A peptide encoding resistance to kiwifruit canker and its application

Abstract

This invention relates to the field of plant molecular genetic engineering, and more particularly to an encoded peptide that regulates resistance to kiwifruit bacterial canker and its applications. The encoded peptide is miPEP157a-2, which can be used to regulate the resistance of kiwifruit plants to kiwifruit bacterial canker. Spraying kiwifruit tissue culture seedlings with the encoded peptide solution, or culturing kiwifruit tissue culture seedlings with a medium containing the encoded peptide, can yield kiwifruit lines with enhanced resistance to kiwifruit bacterial canker. This application provides evidence supporting the participation of miPEP157a-2 and its encoded Ac-miR157a in regulating plant-pathogen interactions, and provides candidate genes for molecular breeding work to provide kiwifruit plants with high resistance to kiwifruit bacterial canker.

Description

Technical Field

[0001] This invention relates to the field of plant molecular genetic engineering technology, and in particular to a encoded peptide that regulates resistance to kiwifruit canker and its application. Background Technology

[0002] kiwi( Actinidia Kiwifruit (Ligusticum striatum) is a fruit with high economic value. However, in the development of the kiwifruit industry, bacterial canker can severely affect kiwifruit yield and hinder high-quality and increased production. Infection with kiwifruit canker not only leads to a decrease in yield but also affects quality, resulting in smaller fruits, thicker skin, and a sour taste. Moreover, kiwifruit canker can spread rapidly, destroying entire kiwifruit-producing areas in a short period, causing enormous losses to kiwifruit production.

[0003] Kiwifruit has a long juvenile period and a complex genetic background, making it difficult to cultivate disease-resistant new varieties using traditional hybridization breeding methods. Currently, the prevention and control of kiwifruit canker mainly relies on agricultural control (quarantine, pruning, etc.), chemical control (copper-based pesticides, antibiotics, etc.), and biological control (inoculants containing actinomycetes, Bacillus, Streptomyces, etc.). However, these control measures are either ineffective or harmful to humans or the environment.

[0004] With the development of technology, genetic engineering breeding technology has been applied to various fields, avoiding the problems of reproductive isolation between species and greatly shortening the breeding cycle. It is an effective method for breeding disease-resistant varieties. Studying the role of resistance-related genes in kiwifruit canker resistance is of guiding significance for gene-based prevention and control. In fruit trees, studies have shown that miRNAs can negatively regulate the expression of target genes and affect their resistance to pathogens. Finding miRNA-mediated kiwifruit canker resistance has profound significance for the genetic improvement of kiwifruit varieties. Currently, there are no reports on miRNA-encoded peptides (miPEPs) related to kiwifruit canker resistance. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention first provides a encoded peptide that regulates resistance to kiwifruit canker disease, the encoded peptide being miPEP157a-2, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention further provides the application of the above-mentioned encoded peptide miPEP157a-2 or biological material containing the above-mentioned gene encoding miPEP157a-2 in regulating the resistance of kiwifruit plants to kiwifruit canker disease.

[0007] Preferably, the biomaterial is any one of expression cassettes, vectors, microorganisms, cell lines, plant tissues, and plant organs.

[0008] Preferably, by increasing the activity of miPEP157a-2, the resistance of kiwifruit plants to kiwifruit canker is improved.

[0009] This invention also provides a method for improving the resistance of kiwifruit plants to kiwifruit canker disease. The method involves spraying kiwifruit tissue culture seedlings with a solution containing the encoded peptide miPEP157a-2 as described above, or culturing kiwifruit tissue culture seedlings using a culture medium containing the encoded peptide miPEP157a-2 as described above, thereby obtaining kiwifruit lines with enhanced resistance to kiwifruit canker disease, which are kiwifruit plants with high resistance to kiwifruit canker disease.

[0010] Preferably, the total amount of the encoded peptide miPEP157a-2 sprayed on each kiwifruit tissue culture seedling in the spraying treatment is 0.1~2 μmol.

[0011] Preferably, the concentration of the peptide miPEP157a-2 added to the culture medium is 0.5 μM.

[0012] The beneficial effects of this invention are as follows:

[0013] This application uses the disease-resistant kiwifruit variety 'Jin Kui' and the susceptible variety 'Hong Yang' as experimental materials, and the kiwifruit canker pathogen (… Pseudomonas syringae pv. actinidiae , Psa Post-inoculation small RNA sequencing results identified Ac-miR157a as being associated with resistance to kiwifruit canker, and this was further confirmed through kiwifruit genetic transformation. AcMIR157a Transgenic plants, using inoculation Psa The method demonstrated the disease resistance function of Ac-miR157a, which positively regulates the resistance of kiwifruit plants to kiwifruit canker.

