An Agrobacterium tumefaciens-mediated gene knockout method for Cordyceps cicadae

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a genetic transformation method for gene knockout in Cordyceps cicadae mediated by Agrobacterium tumefaciens. The present invention discloses the construction of a knockout vector for a target gene beas, and also simultaneously discloses a method for knocking out genes in Cordyceps cicadae by Agrobacterium tumefaciens mediation. The present invention uses molecular biological methods to knock out the key synthetic gene of beauvericin in Cordyceps cicadae fungi, so that beauvericin is not expressed or has a very low content in the mycelium harvested after culturing the strain for 24 days. The strain after knocking out the key synthetic gene of beauvericin has genetic stability of not producing beauvericin. It provides a basis for the development of new drugs for Cordyceps cicadae fungi that do not produce beauvericin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a genetic transformation method for gene knockout in Cordyceps cicadae mediated by Agrobacterium tumefaciens. Background Art

[0002] Cordyceps cicadae Miquel, also known as Jinchanhua, Chanrong, or Chan Cao, etc., is a complex of fungal stroma and nymph cadavers formed after the fungus Isaria cicadae infects cicada nymphs. Research has found that Cordyceps cicadae contains various bioactive components such as polysaccharides, nucleoside substances, myriocin, ergosterol and its peroxide, and cordycepic acid, etc., and has the effects of regulating immunity and metabolism, improving renal function, and anti-tumor, etc. Therefore, as a tonic food, the market demand for Cordyceps cicadae is increasing day by day. However, it has been found in the research that cyclic peptide compounds such as beauvericin (BEA), bassiatin, and bassiatin A can be detected in the cultures or stroma of Cordyceps cicadae. Among them, BEA is a cyclic hexaester peptide fungal toxin, belonging to the emerging fusarium toxin, and was first isolated from the entomopathogenic fungus Beauveria bassiana. The hexaester peptide fungal toxins include BEA and enniatins (ENNs), etc. The European Food Safety Authority believes through risk assessment that the risks of chronic exposure of humans to BEA and ENNs in the diet are worthy of attention.

[0003] There are generally the following several methods for genetic transformation of fungi: protoplast transformation method, liposome transformation method, electroporation transformation method, restriction enzyme-mediated integration method, Agrobacterium tumefaciens-mediated transformation method (ATMT), etc. The Agrobacterium tumefaciens-mediated transformation method has significant advantages compared with other transformation methods. First of all, this technology has achieved genetic transformation of materials such as fungal spores, mycelia, gills, protoplasts, and fruiting bodies, avoiding the cumbersome preparation of protoplasts and permeability-sensitive cells, greatly simplifying the transformation process and reducing the transformation difficulty; secondly, the transformation efficiency is high, which is 140 - 1000 times that of traditional transformation methods; the genetic stability of the transformants is high, and the genetic characteristics of the offspring transformants still remain 85% - 98%; the proportion of single-copy insertion of T-DNA in the transformants is high, which is 66% - 96%. The Agrobacterium-mediated genetic transformation method is already one of the most widely used methods for realizing fungal genetic transformation. More than 100 species of fungi have been successfully transformed by this method, and mutant libraries containing rich types of transformants have been constructed for many fungi, and specific genes are screened through the phenotypes of the mutants.

[0004] There are mainly two types of Agrobacterium, namely Agrobacterium tumefaciens and Agrobacterium rhizogenes. They are Gram-negative bacteria commonly present in the soil. Attracted by plant phenolic substances such as Acetosyringone (AS), they infect host cells through wounds. Subsequently, the virulence genes of the Tumor-Inducing (Ti) plasmid start to express, transferring the T-DNA of Agrobacterium to the host cells to complete the transgenic process. The Vir (Virulence) gene region on the Ti plasmid in Agrobacterium can specifically bind to a highly conserved approximately 25bp base sequence at the T-DNA boundary, enabling the T-DNA to be unaffected by sequence specificity during transformation. Therefore, genetic transformation can be achieved by replacing the endogenous T-DNA with foreign genes using molecular cloning techniques, obtaining plants or fungi that express foreign genes.

[0005] Currently, no gene knockout method for Cordyceps cicadae fungi has been published. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for knocking out the genes of Cordyceps cicadae fungi mediated by Agrobacterium tumefaciens.

[0007] The present invention constructs a knockout vector for knocking out the beas gene of beauvericin non-ribosomal cyclic peptide synthetase (ISF_00179) in Cordyceps fungi.

