Cloning of tomato late blight resistant gene SlprePIP2 and application of tomato late blight resistant gene SlprePIP2 and small peptide SlPIP2

Abstract

The invention provides clone of a tomato anti-late blight gene SlprePIP2 and application of the tomato anti-late blight gene SlprePIP2 and a small peptide SlPIP2, the nucleotide sequence of the tomato anti-late blight gene SlprePIP2 is as shown in SEQ ID NO.1, the amino acid sequence of a small peptide SlPIP2 precursor protein coded by the tomato anti-late blight gene SlprePIP2 is as shown in SEQ ID NO.2, and the small peptide SlPIP2 is derived from the C tail end of the SEQ ID NO.2 and is as shown in SEQ ID NO.3. The invention further provides a preparation method of the tomato anti-late blight gene SlprePIP2. According to the invention, the SlprePIP2 gene which responds to induction of phytophthora infestans and is obviously up-regulated is screened, a cloning method of the gene is provided, a PBI121-SlprePIP2 overexpression vector is constructed, and overexpression of the SlprePIP2 can improve the resistance of plants to phytophthora infestans. Besides, small peptide SlPIP2 is sprayed to tomato plants, the disease resistance of the plants can be further enhanced by improving the transcriptional level of disease-resistant protein PR, the disease-resistant gene of novel plant disease-resistant polypeptide can be excavated, and a new thought is provided for development of work for preventing and treating late blight.

Description

Technical Field

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

[0002] Tomato is one of the most widely grown and popular fruits and vegetables in the world. It is rich in nutrition and has a unique flavor. Late blight caused by late blight bacteria greatly affects the yield and quality of tomatoes. Although chemical pesticides have a certain control effect on the occurrence and spread of late blight, the environmental pollution, food safety and other problems caused by it have brought great troubles to agricultural production. Therefore, the study of the interaction mechanism between tomato and late blight and the use of molecular biology methods to improve the resistance of tomatoes to late blight have become a better research direction.

[0003] Although the traits and flavor of tomatoes have been improved during the long-term domestication process, continuous artificial selection has reduced its genetic diversity, causing many resistance genes in the tomato to be almost lost, which has led to a decrease in the resistance of some cultivated varieties to pathogens. Fortunately, these valuable resistance gene resources are still preserved in wild tomato strains. Therefore, the relationship between wild and cultivated tomatoes provides a new idea for people to explore key regulatory factors, that is, to introduce wild species' genetic resources into cultivated tomatoes, which is expected to improve the latter's resistance. Previous studies have found that the wild currant tomato strain Solanum pimpinellifolium "L3708" (Sp3708 for short) and the cultivated tomato variety Solanum lycopersicum "Zaofen No.2" (Zaofen2 for short) show obvious differences in resistance to late blight. The former has a high degree of disease resistance, while the latter is relatively susceptible. This means that mining 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 the survival of plants, and plants are often able to use some exogenous or endogenous signal molecules to identify and effectively respond to these stresses. These signal molecules include pathogen-associated molecular patterns (PAMPs) derived from pathogens and damage-associated molecular patterns (DAMPs) derived from plants themselves. So far, many DAMPs have been identified, and their functions in plant immunity have been partially understood. DAMPs in plants mainly include cytoplasmic proteins, peptides, nucleotides and amino acids, which are released from damaged cells or secreted by intact cells that have undergone pathogen invasion.

[0005] In recent years, a new type of small peptide PIPs (PAMP-induced secreted peptides) has been discovered in plants, which can act as a DAMP molecule to regulate plant immunity. PIPs are widely present in most angiosperms, including some important crops such as potatoes. In previous studies, many evidences have identified the key role of PIPs in plant resistance. For example, Arabidopsis PIPs are induced by flg22 and chitin. Studies have found that Arabidopsis overexpressing PIP1 and PIP2 precursor genes has enhanced defense against F.oxysporum f.sp.conglutinans strain 699 (Foc699). Exogenous application of mature PIP1 and PIP2 can not only increase Arabidopsis's resistance to Pst DC3000, but also induce the transcription of defense response genes such as FRK1, WRKY30 and PR1. Different from the functions of PIP1 and PIP2 precursor genes, overexpression of PIP3 precursor gene leads to increased sensitivity of Arabidopsis to Botrytis cinerea (B. cinerea) and Pseudomonas syringae (P. syringae). Overexpression of potato StPIP1 reduces the sensitivity of plants to Potato virus Y. After exogenous spraying of synthetic StPIP1 on potato leaves and stem nodes, the ROS level in the plant increased, and the expression of defense-related genes also increased, thus triggering the early defense response of potato.

