Actinidia chinensis bacterial canker disease susceptible gene acadcl and application thereof

Abstract

The application discloses a kiwi bacterial canker disease susceptible gene AcADC1 and application thereof, and belongs to the technical field of genetic engineering. The nucleotide sequence of the AcADC1 gene is shown in SEQ ID NO:1, and the arginine decarboxylase (ADC) synthesized by the gene is encoded, and the amino acid sequence is shown in SEQ ID NO:2, and the arginine (Arg) is responsible for catalyzing the formation of putrescine. The resistance of the kiwi plant with the silenced AcADC1 gene to the bacterial canker disease is improved, the resistance of the kiwi plant with the overexpressed AcADC1 gene to the bacterial canker disease is reduced, the AcADC1 gene plays a negative regulation role in the immune response of the kiwi to the bacterial canker disease, the gene editing technology can be used for editing the AcADC1 gene, and a theoretical basis and gene reserve are provided for kiwi disease-resistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the AcADC1 gene, which is susceptible to bacterial canker in kiwifruit, and its application. Background Technology

[0002] Kiwifruit canker, caused by *Pseudomonas syringaepv. Actinidiae* (Psa), is a common disease of kiwifruit. It affects both the above-ground parts and the root system, primarily the trunk, branches, leaves, and flowers. The main symptoms are cankers on branches and trunks, leaf spots, and flower rot. When branches or twigs are infected, the bark softens and the surface slightly bulges. The affected area then cracks, developing cankers. The phloem rots, eventually leading to dark brown lesions in the xylem, hindering nutrient transport and absorption and ultimately resulting in an unhealthy tree. When twigs are infected, the petioles and pedicels turn brown inside, and the leaves and buds wither and may even die, ultimately causing tree death and orchard destruction, resulting in significant losses for the kiwifruit industry.

[0003] Polyamines (PAs) are low-molecular-weight nitrogen-containing aliphatic compounds widely found in eukaryotic and prokaryotic cells. In higher plants, the most common polyamines include diamines such as putrescine (Put) and cadaverine (Cad), triamines such as spermidine, tetraamines such as spermine, and its isomer, thermo-spermine. Putrescine (Put) is the most abundant in nature and is the central product of the PA biosynthetic pathway. It is mainly synthesized via two pathways: arginine decarboxylase (ADC) catalyzing arginine (Arg) and ornithine decarboxylase (ODC) catalyzing ornithine (Orn). However, the activities of ADC and ODC can be irreversibly inhibited by difluoromethylarginine (DFMA) or difluoromethylornithine (DFMO), respectively. Putrescine plays an important role in regulating plant growth and development, controlling morphogenesis, delaying senescence, and enhancing resistance to disease, cold, drought, and salt. Evidence suggests that the accumulation of putrescine within the host enhances disease resistance when exposed to pathogens. For example, when a virus infects tobacco highly resistant to TMV, the expression of putrescine synthase is significantly upregulated, but not in susceptible varieties; putrescine levels increase in corn after infection with corn smut; overexpression of arginine decarboxylase (ADC) increases putrescine levels in Arabidopsis, thereby increasing resistance to gray mold infection; in ADC loss-of-function mutants and silent lines, reduced putrescine levels lead to susceptibility to pathogens; in Arabidopsis, overexpression of the AtADC2 gene and supplementation with putrescine induce the expression of plant defense genes and stimulate local salicylic acid biosynthesis. Furthermore, some pathogens can utilize host putrescine for successful infection. For example, *Ralstonia solanacearum* induces upregulation of putrescine in tomato through the transcription-activator-like effector (TALE) Brg11, thereby gaining a competitive advantage during infection; *Fusarium graminearum* can utilize the large amounts of putrescine induced in wheat during its infection to stimulate DON toxin synthesis. However, it remains unclear whether putrescine in kiwifruit benefits the host's disease resistance or is utilized by the pathogen. Therefore, analyzing the effects of putrescine and the expression levels of its synthetic genes on kiwifruit disease resistance will be of great significance for the breeding of kiwifruit varieties resistant to bacterial canker. Summary of the Invention

[0004] Since there is a lack of research on the effect of putrescine synthesis in kiwifruit plants on kiwifruit disease resistance, this invention starts by measuring the difference in putrescine content among different susceptible and resistant varieties, and studies the effect of putrescine synthesis gene expression level on kiwifruit disease resistance. Based on this, this invention provides the AcADC1 gene, which is susceptible to bacterial canker in kiwifruit, and its application, thus realizing the breeding of disease-resistant kiwifruit varieties.

