Application of transcription factor AcWRKY20 in resisting kiwifruit bacterial canker

Abstract

The invention belongs to the technical field of genetic engineering, and relates to application of a transcription factor AcWRKY20 in resisting kiwifruit bacterial canker. Through gene cloning, phylogenetic analysis, subcellular localization, transgenic function verification and other technologies, it is proved for the first time that the transcription factor AcWRKY20 has the molecular characteristic of remarkably enhancing the disease resistance of kiwi fruits. According to the invention, the AcWRKY20 gene is respectively silenced and overexpressed and then is inoculated with Psa, and phenotypic observation shows that the gene is a disease-resistant gene and is suitable for canker-resistant breeding of kiwi fruit.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to application of transcription factor AcWRKY20 in resisting kiwifruit bacterial canker. Background Art

[0002] Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a devastating bacterial disease. The disease spreads rapidly and is difficult to control. In severe cases, it can cause plant death, significantly impacting kiwifruit yield. Currently, the prevention and control of kiwifruit bacterial canker relies heavily on copper preparations and agricultural antibiotics, but the long-term application of such agents is not conducive to sustainable ecological development. It is crucial to develop environmentally friendly and effective methods and technologies to control plant diseases. In the evolutionary process of plant response to biotic stresses, multi-level regulatory networks and dynamic signal transduction systems have been formed, among which transcriptional reprogramming is a core strategy for activating defense responses. Plants precisely regulate the spatiotemporal expression and activity of transcription factors (TFs) to coordinate the rapid induction of defense-related genes, thereby establishing an immune barrier. Transcription factors, represented by the WRKY family, play a central regulatory role in this process: their unique WRKY domains specifically bind to the W-box element (TTGACC / T) in target gene promoters, directly activating the expression of defense effector molecules such as pathogenesis-related proteins (PRs), phytoalexin synthases, and reactive oxygen species (ROS) metabolizing enzymes. This transcription factor-driven modular regulatory network not only enables precise plant responses to pathogen attack but also provides key molecular targets for disease-resistant breeding. Therefore, the identification of genes associated with resistance to kiwifruit bacterial canker, the study of resistance mechanisms, and the selection of resistant varieties are crucial for the prevention and control of kiwifruit bacterial canker. Summary of the Invention

[0003] Based on this, the purpose of the present invention is to provide a transcription factor AcWRKY20 that can improve the disease resistance of kiwifruit, which is used to enhance the disease resistance of kiwifruit to ulcer disease, provide new genetic resources for kiwifruit disease-resistant breeding, and contribute to the green and sustainable development of the kiwifruit industry.

[0004] To ensure a complete and unambiguous understanding of the technical solutions of the present invention, it should be noted that the AcWRKY20 protein described herein is represented by the non-italic "AcWRKY20" font, and the AcWRKY20 gene is represented by the italic "AcWRKY20" font. Of course, those skilled in the art will clearly and completely understand the meaning and representation of the relevant genes and their encoded proteins based on the description of the present invention.

[0005] In one aspect, the present invention relates to a method for obtaining a plant resistant to kiwifruit bacterial canker, comprising: increasing the transcription or translation of the AcWRKY20 gene in the plant, or increasing the expression level of the AcWRKY20 protein in the plant;

[0006] The AcWRKY20 gene encodes the AcWRKY20 protein;

[0007] The amino acid sequence of the AcWRKY20 protein is shown in SEQ ID NO: 1.

[0008] Furthermore, in the method for producing plants resistant to kiwifruit bacterial canker provided by the present invention, the coding region sequence of the AcWRKY20 gene is shown in SEQ ID NO: 2.

[0009] SEQ ID NO: 1 is as follows:

[0010] MVTLGEVVQDAVASDKSQHRDSPDHESQSNQEGSTLSVLADEGPGGLHKTQSANSKVSASKCNEEGNTLSMITEEVSDNVQVRQGSDITSREQTAQSDENSPSVIPIRESHNLQQSQSPSSII GDHFSQANQEGTSLSKIPAQDSDNLQERHGSDIVLHASESIKKESSLSAIPEKVPDILQLTQTPNTGSHLLHRDQEGKNFSRTPDKASEDGYNWRKYGQKLVKGNEFTRSYYRCTHPNCPAKR QVERLLDGQITDTIYLGKHEHPKPQPSAQISVSFVQPIQAIRPEETSLDTGQGKACNAHGLASHHAKPAESPKLSTIAPNDDAVEGASSLSNRTNDEVDHSADPDSKRQKKDITNISEIAVEK PNGDPRVVVQTTSEVDIVNDGYRWRKYGQKLVKGNPNPRSYYRCSNAGCPVKKHVERASHDLKVVITTYEGQHDHDKPPARTVTHNAAGADSNITTHNSESRSRPEESRALGLEMAVHTSAN.