[0014] Then, by predicting and synthesizing the potential coding peptide sequence of Ac-miR157a, the coding peptide miPEP157a-2, which encodes the Ac-miR157a gene and can regulate the resistance of kiwifruit plants to kiwifruit canker by regulating the Ac-miR157a gene, was obtained.

[0015] This application clarifies that the encoded peptide miPEP157a-2 and the encoded Ac-miR157a provide evidence to support the regulation of plant-pathogen interactions, and provides candidate genes for molecular breeding work to provide kiwifruit plants with high resistance to kiwifruit canker. Attached Figure Description

[0016] Figure 1 Inoculation of kiwifruit PsaDifferentially expressed miRNAs before and after the experiment are shown in Figure A, which is a statistical graph of differentially expressed miRNAs, and Figure B is a volcano plot analysis of gene expression in the control group and the experimental group.

[0017] Figure 2 The diagram shows the secondary structure folding of the Ac-miR157a precursor. The portion below the black line represents the mature Ac-miR157a sequence.

[0018] Figure 3 This is a schematic diagram of the STTM157a vector construction.

[0019] Figure 4 To overexpress in kiwifruit AcMIR157a As a result, A in the figure is AcMIR157a Positive PCR identification of transgenic kiwifruit; the numbers in the image represent plant numbers; B shows the expression level of Ac-miR157a in transgenic kiwifruit detected by real-time quantitative PCR; C shows the growth status of wild-type tissue culture seedlings and tissue culture seedlings of three transgenic lines, with the leftmost image being wild-type kiwifruit and the others being... AcMIR157a Genetically modified kiwifruit.

[0020] Figure 5 To show the results of STTM157a overexpression in kiwifruit, Figure A shows the positive PCR identification of STTM157a-transgenic kiwifruit, and the numbers in the figure represent plant numbers; Figure B shows the expression level of Ac-miR157a in transgenic kiwifruit detected by real-time quantitative PCR; and Figure C shows the tissue culture seedlings of the transgenic line.

[0021] Figures 6-7 for AcMIR157a Results of disease resistance experiments on transgenic kiwifruit leaves:

[0022] Figure 6 Soaking of tissue culture seedling leaves for stereofluorescence microscopy and laser confocal microscopy observation Psa Fluorescence after (GFP).

[0023] Figure 7 A in the middle is AcMIR157a Inoculation of kiwifruit rooted seedlings Psa The phenotype of the rear blade is given by B, which is the result of taking the logarithm of the spot count for different grinding and dilution ratios.

[0024] Figure 8 The results of the disease resistance experiment on STTM157a transgenic kiwifruit leaves are shown in the figure. Figure A shows the inoculation of STTM157a transgenic kiwifruit seedlings. Psa The phenotype of the rear blade is given by B, which is the result of taking the logarithm of the spot count for different grinding and dilution ratios.

[0025] Figure 9 After exogenously treating kiwifruit with miPEP157a-2 AcMIR157a The results of the relative expression level detection.

[0026] Figure 10 for AcMIR157a Changes in target gene expression levels.

[0027] Figure 11 Agrobacterium-mediated AcMIR157a Transient transformation of tobacco leaves with the target gene; the top row shows fluorescence under UV light, and the bottom row shows fluorescence under sunlight; the left side shows injection of only the target gene, and the right side shows injection of both the target gene and... AcMIR157a Mixed injection.

[0028] Figure 12 The results show the relative expression levels of miR157a target genes in kiwifruit after treatment with 0.5 μM miPEP157a-2 for 0.5 h.

[0029] Figure 13 This experiment demonstrates the disease resistance of kiwifruit treated with exogenous miPEP157a-2 in different ways. In experiment A, kiwifruit was treated with exogenous miPEP157a-2 and then inoculated. Psa Different dilutions of the powder were used for TLC staining. B represents the TLC count result obtained by taking the logarithm of A; C represents the kiwifruit culture medium after adding miPEP157a-2 and then inoculating the sample. Psa Grind and dilute the spot plates at different ratios, and calculate the spot plate count by taking the logarithm of C with D as the value of C.

[0030] Figure 14 The results of overexpression of miPEP157-2 in kiwifruit are shown in Figure A, which is the positive PCR identification of miPEP157-2 transgenic kiwifruit; Figure B is the expression level of Ac-miR157a in transgenic kiwifruit detected by real-time quantitative PCR; and Figure C is the tissue culture seedling of the transgenic line.