[0008] To solve the above technical problem, the present invention provides the construction of a knockout vector for the target gene beas, including the following steps:

[0009] 1) Obtaining the upstream and downstream combined fragment genes of the beas gene:

[0010] Amplify the upstream and downstream fragments of the open reading frame of the beas gene, which is the target gene, from the wild-type genome of Cordyceps fungi; the sequence of the upstream combined fragment of the beas gene is as shown in SEQ ID NO:2, and the sequence of the downstream combined fragment of the beas gene is as shown in SEQ ID NO:3;

[0011] 2) Recombine the upstream combined fragment sequence of the beas gene and the downstream combined fragment sequence obtained in step 1) with the plasmid pPK2-bar-GFP; transform the recombinant product into Eco.li DH5α competent cells to obtain the knockout vector for the target gene beas.

[0012] As an improvement to the construction of the knockout vector plasmid for the target gene beas of the present invention, step 2) is:

[0013] 2.1), ligate the upstream fragment with pPK2-bar-GFP and transfer it into Escherichia coli DH5α. The resulting recombinant plasmid is named 5'-pPK2-bar-GFP;

[0014] 2.2), ligate 5'-pPK2-bar-GFP with the downstream fragment and transfer it into Escherichia coli DH5α. The resulting recombinant plasmid is named 5'-pPK2-bar-GFP-3'; the 5'-pPK2-bar-GFP-3' is the knockout vector of the target gene beas.

[0015] The sequence of the beas gene is as described in SEQ ID NO:1.

[0016] The present invention also simultaneously provides a method for knocking out the gene of Cordyceps cicadae by Agrobacterium tumefaciens-mediated transformation, which is characterized by including the following steps:

[0017] I. Prepare the recombinant plasmid 5'-pPK2-bar-GFP-3';

[0018] II. Transfer 5'-pPK2-bar-GFP-3' into Agrobacterium tumefaciens to obtain the engineered bacteria; perform genetic transformation of fungi with the engineered bacteria to obtain positive single colonies of Agrobacterium tumefaciens AGL1;

[0019] That is, the present invention is as follows:

[0020] First, obtain the wild-type strain of Cordyceps cicadae and its genomic DNA;

[0021] Secondly, the construction of the pPK2-bar-GFP double-label screening system engineered bacteria is as follows: amplify the upstream and downstream fragments of the open reading frame of the target gene from the wild-type genome of Cordyceps fungi, and recombine the above PCR products with the plasmid pPK2-bar-GFP respectively. The recombinant products are transformed into Escherichia coli DH5α competent cells, and the knockout vector of the target gene is obtained through PCR verification. Transfer the knockout vector into Agrobacterium tumefaciens to obtain the engineered bacteria, and perform genetic transformation of fungi.

[0022] Then, perform the following steps:

[0023] III. Fungal activation culture:

[0024] Take the Cordyceps fungi liquid on a PDA plate to streak and activate Cordyceps cicadae, and prepare a spore suspension;

[0025] IV. Engineered bacteria activation culture:

[0026] Inoculate the positive single colony of Agrobacterium tumefaciens AGL1 obtained in Step 2 into an LB liquid medium containing the corresponding antibiotic and culture it to a certain concentration to obtain an engineered bacterial solution; culture it in the dark with shaking in an IMAS liquid medium containing the antibiotic and acetosyringone (AS) to obtain an infection solution;

[0027] Step 5. Agrobacterium infection and co-culture:

[0028] Mix the infection solution obtained in Step 4 with the spore suspension obtained in Step 3 at a ratio of 1:1, and co-culture them on a solid induction medium containing the corresponding antibiotic until white fluffy mycelia grow out;

[0029] The induction medium: IMAS basal medium, 400 mL·L-1 2.5×MM salts, 0.9 g·L-1 Glucose, 10 mL·L-1 50% Glycerol, 40 mL·L-1 40 mM MES pH 5.8, 20 mL·L-1 10 mM AS, 15 g·L-1 Agar;

[0030] Step 6. Differentiation and screening:

[0031] Transfer the white fluffy mycelia grown in Step 5 to a screening medium, and then pour a screening medium containing the corresponding antibiotic on it to form a sandwich model, and continue to culture until white clustered fluff grows out;

[0032] The screening medium: M-100 basal medium, 62.5 mL·L-1 M-100 Salt solution, 10 g·L-1 Glucose, 3 g·L-1 KNO3, 15 g·L-1 Agar; 2 mL·L -1 200 mg / mL cef, 1.5 mL·L -1 200 mg / mL ppt;

[0033] Step 7. Secondary screening:

[0034] Pick the transformants grown in Step 6 onto an M-100 screening medium containing the corresponding antibiotic for secondary screening, screen out possible positive transformants, and transfer them to a PDA medium and continue to culture until strains grow out;

[0035] Step 8: Verify by two-step PCR to obtain positive mutant strains.

[0036] The present invention is mediated by Agrobacterium tumefaciens, and for the first time, genes of Cordyceps cicadae are knocked out through the Agrobacterium tumefaciens system to obtain mutant strains.

[0037] The present invention is the first attempt to knockout the genes of Cordyceps cicadae fungus.

