Preparation method and application of pine resin canker pathogen protoplast
By optimizing enzymatic hydrolysis conditions and constructing a genetic transformation system for pine resin canker pathogens, the problem of gene function research of pine resin canker pathogens was solved, achieving efficient and stable genetic transformation and gene expression, which supports research on pathogenic mechanisms and drug molecular targets.
Patent Information
- Application Number
- CN202511267362.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-05
- Publication Date
- 2025-12-12
AI Technical Summary
The lack of an efficient and stable genetic transformation system for pine resin canker pathogens in existing technologies has led to slow progress in the study of gene function and pathogenic mechanism of pine resin canker pathogens.
Using a PEG/CaCl2-mediated protoplast transformation method, high-concentration pine resin canker pathogen protoplasts were prepared by optimizing enzymatic hydrolysis conditions and using a specific ratio of wall-dissolving enzymes, wall-breaking enzymes, and snail enzymes. A genetic transformation system for pine resin canker pathogens was then constructed to achieve stable integration and expression of exogenous genes.
High-quality protoplasts of pine resin canker pathogens were obtained, enabling stable integration and expression of exogenous genes. Stable transformants were provided for studying pathogenic molecular mechanisms and drug targets, reducing operating costs and avoiding the problems of high heterokaryotic ratios and low gene silencing efficiency in existing methods.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic transformation technology, and more specifically, relates to the preparation method and application of pine resin canker bacteria protoplasts. Background Technology
[0002] *Fusarium circinatum*, the causal agent of pine resin canker, is a quarantine pathogen that seriously damages pine trees. Pine resin canker (PPC) is a severe plant disease caused by this fungus, leading to trunk canker and shoot dieback in pine trees, posing a significant threat to forestry ecosystems and the economy. This disease has spread to many parts of the world, widely distributed in the Americas, Europe, Asia, and Africa. Currently, research on the pathogenic mechanism, host interaction, and gene function of *Fusarium circinatum* is progressing slowly due to the lack of efficient genetic transformation systems. Current research on the functional genes of this pathogen is still in its early stages, with slow progress at the molecular level, limiting a deeper understanding of the infection and pathogenic mechanisms of *Fusarium circinatum*.
[0003] Genetic transformation is a fundamental method and tool for studying the growth, development, and molecular mechanisms of pathogenic bacteria. Currently, various genetic transformation techniques have been applied in Phytophthora research, including PEG / CaCl2-mediated protoplast transformation, microparticle bombardment, Agrobacterium tumefaciens method, and electroporation. While microparticle bombardment produces high transformation efficiency, the resulting transformants are mostly heterokaryotic. Because silenced genes are easily replaced and restored by genes in neighboring nuclei that are not silenced, the gene silencing efficiency is very low. Furthermore, this method requires a high-value gene gun, making its widespread application difficult. While the *Agrobacterium tumefaciens* method can produce high transformation rates, it results in a high proportion of transformants containing heterokaryotic bodies, leading to low gene silencing efficiency. Electroporation transformation yields stable transformants with higher efficiency than PEG / CaCl2-mediated protoplast transformation, but the probability of gene silencing is low, and different research groups often obtain contradictory results. Among existing fungal genetic transformation methods, PEG / CaCl2-mediated protoplast transformation (PMT) is widely used in the study of filamentous fungi such as *Fusarium graminearum* and *Fusarium oxysporum* due to its simplicity, low cost, lack of expensive equipment, and efficient integration of exogenous genes. However, the complex cell wall composition of *P. pine* resin canker pathogens, the difficulty in protoplast preparation, and the lack of a mature transformation system restrict the study of its gene function and the elucidation of its pathogenic mechanisms. Establishing an efficient and stable genetic transformation system for pine resin canker pathogens will help to further study its biology, pathogenic molecular mechanisms, and explore potential drug molecular targets, providing a theoretical basis for the prevention and control of pine resin canker pathogens. Summary of the Invention
[0004] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for preparing protoplasts of *Pinus pine resin canker*. Another technical problem to be solved by the present invention is to provide applications of *Pinus pine resin canker* protoplasts for achieving stable and efficient genetic transformation of *Pinus pine resin canker* protoplasts.