[0031] Figure 15 These are the results of a disease resistance experiment on leaves of miPEP157-2 transgenic kiwifruit. A represents the inoculation of tissue culture seedlings. Psa After grinding and diluting, different multiples of the spot plates were used. B is the result of the spot plate count calculated by taking the logarithm. Detailed Implementation

[0032] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to experiments.

[0033] Example 1

[0034] 1. Test materials

[0035] Kiwifruit tissue culture seedlings were donated by the Anhui West Kiwifruit Research Institute, including the disease-resistant variety 'Jin Kui' and the susceptible variety 'Hong Yang', along with kiwifruit canker pathogen strains (Actinidia kiwifruit canker pathogens). Pseudomonas syringae pv. actinidiae , Psa ) is JF8 ( previously isolated and identified in the laboratory) Psa-V , CCTCC AB2018305) strain.

[0036] The *Escherichia coli* strain DH5α, *Agrobacterium* strain EHA105, *Agrobacterium* strain GV3101, the template plasmids used to construct the overexpression vectors were pK7WG2D, pCAMBIA1300, and pMDC43, which were purchased.

[0037] 2. Screening for the miR157 gene

[0038] 2.1 Preliminary screening of miRNAs responding to kiwifruit canker infection

[0039] To investigate the conservation of plant miR157 family members in evolution, all plant miR157 family members were downloaded from the miRbase V21 database (http: / / www.mirbase.org / ), multiple sequence alignment was performed, and their conservation was analyzed using WebLogo online software.

[0040] Small RNA sequencing was performed on 'Jin Kui' kiwifruit inoculated with kiwifruit canker disease, such as... Figure 1 The results showed that 191 miRNAs were detected during vaccination. Psa Differential expression was observed before and after inoculation, with 69 miRNAs showing significant differences, including 21 upregulated and 48 downregulated. This suggests that these miRNAs may play a regulatory role in resistance to kiwifruit canker. Given previous studies reporting that the miR157 gene family is highly conserved and participates in disease resistance pathways in other species, Ac-miR157a, which showed significant post-inoculation changes, was selected for further investigation.

[0041] Specific primers targeting Ac-miR157a were designed to detect the expression activities of its precursor and mature form in different kiwifruit tissues. Actin was used as an internal control, and qRT-PCR was employed to detect the expression level of the Ac-miR157a precursor. AcMIR157a The expression level of Ac-miR157a was high in kiwifruit stems and leaves, but weak in fruit. Using U6 as an internal control, qRT-PCR was used to detect the relative expression levels of mature Ac-miR157a in different kiwifruit tissues. The results showed that the expression trend of mature Ac-miR157a in various kiwifruit tissues was basically consistent with that of its precursor.

[0042] 2.2 Cloning of the Ac-miR157a precursor sequence from kiwifruit

[0043] The Ac-miR157a sequence obtained from small RNA-seq of kiwifruit was compared with the kiwifruit genome database (Red 5), revealing that Ac-miR157a is located on chromosome 5. Using genomic DNA from 'Jin Kui' kiwifruit leaves as a template, cloning primers were used to amplify the sequence. AcMIR157a The sequence, reaction system and conditions are as described in 3.4.3. The product was detected by 1% gel electrophoresis, and the expected product sequence length is 300 bp (SEQ ID NO.3).

[0044] The precursor sequence of Ac-miR157a was analyzed using RNAfold software, and its secondary fold structure is shown below. Figure 2 Its precursor exhibits the typical stem-loop structure of miRNA. The black line indicates the mature sequence: Ac-miR157a 5'-UUGACAGAAGAUAGAGAGCAC-3' (SEQ ID NO.2), both 21 nt in length.

[0045] 3. AcMIR157a Gene extraction and cloning

[0046] RNA was extracted from kiwifruit samples using the Novizan RNA Extraction Kit (FastPure Universal Plant Total RNA Isolation Kit). The extracted RNA can be used directly in downstream experiments or stored at -80°C. DNA was extracted using the following methods:

[0047] (1) Take an appropriate amount of fresh plant tissue 100 mg and grind it into fine powder in a mortar with liquid nitrogen.

[0048] (2) Transfer the fine powder to the 1.5 mL centrifuge tube prepared earlier, add 400 μL of buffer AP1 and 4 mL of RNase A (10 mg / mL), vortex and mix thoroughly to help with lysis.

[0049] (3) Heat the centrifuge tube in a 65 ℃ water bath for 10 minutes. During the water bath, the centrifuge tube can be inverted 2-3 times to mix the sample.

[0050] (4) Add 130 μL of buffer AP2, vortex to mix for 1 min, place on ice for 5 min, centrifuge at 13,000 rpm for 5-10 min, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, being careful not to aspirate any precipitate.

[0051] (5) Calculate the amount of supernatant, add 1.5 times the volume of AP3 / E, and immediately shake to mix.

