Gene knockout system based on basidiomycetes and knockout method and application thereof

Abstract

The invention discloses a gene knockout system based on basidiomycetes and a knockout method and application thereof, and belongs to the field of gene engineering. According to the invention, a knockout vector is constructed based on a knockout empty vector pCHPH, the knockout vector is converted into agrobacterium and co-cultured with a basidiomycetes protoplast, and a gene knockout system and a knockout method aiming at the basidiomycetes Athelia bombacina are constructed. The PCR and qPCR verification experiment results show that the gene knockout system provided by the invention can efficiently and thoroughly knock out the Athelia bombacina gene, the knockout speed is high, and the positive transformant probability is high. When the knockout system provided by the invention is used for prevention or experimental research of Athelia bombacina, the requirements can be fully met, the experimental time is greatly shortened, and the knockout system has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a basidiomycete-based gene knockout system and a knockout method and application thereof. Background Art

[0002] In recent years, a new disease has been discovered during the fruit storage period. The pathogen is Athelia bombacina, which causes great economic losses during the storage period. The inventor's team previously conducted relevant research on the pathogenicity of A. bombacina and found that the LAC gene may be involved in the pathogenicity of the fungus. However, as a new pathogen, the research on its pathogenicity mechanism is still blank.

[0003] At present, gene knockout technology is one of the most commonly used methods to study gene function in fungi. By knocking out key pathogenic genes through homologous recombination, the function of a gene can be directly and effectively clarified, thereby clarifying its pathogenic process. However, a gene knockout system for A. bombacina has not yet been established. Summary of the invention

[0004] The purpose of the present invention is to provide a gene knockout system based on basidiomycetes and its knockout method and application, so as to solve the problems existing in the above-mentioned prior art. The gene knockout system provided by the present invention can be used to edit the Atheliabombacina gene, which can fully meet the needs of various experimental studies and greatly shorten the experimental time.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a gene knockout system based on basidiomycetes, comprising a knockout empty vector pCHPH, a complementing vector pCNEO, Agrobacterium EHA105 and a protoplast extraction reagent.

[0007] Preferably, the basidiomycete is Athelia bombacina.

[0008] The present invention provides a method for knocking out a basidiomycete gene, comprising the following steps:

[0009] Based on the gene knockout system, the target gene sequence is obtained, the restriction enzyme cutting sites of the knockout empty vector pCHPH are analyzed, a knockout vector is constructed, the Agrobacterium EHA105 is introduced, the Agrobacterium EHA105 containing the plasmid is collected, and the protoplasts of the basidiomycete are infected to achieve the knockout of the basidiomycete gene.

[0010] Preferably, the basidiomycete is Athelia bombacina; and the gene is Lac4 gene.

[0011] Preferably, the method comprises the following steps:

[0012] The upstream fragment of AbLac4 gene was amplified and named AbLac4-up, and the downstream fragment of AbLac4 gene was amplified and named AbLac4-dn;

[0013] The knockout empty vector pCHPH was digested with HindⅢ and connected to AbLac4-dn to obtain AbLac4-dn-pCHPH; the AbLac4-dn-pCHPH was digested with Kpn I to connect to AbLac4-up to obtain AbLac4-up-dn-pCHPH vector;

[0014] Preparation of protoplasts of Athelia bombacina;

[0015] The AbLac4-up-dn-pCHPH vector is transformed into the Agrobacterium EHA105 strain, and the Agrobacterium EHA105 cells containing the recombinant plasmid are co-cultured with the protoplasts of the Athelia bombacina to achieve the knockout of the Lac4 gene.

[0016] Preferably, the process of preparing the AbLac4-up-dn-pCHPH vector further includes the steps of bacterial liquid PCR verification and plasmid extraction and sequencing.

[0017] Preferably, the method further comprises the steps of PCR verification and qPCR verification.

[0018] The present invention also provides an application of the gene knockout system in preparing a product for editing the Athelia bombacina gene.

[0019] The present invention also provides an application of the gene knockout system in preventing and controlling plant diseases caused by Athelia bombacina.

[0020] The invention also provides a product for preventing and treating plant diseases caused by Athelia bombacina, comprising the gene knockout system.