[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0006] A method for preparing protoplasts of *Pinus pineus*, a pathogen causing resin canker, includes the following steps:
[0007] 1) Collecting mycelium: The pine resin canker pathogen Fusarium circinatum NAUA-1 strain was inoculated on PDA solid medium and cultured in the dark at 28℃ for 5 days. Mycelial blocks were then cut and transferred to PDB liquid medium and cultured with shaking at 28℃ for 24 hours. The spore liquid was obtained by filtration, centrifugation was performed, and the precipitate was transferred to YEPD liquid medium and cultured with shaking at 28℃ and 90 rpm for 12-16 hours. The mycelium was collected by filtration through a double-layer filter cloth.
[0008] 2) Preparation of protoplasts by enzymatic hydrolysis: Wash the hyphae with 1.2M KCl and dry them. Place the hyphae in a centrifuge tube containing the enzymatic hydrolysate and hydrolyze at 30℃ and 90rpm for 3.5h.
[0009] 3) Purification and resuspension: After enzymatic hydrolysis, the solution was filtered through two layers of filter cloth. The filtrate was centrifuged and the supernatant was discarded. The precipitate was resuspended in STC solution. After multiple centrifugations and washings, the protoplasts of pine resin canker bacteria were obtained.
[0010] Furthermore, the protoplasts of the pine resin canker pathogen are 1–2 × 10⁻⁶. 7 per mL.
[0011] Furthermore, the STC solution is formulated with 145.744g sorbitol, 6.057g Tris, and 5.5495g CaCl2, and ddH2O is added to bring the volume to 1L.
[0012] Furthermore, the enzymatic hydrolysate is prepared by dissolving 5 mg / mL of cell wall lysing enzyme, 12.5 mg / mL of cell wall breaking enzyme, and 7.5 mg / mL of snail enzyme in 20 mL of 1.2 M KCl and filtering through a 0.22 μm filter membrane.
[0013] Application of pine resin canker pathogen protoplasts in constructing a genetic transformation system for pine resin canker pathogen.
[0014] A method for establishing a genetic transformation system for pine resin canker pathogens, comprising:
[0015] 1) The GFP reporter gene was amplified by PCR and homologously recombinated with the PKD1 vector to construct the PKD1-GFP plasmid;
[0016] 2) Set the final concentration to 1-2 × 10⁻⁶. 7 Protoplasts per mL were mixed with 30 μg PKD1-GFP plasmid and 30 μL LSPTC solution. After standing at room temperature for 30 min, 1 mL SPTC solution was added and incubated at room temperature for 20 min. Then, TB3 liquid medium was added to bring the volume up. After adding kanamycin, the mixture was cultured for 3 h. The mixture was then mixed with TB3 solid medium containing HPH, poured into plates, covered with TB3 solid medium containing a higher concentration of HPH, and transformants were screened.
[0017] 3) Transformants were screened in PDA solid medium containing HPH to obtain the stably genetically transformed strain FcGFP-1.
[0018] Furthermore, the SPTC solution is formulated as follows: 40g PEG4000 is added to STC solution and the volume is adjusted to 100mL.
[0019] Further, in step 2), the HPH concentration in the HPH-containing TB3 solid culture medium is 100 μg / mL.
[0020] Furthermore, in step 2), the HPH concentration in the TB3 solid culture medium containing a higher concentration of HPH is 200 μg / mL.
