Biological products for promoting expression of pxap1 gene and its protein in improving citrus disease resistance

Abstract

The present application belongs to the technical field of plant genetic engineering, and more particularly relates to application of biological products for promoting expression of a PxAP1 gene and a protein thereof in improving citrus disease resistance. The amino acid sequence of the effector protein PxAP1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. Transient expression of PxAP1 in Nicotiana benthamiana leaves can stimulate active oxygen, increasing the content of hydrogen peroxide. The ROS level in transgenic Poncirus trifoliate leaves overexpressing PxAP1 is higher than that in wild-type Poncirus trifoliate, and after inoculation with Xanthomonas campestris, the transgenic Poncirus trifoliate exhibits stronger disease resistance than the wild type. The present application provides a new gene resource and technical means for citrus breeding against bacterial blight, and helps to cultivate new citrus varieties with high resistance to bacterial blight.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the application of biological products that promote the expression of the PxAP1 gene and its protein in improving the disease resistance of citrus. Background Technology

[0002] The citrus industry faces threats from various diseases, among which citrus canker is caused by Xanthomonas aureus, a citrus pathogen. Xanthomonas citri subsp. citri Citrus canker is a global and devastating bacterial disease caused by bacterial infection. It severely impacts citrus yield and quality, leading to damaged fruit appearance, significant fruit drop, and substantial economic losses for citrus growers. Currently, control of this disease mainly relies on chemical fungicides and agricultural measures, but these methods suffer from environmental pollution, increased pathogen resistance, and high costs. Therefore, breeding disease-resistant varieties is widely recognized as the most economical, effective, and environmentally friendly fundamental strategy. However, current technologies still fall short in identifying functional genes related to citrus canker resistance, lacking gene resources and related regulatory mechanisms that can be efficiently applied to citrus canker resistance breeding, thus failing to meet the practical needs of citrus disease resistance breeding and green control.

[0003] Over long periods of evolution, plants have developed complex immune systems to defend against pathogen invasion. Effector proteins are proteins secreted by pathogens during plant infection; they can interfere with normal physiological processes, aiding in pathogen infection. However, some effector proteins can also be recognized by plants, triggering immune responses. Research has found that certain effector proteins play a crucial role in plant immune responses, enhancing plant resistance to pathogens by regulating signaling pathways and activating the expression of disease-related genes. Candidatus Liberibacter asiaticus (CLas) is a Gram-negative bacterium that severely damages the citrus industry, commonly known as Huanglongbing (HLB), causing citrus HLB. Studies have found that effector proteins secreted via the HLB's Sec secretion pathway inhibit plant immunity by targeting important immune-related proteins in citrus; for example, SDE5 targets the PUB21-MYC2 module, and SDE19 targets citrus Sec12 to inhibit plant immunity. Effector proteins of Huanglongbing that can stimulate plant immunity are relatively rare, and the function, potential and application prospects of these proteins in citrus canker resistance are completely unknown. Summary of the Invention

[0004] The purpose of this invention is to provide the application of biological products that promote the expression of the PxAP1 gene and its protein in improving the disease resistance of citrus, so as to solve the above-mentioned technical problems.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of a biological product that promotes the expression of the PxAP1 gene and its protein in improving the disease resistance of citrus fruits. The nucleotide sequence of the PxAP1 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein is shown in SEQ ID NO.1.

[0006] This invention addresses the challenge of controlling citrus canker by providing a biological product overexpressing the PxAP1 gene to enhance citrus disease resistance, thus solving the aforementioned technical problems. The core idea is to activate the immune response in citrus by overexpressing the effector protein gene PxAP1, thereby increasing resistance to the canker pathogen. Specifically, the PxAP1 gene is first cloned, its plant overexpression vector is constructed, and then introduced into citrus using Agrobacterium-mediated genetic transformation to obtain transgenic plants. Experiments show that PxAP1 expression in plants can trigger a burst of reactive oxygen species, increase hydrogen peroxide levels, and enhance the immune response. Further disease resistance evaluation shows that after inoculation with the canker pathogen, the colonization rate of the transgenic citrus is significantly reduced, and the lesion area is smaller, confirming that PxAP1 overexpression can effectively improve citrus resistance to canker. This invention provides a new gene resource and feasible technical pathway for breeding new disease-resistant citrus varieties.

[0007] Furthermore, the disease resistance includes resistance to citrus canker.