[0006] However, to date, there has been no relevant research on SlprePIP2 and its mature peptide SlPIP2 in the interaction between tomato and late blight. Summary of the invention

[0007] In order to solve the problems existing in the background technology, the present invention provides a tomato late blight resistance gene SlprePIP2 and a cloning method and application thereof, as well as a tomato late blight resistance peptide SlPIP2 and an application thereof.

[0008] In order to achieve the above object, the present invention is implemented by the following technical solutions:

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

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

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

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

[0013] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0014] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0015] Sa2, connecting the obtained PCR product with the pMD-19T cloning vector to obtain the connection product pMD-19T-SlprePIP2;

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

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

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

[0019] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0020] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0021] Sb2, connecting the obtained PCR product with the pMD-19T cloning vector to obtain the connection product pMD-19T-SlprePIP2;

[0022] Sb3, double digesting the pMD-19T-SlprePIP2 plasmid with correct sequencing with restriction endonucleases BamH I and Sac I, recovering the target fragment, connecting the target fragment with the pBI121 expression vector without the GUS gene, transforming the obtained ligation product into Escherichia coli DH5α and spreading it on LB solid medium containing kanamycin, picking a single colony and shaking the bacteria to extract the plasmid, performing plasmid PCR identification, and sending the plasmid for sequencing. The one with correct sequencing is the recombinant expression vector pBI121-SlprePIP2;

[0023] Sb4, introducing the recombinant expression vector into Agrobacterium GV3101 competent cells and spreading them on LB solid medium containing kanamycin, gentamicin and rifampicin, picking a single colony for bacterial liquid PCR verification, and obtaining a positive engineering bacterial liquid;

[0024] Sb5. Take five-leaf-stage tomatoes as treatment objects, and inject the prepared positive engineering bacteria solution into the leaves of the five-leaf-stage tomatoes to achieve overexpression of SlprePIP2.

[0025] In a preferred embodiment, the components of the LB medium are 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 components of LB medium are 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, and ultrapure water is diluted to 1 L.

[0027] In a fifth aspect, the present invention provides an application of the above-mentioned tomato late blight resistance peptide S1PIP2, any of the following:

[0028] (1) Used to prevent / treat tomato late blight;

[0029] (2) As a crop immunostimulant.

[0030] In a preferred embodiment, the application of the above-mentioned tomato late blight resistance peptide S1PIP2 is achieved by spraying the tomato late blight resistance peptide S1PIP2 on tomato plants.

[0031] Beneficial effects of the present invention

[0032] 1. The present invention constructs a recombinant expression vector of SlprePIP2. By overexpressing SlprePIP2 of the present invention, regulation of tomato late blight resistance genes can be achieved. Specifically, the expression level of SlprePIP2 in the transient overexpression plant obtained by the method of the present invention is higher than that in the empty-loaded plant, and the late blight resistance effect is better. The present invention enhances the resistance of tomatoes to late blight by overexpressing the SlprePIP2 gene, which is of great significance for breeding late blight-resistant tomato varieties.

[0033] 2. Since the SlPIP2 peptide precursor protein encoding gene SlprePIP2 can enhance the resistance of tomatoes to late blight, it is further analyzed whether the mature SlPIP2 peptide at its C-terminus also has the function of promoting the resistance of tomatoes to late blight. The present invention also provides a tomato late blight resistance peptide SlPIP2, which can enhance tomato immunity and improve the resistance of tomato plants to late blight by spraying tomato plants, which is helpful for the protection of tomato drug resistance and the development of the tomato industry.

[0034] 3. SlPIP2 can increase the transcriptional accumulation of disease resistance proteins PR in plants, which can directly affect the disease resistance of plants. It can be used as a widely useful crop immunostimulant for tomatoes against a variety of pathogens. Therefore, our research will provide new ideas for crop protection and pesticide development. In addition, 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. The SlprePIP2 overexpression plasmid was constructed using the PBI121 vector, and the transiently expressed PBI121 empty vector was used as the control group. The SlprePIP2 overexpression plasmid and the PBI121 empty vector were transiently expressed in tomato leaves, respectively. After 3 days, the transcriptional activity of SlprePIP2 was 5.3 times that of the transiently expressed PBI121 empty vector (EV) in tomato leaves. Then, an in vitro leaf inoculation test was performed. Five days after the transient expression site was infected with late blight, the lesion degree of the SlprePIP2 overexpression group was lower and the lesion diameter was smaller than that of the EV group. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the expression level of SlprePIP2 gene at each time point of infection by late blight pathogen;