[0021] Application of FcGFP-1, a transformed strain of *Pinus pineus*, in the control of pine resin canker disease.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] 1) This invention, by optimizing enzymatic hydrolysis conditions (using a specific ratio of lysing enzyme, cell-breaking enzyme, and snail enzyme, and an enzymatic hydrolysis time of 3.5 hours, etc.), can obtain high concentrations (1-2 × 10⁻⁶). 7 The protoplasts are of high quality (number per mL), and their release and regeneration processes are excellent, laying a solid foundation for subsequent genetic transformation. The procedure is simple and low-cost: no expensive equipment is required; it can be completed through routine shaking culture, centrifugation, and filtration, making it suitable for a wide range of applications.
[0024] 2) This invention utilizes a PEG / CaCl2-mediated protoplast transformation method to successfully transform the PKD1-GFP plasmid into the protoplasts of *Pinus pineus*, the causal agent of pine resin canker. Through screening, a stably genetically transformed strain, FcGFP-1, was obtained. PCR identification and fluorescence observation showed that the GFP gene was stably expressed in the hyphae and conidia of the transformed strain, indicating that this transformation system can efficiently achieve stable integration and expression of exogenous genes. This method avoids the problems of high heterokaryotic ratios and low gene silencing efficiency in transformants obtained by particle bombardment and *Agrobacterium tumefaciens* methods. The transformed strains are stable, providing reliable materials for gene function studies.
[0025] 3) This invention measured the growth rate, aerial hyphae formation, pigment deposition, conidia morphology and yield, and growth under different stress conditions of the transformed strain. The results showed no significant difference between the transformed strain and the wild-type strain. Pathogenicity assays showed no statistically significant difference in lesion size between the transformed strain and the wild-type strain after inoculation of pine seedlings, indicating that the introduction of the GFP gene into the protoplasts had no effect on pathogenicity, ensuring the usability of the transformed strain in subsequent studies. The stably GFP-expressing transformed strain can be used to observe the growth, reproduction, and infection process of the pathogen in the host plant in real time, which helps to further study the pathogenic molecular mechanism of pine resin canker pathogen and its interaction with the host, providing a theoretical basis for exploring potential drug molecular targets and disease control. Attached Figure Description
[0026] Figure 1 Morphological observation diagrams of the protoplast release and regeneration process (A: purified protoplast; B: protoplast germination (2h); C: protoplast germination (4h); D: protoplast regeneration forming hyphae).
[0027] Figure 2 Figure 1 shows the preparation results of pine resin canker protoplasts under different conditions (A shows the preparation results of pine resin canker protoplasts under different enzyme combination concentrations; B shows the preparation results of pine resin canker protoplasts under different enzymatic hydrolysis times).
[0028] Figure 3 The image shows the results of HPH resistance testing for NAUA-1, the pine resin canker bacterium.
[0029] Figure 4 Diagrams showing the identification of green fluorescent protein (GFP) transformants of *Pinus pineus* (A: identification of the GFP gene in the transformants; B: identification of the hyg gene in the transformants; M: 2000 Marker; G: PKD1-GFP vector; W: NAUA-1 wild-type strain; CK: control strain; 1–31: obtained FcG FP 1–31 transformants).
[0030] Figure 5 Fluorescence observation images of hyphae, conidiophores, and conidia (microspores) of the transformant of pine resin canker pathogen;
[0031] Figure 6 The biological phenotypic diagrams of the transformants of *Pinus pineus* are shown below (A: Colony morphology of wild-type NAUA-1 and transformant FcGFP-1 strains after 5 days of growth on PDA medium; B: Colony diameter statistics from 1 to 5 days; C: Dry weight statistics of wild-type NAUA-1 and transformant FcGFP-1 strains; D: Sporulation statistics of wild-type NAUA-1 and transformant FcGFP-1 strains from 2 to 12 hours; EH: Growth statistics of wild-type NAUA-1 and transformant FcGFP-1 strains under various stresses).
[0032] Figure 7 Figure 1 shows the pathogenicity of wild-type (WT) pine resin canker fungus strain NAUA-1 and transformed strain FcGFP-1 on 2-week-old and 1-month-old pine seedlings (AB represents the disease results of 2-week-old pine seedlings; C represents the disease results of 1-month-old pine seedlings).