[0038] The present invention uses molecular biology methods to knockout the key synthetic genes of beauvericin in Cordyceps cicadae fungus, so that beauvericin is not expressed or has extremely low content in the mycelium harvested after culturing the strain for 24 days, that is, beauvericin cannot be detected. The strain after knocking out the key synthetic genes of beauvericin should have genetic stability of not producing beauvericin. It provides a basis for the development of new drugs for Cordyceps cicadae fungus that does not produce beauvericin. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings.

[0040] Figure 1 PCR electrophoresis diagrams of the upstream and downstream fragments of the non-ribosomal cyclic peptide synthetase (beas) gene of beauvericin;

[0041] Figure 1-1 Is the upstream fragment (1194bp) of the non-ribosomal cyclic peptide synthetase (beas) gene of beauvericin, Figure 1-2 Is the downstream fragment (1319bp) of the beauvericin (beas) gene.

[0042] Figure 2 Electrophoresis diagrams of the upstream and downstream fragments ligated to the vector plasmid respectively;

[0043] Figure 2-1 Is the upstream fragment ligated to the pPK2-bar-GFP vector plasmid, Figure 2-2 Is the downstream fragment ligated to the vector plasmid that has been ligated to the upstream fragment 5'-pPK2-bar-GFP.

[0044] Figure 3 Electrophoresis diagram for verifying the transfer of the knockout vector plasmid 5'-pPK2-bar-GFP-3' into Agrobacterium;

[0045] Lanes 1, 3, 4, 5, 6, 7, 8 are false positive transformants, and lane 2 has a target fragment band (1300bp), which is a positive transformant.

[0046] Figure 4 Electrophoresis diagram for "NO" verification of colony PCR of the transformants after knocking out the beas gene;

[0047] Lane 0 is the wild-type cordyceps fungus control group, lanes 1, 2, 4, 6, 7, 8, 9, 10 are false positive transformants, and lanes 3, 5 have no target fragment band (1639bp).

[0048] Figure 5 Electrophoresis diagram for secondary verification of the 3rd and 5th strains screened for the first time, with "YES" and "NO" verified simultaneously;

[0049] Lanes 1, 2, and 3 are the YES verifications of the wild type, mutant strain No. 3, and mutant strain No. 5 in sequence. There is no target band (about 1500 bp) in the wild type, while there are target bands in mutant strains No. 3 and No. 5. Lanes 4, 5, and 6 are the NO verifications of the wild type, mutant strain No. 3, and mutant strain No. 5 in sequence. There is a target fragment band in the wild type, and no target fragment band in the mutant strains.

[0050] Figure 6 It is the liquid chromatogram obtained by HPLC detection of the beauvericin content in the two mutant strains obtained;

[0051] Figure 6-1 It is the standard product diagram; Figure 6-2 -1~6-2-2 is the injection detection diagram of mutant strain No. 3, with 2 parallels; Figure 6-3 -1~6-3-2 is the injection detection diagram of mutant strain No. 5, with 2 parallels. As shown in the figure, both mutant strains do not produce or have extremely low content. Specific implementation manners

[0052] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0053] The cordyceps fungus of the present invention is derived from Cordyceps cicadae informed in the literature Biological characteristics, bioactive components and antineoplastic properties of sporoderm-broken spores from wild Cordyceps cicadae Phytomedicine, 2017, 36: 217–228.

[0054] Example 1. Construction of the knockout vector plasmid

[0055] 1. Obtaining of the combined fragment gene of the upstream and downstream of the beas gene

[0056] Take the original Cordyceps militaris bacterial liquid from a -80°C refrigerator. Dip the bacterial liquid with an inoculation loop and streak it on a PDA solid medium to activate the fungus, and incubate it at a constant temperature of 28°C for about 2 weeks. Collect the mycelia of Cordyceps cicadae from the PDA plate, place them in a pre-cooled mortar, quickly freeze them in liquid nitrogen, and then grind the mycelia into powder. Take an appropriate amount of the ground sample (about 0.2 g) into a 1.5 mL centrifuge tube, add 400 μL of DNA lysis buffer, and mix well by inverting up and down. Add an equal volume of balanced phenol solution (pH 7.0) (Sangon Biotech, Shanghai, China), shake vigorously for 3 - 5 min, and centrifuge at room temperature (12000 rpm) for 10 min. Take 350 μL of the supernatant into a new centrifuge tube, add 5 μL of RNase, and incubate in a water bath at 37°C for 1 h. Then add an equal volume of balanced phenol solution (pH 7.0), and centrifuge at room temperature (12000 rpm) for 10 min. Take 300 μL of the supernatant into a new centrifuge tube, add 2.5 times the volume of absolute ethanol, place it at -20°C for 30 min to fully precipitate the DNA. Centrifuge at room temperature (14000 rpm) for 10 min, discard the supernatant, add 300 μL of 70% ethanol. Centrifuge at room temperature (14000 rpm) for 2 min, discard the supernatant, place it at 37°C to dry for 1 h to fully volatilize the ethanol, add an appropriate amount (60 μL) of ddH2O to dissolve the precipitate, and the obtained product is genomic DNA, whose sequence is as described in SEQ ID NO:1. Using the genomic DNA as a template, cc-beas-5-1 and cc-beas-5-2 are used as primers to amplify the upstream fragment of the beas gene, and cc-beas-3-1 and cc-beas-3-2 are used as primers to amplify the downstream fragment of the beas gene.

