Genetic transformation method of arthrochytrium limacinum

Abstract

The invention discloses an agrobacterium tumefaciens-mediated genetic transformation method of Archeustochytrium cidebbufense, which comprises the following steps: firstly, inducing and collecting zoospore of Archeustochytrium cidebbufense; secondly, preparing agrobacterium tumefaciens containing a hygromycin resistance gene and a target gene plasmid; then, activating and inducing agrobacterium tumefaciens; creating a groove area on an induction culture medium plate, and co-culturing the zoospore and the induced agrobacterium tumefaciens in a groove; and finally, screening the Arteustochytrium limacinum transformant on a selective culture medium containing 25 mg / L of hygromycin, 200 mg / L of tiaculosporin and 300 mg / L of ampicillin. According to the invention, the efficient genetic transformation method of the Arteustochytrids cidebbuki is established for the first time, the method has the advantages of high transformation rate, stable inheritance and the like, an important technical means is provided for deeply researching the pathogenesis of the Arteustochytrids cidebbuki, and meanwhile, a foundation is laid for developing a new strategy for preventing and controlling microalgae diseases.

Description

Technical Field

[0001] The invention belongs to the fields of mycology and microalgae biotechnology, and particularly relates to a genetic transformation method of an economic microalgae pathogenic fungus, Paraphysoderma sedebokerense. Background Art

[0002] Paraphysoderma sedebokerense, a fungal species belonging to the phylum Blastocladiomycota, is a zoosporic fungus located at the base of the fungal phylogenetic tree and holds important scientific research value for revealing the origin and evolution of fungi. Furthermore, as a microalgae parasitic fungus, Paraphysoderma sedebokerense can infect a variety of economically important microalgae, including Haematococcus pluvialis, Chlorella zofingiensis, and Scenedesmus dimorphus, posing a serious threat to the development of the microalgae industry. In recent years, Paraphysoderma sedebokerense infections have frequently occurred in large-scale cultivation of Haematococcus pluvialis worldwide, resulting in a significant decrease in the yield and quality of astaxanthin produced by Haematococcus pluvialis, and causing enormous economic losses. However, the pathogenic mechanism of A. sidbürychnitzschiae is poorly understood, particularly its mechanism of specific infection of host algal cells, which remains incompletely elucidated. Consequently, safe and effective disease control technologies have yet to be established in practical production. Furthermore, the lack of effective genetic manipulation tools, particularly mature genetic transformation systems, has hindered functional research on key pathogenic genes in this fungus, presenting a major technical bottleneck in uncovering its pathogenic mechanisms. Therefore, establishing an efficient and stable genetic transformation system for A. sidbürychnitzschiae is of great theoretical and practical significance.

[0003] To address the above issues, the present invention successfully constructed a genetic transformation system for Arthrochytrid sibiricum using zoospores of Arthrochytrid sibiricum as the receptor material for the first time, utilizing an expression vector carrying hygromycin resistance and a fluorescent marker, and through Agrobacterium tumefaciens-mediated genetic transformation. The establishment of this method provides an important technical platform for in-depth research on the key pathogenic genes and their functions of this fungus. It not only helps to elucidate the biological characteristics and pathogenic mechanisms of Arthrochytrid sibiricum, but also provides a theoretical basis and technical support for the development of efficient and safe microalgae disease prevention and control strategies. At the same time, this method also provides an important technical reference and reference for the establishment of genetic transformation systems for other zoospore-forming lower fungi. Summary of the Invention

[0004] The object of the present invention is to provide a method for constructing genetic transformation of Arthrochytrid sidbukeii, which is simple to operate and genetically stable. The present invention uses zoospores of Arthrochytrid sidbukeii as receptor materials and uses Agrobacterium tumefaciens containing a fluorescent marker and hygromycin resistance on an expression vector to carry out genetic transformation research, thereby constructing a stable genetic transformation method for Arthrochytrid sidbukeii, facilitating subsequent research on the pathogenic mechanism of Arthrochytrid sidbukeii, and also providing a new solution for other zoospore-type lower fungi that are difficult to transform.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides an Agrobacterium-mediated genetic transformation method using Paraphysoderma sedebokerense zoospores as receptor materials, comprising the following steps:

[0007] 1) Induce and collect zoospores of Arthrochytrid sidbukoides on BGM plates;

[0008] 2) preparing Agrobacterium tumefaciens containing a hygromycin resistance gene and a target gene plasmid;

[0009] 3) Activation and induction of Agrobacterium tumefaciens;

[0010] 4) creating a groove area on the co-cultivation induction medium plate, and co-culturing the recombinant Agrobacterium tumefaciens and the zoospores of Arthrochytrid sidbukoides in the groove;

[0011] 5) The co-cultured mixed bacterial liquid was cultured on BGM medium containing resistance selection markers, and transformants of the chytrid fungus were screened and positive transformants were obtained by identification. The resistance selection markers were 25 mg / L hygromycin, 200 mg / L cefotaxime, and 300 mg / L ampicillin.