[0021] The present invention discloses the following technical effects:

[0022] The present invention constructs a knockout vector based on the knockout empty vector pCHPH, and constructs a gene knockout system and a knockout method for the basidiomycete Athelia bombacina by transforming the knockout vector into Agrobacterium and co-culturing with basidiomycete protoplasts. PCR and qPCR validation experimental results show that the gene knockout system provided by the present invention can efficiently and thoroughly knock out the gene of Atheliabombacina, with a fast knockout speed and a high probability of positive transformants. The knockout system provided by the present invention is used for the prevention and control or experimental research of Atheliabombacina, which can fully meet the needs and greatly shorten the experimental time. The present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 It is a schematic diagram of the knockout principle of the present invention;

[0025] Figure 2 It is the electrophoresis diagram of linearized recombinant vector and lower arm amplification of target gene;

[0026] Figure 3 This is the electrophoresis diagram of the positive clones of the recombinant vector verified by PCR in the bacterial solution;

[0027] Figure 4 This is the result of sequencing comparison of recombinant vectors;

[0028] Figure 5 The electrophoresis diagram is the linearized knockout vector and the upper arm amplification of the target gene;

[0029] Figure 6 This is the electrophoresis diagram of the knockout vector positive clone verified by bacterial solution PCR;

[0030] Figure 7 This is the knockout vector sequencing comparison result diagram;

[0031] Figure 8 This is the electrophoresis diagram of the knockout vector sequence amplification results;

[0032] Fig. 9 This is a diagram showing the results of DNA and RNA verification of positive transformants;

[0033] Fig.10 Electropherograms for verification of AbLac1, AbLac2 and HYG fragments;

[0034] Fig.11 Statistical graph for qPCR experiment validation;

[0035] Fig.12 This is the electropherogram of PCR verification of Agrobacterium-mediated spore experiment. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] In the present invention, A. bombacina is used as the abbreviation of Athelia bombacina; dn is used as the abbreviation of down (downstream); it can be clearly judged that the above abbreviations in the present invention and the original terms have exactly the same meanings.

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Test materials

[0045] 1.1.1 Test reagents

[0046] Table 1 Test reagent information

[0047]

[0048]

[0049] 1.1.2 Knockout vector

[0050] The gene knockout empty vector pCHPH (containing hygromycin resistance) was kindly donated by Professor Bi Yang of the Postharvest Biology and Technology Laboratory of Gansu Agricultural University.

[0051] 1.2 Test methods

[0052] 1.2.1 Principles of gene knockout vector construction

[0053] In order to knock out the Lac4 gene, the present invention uses homologous recombination to construct a knockout vector using a gene knockout empty vector pCHPH. Figure 1 Based on the whole genome sequencing results, the target gene sequence containing 2000bp above and below was obtained by TBtools software, the restriction enzyme cutting sites of the vector were analyzed by DNAMAN software, the knockout vector was constructed by cloning technology, and then the protoplasts were infected with Agrobacterium to introduce the exogenous gene to achieve the knockout of the target gene.

[0054] The present invention firstly digests the gene knockout empty vector pCHPH with HindⅢ to connect the fragment to AbLac4-down to obtain AbLac4-dn-pCHPH; then digests it with KpnI to connect AbLac4-up to obtain AbLac4-up-dn-pCHPH recombinant knockout vector (the above connection results are all verified by bacterial liquid PCR and extracted plasmid sequencing).

[0055] Secondly, the knockout vector AbLac4-up-dn-pCHPH was transformed into the Agrobacterium EHA105 strain, and the Agrobacterium cells containing the recombinant plasmid were co-cultured with A. bombacina protoplasts. The hph gene containing the homologous arm was replaced with the Lac4 gene in A. bombacina by the homologous replacement method, thereby achieving the knockout of the Lac4 gene.