[0052] (6) Add the mixture obtained in the previous step (including any possible precipitate) to an adsorption column AC (place the adsorption column in the collection tube), centrifuge at 13,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

[0053] (7) Add 600 μL of rinsing buffer WB, centrifuge at 13,000 rpm for 30 sec, discard the waste liquid, and repeat once.

[0054] (8) Place the adsorption column AC back into the empty collection tube and centrifuge at 13,000 rpm for 2 min to remove as much of the washing liquid as possible, so as to avoid the residual ethanol in the washing liquid inhibiting the downstream reaction.

[0055] (9) Remove the adsorption column AC and place it in a clean centrifuge tube. Add 50 μL-100 μL of elution buffer EB to the middle of the adsorption membrane, incubate at room temperature for 3-5 min, and centrifuge at 13,000 rpm for 1 min. Add the resulting solution back into the adsorption column, incubate at room temperature for 2 min, and centrifuge at 13,000 rpm for 1 min.

[0056] 3.1 Gene Cloning

[0057] 1) Using DNA as a template, perform 2×Fast Taq enzyme temperature gradient PCR to determine the optimal amplification temperature, and then perform PCR amplification with a high-fidelity enzyme at the optimal temperature. AcMIR157a The PCR system and procedure are as follows:

[0058] Table 1 PCR amplification system

[0059] ;

[0060] Table 2 PCR amplification program

[0061] ;

[0062] 2) Agarose gel electrophoresis was used to detect and recover the PCR products. The recovered DNA products were ligated into blunt-ended Blunt Simple vectors (Zero Background pTOPO-Blunt Simple Cloning Kit) to determine the gene's base sequence. The reaction system is as follows:

[0063] Table 3 Connecting Transformation Reaction System

[0064] ;

[0065] Reaction conditions: 37 ℃ for 5 min (for longer fragments, the reaction time can be increased to 10-15 min).

[0066] Note: The amount of PCR product used should be determined based on the size of the insert fragment.

[0067] 3) Transformation of E. coli competent cells and cloning identification and sequencing

[0068] (1) Place the competent cells and the above-mentioned binding solution on ice at the same time. After 2-3 min, add the binding solution and competent E. coli into a 200 μL centrifuge tube, mix well, and place on ice for 30 min.

[0069] (2) Heat shock: Place the centrifuge tubes in a water bath at 42 ℃ for 90 seconds. Keep the centrifuge tubes stationary;

[0070] (3) Ice shock: Quickly place the centrifuge tubes into ice for 2-3 minutes and keep them still;

[0071] (4) Recovery: Add the mixture to 700 µL LB medium, incubate at 37 ℃ and 200 r / min for 45-60 min;

[0072] (5) Spreading: Take the culture medium from (4) and spread it evenly onto LB agar plates containing 100 mg / L ampicillin. Incubate overnight at 37 ℃ and observe the growth of single colonies;

[0073] (6) Pick about 8 single colonies from the plate culture medium and culture them in liquid LB medium containing 100 mg / L ampicillin. Incubate at 37 ℃, 200 r / min for 4-5 h, and then perform bacterial PCR detection. Select the correct bands and send them to the biotechnology company for sequencing. If the sequencing results are correct after alignment, they can be used for further subsequent experiments.

[0074] 4. AcMIR157a Gene vector construction and stable transformation

[0075] 4.1 Construction and Stable Transformation of Overexpression Vectors

[0076] The cloned Ac-miR157a precursor sequence was inserted after the 35S promoter of the pCAMBIA1300 vector (p1300) using homologous recombination to construct an overexpression vector. 35S:AcMIR157 The sequence is shown in SEQ ID NO.4.

[0077] (1) Carrier linearization

[0078] The overexpression vector pCAMBIA1300 was double-digested using restriction endonucleases XbaⅠ and BamHⅠ. The correctness of the double-digested bands was detected by agarose gel electrophoresis. The correct bands were then selected for gel extraction and recovery. The concentration of the gel-recovered products was determined using a nucleic acid analyzer. The products were stored at -20 °C.

[0079] The clone obtained from the above AcMIR157a PCR amplification was performed using the template. After amplification, 10 μL of 5× Loading Buffer was added to the PCR tube, and the mixture was thoroughly mixed with a pipette before agarose gel electrophoresis. Once the bands were correctly detected, the gel was cut and recovered. The concentration of the recovered product was determined using a nucleic acid analyzer, and the product can be stored at -20 °C for future use.