[0057] The DNA lysis buffer consists of 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM EDTA-Na, 2% CTAB, 2% PVP, 1% (V / V) β-mercaptoethanol, and the pH value is 8.0.

[0058] The amplification systems are respectively:

[0059] (1) 1 μL of KOD-Plus-Neo, 5 μL of 2 mM dNTPs (10 mM each), 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 4 μL of DNA (100 ng / μL), 29 μL of ddH2O, 1.5 μL of cc-beas-5-1, and 1.5 μL of cc-beas-5-2.

[0060] (2) 1 μL of KOD-Plus-Neo, 5 μL of 2 mM dNTPs (10 mM each), 5 μL of 10× PCR Buffer for KOD-Plus-Neo, 4 μL of DNA (100 ng / μL), 29 μL of ddH2O, 1.5 μL of cc-beas-3-1, 1.5 μL of cc-beas-3-2.

[0061] The high-fidelity enzymes used above are all from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0062] The amplification conditions were 2 min at 94°C, 10 s at 98°C, 30 s at 55°C, 1 min at 68°C, for 30 cycles; 5 min at 68°C; The PCR products were subjected to agarose gel electrophoresis detection and the target bands were selected for gel cutting and recovery. The results were as Figure 1 shown. There was a 1194 bp band for the upstream fragment and a 1319 bp band for the downstream fragment.

[0063] The purified PCR products were sequenced, and the obtained sequences were consistent with the upstream and downstream combined fragment sequences of the beas gene. The primer sequences were as follows:

[0064] cc-beas-5-1: 5'-ATATCTCTCAGGTCGGCTG-3'

[0065] cc-beas-5-2: 5'-ACGTAGAGAGTGGTAAG-3'

[0066] cc-beas-3-1: 5'-ACCACGTGCTGTCCATC-3'

[0067] cc-beas-3-2: 5'-CAAGATGTCCTTGATAC-3'.

[0068] The upstream combined fragment sequence of the beas gene is as described in SEQ ID NO:2, and the downstream combined fragment sequence of the beas gene is as described in SEQ ID NO:3.

[0069] 2. Construction of the recombinant plasmid vector 5’-pPK2-bar-GFP-3’

[0070] 2.1), The upstream combined fragment of the beas gene and the pPK2-bar-GFP plasmid were digested with restriction enzymes XbaI and EcoRI at 37°C for more than 2 hours, and then T4 DNA ligase was used to ligate the two. The ligation product was transformed into competent Eco.li DH5α cells and spread on an LB plate containing 20 μg / mL kanamycin (Km). The plate was inverted and cultured in a 37°C constant temperature incubator for one day. Randomly select 8 transformants for colony PCR identification.

[0071] The identification primers were cc-beas-5-1 and Bar-up. The system was 10 μL of 2×Taq Plus Master Mix II, 1 μL of upstream primer, 1 μL of downstream primer, and 8 μL of ddH2O. All the enzymes used above were from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0072] The reaction conditions were 94°C for 1.5 min; 94°C for 20 s, 55°C for 20 s, 72°C for 2 min, for 30 cycles; 72°C for 5 min; The results were as Figure 2-1 shown: Both the 1st and 2nd transformants had bands around 1300 bp, and the remaining 6 strains without corresponding bands were false positives.

[0073] Therefore, it was shown that the 1st and 2nd transformants were positive clones. One of them was selected for sequencing, and the obtained sequence was consistent with the sequence of the upstream combined fragment of the beas gene;

[0074] The results showed that the upstream fragment was successfully ligated to pPK2-bar-GFP and transferred into Eco.li DH5α. This recombinant plasmid was named 5’-pPK2-bar-GFP.

[0075] The above pPK2-bar-GFP plasmid was described, for example, in "A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii".

[0076] 2.2), Then the downstream combined fragment of the beas gene and the 5’-pPK2-bar-GFP plasmid were digested with restriction enzymes SpeI and EcoRⅤ at 37°C for more than 2 hours, and then T4 DNA ligase was used to ligate the two; The subsequent operation method was the same as that in 2.1) above; that is, the ligation product was transformed into competent Eco.li DH5α cells and spread on an LB plate containing 20 μg / mL kanamycin (Km). The plate was inverted and cultured in a 37°C constant temperature incubator for one day. Randomly select 8 transformants for colony PCR identification.