[0011] SEQ ID NO: 2 is as follows:

[0012]

[0013] Furthermore, in the method for producing plants resistant to kiwifruit bacterial canker provided by the present invention, the plant is kiwifruit.

[0014] Furthermore, in the method for producing plants resistant to kiwifruit bacterial canker provided by the present invention, the pathogen of kiwifruit bacterial canker is Pseudomonas syringaepv.actinidiae.

[0015] Furthermore, in the method for producing plants resistant to kiwifruit bacterial canker provided by the present invention, a vector overexpressing the AcWRKY20 gene is constructed, the vector is transformed into Agrobacterium, and the Agrobacterium is used to infect the plant.

[0016] Furthermore, in the method for producing plants resistant to kiwifruit bacterial canker provided by the present invention, the pICH86988 vector is used in the vector construction.

[0017] On the other hand, the present invention relates to the use of the AcWRKY20 gene in preventing and treating kiwifruit bacterial canker, increasing the transcription or translation of the AcWRKY20 gene in the plant, or increasing the expression level of the AcWRKY20 protein in the plant, thereby improving the resistance of the plant to kiwifruit bacterial canker;

[0018] The AcWRKY20 gene encodes the AcWRKY20 protein;

[0019] The amino acid sequence of the AcWRKY20 protein is shown in SEQ ID NO: 1.

[0020] Furthermore, in the use of the AcWRKY20 gene provided by the present invention in preventing and treating kiwifruit bacterial canker, the coding region sequence of the AcWRKY20 gene is shown in SEQ ID NO: 2.

[0021] Furthermore, in the use of the AcWRKY20 gene provided by the present invention in preventing and treating kiwifruit bacterial canker, the plant is kiwifruit.

[0022] Furthermore, in the use of the AcWRKY20 gene provided by the present invention in preventing and treating kiwifruit bacterial canker, the pathogen of kiwifruit bacterial canker is Pseudomonas syringae pv. actinidiae.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] Through comparative transcriptome analysis, the present invention found that the AcWRKY20 gene was significantly upregulated by Psa infection, suggesting that this gene may play a unique role in the disease resistance mechanism. In order to systematically analyze the regulatory function of WRKY family genes in canker resistance, the present invention focuses on the AcWRKY20 gene. Through gene cloning, phylogenetic analysis, subcellular localization and transgenic function verification, it is confirmed for the first time that this gene has the molecular characteristics of significantly enhancing the disease resistance of kiwifruit. Psa infection experiments showed that AcWRKY20 showed a specific expression pattern under pathogen stress, and its dynamic regulation was highly correlated with the disease resistance phenotype. This discovery not only reveals a new functional module of WRKY transcription factors in kiwifruit-pathogen interactions, but also provides a key theoretical basis for the development of disease-resistant molecular markers, gene editing target screening and directional breeding of resistant varieties, which has important practical value for promoting the green and sustainable development of the kiwifruit industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a diagram of the AcWRKY20 protein domain analysis.

[0027] Figure 2 The subcellular localization map of AcWRKY20 in tobacco leaves.

[0028] Figure 3 Phylogenetic analysis of AcWRKY20 homologous proteins in different species.

[0029] Figure 4 This is a diagram showing the expression of AcWRKY20 gene induced by Psa.

[0030] Figure 5 This is the electrophoresis diagram of the AcWRKY20 gene clone.

[0031] Figure 6 Schematic diagram of the acquisition of AcWRKY20 transgenic kiwifruit plants. A shows the stable genetic transformation process of AcWRKY20; B shows the phenotypes of WT and AcWRKY20 overexpressing plants (OE#2 and OE#18); C shows the expression level of AcWRKY20 in transgenic lines by qRT-PCR, using AcActin as an internal reference; D shows the protein detection of AcWRKY20 transgenic plants.

[0032] Figure 7This figure demonstrates the disease resistance of kiwifruit plants stably overexpressing AcWRKY20. A shows the disease phenotype of Psa-infected leaf discs; B shows the leaf disc lesion area; and C shows the Psa biomass. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0034] In the following examples, the primer information used is shown in the following table.