[0008] Furthermore, the bioproduct includes a recombinant overexpression vector containing the PxAP1 gene.

[0009] Furthermore, the biological product also includes recombinant Agrobacterium EHA105 containing the recombinant overexpression vector.

[0010] Furthermore, the recombinant overexpression vector is formed by inserting the PxAP1 gene into the pCAMBIA1380GFP vector. Xba I and Kpn Obtained from the I restriction site.

[0011] Furthermore, the recombinant Agrobacterium is obtained by introducing the recombinant overexpression vector into Agrobacterium EHA105.

[0012] Furthermore, the recombinant Agrobacterium was cultured to obtain an infecting solution, which was then used to infect the epicotyl of citrus fruit, thereby achieving heterologous overexpression of the PxAP1 gene in citrus fruit.

[0013] The present invention also provides a method for cultivating citrus resistant to citrus canker, comprising the following steps: increasing the expression level of the PxAP1 gene or the protein encoded by the PxAP1 gene in citrus to improve the disease resistance of citrus.

[0014] The present invention has the following beneficial effects: In transgenic trifoliate orange leaves overexpressing PxAP1 protein, the colonization of causal agent of leaf canker was significantly reduced, and the area of ​​canker lesions formed by causal agent infection was significantly reduced. Attached Figure Description

[0015] Figure 1 This is a subcellular localization map of PxAP1-GFP in plant cells.

[0016] Figure 2 The graph shows the reactive oxygen species (ROS) induced by PxAP1 in tobacco leaves, where A is the leaf staining graph and B is the hydrogen peroxide content graph.

[0017] Figure 3 The images show PxAP1 transgenic trifoliate orange and its DAB staining. In the images, A is the GFP fluorescence image of the transgenic trifoliate orange, B is the electrophoresis image of the PxAP1 gene, and C is the DAB staining image of the transgenic trifoliate orange leaves.

[0018] Figure 4 The images show the lesions and proliferation curves of *Bacillus cankerans* on the leaves of PxAP1 transgenic trifoliate orange. In the image, A shows the leaf lesions, and B shows the proliferation curve. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0020] Example 1 The amino acid sequence of the effector protein PxAP1 is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0021] SEQ ID NO.1: RQQAIVTRFGKIHATYREPGIYFKMPFSFMNVDRVKYLQKQIMRLNLDNIRVQVSDGKFYEVDAMMTYRIIDPSLFCQSVSCDRIAAESRLRTRLDASIRRVYGLRRFDDALSKQREKMMMEVCEDLRYDAEKL GISIEDVRVLRTDLTQEVSQQTYDRMKAERLAEAEFIRARGREEGQKRMSIADRKATQILSEARRDSEINYGKGEAERGRILSNVFQKDPEFFEFYRSMRAYTDSLASSDTFLVLSPDSDFFKYFDRFQERQKNYRKEY.

[0022] SEQ ID NO.2: CGTCAACAAGCGATTGTTACTCGATTTGGAAAGATTCATGCTACCTATAGAGAGCCAGGCATTTATTTTAAAATGCCATTTTCTTTTATGAATGTTGATAGAGTTAAATATCTGCAAAAACAAATTATGCGTCTAAATCTTGATAATATTAGAGTACAAGTTTCTGATGGGAAATTCTATGAAGTTGATGCTATGATGACTTATAGAATCATTGATCCCTCTTTATTTTGTCAATCTGTTTCGTGTGATCGTATTGCTGCAGAATCTCGCTTACGTACTCGTCTTGATGCATCTATCCGGCGTGTTTATGGATTACGTCGTTTTGATGATGCTTTATCCAAACAACGTGAAAAAATGATGATGGAGGTATGTGAAGATTTGCGCTACGATGCTGAAAAATTAGGAATTAGTATAGAAGATGTGCGCGTTCTACGTACTGATTTAACACAGGAAGTTTCTCAACAAACATATGATCGGATGAAAGCAGAACGCTTAGCAGAGGCTGAGTTTATCCGTGCTAGAGGAAGAGAAGAGGGACAAAAACGCATGTCTATTGCTGATCGTAAAGCAACTCAGATTTTATCTGAAGCGCGACGTGATTCTGAAATAAATTATGGGAAGGGTGAAGCGGAAAGGGGGCGTATTCTCTCTAATGTATTTCAGAAAGATCCTGAATTTTTCGAATTTTATCGTTCTATGAGAGCATATACGGATTCTTTGGCTTCATCAGATACCTTTTTGGTTCTTTCGCCAGATTCGGATTTTTTCAAGTATTTTGATCGCTTCCAAGAAAGACAGAAAAATTATAGAAAAGAATATTGA。

[0023] 1. Subcellular localization of PxAP1-GFP.