[0033] Figure 8 The following is a statistical chart showing the size of lesions on 2-week-old and 1-month-old pine seedlings of the wild-type (WT) strain NAUA-1 and the transformed strain FcGFP-1 of pine resin canker pathogen (A is the average lesion length of 2-week-old pine seedlings; B is the average lesion length of 1-month-old pine seedlings). Detailed Implementation
[0034] The present invention will be further described below with reference to specific embodiments, but these embodiments do not limit the form of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of the present invention. Experimental methods and reagents not specified in the embodiments are performed according to conventional conditions in the art.
[0035] PDB liquid culture medium: 200g peeled yellow-fleshed potatoes, 20g anhydrous glucose, add ddH2O to make up to 1L.
[0036] PDA solid culture medium: 200g peeled yellow-fleshed potatoes, 20g anhydrous glucose, 15g agar powder, add ddH2O to make up to 1L.
[0037] TB3 medium: 200g sucrose, 3g yeast extract, 3g casamino acids, 7.5g agar powder, add ddH2O to bring the volume to 1L.
[0038] TB3 solid medium: 200g sucrose, 3g yeast extract, 3g casamino acids, 7.5g agar powder, add ddH2O to bring the volume to 1L.
[0039] TB3 liquid culture medium: 200g sucrose, 3g yeast extract, 3g casamino acids, add ddH2O to bring the volume to 1L.
[0040] 1.2M KCl solution: 89.4g KCl, add ddH2O to make up to 1L.
[0041] STC solution: 145.744g sorbitol, 6.057g Tris, 5.5495g CaCl2, add ddH2O to bring the volume to 1L.
[0042] SPTC solution: 40g 4000PEG, add STC solution to make up to 100mL.
[0043] YEPD liquid medium: 10g yeast extract, 10g peptone, 20g anhydrous glucose, add ddH2O to bring the volume to 1L.
[0044] Enzymatic hydrolysate: 5 mg / mL cell wall lysin, 12.5 mg / mL cell wall breaking enzyme, and 7.5 mg / mL snail enzyme, dissolved in 20 mL of 1.2 M KCl and filtered through a 0.22 μm filter membrane.
[0045] The pine resin canker pathogen Fusarium circinatum NAUA-1 used in this application is disclosed in Chinese patent application No. 2006100400353 and originates from NJAU.
[0046] Example 1: Preparation of protoplasts
[0047] 1. Collect mycelium
[0048] Filter paper discs of the preserved pine resin canker pathogen NAUA-1 strain were affixed to PDA solid medium and incubated in the dark at 28℃ for 5 days. Ten mycelial fragments, approximately 2mm × 2mm in size, were cut along the colony edge using a sterile scalpel and transferred to PDB liquid medium. After shaking and incubating for 24 hours at 28℃, the fragments were filtered through three layers of clean paper to obtain the original spore solution. 2 mL of the spore solution was transferred to a centrifuge tube, centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded. The precipitate was transferred to YEPD liquid medium and mixed by shaking. The mixture was then incubated at 28℃ and 90 rpm for 12–16 hours to obtain a large number of young spore mycelial balls, which were collected by filtration through a double-layer filter cloth.