[0035]

[0036]

[0037] In the following examples, the test strain Pseudomonas syringae pv. actinidiae, Psa, was isolated from a diseased plant.

[0038] Example 1

[0039] This example provides bioinformatics analysis and vector construction of the AcWRKY20 gene.

[0040] The coding region sequence of the AcWRKY20 gene was obtained from the Actinidia genome V3.0 data on the Actinidia genome website. The full length is 1476 bp, encoding 491 amino acids with a molecular weight of 53.71 kDa. The SMART database and other predictions found that the gene is located on chromosome 19 of Actinidia and has two typical WRKY domains ( Figure 1 The recombinant plasmid AcWRKY20-mCherry carrying the fusion mCherry tag was transformed into Agrobacterium and transiently expressed in tobacco. Subcellular localization experiments revealed that AcWRKY20 was localized in the cell nucleus ( Figure 2 ).

[0041] The homologous genes of this gene in 23 different plants, including kiwi, camellia, cherry, pear, green ramie, tomato, Arabidopsis, and tobacco, were compared in the NCBI Protein BLAST database. MEGAX was used to construct a phylogenetic tree based on the maximum likelihood method ( Figure 3), and found that this gene is highly conserved in plants and clusters into a branch with CfWRKY20 in tea (Camellia sinensis) and large-flowered four-flowered ophthalmology (Cornus florida), indicating that this gene may have potential broad-spectrum application value.

[0042] Kiwifruit leaves were inoculated with the canker pathogen M228 using the leaf disc vacuum infiltration method. Samples were collected at 0, 12, 24, and 48 hours after inoculation. RNA was extracted from the leaves using an RNA kit (Beijing Huayueyang Biotechnology Co., Ltd., Cat. No. 0416-50). cDNA was obtained using a reverse transcription kit (Thermo Fisher Scientific, Cat. No. K1162). qRT-PCR analysis revealed that AcWRKY20 expression began to increase 24 hours after M228 infection ( Figure 4 ), indicating that AcWRKY20 plays a role in the interaction between kiwifruit and Psa.

[0043] (1) Target gene cloning

[0044] RNA was extracted from 'Hongyang' kiwifruit leaves using an RNA kit (Beijing Huayueyang Biotechnology Co., Ltd., Catalog No. 0416-50); cDNA was obtained using a reverse transcription kit (Thermo Fisher Scientific, Catalog No. K1162). Full-length primers for the CDS region were designed using Primer Plus 3.0 software (Appendix 3). PCR amplification was performed using cDNA as a template. The target fragment PCR amplification reaction system consisted of 25 μL of 2× Phanta Flash Master Mix (Dye Plus), 2 μL of AcWRKY20-F (10 μM), 2 μL of AcWRKY20-R (10 μM), 4 μL of cDNA, and 17 μL of ddH2O. The PCR reaction procedure was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 55°C annealing for 15 s, and 72°C extension for 15 s, for 35 cycles, followed by 72°C extension for 5 min. The amplified PCR products were identified by 1% gel electrophoresis. The electrophoresis results are shown in Figure 2. Figure 5 The positive target band was purified by gel recovery (Magen gel recovery kit HiPure Gel Pure DNA Mini Kit), and the sequencing results of the recovered product were consistent with the genomic coding region sequence.

[0045] (2) Construction of overexpression recombinant vector

[0046] Upstream and downstream primers were designed for the overexpressed gene fragment and added to the ClaI and SpeI, SalI and SmaI restriction sites. The amplification primers were mCherry-WRKY20-F, mCherry-WRKY20-R, pICH86988eGFP-WRKY20-F, and pICH86988eGFP-WRKY20-R. The PCR amplification reaction system of the target fragment was the same as the PCR reaction procedure above, and gel recovery was performed.