[0005] In a first aspect, the present invention provides a kiwifruit bacterial canker susceptibility gene AcADC1, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] Furthermore, in the kiwifruit bacterial canker susceptibility gene AcADC1 provided by the present invention, the kiwifruit bacterial canker susceptibility gene AcADC1 is silenced, thereby improving the resistance of kiwifruit to kiwifruit bacterial canker.

[0007] Furthermore, in the kiwifruit bacterial canker susceptibility gene AcADC1 provided by the present invention, the nucleotide sequence of the specific fragment of the kiwifruit bacterial canker susceptibility gene AcADC1 is shown in SEQ ID NO:3.

[0008] Furthermore, in the kiwifruit bacterial canker susceptibility gene AcADC1 provided by the present invention, the primer sequences for PCR amplification of the specific fragment of the kiwifruit bacterial canker susceptibility gene AcADC1 are shown in SEQ ID NO:4-5.

[0009] Furthermore, the arginine decarboxylase encoded by the kiwifruit bacterial canker susceptibility gene AcADC1 provided by the present invention has the amino acid sequence shown in SEQ ID NO:2.

[0010] Secondly, the present invention provides a method for breeding kiwifruit varieties resistant to bacterial canker, which involves silencing the kiwifruit bacterial canker susceptibility gene AcADC1 or silencing a specific fragment of the kiwifruit bacterial canker susceptibility gene AcADC1 in kiwifruit plants.

[0011] The nucleotide sequence of AcADC1, the susceptibility gene for bacterial canker in kiwifruit, is shown in SEQ ID NO:1;

[0012] The nucleotide sequence of a specific fragment of the AcADC1 gene, which is susceptible to bacterial canker in kiwifruit, is shown in SEQ ID NO:3.

[0013] Furthermore, the present invention provides a method for cultivating kiwifruit varieties resistant to bacterial canker, comprising: constructing a vector containing a gene AcADC1 silencing fragment, transferring the vector into Agrobacterium, and then transfecting kiwifruit with the obtained Agrobacterium to obtain the kiwifruit.

[0014] Furthermore, in a method for cultivating kiwifruit varieties resistant to bacterial canker, the present invention provides a method in which the Agrobacterium type is GV3101.

[0015] Thirdly, this invention provides the application of the AcADC1 gene, a susceptibility gene to bacterial canker in kiwifruit, in kiwifruit resistance to bacterial canker.

[0016] Furthermore, in the application provided by the present invention, the AcADC1 gene, which is susceptible to bacterial canker in kiwifruit, is silenced, thereby increasing the resistance of kiwifruit to bacterial canker.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0018] The present invention relates to the putrescine synthesis gene AcADC1 in kiwifruit plants, which plays a role in the interaction between kiwifruit and Psa and has a negative regulatory effect on the immune response of kiwifruit against bacterial canker.

[0019] This invention clarifies the function of the AcADC1 gene: transient overexpression of the AcADC1 gene results in susceptibility to Psa, with a significant increase in lesion area and pathogen biomass; silencing the AcADC1 gene results in resistance to Psa, with a significant decrease in lesion area and pathogen biomass. The AcADC1 gene is a susceptibility gene.

[0020] Based on the negative regulatory role of the AcADC1 gene in the immune response of kiwifruit plants to bacterial canker, the AcADC1 gene can be used to create kiwifruit varieties resistant to bacterial canker. The specific method involves silencing the AcADC1 gene in kiwifruit plants; the method for silencing the AcADC1 gene is not limited to Agrobacterium-mediated transformation.