[0080] After obtaining the two recycled gum products, a recombination reaction can be carried out. The specific system is as follows:

[0081] Table 4 Recombination Reaction System

[0082] ;

[0083] Reaction conditions: 50 ℃ for 20 min

[0084] Finally, the cells were quickly transferred to ice and allowed to stand for 5 minutes. After that, 5 μL of the solution was used to transform E. coli DH5α competent cells. Positive single clones were identified by colony PCR and sent to a biotechnology company for sequencing verification. Plasmids were extracted from positive single clones with correct sequences and no mutations. The extracted plasmids were then transformed into Agrobacterium GV3101 competent cells. Two days later, single clones were picked and tested for positive single clones again. After shaking, the bacterial solution of the positive single clones was added to an equal volume of 50% sterile glycerol, mixed well, and stored at -80 ℃.

[0085] 4.2 Construction of the silencing vector STTM-miR157a

[0086] The miR157a interference vector was constructed using short tandem target mimic (STTM) technology (Tang, G., Yan, J., Gu, Y., Qiao, M., Fan, R., Mao, Y., & Tang, X. (2012). Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods (San Diego, Calif.), 58(2), 118–125. https: / / doi.org / 10.1016 / j.ymeth.2012.10.006). The U in the mature Ac-miR157a sequence was replaced with T to construct the STTM157a element. See [link to documentation]. Figure 3 The two ends of the TM bridge are connected by an 88-nt fixed base, with the TM at both ends serving as the binding sites for miR157. The raised CTA portion is used to maintain structural stability and allows specific miRNAs to bind but cannot be cleaved. STTM157a was then constructed into the overexpression vector PHB via enzyme digestion and ligation, and named... 35S: STTM157a .

[0087] 5. Experiment

[0088] 5.1 Identification of transgenic positive plants

[0089] Successfully converted using the above method AcMIR157a Agrobacterium-mediated transformation of kiwifruit tissue culture seedlings yields regenerated kiwifruit seedlings. The steps are as follows:

[0090] (1) Pre-culture of leaf discs: Select 'Hongyang' kiwifruit tissue culture seedlings with a growth cycle of about 45 days, select suitable leaves and cut them into leaf discs of about 5 mm × 5 mm in size, and lay them flat on MS medium containing 3 mg / L ZT + 3 mg / L 6-BA + 1 mg / L NAA, and culture them in the dark for 2-3 days.

[0091] (2) Co-culture with bacterial suspension: Fresh Agrobacterium tumefaciens containing the target fragment was resuspended in MS resuspension to a suitable concentration as the infection solution. The pre-cultured leaf discs were then removed and placed in the infection solution for about 15-20 min, shaking them as much as possible during this period. Afterward, the leaves were placed in a filter paper dish to dry and then transferred to a symbiotic culture medium in the dark for 2-3 days.

[0092] (3) Rotary disc screening: Take out the co-cultured leaf discs, wash them with sterile water about 2-3 times to wash off excess bacterial solution, then place them in filter paper trays to dry, and transfer them to kiwi fruit screening medium containing MS + 2 mg / L NAA + 1 mg / L ZT + 400 mg / L Cef + 3 mg / L Hyg, and wait for them to produce callus tissue and new shoots (the medium should be changed according to their growth status during this period).

[0093] (4) Tissue culture rooting: Select plants with good growth and transfer them to rooting medium for culture (1 / 2MS + 1 mg / L IBA).

[0094] (5) Soil acclimatization: Place the rooted tissue culture seedlings in a light incubator with the lid of the tissue culture bottle open. After the kiwifruit tissue culture seedlings have stabilized, remove them and soak them in sterile water for about 3 days before placing them in soil for cultivation. During the transplanting period, ensure that the nutrient soil is adequately moist.

[0095] The obtained kiwifruit regenerated seedlings were numbered, and the forward primers on the vector were used to... AcMIR157a The reverse primer was used as a specific detection primer for PCR identification, see [link to relevant documentation]. Figure 4 In section A, further analysis using stem-loop RT-qPCR was performed; see [link to relevant documentation]. Figure 4 In the OE#1, OE#2 and OE#8 lines, the expression level of Ac-miR157 was significantly higher than that of wild-type kiwifruit, about 6-8 times higher, indicating that Ac-miR157a was successfully overexpressed in kiwifruit.

[0096] Identification of STTM157a transgenic kiwifruit plants, see [reference]. Figure 5 The expression levels of Ac-miR157a in STTM157a-1 and STTM157a-2 decreased compared to the wild type, indicating that STTM157a was successfully expressed in kiwifruit.