[0049] 2. Preparation of protoplasts
[0050] The obtained mycelia were washed with 5 mL of 1.2 M KCl. The washed mycelia were then blotted dry with absorbent paper. The dried mycelia were then placed in a 50 mL centrifuge tube containing 20 mL of enzymatic hydrolysis solution (5 mg / mL lysozyme, 12.5 mg / mL cell wall-breaking enzyme, and 7.5 mg / mL snailase dissolved in 20 mL of 1.2 M KCl, filtered through a 0.22 μm filter membrane). The centrifuge tube was placed horizontally and incubated at 30 °C and 90 rpm for 3.5 h with shaking. After hydrolysis, the protoplasts were collected by filtering through two layers of filter cloth. The filtrate was transferred to a new 50 mL centrifuge tube and centrifuged at 3500 rpm for 5 min. The supernatant was discarded after centrifugation. 2 mL of STC was added to the precipitate and the protoplasts were gently resuspended by pipetting. The resuspended protoplasts were transferred to two 1.5 mL centrifuge tubes and centrifuged at 4 °C and 3500 rpm for 5 min. The supernatant was discarded after centrifugation. 1 mL of ST was added... C. Gently resuspend the protoplasts and centrifuge at 1500 rpm for 4 min at 4 °C; collect the protoplasts, discard the supernatant, add an appropriate amount of STC, and count using a hemocytometer. Randomly aspirate three samples for further analysis to achieve a final protoplast concentration of 1–2 × 10⁻⁶. 7 Protoplast release during the enzymatic hydrolysis of pine resin canker bacteria was observed using an optical microscope. Protoplasts were added to TB3 regeneration medium and incubated at 28°C for 12 hours. Samples were taken at regular intervals, and the protoplast regeneration process was observed under an optical microscope.
[0051] The results are as follows Figure 1 As shown, the protoplasts produced by the pine resin canker bacterium NAUA-1 after release and regeneration are of high quality.
[0052] Example 2: Preparation of protoplasts of pine resin canker pathogens under different conditions
[0053] To further establish a better system, enzymatic hydrolysis experiments were conducted using lysozyme, cell-wall-breaking enzyme, and snail enzyme. Four groups of enzymatic hydrolysates with different enzyme ratios were set up (Group A: 5 mg / mL lysozyme, 12.5 mg / mL cell-wall-breaking enzyme, 7.5 mg / mL snail enzyme; Group B: 12.5 mg / mL cell-wall-breaking enzyme, 10 mg / mL lysozyme; Group C: 7.5 mg / mL cell-wall-breaking enzyme, 20 mg / mL lysozyme; Group D: 7.5 mg / mL cell-wall-breaking enzyme, 10 mg / mL lysozyme; all enzymes were dissolved in 20 mL of 1.2 M KCl). Six time points were set for enzymatic hydrolysis (1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h) to find the optimal enzymatic hydrolysis conditions.
[0054] The results are as follows Figure 2 As shown, the highest protoplast yield was observed under treatment group A. Using the enzyme system of group A, extending the enzyme solution time resulted in a continuous increase in the number of protoplasts, reaching 1.82 × 10⁻⁶ at 3 hours.7 cells / mL ( Figure 2 (A). There was no statistically significant difference in the number of protoplasts released at enzymatic hydrolysis times of 3h, 4h, and 5h. Therefore, to obtain the optimal speed and efficiency, the enzymatic hydrolysis experiment used the A group enzymatic hydrolysis system, which was shaken at 80rpm for 3h at 30℃. Figure 2 (B).
[0055] Example 3: Sensitivity of NAUA-1, the causal agent of pine resin canker, to HPH and G418.
[0056] After inoculating *Pinus pineus* NAUA-1 onto PDA solid medium and culturing for 5 days, several mycelial blocks were collected along the edge of the colony using a 5 mm diameter punch. These mycelial blocks were then inoculated onto PDA medium containing different concentrations of HPH using sterile toothpicks. The HPH concentration gradients used in the experiment were 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 μg / mL. The medium was incubated at 28℃ for 5 days, and colony growth and size were observed and measured.
[0057] The results are as follows Figure 3 As shown, *Pinus pineus* NAUA-1, after being cultured for 5 days in the dark at 28°C, exhibited normal hyphal growth phenotype in PDA solid medium without HPH. However, in selective media containing different concentrations of HPH, the degree of inhibition of hyphal growth increased with increasing drug concentration. When the HPH concentration reached 80 μg / mL, hyphal growth completely ceased, indicating that this concentration was the minimum effective concentration (MIC) for completely inhibiting the growth of strain NAUA-1. Based on this, in the subsequent optimization of the genetic transformation system, the HPH concentration of the initial screening medium was set at 100 μg / mL (higher than the MIC to ensure strict screening), while a HPH concentration of 200 μg / mL was used for plate screening to further exclude false positive transformants.
