Preparation method of Ilyonectria robus fungus protoplast and construction method of genetic transformation system

Abstract

The invention relates to the field of microbial genetic transformation, and discloses a preparation method of Ilyonectria robus fungus protoplast and a construction method of a genetic transformation system.The preparation method comprises the following steps that S11, I.robus hyphae are prepared, after I.robus is cultured on a PDA solid culture medium for 14 days, the I.robus hyphae are inoculated to a CMC liquid culture medium to be subjected to shake-flask culture, and spores are filtered and collected; diluting the spore concentration, inoculating into a YEPD liquid culture medium, carrying out shake-flask culture, filtering and collecting hyphae; and S12, preparing enzymatic hydrolysate, namely adding the hyphae prepared in the step S11 into the enzymatic hydrolysate prepared in the step S12, carrying out shake flask enzymolysis, filtering, centrifuging filtrate, discarding supernate, adding a 0.7 M NaCl solution, centrifuging, and discarding the supernate, so as to obtain the I.robus fungus protoplast. The protoplasts with proper quantity and good quality can be efficiently obtained, and the transformation efficiency of a subsequent genetic transformation system is improved; and the constructed genetic transformation system can be used for construction of a mutant library of the I.robusta strain, interaction of a host plant and the I.robusta strain and research on the pathogenesis of the I.robusta strain.

Description

Technical Field

[0001] The invention relates to the technical field of microbial genetic transformation, and in particular to a method for preparing Ilyonectria robusta fungal protoplasts and a method for constructing a genetic transformation system. Background Art

[0002] Ilyonectria fungi are soil-dwelling fungi with a wide host range, and most species are plant pathogens that can cause plant root, stem, and leaf diseases. I.robusta has a wide host range and can parasitize on a variety of plants, including crops, ornamental plants, and medicinal plants. This fungus is one of the main pathogens that causes root rot in plants such as ginseng, American ginseng, and Coptis chinensis, as well as apple replant disease.

[0003] At present, there are few reports on the infection mode and pathogenic mechanism of Ilyonectria fungi. Therefore, the establishment of an efficient and stable PEG-mediated I. robusta protoplast genetic transformation system can be used to construct a mutant library of I. robusta strains, laying the foundation for subsequent research on the pathogenic genes of the fungus, and helping to conduct in-depth research on the infection and pathogenic mechanisms, providing new clues for the interaction mechanism with the host and biological control research.

[0004] Based on this, the present invention proposes a method for preparing protoplasts of Ilyonectria robusta fungus and a method for constructing a genetic transformation system. Summary of the invention

[0005] The invention provides a method for preparing Ilyonectria robusta fungal protoplasts and a method for constructing a genetic transformation system. The method for preparing I. robusta fungal protoplasts can be used to prepare I. robusta fungal protoplasts with a suitable quantity and capable of improving the transformation efficiency of a subsequent genetic transformation system.

[0006] In order to achieve the above object, the present invention provides a method for preparing protoplasts of Ilyonectria robusta fungus, comprising the following steps:

[0007] S11. Preparation of I. robusta mycelium

[0008] After culturing I. robusta on PDA solid medium for 14 days, the strain was inoculated into CMC liquid medium for shake flask culture for 4 days, filtered, and the filtrate was collected. The filtrate was centrifuged at 10000 rpm for 10 minutes to collect spores; the spore concentration was diluted and inoculated into YEPD liquid medium for shake flask culture for 2 days, and the hyphae were collected by filtration;

[0009] S12. Prepare enzymatic solution

[0010] The enzymatic solution is prepared by dissolving 1-3 wt% of the lysozyme and 1-2 wt% of the wall-dissolving enzyme in a 0.7 M NaCl solution, filtering and sterilizing for later use;

[0011] S13. Preparation of I. robusta protoplasts

[0012] The mycelium prepared in step S11 was added to the enzymolysis solution prepared in step S12 for enzymolysis in a shake flask, filtered, and the filtrate was collected. The filtrate was centrifuged at 4°C, the supernatant was discarded, and 0.7 M NaCl solution was added to continue centrifugation, and the supernatant was discarded to obtain I. robusta protoplasts.