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

[0038] Figure 3 This is the disease resistance phenotype of tomatoes after spraying with SlPIP2 peptide and the changes in the transcription level of PR genes. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

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

[0041] A tomato late blight resistance gene SlprePIP2, whose nucleotide sequence is shown in SEQ ID NO.1, the amino acid sequence of the encoded precursor protein is shown in SEQ ID NO.2, and 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 a tomato late blight resistance gene SlprePIP2 comprises the following steps:

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

[0044] SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC

[0045] SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA

[0046] Sa2, connecting the obtained PCR product with the pMD-19T cloning vector to obtain the connection product pMD-19T-SlprePIP2;

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

[0048] An application of a tomato late blight resistance gene SlprePIP2 comprises the following steps:

[0049] S1. Construction of a recombinant expression vector of SlprePIP2;

[0050] S2, introducing the recombinant expression vector into Agrobacterium GV3101 competent cells and spreading them on LB solid medium containing kanamycin, streptomycin and rifampicin, picking a single colony for bacterial liquid PCR verification, and obtaining a positive engineering bacterial liquid;

[0051] S3. Take five-leaf stage tomatoes as treatment objects, and introduce the prepared positive engineering bacteria solution (Agrobacterium instant infection solution) into the leaves of the seven-leaf stage tomatoes by injection, thereby achieving overexpression of SlprePIP2.

[0052] The preparation method for constructing the recombinant expression vector encoding the gene SlprePIP2 includes using restriction endonucleases BamHI and SacI to double-digest the pMD-19T-SlprePIP2 plasmid with correct sequencing, recovering the target fragment, connecting the target fragment with the pBI121 expression vector with the GUS gene removed, transforming the obtained connection product into Escherichia coli DH5α and coating it on an LB solid culture medium containing kanamycin, picking a single colony and shaking the bacteria to extract the plasmid, performing plasmid PCR identification, and sending the plasmid for sequencing. The plasmid with correct sequencing is the recombinant expression vector pBI121-SlprePIP2. The pMD-19T-SlprePIP2 plasmid is derived from the cloning method, that is, its preparation process is the same as the above steps Sa1 to Sa2.

[0053] A tomato late blight resistance peptide S1PIP2, whose amino acid sequence is shown in SEQ ID NO.3.

[0054] The present invention also provides the tomato late blight resistance peptide S1PIP2 for use in tomato late blight resistance. The tomato peptide S1PIP2 is sprayed on tomato plants, and then a spore suspension of late blight fungus is sprayed on the tomato plants, and the pathological changes of the plants are observed. The reaction induced by S1PIP2 can produce effective tomato immunity.

[0055] The tomato late blight resistance peptide S1PIP2 is used as a crop immunostimulant.

[0056] Example 1

[0057] Acquisition of SlprePIP2 gene

[0058] Firstly, the transcriptome library of tomato infected with late blight fungus (P. infestans P12103) was used to screen genes induced by late blight fungus, and polypeptides encoding amino acids less than 150 were selected to find the gene encoding the SpPIP peptide of interest. Real-time fluorescence quantitative PCR was used to further screen out the gene SlprePIP2 induced by late blight fungus, and its mature peptide was named SlPIP2.

[0059] 1. Cultivation of Biological Materials and Inoculation of Tomatoes with Pseudomonas infestans

[0060] The seeds of Sp3708 tomato were soaked and germinated at 28℃, then sown in soil. The tomato plants at 4-5 leaf stage were used as experimental materials under the condition of 28±2℃ with 16h light and 8h dark.

[0061] Inoculate the late blight pathogen onto oat solid medium and culture at 20°C for 3-4 weeks. Add 10 mL of sterile water to the medium covered with hyphae, gently scrape off the hyphae on the surface of the medium and place it at 4°C to promote the release of zoospores. Collect the filtrate with 3 layers of sterile gauze and count the number of spores using a hemocytometer. Adjust the suspension concentration to 1×10 / mL with sterile water. 6 Spores.