[0021] The clarification of the AcADC1 gene function described in this invention provides a new direction for the breeding of kiwifruit varieties resistant to kiwifruit canker and also provides an important theoretical basis for the safe and sustainable prevention and control of kiwifruit canker. Attached Figure Description

[0022] Figure 1This study depicts the determination of putrescine content in resistant kiwifruit varieties and the occurrence of leaf canker in Hongyang kiwifruit after exogenous addition of different concentrations of putrescine (Put). Figure A shows the results of putrescine content determination in resistant kiwifruit varieties; Figure B shows the occurrence of leaf canker in Hongyang kiwifruit after exogenous addition of different concentrations of putrescine; Control represents the blank control; 10 μM Put, 100 μM Put, and 200 μM Put represent different concentrations of putrescine; H2O is the negative control.

[0023] Figure 2 The disease symptoms of *Actinidia chinensis* leaves 5 days after inoculation with Psa M228, an inhibitor of putrescine synthesis. H2O served as a negative control; Psa M228 represented inoculation with Psa M228 alone; and Psa M228+DMFA represented inoculation with both Psa M228 and DMFA, where DMFA is an inhibitor of putrescine synthesis.

[0024] Figure 3 This study investigated the disease incidence and relative expression level of AcADC1 gene silencing in *Actinidia chinensis* leaves 5 days after inoculation with Psa. In this data, A represents the disease incidence 5 days after AcADC1 gene silencing in *Actinidia chinensis* leaves inoculated with Psa; B represents the relative expression level of AcADC1 gene silencing; TRV:00 represents *Agrobacterium* carrying the pTRV1:pTRV2 vector; TRV:AcADC1 represents *Agrobacterium* carrying the pTRV1:pTRV2-AcADC1 vector.

[0025] Figure 4 This study investigated the disease incidence and relative expression level of AcADC1 gene in *Actinidia chinensis* leaves 5 days after transient overexpression of the AcADC1 gene followed by inoculation with Psa. In the figures, A represents the disease incidence 5 days after Psa inoculation; B represents the relative expression level of AcADC1 gene; H2O is the negative control; CK represents *Agrobacterium* carrying the overexpression vector pCAMBIA1302; and OE-AcADC1 represents transient overexpression of the AcADC1 gene. Detailed Implementation

[0026] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0027] The pTRV1, pTRV2, pCAMBIA1302 vectors and the pathogen of bacterial canker in kiwifruit, *Pseudomonas syringae* pv. actinidae (Psa), were all provided by Northwest A&F University. The highly susceptible kiwifruit variety "Hongyang," the susceptible kiwifruit variety "Nongda Jinmi," the moderately susceptible varieties "Xuxiang" and "Cuixiang," the resistant variety "Hayward," and the highly resistant variety "Longcheng No. 2" were all collected from Yangling Xintiandi Orchard.

[0028] Example 1

[0029] This embodiment provides a method for determining the putrescine content in different kiwifruit varieties.

[0030] 1g of fresh kiwifruit leaves from different varieties (including 'Hongyang', 'Nongda Jinmi', 'Xuxiang', 'Cuixiang', 'Hayward', and 'Longcheng No. 2') were weighed and ground into powder in liquid nitrogen. 4mL of 5% (v / v) perchloric acid aqueous solution was added, and the mixture was homogenized and incubated in an ice bath for 1 hour. Then, the mixture was centrifuged at 10000g for 40 minutes at 4℃. 2mL of the supernatant was transferred to a new centrifuge tube, and 2mL of 4M NaOH was added to neutralize the perchloric acid. Then, 7μL of benzoyl chloride was added, and the mixture was incubated at 37℃ for 25 minutes to derivatize putrescine. 2mL of saturated NaCl solution and diethyl ether were added for extraction. The diethyl ether containing polyamines was dried using a nitrogen evaporator, redissolved in methanol, and filtered through a 0.22μm filter membrane (organic phase) before analysis. Putrescine standards were prepared using the same method, except for the perchloric acid extraction procedure described above. Quantitative analysis was performed using a high-performance liquid chromatography (HPLC) system (Waters, W2690 / 5 HPLC pump / 2998 UV detector, USA). The sample injection volume was 10 μL, and a Waters SymmetryShield™ RP18 column (3.9 × 20 mm, 5 μm) was used; the column temperature was 30 °C; the mobile phase A was methanol (51%), and the mobile phase B was ultrapure water (49%); the flow rate was 0.7 mL / min; the run time was 20 min, and the putrescine signal of the sample was recorded at 230 nm. The detection results are as follows: Figure 1 As shown in A, the putrescine content in the highly susceptible variety "Hongyang" and the susceptible variety "Nongda Jinmi" is higher than 100 μmol / g, the putrescine content in the moderately susceptible varieties "Xuxiang" and "Cuixiang" is between 50 and 100 μmol / g, while the putrescine content in the resistant variety "Hayward" and the highly resistant variety "Longcheng No. 2" is almost undetectable.