[0058] Example 4: Protoplast Transformation and Transformant Screening
[0059] 1. Constructing the PKD1-GFP plasmid
[0060] PCR amplification of the GFP reporter gene was performed using a 50 μL reaction mixture: 1 μL template DNA, 2 μL each of forward and reverse primers, 25 μL high-fidelity enzyme, and ddH2O to a final volume of 50 μL. Reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for 33 cycles; and a final extension at 72℃ for 10 min. The product was subjected to electrophoresis, and the target gene fragment was recovered using a gel extraction kit.
[0061] Take 3 μL of the purified GFP gene fragment, 1 μL of PKD1 vector, and 5 μL of homologous recombinase and add them to a PCR tube, then add ddH2O to a final volume of 10 μL. Incubate the mixture at 50 °C for 30 min.
[0062] Remove competent *E. coli* cells (DH5α) from a -80°C freezer and thaw naturally on ice. After thawing, transfer 5 μL of yeast plasmid to the competent *E. coli* cells and mix gently. Incubate the mixture on ice for 25 min. Immediately after the ice bath, transfer the mixture to a 42°C metal bath for 45 s heat shock, then incubate on ice again for 2 min. Add 700 μL of antibiotic-free LB broth to the mixture, mix gently, and then incubate at 37°C and 200 rpm for 1 h. Remove the bacterial culture, centrifuge at 5000 rpm for 5 min, discard the supernatant, leaving approximately 100 μL of liquid, mix gently, and transfer the bacterial culture to an LB agar plate containing Amp. Spread the bacterial culture evenly with a spreader and incubate overnight at 37°C. Select positive single colonies (with Amp resistance) for sequencing; if the GFP gene sequence is correct, the PKD1-GFP plasmid has been successfully constructed. Plasmids of the corresponding E. coli positive transformants were extracted for further transformation experiments.
[0063] 2. Protoplast transformation
[0064] Dispense the solution into 15 mL sterile centrifuge tubes to a final concentration of 1–2 × 10⁻⁶. 7 Protoplasts were added at a density of 100 μL / mL to each tube. Then, 30 μg of PKD1-GFP plasmid and 30 μL of SPTC solution were mixed and incubated at room temperature for 30 min. Another 1 mL of SPTC solution was added, and the mixture was incubated at room temperature for 20 min. TB3 liquid medium was added to a final volume of 10 mL, followed by 10 μL of kanamycin solution (50 mg / mL). The mixture was incubated at 90 rpm and 28 °C for 3 h. The liquid was transferred to a new 50 mL centrifuge tube, and 40 mL of TB3 solid medium containing 100 mg / mL HPH was added. The mixture was mixed and poured into a petri dish. After the plate solidified, a layer of 10 mL of TB3 solid medium containing 200 mg / mL HPH was added, and the plate was incubated in the dark at 28 °C for 4–6 days until transformants appeared.
[0065] 3. Transformant screening
[0066] Transformants were picked and placed in PDA solid medium containing 100 mg / mL HPH, and cultured in the dark at 28°C for three generations. Transformants with stable traits were screened; these were the protoplast-transformed pine resin canker pathogens. Genomic DNA was extracted from the transformants using a kit, and a pair of specific primers was designed based on the sequence of the introduced green fluorescent protein gene.
[0067] GFP-F: 5'-GACGACGGCAACTACAAG-3'
[0068] GFP-R: 5'-GAACTCCAGCAGGACCAT-3'
[0069] HPH-F: 5'-GGAGGTCAACACATCAATG-3'
[0070] HPH-R: 5'-CTCTATTCCTTTGCCCTCG-3'
[0071] The extracted DNA was used as a template for PCR amplification, and the PCR products were detected by 1% agarose gel electrophoresis.