[0012] Furthermore, the step of inducing and collecting zoospores of the chytrid fungus sibiricum in step 1) is: treating the chytrid fungus culture plate at a low temperature of 4°C, and then using a salt solution to induce and promote the release of zoospores in the sporangium; the components of the BGM culture medium are shown in Table 1, and the components of the salt solution are shown in Table 5.

[0013] Furthermore, in step 3), the induction culture medium of Agrobacterium tumefaciens contains 200 μM acetosyringone, 40 mM morpholineethanesulfonic acid and 10 mM glucose, preferably Agrobacterium tumefaciens EHA105.

[0014] Furthermore, in step 4), the co-cultivation induction medium contains 200 μM acetosyringone, 40 mM morpholineethanesulfonic acid and 5 mM glucose; the volume ratios of the co-cultivated Agrobacterium tumefaciens solution, the zoospore suspension of the chytrid fungus syringae and the induction medium are 1:1:2, 1:2:1, 2:1:1 and 1:1:0, and the concentration of the zoospore suspension is 2-3×10 7 Transformants can be screened only when at least one ratio of the two cultures shows growth after co-cultivation. The preferred co-cultivation condition is 24°C for 1-3 days.

[0015] The present invention provides a construction method for genetic transformation of Arthrochytrid siddbeckii with simple operation and genetic stability. The method uses low temperature and salt solution to induce to obtain a suspension of Arthrochytrid siddbeckii zoospores, then Agrobacterium tumefaciens containing a hygromycin marker gene and a target gene plasmid is mixed with the Arthrochytrid siddbeckii zoospores in proportion, and then co-cultivated on a co-cultivation induction culture medium plate with prefabricated grooves. Transformants are obtained through resistance screening, thereby overcoming the problem of difficulty in genetic transformation of zoospore-type lower fungi.

[0016] The present invention successfully transformed the pCAMBgfp plasmid into the chytrid fungus Arthrochytrium sieberii and screened a series of integrated hygromycin resistance genes. PCR identification results showed that all tested transformants were able to amplify fragments of the expected size, indicating that the hygromycin resistance gene fragment had been successfully inserted into the genome of Arthrochytrium sieberii. The present invention used 2-3×10 7 Using zoospores of Arthrochytrid sidbukii of 1,000 cells / mL as the material, 83 positive transformants were screened through Agrobacterium tumefaciens-mediated infection, and they had high genetic stability.

[0017] The present invention also successfully introduced the exogenous green fluorescent protein encoding gene into the chytrid fungus sibiricum, and the transformant showed obvious green fluorescence under excitation light, indicating that the exogenous GFP gene has been transferred into the chytrid fungus sibiricum genome and expressed. It can be used for subsequent real-time, in situ, dynamic process analysis of the infection of chytrid fungus sibiricum cells into Haematococcus pluvialis, and is also an important tool for studying algae-fungus interactions.

[0018] The present invention has the following advantages and positive effects:

[0019] (1) Currently, there are relatively few studies on the genetic transformation of zoosporic lower fungi, and there is only one successful case of electroporation transformation of Batrachochytrium dendrobatidis. However, the electroporation transformation method has some limitations and defects, such as the instability of the fluorescent signal, which disappears after about 6 hours. To date, there have been no reports of the successful transformation of other zoosporic lower fungi using this method. Therefore, the present invention establishes a genetic transformation method for the Sidbuk-type chytrid fungus, filling the gap in the genetic transformation of microalgae disease fungi, and also provides strong technical support for the establishment of genetic transformation systems for other zoosporic lower fungi.

[0020] (2) The method of the present invention utilizes BGM culture medium and can prepare a large amount of zoospore suspension of the chytrid fungus of the class Buk by low temperature and salt solution induction method, meeting the needs of constructing genetic transformation operations.

[0021] (3) Traditionally, the transformation of fungi is usually carried out by co-cultivation using sterile cellophane. However, we found that the traditional cellophane method is not suitable for the chytrid fungus. The method of the present invention uses the zoospores of the chytrid fungus as the receptor material, and uses Agrobacterium tumefaciens to mediate and co-cultivate on a Co-IM plate with prefabricated grooves. Transformants containing the hygromycin resistance gene and the GFP reporter gene are successfully constructed, achieving efficient genetic transformation of the chytrid fungus. This method not only has the advantages of simple operation, high transformation rate and stable inheritance, but also lays the foundation for subsequent exploration of the pathogenic mechanism of the chytrid fungus. This is the first report of a genetic transformation method for the chytrid fungus in China and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A map of plasmid pCAMBgfp is shown.