[0031] This embodiment also verified the incidence of leaf canker in "Hongyang" kiwifruit after the addition of exogenous putrescine. The specific measurement method included: soaking healthy "Hongyang" kiwifruit leaves in a 0.6% sodium hypochlorite solution for 10 minutes for surface disinfection, rinsing three times with sterile water, and air-drying. Subsequently, leaf discs were prepared using an 11mm diameter perforator (avoiding leaf veins and large lateral veins), and placed in 60mL of 10 4 In 100 mL centrifuge tubes containing CFU / mL Psa M228, the leaf discs were immersed three times using a vacuum pump (0.1 MPa) until the underside of the leaf was more than 90% moistened. Finally, the leaf discs containing the vacuum-infiltrated Psa M228 were neatly arranged face down on water agar plates, and on water agar plates containing 10 μM, 100 μM, and 200 μM respectively. They were incubated at 16°C under light for 16 h, followed by 8 h in darkness. Disease incidence was assessed 5 days post-inoculation. Vacuum-infiltrated H2O served as a negative control. The experimental results are as follows: Figure 1 As shown in B in the figure. Without the addition of exogenous putrescine (Put), the brown area on kiwifruit leaves is small; when the concentration of exogenous putrescine increases from 10 μM to 200 μM, the brown area on kiwifruit leaves increases with the increase of putrescine concentration.

[0032] Example 2

[0033] This embodiment provides a method for determining the disease incidence of kiwifruit inoculated with Psa after using putrescine synthesis inhibitors.

[0034] Healthy 'Hongyang' kiwifruit leaves were disinfected by soaking them in a 0.6% sodium hypochlorite solution for 10 minutes, followed by rinsing three times with sterile water and air drying. Leaf discs were then prepared using an 11mm diameter perforator (avoiding the veins and large lateral veins) and placed in 60mL of 10... 4 CFU / mL Psa M228, 60mL 10 4 In 100 mL centrifuge tubes containing CFU / mL Psa M228 and 2 mM DFMA (a putrescine synthesis inhibitor), the leaf discs were immersed three times under vacuum (0.1 MPa) until the underside of the leaves was more than 90% moistened. Finally, the leaf discs containing Psa M228 and Psa M228+DFMA were neatly arranged face down on water agar plates, and on water agar plates containing 2 mM DFMA, respectively. They were incubated at 16°C under light for 16 h and under darkness for 8 h. Disease incidence was assessed 5 days post-inoculation. Vacuum-infused H2O served as a negative control. The experimental results are as follows: Figure 2 As shown, the use of putrescine synthesis inhibitors is mainly to inhibit the activity of ADCs, and the use of putrescine synthesis inhibitors can reduce the brown area of ​​kiwifruit after inoculation with Psa.

[0035] Example 3

[0036] This embodiment provides a method for obtaining the AcADC1 gene.

[0037] The nucleotide sequence of the AcADC1 gene obtained based on the kiwifruit genome V3.0 is shown in SEQ ID NO:1, and the amino acid sequence of the kiwifruit arginine decarboxylase (ADC) encoded by the AcADC1 gene is shown in SEQ ID NO:2.

