rpm for 3 hours.

[0127] (2) Instantaneous transformation of tomato plants

[0128] ① Select tomato leaves with uniform growth at the five-leaf stage as experimental materials. After placing them under weak light for 1-2 hours, use sandpaper to polish and remove the needle from the disposable syringe tip, and draw 200μL of activated Agrobacterium transient infection solution.

[0129] ② The treated tomatoes were cultured at 25℃ for 16 hours of light and 8 hours of darkness. Three tomato plants with the same growth were selected for each group of experiments. The experiment was repeated three times, and each Agrobacterium tumefaciens infiltration experiment was performed 9 times.

[0130] ③ Three days after injection, tomato leaves from the same location were harvested, ground into powder in a mortar, and stored in 1 mL of Trizol solution. Total RNA was extracted from the tomato leaves injected with EV and pBI121-SlprePIP2, respectively. Reverse transcription was then performed, and the expression level was measured to confirm successful vector transfer. Figure 2 As shown, the expression level of transient overexpression of SlprePIP2 (TOEprePIP2) was almost 5.3 times that of transient expression of the empty vector PBI121 in tomato leaves (EV group).

[0131] (3) Inoculation of detached leaves with Phytophthora infestans

[0132] ① Three days after injection, use a sterile toothpick to make small wounds in the middle of the leaves of EV and pBI121-SlprePIP2 tomatoes, and place them on sterile filter paper in a petri dish;

[0133] ② Apply 20 μL of late blight spore suspension to the wound on the leaf, cover the petri dish with plastic wrap to maintain humidity, and incubate in the dark at 20℃ for 5 days. Observe the disease development. Results are as follows: Figure 2 As shown, compared with the EV group, the SlprePIP2 overexpression group (TOEprePIP2) had lower lesion severity and smaller lesion diameter.

[0134] Example 3

[0135] Application of Tomato Peptide SlPIP2

[0136] Since its precursor gene can enhance tomato resistance to late blight, we further analyzed whether its C-terminal mature peptide also promotes tomato resistance to late blight. We chemically synthesized a 23-amino acid peptide corresponding to the predicted active peptide sequence of SlPIP2, as shown in SEQ ID NO.3. The peptide was diluted in water to the final concentration and used for activity assay.

[0137] Functional study of tomato peptide SlPIP2

[0138] This invention uses a 1 μM water-soluble peptide, SlPIP2, sprayed onto 4-week-old tomato plants for 24 hours, with watered leaves serving as a control (CK). 20 μL of late blight spore suspension was added dropwise to the leaf wounds, and the culture dishes were covered with plastic wrap to maintain humidity. The plants were then incubated in the dark at 20°C for 5 days, and disease development was observed. Results are as follows... Figure 3 As shown, tomato leaves sprayed with the small peptide SlPIP2 exhibited significantly milder disease symptoms compared to the control (CK) leaves. This invention demonstrates that the SlPIP2-induced response may produce effective tomato immunity. Furthermore, the expression levels of disease resistance-related genes SlPR1, SlPR2, and SlPR5 in tomato plants treated with the small peptide (SlPIP2) were significantly higher than those in the CK, indicating that the tomato plants treated with the small peptide have high disease resistance. This experimental method is simple and explores a potential plant protectant to replace traditional pesticides, which is expected to enhance plant disease resistance.

[0139] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0140]

Claims

1. A tomato anti- late blight small peptide SlPIP2, characterized in that, The small peptide SlPIP2 is derived from a gene. SlprePIP2 The C-terminus of the gene encodes the small peptide SlPIP2 precursor protein. SlprePIP2 The nucleotide sequence of the small peptide SlPIP2 precursor protein is shown in SEQ ID NO.1; the amino acid sequence of the small peptide SlPIP2 precursor protein is shown in SEQ ID NO.2; and the amino acid sequence of the small peptide SlPIP2 is shown in SEQ ID NO.

3.

2. Use of the tomato anti- late blight small peptide SlPIP2 according to claim 1, characterized in that, For preventing tomato late blight.

3. The use of the tomato anti- late blight small peptide SlPIP2 according to claim 2, characterized in that, By spraying tomato plants with the tomato anti-late blight small peptide SlPIP2.

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