[0097] 5.2 Pathogen inoculation

[0098] Kiwi fruit canker pathogen was inoculated using the tissue culture seedling soaking method. Wild-type tissue culture seedlings with the same growth cycle and AcMIR157a Tissue culture seedlings of overexpressing transgenic lines were simultaneously immersed in pathogenic bacteria (with GFP tags), and then cultured under normal conditions for 6 days before sampling. The fluorescence intensity between the two was observed using a stereofluorescence microscope and laser confocal microscopy.

[0099] 1) The steps for the tissue culture seedling soaking method are as follows:

[0100] (1) Take out the kiwifruit canker strain JF8 stored at -80 ℃, thaw it at room temperature, and then streak it on LB medium to activate the pathogen.

[0101] (2) After 2 days of plate culture, single clones were picked and added to LB liquid culture tubes with 2 mL centrifuge tubes. The culture was carried out overnight at 28°C with shaking at 200 r / min.

[0102] (3) Add the above bacterial solution to fresh LB liquid at a ratio of 1 / 100, and culture at 28℃ and 200 r / min for 12h.

[0103] (4) Centrifuge the activated JF8 bacterial culture at 6000 rpm for 5 min and collect the bacterial cells. Then resuspend the cells in sterilized 10 mg / L MgSO4 and adjust to OD200. 600nm =0.1 and add 0.05% Silwet-77 to it and mix well for later use.

[0104] (5) Select wild-type and transgenic kiwifruit tissue culture seedlings with uniform growth at 8-12 weeks of age, pour the above-mentioned infection solution into the tissue culture bottle, and use the soaking method to infect the kiwifruit tissue culture seedlings. After soaking for 5 minutes, remove the entire tissue culture seedling and replace it with a new culture medium.

[0105] (6) The infected tissue culture seedlings were placed in a growth environment with a 16-hour light (24 ℃) and 8-hour dark (22 ℃) cycle, and a light density of 150 μmol / m³. -2 s -1 The plants were cultured in a plant growth incubator with 50% humidity for 6 days, and samples were taken for colony counting.

[0106] (7) Colony counting: ① Take the same amount of inoculated wild-type and transgenic kiwifruit leaves, add 1 mL of sterile PBS solution, grind them into a homogenate using a mortar, and then transfer them to 1.5 mL centrifuge tubes. ② Dilute the above homogenate with resuspension solution in a serial dilution, ensuring thorough mixing to prevent bacterial cells from adhering to the bottom of the centrifuge tube. ③ Take 5 µL of sample solution from each concentration gradient, slowly spot the sample solution onto an LB culture plate, let it stand until the surface dries, seal the plate, and invert it in a 28 ℃ incubator for 2 days. ④ Take out the culture plate after 2 days of incubation, photograph it, and count the colonies using ImageJ software.

[0107] 2) The steps for inoculating detached leaf discs (vacuum infiltration) are as follows:

[0108] (1) Take out the kiwifruit canker strain JF8 stored at -80 ℃, thaw it at room temperature, and then streak it on LB medium to activate the pathogen.

[0109] (2) After 2 days of plate culture, single clones were picked and added to LB liquid culture tubes with 2 mL centrifuge tubes. The culture was carried out overnight at 28 °C with shaking at 200 r / min.

[0110] (3) Take the above bacterial solution and add it to fresh LB liquid at a ratio of 1 / 100. Incubate at 28 °C and 200 r / min for 12 h.

[0111] (4) Centrifuge the activated bacterial solution at 6000 rpm for 5 min, remove the supernatant and collect the bacterial cells. Resuspend the bacterial solution in pre-sterilized PBS, gently break up the precipitate with a pipette tip to completely suspend the bacterial cells, and measure the OD600nm of the bacterial solution to be 0.1. Then add Silwet-77 (concentration 0.05%) and mix well for later use.

[0112] (5) Using a hole punch (1 cm in diameter), take leaf tissues of the same size from the leaves of wild-type and transgenic kiwifruit plants, and put them into the above-mentioned bacterial solution. Use the vacuum permeation method to let the bacterial solution enter the kiwifruit leaves. During this process, vacuum can be drawn multiple times until the leaf disc is filled with bacterial solution at about 60%-80%.

[0113] (6) Place the treated kiwifruit leaf discs on filter paper to dry, then place the front side of the discs on 0.6% agar medium and incubate them in a light incubator for 6 days. Observe the disease situation and count the colonies.

[0114] 5.3 Results

[0115] See Figure 6 As you can see, AcMIR157a The leaf area of ​​overexpressing plants was reduced compared to wild-type (WT) plants, indicating that... AcMIR157a Overexpression may hinder Psa The infection process. The collected samples were subjected to grinding point counting (…). Figure 7 Actinopterygium indicum (A), wild-type (WT) kiwifruit plants and overexpression AcMIR157a The bacterial count in kiwifruit plants is as follows Figure 7 As shown in Figure B, overexpression AcMIR157a The amount of mycelium in tissue culture seedlings was significantly reduced compared to wild-type seedlings 6 days after inoculation with the pathogen of kiwifruit canker.