[0038] RNA was extracted from the leaves of the "Hongyang" kiwifruit variety using an RNA kit (Beijing Huayueyang Biotechnology Co., Ltd., catalog number 0416-50), and cDNA was obtained using a reverse transcription kit (ThermoFisher Scientific, catalog number K1162). Primers were designed for the full-length sequence of the AcADC1 gene using cDNA as a template, and PCR amplification was performed.

[0039] Primer sequences for PCR amplification of the AcADC1 gene:

[0040] Upstream primer: (5'-ATGCCGGCCCTCGCTTGTTTC-3');

[0041] Downstream primer: (5'-TCAACGCTTAAGTGAGTAC-3').

[0042] The PCR amplification system consisted of: 25 μL of 2×Phanta Max Master Mix, 1 μL of template cDNA, and water to a final volume of 50 μL.

[0043] PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; final extension at 72℃ for 5 min.

[0044] Electrophoresis was performed on an agarose gel. The PCR products were purified using the Magen HiPure Gel Pure DNA MiniKit.

[0045] Example 4

[0046] This embodiment provides a method for constructing an AcADC1 gene silencing vector.

[0047] Total RNA was extracted from kiwifruit and cDNA was synthesized by reverse transcription. Primers were designed based on the AcADC1 gene sequence (SEQ ID NO:1) to amplify a partial fragment for the construction of the AcADC1 gene silencing vector. The partial fragment of the AcADC1 gene was amplified by PCR to obtain a nucleotide sequence of 407 bp (SEQ ID NO:3), which is the specific fragment of the AcADC1 gene.

[0048] Primers used to amplify the nucleotide sequence shown in SEQ ID NO:3:

[0049] Upstream primer (SEQ ID NO:4): (5'-GTGAGCTCGGTACCGGATCCGAGGG GCTCGCAGAGGATGC-3');

[0050] Downstream primer (SEQ ID NO:5): (5'-TGAGTAAGGTTACCGAATTCTGCAACGGCAAGCTCGAT-3').

[0051] PCR amplification program: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, 35 cycles; final extension at 72℃ for 5 min.

[0052] PCR products were analyzed by electrophoresis on a 1% agarose gel and purified using the Magen HiPure GelPure DNA Mini Kit. The purified PCR products were ligated into the pTRV2 vector digested with BamHI / EcoRI according to the ClonExpress II One Step Cloning Kit (Vazyme) procedure to obtain the recombinant plasmid pTRV2-AcADC1, which silences the AcADC1 gene. This recombinant plasmid was transformed into *E. coli* DH5α competent cells, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated at 37°C for 16 h. Colony PCR was then performed for verification. Three clones were picked, and the TRV2-AcADC1 plasmid was extracted using a plasmid extraction kit (Takara). The sequence of the TRV2-AcADC1 plasmid is shown in SEQ ID NO:3. The correctly sequenced plasmid was transformed into Agrobacterium GV3101 by heat shock, plated on LB agar plates (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated at 28°C for 48 h. Colony PCR was then performed to verify the colony, and the correct clones were selected for subsequent experiments.

[0053] Example 5

[0054] This embodiment provides a method for constructing a transient overexpression vector for the AcADC1 gene.

[0055] The nucleotide sequence of the AcADC1 gene obtained based on the kiwifruit genome V3.0 is shown in SEQ ID NO:1, and the amino acid sequence encoded by the AcADC1 gene is shown in SEQ ID NO:2.

[0056] RNA was extracted from the leaves of the "Hongyang" kiwifruit variety using an RNA kit (Beijing Huayueyang Biotechnology Co., Ltd., catalog number 0416-50), and cDNA was obtained using a reverse transcription kit (ThermoFisher Scientific, catalog number K1162). Primers were designed for the full-length sequence of the AcADC1 gene using cDNA as a template, and PCR amplification was performed.

[0057] Primer sequences for PCR amplification of the AcADC1 gene:

[0058] Upstream primer: (5'-GGGGACTCTTGACCATGGATGCCGGCCCTCGCTTGTT TC-3');

[0059] Downstream primer: (5'-CTCACCATCCTAGGACTAGTTCAACGCTTAAGTGAGT AC-3').