[0056] 1.2.2 Extraction of A. bombacina genomic DNA

[0057] Inoculate A. bombacina into a PDA medium covered with cellophane and culture for 4-5 days. Take 0.1g of mycelium and place it in a 1.5mL centrifuge tube. Add 0.5mL of extraction buffer Tris-HCl and 200μL of 10M ammonium acetate, invert and mix, place in an ice water bath for 8min, and centrifuge at 13000rpm for 10min to obtain the supernatant. Then extract the supernatant twice with an equal volume of chloroform and isoamyl alcohol (24:1), mix well, and centrifuge for 5min. Take the supernatant phase and place it in another clean centrifuge tube to remove impurities. Take the upper aqueous phase and place it in another centrifuge tube, add 0.5-0.6 times the volume of isopropanol precooled at -20℃, invert and mix well, and place at -20℃ for 30min to precipitate DNA. Finally, centrifuge at 13000rpm for 15min. The supernatant was discarded, the precipitate was washed twice with 1 mL of 75% ethanol, the precipitate was dissolved with 50 μL of sterile water, and the extracted DNA was detected by gel electrophoresis and stored at -20°C for future use.

[0058] 1.2.3 Primer design and amplification of upstream and downstream fragments of AbLac4 gene

[0059] Using A. bombacina wild-type DNA as a template, primers Lac4-KpnI-F and Lac4-KpnI-R and Lac4-Hind III-F and Lac4-Hind III-R were used to amplify the upstream and downstream fragments of the AbLac4 target gene, respectively. The primer sequences are shown in Table 2. The PCR product was subjected to electrophoresis, and the target gene band was recovered using a gel recovery kit. The recovery steps refer to the instructions of the gel recovery kit.

[0060] Table 2 PCR primer sequences

[0061]

[0062] Table 3 PCR amplification system

[0063]

[0064] PCR reaction program: 95°C for 3 min, 95°C for 15 s, screening temperature for 30 s, 72°C for 1 min 30 s, 33 cycles; 72°C for 5 min; and storage at 4°C.

[0065] 1.2.4 Preparation of linearized vector and recombination reaction

[0066] After analysis by DNAMAN software, the construction of the recombinant vector needs to follow the principle of connecting the lower arm first and then the upper arm. The recombination reaction can be completed after reacting at 37°C for 30 minutes to achieve the in vitro circularization of the two linearized DNAs. The reaction system is as follows: the target gene reaction system is: 6μL target fragment, 2μL linear vector, 4μL 5×CE II buffer, 2μL Exnase II, 6μL ddH2 O.

[0067] 1.2.5 Screening of positive clones and extraction of plasmids

[0068] Inject 10 μL of the above amplification product into 100 μL of freshly thawed DH5α competent cells, let stand on ice for 30 min, gently mix, heat shock in a 42°C water bath for 30 s, immediately freeze on ice for 2-3 min, add 900 μL LB liquid culture medium (without antibiotics), culture at 37°C, 200 rpm, for 1 h, centrifuge at 5000 rpm for 5 min, discard the supernatant, aspirate the remainder into LB solid culture medium (containing kanamycin), spread the plate with a sterile applicator, and culture inverted at 37°C for 12-16 h.

[0069] Single clones were picked for bacterial liquid PCR identification of positive clones, and the positive clone bacterial liquid was sent to Sangon Biotechnology Co., Ltd. for sequencing. The sequenced sequence was compared with the target theoretical sequence by DNAMAN software. 100 μL of the correctly sequenced strain was taken and expanded in 30 mL of culture medium for 24 hours. The plasmid was extracted using the Novizan plasmid extraction kit. The specific steps refer to the kit instructions. After obtaining the recombinant vector connected to the lower arm, repeat this step to connect the upper arm. The positive screening and plasmid extraction steps are the same as above.

[0070] 1.2.6 Freeze-thaw transformation of Agrobacterium EHA105

[0071] Add 3 μL of the recombinant plasmid connected with the upper and lower homologous arms to 200 μL of competent cells and mix gently. Then put the above mixture in an ice bath for 30 minutes, freeze it in liquid nitrogen for 2 minutes, and quickly place it in a 37°C water bath for 5 minutes to thaw the competent cells. Add 1 mL of LB liquid culture medium without antibiotics to the thawed competent cells and culture them at 28°C and 100 rpm for 4-5 hours. After the recovery culture is completed, centrifuge the competent cells at room temperature at 4000 rpm for 5 minutes, gently pour off part of the supernatant, and resuspend the bacteria in the remaining approximately 100 μL of liquid. Evenly spread the bacterial liquid on the LB plate containing kana antibiotics and culture it upside down in a 28°C incubator for 2-4 days.