[0023] Figure 2 Micrograph of zoospores of Arthrochytrid sidbukoides collected for induction.

[0024] Figure 3 The results show the inhibitory effect of different concentrations of hygromycin on the growth of Buk-type chytrid fungus.

[0025] Figure 4 Images of the pre-made grooves on the Co-IM plate and the Co-IM plate after co-culture. a: Grooves on the Co-IM plate; b: Opaque areas appear on the Co-IM plate after 2 days of co-culture.

[0026] Figure 5 The effect of co-cultivation time on the transformation rate of B.

[0027] Figure 6 These are transformant colonies of the Sidbury's chytrid fungus.

[0028] Figure 7 The electrophoresis pattern of PCR amplification of the hygromycin resistance gene in transformants of Arthrochytrium siddenkoides. M: marker D2000, 1: pCAMBgfp plasmid, 2: sterile water blank control, 3: WT control, 4-9: transformants.

[0029] Figure 8 Fluorescence micrographs of wild-type strains and transformants of the German Arthrochytrid fungus. WT represents the German Arthrochytrid fungus wild-type strain EPG03, and T-17 represents the transformant.

[0030] Figure 9 In vivo fluorescence imaging of wild-type strains and transformant colonies of the German book-like chytrid fungus.

[0031] Figure 10 This test examines the genetic stability of transformants of the Arthrochytrium buxiformis strain EPG03. WT indicates wild-type Arthrochytrium buxiformis strain EPG03. S indicates subculture on plates containing selective antibiotics, N indicates subculture on plates without antibiotics, 06, 07, and 08 indicate transformant numbers, and 1×, 5×, 10×, 20×, and 40× indicate dilution multiples. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below through specific examples.

[0033] 1. Experimental Materials

[0034] Paraphysoderma sedebokerense EPG03 was isolated and purified from the Microalgae Biotechnology Laboratory of Wuhan Botanical Garden, Chinese Academy of Sciences, and used as the receptor material in subsequent experiments and cultured at 28°C.

[0035] Agrobacterium tumefaciens EHA105, carrying rifampicin resistance genes and kanamycin resistance genes, was kindly provided by Jodie Taylor, The Sainsbury Laboratory, Norwich, UK.

[0036] Escherichia coli DH5α was cultured statically at 37°C in LB solid medium or with shaking in liquid medium, and stored at -80°C in LB liquid medium containing 20% glycerol.

[0037] The binary plasmid pCAMBgfp (containing the hygromycin resistance gene and the GFP gene) Figure 1) was kindly donated by the Kiwifruit Germplasm Resources and Breeding Group of Wuhan Botanical Garden, Chinese Academy of Sciences. Construction method: gfp and hph fragments were ligated with the pCAMBIA1300 vector double-digested with XhoⅠ and EcoRⅠ, and then transformed into Agrobacterium EHA105.

[0038] 2. Key culture medium

[0039] Table 1 BGM (Blastoclads Growth Medium) components

[0040]

[0041] Table 2 Induction medium (IM) components

[0042]

[0043] Table 3 Co-culture induction medium (Co-IM) components

[0044]

[0045] Table 4 2.5×Minimal Salts Stock Solution Components

[0046]

[0047] Table 5 Dilute Salts Solution (DS, 1×) Components

[0048]

[0049] Example 1 Induction and Collection of Zoospores of Arthrochytrid spp.

[0050] A method for repeatedly inducing and collecting zoospores of Arthrochytrid-like fungi, comprising the following steps:

[0051] (1) Plate selection: Select plates with good growth of the chytrid fungus within 10 days after inoculation;

[0052] (2) Low temperature induction treatment: The selected A. sidbukeii plates were placed in a 4°C refrigerator for 30 minutes to promote the production of zoospores in the sporangium;

[0053] (3) Salt solution induction treatment: After taking the plate out of the refrigerator, gently rinse the surface of the above-mentioned Sidbuk-like chytrid plate with 1× diluted salt solution (DS) or induction medium (IM) solution to promote the release of zoospores in the sporangium. Repeat at least 3 times to ensure that all zoospores are thoroughly collected;

[0054] (4) Filtration and collection: Filter the collected cell suspension using Whatman filter paper with a pore size of 8 μm to obtain pure and viable zoospores of the chytrid fungus. Transfer the collected cell suspension to a 50 mL centrifuge tube.

[0055] (5) Microscopic examination: The zoospores of the induced and collected parachytrid fungus were examined under a microscope to observe the zoospore morphology ( Figure 2 ) and calculate the quantity for future use.

