Application of cselk gene and its protein in improving resistance to citrus bacterial canker

Abstract

The application discloses application of CsELK gene and protein in improving citrus canker resistance, belongs to the technical field of genetic engineering, and particularly relates to application of the CsELK gene in improving citrus canker resistance, wherein the nucleotide sequence of the CsELK gene is shown as SEQ ID NO. 1, and the citrus canker resistance is improved by overexpressing the CsELK gene. The application improves the expression level of the CsELK gene by overexpressing the CsELK gene, effectively improves the citrus canker resistance, and obtains a transgenic citrus strain with significant resistance to citrus canker.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of the CsELK gene and its protein in improving resistance to citrus canker. Background Technology

[0002] Citrus is one of the pillar economic crops, and with the expansion of the industry, its disease problems have become increasingly prominent. Among them, citrus canker (Citrus bacterial canker, CBC) is a common and serious disease. Citrus canker is caused by Xanthomonas citrus subsp. citrus (…). Xanthomonas citri subsp. Citri , Xcc This disease is caused by bacterial canker and primarily infects the above-ground parts of the plant, including leaves, fruits, shoots, and sepals. Infected fruits often exhibit symptoms such as chlorosis, premature drop, and damage to their appearance, affecting not only yield but also significantly reducing their commercial value and resulting in substantial economic losses. Studies have shown that different citrus varieties exhibit significant differences in resistance to bacterial canker: sour oranges and sweet oranges are the most susceptible, followed by lemons, pomelos, and grapefruits, while kumquats show relatively strong resistance.

[0003] Currently, there is no effective way to completely eradicate citrus canker, and prevention in actual production mainly relies on physical, chemical, and integrated control measures. However, long-term reliance on chemical agents can easily lead to environmental pollution risks, and while strict quarantine systems can delay the spread of the disease, they are difficult to completely eliminate infected plants. Therefore, promoting the planting of disease-resistant varieties in epidemic areas has become one of the more sustainable control strategies. Traditional hybridization breeding is difficult to respond quickly to industry needs due to its long cycle and low efficiency, while the development of molecular biology has opened up new paths for disease-resistant breeding. In recent years, with the successive identification of resistance-related genes, citrus genetic engineering breeding has shown broad prospects. For example, overexpression of the transcription factor CsBZIP40 can enhance the plant's resistance to canker by regulating the synthesis of salicylates (SA) and related genes in its signaling pathway, activating pathogenesis-related (PR) protein genes. Similarly, overexpression or interference of transcription factors such as CitMYB20, CsWRKY61, CsAP2-09, and CsLOB1 in transgenic citrus also affected resistance to citrus canker, suggesting their regulatory role in disease response. Furthermore, editing the CsWRKY22 gene or interfering with CsDMR6 expression using CRISPR / Cas9 technology significantly reduced citrus susceptibility to citrus canker, providing a feasible strategy for precision disease-resistant breeding.

[0004] ELK (Ets-like) transcription factors are members of the ETS (E26 transformation-specific) superfamily, characterized by a highly conserved ETS domain responsible for recognizing and binding to specific DNA sequences in the promoter regions of target genes. However, research on ELK transcription factors of the ETS family in plants remains very limited, and studies on their application in citrus disease resistance breeding are currently lacking; there are no reports of using ELK to improve citrus resistance to bacterial canker. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of the CsELK gene and its protein in enhancing resistance to citrus canker. By overexpressing the CsELK gene, this invention increases the expression level of the CsELK gene, effectively enhancing the resistance of citrus to canker and obtaining transgenic citrus lines with significant resistance to citrus canker.

[0006] To achieve the above objectives, the present invention provides the application of the CsELK gene in improving resistance to citrus canker, wherein the nucleotide sequence of the CsELK gene is shown in SEQ ID NO.1, and resistance to citrus canker is improved by overexpressing the CsELK gene.

[0007] Preferably, the improvement of citrus canker resistance specifically means significantly reducing the lesion area of ​​citrus bacterial canker and alleviating the severity of citrus canker.

