Beauvericin synthesis regulatory gene CcWC-1 and application thereof

Abstract

The invention belongs to the technical field of fungus molecular biology, and particularly relates to a beauvericin synthesis regulatory gene CcWC-1 and application thereof. The nucleotide sequence of the beauveria bassiana synthesis regulation gene CcWC-1 is as shown in SEQ ID NO. 1. The invention provides the cordyceps sobolifera CcWC-1 gene related to regulation and control of beauveria bassiana synthesis as well as the encoded protein and application thereof for the first time. By constructing a CcWC-1 gene knockout strain, the result shows that compared with a wild strain, the content of beauveria bassiana in the knockout strain is remarkably changed. Under an illumination culture condition, the beauveria bassiana content of the CcWC-1 knockout strain is obviously higher than that of a wild type. The invention discloses a regulation and control mechanism of illumination on the synthesis of the beauveria bassiana, provides a new gene resource and a production strategy for the artificial efficient production of the beauveria bassiana, and is beneficial to improving the yield of the beauveria bassiana.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal molecular biology, and specifically relates to a beauvericin synthesis regulatory gene CcWC-1 and an application thereof. Background Art

[0002] Cordyceps chanhua is an important medicinal fungus with a long history of application in traditional Chinese medicine. Modern research shows that Cordyceps chanhua contains a variety of biologically active ingredients, including nucleosides, cordycepic acid, polysaccharides, ergosterol and its derivatives, cyclic peptide compounds, etc. These active ingredients give Cordyceps chanhua a variety of pharmacological effects, such as anti-fatigue, sedation and analgesia, lowering blood pressure, lowering blood sugar, and anti-tumor. Among them, the Latin name of Cordyceps chanhua is Cordyceps chanhua.

[0003] Beauvericin is an important cyclic peptide active substance in cicada fungus. Beauvericin was originally isolated from Beauveria bassiana and later found in a variety of fungi. As a compound with multiple biological activities, beauvericin has attracted widespread attention from researchers. Studies have shown that beauvericin has multiple biological activities such as antibacterial, antiviral, anti-inflammatory, antioxidant and anti-tumor. Especially in the anti-tumor aspect, beauvericin exhibits growth inhibition effects on a variety of tumor cells and can induce tumor cell apoptosis. In addition, beauvericin also has potential immunomodulatory effects. Among them, the English name of beauvericin is Beauvericin, BEA. The Latin name of Beauveria bassiana is Beauveria bassiana.

[0004] Light is an important environmental factor that affects fungal growth and secondary metabolism. Studies have shown that light can regulate the growth, morphogenesis, and synthesis of secondary metabolites of a variety of fungi. For example, in Aspergillus flavus, light can affect the synthesis of aflatoxin; in Cordyceps militaris, white light and blue light can promote the synthesis of carotenoids. However, there are few studies on the effect of light on the synthesis of beauvericin in Cicadae.

[0005] The light response of fungi involves complex photosensitivity and signal transduction systems. Among them, WC-1 (White Collar-1) encodes an important blue light receptor, which is involved in regulating light-dependent physiological processes in many fungi. At the same time, as an important blue light receptor and transcription factor in fungi, WC-1 plays a key role in regulating fungal metabolism. For example, in many filamentous fungi, the loss of WC-1 leads to a reduction in pigment synthesis. Therefore, in-depth research on the effects of light on the synthesis of beauvericin in Cicadae ambrosia and its molecular mechanism will not only help to understand the relationship between fungal light response and secondary metabolism, but also provide a theoretical basis and technical support for regulating beauvericin production and improving the medicinal value of Cicadae ambrosia. At present, there is a lack of understanding of the key genes that regulate the synthesis of beauvericin in Cicadae ambrosia. Summary of the invention

[0006] In order to solve the problem that the prior art lacks knowledge of the key gene regulating the synthesis of beauvericin in Cicadae, the present invention provides a beauvericin synthesis regulatory gene CcWC-1 and its application. To achieve the above purpose, the present invention adopts the following technical scheme:

[0007] The present invention provides a beauvericin synthesis regulatory gene CcWC-1, and the nucleotide sequence of the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.1.