[0077] The identification primers were Bar-down and cc-beas-3-2. The reaction system was composed of 10 μL of 2× Taq Plus Master Mix II, 1 μL of upstream primer, 1 μL of downstream primer, and 8 μL of ddH2O. All the enzymes used above were from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0078] The reaction conditions were as follows: 94°C for 1.5 min; 94°C for 20 s, 55°C for 20 s, 72°C for 2 min, for 30 cycles; 72°C for 5 min. The results were as Figure 2-2 shown: Bands around 1400 bp were observed in the transformants No. 3, 4, and 6, while no corresponding bands in the others indicated false positives.

[0079] Therefore, it was shown that the transformants No. 3, 4, and 6 were positive clones. One of them was selected for sequencing, and the obtained sequence was consistent with the sequence of the combined fragment downstream of the beas gene.

[0080] The results showed that the connection between 5’-pPK2-bar-GFP and the downstream fragment was completed and successfully transferred into Eco.li DH5α. This recombinant plasmid was named 5’-pPK2-bar-GFP-3’.

[0081] The primer sequences were as follows:

[0082] Bar-up: 5'-AGGCATTCATTGTTGAC-3'

[0083] Bar-down: 5'-TCAGCCTGCCGGGTACCGC-3'.

[0084] Example 2. Agrobacterium tumefaciens-mediated fungal transformation

[0085] 1) Take the original fungal solution of Cordyceps militaris from the -80°C refrigerator. Use an inoculation loop to dip the fungal solution and streak it on a PDA solid medium to activate the fungus, and incubate it at 28°C for about 2 weeks.

[0086] 2) Transfer the recombinant plasmid 5’-pPK2-bar-GFP-3’ obtained in Example 1 into AGL1 Agrobacterium competent cells, and plate culture (LB plate containing 20 μg / mL Km, incubate it upside down in a 28°C constant temperature incubator for 2 - 3 days).

[0087] Randomly select 8 positive single colonies, inoculate them into 1 mL of LB liquid medium containing kanamycin (Km) antibiotic, and culture them in a shaker (28°C, 250 rpm) for 16 - 20 h for bacterial liquid PCR identification.

[0088] The identification primers are cc-beas-5-1 and Bar-up. The reaction system is as follows: 10 μL of 2× Taq Plus Master Mix II, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of bacterial solution, and 7 μL of ddH2O. All the enzymes used above are from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0089] The reaction conditions are as follows: 94°C for 1.5 min; 94°C for 20 s, 55°C for 20 s, 72°C for 2 min, for 30 cycles; 72°C for 5 min.

[0090] The results are as Figure 3 shown: It was verified that the obtained Agrobacterium tumefaciens No. 2 had a band of about 1300 bp, indicating that the plasmid of the positive single colony knockout vector had been transferred into Agrobacterium tumefaciens, and the remaining 7 strains without corresponding bands were false positives.

[0091] It was determined that the plasmid had been transferred into Agrobacterium tumefaciens; the positive single colony of this Agrobacterium tumefaciens AGL1 was expanded in culture, inoculated into 4 mL of LB liquid medium containing the antibiotic kanamycin (Km), and cultured in a shaker (28°C, 250 rpm) for 16 - 20 h. Then, 1 mL of the bacterial solution was mixed with the corresponding volume of 50% glycerol and stored at -80°C for bacterial preservation.

[0092] 3) Pick the positive single colony of Agrobacterium tumefaciens AGL1 obtained in step 2) above, inoculate it into 10 mL of LB liquid medium containing the antibiotic kanamycin (Km), and culture it in a shaker (28°C, 250 rpm) for 16 - 20 h until the OD660 reaches 1.0 - 1.5; dilute the bacterial solution to OD660 = 0.15 with Induction Medium (IMAS solution) containing 40 mM MES and 200 μL of 10 mM Acetosyringone (AS), and culture it in a shaker (28°C, 250 rpm) until the OD660 reaches 0.4 - 0.5 (about 4 h); obtain the Agrobacterium tumefaciens induction culture solution.

[0093] The IMAS solution containing 40 mM MES is: 400 mL·L -12.5×MM salts, 1.8 g·L-1 Glucose, 10 mL·L-1 50% Glycerol, 40 mL·L-1 40 mM MES pH 5.8. That is, the preparation method is as follows: To prepare 100 ml of IMAS solution, 40 ml of 2.5×MM salts, 0.18 g of Glucose, 1 ml of 50% Glycerol are required, and the remaining volume is made up to 96 ml with ddH2O. After autoclaving, add 4 ml of 40 mM MES (19.52 g to 100 mL ddH2O, adjust the pH to 5.3 with 5 M KOH, filter and sterilize with a 0.22 μm microporous membrane, and store at -20°C for later use.