[0008] The present invention also provides the application of the protein encoded by the CsELK gene in improving resistance to citrus canker, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Preferably, by increasing the expression level of the protein encoded by the CsELK gene, the resistance to citrus canker is improved, the lesion area of ​​bacterial citrus canker is significantly reduced, and the severity of citrus canker is alleviated.

[0010] The present invention also provides an application of a vector overexpressing the CsELK gene in improving resistance to citrus canker. The vector, by overexpressing the CsELK gene, improves resistance to citrus canker, significantly reduces the lesion area of ​​bacterial citrus canker, and alleviates the severity of citrus canker.

[0011] The present invention also provides the application of a strain containing the CsELK gene in improving resistance to citrus canker. The strain improves resistance to citrus canker by overexpressing the CsELK gene, significantly reduces the lesion area of ​​bacterial citrus canker, and alleviates the severity of citrus canker.

[0012] The present invention also provides a method for improving resistance to citrus canker using the CsELK gene, comprising the following steps: 1) Clone the CsELK gene; 2) Construct the overexpression vector of the CsELK gene obtained in step 1); 3) Using the CsELK gene overexpression vector obtained in step 2), citrus was transformed with Agrobacterium tumefaciens to obtain transgenic plants. The transgenic plants were evaluated for resistance to citrus canker and transgenic citrus plants with improved resistance to citrus canker were obtained.

[0013] Preferably, cloning the CsELK gene in step 1) specifically involves: extracting total RNA from citrus fruits, reverse transcribing it into cDNA as a template, performing PCR amplification using primers OE-CsELK-F and OE-CsELK-R, and recovering the DNA fragment of the CsELK gene. The nucleotide sequence of primer OE-CsELK-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer OE-CsELK-R is shown in SEQ ID NO.4.

[0014] Preferably, in step 2), the CsELK gene overexpression vector is constructed by: ligating the CsELK gene DNA fragment recovered by BamHI and EcoRI digestion into the pLGNe vector recovered by BamHI and EcoRI digestion, thus constructing the overexpression vector pLGNe-CsELK.

[0015] Preferably, in step 3), the CsELK gene overexpression vector is used to transform citrus through Agrobacterium tumefaciens. Specifically, the CsELK gene overexpression vector is used to transform citrus explants through Agrobacterium tumefaciens. The genetically transformed citrus explant cells are identified by GUS staining, PCR, and qRT-PCR analysis to determine the CsELK gene expression level.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides the application of the CsELK gene and its protein in improving resistance to citrus canker. By overexpressing the CsELK gene, this invention effectively enhances the expression level of the CsELK gene, thereby increasing the resistance of citrus to canker and obtaining transgenic citrus lines with significant resistance to the disease. This invention involves cloning the citrus CsELK gene, constructing an overexpression vector, and then transforming citrus into transgenic plants. The resulting transgenic plants reduce the severity of citrus canker, improve resistance to the disease, and significantly reduce the lesion area of ​​bacterial canker in citrus. This has significant value for molecular breeding of citrus canker resistance, lays the foundation for genetic breeding of citrus canker resistance, and greatly promotes the development and application of citrus canker resistance genetic engineering. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The bioinformatics features of the CsELK gene in this invention are shown in the figure. In the figure, A is the chromosomal location of the citrus CsELK gene, bp represents a base, B is the gene structure of citrus CsELK, C is the conserved domain of the citrus CsELK gene, and aa represents an amino acid. Figure 2 This is a structural diagram of the CsELK gene overexpression vector of the present invention. In the diagram, GUS represents the β-glucosidase gene, 35S represents the plant constitutive promoter derived from cauliflower mosaic virus, and NOS represents the crown gall synthase gene terminator. Figure 3 This is a graph showing the expression level of the CsELK gene in the transgenic plants of this invention. The graph shows… "This indicates a significant difference compared to the WT group (P<0.05)," "This indicates a highly significant difference compared to the WT group (P < 0.01)." "This indicates that the difference compared with the WT group was extremely significant (P < 0.001)." “" indicates that the difference between the same group and the WT group is extremely significant (P<0.0001). WT represents wild-type plants, OE-CsELK-1 represents transgenic plant 1, OE-CsELK-2 represents transgenic plant 2, and OE-CsELK-3 represents transgenic plant 3. Figure 4 The images show the symptoms of each group of plants after 10 days of inoculation with ulcer pathogen. In this invention, A represents the wild-type plant group, and B to D represent the transgenic plant groups. Figure 5 This is a statistical chart showing the size of lesions on the leaves of plants from different groups of plants 10 days after inoculation with the causal agent of canker. The chart includes the symbols "". "" indicates that the difference with the WT group is extremely significant (P<0.001). WT represents wild-type plants, OE-CsELK-1 represents transgenic plant 1, OE-CsELK-2 represents transgenic plant 2, and OE-CsELK-3 represents transgenic plant 3. Figure 6 This is a statistical chart showing the disease index of the leaves of each group of plants in this invention 10 days after inoculation with ulcer pathogen. The chart shows the disease index of each group of plants. "" indicates that the difference with the WT group is extremely significant (P<0.001). WT represents wild-type plants, and OE-CsELK represents transgenic plants. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. This specification and its embodiments are exemplary only. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended, meaning they include but are not limited to.