[0008] Beauvericin synthesis regulatory gene CcWC-1 is a key gene regulating the synthesis of beauvericin in cicada fungus. The present invention provides a beauvericin synthesis regulatory gene CcWC-1 by studying the gene and its mechanism of action, solving the problem of lack of knowledge of key genes regulating the synthesis of beauvericin in cicada fungus in the prior art. The present invention conducts in-depth research on the regulatory mechanism of beauvericin synthesis in cicada fungus, and provides the possibility of increasing the yield of beauvericin in cicada fungus by genetic engineering means.

[0009] Preferably, the protein expressed by the beauvericin synthesis regulatory gene CcWC-1 is the protein shown in (1) or (2) below:

[0010] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2.

[0011] (2) A protein derived from (1) by replacing and / or adding one to ten amino acid residues in the amino acid residue sequence as shown in SEQ ID NO. 2 and having the function of the beauvericin synthesis regulatory gene CcWC-1.

[0012] The present invention also provides application of the beauvericin synthesis regulatory gene CcWC-1 in regulating fungal metabolism.

[0013] Wherein, the beauvericin synthesis regulatory gene CcWC-1 is used to regulate the synthesis of beauvericin.

[0014] Preferably, genetic engineering is used to knock out the beauvericin synthesis regulatory gene CcWC-1, or reduce the expression level of the beauvericin synthesis regulatory gene CcWC-1, to obtain a cicada fungus mutant with a high beauvericin content, thereby regulating fungal metabolism.

[0015] The present invention also provides a fungal gene knockout vector, wherein the fungal gene knockout vector is a knockout vector targeting the beauvericin synthesis regulatory gene CcWC-1;

[0016] The fungal gene knockout vector comprises homologous recombination arms of 1 kb to 1.5 kb in the upstream and downstream ORF regions of the beauvericin synthesis regulatory gene CcWC-1, and a resistance screening marker gene Bar.

[0017] Preferably, the fungal gene knockout vector is based on the pDHt-SK-Bar plasmid and is constructed by recombination of Pst I and Xba I restriction sites.

[0018] Specifically, the fungal gene knockout vector selects 1kb to 1.5kb sequences as homologous recombination arms in the upstream and downstream ORF regions of the beauvericin synthesis regulatory gene CcWC-1, respectively, and recombines them into the pDHt-SK-Bar plasmid using multi-fragment ligase through the restriction sites of PstI and Xba I. The host cell structure finally constructed is: pDHt-SK-Bar skeleton-PstI-upstream homologous arm-Bar gene-downstream homologous arm-Xba I.

[0019] The nucleotide sequence of the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.1:

[0020]

[0021] The amino acid sequence of the protein expressed by the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.2:

[0022] MEGYYPSNQLPTQEQQRQRQNNISNTTQQQQQQQQQQQMPPSMSMPHGNTFTSQMMGNPTLLQRRASASLGFGARQLSDEVDARRMSLAVEQGAMNPPSTTGPGNFMGFQPSAQHLNNFSMLDTSNMGSMMPDGDGFPNLSPDAMGNLVSAQFANINMGAMFPSSSSAILGTQTSPTVIQLSMPSNDTIHFSNTAEYGAFTNPRDNTVPAPLQMSQLETPTNPSHDPRLGATAVGVDQSISSIPHHESSACSAASPVQEKSSSTGPTQPSTSSVATTPNAPATSRDLRERSIYSKSGFDMLKALWLVASRKNPSIDLGAVDMSCAFVVCDITMNDCPIIYVSDNFQNLTGYSSHDIVGQNCRFLQAPDGKVEAGTKREFVDNGAVFNLKKMIQERREVQQSLINYRKGGKPFLNLLTMIPIPWETDEIRYFIGFQIDLVECPDAIAGSEFGGVAVNYKHSDIGQYIWTPPSSAFWEADNGQTLGVDDVSTILQQFNAKGIASDWHRQSWDKMLLENCDDVIHVLSLKGLFLYLSPACKRVLEYDAAELVGNSLSSICHPSDIVPVTRELKDATTGDQVNIVFRIRRKQSGYTWFESHGSLYIEQGKGRKCIILVGRKRPVFSISRRNIESNGGIGDSELWTKLSTSGTFLYVSSNVRSLLDLQPESLVATSIQELMRKDSRPEFGRTLEKARRGKIVTCKHEVLNRRGQGLQAQTTLYPGDATEGQKPSFLLAQTKLLKASSRALAPAISTAGSTAGRSVHGQISQPGGGGLALGNQDEALASDDNIFDELRTTKCSSWQFELRQMEKVNRILAEELGGLLSSKKKRKRRKGVGNVVRDCANCHTRNTPEWRRGPSGQRDLCNSCGLRWAKQVSPPKSSVCPAMMRLTSLMSDGSSVTAQLCTQ*RFPKPKVCVTHTFITSAQGGICRRKLN*S*RQEISDNRNFGFRRFHILVYHQPAHEPHCQCWDSDGFNSRRARN*PT。