[0056] Example 2 Establishment of a Genetic Transformation Method of Arthrochytrid sibiricum Mediated by Agrobacterium tumefaciens

[0057] 1. Sensitivity testing of B. sylvestris to different antibiotics

[0058] To ensure the smooth progress of the selection culture, it is necessary to determine in advance the type and concentration of antibiotics suitable for the German chytrid fungus, so as to establish a stable resistance screening marker. The screening method is as follows: First, three antibiotics widely used in fungi were selected, including hygromycin, blasticidin, and zeocin. Then, a concentration gradient was set up to test the effective concentration of the three antibiotics to inhibit the German chytrid fungus. The antibiotics were added to the BGM culture medium, and antibiotic plates with different concentrations were prepared to test the growth inhibition effect of the German chytrid fungus on solid culture medium. The cell growth, colony formation, and zoospore release of the German chytrid fungus were evaluated using an optical microscope. In order to verify the long-term inhibitory effect of the antibiotics on the solid culture medium, we conducted a 14-day observation to ensure that no new colonies were formed, thereby confirming the effectiveness of the antibiotics.

[0059] As shown in Table 6, all three antibiotics tested inhibited the growth and colony formation of Achytrium sidbukoides on solid culture medium. The minimum effective inhibitory concentration (MIC) for hygromycin was 25 μg / mL, that for blasticidin was 100 μg / mL, and that for bleomycin was 200 μg / mL. Hygromycin exhibits excellent inhibitory effects even at low concentrations, making it the preferred choice for genetic screening in this invention.

[0060] Table 6 Effective concentrations of different antibiotics in inhibiting the growth of Parachytrid

[0061]

[0062] 0: indicates no colonies grow on the medium with the antibiotic concentration; +: indicates a few colonies grow;

[0063] ++: indicates the same performance as the no-antibody control.

[0064] 2. Sensitivity test of B. sylvestris to hygromycin

[0065] Hygromycin was selected as the selective antibiotic and its effective concentration was verified again. The screening steps were as follows: colonies were picked from a fresh plate of the chytrid fungus genus Sidbeckia spp., mixed with an appropriate amount of sterile water, and then filtered through a filter with a pore size of 40 μm. The cell concentration was controlled at 1-3×10 7 After 100 μg / mL of culture medium, inoculate onto BGM plates containing hygromycin at concentrations of 1, 15, 25, 35, 50, and 100 μg / mL, along with a control containing no antibiotic. Wrap the antibiotic-containing plates with tin foil to prevent light degradation. Incubate in a 28°C incubator and observe daily to determine the optimal hygromycin concentration. Inoculate 200 μL per plate, with three replicates per treatment.

[0066] like Figure 3 As shown, with the increase of hygromycin concentration, the colony growth time is delayed and the growth slows down. On the BGM plate without hygromycin, the chytrid fungus grows normally. Colony growth was observed on the plate containing 1μg / mL hygromycin and the control without antibiotics at the same time after 48h, which shows that 1μg / mL hygromycin has almost no inhibitory effect on the chytrid fungus. Colonies were observed on the plate containing 10μg / mL hygromycin on the 10th day after inoculation, and colonies were observed on the plate containing 15μg / mL hygromycin on the 12th day after inoculation, while no chytrid fungus colonies grew on the 25μg / mL hygromycin plate. Therefore, the present invention uses 25μg / mL as the minimum screening concentration of hygromycin to inhibit the chytrid fungus, that is, the appropriate concentration for screening positive transformants.

[0067] 3. Sensitivity testing of Agrobacterium tumefaciens to different antibiotics

[0068] To successfully screen transformants, we needed to inhibit the growth of Agrobacterium tumefaciens without inhibiting the growth of Arthrochytrid sidbukoids. We tried the following five antibiotics: carbernicillin, tetracycline, ampicillin, cefotaxime, and ceftriaxone sodium. First, a single colony of activated Agrobacterium tumefaciens EHA105 harboring the binary vector pCAMBgfp was selected and inoculated into LB liquid medium (containing 50 μg / mL Kan) and cultured with shaking at 225 rpm. Set up plates containing different concentrations of ampicillin (100, 200, 300 μg / mL), cefotaxime (200, 300 μg / mL), tetracycline (50, 100 μg / mL), and ceftriaxone (100, 200, 300 μg / mL). 600 When the value was 0.5, plates were evenly coated with 200 μL of bacterial solution per plate and incubated at 28°C. The growth of Agrobacterium tumefaciens was recorded and observed, and the lowest concentration at which Agrobacterium did not grow was used for transformation. Each treatment was repeated three times. Next, a mixed culture of Agrobacterium tumefaciens and Achytrium sidbukei containing the plasmid pCAMBgfp, harvested from the co-culture, was inoculated onto BGM solid medium containing the antibiotics cefotaxime, tetracycline, carbenicillin, ampicillin, ceftriaxone sodium, or a combination thereof. The concentration that inhibited the growth of Agrobacterium while not inhibiting the growth of Achytrium sidbukei was used for transformation screening. Each treatment was repeated three times.

