Method for improving resistance to bacterial canker disease of kiwifruit by modifying acLFYL1 gene and application thereof

Abstract

The application provides a method for improving the resistance of kiwi to bacterial canker by modifying AcLFYL1 gene, and overexpressing the AcLFYL1 gene through a genetic engineering approach, wherein the nucleotide sequence of the AcLFYL1 gene is shown as SEQ ID NO. 1. The application also provides an application of the method for improving the resistance of kiwi to bacterial canker by modifying the AcLFYL1 gene in the improvement and disease-resistant breeding of kiwi bacterial canker resistance. In the research, the AcLFYL1 gene having a positive regulation on the resistance of kiwi to bacterial canker is screened, the AcLFYL1 gene is overexpressed through a genetic engineering approach, and the kiwi plant having stronger resistance to bacterial canker can be obtained, thereby laying a foundation for the improvement and disease-resistant breeding of kiwi bacterial canker resistance.

Description

Technical Field

[0001] This invention relates to the field of plant molecular genetic engineering, and in particular to a method and application for improving the resistance of kiwifruit to bacterial canker by modifying the AcLFYL1 gene. Background Technology

[0002] Kiwifruit is a dioecious perennial deciduous vine. Because its fruit is rich in vitamin C, minerals, and various nutrients, it is known as the "King of Fruits." Kiwifruit is very popular with consumers and has high economic value.

[0003] Kiwifruit canker is a highly devastating bacterial disease. The pathogen is *Pseudomonas syringae* pv. Actinidiae (PSA). PSA can colonize multiple parts of the kiwifruit tree, including leaves, branches, flowers, fruits, and roots, spreading throughout the entire tree and ultimately causing its death. Due to its insidious nature, rapid spread, high mortality rate, and difficulty in control, kiwifruit canker has been listed as a quarantine disease for forest plants in China.

[0004] Currently, kiwifruit canker has become a common problem in the global kiwifruit industry, severely restricting its development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for improving the resistance of kiwifruit to ulcer disease by modifying the AcLFYL1 gene.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A method for improving the resistance of kiwifruit to pyrethroid disease by modifying the AcLFYL1 gene involves overexpressing the AcLFYL1 gene through genetic engineering; wherein the nucleotide sequence of the AcLFYL1 gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the AcLFYL1 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the genetic engineering approach refers to:

[0010] The AcLFYL1 gene fragment shown in SEQ ID NO.1 was inserted into a eukaryotic expression vector to obtain a eukaryotic recombinant expression vector; then the eukaryotic recombinant expression vector was transformed into Agrobacterium and the transformation into the target plant was mediated by Agrobacterium.

[0011] As one of the preferred embodiments of the present invention, the eukaryotic expression vector is selected as pHB vector.

[0012] As one of the preferred embodiments of the present invention, the Agrobacterium is selected as Agrobacterium EHA105.

[0013] As one of the preferred embodiments of the present invention, the AcLFYL1 gene expression product is used to positively regulate the kiwifruit resistance to canker in the target plant.

[0014] The above-mentioned method of improving the resistance of kiwifruit to bacterial canker by modifying the AcLFYL1 gene is applied in the improvement of kiwifruit resistance to bacterial canker and disease-resistant breeding.

[0015] The advantages of this invention compared to the prior art are as follows: In this study, the AcLFYL1 gene, which has a positive regulatory effect on the resistance of kiwifruit to bacterial canker, was screened. By overexpressing the AcLFYL1 gene through genetic engineering, kiwifruit plants with stronger resistance to bacterial canker can be obtained, laying the foundation for the improvement of kiwifruit resistance to bacterial canker and disease-resistant breeding. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the pHB-AcLFYL1 overexpression vector in Example 2;

[0017] Figure 2 This is a diagram showing the positive identification results of DNA level in the transgenic plant overexpressing the Chinese kiwifruit "Donghong" in Experiment Example 1 (in the diagram, "+" indicates the positive control, "-" indicates the negative control, and "DH OE-AcLFYL1" indicates the transgenic plant sample overexpressing the Chinese kiwifruit "Donghong").