[0023] The present invention also provides a method for increasing the content of beauvericin in Cicadae Convolvulus, wherein the fungal gene knockout vector is introduced into Cicadae Convolvulus strains by using Agrobacterium-mediated genetic transformation to obtain Cicadae Convolvulus transgenic strains with high beauvericin content.

[0024] Preferably, a T-DNA vector containing a knockout sequence of the beauvericin synthesis regulatory gene CcWC-1 is constructed; the constructed T-DNA vector is transformed into Agrobacterium; the Agrobacterium carrying the T-DNA vector is co-cultured with a cicada confusa strain; the transformed cicada confusa strain is screened on a selective culture medium; and the cicada confusa transgenic strain with a high beauvericin content is regenerated to obtain the cicada confusa transgenic strain.

[0025] Among them, the cicada fungus transgenic strain with high beauvericin content is also called the cicada fungus transgenic strain with the beauvericin synthesis regulatory gene CcWC-1 knocked out, the cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1, or the cicada fungus mutant strain with the beauvericin synthesis regulatory gene CcWC-1 knocked out.

[0026] The co-culture conditions were 28° C. in the dark for 48 hours.

[0027] The selective culture medium is prepared as follows: 10 g glucose, 3 g KNO 3 , 15g agar powder, add 62.5mL M-100 salt solution, add 0.16g glufosinate ammonium, 0.27g thiophanate-methyl, and then add pure water to make up to 1L.

[0028] The present invention constructs a cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 by gene knockout technology, and compares the related traits of beauvericin synthesis of the cicada fungus with those of the cicada fungus wild-type strain. The results show that the beauvericin content of the cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 is significantly changed compared with the cicada fungus wild-type strain. Under light culture conditions, the beauvericin content of the cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 is significantly higher than that of the cicada fungus wild-type strain.

[0029] The present invention also provides a genetically engineered host cell, which is a cicada fungus mutant in which the beauvericin synthesis regulatory gene CcWC-1 is knocked out, and the beauvericin synthesis regulatory gene CcWC-1 is knocked out in its genome.

[0030] Specifically, the host cell is a cicada fungus strain transformed by gene knockout technology, wherein the beauvericin synthesis regulatory gene CcWC-1 is specifically knocked out and a Bar resistance marker gene is introduced.

[0031] The present invention also provides the use of the host cell in increasing the yield of beauvericin. Specifically, the host cell is cultured under light culture conditions to increase the yield of beauvericin in the host cell.

[0032] Among them, the light culture conditions are 12 hours of light and 12 hours of darkness alternating under 850lx white light, blue light or red light, and the culture temperature is controlled at 25°C.

[0033] The present invention also provides a primer pair for cloning the upstream and downstream homologous arms of the beauvericin synthesis regulatory gene CcWC-1.

[0034] The primer pair comprises an upstream homology arm primer pair and a downstream homology arm primer pair, and the upstream homology arm primer pair comprises an upstream homology arm primer-F and an upstream homology arm primer-R.

[0035] The nucleotide sequence of the upstream homology arm primer-F is shown in SEQ ID NO.3:

[0036] 5'-TTGATATCGAATTCCTGCAGCGATGGCTGTGTTGCATATG-3'.