[0024] Insert the PxAP1 gene into the pCAMBIA2300GFP vector Sac I and XbapCAMBIA2300-PxAP1-GFP was constructed between the I restriction sites, and the correctly sequenced plasmid was transformed into Agrobacterium Gv3101 strain. Agrobacterium carrying the PxAP1 gene was added to 3 ml of LB medium containing kanamycin and cultured overnight at 28°C with shaking at 200 rpm. The cells were collected by centrifugation at 3500 g for 5 min, resuspended in 10 mM MES buffer (10 mM MES, 10 mM MgCl2, 200 μM acetylsyl eugenol), and its OD200 was measured. 600 After adjusting to 0.4, let stand for 1 hour. Inject 1 ml of the injection solution into the underside of the tobacco leaf using a needleless syringe. Two days later, prepare a slide from the injected area and observe GFP fluorescence under a Leica SP8 confocal microscope at 488 nm excitation light. The results showed that it co-localized with the peroxisome, such as... Figure 1 As shown.

[0025] II. PxAP1-GFP stimulates plant ROS.

[0026] 1. DAB staining to detect ROS bursts in tobacco leaves PxAP1-GFP and GFP were transiently expressed in *Nicotiana benthamiana* according to the method described above. 0.1 g of DAB powder (Beijing Coollab Technology Co., Ltd.) was dissolved in 100 mL of sterile water, and the pH was adjusted to 3.8 to prepare a 0.1% (w / w) DAB solution. Leaves transiently expressing PxAP1-GFP and GFP were placed in a 50 mL syringe, and DAB solution was added until the leaves were submerged. The injection hole was held down with the thumb, and the syringe was manually vacuumed to draw the DAB solution into the leaves. The leaves were kept submerged and left to stand overnight in the dark at room temperature. Subsequently, the leaves were destained using 95% (v / v) ethanol in an 85°C water bath. For ease of observation, after complete decolorization, the leaves were washed twice with 70% (v / v) ethanol and 30% (v / v) ethanol, respectively. The DAB staining results were photographed and recorded. The results are as follows: Figure 2 As shown in Figure A, compared to GFP, leaves overexpressing PxAP1-GFP showed deeper DAB staining, indicating that PxAP1-GFP stimulates ROS in plant leaves.

[0027] 2. Determination of H2O2 content Weigh 0.1g of tobacco leaves overexpressing PxAP1-GFP and GFP, flash freeze in liquid nitrogen, add 1mL of acetone and grind, then bring the homogenate to a final volume of 1mL with acetone. Centrifuge at 12000g, 4℃ for 10min, collect the supernatant and place on ice. Subsequently, determine the H2O2 content using a hydrogen peroxide content assay kit (Suzhou Grees Biotechnology Co., Ltd.), reading the absorbance at 415nm. Figure 2 As shown in Figure B, the results indicate that the H2O2 content in leaves overexpressing PxAP1-GFP is higher than that in the control GFP.

[0028] III. Preparation of transgenic citrus materials overexpressing PxAP1.

[0029] Remove the outer seed coat from the trifoliate orange seeds, disinfect them by soaking them in a 2% sodium hypochlorite solution for 15 minutes, and then rinse them three times with sterile water for 5 minutes each time. After blotting the seeds dry with sterile filter paper, sow them in test tubes containing MS medium and culture them in the dark for about 35 days. Select uncontaminated, 15cm long etiolated citrus seedlings and culture them under light for 7 days. Cut the epicotyl into stem segments of about 1.5cm.