[0062] Tomatoes were treated with late blight by spray inoculation. The pathogen spore suspension was evenly sprayed onto the surface and stem of each tomato leaf using a sprayer. After inoculation, the plants were cultured in the dark at 90% to 100% humidity and 20±1°C for 24 hours, and then cultured in the dark at 16 hours of light and 8 hours of darkness. Tomato leaves were taken at 0, 3, 6, 12, 24, 48, 72, and 96 hours after infection and quickly frozen in liquid nitrogen for later use.

[0063] 2. Total RNA Extraction and Reverse Transcription

[0064] Using RNAiso Plus, extract total RNA from all tomato leaves. Select high-quality total RNA from uninoculated and inoculated tomatoes for transcriptome sequencing. The specific steps are as follows:

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

[0066] ① The mortar and pestle should be baked in an oven. Liquid nitrogen should be poured into the mortar before grinding the sample to fully pre-cool it. During extraction, all gun tips and centrifuge tubes should be treated with RNase-free treatment.

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

[0068] ③ Add 250 μL of chloroform solution to the centrifuge tube, mix thoroughly by inversion, let stand for 5 minutes, and centrifuge at 4°C and 12,000 rpm for 10 minutes;

[0069] ④ After centrifugation, aspirate the colorless supernatant and transfer it to a new centrifuge tube. 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 Pour off the supernatant, keep the precipitate, add 1 mL of 75% ethanol, carefully resuspend and wash the precipitate, centrifuge at 4°C, 12000 rpm for 5 min; then repeat this step again;

[0071] ⑥ After discarding the supernatant, open the lid and leave it for a while until the ethanol evaporates, then add 20 μL 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, each reagent was added to a 200 μL RNase-free PCR tube according to the reverse transcription system in the instruction manual of Takara (Code No. RR036A), and then placed in a PCR instrument and reacted according to the conditions of 37°C, 15min, 85°C, 5s, and finally tomato cDNA was obtained. Finally, the sample was stored in a refrigerator at -20°C for subsequent RT-qPCR experiments (real-time polymerase chain reaction).

[0074] 3. Acquisition of SlprePIP2 gene

[0075] like Figure 1 As shown, the transcription level of the gene encoding the screened PIP polypeptide precursor protein was detected by real-time fluorescence quantitative PCR technology, and a gene whose transcription level was significantly upregulated after infection by late blight fungus was obtained. The gene was compared with the PIP polypeptide encoding sequences in Arabidopsis and potato, and the encoding gene was named SlprePIP2, and its mature small peptide was named SlPIP2.

[0076] The above-mentioned real-time fluorescence quantitative PCR reaction system is:

[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, a SlprePIP2 overexpression plasmid 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 transient expression of PBI121 empty vector (EV) in tomato leaves. Then, an in vitro leaf inoculation test was performed. Five days after the transient expression site was infected with late blight, the lesion degree of the SlprePIP2 overexpression group was lower and the lesion diameter was smaller than that of the EV group.

[0081] 1. Cloning of SlprePIP2 gene

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

[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 the PCR products were detected by 1% agarose gel electrophoresis, the PCR products that matched the target fragment size were recovered using a gel excision recovery kit (purchased from Takara).

[0090] 3) Connect the target fragment to the cloning vector

[0091] The target fragment recovered above was connected to the cloning vector pMD-19T (purchased from Takara), and the reaction system was as follows:

[0092]

[0093] The ligation product pMD-19T-SlprePIP2 was obtained by ligation at 16°C for 8 h.

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

[0095] (1) Add 10 μL of the ligation product to the E. coli DH5α competent cells, pipette to mix, and place 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, ice-water bath again for 2 minutes;

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

[0098] (4) The bacterial solution was centrifuged at 12000 r / min, the supernatant was discarded, and a small amount of bacterial solution was kept. The suspension was fully suspended by pipetting, and then spread on an LB plate containing 50 μg / L ampicillin, and cultured in a constant temperature incubator at 37°C overnight;

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

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

[0101] According to the instructions of the plasmid mini-extraction kit (purchased from TIANGEN), the pMD-19T-SlprePIP2 plasmid contained in the above bacterial solution was extracted. 5 μL of the plasmid sample was taken for 1% agarose gel electrophoresis detection.

[0102] 6) Plasmid PCR detection

[0103] The extracted pMD-19T-SlprePIP2 plasmid was used as a template, and PCR was performed using the SlprePIP2-FP and SlprePIP2-RP primers. The plasmid was sent for sequencing, and the one that was consistent with SEQ ID NO.1 was the correct plasmid for connecting to pMD-19T (pMD-19T-SlprePIP2 plasmid).