[0037] The nucleotide sequence of the upstream homology arm primer-R is shown in SEQ ID NO.4:

[0038] 5'-GATCCCCCGGGCTGCAGCTGGGAGCTGATAACGCATAGGA-3'.

[0039] The downstream homology arm primer pair includes downstream homology arm primer-F and downstream homology arm primer-R.

[0040] The nucleotide sequence of the downstream homology arm primer-F is shown in SEQ ID NO.5:

[0041] 5'-CCGCCACCGCGGTGGAGCTCCTGCATTACCCAGCATACGA-3'.

[0042] The nucleotide sequence of the downstream homology arm primer-R is shown in SEQ ID NO.6:

[0043] 5'-GGGAACAAAAGCTGGAGCTCGCGGACTATTGACCTTTGCA-3'.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention provides a beauvericin synthesis regulatory gene CcWC-1 and its application. The present invention provides for the first time a Cicada Flower CcWC-1 gene related to the regulation of fungal metabolism, its encoded protein and application. The knockout vector of the beauvericin synthesis regulatory gene CcWC-1 was transferred into the wild-type Cicada Flower by Agrobacterium-mediated genetic transformation. The results showed that the content of the metabolite beauvericin in the knockout strain was significantly changed compared with the wild-type strain. The results provide a theoretical basis for studying how the fungal light sensing system regulates secondary metabolism, and also provide a theoretical basis and technical support for regulating the yield of beauvericin and improving the medicinal value of Cicada Flower. The present invention provides a beauvericin synthesis regulatory gene CcWC-1 by studying the gene and its mechanism of action, which solves the problem of the lack of recognition of the key gene regulating the synthesis of beauvericin in Cicada Flower in the prior art. The present invention conducts an in-depth study on the regulatory mechanism of beauvericin synthesis in Cicada Flower, and provides the possibility of increasing the yield of beauvericin in Cicada Flower by genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram showing the structural domain division of the protein encoded by the beauvericin synthesis regulatory gene CcWC-1 in the present invention.

[0047] Figure 2 The invention is to screen for the knockout strain of the beauvericin synthesis regulatory gene CcWC-1; wherein, Figure 2 Figure A in the figure is: PCR detection of target gene length; ΔCcWC-1: positive transformant of target gene knockout; C-ΔCcWC-1: positive transformant of target gene knockout complementation; WT: wild type; Bar: vector plasmid;

[0048] Figure 2 Figure B in the figure is: PCR detection of the target gene. ΔCcWC-1: positive transformant of target gene knockout; C-ΔCcWC-1: positive transformant of target gene knockout complementation: WT: wild type.

[0049] Figure 3 This is the phenotypic analysis of the knockout strain of the beauvericin synthesis regulatory gene CcWC-1 in the present invention; wherein, Figure 3 Figure A shows the effect of the beauvericin synthesis regulatory gene CcWC-1 on the mycelial growth of Cicadae confusa. Figure 3 Figure B shows the effect of the beauvericin synthesis regulatory gene CcWC-1 on the synthesis of beauvericin in Cicadae. DETAILED DESCRIPTION

[0050] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0051] The present invention provides a genetically engineered host cell, which is a Cordyceps sinensis cell, in which the beauvericin synthesis regulatory gene CcWC-1 is knocked out and contains a Bar resistance marker gene. The Cordyceps sinensis cell is also called a Cordyceps sinensis strain.

[0052] The beauvericin synthesis regulatory gene CcWC-1 provided by the present invention is a cicada light-sensitive gene. The beauvericin synthesis regulatory gene CcWC-1 is also called CcWC-1 gene, and its nucleotide sequence is shown in SEQ ID NO.1:

[0053]

[0054] The protein encoded by the CcWC-1 gene is the protein described in (1) or (2) below:

[0055] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2;

[0056] (2) A protein derived from (1) by replacing and / or adding one to ten amino acid residues in the amino acid residue sequence shown in SEQ ID NO. 2 and having the function of the CcWC-1 gene.