[0116] The grinding spots on the sample were counted. Figure 8 Chinese A), wild-type (WT) kiwifruit plants and AcMIR157a The bacterial count in kiwifruit plants from the silent transgenic line (STTM157a) is as follows: Figure 8 As shown in Figure B, the amount of wild-type bacteria in STTM157a tissue culture seedlings increased significantly 6 days after inoculation with the pathogen of kiwifruit canker.

[0117] It is evident that after inoculation with peptic ulcer bacteria, overexpression... AcMIR157aCompared with the wild type, the lesions of the strains were smaller and the number of colonies was reduced. The number of colonies of the Ac-miR157a silent transgenic strain (STTM157a) was increased compared with the wild type, indicating that Ac-miR157a gene expression is positively correlated with the resistance of kiwifruit plants to kiwifruit canker.

[0118] Example 2

[0119] 1. Prediction and synthesis of potential coding peptide sequences of miR157a

[0120] Previous studies have shown that miRNAs are transcribed in the cell nucleus under the action of RNA polymerase II, and their transcription mechanism has certain similarities to the transcriptional regulation of protein-coding genes. Based on Example 1, the promoter transcription start site prediction websites TSSP and BDGP were used to predict promoter transcription sites within a 500bp upstream sequence of the miR157a precursor (see Table 5). Four possible ORFs encoding peptides meeting the criteria were obtained. These predicted peptides were sent to Dankang Biotechnology Co., Ltd. for synthesis, ultimately yielding the peptide miPEP157a-2 encoding the Ac-miR157a gene.

[0121] Table 5. Predicted miPEP157a

[0122] .

[0123] 2. miPEP157a-2 in kiwifruit AcMIR157a Expression pattern analysis

[0124] Overexpression or exogenous application of miPEP will enhance... MIRNA The transcriptional specificity of genes increases the expression level of their corresponding miRNAs. MiPEP157a-2 with a synthetic purity ≥90% was dissolved and used as an exogenous treatment for wild-type kiwifruit tissue culture seedlings. The optimal treatment concentration and time were first screened by setting concentration gradients (0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM) and spraying time gradients (0 h, 0.5 h, 40 min, 1 h, 1.5 h, 2 h). Figure 9 As shown, the concentrations of miPEP157a-2 treated kiwifruit for 0.5 h were different. AcMIR157a All showed an upward trend, with the highest expression level observed in the 0.5 μM concentration group.

[0125] To further clarify the role of miPEP157a-2 in... AcMIR157a To study the influence of miR157a expression patterns, bioinformatics methods were used to predict target genes of miR157a, resulting in a total of 22 genes. SPLsTen miR157a binding sites with high scores and significant differences in expression after Psa infection were selected. SPLs Further validation was conducted. The expression levels of the corresponding target gene in wild-type and overexpressing Ac-miR157a kiwifruit were detected using real-time quantitative PCR. This study targeted the overexpression of... AcMIR157a Three strains with high expression levels were selected for further analysis. Quantitative real-time PCR was used to detect changes in the expression levels of the 10 target genes screened above, using kiwifruit tissue culture seedlings as material. Figure 10 It was shown in plants overexpressing miR157a AcSPL15 , AcSPL6c , AcSPL1 , AcSP12b , AcSPL6d , AcSPL17b The relative expression levels decreased, with AcSPL1 showing the most significant decrease.

[0126] The predicted target region sequences of the six target genes were constructed into the pICH86988 vector (carrying green fluorescence), and AcMIR157a Transient co-transformation of tobacco was performed via Agrobacterium-mediated transformation using a fluorescent PHB expression vector. Since some target genes share the same target sequence, only one of these genes needs to be selected for vector construction and tobacco injection. The final injection combinations are shown in Table 6, where + indicates injection and - indicates no injection.

[0127] Table 6 AcMIR157a Combined treatment with injection of different target genes

[0128] ;

[0129] See results Figure 11 Under handheld fluorescence, the uninjected portions of tobacco leaves appear reddish-purple under ultraviolet light (the plant's chloroplasts emit red light under ultraviolet light, but the exposure time and other factors cause them to appear purplish-red), while leaves injected only with the target gene show green fluorescence around the injection well (the color of GFP under ultraviolet light). AcMIR157a and AcSPL1 , AcSPL6c ( AcSPL6d ), AcSPL15 ( AcSPL12b No green fluorescence was observed after mixing and co-rotating separately, compared with AcSPL17b The co-transformation fluorescence intensity decreased. The reason for the disappearance or weakening of this fluorescence is that Ac-miR157a can bind to the target gene, thereby cleaving the target gene or inhibiting its translation. These results indicate that Ac-miR157a can inhibit the expression of its target gene through a base-complementary binding mechanism.