[0069] As shown in Table 7, in experiments treating Agrobacterium tumefaciens alone, certain concentrations of cefotaxime, tetracycline, carbenicillin, ampicillin, and ceftriaxone sodium were all able to inhibit the growth of Agrobacterium tumefaciens. As shown in Table 8, in experiments treating a mixed culture of Agrobacterium tumefaciens and Achytrium sidobium, only the antibiotic combination of 300 μg / mL ampicillin and 200 μg / mL cefotaxime performed well, completely preventing Agrobacterium tumefaciens colonies from growing and not inhibiting the growth of Achytrium sidobium, thus meeting the screening requirements. Therefore, the present invention uses 300 μg / mL ampicillin and 200 μg / mL cefotaxime as the minimum screening concentration for inhibiting the growth of Agrobacterium tumefaciens, i.e., the appropriate concentration for screening positive transformants.

[0070] Table 7 Effective concentrations of different antibiotics in inhibiting the growth of Agrobacterium

[0071]

[0072] 0: indicates that no colonies grow on the culture medium with this concentration of antibiotics; + indicates that a few colonies grow.

[0073] Table 8 Effective concentrations of different antibiotic combinations to inhibit Agrobacterium growth

[0074]

[0075] 0: No colonies grow on the medium with the antibiotic concentration within 5 days; +: A few colonies can be observed growing within 5 days; ++: Clear colonies can be observed within 3 days

[0076] 4. Preparation of Agrobacterium tumefaciens containing antibiotic marker gene and target gene plasmid

[0077] Chemical transformation of the plasmid pCAMBgfp into Agrobacterium tumefaciens EHA105 was performed. Chemical transformation experiments should be started at least four days before the planned transformation date to ensure sufficient time to obtain viable single colonies for subsequent experiments. Colonies should be transferred to liquid culture for pre-cultivation the night before transformation. First, centrifuge the pCAMBgfp plasmid powder at 10,000-12,000 rpm for 2-3 minutes. Then, add 40 μL of sterile water and dissolve at room temperature for 1 minute. Remove competent EHA105 from the -80°C freezer and thaw on ice. Add 1.5 μL of plasmid and 50 μL of EHA105 bacterial suspension to a sterile 1.5 mL microcentrifuge tube and gently swirl to mix. Place the microcentrifuge tube on ice, then in a liquid nitrogen-37°C water bath, and then on ice for 5 minutes in each condition. Then, add 300-600 μL of antibiotic-free LB liquid culture medium to the centrifuge tube, and evenly spread the Agrobacterium tumefaciens cell solution on an LB culture dish containing 50 μg / mL kanamycin and 25 μg / mL rifampicin. Seal and invert the culture dish and place it in a constant temperature incubator at 28°C for culture.

[0078] After approximately 48 hours of incubation, single colonies should be observed. Subsequently, the positively transformed Agrobacterium should be streaked again onto LB medium plates containing kanamycin. After incubation at 28°C for 48 hours, new monoclonal Agrobacterium tumefaciens colonies should be obtained. Select a single colony and extract the plasmid using the TIANGEN Plasmid Miniprep Kit (DP106-02) and verify by PCR. This confirms that pCAMBgfp has been successfully transformed into Agrobacterium tumefaciens and can be used for subsequent transformation experiments.

[0079] 5. Create a grooved area on the induction culture plate

[0080] In the process of Agrobacterium-mediated genetic transformation, the choice of co-cultivation method is crucial. Traditionally, fungal transformation is usually carried out using sterile cellophane co-cultivation. However, we found that the traditional cellophane method is not suitable for the chytrid fungus genus Sidbury. Experiments have shown that after co-cultivation using cellophane, sporangia need to be recovered from the cellophane. This process is cumbersome, has poor reproducibility, reduces the sporangium yield, and no transformed colonies are observed on the selective culture medium. Therefore, we tried the groove co-cultivation method, in which Agrobacterium and zoospores are mixed and directly inoculated into the grooves of the solid culture medium for co-cultivation. The groove design helps to maintain the local concentration of cells, thereby significantly improving the transformation efficiency. The successful application of this method not only improves the transformation efficiency of the chytrid fungus genus Sidbury, but also provides a new solution for other difficult-to-transform zoospore-forming lower fungi.