[0072] 1.2.7 Preparation of protoplasts

[0073] The preparation method of protoplasts was carried out according to the method of Jia et al. Take a PDA plate of A. bombacina colonies that have been cultured for 5 days, use a sterile puncher to evenly punch 8 bacterial cakes on the edge of the colony, then add 100mL YEPD medium, and culture at 25℃, 150rpm for 30h. Use 4 layers of sterile filter cloth to filter and collect the mycelium, rinse with sterile water 2-3 times, then transfer the mycelium to a 50mL centrifuge tube containing 10mL of enzymatic solution, wrap the tube cover with sealing film to prevent enzyme leakage, place it horizontally on a shaker, and enzymatically hydrolyze the mycelium at 30℃, 90rpm. After 90-120min, absorb the enzyme solution and observe the release of protoplasts under a microscope. When the number of protoplasts no longer increases, fold a layer of lens paper into a funnel shape to filter the enzyme solution, collect the filtrate into a 50mL centrifuge tube, and place it at -20℃ for use.

[0074] 1.2.8 Transfection of A. bombacina with Agrobacterium tumefaciens EHA105

[0075] Pick a single colony of Agrobacterium into 5 mL of LB liquid medium containing rifamycin and kanamycin, and culture at 28°C, 220 rpm for 24 h. After the culture is completed, centrifuge at 3000 rpm for 5 min, discard the supernatant, wash the precipitate twice with IM liquid medium, and resuspend it to OD 600 =0.15, take 10 mL of bacterial solution, add 20 μL AS (200 μmol / L), and culture at 28°C, 220 rpm for 6 h until OD 600 =0.5. After the culture was completed, centrifuge at 8000 rpm for 2 min, remove part of the supernatant, and use the remaining supernatant to resuspend the precipitated bacteria to a value of OD 600 =0.9. Meanwhile, a certain concentration of A. bombacina protoplasts was inoculated into 10 mL IM liquid medium and cultured at 25°C, 100 rpm for 4 h. Finally, 250 μL of protoplasts and Agrobacterium were spread on IM solid medium containing AS, cultured in a 25°C incubator in the dark for 2 days, and then transferred to CYA solid medium containing HYG and CR. After cultured at 25°C in the dark for 5 days, transformants were picked for verification.

[0076] 1.2.9 Validation of knockout mutants

[0077] The wild-type and mutant strains were identified by amplifying fragments. The primer KLLac-4-F of the target gene was used to amplify the total DNA sequence of the wild-type and mutant strains. The total DNA extraction method was the same as above. Whether the mutant strain could amplify the band by the target gene primer was used to determine whether the target gene was initially knocked out. Based on the PCR verification, the total RNA was extracted using the TRNzolUniversal total RNA extraction reagent for RNA verification. The PrimeScripTM RT regent Kit with gDNA Eraser Kit was used to remove genomic DNA and reverse transcription reaction, and the cDNA samples were stored in a refrigerator at -20°C for use. Actin was used as an internal reference, and the gene expression was determined using a fluorescent quantitative PCR kit (SYBR Green). The specific method was referred to the kit instructions.

[0078] 1.2.10 Data Processing

[0079] The data obtained in the present invention were sorted using Excel 2010 and Origin 2018, SPSS18.0 software was used for data processing and analysis, TBtools software was used for data visualization, and Duncan's multiple comparison method was used to analyze the significant differences of the data (P < 0.05).

[0080] 2. Results and Analysis

[0081] 2.1 Amplification of the lower arm of the target gene and construction of the recombinant vector

[0082] 2.1.1 Amplification of the lower arm of the target gene and linearization of the vector

[0083] Using wild-type A. bombacina as a template, specific primers were used to amplify the lower arm of AbLac4, and a 1069 bp amplification product was obtained. The amplification product was detected by 1% agarose gel electrophoresis, and a clear and bright single band was obtained (such as Figure 2 ).