[0018] Figure 3 This is an image of the transgenic Chinese kiwifruit "Donghong" overexpressed plant obtained based on the method of this invention in Experiment Example 1;

[0019] Figure 4 These are the appearance images of the wild-type and "Donghong" positive overexpression transgenic plants OE-AcLFYL1-1, OE-AcLFYL1-2 and OE-AcLFYL1-3 in Experiment Example 2;

[0020] Figure 5 This is a graph showing the AcLFYL1 protein content in transgenic plants analyzed by Western Blot in Experiment Example 2;

[0021] Figure 6 This is a diagram showing the changes in the structural morphology of AcLFYL1 transgenic kiwifruit leaves under microscopic analysis in Experiment Example 2.

[0022] Figure 7The diagram shows the changes in stem structure of AcLFYL1 transgenic kiwifruit under microscopic analysis in Experiment Example 2 (Figure a shows the stem microstructure of wild-type "Donghong", Figure b shows the stem microstructure of OE-AcLFYL1-1, Figure c shows the stem microstructure of OE-AcLFYL1-2, and Figure d shows the stem microstructure of OE-AcLFYL1-3).

[0023] Figure 8 This is a graph showing the lignin content of AcLFYL1 overexpressing transgenic plants in Experiment Example 3;

[0024] Figure 9 The figure shows the effect of AcLFYL1 on the resistance of kiwifruit to bacterial canker in Experiment Example 3 (Figure a shows the phenotype of AcLFYL1 overexpressing transgenic plants after vacuum inoculation with PSA bacterial canker, Figure b shows the leaf lesion area statistics, and Figure c shows the number of colonies per unit area).

[0025] Figure 10 This is a diagram of the bacterial load per unit area in the leaves of WT and AcLFYL1 overexpressing transgenic plants under a laser confocal microscope in Experiment Example 3.

[0026] Figure 11 This is a diagram showing that AcLFYL1 can activate CSE expression in yeast one-hybrid assay in Experiment Example 4. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the materials used in the following embodiments are conventional materials in the art; unless otherwise specified, the experimental conditions used are conventional conditions well known to those skilled in the art.

[0028] Meanwhile, the main experimental solutions in the following examples were prepared as follows:

[0029] Table 1. Main antibiotic preparations

[0030]

[0031] Table 2 Main Culture Medium Formulations

[0032]

[0033] Example 1: Cloning of the AcLFYL1 gene:

[0034] I. Total RNA extraction from kiwifruit

[0035] Total RNA was extracted from the leaves of *Actinidia chinensis* 'Donghong' (Anhui Agricultural University Nongcuiyuan) using the "BIOFIT Plant Total RNA Extraction Kit." The specific steps were followed as described in the instruction manual and optimized appropriately. After RNA extraction, 2 μL of the RNA sample was analyzed using 2% agarose gel denaturing electrophoresis to determine RNA quality. Simultaneously, 1 μL of RNA was used in a micro-volume nucleic acid analyzer to determine concentration and purity. The RNA sample was then promptly stored at -80°C for later use.

[0036] II. Synthesis of the first strand of cDNA

[0037] Following the kit instructions, the total RNA from the kiwifruit samples that passed the integrity and quality tests in the previous step was reverse transcribed into one-stranded cDNA. The steps are as follows:

[0038] (1) Take 1 μL of RNA sample, add 14 μL of Nuclease-Free Water, 4 μL of 5×RTⅢAll-in-OneMix and 1 μL of dsDNase, for a total volume of 20 μL;

[0039] (2) Use a PCR instrument to incubate at 37°C for 2 min, then at 55°C for 15 min, and finally at 85°C for 5 min.

[0040] (3) Store the cDNA sample in the -20℃ space of the refrigerator.

[0041] III. Extraction of Plant Genomic DNA

[0042] In this study, the main method for extracting plant genomic DNA was the "BIOFIT Plant DNA Extraction Kit." The specific steps were based on the instruction manual and appropriately optimized. After DNA extraction, the concentration and purity were measured using 1 μL of DNA with an ultra-micro nucleic acid analyzer. The DNA samples were then promptly stored at -20°C for later use.

[0043] IV. Primer Design and Gene Amplification

[0044] The AcLFYL1 gene sequence (as shown in SEQ ID NO.1) was obtained from the Kiwifruit genome database (http: / / kiwifruitgenome.org / ) and the NCBI database. Cloning primers were then designed using Primer 5.0 software, and suitable restriction enzyme sites and protective bases were selected and added to the primers by comparing them with the vector. PCR amplification was then performed.