[0013] Preferably, in step S11, the shake flask culture is carried out in a shaker at 20°C and 200 rpm.

[0014] Preferably, in step S13, the mass volume ratio of the mycelium to the enzymatic hydrolysate is 0.1-0.2 g:10 mL.

[0015] Preferably, in step S13, the shake flask enzymolysis is performed in a shaker at a temperature of 30° C. and a rotation speed of 100 rpm;

[0016] Preferably, in step S13, the centrifugation condition is 5000 rpm for 30 min.

[0017] The present invention also provides application of the preparation method in constructing an I. robusta fungal genetic transformation system.

[0018] The present invention also provides a method for constructing a genetic transformation system of Ilyonectria robusta fungi, comprising the following steps:

[0019] S21, adding STC buffer to the I. robusta protoplasts prepared by the above preparation method to obtain a protoplast suspension;

[0020] S22, fully mixing the protoplast suspension prepared in step S21 and the plasmid containing hygromycin B resistance (hyg) and green fluorescent protein gene (GFP) markers, and placing in an ice bath for 20 to 30 minutes to obtain a first protoplast solution;

[0021] S23, adding PTC buffer to the first protoplast solution, and ice bathing for 20-30 min to obtain a second protoplast solution;

[0022] S24, add TB3 regeneration medium to the second protoplast solution, suspend the second protoplast solution in the regeneration medium and recover, incubate overnight at a speed of 100 rpm in a temperature shaking incubator at 20° C. to obtain a preliminary recovered protoplast solution;

[0023] S25, centrifuging the initially revived protoplast solution at 5000 rpm for 30 min, retaining 1-2 mL of the supernatant to resuspend the precipitate after centrifugation to obtain a protoplast resuscitation solution;

[0024] S26. The protoplast recovery solution was fully mixed with a regeneration medium containing 10 μg / mL hygromycin B, poured into a plate and cultured at 20° C. in the dark for 7 to 14 days to obtain I. robusta fungal transformants.

[0025] Preferably, in step S22, the concentration of I. robusta fungal protoplasts in the protoplast suspension is (4-6)×10 7 / mL; the plasmid is a fungal expression plasmid pCT74-hygr-GFP containing a ToxA promoter and carrying a hygromycin resistance gene (hyg) and a green fluorescent protein gene (GFP).

[0026] Preferably, the mass volume ratio of the plasmid and protoplast suspension is 10 μg:200 μL, the volume ratio of the protoplast suspension, PTC buffer solution and TB3 regeneration medium is 1:5:25, and the volume ratio of the protoplast recovery solution and TB3 regeneration solid medium containing 10 μg / mL hygromycin B is 1:25.

[0027] Preferably, the STC buffer is obtained by weighing 40 g of mannitol, 1.47 g of CaCl2·2H2O, 10 mL of 1M Tris-Cl, diluting to 200 mL with distilled water, and sterilizing under high pressure at 121°C; the PTC buffer is obtained by weighing 40 g of polyethylene glycol 4000 (PEG4000), dissolving it in 100 mL of STC buffer, and filtering and sterilizing.

[0028] In summary, the present invention has the following beneficial effects:

[0029] The present invention can efficiently prepare I.robusta fungal protoplasts of suitable quantity and good quality, and the concentration of the protoplasts can be (4-6)×10 710 mL of I. robusta fungal protoplasts with 1000 cells / mL were prepared; a complete genetic transformation of I. robusta fungus mediated by PEG-CaCl2 and a screening system with hygromycin as a resistance marker were established; 1000 positive transformants / mL DNA were obtained, and the transformation efficiency of the subsequent genetic transformation system could be improved; the genetic transformation system constructed by the present invention could be used for the construction of an I. robusta strain mutant library, the interaction between host plants and I. robusta, and the study of the pathogenic mechanism of I. robusta strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the amount of protoplasts obtained under different enzymolysis solutions in Example 1 (a is enzymolysis solution 1, b is enzymolysis solution 2, and c is enzymolysis solution 3);