[0060] The PCR amplification system consisted of: 25 μL of 2×Phanta Max Master Mix, 1 μL of template cDNA, and water to a final volume of 50 μL.

[0061] PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; final extension at 72℃ for 5 min.

[0062] PCR products were analyzed by electrophoresis on a 1% agarose gel and purified using the Magen HiPure GelPure DNA Mini Kit. The purified product was then ligated into the NcoI / SpeI-digested pCAMBIA1302 vector using the ClonExpress II One Step Cloning Kit (Vazyme) according to the kit instructions to obtain the recombinant 1302-AcADC1 plasmid.

[0063] The plasmid was transformed into *E. coli* DH5α competent cells, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated at 37°C for 16 h. Colony PCR was then performed for verification. Three clones were selected, and the plasmid was extracted and sequenced using a plasmid extraction kit (Takara) (Shanghai Sangon Biotech Co., Ltd.). The sequence is shown in SEQ ID NO:1. The correctly sequenced plasmid was then heat-transformed into *Agrobacterium* GV3101 cells, plated on LB agar plates (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated at 28°C for 48 h. Colony PCR was then performed for verification, and the correct clones were selected for subsequent experiments.

[0064] Example 6

[0065] This example provides information on the susceptibility of kiwifruit after silencing and overexpressing the AcADC1 gene.

[0066] 1. Agrobacterium culture

[0067] Two days prior to inoculation, GV3101 Agrobacterium species containing TRV1:TRV2 (v:v = 1:1), TRV1:TRV2-AcADC1 (a TRV2 vector containing the AcADC1 gene silencing fragment), pCAMBIA1302, and pCAMBIA1302-AcADC1 (a pCAMBIA1302 vector containing the AcADC1 gene) were inoculated into LB medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and cultured at 28°C and 220 rpm on a shaker until OD reached. 600 The value was 0.5. Then, it was washed three times with 10 mM MgCl2 buffer, and resuspended in freshly prepared Agrobacterium infection medium MMA (containing 0.2 mM AS (acetylsyleugenol), 10 mM MgCl2, 10 mM MES, pH 5.6). The GV3101 Agrobacterium samples containing different plasmids were wrapped in aluminum foil and incubated at room temperature in the dark for 3 hours before use.

[0068] 2. Agrobacterium leaf disc vacuum permeation method

[0069] Take several healthy leaves from a two-year-old 'Hongyang' kiwifruit variety susceptible to disease. The leaves should be uniform in size and growth. Rinse thoroughly with tap water, then surface disinfect with 0.6% sodium hypochlorite for 10 minutes. Rinse three times with sterile water until there is no pungent odor. Finally, blot dry any remaining moisture with sterile filter paper. Use a sterile perforator. Leaf discs were prepared, with 30-50 discs per tube containing 60 mL of Agrobacterium GV3101 containing different plasmids. Leaves from the 'Hongyang' variety were used as experimental materials for both gene silencing and overexpression. Agrobacterium GV3101 containing the pTRV1:pTRV2 (v:v = 1:1) plasmid served as the control for AcADC1 gene silencing, while Agrobacterium GV3101 containing the pCAMBIA1302 plasmid served as the control for AcADC1 gene overexpression. A 0.1 MPa vacuum pump was used to penetrate the underside of the leaves until they were more than 90% wetted (leaf discs that did not meet the infection requirements were discarded). The surface moisture of the leaf discs was blotted dry with sterile filter paper, and the leaf discs were placed face down on 0.5%-0.8% water agar plates and incubated at 28°C.

[0070] 3. Silencing and Overexpression Efficiency Detection

[0071] Total RNA was extracted from *Actinidia chinensis* leaves infected with *Agrobacterium* vectors pTRV1:pTRV2 (v / v = 1:1), pTRV1:pTRV2-AcADC1 (v / v = 1:1), pCAMBIA1302, and pCAMBIA1302-AcADC1, respectively. Using cDNA obtained from RNA reverse transcription as a template, three biological replicates and three experimental replicates were performed for each treatment. Actin was used as an internal control gene, and the expression level of the AcADC1 gene was detected by qRT-PCR.