[0030] The PxAP1 gene was inserted into the pCAMBIA1380GFP vector. Xba I and Kpn The pCAMBIA1380GFP-PxAP1 vector was constructed between the I restriction sites and transformed into Agrobacterium EHA105 strain. The recombinant strain was cultured overnight in resistant LB broth, collected by centrifugation, and resuspended in CCM (20 mg / L acetylsuccinone). OD 600 Adjust to 0.5. Soak the cut stem segments in Agrobacterium CCM suspension for 10 minutes, then discard the suspension and aspirate excess liquid. Place the stem segments on CCM solid medium and culture in the dark. After 2 days, transfer the stem segments to RM medium and culture in the dark for 5 days, then transfer them to light until budding. Buds exhibiting GFP fluorescence are grafted for propagation. Semi-quantitative PCR detection confirmed the expression of the PxAP1 gene in the transgenic buds, obtaining transgenic trifoliate orange overexpressing PxAP1. Following the DAB staining method for tobacco leaves, DAB staining was performed on the leaves of transgenic trifoliate orange overexpressing PxAP1 and wild-type trifoliate orange. The results showed that the leaves overexpressing PxAP1 stained more deeply with DAB, indicating that PxAP1 stimulates ROS in citrus leaves. Figure 3 As shown.

[0031] Overexpression of PxAP1 improved the resistance of trifoliate orange to ulcerative colitis pathogens. Ulcerative bacteria Xanthomonas citri subsp. citri(Xcc) has been published in the paper “Huang G, Chang X, Hu Y, Li F, Wang N, Li R. SDE19, a SEC-dependent effector from 'Candidatus Liberibacter asiaticus' suppresses plant immunity and targets Citrus sinensis Sec12 to interfere with vesicle trafficking. PLoS Pathog. 2024 Sep 10;20(9):e1012542. doi: 10.1371 / journal.ppat.1012542. PMID: 39255299; PMCID:PMC11414923.”

[0032] Activate *Pseudomonas ulcerans* Xcc by streaking on NA medium and incubate at 28°C for 2 days. Scrape colonies from the NA medium and transfer them to 2 mL sterile centrifuge tubes. Add 1 mL of sterile water and vortex or pipette for diffusion. Collect the bacterial culture at 8000 g for 5 min, discard the supernatant, repeat twice to remove the gelatinous substance, and then dilute with sterile water. Measure the OD using a UV spectrophotometer. 600 Adjust the final concentration of Xcc to OD 600 The value was approximately 0.003. A small incision was made on the underside of the leaf using a syringe needle, and the bacterial solution was injected into the lower epidermis of the leaf. The leaf was then cultured in a culture chamber (20-25℃, 14h light / 10h dark). Leaves were collected using a perforator on days 1, 4, 6, 8, and 11 and ground in 500μL of sterile water. The grinding solution was diluted with sterile water at a ratio of (10...)... 2 10 3 10 4 10 5 10 6 Serial dilutions were performed, with 5 μL of each dilution applied as spot treatments. The samples were then incubated at 28°C for 3 days. Afterward, the colony counts for each dilution were tallied, and the bacterial colony density (cfu / cm³) was calculated. 2 The result is as follows: Figure 4 As shown, compared with wild-type trifoliate orange, the Xcc colonization of transgenic trifoliate orange overexpressing PxAP1 was significantly lower than that of wild-type trifoliate orange on days 4, 6, 8 and 11 after inoculation with citrus canker pathogen. The area of ​​canker lesions formed by citrus canker pathogen infection of leaves was significantly reduced, indicating that overexpression of the PxAP1 gene enhanced the resistance of trifoliate orange to citrus canker pathogen.

[0033] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0034] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0035] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of biological products that promote the expression of the PxAP1 gene and its protein in improving the resistance of citrus to citrus canker, characterized in that, The nucleotide sequence of the PxAP1 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein is shown in SEQ ID NO.1; The biological product includes a recombinant overexpression vector containing the PxAP1 gene and recombinant Agrobacterium EHA105.

2. The application according to claim 1, characterized in that, The recombinant overexpression vector is formed by inserting the PxAP1 gene into the pCAMBIA1380GFP vector. Xba I and Kpn Obtained from the I restriction site.

3. The application according to claim 1, characterized in that, The recombinant Agrobacterium was obtained by introducing the recombinant overexpression vector into Agrobacterium EHA105.

4. The application according to claim 3, characterized in that, The recombinant Agrobacterium was cultured to obtain an infecting solution, which was then used to infect the epicotyl of citrus fruits, thereby achieving overexpression of the PxAP1 gene in citrus fruits.

5. A method for cultivating citrus fruits resistant to citrus canker, characterized in that, Includes the following steps: Increasing the expression level of the PxAP1 gene or the protein described in claim 1 in citrus fruits can enhance the resistance of citrus fruits to citrus canker.

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