[0057] Wherein, the amino acid sequence shown in SEQ ID NO.2 is as follows:

[0058] MEGYYPSNQLPTQEQQRQRQNNISNTTQQQQQQQQQQQMPPSMSMPH

[0059] GNTFTSQMMGNPTLLQRRASASLGFGARQLSDEVDARRMSLAVEQGAMNP

[0060] PSTTGPGNFMGFQPSAQHLNNFSMLDTSNMGSMMPDGDGFPNLSPDAMGN

[0061] LVSAQFANINMGAMFPSSSSAILGTQTSPTVIQLSMPSNDTIHFSNTAEYGAFT

[0062] NPRDNTVPAPLQMSQLETPTNPSHDPRLGATAVGVDQSISSIPHHESSACSAA

[0063] SPVQEKSSSTGPTQPSTSSVATTPNAPATSRDLRERSIYSKSGFDMLKALWLVA

[0064] SRKNPSIDLGAVDMSCAFVVCDITMNDCPIIYVSDNFQNLTGYSSHDIVGQNC

[0065] RFLQAPDGKVEAGTKREFVDNGAVFNLKKMIQERREVQQSLINYRKGGKPF

[0066] LNLLTMIPIPWETDEIRYFIGFQIDLVECPDAIAGSEFGGVAVNYKHSDIGQYI

[0067] WTPPSSAFWEADNGQTLGVDDVSTILQQFNAKGIASDWHRQSWDKMLLEN

[0068] CDDVIHVLSLKGLFLYLSPACKRVLEYDAAELVGNSLSSICHPSDIVPVTRELK

[0069] DATTGDQVNIVFRIRRKQSGYTWFESHGSLYIEQGKGRKCIILVGRKRPVFSIS

[0070] RRNIESNGGIGDSELWTKLSTSGTFLYVSSNVRSLLDLQPESLVATSIQELMRK

[0071] DSRPEFGRTLEKARRGKIVTCKHEVLNRRGQGLQAQTTLYPGDATEGQKPSF

[0072] LLAQTKLLKASSRALAPAISTAGSTAGRSVHGQISQPGGGGLALGNQDEALA

[0073] SDDNIFDELRTTKCSSWQFELRQMEKVNRILAEELGGLLSSKKKRKRRKGV

[0074] GNVVRDCANCHTRNTPEWRRGPSGQRDLCNSCGLRWAKQVSPPKSSVCPA

[0075] MMRLTSLMSDGSSVTAQLCTQ*RFPKPKVCVTHTFITSAQGGICRRKLN*S*R

[0076] QEISDNRNFGFRRFHILVYHQPAHEPHCQCWDSDGFNSRRARN*PT.

[0077] The present invention provides the application of the CcWC-1 gene in regulating fungal metabolism, and specifically the application of the CcWC-1 gene in regulating the physiological, metabolic and light sensing processes of Cicadae.

[0078] The fungal gene knockout vector provided by the present invention is a pDHt-SK-Bar-CcWC-1 vector in which a CcWC-1 gene upstream homology arm, a Bar resistance marker gene, and a CcWC-1 gene downstream homology arm are sequentially connected in a multiple cloning site region.

[0079] The upstream and downstream homology arms were 1 to 1.5 kb sequences upstream and downstream of the ORF region of the CcWC-1 gene, respectively. These fragments were connected to the pDHt-SK-Bar plasmid backbone through the restriction sites of Pst I and Xba I.

[0080] The present invention also provides a host bacteria containing the gene knockout vector of the CcWC-1 gene, wherein the gene knockout vector of the CcWC-1 gene is a knockout vector targeting the CcWC-1 gene.

[0081] The present invention is used for cloning the primer pair of upstream and downstream homology arms of the CcWC-1 gene, and the primer pair comprises an upstream homology arm primer pair and a downstream homology arm primer pair.

[0082] Wherein, the upstream homology arm primer pair includes upstream homology arm primer-F and upstream homology arm primer-R.

[0083] The nucleotide sequence of the upstream homology arm primer-F is shown in SEQ ID NO.3:

[0084] 5'-TTGATATCGAATTCCTGCAGCGATGGCTGTGTTGCATATG-3'.