[0021] The late-ripening oranges used in this invention were sourced from the Citrus Research Institute of the Chinese Academy of Agricultural Sciences.

[0022] Example 1 I. Bioinformatics analysis of the CsELK gene in citrus: The citrus CsELK gene is located on citrus chromosome 3, between 9349681 bp and 9353519 bp. This chromosome is 1.23 kbp in length, and its CDS sequence is 1005 bp, encoding 334 amino acids. Analysis of the protein amino acid sequence revealed the presence of a distinct ELK_2 structural functional domain (e.g., ...). Figure 1 China A Figure 1 China B and Figure 1 (As shown in C).

[0023]

[0024] The amino acid sequence of the protein encoded by the CsELK gene is shown in SEQ ID NO.2, SEQ ID NO.2: MEDLYKRLDDPAAATVSYSSDDFLRVENFSLGNFTANTTTAGELYNPPCVDSMLQFNSEITAAAGSDNISDDLIKARIANHPRFPNLLSAYIDCQKVGVPSEVAFLLEEICRENNTTRCSSEIGADPELDEFMESYSEVLRRYKEELAKPFDEATTFLSNIELQL SNLCNGEFTRTLDYPSDEAAGTSEEELSYEEAEPLDCQDFLNASSGPADDQDIKGMLMRKYSGYLSSLKKEFLKKKKKGKLPKDAKTTLMDWWNEHYRWPYPTEDDKLKLSKETGLDQKQISNWFINQRKRHWKPSEDMRFALLEGATLGGNYSSNDRGPMFLNTPDFN.

[0025] II. Cloning of the CsELK gene coding sequence in citrus: 1. RNA extraction and cDNA synthesis: Total RNA was extracted from citrus (Late Orange) leaves using a plant total RNA extraction kit (Adley, CAT: RN09). RNA quality was verified by agarose gel electrophoresis, and its concentration was determined using a concentration meter. cDNA was synthesized using a reverse transcription kit PrimeScript RTMaster Mix (TaKaRa, CAT: RR036A) for subsequent experiments.

[0026] 2. PCR amplification of the CsELK gene coding sequence: A DNA fragment encoding the CsELK sequence was amplified from citrus cDNA using primers OE-CsELK-F (SEQ ID NO.3), OE-CsELK-R (SEQ ID NO.4), and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q). The fragment was 1005 bp in length and was sequenced to identify the citrus CsELK gene coding sequence (SEQ ID NO.1). Under UV light, agarose gel blocks containing the target fragment were cut with a clean blade, and the DNA fragment was recovered using a gel recovery kit (BioFlux, CAT: BSC02M1).

[0027] The nucleotide sequence of primer OE-CsELK-F (containing restriction enzyme sites) is shown in SEQ ID NO.3, SEQ ID NO.3: ACAGGGTACCCGGGGATCCATGGAAGATTTGTACAAGAGACTTGATG.

[0028] The nucleotide sequence of primer OE-CsELK-R (containing restriction enzyme sites and a 3×flag tag sequence) is shown in SEQ ID NO.4, SEQ ID NO.4: GTCGTCATCCTTGTAATCCTGGAGTATTTAAGAACATGGGTCCC.