[0094] The preparation method of 100 ml of 10 mM AS is as follows: Dissolve 0.1962 g of AS in ddH2O, make up the volume to 100 ml, adjust the pH to 8 with 5 M KOH, filter and sterilize with a 0.22 μm microporous membrane, and store at -20°C in the dark for later use.

[0095] 4), From the mature mycelium of Cordyceps cicadae obtained in step 1) above, add 16 ml of 0.05% Triton-X-100 (v / v) to a 50 ml EP tube, scrape out the fungal mycelium from 3 plates, vortex, and filter the above liquid with a pipette tip with glass wool to obtain a spore suspension. Take 25 μl of the spore suspension + 975 μl of 0.05% Triton in a 1.5 ml EP tube and mix well, and count the spores under a microscope. Gradient dilute it to 1×10 6 spores / mL with an IMAS liquid medium containing 40 mM MES and 200 μM AS (note to avoid light).

[0096] 5), Take 100 μL of the diluted spore suspension obtained in step 4) and mix it 1:1 with an equal volume of the Agrobacterium induction culture solution obtained in step 3), gently pipette and mix well, and evenly coat it on Induction Medium Plates (IMAS Plates) containing 40 mM MES and 200 μM AS and covered with a black sterile filter paper (Neumann, Germany), and incubate the plate upright in a 28°C constant temperature incubator for 2 days;

[0097] IMAS Plates containing 40 mM MES and 200 μM AS are: 400 mL·L -12.5×MM salts, 0.9 g·L-1 Glucose, 10 mL·L-1 50% Glycerol, 15 g·L-1 Agar, 40 mL·L-1 40 mM MES pH 5.8, 20 ml 10 mM AS. That is, the preparation method is as follows: To prepare 100 ml of IMAS Plates, 40 ml of 2.5×MM salts, 0.09 g of Glucose, 1 ml of 50% Glycerol are needed, and the remaining volume is made up to 94 ml with ddH2O. After autoclaving, when pouring the plates, add 4 ml of 40 mM MES (19.52 g to 100 mL ddH2O, adjust the pH to 5.3 with 5 M KOH, filter and sterilize with a 0.22 μm microporous membrane, store at -20°C for later use) and 2 ml of 10 mM AS

[0098] The preparation method of 10 mM AS is as follows: 0.1962 g to 100 mL ddH2O, adjust the pH to 8 with 5 M KOH, filter and sterilize with a 0.22 μm microporous membrane, store at -20°C in the dark for later use.

[0099] Transfer the black filter paper with white fluffy mycelia grown in the induction medium to the M-100 screening medium plate (containing 400 μg / mL cefotaxime and 300 μg / mL ppt antibiotics). Then, pour 12 mL - 14 mL of M-100 screening medium containing the corresponding antibiotics above the black filter paper to form a sandwich model (that is, both sides of the black filter paper are M-100 screening medium). After the M-100 screening medium solidifies, incubate the plate upside down in a constant temperature incubator at 28°C for 5 - 10 days; Pick the transformants with toothpicks onto the M-100 screening medium containing the same concentration of antibiotics for secondary screening, and incubate the plate upside down in a constant temperature incubator at 28°C for about 2 days;

[0100] If the selected transformants can grow again on the M-100 screening medium for secondary screening, transfer the transformants that can grow again to the PDA medium and incubate upside down (incubate at a constant temperature of 28°C). After 3 - 5 days, further screen by PCR to finally determine the positive transformants. The screening method is specifically shown in Example 3 below.

[0101] On the M-100 screening medium (containing 400 μg / mL cefotaxime and 300 μg / mL ppt antibiotics): 62.5 mL·L-1 M-100 Salt solution, 10 g·L-1 Glucose, 3 g·L-1 KNO3, 15 g·L-1 Agar; 2 mL·L-1 200 mg / mL cef, 1.5 mL·L-1 200 mg / mL ppt;

[0102] Preparation method of M-100 Salt solution (1L): 16 g of KH2PO4, 4 g of Na2SO4, 8 g of KCl, 2 g of MgSO4·7H2O, 1 g of CaCl2, 8 ml of M-100 Trace Element Solution;

[0103] Preparation method of M-100 Trace Element Solution (500 ml): 30 mg of H3BO3, . 70 mg of MnCl2·4H2O, 200 mg of ZnCl2, . 20 mg of Na2MoO4·2H2O, . 50 mg of FeCl3·6H2O, . 200 mg of CuSO4·5H2O.