[0085] The nucleotide sequence of the upstream homology arm primer-R is shown in SEQ ID NO.4:

[0086] 5'-GATCCCCCGGGCTGCAGCTGGGAGCTGATAACGCATAGGA-3'.

[0087] The downstream homology arm primer pair includes downstream homology arm primer-F and downstream homology arm primer-R.

[0088] The nucleotide sequence of the downstream homology arm primer-F is shown in SEQ ID NO.5:

[0089] 5'-CCGCCACCGCGGTGGAGCTCCTGCATTACCCAGCATACGA-3'.

[0090] The nucleotide sequence of the downstream homology arm primer-R is shown in SEQ ID NO.6:

[0091] 5'-GGGAACAAAAGCTGGAGCTCGCGGACTATTGACCTTTGCA-3'.

[0092] The genetically engineered host cell of the present invention has the following characteristics: the CcWC-1 gene is specifically knocked out; it contains a Bar resistance marker gene, so that the cell can survive in a culture medium containing a specific antibiotic; and the cell's ability to respond to light is changed.

[0093] Example 1

[0094] 1. Materials

[0095] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0096] The cicada fungus strain used in this example is the RCEF5833 strain, which was isolated from the bamboo cicada (Platylomiapieli Kato) collected from Jingting Mountain in Xuancheng, Anhui Province, China, and is stored in the China Microbiological Culture Collection Center with a storage number of CGMCCNO.40986. The storage and culture information of the RCEF5833 strain is detailed in "Zhao C, Bu H, Zhu J, et al. Integration of untargeted metabolomics with transcriptomics provides insights into beauvericin biosynthesis in Cordyceps chanhua under H 2 O 2 -induced oxidativestress[J].Journal of Fungi,2022,8(5):484".

[0097] Among them, the Latin name of bamboo cicada is PlatylomiapieliKato.

[0098] The expression vector used in this example is pDHt-SK-Bar, which comes from the Key Laboratory of Insect Development and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Escherichia coli Top10 used for expression vector and Agrobacterium tumefaciens AGL1 for DNA homology replacement were purchased from Weidi Biotechnology.

[0099] The solution required for this embodiment and its preparation method are as follows:

[0100] (1) 2.5×MM salt solution for induction medium: accurately weigh 5.125 g K 2 H 2 PO 4 3.625g KH 2 PO4 、1.250g MgSO 4 7H 2 O, 0.375 g NaCl, 0.165 g CaCl 2 ·H 2 O, 0.0062 g FeSO 4 7H 2 O, 1.250 g (NH 4 ) 2 SO 4 Place in a beaker and add ddH 2 O, stir evenly, dilute to 1 L, store in a reagent bottle with a cover, and stir with a magnetic stirrer for 12 h.

[0101] 2.5× MM induction medium with saline solution is used to prepare IM medium.

[0102] (2) M-100 Trace Element Solution: Accurately weigh 0.03 g H 3 BO 3 , 0.07 g MnCl·4H 2 O, 0.20g ZnCl 2 , 0.02g Na 2 MoO 4 ·2H 2 O, 0.02 g FeCl 3 6H 2 O, 0.20 g CuSO 4 ·5H 2 O was placed in a beaker and ddH 2 O, stir evenly, dilute to 0.5L, store in a reagent bottle with a cover, and stir with a magnetic stirrer for 12h. M-100 trace element solution is used to prepare M-100 salt solution.

[0103] (3) M-100 Salt Solution: Accurately weigh 16 g KH 2 PO 4 4gNa 2 SO 4 , 8g KCl, 2g MgSO 4 7H 2 O, 1g CaCl 2 Place in a beaker and add 8 mL of M-100 trace element solution and ddH 2 O, stir evenly, dilute to 1 L, store in a reagent bottle with a cap, and stir with a magnetic stirrer for 12 h. M-100 salt solution is used to prepare M-100 culture medium.