[0031] Figure 2 The effect of different enzymolysis times on the number of protoplasts obtained in Example 1;

[0032] Figure 3 It is the influence of different STC buffers on protoplast morphology in Example 2 (a is STC Buffer1, and b is STCBuffer2);

[0033] Figure 4 is the resistance of I. robusta strain YJ56 to different hygromycin B concentrations in Example 2;

[0034] Figure 5 These are the observation results of the colonies of I. robusta that failed to transform and successfully transformed in Example 3 (a is the failed transformation of R-2, and b is the successful transformation of R-3);

[0035] Figure 6 The mycelium of the transformant successfully transformed in Example 3 was examined under a microscope;

[0036] Figure 7 This is the gel electrophoresis diagram of the wild type and transformant of I. robusta strain YJ56 in Example 3;

[0037] Figure 8 This is the result of comparing the pathogenicity of the wild type and transformant of I. robusta strain YJ56 in Example 4. DETAILED DESCRIPTION

[0038] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0039] The term "PDA solid medium" herein refers to potato dextrose agar, which is based on water as solvent, 20wt% potato juice, 2wt% glucose, 2wt% agar, natural pH, and autoclaved at 121°C for 20min.

[0040] The term "CMC liquid culture medium" herein refers to 1 g of NH4NO3, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 1 g of yeast extract, distilled water to 1 L, and autoclaved at 121°C for 20 min.

[0041] The term "YEPD liquid medium" herein refers to 3 g of yeast extract, 10 g of peptone, 20 g of glucose, distilled water to 1 L, and high-pressure sterilization at 121°C for 20 min.

[0042] The term "TB3 regeneration medium" herein refers to 3 g of yeast extract, 3 g of acid hydrolyzed casein, 200 g of mannitol, distilled water to 1 L, and autoclaved at 121°C for 20 min.

[0043] The term "TB3 regeneration medium containing 10 μg / mL hygromycin B" herein means that 10 μg / mL hygromycin B is added to the TB3 regeneration medium.

[0044] The term "0.7 M NaCl solution" herein refers to weighing 58.44 g of NaCl, making the volume to 1 L with distilled water, and sterilizing by high pressure at 121° C. for 20 min.

[0045] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0046] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0047] Embodiment 1:

[0048] A method for preparing protoplasts of Ilyonectria robusta fungus, comprising the following steps:

[0049] S11. Preparation of I. robusta mycelium

[0050] After culturing I. robusta strain YJ56 on PDA solid medium for 14 days, it was inoculated into CMC liquid medium and cultured in a shaker at 200 rpm at 20°C for 4 days. The filtrate was collected by filtering with a double-layer Miracloth filter cloth, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded to obtain conidia. The concentration of the obtained conidia was adjusted to 10 with sterile water. 7 / mL, and inoculated into YEPD liquid medium, cultured at 20°C in a shaker at 200 rpm for 2 days. Filter and collect the mycelium.

[0051] S12. Preparation of I. robusta protoplasts

[0052] Prepare enzymatic hydrolysis solution, add the mycelium prepared in step S11 to the enzymatic hydrolysis solution after filtration and sterilization, perform enzymatic hydrolysis at 100 rpm in a shaker at 30°C for 2 to 5 hours, filter, collect the filtrate, centrifuge the filtrate at 4°C and 5000 rpm for 30 minutes, and discard the supernatant.

[0053] The cells were resuspended in 0.7 M NaCl solution and centrifuged at 5000 rpm for 30 min. The supernatant was discarded to obtain I. robusta protoplasts, which were counted using a hemocytometer.