[0072] The PCR reaction system consisted of: 1 μL gDNA template, 10 μL 2×Rapid Taq Master Mix, 1 μL each of forward and reverse primers, and ddH2O added to a final volume of 25 μL.
[0073] The PCR reaction program was as follows: 94℃ for 5 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, for 33 cycles; 72℃ for 10 min.
[0074] The results are as follows Figure 4 As shown, wild-type *Phytophthora indicum* without GFP transformation and blank vector were used as negative controls. Negative controls could not amplify the target band. PKD1-GFP vector, as a positive control, and other transformants could amplify GFP bands of approximately 500 bp, consistent with the expected fragment size. The transformation was successful, and the positive strain was named FcGFP-1.
[0075] 4. Fluorescence observation
[0076] Positive strains were examined under a fluorescence microscope (Carl Zeiss Microscopy, LLC, White Plains, NY, USA) to observe GFP expression in different trophic structures such as hyphae and medullary conidia.
[0077] The results are as follows Figure 5 As shown, under a fluorescence microscope, the hyphae and conidia of the transformants exhibited strong green fluorescence, indicating that GFP protein was stably expressed in FcGFP-1 hyphae and conidia. Strains that normally expressed green fluorescent protein were transferred to PDA solid medium containing the screening agent HPH and stored at -20°C using filter paper.
[0078] Example 5 Functional determination of transformed strains
[0079] 1. Growth Measurement
[0080] Transformants were inoculated onto new PDA solid plates and cultured at 28°C for 3 days.
[0081] The results are as follows Figure 6 As shown, compared with the wild type, strain FaGFP-1 showed no significant differences in growth rate, aerial hyphae formation, or pigment deposition. Figure 6 (A and B). Furthermore, after culturing in PDA liquid medium for 24 hours, there was no significant difference in dry weight between the two strains (A and B). Figure 6 (C).
[0082] 2. Conidia determination
[0083] After culturing in MBM liquid medium for 2h, 4h, 6h, 8h and 12h, 10μL of wild-type strain and FaGFP-1 conidial suspension were collected for analysis.
[0084] The results are as follows Figure 6 As shown in D, compared with the wild type, the conidia of the transformed strain FcGFP-1 had normal morphology, and there was no significant difference in spore yield at each time point.
[0085] 3. Stress Measurement
[0086] The results are as follows Figure 6 As shown in the EH, the vegetative growth of GFP transformants was not significantly different from that of wild-type strains on plates containing different concentrations of NaCl, CongoRed, H2O2, SDS, and Sorbitol.
[0087] 4. Pathogenicity test
[0088] Conidial suspension inoculation was used, with wild-type strain NAUA-1 as a control. Conidial suspensions of the transformed strain and the wild-type strain were inoculated onto the stems of 2-week-old pine seedlings, respectively. After five days of inoculation in the dark at 28°C, the length of lesions was observed and measured, and statistical analysis was performed.
[0089] The results are as follows Figure 7 As shown, all inoculated slash pine seedlings exhibited obvious disease symptoms, with rot and shriveling at the inoculation site. There was no statistically significant difference in the average lesion size between the wild-type strain and the transformed strain FcGFP-1, indicating that the pathogenicity of the transformed strain FcGFP-1 was consistent with that of the wild-type.
[0090] Further, one-month-old slash pine seedlings were selected, and approximately 0.5 cm of the top portion of the seedling was removed using a sterile scalpel. 10 μL of a 10% concentration was then extracted. 5Inoculate the seedling apex with conidial solution at a concentration of / mL. After culturing for 20 days at 28℃ under alternating light and dark conditions (12 hours per day), observe the seedling disease status. The seedlings showed obvious disease, withering downwards from the inoculation point at the apex; the length of the diseased area was measured with a ruler.