[0104] (4) 1 mol / L acetosyringone solution: Accurately weigh 0.1962 g of acetosyringone, add 1 mL of dimethyl sulfoxide, shake well, filter using an organic filter membrane, and store at -20°C to obtain the solution. The English name of acetosyringone is Acetosyringone, AS. The English name of dimethyl sulfoxide is Dimethyl Sulphoxide, DMSO.

[0105] (5) Glufosinate reagent: Accurately weigh 0.3 g of glufosinate and add 1 mL of ddH 2 O and shake well, filter using an organic filter membrane to obtain the glufosinate-ammonium reagent, and store it at -20°C.

[0106] (6) Thioproterenol reagent: Accurately weigh 0.3 g of thioproterenol and add 1 mL of ddHO. 2 O and shake well, and filter using an organic filter membrane to obtain the product. Store the thiosporin reagent at -20°C.

[0107] (7) Kanamycin reagent: Accurately weigh 0.5 g of kanamycin and add 10 mL of ddHO 2 O and shake well, and filter using an organic filter membrane to obtain the kanamycin reagent. Store the kanamycin reagent at -20°C.

[0108] (8) Benomyl reagent: Accurately weigh 0.5 g of benomyl and add 10 mL of ddHO 2 O and shake well, and filter using an organic filter membrane to obtain the product. Store the benomyl reagent at -20°C.

[0109] The culture medium and its preparation method required for this embodiment are as follows:

[0110] (1) Luria-Bertani medium, LB medium: Weigh 10 g of tryptone, 10 g of yeast extract powder, 10 g of NaCl, and 15 g of agar powder respectively, add pure water to make up to 1 L, and autoclave at 121°C for 20 min.

[0111] Peptone medium, YEB medium: weigh 10g tryptone, 1g yeast extract powder, 5g sucrose, 0.5g MgSO 4 7H 2 O. Add 15g agar powder to 1L with pure water and sterilize in an autoclave at 121℃ for 20min.

[0112] Sabouraud glucose medium, SDAY medium: weigh 10g of peptone, 10g of yeast extract powder, 40g of glucose, and 15g of agar powder respectively, add pure water to make up to 1L, and autoclave at 121℃ for 20min.

[0113] Induction medium, IM: Weigh 0.36 g glucose and 3.154 g MES respectively, add 80 mL 2.5×MM induction medium salt solution, 1 mL 50% glycerol by volume, add pure water to make up to 200 mL, and autoclave at 121°C for 20 min.

[0114] Induction Medium, IM: Weigh 0.18 g glucose, 3.154 g MES, and 3 g agar powder respectively, add 80 mL 2.5×MM induction medium salt solution, 0.50 mL 50% glycerol by volume, add pure water to make up to 200 mL, autoclave at 121°C for 20 min, add 40 μL 1 mol / L AS solution when cooled to 45°C.

[0115] M-100 medium: weigh 10 g glucose, 3 g KNO 3 , 15g agar powder, add 62.5mL M-100 salt solution, add pure water to make up to 1L, and autoclave at 121℃ for 20min.

[0116] 2. Methods

[0117] 2.1 Cicada fungus strain culture

[0118] Pipette 100 μL of the cicada fungus strain RCEF5883 at a concentration of 1×10 7 The spore suspension of 1000 / mL was evenly spread on a SDAY plate covered with cellophane, and the plate was placed under 850lx white light, and cultured for 6 days with 12h light and 12h dark alternately, and the culture temperature was controlled at 25°C. The control group was cultured in the dark under the same conditions.

[0119] 2.2 Transcriptome library construction and sequencing

[0120] The total RNA was treated by mRNA enrichment method, the obtained RNA was fragmented by shearing buffer, reverse transcribed by random N6 primer, and then the cDNA second strand was synthesized to form double-stranded DNA. The ends of the synthesized double-stranded DNA were filled and phosphorylated at the 5' end, and a sticky end with a protruding "A" was formed at the 3' end, and then a bubble-shaped connector with a protruding "T" at the 3' end was connected. The connection product was PCR amplified by specific primers. The PCR product was heat-denatured into a single strand, and then a bridge primer was used to circularize the single-stranded DNA to obtain a single-stranded circular DNA library, which was then sequenced on the machine.