[0084] 2.1.2 Preparation of AbLac4-dn-pCHPH recombinant vector and verification of sequencing results

[0085] The gene knockout empty vector pCHPH was digested with HindIII and connected to AbLac4-down to obtain the AbLac4-dn-pCHPH recombinant vector, which was introduced into Escherichia coli for transformation to obtain a batch of positive clone strains. Eight strains with smooth surface and regular shape were selected for bacterial liquid PCR verification and recorded as pC-down. The primers Lac4-HindIII-F and Lac4-HindIII-R were used to amplify the lower arm of pC-down to obtain 6 positive clone strains. The amplified products were detected by 1% agarose gel electrophoresis to obtain clear and bright single bands as shown in Figure 3 Then one of the monoclonal strains was selected for sequencing verification. The sequencing results were as follows Figure 4 .

[0086] 2.2 Amplification of the upper arm of the target gene and construction of the knockout vector

[0087] 2.2.1 Amplification of the upper arm of the target gene and vector linearization

[0088] Using wild-type A. bombacina as a template, specific primers were used to amplify the lower arm of AbLac4, and a 1020bp amplification product was obtained. Plasmids were extracted from the bacterial solution of pC-down to obtain AbLac4-dn-pCHPH, which was linearized using KPnI with this vector as a template. The amplification products were detected by 1% agarose gel electrophoresis, and clear and bright single bands were obtained (such as Figure 5 ).

[0089] 2.2.2 Preparation of AbLac4-up-dn-pCHPH knockout vector and verification of sequencing results

[0090] AbLac4-dn-pCHPH was digested with KPnI to connect AbLac4-up to obtain the AbLac4-up-dn-pCHPH recombinant vector, which was introduced into Escherichia coli for transformation to obtain a batch of positive clone strains. Twelve strains with smooth surface and regular shape were selected for bacterial liquid PCR verification and recorded as pC-up. The lower arm of pC-up was amplified using primers Lac4-KpnI-F and Lac4-KpnI-R to obtain 11 positive clone strains. The amplified products were detected by 1% agarose gel electrophoresis to obtain clear and bright single bands as shown in Figure 6 Then one of the monoclonal strains was selected for sequencing verification. The sequencing results were as follows Figure 7 .

[0091] 2.3 Knockout vector construction and verification

[0092] In order to further verify the correctness of the construction of AbLac4-up-dn-pCHPH recombinant vector, HPH vector universal primers were used to amplify the pC-up-dn and pC fragments respectively. The amplified products were detected by 1% agarose gel electrophoresis to obtain a clear and bright single band as shown in Figure 8 .

[0093] 2.4 Screening and identification of mutants

[0094] like Fig. 9As shown, △Lac4 was specifically amplified using cloning primers KLLac-4-F and KLLac-4-F, and the amplified products were detected by 1% agarose gel electrophoresis to obtain 8 transformants, which were named △Lac4-1-△Lac4-8, and were quantitatively determined respectively. The one with the most significant silencing was selected as the final transformant △Lac4.

[0095] Comparative Example 1 Protoplast transformation

[0096] In order to find out a gene knockout system suitable for A. bombacina, the inventors conducted relevant tests on the current conventional gene knockout method (Comparative Example 1 and Comparative Example 2), and found that the gene knockout method provided by the present invention is faster and has a higher probability of positive transformants. The protoplast transformation used in this comparative example has the problem of incomplete knockout and false positives.

[0097] 1. Amplification of upstream and downstream fragments of target genes (AbLac1 and AbLac2)

[0098] Using wild-type DNA of Athelia bombacina (Link) Pers as template, primers Lac-Up-F and Lac-Up-R and Lac-Dn-F and Lac-Dn-R were used to amplify the upstream and downstream fragments UP-AbLac1 and Dn-AbLac1 of the target genes AbLac1 and AbLac2, respectively. The PCR products were subjected to electrophoresis, and the target gene bands were recovered using a gel recovery kit. The recovery steps refer to the instructions of the gel recovery kit.