[0029] PCR amplification program: 94℃, 5 min; 94℃, 30 s, 58℃, 30 s, 72℃, 1.5 min, 35 cycles; extension at 72℃ for 3 min.

[0030] III. Construction of CsELK gene overexpression vector and transformation of Agrobacterium: 1. Construction of overexpression vectors: (1) Based on the PCR cloning verification of the correct CsELK gene sequence, restriction endonuclease sites were screened by sequence alignment.

[0031] (2) The 5' end of the seamless cloning PCR primers must contain a 15-25 nt sequence homologous to the end of the adjacent fragment (insert fragment or vector). The primer design is as follows: 5'—Upstream vector terminal homologous sequence + restriction enzyme site + gene-specific forward amplification sequence—3'; 3'—Gene-specific reverse amplification sequence + restriction enzyme site + downstream vector terminal homologous sequence—5'.

[0032] (3) DNA fragment amplification was performed using high-fidelity polymerase. The PCR product was identified by agarose gel electrophoresis and purified before the concentration was determined. The empty vector was subjected to double enzyme digestion (37℃, 1 h). The original vector plasmid was set as a negative control. The enzyme digestion efficiency was confirmed by electrophoresis (Note: The migration rate of the digested vector fragment was significantly slower than that of the original plasmid).

[0033] (4) The purified linearized vector and the target fragment were ligated using a homologous recombinase-mediated reaction. The following reaction system was prepared in an ice-water bath: 5 μL of seamless cloning mixed enzyme; 50-200 ng of linearized vector; 50-200 ng of insert fragment; and ddH2O was added to bring the reaction system to 10 μL. The reaction system was placed at 50℃ and reacted for 45 min.

[0034] (5) Transformed into DH5α competent Escherichia coli cells, after 18 h of culture, single colonies were screened for propagation and sequencing analysis. Plasmids of positive clones were extracted using a plasmid extraction kit (Omega, CAT: D6942) to obtain the CsELK gene overexpression vector pLGNe-CsELK (e.g., pLGNe-CsELK). Figure 2 (As shown).

[0035] 2. Transformation of Agrobacterium with overexpression vector: The recombinant plasmid was used to transform EHA105 Agrobacterium competent cells. After the colonies grew, single colonies were verified by PCR using primers OE-CsELK-F (SEQ ID NO.3) and OE-CsELK-R (SEQ ID NO.4).

[0036] PCR reaction conditions: 94℃ for 3 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min.

[0037] IV. Genetic transformation of citrus using CsELK gene overexpression vector: 1. Pretreatment of experimental materials: Three days prior to the event, Agrobacterium tumefaciens containing the target gene overexpression vector, stored at -80℃, was activated. After streaking on LK solid medium, the culture was placed in a 28℃ incubator in the dark for 48 hours. Ten morphologically sound single colonies were selected and transferred using a sterile pipette tip to 50mL Erlenmeyer flasks containing 20mL LK liquid medium (containing 26.8µL AS inducer), and then incubated overnight at 28℃ on a shaker (220 r / min).

[0038] 2. Preparation of plant materials: On the day of the experiment, healthy leaves of wild-type Late Orange (WT) were collected, rinsed with running tap water for 30 seconds, and then subjected to surface disinfection treatment in sequence: soaked in 75% ethanol for 30 seconds, rinsed with sterile water 3 times (1 minute each time), and finally wiped off the surface moisture with sterile degreased cotton for later use.

[0039] 3. Preparation of bacterial suspension: Transfer the overnight culture to a 50 mL sterile centrifuge tube, centrifuge at 5000 r / min for 10 min, and discard the supernatant in a clean bench. Resuspend the cells in an appropriate amount of AIM liquid medium, and adjust the bacterial concentration to OD using a spectrophotometer. 600 =0.6±0.02, and then pre-incubated at 28℃ in a shaker (220 r / min) for 60 min for activation.