[0104] Example 3: Verification of positive transformants by two groups of PCR

[0105] First-step PCR verification:

[0106] "NO" verification: Select possible positive transformants that can grow again on the PDA plate. Pick a small amount of mycelium as the template. Use a segment intercepted from the internal fragment of the target gene as the upstream primer (CF-1), and use a segment intercepted from about 100 bp downstream of the downstream fragment of the target gene as the downstream primer (CF-2) for verification. Set a negative control group with the mycelium of the wild-type strain of Cordyceps cicadae as the template. The PCR system is: 0.4 μL of KOD FX DNA Polymerase, 4 μL of 2 mM dNTPs, 10 μL of 2×PCR Buffer for KODFX, 4.4 μL of ddH2O, 0.6 μL of CF-1, 0.6 μL of CF-2; all the enzymes used above are from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0107] The amplification conditions are 5 min at 94°C, 10 s at 98°C, 45 s at 56°C, 2 min at 68°C, for 30 cycles; 5 min at 68°C; Detect the PCR products by agarose gel electrophoresis.

[0108] Upstream primer (CF-1): GACTGGCTGATGGCCAC

[0109] Downstream primer (CF-2): GAGAAGTCGCGGTACTG

[0110] Select 10 transformants for PCR verification. The result should be that the wild-type control group has a target band of 1639 bp, and the experimental group has no target band. The actual result is as Figure 4Shown as follows: There is a target band in the wild-type control group, and there is no 1639bp band in transformants No. 3 and No. 5.

[0111] The above verification fragment (the target band of 1639bp) after knocking out the mutant strain is as described in SEQ ID NO:4.

[0112] The second-step PCR verification:

[0113] For the "NO" and "YES" verifications, select the possible positive transformants No. 3 and No. 5 in the first-step PCR verification, pick a little mycelium as the template, use Bar-down as the upstream primer and CF-2 as the downstream primer for the "YES" verification, and use CF-1 as the upstream primer and CF-2 as the downstream primer for the "NO" verification. Set the negative control group with the mycelium of the wild-type strain as the template. The PCR system is as follows:

[0114] "YES" verification: 0.4μL of KOD FX DNA Polymerase, 4μL of 2mM dNTPs, 10μL of 2×PCR Buffer for KOD FX, 4.4μL of ddH2O, 0.6μL of CF-2, 0.6μL of Bar-down;

[0115] "NO" verification: 0.4μL of KOD FX DNA Polymerase, 4μL of 2mM dNTPs, 10μL of 2×PCR Buffer for KOD FX, 4.4μL of ddH2O, 0.6μL of CF-1, 0.6μL of CF-2; All the enzymes used above are from Hangzhou Shuomeng Biotechnology Co., Ltd.

[0116] The amplification conditions are 5min at 94℃, 10s at 98℃, 45s at 56℃, 2min at 68℃, for 30 cycles; 5min at 68℃; The PCR products are detected by agarose gel electrophoresis.

[0117] The "YES" result should be that there is no target band in the wild-type control group, and there is a target band of about 1500bp in the experimental group. The results are as shown in Figure 5 lanes 1, 2, and 3: There is no target band in the wild-type, and there is a band of about 1500bp in both transformants No. 3 and No. 5.

[0118] The above verification fragment (the target band of about 1500bp) for the "YES" verification of the knocked-out mutant strain is as described in SEQ ID NO:5.

[0119] The "NO" result should be that there is a target band of 1639bp in the wild-type control group, and there is no target band in the experimental group. The results are as shown in Figure 5As shown in lanes 4, 5, and 6: The wild type has the target band, and neither the transformants of No. 3 and No. 5 have the 1639 bp band.

[0120] The verification fragment (the target band of 1639 bp) of the above knockout mutant "NO" is as described in SEQ ID NO:4.

[0121] That is, the judgment method is: when the result of "YES" is that the wild type control group has no target band and the experimental group has the target band, while the result of "NO" is that the wild type control group has the target band and the experimental group has no target band, it is determined that the target gene fragment has been knocked out; on the contrary, when the result of "NO" in the first step of verification is that the wild type control group has the target band and the experimental group also has the target band, it is determined that the knockout fails.

[0122] The results show that both the transformants of No. 3 and No. 5 are positive mutants.

[0123] Example 4: The positive mutants obtained in Example 3 were cultured at a constant temperature of 28 °C in a PDA medium for 24 days, and the fruiting bodies or mycelia harvested were detected for the content of beauvericin by HPLC high performance liquid chromatography.

[0124] The liquid chromatogram obtained by HPLC detecting the content of beauvericin in the fruiting body; The results are as Figure 6 shown;

[0125] Figure 6-1 is the standard product map; Figure 6-2 is the injection detection map of the No. 3 mutant, with 2 parallels; Figure 6-3 is the injection detection map of the No. 5 mutant, with 2 parallels. As Figure 6 shown, the two mutants do not produce beauvericin or the content of beauvericin is extremely low.