[0104] 2. Construction of 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) to recover the target fragment. The enzyme digestion reaction system and method are as follows:

[0107]

[0108] The enzyme digestion was carried out at 37°C for 2 h, and the digestion products were detected by 1% agarose gel electrophoresis.

[0109] 2) Double restriction enzyme digestion of pBI121 plasmid

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

[0111]

[0112] The enzyme digestion was carried out at 37°C for 2 h, and the digestion products were detected by 1% agarose gel electrophoresis.

[0113] 3) Connect the target fragment to the pBI121 vector

[0114] Use T4 DNA ligase (purchased from Takara) to connect the target fragment and the pBI121 vector with the GUS gene cut out. The reaction system is as follows:

[0115]

[0116] The cells were connected at 16°C for 4 hours, and the ligation product was transformed into E. coli DH5α competent cells (same as the transformation process of cloning the SlprePIP2 gene). After transformation, the cells were spread on LB solid culture medium containing kanamycin, and a single colony was picked and shaken to extract the plasmid. After PCR identification, the plasmid was sent for sequencing. The one with correct sequencing was the recombinant expression vector pBI121-SlprePIP2.

[0117] 3. Application of SlprePIP2

[0118] 1) Preparation of pBI121-SlprePIP2 Agrobacterium engineering bacteria

[0119] The correctly sequenced recombinant plasmid pBI121-SlprePIP2 was introduced into the competent cells of Agrobacterium strain GV3101 by freeze-thaw method and mixed gently. Freeze in liquid nitrogen for 2 minutes, water bath at 37°C for 5 minutes, add 1mL LB liquid medium, and culture at 28°C, 180rpm for 2-3 hours. After centrifugation, aspirate 800μL, mix the remaining bacterial solution, and spread the bacterial solution on the LB solid medium containing 100mg / L Str (streptomycin), 100mg / L Rif (rifampicin) and 50mg / L Kana (kanamycin) with a coating rod. After the bacterial solution is absorbed, transfer it to a 28°C constant temperature incubator and invert it for 18-24 hours.

[0120] 2) Identification of positive clones

[0121] Pick a single colony from the plate, inoculate it into LB liquid medium containing antibiotics, and culture it at 28°C, 180rpm for 12-16 hours. Use specific primers for PCR verification. Add an equal volume of glycerol to the positive clone solution for storage.

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

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

[0124] ① Inoculate the activated Agrobacterium culture solution containing transiently expressed pBI121 empty vector (EV group) and pBI121-SlprePIP2 into 5 mL of LB liquid medium containing 50 mg / L Gent, 100 mg / L Rif and 50 mg / L Kana, and culture at 28°C with shaking at 180 rpm for 12 to 16 h;

[0125] ② Add 50mg / L Gent, 100mg / L Rif, 50mg / L Kana, 10mmol / L MES (morpholineethanesulfonic acid monohydrate), 20μmol / L AS (acetosyringone) and 2mmol / L MgSO4 to LB, and adjust the pH to 5.6. Pipette 1mL of activated bacterial liquid and add it to the liquid culture medium. The culture conditions are the same as above;

[0126] ③4℃, 4000rpm, centrifuge for 10min to collect the cells. Resuspend the cells in MMA solution (containing 10mmol / L MgCl2, 10mmol / LMES, 20μmol / L AS, pH 5.6) and measure the OD with an enzyme marker 600 , adjusted to 1.0, 28°C, shaking at 180 rpm for 3 h.

[0127] (2) Transient transformation of tomato plants

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

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

[0130] ③ 3 days after injection, pick the tomato leaves at the same position, grind them into powder in a mortar, and store them in 1 mL Trizol solution. Extract the total RNA of the tomato leaves injected with EV and pBI121-SlprePIP2, respectively, and then reverse transcribe and measure the expression level to confirm that the expression vector has been successfully transferred. Figure 2 As shown, the expression level of transient overexpression of SlprePIP2 (TOEprePIP2) was almost 5.3 times that of transient expression of PBI121 empty vector (EV group) in tomato leaves.

[0131] (3) Inoculation of detached leaves with late blight

[0132] ① 3 days after injection, use a sterile toothpick to poke a small wound in the middle of the EV and pBI121-SlprePIP2 tomato leaves and place them on sterile filter paper in a culture dish;

[0133] ② Take 20 μL of the spore suspension of late blight fungus and drip it on the wound of the leaf, cover the culture dish with plastic wrap to maintain humidity, culture it in the dark at 20℃ for 5 days, and observe the disease situation. Figure 2 As shown, compared with the EV group, the SlprePIP2 overexpression group (TOEprePIP2) had a lower degree of lesions and a smaller lesion diameter.