[0045] The primers used for amplifying the AcLFYL1 gene were AcLFYL1 F / R, the template was cDNA single strand, and the enzyme used was PrimeSTAR. A 20 μL reaction system was designed, and the components are shown in Table 3. The reaction conditions are shown in Table 4. The AcLFYL1 F primer sequence (5'-3') was “ggactagtATGGACCCGGACGCCTTC”, and the AcLFYL1 R primer sequence (5'-3') was “aactgcagGAAGGGCAGGTGCTCAGGCTC”.

[0046] Table 3 AcLFYL1 gene PCR amplification system

[0047] 2×PrimeSTAR mix 10μL AcLFYL1 F 0.5μL AcLFYL1 R 0.5μL template 1μL <![CDATA[ddH2O]]> 8μL Total 20μL

[0048] Table 4 PCR reaction conditions for AcLFYL1 gene

[0049]

[0050] V. Cloning Vector Ligation, Transformation and Validation

[0051] The PCR products from the above steps were detected by 1.2% agarose gel electrophoresis. If the bands were single, it indicated that the amplification efficiency was high and the effect was good.

[0052] Connection: Use a pipette to transfer 3 μL of PCR product into a new PCR tube, add 1 μL of... After Blunt Simple, mix it thoroughly and then use a PCR instrument to perform ligation at 25°C for 30 minutes.

[0053] Then proceed with the conversion:

[0054] (1) Take out the previously aliquoted Trans-T1 competent cells and quickly place them on ice to allow them to thaw slowly and naturally. After they have thawed, transfer 50 μL of competent cells into a new centrifuge tube.

[0055] (2) Add all the ligation products to the competent cells, mix them thoroughly with a pipette (be careful to be very gentle), then insert the centrifuge tube into ice and place it on ice for 30 minutes.

[0056] (3) Take out the centrifuge tube, insert the float into the 42℃ water bath and heat for 30 seconds. Once the time is up, take it out immediately, insert it into ice, and let it sit for 2 minutes.

[0057] (4) Add 800 μL of LB liquid medium without any antibiotics to the centrifuge tube, shake it well by hand, and then place it in a shaker at 37°C for 1 hour to allow the bacteria to recover and be subcultured.

[0058] (5) Add 40 μL of X-gal and 8 μL of LPTG to LB solid culture medium containing kanamycin sulfate (Kana) resistance, and spread them evenly on the culture medium using a glass spreader. Seal the culture medium and place it in a 37°C constant temperature incubator to allow it to be fully absorbed.

[0059] (6) Centrifuge at 4000 r / min for 3 min to precipitate and enrich the bacterial cells. Discard the supernatant and keep about 100 μL of culture medium. Mix the bacterial solution with a pipette and transfer it all to the LB solid culture medium treated in the previous step. Spread it evenly with a glass spreader.

[0060] (7) After the bacterial solution is completely adsorbed onto the culture medium, seal the culture medium and incubate it upside down in a 37°C constant temperature incubator for 12 hours.

[0061] (8) After 12 hours, blue and white monoclonal colonies will grow on the culture medium. First, add an appropriate amount (about 10 μL) of LB liquid culture medium to the PCR tube, and then select a suitable size and white monoclonal colony to add to it.

[0062] PCR validation: Using the selected single colony as a template, a 20 μL reaction system was designed, containing: bacterial culture, enzyme (2×Rapid Taq Master Mix, amplification efficiency 1 kb / 60 ​​s), forward and reverse primers (M13F / M13R, 10 μm), and sterile ddH2O. The specific reaction system is shown in Table 5, and the reaction conditions are shown in Table 6. The M13F primer sequence (5'-3') is “TGTAAAACGACGGCCAGT”, and the M13R primer sequence (5'-3') is “CAGGAAACAGCTATGACC”.

[0063] Table 5 PCR amplification system

[0064] 2×Rapid Taq Master mix 10μL upstream primer M13F 0.5μL Downstream primer M13R 0.5μL template 1μL <![CDATA[ddH2O]]> 8μL Total 20μL

[0065] Table 6 PCR Reaction Conditions

[0066]

[0067]

[0068] The PCR products were then detected. After PCR verification, single colonies with a single band and the correct size were selected and placed in LB liquid medium (approximately 4 mL) containing Kana penicillin resistance, and then incubated in a shaker at 37°C for 12 h.