[0126] Beauvericin is a toxin. The positive mutant strains of the Cordyceps cicada fungi obtained in the present invention do not produce beauvericin or the content of beauvericin is extremely low. Therefore, the present invention provides a basis for the development of new drugs for Cordyceps cicada fungi that do not produce beauvericin.

Claims

1. A method for knocking out genes of Cordyceps cicadae by Agrobacterium tumefaciens-mediated transformation, characterized in that It includes the following steps: I. Target gene beas Construction of the knockout vector 5'-pPK2-bar-GFP-3' includes the following steps: 1). beas Obtaining of gene fragments of upstream and downstream combined genes: The upstream and downstream fragments of the open reading frame of the gene, which is used as the target gene, are amplified from the wild-type genome of Cordyceps sinensis beas ; beas The sequence of the upstream combined fragment of the gene is as described in SEQ ID NO: 2 beas ; the sequence of the downstream combined fragment of the gene is as described in SEQ ID NO: 3 2), the beas upstream combined fragment sequence of the gene, beas downstream combined fragment sequence of the gene is recombined with plasmid pPK2-bar-GFP; the recombinant product is transformed into Eco.li DH5α competent cells to obtain the knockout vector 5'-pPK2-bar-GFP-3' of the target gene beas ; Second, transfer 5’-pPK2-bar-GFP-3’ into Agrobacterium tumefaciens to obtain engineering bacteria; perform genetic transformation of fungi on the engineering bacteria to obtain positive single colonies of Agrobacterium tumefaciens AGL1; Third, activate and culture the fungi: Take the Cordyceps militaris liquid on a PDA plate and streak it to activate Cordyceps cicadae, and prepare a spore suspension; Fourth, activate and culture the engineering bacteria: Inoculate the positive single colonies of Agrobacterium tumefaciens AGL1 obtained in the second step into an LB liquid medium containing the corresponding antibiotics and culture to a certain concentration to obtain an engineering bacteria solution; perform dark shaking culture in an IMAS liquid medium containing antibiotics and acetosyringone to obtain an infection solution; Fifth, infect with Agrobacterium tumefaciens and co-culture: Mix the infection solution obtained in the fourth step with the spore suspension obtained in the third step at a ratio of 1:1, and co-culture on a solid induction medium containing the corresponding antibiotics until white fluffy mycelia grow; Sixth, differentiation and screening: Transfer the white fluffy mycelia grown in the fifth step to a screening medium, and then pour a screening medium containing the corresponding antibiotics on it to form a sandwich model, and continue to culture until white clustered fluff grows; Seventh, secondary screening: Pick the transformants grown in the sixth step onto an M-100 screening medium containing the corresponding antibiotics for secondary screening, screen out possible positive transformants, and transfer them to a PDA medium and continue to culture until strains grow; Eighth: Use two-step PCR verification to obtain positive mutant strains.

2. The method for knocking out genes of Cordyceps cicadae by Agrobacterium tumefaciens-mediated transformation according to claim 1, wherein Step 2) is to perform the following steps in sequence: 2.1), After ligating the upstream fragment with pPK2-bar-GFP and transferring it into Eco.li DH5α, the resulting recombinant plasmid was named 5'-pPK2-bar-GFP; 2.2), After ligating 5'-pPK2-bar-GFP with the downstream fragment, it was transformed into Eco.li DH5α, and the resulting recombinant plasmid was named 5'-pPK2-bar-GFP-3'.

3. The method for knocking out the gene of Cordyceps cicadae mediated by Agrobacterium tumefaciens according to claim 1 or 2, characterized in that: Induction medium: IMAS basal medium, 400 mL·L -1 2.5×MM salts, 0.9 g·L -1 Glucose, 10 mL·L -1 50% Glycerol, 40 mL·L -1 40 mM MES pH5.8, 20 mL·L -1 10 mM AS, 15 g·L -1 Agar; Screening medium: M-100 basal medium, 62.5 mL·L -1 M-100 Salt solution, 10 g·L -1 Glucose, 3 g·L -1 KNO3, 15 g·L -1 Agar; 2 mL·L -1 200 mg / mL cef, 1.5 mL·L -1 200 mg / mL ppt; The preparation method of 1 L of M-100 Salt solution: 16 g of KH2PO4, 4 g of Na2SO4, 8 g of KCl, 2 g of MgSO4·7H2O, 1 g of CaCl2, 8 ml of M-100 Trace Element Solution; Preparation method of 500 ml of M-100 Trace Element Solution: 30 mg of H3BO3, MnCl2 . 4H2O 70 mg, 200 mg of ZnCl2, Na2MoO4 . 2H2O 20 mg, FeCl3 . 6H2O 50 mg, CuSO4 . 5H2O 200 mg; cef represents cefotaxime, and ppt represents ppt antibiotic.

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