[0054] The commonly used enzymes for enzymolysis of fungal cell walls include lysing enzyme, driselase, chitinase, kitalase, cell wall lysing enzyme, snail enzyme + cellulase + lysozyme, etc. The type, concentration and enzymolysis time of suitable enzymes are the key factors leading to the success of protoplast preparation. Therefore, in this embodiment, the present invention investigates the influence of different enzymolysis solutions on the amount of protoplast acquisition, and the three enzymolysis solutions of configuration are as follows:

[0055] Enzyme solution 1: Dissolve 5 wt% of the enzyme in 0.7 M NaCl solution, filter and sterilize for later use;

[0056] Enzyme solution 2: Take 2.5wt% snail enzyme, 1wt% cellulase, and 1wt% lysozyme, dissolve in 0.7M NaCl solution, filter and sterilize for later use;

[0057] Enzyme hydrolysate 3: 2 wt % lysozyme (Sigma) and 1 wt % lysozyme (Sigma) were dissolved in 0.7 M NaCl solution, filtered and sterilized for later use.

[0058] Among them, lytic enzyme and lysozyme were purchased from Sigma.

[0059] Figure 1The results showed that only a small amount of I. robusta protoplasts were released from both enzymatic hydrolysates 1 and 2. Enzymatic hydrolysate 1 could obtain a concentration of 1.25×10 6 / mL protoplasts 500μL ( Figure 1 a), the concentration of enzymatic hydrolysate 2 was 2.67×10 7 / mL protoplasts 2mL ( Figure 1 b), while the concentration of enzymatic hydrolysate 3 was (4.36~5.83)×10 7 / mL protoplasts 10mL ( Figure 1 c), high yield of I. robusta fungal protoplasts can be obtained.

[0060] The optimal enzymatic hydrolysis solution 3 was used to further investigate the effect of different enzymatic hydrolysis times on the amount of protoplasts obtained. Figure 2 The results showed that the number of I. robusta fungal protoplasts obtained after 2 h of enzymatic hydrolysis was the largest, and as the enzymatic hydrolysis time increased, the number of I. robusta fungal protoplasts decreased.

[0061] In summary, the method for preparing Ilyonectria robusta fungal protoplasts provided by the present invention can prepare high-yield I. robusta fungal protoplasts by hydrolyzing the prepared I. robusta mycelium with an enzymatic hydrolysis solution for 2 h, and the obtained concentration is (4.36-5.83)×10 7 10mL of protoplasts with a concentration of cells / mL.

[0062] Embodiment 2:

[0063] A method for constructing a genetic transformation system of Ilyonectria robusta fungi comprises the following steps:

[0064] S21, adding STC buffer to the I. robusta fungal protoplasts prepared by using the optimal parameters of Example 1 to obtain a concentration of (4.36-5.83)×10 7 / mL of protoplast suspension;

[0065] S22, adding 1-30 μg of a plasmid containing an antibiotic marker gene (hyg) and a green fluorescent protein gene (GFP) to the 200 μL protoplast suspension prepared in step S21, mixing well, and placing in an ice bath for 20-30 min to obtain a first protoplast solution;

[0066] S23, adding 1 mL of PTC buffer solution to the first protoplast solution, and placing it in an ice bath for 20 to 30 min to obtain a second protoplast solution;

[0067] S24, adding 5 mL of TB3 regeneration medium to the second protoplast solution, suspending the second protoplast solution in the regeneration medium to recover, and culturing overnight at a rotation speed of 100 rpm in a shaking incubator at 20° C. to obtain a preliminarily recovered protoplast solution;

[0068] S25, centrifuging the initially revived protoplast solution at 5000 rpm for 30 min, retaining 1-2 mL of supernatant to resuspend the precipitate after centrifugation to obtain a protoplast resuscitation solution;

[0069] S26, the protoplast recovery solution was fully mixed with 25 mL of TB3 regeneration solid medium containing 10 μg / mL hygromycin B, poured into a plate, and cultured in the dark at 20° C. for 7 to 14 days to obtain I. robusta fungal transformants;

[0070] S27. Subculture for five consecutive generations on resistant PDA plates containing 10 μg / mL hygromycin B and observe the growth of transformants.