[0069] VI. Plasmid Extraction and Enzyme Digestion Verification

[0070] Plasmid extraction: High-quality plasmids were extracted using a kit (FastPure Plasmid Mini Kit);

[0071] Enzyme digestion verification: The extracted plasmids were digested with the corresponding restriction endonucleases (SpeI, PstI) and detected by 1.2% agarose gel electrophoresis.

[0072] Among them, the plasmid of the verified single-clone strain was sent to Sangon Biotech for sequencing.

[0073] Example 2: Construction of pHB-AcLFYL1 overexpression vector and recombinant bacteria:

[0074] I. Enzyme digestion, vector, and gel recovery

[0075] E. coli (containing pHB carrier and 50% glycerol) was quickly placed on ice to thaw slowly and naturally. After surface sterilization, it was placed in a clean bench. In a clean, sterilized glass tube, approximately 3 mL of LB liquid medium (containing Kana resistance) was added, followed by a pipette to add 45 μL of the E. coli glycerol culture. The tube was incubated at 37°C in a shaker for 12 hours to allow for mass proliferation. Once the bacterial culture reached a certain concentration, the plasmid was extracted and analyzed by 1% agarose gel electrophoresis. If the analysis was successful, the plasmid was used for further analysis.

[0076] The plasmids of the correctly sequenced monoclonal strains and the plasmids detected by the pHB vector were double-digested using specific restriction endonucleases SpeI and PstI. The plasmid concentration was determined before digestion to establish the optimal digestion system (1 μg DNA / 1 μL enzyme). The digestion system is shown in Table 7.

[0077] Table 7 Double Enzyme Digestion System

[0078] 10×FD buffer 5μL plasmid 25μL Restriction endonuclease SpeI 1.5μL restriction endonuclease PstI 1.5μL <![CDATA[ddH2O]]> 17μL Total 50μL

[0079] After mixing the enzyme digestion system, the enzyme digestion reaction was carried out at 37°C for 60 min using a PCR instrument. After the enzyme digestion was completed, the enzyme digestion product was checked by gel electrophoresis to see if it was correct.

[0080] Recover and purify the correct target fragment. Use the TIANgel Midi Purification Kit (Agarose Gel DNA Recovery Kit) for recovery and purification, following the instructions in the manual. Use a micro-volume nucleic acid analyzer to check the concentration and purity of the final DNA fragment solution with 1 μL. Store the DNA fragment at -20°C.

[0081] II. Connection, Validation and Initial Transformation

[0082] The vector fragment obtained in the previous step was ligated with the gene fragment using T4 DNA ligase. A 10 μL ligation system was designed, and the components are shown in Table 8.

[0083] Table 8 Connection System

[0084]

[0085]

[0086] The ligation system was placed in a PCR instrument and ligated at 25°C for 30 min to obtain the pHB-AcLFYL1 overexpression vector. Figure 1 As shown.

[0087] After ligation is complete, the ligation product is transformed into E. coli.

[0088] Successfully transformed monoclonal colonies were selected from the culture medium and verified by PCR. After successful verification, the corresponding glycerol bacteria were preserved, and Agrobacterium-mediated transformation was performed simultaneously.

[0089] III. Agrobacterium-mediated transformation

[0090] (1) Take out competent cells EHA105 and place them on ice to cool naturally. Add 50 μL of thawed competent cells to a new 1.5 mL centrifuge tube, then add 5 μL of recombinant plasmid pHB-AcLFYL1, gently mix them, and immediately place them on ice for 10 min.

[0091] (2) Transfer the centrifuge tubes to liquid nitrogen and freeze for 5 minutes.

[0092] (3) Immediately take it to a 37℃ water bath for 5 minutes.

[0093] (4) Immediately after the water bath heat shock, insert into ice bath for 5 minutes.

[0094] (5) Add 1 mL of YEP (antibiotic-free) liquid culture medium and place it in a shaker at 28°C for 3 hours to activate and culture.