[0081] First, draw a cross on the bottom of the petri dish to divide the circular petri dish into four quadrants. Then draw a circle with a diameter of approximately 2.5 cm in each quadrant, and make sure that the circles do not overlap. Dip the bottom of a heatable glass test tube into anhydrous ethanol solution about 2.5 cm high, heat it over the flame of an alcohol lamp to burn the ethanol solution, and then let the glass test tube cool naturally in a sterile area. Repeat this step 3-5 times to ensure that the bottom of the test tube is sterile. On a sterile operating table, gently press the warm (but not enough to melt the agar) sterile glass test tube into the Co-IM plate in the center of a circle drawn previously, keep the test tube vertical and rotate slowly until a groove roughly the same size as the drawn circle is formed ( Figure 4 a) Ensure that the four grooves on the culture plate do not overlap and can accommodate approximately 200 μL of liquid. Follow the above steps to create grooves on each desired Co-IM plate.

[0082] Example 3 Agrobacterium tumefaciens-mediated genetic transformation of Arthrochytrid sibiricum

[0083] 1. Preparation of Zoospore Suspension of Arthrochytrid sidbuk

[0084] In the entire genetic transformation process, the state and quantity of Arthrochytrid zoospores are very important. Since Agrobacterium tumefaciens needs to infect Arthrochytrid zoospores to complete the purpose of introducing foreign genes, and the sporangium has a thicker cell wall, which makes it impossible for Agrobacterium tumefaciens to enter, in order to improve the efficiency of genetic transformation, it is necessary to culture more synchronous Arthrochytrid zoospores, and at least two generations of Arthrochytrid zoospores need to be cultured on BGM medium without antibiotics to ensure that there are no antibiotic residues in the culture on the day of genetic transformation. Four days before transformation, the method of induction and collection of Arthrochytrid zoospores in Example 1 was used to obtain pure and active Arthrochytrid zoospores, which were then re-spread on BGM plates and cultured at 28°C for 4 days. On the day of transformation, the above steps were repeated to ensure that as many zoospores as possible were obtained, and the concentration of the spore solution was adjusted to 2-3×10 7 pieces / mL.

[0085] 2. Activation and Induction of Agrobacterium tumefaciens

[0086] (1) On the eve of genetic transformation, a single colony of Agrobacterium tumefaciens EHA105 containing the pCAMBgfp plasmid was inoculated into LB liquid medium and cultured overnight at 28°C with a shaking speed of 225 rpm;

[0087] (2) At 8:00 am on the day of transformation, the concentration of Agrobacterium tumefaciens liquid was measured. 600 After centrifuging the Agrobacterium tumefaciens solution at 4500 rpm for 5 minutes, dilute it to OD 600 =0.15-0.20, 28°C, 225rpm shaking culture. After about 8 hours, OD 600 Agrobacterium tumefaciens bacterial suspension with a value between 0.45 and 0.5 was used for subsequent co-cultivation experiments.

[0088] 3. Co-culture on Co-IM Plates with Pre-made Grooves

[0089] In order to evaluate the effects of co-cultivation time and temperature on the transformation efficiency of zoospores of Buk-like chytrids, we conducted an experimental study. The results showed that under the condition of 19°C, only one transformant colony was obtained in three replicates of co-cultivation for two days, and the transformation efficiency was extremely low, so this temperature was no longer considered in subsequent experiments. In contrast, under the condition of 24°C, the number of transformants increased significantly. After the mixture was applied to the selective medium for 12 days, transformant colonies were observed on the selective medium for 1 day and 2 days of co-cultivation. We used the number of colonies of Buk-like chytrids EPG03 on the control BGM plate and the selective medium plate as the standard to calculate the transformation efficiency of different co-cultivation days at 24°C ( Figure 5The results showed that the number of transformants was low when the co-cultivation time was one day, but increased after two days. However, no transformants appeared on the selective medium plates after three days of co-cultivation, suggesting that excessive co-cultivation time may be detrimental to the transformation process. Therefore, the optimal co-cultivation conditions for Agrobacterium tumefaciens and Achytrium sidbücherii zoospores are 24°C for two days, achieving the highest transformation efficiency and making them suitable for subsequent experimental procedures.

[0090] (1) Prepare and label four 1.5 mL sterile centrifuge tubes. Add Agrobacterium tumefaciens (OD 600 =0.45-0.5) and zoospore suspension of Buk-like chytrid fungus (2-3×10 7 Transfer the bacterial suspension in the microcentrifuge tube to the same microcentrifuge tube. Use a pipette to gently pipette to mix the bacterial suspension in the microcentrifuge tube.

[0091] Table 9 Different ratios of the four quadrants in IM culture medium

[0092]

[0093] (2) Carefully transfer all 200 μL of the co-culture solution to the pre-made grooves on the room temperature Co-IM plate. All four co-culture tubes of the same plasmid can be placed on one Co-IM plate, one tube per quadrant, starting from the left and moving to the right to reduce the risk of contamination.