[0040] 4. Genetic transformation operation: Using a sterile syringe needle, make a small incision of about 2 mm on the back of the leaf along the vein direction (ensuring that the leaf tissue is not penetrated). Inject the activated bacterial solution evenly into the incision site. After absorbing the overflowing bacterial solution with sterile gauze, lay the treated leaf flat on the surface of AIM solid culture medium and induce transformation in a dark incubator at 28°C.

[0041] 5. Sample collection and analysis: 1 cm samples were taken from the inoculation site 0 days (immediately) and 5 days after infection treatment. 2 Tissue samples were flash-frozen in liquid nitrogen and immediately transferred to an ultra-low temperature freezer at -80°C for storage. Simultaneously, 0.5 g of fresh samples (n=3, OE-CsELK-1, OE-CsELK-2, OE-CsELK-3) were collected on day 5 for gene expression analysis.

[0042] V. Validation of CsELK gene overexpression transgenic plants: 1. qRT-PCR analysis of transgenic plants: Total RNA (Adelaide, CAT No: RN09) was extracted from leaves of transgenic plants and cDNA was synthesized using the PrimeScript RT Master Mix reverse transcription kit (TaKaRa, CAT: RR036A). The expression level of the target gene was detected by qRT-PCR. The detection primers were RT-CsELK-F (SEQ ID NO.5) and RT-CsELK-R (SEQ ID NO.6). Two... -△△Ct The relative expression level of the CsELK gene in transgenic plants was calculated as follows: The water-treated sample was defined as the reference factor, with a CsELK expression level of 1. The fold increase in gene expression relative to the reference factor in transgenic citrus was then calculated as 2. -△△Ct , which is its relative expression level.

[0043] The nucleotide sequence of primer RT-CsELK-F is shown in SEQ ID NO.5, SEQ ID NO.5: GCACGGATCGCAAATCATCC.

[0044] The nucleotide sequence of primer RT-CsELK-R is shown in SEQ ID NO.6, SEQ ID NO.6: GAAATGCCACTTCGGAGGGA.

[0045] qRT-PCR reaction conditions: 95℃ for 3 min, 94℃ for 10 s; 56℃ for 10 s, 72℃ for 10 s, 40 cycles; 72℃ for 10 min.

[0046] The results showed that the CsELK gene was expressed at a high level in transgenic plants (OE-CsELK-1, OE-CsELK-2, OE-CsELK-3) compared to wild-type plants (WT) (up to 421 times higher than the control). Figure 3 (As shown).

[0047] VI. Evaluation of resistance in transgenic plants overexpressing the CsELK gene: Mature leaves of transgenic plants (OE-CsELK-1, OE-CsELK-2, OE-CsELK-3) were washed, disinfected with 75% alcohol, and rinsed with sterile water before being placed in a clean bench. Acupuncture was performed centered on the leaf veins, and 1 μL (1 × 10⁻⁶) of ulcer-causing bacterial solution was pipetted into each well. 5 CFU / mL); cultured in a constant temperature and light incubator at 28℃ (16 h light / 8 h dark); photographed leaves 10 days after inoculation, and the area of ​​lesions was counted using ImageJ V1.47 software.

[0048] The disease is classified into grades 0-7 based on the area of ​​the lesions, with the letter R representing the lesion area, grade 0 (R ≤ 0.25 mm). 2 Level 1 (0.25 mm) 2 <R≤0.5 mm 2 Level 2 (0.5 mm) 2 <R≤0.75 mm 2 Level 3 (0.75 mm) 2 <R≤1 mm 2 Level 4 (1.0 mm) 2 <R≤1.25 mm 2 Level 5 (1.25 mm) 2 <R≤1.5 mm 2 Level 6 (1.5 mm) 2 <R≤1.75 mm 2 Level 7 (R > 1.75 mm) 2 The disease index is calculated according to the formula: DI = 100 × Σ[number of lesions at each level × corresponding level value] / (total number of lesions × maximum level).