[0095] (6) Centrifuge at 4000 r / min for 5 min to enrich the bacterial cells. Discard the culture medium, leaving about 100 μL. Mix the bacterial cells evenly with the remaining culture medium. Then, use a pipette to transfer the bacterial solution onto YEB solid medium containing Kan & Rif and spread it evenly with sterilized small glass beads. Incubate at 28℃ for 36 h.

[0096] After the bacterial colonies of suitable size have grown, they are picked and placed in YEP liquid medium (containing Kan & Rif), and then incubated overnight at 28°C in a shaker. The next day, the bacterial culture is used as a template for PCR identification using AcLFYL1F / AcLFYL1 R primers. The electrophoretic bands are observed; if the band size matches the description, the identification is considered successful. 800 μL of the successfully identified single-clone bacterial culture is transferred to a cryovial, and an equal volume of 50% glycerol is added. The cryovial is then stored in an ultra-low temperature freezer.

[0097] Example 3: Genetic transformation of kiwifruit:

[0098] Genetic transformation of kiwifruit was performed using the Agrobacterium-mediated transformation method, and the entire procedure was carried out under aseptic conditions. The method is as follows:

[0099] (1) Activate Agrobacterium pHB-AcLFYL1 by streaking it onto a Kan&Rif YEP solid medium plate and incubating the plate at 28°C for 48 hours. Add 1 mL of YEP liquid medium to a clean centrifuge tube, pick a suitable single colony and add it to the tube, and incubate at 28°C in a shaker. Perform PCR verification the next day, selecting the correctly verified single colonies for expansion culture (100 μL is added to 10 mL of Kan&Rif YEP medium), and incubate at 28°C in a shaker at 200 rpm for 4-6 hours until the OD value of the bacterial culture is approximately 1.

[0100] (2) Cut healthy and tender leaves from tissue culture seedlings of Chinese kiwifruit “Donghong” and hairy kiwifruit “Huate”, cut them into pieces of about 0.5*0.5cm, and cut the petioles into pieces of about 0.5cm. Culture them on a pre-culture medium for 2 to 5 days.

[0101] (3) Centrifuge the bacterial culture (5000 r / min, 10 min) and transfer all the precipitated bacterial cells to 100 mL of induction medium. Remove the leaves and petioles from the pre-culture medium and immerse them completely in the induction medium (Agrobacterium pHB-AcLFYL1 infects the leaves of "Donghong" and "Huate"). Immerse for about 15 min, gently shaking the medium occasionally during the immersion process. After immersion, use sterilized filter paper to absorb the moisture from the surface of the leaves and petioles, and then transfer the leaves and petioles to the co-culture medium for co-culture for 2 days.

[0102] (4) After co-culture, the leaves and petioles were transferred to a sterile solution containing carboxybenzyl (0.25 mg / L) and cephalosporin (0.1 mg / L) for washing, and then rinsed twice with sterile water. During the rinsing process, the leaves and petioles were shaken vigorously to remove excess Agrobacterium on the surface of the leaves and petioles. After rinsing, the leaves and petioles were dried with sterile filter paper and neatly placed on the selection medium. After culturing in the selection medium for 14 days, they were transferred to a higher concentration of selection medium and subcultured every 14 days thereafter.

[0103] (5) The explant callus in the screening medium gradually turns green and grows buds, and is then transferred to the meristematic medium; after the seedlings grow, they are transferred to the rooting medium to grow into complete plants.

[0104] (6) After the positive transgenic plants have grown well and are robust, harden them off. Every day, open the cover of the transgenic seedlings to be hardened off and place them in a room temperature environment for two hours. During this period, spray the transgenic seedlings with an appropriate amount of water from time to time to keep their leaves moist. Continue for three days, then wash the culture medium and rotten callus tissue on the roots of the transgenic seedlings with clean water. After cleaning, transplant them into nutrient soil.

[0105] Experimental Example 1: Identification of transgenic positive kiwifruit plants:

[0106] I. Experimental Methods

[0107] After the callus tissue in Example 3 has completely differentiated into leaves and buds on the screening medium, DNA is extracted from the leaves, and positive identification is performed using vector primers and target fragment primers.

[0108] II. Experimental Results

[0109] The results are as follows Figure 2 As shown in the figure ("+" indicates a positive control, "-" indicates a negative control, and "DH OE-AcLFYL1" indicates a transgenic plant sample overexpressing the Chinese kiwifruit "Donghong").