[0091] The results are as follows Figure 8 As shown, there was no statistically significant difference in the average pathogenesis length between the wild-type strain and the transformed strain FcGFP-1, indicating that the introduction of the GFP gene into the protoplast had no effect on pathogenicity.
[0092] The embodiments described above are merely illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A method for preparing protoplasts of pine resin canker pathogens, characterized in that, Includes the following steps: 1) Collecting mycelium: The pine resin canker pathogen Fusarium circinatum NAUA-1 strain was inoculated on PDA solid medium and cultured in the dark at 28℃ for 5 days. Mycelial blocks were then cut and transferred to PDB liquid medium and cultured with shaking at 28℃ for 24 hours. The spore liquid was obtained by filtration, centrifugation was performed, and the precipitate was transferred to YEPD liquid medium and cultured with shaking at 28℃ and 90 rpm for 12-16 hours. The mycelium was collected by filtration through a double-layer filter cloth. 2) Preparation of protoplasts by enzymatic hydrolysis: Wash the hyphae with 1.2M KCl and dry them. Place the hyphae in a centrifuge tube containing the enzymatic hydrolysate and hydrolyze at 30℃ and 90rpm for 3.5h. 3) Purification and resuspension: After enzymatic hydrolysis, the solution was filtered through two layers of filter cloth. The filtrate was centrifuged and the supernatant was discarded. The precipitate was resuspended in STC solution. After multiple centrifugations and washings, the protoplasts of pine resin canker bacteria were obtained.
2. The method for establishing the genetic transformation system of pine resin canker pathogen according to claim 1, characterized in that, The protoplasts of the pine resin canker pathogen are 1–2 × 10⁻⁶. 7 per mL.
3. The method for establishing the genetic transformation system of pine resin canker pathogens according to claim 1, characterized in that, The STC solution was formulated with 145.744g sorbitol, 6.057g Tris, and 5.5495g CaCl2, and ddH2O was added to bring the volume to 1L.
4. The method for establishing the genetic transformation system of pine resin canker pathogens according to claim 1, characterized in that, The enzymatic hydrolysate was prepared by dissolving 5 mg / mL of cell wall lysing enzyme, 12.5 mg / mL of cell wall breaking enzyme, and 7.5 mg / mL of snail enzyme in 20 mL of 1.2 M KCl and filtering through a 0.22 μm filter membrane.
5. The application of the protoplasts described in claim 1 in constructing a genetic transformation system for pine resin canker pathogens.
6. A method for establishing a genetic transformation system for pine resin canker pathogens, characterized in that, include: 1) The GFP reporter gene was amplified by PCR and homologously recombinated with the PKD1 vector to construct the PKD1-GFP plasmid; 2) Set the final concentration to 1-2 × 10⁻⁶. 7 Protoplasts per mL were mixed with 30 μg PKD1-GFP plasmid and 30 μL LSPTC solution. After standing at room temperature for 30 min, 1 mL SPTC solution was added and incubated at room temperature for 20 min. Then, TB3 liquid medium was added to bring the volume up. After adding kanamycin, the mixture was cultured for 3 h. The mixture was then mixed with TB3 solid medium containing HPH, poured into plates, covered with TB3 solid medium containing a higher concentration of HPH, and transformants were screened. 3) Transformants were screened in PDA solid medium containing HPH to obtain the stably genetically transformed strain FcGFP-1.
7. The method for establishing according to claim 5, characterized in that, The SPTC solution was formulated by adding 40g of PEG4000 to STC solution and bringing the volume to 100mL.
8. The method for establishing according to claim 5, characterized in that, In step 2), the HPH concentration in the TB3 solid culture medium containing HPH is 100 μg / mL.
9. The method for establishing according to claim 5, characterized in that, Step 2) The HPH concentration in the TB3 solid culture medium containing a higher concentration of HPH is 200 μg / mL.
10. Application of FcGFP-1, a transformed strain of *Pinus pineus*, in the prevention and control of pine resin canker disease.
Citation Information
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