[0049] The results showed that 10 days after inoculation with the causal agent of bacterial canker, both the overexpressing plants (OE-CsELK) and the wild-type plants grafted at the same time (WT) developed the disease to varying degrees, with some differences in lesion size (e.g., Figure 4 China A and Figure 4(As shown in Figure B). Statistical analysis revealed that the lesion area of ​​transgenic plants (OE-CsELK-1, OE-CsELK-2, OE-CsELK-3) was significantly smaller than that of wild-type plants (WT), ranging from 20.09% to 40.22% of that of wild-type plants (e.g., ...). Figure 5 (As shown). The disease index of the transgenic plant (OE-CsELK) was significantly lower than that of the wild-type plant (WT), reaching 54.54% of the control (e.g.). Figure 6 (As shown in the figure). It can be seen that CsELK gene overexpression can significantly reduce the lesion area of ​​citrus bacterial canker and alleviate the severity of citrus canker.

[0050] Therefore, CsELK gene overexpression can significantly reduce the lesion area of ​​citrus canker and alleviate the severity of the disease. This gene can be used independently for molecular breeding of disease resistance, or it can be used in conjunction with other disease resistance or susceptibility genes for molecular breeding of citrus canker resistance.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the CsELK gene in improving resistance to citrus canker, characterized in that, The nucleotide sequence of the CsELK gene is shown in SEQ ID NO.

1. Overexpression of the CsELK gene enhances resistance to citrus canker.

2. The application according to claim 1, characterized in that, Specifically, improving resistance to citrus canker involves significantly reducing the lesion area of ​​bacterial citrus canker and alleviating the severity of the disease.

3. The application of the protein encoded by the CsELK gene as described in claim 1 in improving resistance to citrus canker, characterized in that, The amino acid sequence of the protein encoded by the CsELK gene is shown in SEQ ID NO.

2. By increasing the expression level of the protein encoded by the CsELK gene, the resistance to citrus canker is improved, the lesion area of ​​bacterial citrus canker is significantly reduced, and the severity of citrus canker is alleviated.

4. The application of a vector overexpressing the CsELK gene as described in claim 1 in improving resistance to citrus canker, characterized in that, The vector, by overexpressing the CsELK gene, enhances resistance to citrus canker, significantly reduces the lesion area of ​​bacterial citrus canker, and alleviates the severity of citrus canker.

5. The application of a strain containing the CsELK gene as described in claim 1 in improving resistance to citrus canker, characterized in that, The strain, Agrobacterium tumefaciens, enhances resistance to citrus canker by overexpressing the CsELK gene, significantly reduces the lesion area of ​​bacterial citrus canker, and alleviates the severity of citrus canker.

6. A method for improving resistance to citrus canker using the CsELK gene described in claim 1, characterized in that, Includes the following steps: 1) Clone the CsELK gene; 2) Construct the overexpression vector of the CsELK gene obtained in step 1); 3) Using the CsELK gene overexpression vector obtained in step 2), citrus was transformed with Agrobacterium tumefaciens to obtain transgenic plants. The transgenic plants were evaluated for resistance to citrus canker and transgenic citrus plants with improved resistance to citrus canker were obtained.

7. The method for improving resistance to citrus canker according to claim 6, characterized in that, The cloning of the CsELK gene in step 1) specifically involves: extracting total RNA from citrus fruits, reverse transcribing it into cDNA as a template, performing PCR amplification using primers OE-CsELK-F and OE-CsELK-R, and recovering the DNA fragment of the CsELK gene. The nucleotide sequence of primer OE-CsELK-F is shown in SEQ ID NO. 3, and the nucleotide sequence of primer OE-CsELK-R is shown in SEQ ID NO.

4.

8. The method for improving resistance to citrus canker according to claim 6, characterized in that, In step 2), the CsELK gene overexpression vector is constructed by: ligating the CsELK gene DNA fragment recovered by BamHI and EcoRI digestion into the pLGNe vector recovered by BamHI and EcoRI digestion, thus constructing the overexpression vector pLGNe-CsELK.

9. The method for improving resistance to citrus canker according to claim 6, characterized in that, Step 3) involves using the CsELK gene overexpression vector to transform citrus through Agrobacterium tumefaciens. Specifically, the CsELK gene overexpression vector is used to transform citrus explants through Agrobacterium tumefaciens. The genetically transformed citrus explant cells are then identified by GUS staining, PCR, and qRT-PCR analysis to determine the CsELK gene expression level.

Images