[0110] Depend on Figure 2 It can be seen that the bands of the positive plants are the same size as those of the positive control, indicating that the AcLFYL1 overexpression vector has been successfully transferred into the "Donghong" kiwifruit plants.

[0111] The morphological appearance of the transgenic kiwifruit "Donghong" overexpressed based on the method of this invention is as follows: Figure 3 As shown.

[0112] Next, in order to facilitate the detection of AcLFYL1 protein levels in overexpressing plants and to conduct detailed research on the mechanism of AcLFYL1 action, the plasmids of the transfected plants in Experiments 2-3 below were pHB-AcLFYL1-FLAG recombinant plasmids, that is, FLAG tags were added after the AcLFYL1 gene, and other steps were the same as those in the above examples.

[0113] Experiment Example 2: Analysis of protein levels and changes in leaf and stem structure in the transgenic positive kiwifruit plant AcLFYL1:

[0114] I. Experimental Methods

[0115] Total protein was extracted from wild-type and "Donghong" positive overexpression transgenic plants OE-AcLFYL1-1, OE-AcLFYL1-2 and OE-AcLFYL1-3, respectively, and Western blotting was performed using FLAG specific antibody. At the same time, the structural changes of leaves and stems of transgenic plants were analyzed.

[0116] II. Experimental Results

[0117] Figure 4 Images show the appearance of wild-type and "Donghong" positive overexpression transgenic plants OE-AcLFYL1-1, OE-AcLFYL1-2, and OE-AcLFYL1-3. Figure 5 The image shows the AcLFYL1 protein content in transgenic plants analyzed by Western blotting. Figure 6 The diagram shows the changes in the structural morphology of AcLFYL1 transgenic kiwifruit leaves under microscopic analysis. Figure 7 Diagram showing changes in stem structure of AcLFYL1 transgenic kiwifruit for microscopic analysis.

[0118] Depend on Figure 5 It can be seen that a specific band of 45 kDa appeared in the protein lane of the plant with added overexpression material, while no band was detected in the total protein lane of the wild-type control plant, indicating that the AcLFYL1:5×FLAG fusion protein was successfully expressed in the plant.

[0119] Depend on Figure 6 It can be seen that, compared with WT, AcLFYL1 overexpression caused significant changes in the cell structure of "Donghong" kiwifruit leaves, with a significant increase in the number of cells and a more compact arrangement, which may be the reason for the leaves becoming harder and thicker.

[0120] Depend on Figure 7 It can be seen that, compared with WT, AcLFYL1 overexpression caused significant changes in the stem structure of "Donghong" kiwifruit, with the cambium and phloem becoming significantly thicker, which may be an important reason for the thicker stem.

[0121] Experiment Example 3: Effects of the AcLFYL1 gene on the regulation of lignin content in kiwifruit and its resistance to kiwifruit canker:

[0122] I. Experimental Materials

[0123] The plant materials were one-year-old wild-type "Donghong" kiwifruit and the AcLFYL1 overexpressing "Donghong" kiwifruit plants from Example 3.

[0124] II. Strains

[0125] Actinobacillus syringae strain PSA CFBP7286-GFPuv.

[0126] III. Reagents and Instruments

[0127] UV spectrophotometer / ELISA reader, micro-quartz cuvettes (10mm diameter) / 96-well UV plates (non-polystyrene), adjustable pipettes, constant temperature water bath, 30-50 mesh sieve, perchloric acid (HClO, MW=100.46, CAS:7601-90-3), glacial acetic acid (CHO, MW=60.05, CAS:64-19-7) and distilled water.

[0128] IV. Experimental Methods:

[0129] 1. Determination of lignin content in kiwifruit

[0130] Remove the lower layer solution, place it in a desiccator, and dry it with nitrogen gas; add 5 mL of potassium hydroxide solution and mix well; add 2 mL of p-aminobenzenesulfonic acid solution and mix well; add 10 mL of n-hexane and mix well; centrifuge to remove the upper layer solution and retain the lower layer; measure the absorbance using a UV spectrophotometer. Data analysis: Calculate the lignin content in the sample using the standard curve method.