[0047] (3) Construction and transformation of fusion expression vector

[0048] The pICH86988-mCherry and pICH86988-eGFP empty vectors were cleaved with TaKaRa restriction enzymes (QuickCut TM ClaI, QuickCut TM SpeI, QuickCut TM SalI, QuickCut TM SmaI) was used for enzyme digestion. The enzyme digestion reaction system was: 1ug of vector plasmid, 1μL of each endonuclease, 2μL of 10X QuickCut Buffer, and ddH2O was added to 20μL. The PCR reaction procedure was: incubation at 37°C for 30min and termination of the reaction at 85°C for 10min. Further, the linearized plasmid and the gel-recovered fragment product were connected under the action of C112 (Nanjing Novozyme Biotechnology Co., Ltd., product number: C112) ligase (see the instructions for specific operations). The ligated plasmid was then rapidly transformed into the competent E. coli by the heat shock method. 5α, the transformation method was referred to Beijing Qingke Biotechnology Co., Ltd. 5αChemically Competent Cell User Manual. Transformed E. coli were plated onto LB solid medium containing 50 μg / mL kanamycin and incubated inverted at 37°C for 12 hours. Single colonies were selected for colony PCR analysis. The primers for subcellular localization detection were mCherry-F and mCherry-R, and the primers for stable overexpression detection were pICH86988-F and pICH86988-R (Appendix 3). The colony PCR amplification reaction system and procedure were the same as those for gene cloning. Successfully transformed positive single colonies were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and comparison. The verified single colony was selected and inoculated into 5 mL of LB medium supplemented with kanamycin (50 μg / mL). The culture was shaken at 37°C and 200 rpm for 14-16 hours. The plasmid was then extracted from the E. coli using a plasmid extraction kit (Beijing Zhuangmeng International Biogene Technology Co., Ltd., Miniquick DNA Purification Kit). The recombinant plasmid was transformed into Agrobacterium GV3101 (GV3101 (pSoup) Chemically Competent Cell, Shanghai Weidi Biotechnology Co., Ltd.). After 48 hours of incubation in the dark at 28°C, single colonies were selected for colony PCR testing using primers mCherry-F, mCherry-R, pICH86988-F, and pICH86988-R. Successful colonies were incubated in LB medium containing kanamycin (50 μg / mL) and rifampicin (50 μg / mL) at 28°C and 220 rpm until the desired experimental conditions were reached and stored for future use.

[0049] Example 2

[0050] This example provides information on obtaining transgenic kiwifruit stably overexpressing AcWRKY20.

[0051] Prepare sterile tissue culture seedlings of the 'Hongyang' variety, approximately 30 days old and in good condition. Select young leaves, cut into 2 mm pieces, and place them snugly on a selection medium. Keep in the dark for 2 days. Construct the target gene in the overexpression vector pICH86988 and transform the constructed vector into Agrobacterium tumefaciens EHA105. Transfer the cells to liquid LB medium containing hygromycin and shake at 220 rpm for 12-16 hours before harvesting. Resuspend the cells in 50 mL of liquid MS and add 100 μM acetosyringone to prepare an infection medium (OD600 = 0.6-0.8). Add the dark-grown leaves to the infection medium and incubate at 28°C and 100 × g for 40 minutes. Filter the infection medium through sterile gauze, harvest the kiwifruit leaves, and rinse them three times with sterile MES (2-(N-morpholino)ethanesulfonic acid) medium containing AS (acetosyringone). Transfer to the differentiation medium and keep it in the dark for 2 days. Induce callus: Spread the above kiwifruit leaves on the kiwifruit callus induction medium containing the corresponding antibiotics, culture in the dark for 3 days and then transfer to the normal light incubator for 30 days. Cut the grown callus tissue and transfer it to the kiwifruit germination medium with the corresponding antibiotics. Transplant the differentiated healthy new shoots to the induction differentiation medium. After co-cultivation, screening and regeneration culture, positive plants ( Figure 6 A).

[0052] Quantitative expression analysis showed that the expression of AcWRKY20 in AcWRKY20-OE#2 and AcWRKY20-OE#18 plants was upregulated by 25- and 30-fold, respectively, compared with the wild type ( Figure 6 B and Figure 6 D). In addition, Western blot analysis was used to further analyze the protein expression of AcWRKY20 in transgenic plants. The results showed that the target protein band was detected in the AcWRKY20#2 and AcWRKY20#18 lines, but not in the wild-type plants, indicating that AcWRKY20 was transcribed and expressed in the overexpressing plants and functioned through protein expression ( Figure 6 C).

[0053] Example 3

[0054] This example provides stable overexpression verification of the disease resistance function of AcWRKY20.

[0055] Pick a single colony of M228-GFP prepared in advance and inoculate it into LB liquid medium. Shake and incubate at 28°C and 220 rpm for 12-16 hours. Centrifuge the resulting bacterial solution at 8000 rpm for 10 minutes to enrich the bacteria. Wash the bacteria three times with sterile water and mix them by pipetting. Measure the concentration of the bacterial suspension using a spectrophotometer and adjust the concentration of the bacterial suspension to OD 600 =0.1, collect the cells and dilute them to a final concentration of 1×10 5cfu / ml. Similarly, vacuum infiltration was used to infect Psa into kiwifruit leaves. After incubation at 16°C for approximately 5 days, photos were taken to observe the pathogen's presence and the area of ​​the lesions was calculated.