[0099] Table 4 PCR primer sequences

[0100]

[0101] Table 5 PCR amplification system

[0102]

[0103] PCR reaction program: 95°C for 3 min; 95°C for 15 s, screening temperature for 30 s, 72°C for 1 min 30 s, 33 cycles; 72°C for 5 min; and storage at 4°C.

[0104] 2. Extraction of pCB1003 plasmid

[0105] The E. coli containing the pCB1003 plasmid frozen at -80°C was taken out and placed on ice for thawing. The thawed E. coli was dipped into the bacterial solution with a sterilized inoculation needle and then streaked. After being cultured in a 37°C constant temperature incubator for 14-15 hours, a single colony was picked up with a sterilized pipette tip and placed in 3mL LB medium containing kanamycin (50μL 100mg / μL kanamycin was added to 100mL LB medium) and cultured in a 37°C 200rpm shaker for 8 hours. Then 1mL of the shaking culture solution was drawn into a sterilized 50mL centrifuge tube (20mL of LB medium containing kanamycin had been added) and shaken again overnight to increase the concentration of the bacterial solution. Finally, the plasmid was extracted using a plasmid extraction kit. The method and steps refer to the instructions of the Tiangen plasmid extraction kit.

[0106] 3. Amplification of upstream and downstream fragments of HYG gene

[0107] Using pCB1003 plasmid as template, primers H1-F and H1-R and H2-F and H2-R were used to amplify the upper and lower arm fragments H1 and H2 of the HYG gene, respectively. The PCR products were run on electrophoresis, and the target bands were recovered using a gel recovery kit.

[0108] Table 6 PCR primer sequences

[0109]

[0110] Table 7 PCR amplification system

[0111]

[0112] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 1 min 30 s, 33 cycles; 72°C for 5 min; and storage at 4°C.

[0113] 4. Fusion PCR

[0114] Taking AbLac1 as an example, the upper and lower arm fragments Lac1-Up and Lac1-Dn of the target gene were amplified using Lac1-Up-F and Lac1-Up-R as well as Lac1-Dn-F and Lac1-Dn-R primers; the H1 and H2 fragments were amplified using H1-F and H1-R as well as H2-F and H2-R primers.

[0115] First, Lac1-Up and H1 as well as H2 and Lac1-Dn were connected by the first round of PCR. The connection system is as follows:

[0116] Table 8 Lac1-Up connection system

[0117]

[0118] Table 9 Connection system of Lac1-Dn

[0119]

[0120] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 1 min 30 s, 33 cycles; 72°C for 5 min; and storage at 4°C.

[0121] Secondly, the obtained Lac1-Up-H1 and H2-Lac1-Dn fragments were amplified by the second round of PCR. Therefore, primers Lac1-Up-F and H1-R as well as H2-F and Lac1-Dn-R were added to the first round of PCR to prepare a 50 μL amplification system as follows:

[0122] Table 10 Amplification system of Lac1-Up

[0123]

[0124] Table 11 Amplification system of Lac1-Dn

[0125]

[0126] PCR reaction program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 1 min 30 s, 33 cycles; 72°C for 5 min; and storage at 4°C.

[0127] Finally, the amplified product was recovered by ethanol precipitation. First, 2 volumes of anhydrous ethanol (precooled at 4°C) were added to the PCR product and placed in a -20°C refrigerator for precipitation overnight. The precipitated sample was centrifuged at 12000rpm for 15min, and the upper waste liquid was discarded. 200μL 75% ethanol was added to the precipitate (no need to shake the precipitate), centrifuged at 12000rpm for 5min, and the waste liquid was discarded. The sample was centrifuged instantaneously, and after the excess 75% ethanol was absorbed by the pipette, the centrifuge tube was inverted on the lens paper and left at room temperature for 10-12min until it was dry. 50μL of sterile water was added to the precipitate and the pipette was repeatedly aspirated until the precipitate was fully dissolved. After measuring the concentration, it was stored at -20°C for use.

[0128] 5. Protoplast Preparation

[0129] The method is the same as in Example 1.

[0130] 6. Transformant Screening

[0131] Transformants that can grow normally on PDA plates containing the hygromycin resistance selection marker were selected and verified by PCR after three generations of stable inheritance.