[0131] 2. Effects of AcLFYL1 on resistance to kiwifruit canker

[0132] The *Actinidia syringae* strain PSA CFBP7286-GFPuv was used to identify WT and AcLFYL1 overexpressing transgenic kiwifruit by inoculation with detached leaf discs without injury. Four days after inoculation, phenotypic analysis, lesion area statistics, and colony count per unit area were performed.

[0133] Further, the *Actinidia syringae* strain PSA CFBP7286-GFPuv was used to identify WT and AcLFYL1 overexpressing transgenic kiwifruit by inoculation with wounded leaves. Four days after inoculation, phenotypic analysis and lesion area statistics were performed.

[0134] V. Experimental Results

[0135] 1. The AcLFYL1 gene regulates the lignin content of kiwifruit.

[0136] like Figure 8 As shown, compared with WT, the lignin content of AcLFYL1 overexpressing plants was significantly increased.

[0137] 2. Effects of AcLFYL1 on resistance to kiwifruit canker

[0138] The lignin content of kiwifruit cell walls is closely related to the disease resistance of kiwifruit. When plants are infected with pathogens, the increased accumulation of lignin in the cell walls provides a basic barrier against pathogen spread. Kiwifruit canker induced by *Pseudomonas syringae* is the most serious bacterial disease of kiwifruit. To clarify whether AcLFYL1 overexpression affects the disease resistance of kiwifruit, this study identified the disease resistance of WT and AcLFYL1-overexpressing transgenic kiwifruit plants. The results showed that, compared with WT, AcLFYL1-overexpressing transgenic kiwifruit exhibited significantly enhanced resistance to canker.

[0139] like Figure 9 As shown in Figure a, four days after inoculation, all leaves of the WT plants showed extensive disease damage, while the OE-AcLFYL1-1, OE-AcLFYL1-2, and OE-AcLFYL1-3 overexpressing transgenic plants showed fewer leaf lesions, and the lesion areas were smaller. Further statistical analysis of the number of colonies per unit area and the lesion area revealed... Figure 9 As shown in b and 9c, the lesion area and colony count per unit area of ​​OE-AcLFYL1-1, OE-AcLFYL1-2, and OE-AcLFYL1-3 overexpressing transgenic plants were significantly less than those of WT. AcLFYL1 overexpressing kiwifruit showed stronger resistance to bacterial canker than WT.

[0140] like Figure 10 As shown, four days after inoculation with PSA labeled with green fluorescence, observation under a laser confocal microscope revealed that the bacterial load per unit area in WT leaves was higher than that in transgenic plants overexpressing OE-AcLFYL1-1, OE-AcLFYL1-2, and OE-AcLFYL1-3.

[0141] Experiment Example 4: AcLFYL1 activates the expression of the key gene CSE in lignin synthesis, increasing cell wall lignin content and enhancing resistance to kiwifruit canker.

[0142] I. Experimental Vectors and Strains

[0143] The vectors used in this experiment were Placzi vector and PJG4-5 vector; the yeast strain was EGY48.

[0144] II. Reagents

[0145] 1. Sterilized pipette tips (1000μL, 200μL, 10μL), spreaders or glass beads, Φ90mm petri dishes, ready for use.

[0146] 2. Incubate the carrier DNA in a water bath at 95–100°C for 5 minutes, then quickly place it on ice. This process can be repeated once more for future use.

[0147] 4. Prepare your own 0.9% physiological saline solution, or you can use sterile ddH2O water instead, for later use.

[0148] 5. Preparation of ordinary plates: Dissolve one culture medium strip in 0.5L of deionized water, without adjusting the pH value, and autoclave (e.g., sterilize at 115℃ for 20min). Liquid culture medium can be stored at 4℃ in a refrigerator; solid culture medium, 20-25mL / plate (Φ90mm), can be stored at 4℃ after solidification.

[0149] 6. Preparation of chromogenic plates: Refer to the instructions for the YK3010 LacZ gene extracellular expression chromogenic kit.

[0150] III. Experimental Methods

[0151] After sequencing PJG 4-5-LFY, PJG 4-5-Empty, pCSE-LacZi, and Empty pLacZi empty vectors (or Mutant BaitpLacZi), the E. coli culture was expanded and cultured, and then plasmids were extracted.