[0056] To investigate the role of the AcWRKY20 gene in disease resistance in kiwifruit, the transgenic lines AcWRKY20-OE#2 and AcWRKY20-OE#18, as well as the wild-type kiwifruit, were inoculated with the pathogen Psa M228. Five days after inoculation, the disease symptoms of AcWRKY20-OE#2 and AcWRKY20-OE#18 plants were significantly milder than those of the wild-type, as evidenced by a reduction in the area of ​​brown lesions on the leaves ( Figure 7 A). The average lesion area of ​​AcWRKY20-OE#2 and AcWRKY20-OE#18 lines decreased by 3 and 3.5 times, respectively ( Figure 7 B). In addition, the biomass in the leaves of the overexpression lines was significantly higher than that of the WT ( Figure 7 C), indicating that AcWRKY20 is a disease resistance-related gene.

[0057] AcWRKY20 is significantly upregulated by the induction of canker pathogens and is closely related to the disease resistance of the variety. The source of the gene was clarified in Example 1. Through phylogenetic analysis with 23 different plants, it was found that AcWRKY20 was clustered alone in one branch, and it was speculated that the gene was a newly discovered WRKY transcription factor in kiwifruit. In Example 2, a transgenic kiwifruit plant that stably overexpressed AcWRKY20 was successfully created. In Example 3, the AcWRKY20 gene was silenced and overexpressed, and then inoculated with Psa. Phenotypic observations, diseased area, and pathogen biomass statistics all showed that the gene is a potential disease-resistant gene. The discovery of the AcWRKY20 gene provides a functional gene with application potential and a new direction for the breeding of kiwifruit resistant to canker disease, and provides an important theoretical basis for the green and sustainable prevention and control of canker disease.

[0058] The embodiments described above are only some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents the preferred embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A method for obtaining plants resistant to kiwifruit bacterial canker, characterized in that: include: increasing the transcription or translation of the AcWRKY20 gene in the plant, or increasing the expression level of the AcWRKY20 protein in the plant; The AcWRKY20 gene encodes the AcWRKY20 protein; The amino acid sequence of the AcWRKY20 protein is shown in SEQ ID NO:

1.

2. The method for treating plants resistant to kiwifruit bacterial canker according to claim 1, wherein: The coding region sequence of the AcWRKY20 gene is shown in SEQ ID NO:

2.

3. The method for treating plants resistant to kiwifruit bacterial canker according to claim 1, wherein: The plant is kiwi fruit.

4. The method for treating plants resistant to kiwifruit bacterial canker according to claim 1, wherein: The pathogen of kiwifruit bacterial canker is Pseudomonas syringaepv.actinidiae.

5. The method for treating plants resistant to kiwifruit bacterial canker according to claim 1, wherein: A vector for overexpressing the AcWRKY20 gene is constructed, the vector is transformed into Agrobacterium, and the Agrobacterium is used to infect the plant.

6. The method for treating plants resistant to kiwifruit bacterial canker according to claim 5, characterized in that: The pICH86988 vector was used for constructing the vector.

7. The use of the AcWRKY20 gene in preventing and treating kiwifruit bacterial canker is characterized by: Increasing the transcription or translation of the AcWRKY20 gene in the plant, or increasing the expression level of the AcWRKY20 protein in the plant, thereby improving the resistance of the plant to kiwifruit bacterial canker; The AcWRKY20 gene encodes the AcWRKY20 protein; The amino acid sequence of the AcWRKY20 protein is shown in SEQ ID NO:

1.

8. The use of the AcWRKY20 gene in preventing and treating kiwifruit bacterial canker according to claim 7, characterized in that: The coding region sequence of the AcWRKY20 gene is shown in SEQ ID NO:

2.

9. The use of the AcWRKY20 gene in preventing and treating kiwifruit bacterial canker according to claim 7, characterized in that: The plant is kiwi fruit.

10. The use of the AcWRKY20 gene in preventing and treating kiwifruit bacterial canker according to claim 7, characterized in that: The pathogen of kiwifruit bacterial canker is Pseudomonas syringae pv. actinidiae.

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