[0071] In this embodiment, the present invention adds 1, 10, 20, and 30 μg of plasmid containing antibiotic marker gene (hyg) and green fluorescent protein gene (GFP) marker to 200 μL protoplast suspension for genetic transformation. The protoplasts added with only 10 μg of plasmid are successfully transformed and the transformants are stably expressed.

[0072] In this embodiment, the present invention investigates the effects of different STC buffers on the protoplast morphology in a protoplast suspension, and the configurations of two different STC buffers are as follows:

[0073] STC Buffer 1: weigh 40 g sucrose, 1.47 g CaCl2·2H2O, 10 mL 1 M Tris-Cl, dilute to 200 mL with distilled water, and sterilize by autoclaving at 121°C;

[0074] STC Buffer 2: weigh 40 g mannitol, 1.47 g CaCl2·2H2O, and 10 mL 1 M Tris-Cl, dilute to 200 mL with distilled water, and sterilize by autoclaving at 121°C.

[0075] Figure 3 The results showed that when the protoplasts were resuspended in STC Buffer, the protoplasts resuspended in STC buffer1 were ruptured ( Figure 3 a), I. robusta protoplasts resuspended in STC buffer 2 have normal morphology ( Figure 3b), therefore, the present invention selects STC Buffer 2 for preparing the protoplast suspension.

[0076] In this example, the present invention also investigated the resistance of I. robusta (strain YJ56) to hygromycin B, and the specific operation was as follows:

[0077] After I. robusta (strain YJ56) was cultured on PDA solid medium for 14 days, it was inoculated on PDA solid medium with hygromycin B concentrations of 0, 5, 10, 25, 50, and 100 μg / mL, respectively, and cultured in the dark at 20°C for 14 days. Three replicates were set for each treatment. The results showed that I. robusta could not grow on PDA solid medium with hygromycin B concentrations of 5-100 μg / mL ( Figure 4 ), therefore, the present invention selects a plasmid containing hygromycin B resistance to prepare the first protoplast solution.

[0078] Further, acquisition of plasmid DNA: the pCT74-hygr-GFP plasmid (carrying hygromycin B resistance) used was purchased from Miaoling plasmid platform. The plasmid contains ToxA promoter, hygromycin resistance gene (hyg) and green fluorescent protein gene (GFP).

[0079] Polyethylene glycol (PEG) is a key factor in fungal genetic transformation. Commonly used PEGs include PEG3000, PEG3350, PEG4000, PEG6000 and PEG8000. The appropriate concentration of PTC buffer can improve the transformation efficiency of fungi. The present invention investigated the effect of 4 PTC buffers on the number of I.robusta transformants. The 4 PTC buffers are as follows:

[0080] PTC buffer 1: weigh 40 g PEG4000 and dissolve it in 100 mL STC buffer 1;

[0081] PTC buffer 2: weigh 40 g PEG8000 and dissolve it in 100 mL STC buffer 1;

[0082] PTC buffer3: weigh 40 g PEG4000 and dissolve it in 100 mL STC Buffer2;

[0083] PTC buffer4: Weigh 40 g PEG8000 and dissolve it in 100 mL STC Buffer2.

[0084] Table 1 Number of I. robusta transformants in different PTC buffers

[0085]

[0086]

[0087] The results in Table 1 show that only under the condition of PTC buffer 3, single colonies grew on the regeneration plate. The single colonies were numbered R-1 to R-11. On the resistance PDA plate of subculture, except R-2, the other 10 colonies grew normally. Under the conditions of the other three PTC buffers, no single colony grew on the regeneration plate. Therefore, PTC buffer 3 was selected for preparing the second protoplast solution.

[0088] In summary, the method for constructing an Ilyonectria robusta fungal genetic transformation system provided by the present invention can obtain I. robusta fungal transformants by selecting STC Buffer 2 for preparing a protoplast suspension, selecting a plasmid containing hygromycin B resistance to prepare a first protoplast solution, and using PTC buffer 3 to prepare a second protoplast solution.