[0072] The primers used for qRT-PCR to detect silencing efficiency and overexpression are:

[0073] AcADC1-F: (5'-GCTCTTTTGGTCTGCAACGG-3');

[0074] AcADC1-R: (5'-TCCGGCTCATGTCAATCACC-3').

[0075] Add 10 μL of 2×ChamQ SYBR qPCR Master Mix, 0.4 μL of AcADC1-F, 0.4 μL of AcADC1-R, 2 μL of Template DNA / cDNA, and 7.2 μL of H2O sequentially to ice to establish a total 20 μL qRT-PCR system and perform amplification. The amplification reaction program is shown in Table 1.

[0076] Table 1. qRT-PCR amplification program

[0077]

[0078]

[0079] The results showed that Agrobacterium carrying pTRV1:pTRV2-AcADC1 reached the silencing requirement 5 days after infection, and the silencing efficiency was 34% compared with the control group pTRV1:pTRV2 (e.g., Figure 3 (As shown in B). Agrobacterium carrying pCAMBIA1302-AcADC1 showed a 2.7-fold increase in AcADC1 expression compared to the control group pCAMBIA1302 after 5 days of infection (as shown in B). Figure 4 As shown in B in the figure, this indicates that the expression was successful.

[0080] 4) Infection by kiwifruit canker bacteria

[0081] After confirming that the AcADC1 gene reached the conditions for silencing or overexpression, the PsaM228 strain of kiwifruit canker pathogen (1×10⁻⁶) was inoculated using the leaf disc vacuum permeation method. 4 The CFU / mL sample was placed on 0.8% water agar plates and incubated in an artificial climate chamber. After 5 days, the infection status of PsaM228 was observed and the lesion area was counted. The results showed that compared with the TRV:00 control group, silencing the AcADC1 gene significantly reduced the disease incidence on leaves (e.g., CFU / mL). Figure 3 As shown in A), the leaf disease incidence was significantly more severe after overexpression of the AcADC1 gene compared to the control group (as shown in A). Figure 4 (As shown in A).

[0082] The above results indicate that putrescine content is high in kiwifruit varieties susceptible to bacterial canker, and high putrescine levels can exacerbate the occurrence of bacterial canker. However, the use of the putrescine synthesis inhibitor DMFA significantly reduced the severity of bacterial canker. Inoculation of Psa M228 with silencing and overexpression of the putrescine synthesis gene AcADC1, respectively, showed that this gene is a susceptibility gene. Clarifying the function of the AcADC1 gene provides a new direction for breeding kiwifruit varieties resistant to bacterial canker and also provides an important theoretical basis for the safe and sustainable control of kiwifruit bacterial canker.

[0083] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A susceptibility gene for bacterial canker in kiwifruit AcADC1 Its characteristics are, The susceptibility gene for bacterial canker in kiwifruit. AcADC1 The nucleotide sequence is shown in SEQ ID NO:

1.

2. A method for breeding kiwifruit varieties resistant to bacterial canker, characterized in that, Silencing the susceptibility gene for bacterial canker in kiwifruit plants AcADC1 Or silence the susceptibility gene for bacterial canker in kiwifruit. AcADC1 Specific fragments; Kiwi fruit bacterial canker susceptibility gene AcADC1 The nucleotide sequence is shown in SEQ ID NO:1; Kiwi fruit bacterial canker susceptibility gene AcADC1 The nucleotide sequence of the specific fragment is shown in SEQ ID NO:

3.

3. The method according to claim 2, characterized in that, include: Constructing a gene AcADC1 The vector for the silent fragment was obtained by transferring the vector into Agrobacterium and then transfecting kiwifruit with the resulting Agrobacterium.

4. The method according to claim 3, characterized in that, The Agrobacterium species is GV3101.

5. The susceptibility gene for bacterial canker in kiwifruit as described in claim 1. AcADC1 Application in the treatment of bacterial canker in kiwifruit.

6. The application according to claim 5, characterized in that, Silent genes associated with bacterial canker in kiwifruit AcADC1 This enhances the resistance of kiwifruit to bacterial canker.

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