[0094] (3) Gently slide the Co-IM plate to a location where it will not be disturbed. Avoid picking up the plate to prevent mixing of the co-culture solution, disrupting the established ratio, and potentially reducing transformation efficiency or success rate.

[0095] (4) Allow the Co-IM plate to air dry at room temperature for 6-12 hours. Seal the plate with parafilm, invert it, and place it in a sealed container. Incubate at 24°C in the dark for 2 days.

[0096] (5) Observe growth. After the co-culture is completed, the opaque, approximately circular area formed on the surface of the Co-IM plate is used as a sign of growth. The experimental results show that the mixing of different proportions of donor and recipient bacteria significantly affects the transformation efficiency. If not all quadrants show signs of growth, it is still normal, but only if at least one quadrant shows signs of growth can the transformants be screened. At this time, the success rate of obtaining screenable transformants may be reduced. When using the plasmid pCAMBgfp for transformation experiments, opaque areas can be observed in the grooves of the four quadrants of the Co-IM plate ( Figure 4 b), indicating that the co-culture process was normal.

[0097] 3. Screening of Transformants

[0098] (1) After the co-cultivation is completed, selective culture screening can be performed to obtain transformants of the chytrid fungus. To select these transformants, cells need to be collected from the grooves of the Co-IM plate. First, prepare a 15 mL centrifuge tube containing 5 mL of IM medium, take out 1 mL of IM medium and evenly distribute it into the grooves of the corresponding Co-IM plate. After incubation at room temperature for 5 minutes, use a sterile cell scraper to gently scrape the surface of the groove and collect the growth at the bottom of the centrifuge tube containing IM medium. Rotate the plate and scrape along the surface of the groove until most of the opaque areas on the plate are removed. Use a pipette tip to scrape any remaining growth from the cell scraper and resuspend it in a 15 mL centrifuge tube containing IM medium. Pipette all the liquid scraped from the plate and add it to the centrifuge tube.

[0099] (2) Oscillate the centrifuge tube for 1-2 seconds using an oscillator. Centrifuge the tube at 2000 × g for 10 minutes at room temperature. Slowly pour off the supernatant with the high side of the pellet facing upward to minimize pellet loss.

[0100] (3) Slowly resuspend the precipitate in 500 μL fresh IM medium, and take 200 μL of the resuspended chytrids from the genus Siebeckii and distribute it on a BGM agar plate containing selective antibiotics. The selection medium is divided into three groups: the Agrobacterium tumefaciens control group, the chytrids from the genus Siebeckii control group, and the experimental group for screening transformants. These three culture media are all BGM culture media, and the types of antibiotics contained in them are different. The culture medium of the Agrobacterium tumefaciens control group contains 25 mg / L hygromycin to inhibit the growth of chytrids from the genus Siebeckii; the culture medium of the chytrids from the genus Siebeckii control group contains 200 mg / L thiophanate-methyl and 300 mg / L ampicillin to inhibit the growth of Agrobacterium tumefaciens; the screening experimental group contains 25 mg / L hygromycin, 200 mg / L thiophanate-methyl and 300 mg / L ampicillin to ensure that only successfully transformed chytrids from the genus Siebeckii transformants can grow colonies.

[0101] (4) Spread the mixed bacterial solution evenly on the selective culture medium, seal and invert the culture dish, and culture it in a constant temperature incubator at 28°C.

[0102] (5) Select successfully transformed B. sidbukei colonies. Gently pick up the colonies from the agar with a sterile 18G needle and resuspend each colony in 50 μl of diluted saline solution (DS). Gently break up the colonies with a pipette and then inoculate the colony suspension onto selective BGM medium. Continue to subculture to obtain enough culture to continue subsequent experiments. If the transformation is successful, B. sidbukei colonies with hygromycin resistance should appear after 9-11 days of growth on the selective medium. These colonies should be rough, white and opaque ( Figure 6 ).

[0103] Example 4. Identification and detection of transformants of the chytrid fungus

[0104] 1. PCR Identification of Transformed Strains

[0105] Genomic DNA from transformants and wild-type Arthrochytrid siddensis EPG03 was extracted using the Solarbio Fungal Genomic DNA Extraction Kit (Cat#D2300). Primers HygF (5′-ATGCCTGAACTCACCGCGAC-3′) and HygR (5′-CTATTCCTTTGCCCTCGGAC-3′) were designed using the hygromycin resistance gene fragment HygB as a template. PCR was used to verify the insertion of the T-DNA fragment, with a target fragment length of 1000 bp. The reaction system consisted of 10 μL of Green Taq Mix, 1 μL of each 10 μmol / L upstream and downstream primers, 2 μL of template, and sterile distilled water to a final volume of 20 μL. The amplification reaction was performed as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 1 min, and 72°C for 5 min. After the reaction, the reaction was detected by 1.2% agarose gel electrophoresis, and then the gel was placed in a gel imager to observe whether a single bright band was generated.