[0089] Embodiment 3:

[0090] Identification of the I. robusta strain YJ56 transformants obtained in Example 2

[0091] 1. Microscopic examination

[0092] Under fluorescent irradiation, observe whether the I. robusta strain YJ56 transformants obtained in Example 2 exhibit green fluorescence. Successfully transformed transformants exhibit green fluorescence (R-3), while unsuccessfully transformed colonies have no green fluorescence (R-2) (see Figure 5 ). The mycelium of the successfully transformed transformants was picked for microscopic examination, and a Zeiss inverted fluorescence microscope was used to observe whether the transformants carried GFP. The results were as follows Figure 6 As shown;

[0093] Figure 6 The results showed that the mycelium of the transformant could produce GFP fluorescence, indicating that the pCT74-hygr-GFP plasmid fragment had been successfully transferred into the I. robusta strain YJ56 and expressed.

[0094] 2. PCR identification

[0095] The transformant DNA was extracted by CTAB method and verified by PCR using GFP primer pair, with the wild-type strain as negative control.

[0096] GFP-F: 5'-GATTGGAATGCATGGAGGAGT-3';

[0097] GFP-R: 5'-GCTTCTCGTTGGGGTCTTTG-3';

[0098] PCR reaction system: PCR SuperMix (+dye) 15 μL, forward primer 1 μL, reverse primer 1 μL, DNA template 1 μL, ddH2O to 30 μL.

[0099] PCR reaction conditions: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 45s (30 cycles); 72℃ extension for 7min; 4℃ storage; 1.0% agarose gel electrophoresis to detect PCR products, and use UV gel imaging system to observe and take pictures. The results are shown in the figure. Figure 7 shown.

[0100] Depend on Figure 7 It can be seen that the transformant band size is about 1000 bp (1-5 represent I. robusta transformants R-3, R-6, R-7, R-9 and R-10, respectively), while the original I. robusta strain YJ56 has no band.

[0101] Based on the above results, the GFP fragment was successfully inserted into the genome of I. robusta strain YJ56.

[0102] Embodiment 4:

[0103] Five successfully transformed I.robusta transformants (R-3, R-6, R-7, R-9 and R-10) were selected from the transformants of I.robusta strain YJ56 obtained in Example 2. American ginseng was used as the host plant. Through in vitro inoculation experiments, the roots were soaked in 75% ethanol and 1% sodium hypochlorite for 30 seconds and 5 minutes, respectively, and then washed with sterile water and placed in a culture dish. The roots of American ginseng were pierced with a dissecting needle and inoculated with 10 μL of spore suspension (10 7 Each root was punctured 3 times, and 5 ginseng roots were treated for each treatment. The disease was observed after culturing at 20℃ for 10 days. The pathogenicity of the transformants was compared to that of the wild type. The results were as follows: Figure 8 As shown, the transformed strains have no effect on pathogenicity and can be used for subsequent pathogenicity studies.

[0104] In summary, the present invention can prepare high-yield I. robusta fungal protoplasts, wherein the concentration can be (4.36-5.83)×10 7The number of I. robusta fungal protoplasts per mL reaches 10 mL; and the I. robusta fungal genetic transformation system constructed can successfully obtain I. robusta fungal transformants; and the obtained I. robusta transformed strains do not affect their pathogenicity and can be used for subsequent pathogenicity studies.

[0105] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing protoplasts of Ilyonectria robusta fungus, characterized in that: The steps include: S11. Preparation of I. robusta mycelium After culturing I. robusta on PDA solid medium for 14 days, the strain was inoculated into CMC liquid medium for shake flask culture for 4 days, filtered, and the filtrate was collected. The filtrate was centrifuged at 10000 rpm for 10 minutes to collect spores; the spore concentration was diluted and inoculated into YEPD liquid medium for shake flask culture for 2 days, and the hyphae were collected by filtration; S12. Prepare enzymatic solution The enzymatic solution is prepared by dissolving 1-3 wt% of the lysozyme and 1-2 wt% of the wall-dissolving enzyme in a 0.7 M NaCl solution, filtering and sterilizing for later use; S13. Preparation of I. robusta protoplasts The mycelium prepared in step S11 was added to the enzymolysis solution prepared in step S12 for enzymolysis in a shake flask, filtered, and the filtrate was collected. The filtrate was centrifuged at 4°C, the supernatant was discarded, and 0.7 M NaCl solution was added to continue centrifugation, and the supernatant was discarded to obtain I. robusta protoplasts.