[0132] 7.PCR Validation

[0133] The wild-type and mutant strains were identified by amplifying fragments. The primers of the target gene and the primers of the HYG vector fragment were used to amplify the total DNA sequences of the wild-type and mutant strains respectively. The total DNA was extracted in the same way as above. Whether the target gene was initially knocked out was judged based on whether the mutant strain could amplify a band using the target gene primers. Whether the fusion fragment was successfully inserted was judged based on whether the mutant strain could amplify a band using the vector fragment primers.

[0134] 8. RT-qPCR Validation

[0135] Total RNA was extracted using TRNzol Universal Total RNA Extraction Reagent, and the RNA concentration and quality were then tested using a concentration meter. PrimeScript was used to remove genomic DNA and perform reverse transcription. TM RT regent Kit with gDNAEraser kit, cDNA samples were stored in a -20℃ refrigerator until use. Actin was used as an internal reference, and the gene expression level was determined using a fluorescent quantitative PCR kit (SYBR Green). The specific method was referred to the kit manual.

[0136] 9. Results

[0137] 9.1PCR verification

[0138] According to the PCR verification results ( Fig.10 ), it can be preliminarily considered that positive transformants were obtained.

[0139] 9.2 qPCR Validation

[0140] Through quantitative verification ( Fig.11 ), it was found that the transformants cultivated using this method all had incomplete knockout or false positive results.

[0141] Comparative Example 2 Agrobacterium-mediated spore

[0142] The only difference from Example 1 is that spores of Athelia bombacina are infected with Agrobacterium instead of protoplasts.

[0143] The results are as follows Fig.12 As shown, bands appeared after amplification, indicating that the knockout was unsuccessful.

[0144] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene knockout system based on basidiomycetes, characterized in that: It includes a knockout empty vector pCHPH, a complementing vector pCNEO, Agrobacterium EHA105 and a protoplast extraction reagent.

2. The gene knockout system according to claim 1, characterized in that The basidiomycete is Athelia bombacina.

3. A method for knocking out a basidiomycete gene, characterized in that: The following steps are involved: Based on the gene knockout system described in claim 1, the target gene sequence is obtained, the restriction enzyme cutting sites of the knockout empty vector pCHPH are analyzed, a knockout vector is constructed, introduced into the Agrobacterium EHA105, the Agrobacterium EHA105 containing the plasmid is collected, and the protoplasts of the basidiomycete are infected to achieve the knockout of the basidiomycete gene.

4. The method according to claim 3, characterized in that The basidiomycete is Athelia bombacina; and the gene is the Lac4 gene.

5. The method according to claim 4, characterized in that The following steps are involved: The upstream fragment of AbLac4 gene was amplified and named AbLac4-up, and the downstream fragment of AbLac4 gene was amplified and named AbLac4-dn; The knockout empty vector pCHPH was digested with HindⅢ and connected to AbLac4-dn to obtain AbLac4-dn-pCHPH; the AbLac4-dn-pCHPH was digested with Kpn I to connect to AbLac4-up to obtain AbLac4-up-dn-pCHPH vector; Preparation of protoplasts of Athelia bombacina; The AbLac4-up-dn-pCHPH vector is transformed into the Agrobacterium EHA105 strain, and the Agrobacterium EHA105 cells containing the recombinant plasmid are co-cultured with the protoplasts of the Athelia bombacina to achieve the knockout of the Lac4 gene.

6. The method according to claim 5, characterized in that The process of preparing the AbLac4-up-dn-pCHPH vector also includes the steps of bacterial liquid PCR verification and plasmid extraction and sequencing.

7. The method according to claim 5, characterized in that After the co-culture, the method further includes the steps of PCR verification and qPCR verification.

8. Use of the gene knockout system as claimed in claim 1 or 2 in the preparation of a product for editing the Athelia bombacina gene.

9. Use of the gene knockout system according to claim 1 or 2 in preventing and controlling plant diseases caused by Athelia bombacina.

10. A product for preventing and controlling plant diseases caused by Athelia bombacina, characterized in that: Comprising the gene knockout system according to claim 1 or 2.

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