[0106] The results are as follows Figure 7 As shown, the pCAMBgfp plasmid and the transformants of the chytrid fungus sibiricum amplified the target band of about 1000 bp, while the wild-type strain EPG03 and the negative control (sterile water) did not amplify the target band, indicating that the hygromycin resistance gene in the positive transformants had been inserted into the genome of the chytrid fungus sibiricum.

[0107] 2. Observation of Fluorescent Protein GFP

[0108] Pick a small amount of cells from the positive transformants and place them on a glass slide, and then place them under a Leica inverted fluorescence microscope DMi8 for fluorescence detection. Figure 8 Under excitation light, the wild-type EPG03 did not produce fluorescence, while the transformant could be observed to have obvious green fluorescence, indicating that the exogenous GFP gene had been transferred into the genome of EPG03 and was expressed.

[0109] At the colony level, the selection culture plate was directly observed using a live fluorescence imager, with the wild-type EPG03 plate of the chytrid fungus Sieb. kohlii as a control. Obvious fluorescence (GFP fluorescence) was observed under an excitation light wavelength of 488 nm and an emission light wavelength of 510 nm. Figure 9 ).

[0110] 3. Genetic Stability Testing of Transformed Strains

[0111] To evaluate the genetic stability of the transformants, we performed a 24-day passage experiment in selective BGM medium (containing 25 mg / L hygromycin) and non-selective BGM medium (without antibiotics). Subsequently, the growth of the transformants under different conditions and their response to antibiotic selection pressure were tested ( Figure 10 The results showed that after no less than 16 generations of continuous culture on non-selective culture plates, the transformants were able to grow normally when transferred to antibiotic plates containing hygromycin, demonstrating stable hygromycin resistance. In addition, when the transformants were cultured continuously for multiple generations on selective culture plates containing antibiotics and then transferred to antibiotic plates, they continued to grow normally. These results indicate that the transformants selected by the transformation method adopted by the present invention have high genetic stability and can maintain stable expression of exogenous genes even in the absence of continuous selection pressure.

[0112] Obviously, the above embodiments are merely examples for clear description and are not intended to limit the implementation methods.

Claims

1. A West German Buk-type chytrid fungus ( Paraphysoderma sedebokerense ) is an Agrobacterium-mediated genetic transformation method, characterized in that The following steps are involved: 1) Induce and collect zoospores of Arthrochytrid sidbukoides on BGM plates; 2) Prepare Agrobacterium tumefaciens containing the hygromycin resistance gene and the target gene plasmid; 3) Activation and induction of Agrobacterium tumefaciens; 4) creating a groove area on the co-culture induction medium plate, and co-culturing the recombinant Agrobacterium tumefaciens and the zoospores of Arthrochytrid sidbukoides in the groove; 5) The co-cultured mixed bacterial liquid was cultured on BGM medium containing resistance selection markers of 25 mg / L hygromycin, 200 mg / L cefotaxime, and 300 mg / L ampicillin to screen for transformants of the chytrid fungus, and positive transformants were identified.

2. The genetic transformation method according to claim 1, characterized in that The components of the BGM culture medium are shown in Table 1.

3. The genetic transformation method according to claim 1, wherein The induced collection in step 1) includes treating the culture plate of the chytrid fungus genus Siederbuk at a low temperature of 4°C, and then inducing it with a salt solution.

4. The genetic transformation method according to claim 3, characterized in that The composition of the salt solution is shown in Table 5.

5. The genetic transformation method according to claim 1, characterized in that The Agrobacterium is Agrobacterium tumefaciens EHA105.

6. The genetic transformation method according to claim 1, characterized in that The induction medium for Agrobacterium tumefaciens in step 3) contains 200 μM acetosyringone, 40 mM morpholineethanesulfonic acid, and 10 mM glucose.

7. The genetic transformation method according to claim 1, characterized in that The co-culture induction medium in step 4) contained 200 μM acetosyringone, 40 mM morpholineethanesulfonic acid, and 5 mM glucose.

8. The genetic transformation method according to claim 1, characterized in that In step 4), the volume ratios of the co-cultured Agrobacterium tumefaciens solution, the Arthrochytrid syringae zoospore suspension, and the induction medium are 1:1:2, 1:2:1, 2:1:1, and 1:1:0, and the concentration of the zoospore suspension is 2-3×10 7 Between 100 and 100 mL.

9. The genetic transformation method according to claim 1, characterized in that The co-culture condition in step 4) is 24° C. for 1-3 days.

Images