2. The method for preparing protoplasts of Ilyonectria robusta fungus according to claim 1, characterized in that: In step S11, the shake flask culture is carried out in a shaker at 20°C and 200 rpm.

3. The method for preparing protoplasts of Ilyonectria robusta fungus according to claim 1, characterized in that: In step S13, the mass volume ratio of the mycelium to the enzymatic hydrolysate is 0.1-0.2 g:10 mL.

4. The method for preparing protoplasts of Ilyonectria robusta fungus according to claim 1, characterized in that: In step S13, the shake flask enzymolysis is carried out in a shaker at a temperature of 30° C. and a rotation speed of 100 rpm for 2 to 5 hours.

5. The method for preparing protoplasts of Ilyonectria robusta fungus according to claim 1, characterized in that: In step S13, the centrifugation condition is 5000 rpm for 30 min.

6. Use of the preparation method according to any one of claims 1 to 5 in constructing a genetic transformation system for I. robusta fungi.

7. A method for constructing a genetic transformation system of Ilyonectria robusta fungus, characterized in that: The steps include: S21, adding STC buffer to the I. robusta protoplasts prepared by the preparation method according to any one of claims 1 to 5 to obtain a protoplast suspension; S22, fully mixing the protoplast suspension prepared in step S21 and the plasmid containing hygromycin B resistance and GFP marker, and placing in an ice bath for 20 to 30 minutes to obtain a first protoplast solution; S23, adding PTC buffer to the first protoplast solution, and ice bathing for 20-30 min to obtain a second protoplast solution; S24, add TB3 regeneration medium to the second protoplast solution, suspend the second protoplast solution in the regeneration medium to recover, and culture overnight at 100 rpm in a temperature shaker at 20° C. to obtain a preliminary recovered protoplast solution; S25, centrifuging the initially revived protoplast solution at 5000 rpm for 30 min, reserving 1-2 mL of supernatant to resuspend the precipitate after centrifugation to obtain a protoplast resuscitation solution; S26. The protoplast recovery solution was fully mixed with TB3 regeneration medium containing 10 μg / mL hygromycin B, poured into a plate and cultured in the dark at 20° C. for 7 to 14 days to obtain I. robusta fungal transformants.

8. The method for constructing a genetic transformation system of Ilyonectria robusta fungus according to claim 6, characterized in that: In step S22, the concentration of I. robusta fungal protoplasts in the protoplast suspension is 4 to 6×10 7 / mL; the plasmid is a fungal expression plasmid pCT74-hygr-GFP containing a ToxA promoter and carrying a hygromycin resistance gene and a green fluorescent protein gene.

9. The method for constructing a genetic transformation system of Ilyonectria robusta fungus according to claim 6, characterized in that: The mass volume ratio of the plasmid and the protoplast suspension is 10 μg:200 μL, the volume ratio of the protoplast suspension, the PTC buffer solution and the TB3 regeneration medium is 1:5:25, and the volume ratio of the protoplast recovery solution and the TB3 regeneration solid medium containing 10 μg / mL hygromycin B is 1:

25.

10. The method for constructing a genetic transformation system of Ilyonectria robusta fungus according to claim 1, characterized in that: The STC buffer solution is obtained by weighing 40 g of mannitol, 1.47 g of CaCl2·2H2O, and 10 mL of 1M Tris-Cl, diluting the volume to 200 mL with distilled water, and sterilizing by high pressure at 121° C.; the PTC buffer solution is obtained by weighing 40 g of PEG4000, dissolving it in 100 mL of STC buffer solution, and filtering and sterilizing.

Images