[0152] 1. Streak yeast strain EGY48 on a YPD plate, invert the plate and incubate at 28-30℃. After 2-3 days, pick a single clone and place it in 1.5 ml of YPDA liquid medium in an EP tube and incubate overnight on a shaker at 28-30℃.

[0153] 2. Expand the yeast strain EGY48 in an Erlenmeyer flask to 20 mL.

[0154] 3. Pour the cultured bacteria into 50mL centrifuge tubes, 10mL per tube, and centrifuge at 4000rpm for 6 minutes.

[0155] 4. Resuspend the bacterial cells in 5 mL ddH2O, centrifuge at 4000 rpm, and discard the supernatant.

[0156] 5. Prepare a 5mL buffer with LiAc:TE:H2O = 1:1:8.

[0157] 6. Add 1 mL of the above buffer to each centrifuge tube, centrifuge at 4000 rpm, and discard the supernatant.

[0158] 7. Add 1 mL of the above buffer to each centrifuge tube to resuspend the contents, then aliquot them into 1.5 mL EP tubes, adding 5 μL of carrier DNA to each 30 μL tube.

[0159] 8. Add 5 μL of plasmid DNA (pCSE-Placzi, PJG 4-5-LFY) to each of the 30 μL competent cells in the EP tube, and then add 5 μL of plasmid DNA (pCSE-Placzi, PJG 4-5 empty vector) to each of the 30 μL competent cells in another EP tube.

[0160] 9. Add 180ul buffer [40% PEG (LiAc:TE:PEG=1:1:8)], place in a 30℃ shaker, 200rpm, 30min.

[0161] 10. After adding 70ul of DMSO to each EP tube, heat shock it in a 42°C water bath for 15 minutes, then in an ice bath for 2 minutes. Finally, take 50ul of the solution and spread it onto the two-cell plate (-ura, -trp).

[0162] IV. Experimental Results

[0163] After successful transformation, fresh single colonies (2-3 mm) were picked from each sample and incubated on a dual-deficiency chromogenic plate (Gal / Raff / x-gal / CM-Ura-Trp) at 28-30°C for 2-3 days. The growth of yeast on the plates and the intensity of the blue color were observed. Results showed that AcLFYL1 could activate the expression of the key lignin synthesis gene CSE (e.g., ...). Figure 11 ).

[0164] In summary, this invention, through overexpression of the AcLFYL1 gene in the Chinese kiwifruit 'Donghong' variety, demonstrated through phenotypic analysis that AcLFYL1 overexpression leads to thicker kiwifruit plants and increased leaf thickness, thereby enhancing the kiwifruit's resistance to bacterial canker. Transcriptome analysis and Chip-seq experiments showed that increased AcLFYL1 expression enhances the expression of the key lignin synthesis gene Caffeoyl shikimate esterase (CSE; Actinidia14681), increasing cell wall thickness and thus improving the mechanism of kiwifruit bacterial canker resistance. This invention lays the foundation for disease-resistant kiwifruit breeding.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 method of modification AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, Through genetic engineering AcLFYL1 Gene overexpression; wherein, the AcLFYL1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The modification according to claim 1 AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, The AcLFYL1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The modification according to claim 1 AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, The genetic engineering approach refers to: The SEQ ID NO.1 shown AcLFYL1 Gene fragments are inserted into eukaryotic expression vectors to obtain eukaryotic recombinant expression vectors; then the eukaryotic recombinant expression vectors are transformed into Agrobacterium and transformed into target plants using Agrobacterium-mediated transformation.

4. The modification according to claim 3 AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, The eukaryotic expression vector selected is the pHB vector.

5. The modification according to claim 3 AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, The selected Agrobacterium is Agrobacterium. EHA105 .

6. The modification according to any one of claims 1 to 5 AcLFYL1 The method of using genes to enhance the resistance of kiwifruit to ulcer disease is characterized by, The AcLFYL1 The gene expression product is used to positively regulate the kiwifruit's resistance to bacterial canker in the target plant.

7. A method by modification as described in any one of claims 1 to 6 AcLFYL1 Application of gene-based methods to enhance kiwifruit resistance to bacterial canker in kiwifruit bacterial canker resistance improvement and kiwifruit bacterial canker resistance breeding.

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