[0134] Example 3

[0135] Application of tomato peptide SlPIP2

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

[0137] Functional study of tomato peptide SlPIP2

[0138] The present invention uses 1 μM of water-soluble small peptide SlPIP2 to spray 4-week-old tomato plants for 24 hours, and the water-supplied leaves are used as the control (CK). Take 20 μL of the spore suspension of late blight fungus and drip it on the wound of the leaves, wrap the culture dish with plastic wrap to maintain humidity, and culture it in the dark at 20°C for 5 days to observe the disease. The results are as follows Figure 3 As shown, tomato leaves sprayed with the small peptide SlPIP2 showed significantly milder disease symptoms than CK leaves. The present invention shows that the reaction induced by SlPIP2 may produce effective tomato immunity. And the expression levels of disease resistance-related genes SlPR1, SlPR2 and SlPR5 in tomato plants (SlPIP2) treated with small peptides are significantly higher than those in CK, indicating that tomato plants treated with small peptides have high disease resistance. This experimental method is simple and explores a potential plant protection agent to replace traditional pesticides, which is expected to enhance plant disease resistance.

[0139] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0140]

Claims

1. A tomato late blight resistance gene SlprePIP2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A tomato late blight resistance peptide S1PIP2, characterized in that: The small peptide SlPIP2 is derived from the C-terminus of the small peptide SlPIP2 precursor protein encoded by the gene SlprePIP2, and the nucleotide sequence of the gene SlprePIP2 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.

3. A method for cloning the tomato late blight resistance gene SlprePIP2 according to claim 1, characterized in that: The steps include: Sa1, using tomato cDNA as a template and specific primers, PCR amplification was performed; the specific primers are as follows: SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA Sa2, connecting the obtained PCR product with the pMD-19T cloning vector to obtain the connection product pMD-19T-SlprePIP2; Sa3. Transform the ligation product pMD-19T-SlprePIP2 into Escherichia coli DH5α and spread it on LB solid culture medium containing ampicillin. Pick a single colony, shake the bacteria and extract the plasmid. Perform plasmid PCR and send the plasmid for sequencing. If the sequencing result is the plasmid with SEQ ID NO.1, the plasmid is correct.

4. An application of the tomato late blight resistance gene SlprePIP2 according to claim 1, characterized in that: The steps include: Sb1, using tomato cDNA as a template and specific primers, PCR amplification was performed; the specific primers are as follows: SlprePIP2-FP:GCGggatccATGAAAATGGGCACAACAAAATC SlprePIP2-RP:GCGgagctcTCAATTGTGTTCATTGTGTACCA Sb2, connecting the obtained PCR product with the pMD-19T cloning vector to obtain the connection product pMD-19T-SlprePIP2; Sb3, double digesting the pMD-19T-SlprePIP2 plasmid with correct sequencing with restriction endonucleases BamHI and SacI, recovering the target fragment, connecting the target fragment with the pBI121 expression vector without the GUS gene, transforming the obtained ligation product into Escherichia coli DH5α and spreading it on LB solid medium containing kanamycin, picking a single colony and shaking the bacteria to extract the plasmid, performing plasmid PCR identification, and sending the plasmid for sequencing. The recombinant expression vector pBI121-SlprePIP2 is the one with correct sequencing; Sb4, introducing the recombinant expression vector into Agrobacterium GV3101 competent cells and spreading them on LB solid medium containing kanamycin, gentamicin and rifampicin, picking a single colony for bacterial liquid PCR verification, and obtaining a positive engineering bacterial liquid; Sb5. Take five-leaf-stage tomatoes as treatment objects, and inject the prepared positive engineering bacteria solution into the leaves of the five-leaf-stage tomatoes to achieve overexpression of SlprePIP2.

5. The use according to claim 4, characterized in that: The components of LB medium are tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L and ultrapure water.

6. A use of the tomato late blight resistance peptide S1PIP2 according to claim 2, characterized in that: Any of the following: (1) Used to prevent / treat tomato late blight; (2) As a crop immunostimulant.

7. The use of the tomato late blight resistance peptide S1PIP2 according to claim 6, characterized in that: This is achieved by spraying tomato plants with the tomato late blight resistance peptide S1PIP2.

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