[0130] Furthermore, kiwifruit treated with 0.5 μM miPEP157a-2 for 0.5 h were selected as templates for further analysis. AcMIR157a The relative expression levels of the target genes were quantitatively analyzed. See [link to relevant documentation]. Figure 12 After exogenous treatment with miPEP157a-2, the expression levels of miR157a target genes in kiwifruit were significantly downregulated. These results indicate that miPEP157a-2 possesses biological activity and can promote… AcMIR157a The expression.

[0131] 3. miPEP157a-2 enhances the resistance of kiwifruit to bacterial canker.

[0132] To further investigate whether miPEP157a-2 plays a role in the infection of kiwifruit by the pathogen of kiwifruit canker, miPEP157a-2 was applied to kiwifruit tissue culture seedlings via two methods: exogenous spraying and addition to the culture medium. In the former case, 0.5 μM miPEP157a was directly sprayed onto the kiwifruit plants, and inoculation was carried out after complete absorption. Psa The latter involves adding miPEP157a-2 to 50 ml of culture medium to a concentration of 0.5 μM, then culturing the kiwifruit tissue culture seedlings in the medium until they stabilize before inoculation. Psa .

[0133] See Figure 13 ,Depend on Figure 13 As shown in A and B, the bacterial count in kiwifruit directly sprayed with miPEP157a-2 was reduced compared to the wild-type control group (without miPEP157a-2 spray); Figure 13 As shown in C and D, the bacterial count in kiwifruit with added miPEP157a-2 in the same culture medium was significantly lower than that in the control.

[0134] 4. Long-term stability verification

[0135] Following the aforementioned method, stable miPEP157 overexpression lines were obtained using the miPEP157-2 encoding gene as the target gene. (See [link to relevant documentation]). Figure 14 PCR identification and real-time quantitative PCR detection confirmed that miPEP157-2 was successfully overexpressed in the transgenic lines.

[0136] The overexpressing strain was inoculated with *Pseudomonas ulcerans* 1 by soaking, and leaf samples were ground and counted using a platelet analyzer. Figure 15 (A represents the results of TLC at different dilutions) The bacterial count in wild-type (WT) kiwifruit plants and kiwifruit plants overexpressing miPEP157 is shown in Figure 1. Figure 15As shown in Figure B, the tissue culture seedlings overexpressing miPEP157-2 showed a significantly reduced amount of bacteria compared to wild-type seedlings 6 days after inoculation with the kiwifruit canker pathogen, further demonstrating that miPEP157 overexpression can significantly enhance the resistance of kiwifruit to canker.

[0137] The above experiments demonstrate that the peptide miPEP157a-2 encoded by the Ac-miR157a gene can enhance the resistance of kiwifruit plants to kiwifruit canker. The mechanism and regulation of this effect are explained below. AcMIR157a It relates to the expression.

[0138] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A peptide encoding resistance to kiwifruit canker, characterized in that, The coding peptide is miPEP157a-2, and the amino acid sequence is shown as SEQ ID NO.

1.

2. Use of the biological material of the coding peptide miPEP157a-2 or the coding gene containing the coding peptide miPEP157a-2 of claim 1 in improving the resistance of kiwifruit plants to kiwifruit canker disease.

3. Use according to claim 2, wherein the compound is ###0002### The biological material is any one of an expression cassette, a vector, a microorganism, and a cell line.

4. A method of increasing the resistance of an actinidia plant to actinidia canker, characterized in that, Spraying the solution of the coding peptide miPEP157a-2 of claim 1 on kiwifruit tissue culture seedlings to obtain kiwifruit lines with enhanced resistance to kiwifruit canker disease, i.e. kiwifruit plants with high resistance to kiwifruit canker disease.

5. The method of claim 4, wherein, In the spraying treatment, the total amount of the coding peptide miPEP157a-2 sprayed on each kiwifruit tissue culture seedling is 0.1-2 μmol.

6. A method of increasing the resistance of an actinidia plant to actinidia canker, characterized in that, Culturing kiwifruit tissue culture seedlings using a medium added with the coding peptide miPEP157a-2 of claim 1 to obtain kiwifruit lines with enhanced resistance to kiwifruit canker disease, i.e. kiwifruit plants with high resistance to kiwifruit canker disease.

7. The method of claim 6, wherein, The concentration of the coding peptide miPEP157a-2 added in the medium is 0.5 μM.

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