[0121] 2.3 Target gene screening and protein conserved functional domain analysis

[0122] The gene sequences obtained by transcriptome sequencing of Cicadae confusa were aligned to the protein databases nr, SwissProt, KEGG and COG / KOG. GO enrichment analysis and KEGG pathway enrichment analysis were combined to identify significantly enriched biological processes and metabolic pathways. The Cicada confusa light-sensitive gene CcWC-1 was screened out, compared and verified on the NCBI website and located on the Cicadae confusa total genome. The conserved functional domains of proteins were analyzed using the software SMART-PFAM.

[0123] Among them, the cicada fungus light-sensitive gene WC-1 is also called the CcWC-1 gene.

[0124] 2.4 Extraction of Cicadae genomic DNA

[0125] Take 0.30g of cicada fungus mycelium, put it in a 1.5mL centrifuge tube, add 2 steel balls, freeze it in liquid nitrogen for 60sec, put it in a shaker and grind it at 60Hz for 50sec, and repeat 3 times. Add 700μL CTAB extraction buffer, shake and mix, and place in a 65℃ water bath for 30min. Add 800μL of a mixed solvent of phenol, chloroform, and isopropanol, mix it upside down, and centrifuge it at 12000r / min for 10min. After centrifugation, transfer the aqueous phase to a new 1.5mL centrifuge tube; add an equal volume of a mixed solvent of chloroform and isopropanol, mix it upside down, and centrifuge it at 12000r / min for 10min. After centrifugation, transfer the supernatant to a new 1.5mL centrifuge tube; add an equal volume of isopropanol, mix it upside down, let it stand at -20℃ for 20min, centrifuge it at 13000r / min for 10min, and remove the supernatant. Add 1 mL of 75% ethanol, mix by inverting, centrifuge at 13,000 rpm for 1 min, remove the supernatant, and repeat once. After the residual ethanol in the centrifuge tube evaporates, add 50 μL of ddH 2 Dissolve DNA in 5% HO and store at -20°C.

[0126] The mixed solvent of phenol, chloroform and isopropanol is a solvent obtained by uniformly mixing phenol, chloroform and isopropanol in a volume ratio of 25:24:1.

[0127] The volume ratio of chloroform to isopropanol in the mixed solvent of chloroform and isopropanol is 24:1.

[0128] 2.5 Construction of knockout vector for target gene

[0129] Two homology arms were designed at 1114bp and 1105bp in the ORF regions upstream and downstream of the target gene at both ends, and two pairs of primers were designed at both ends of the homology arms, and the homologous sequences of PstⅠ and XbaⅠ were added in front of the primers. The genomic DNA of Cicadae confusa was used as a template for PCR amplification. The products were recombined into the pDHt-SK-Bar plasmid through the restriction sites of Pst I and Xba I using multi-fragment ligase, and introduced into the Escherichia coli Top10 strain for transformation. The positive transformants were selected, verified by PCR and DNA sequencing, and then transformed into Agrobacterium tumefaciens AGLT1. The positive transformants were picked, verified by PCR and DNA sequencing, and the bacterial solution was stored in a 4°C refrigerator for standby use.

[0130] Among them, PstⅠ: upper homology arm F / R and XbaⅠ: lower homology arm F / R.

[0131] The upstream homology arm amplification primer pair includes upstream homology arm primer-F and upstream homology arm primer-R.

[0132] The nucleotide sequence of the upstream homology arm primer-F is shown in SEQ ID NO.3:

[0133] 5'-TTGATATCGAATTCCTGCAGCGATGGCTGTGTTGCATATG-3'. The nucleotide sequence of the upstream homology arm primer-R is shown in SEQ ID NO.4: 5'-GATCCCCCGGGCTGCAGCTGGGAGCTGATAACGCATAGGA-3'. The downstream homology arm primer pair includes a downstream homology arm primer-F and a downstream homology arm primer-R. The nucleotide sequence of the downstream homology arm primer-F is shown in SEQ ID NO.5: 5'-CCGCCACCGCGGTGGAGCTCCTGCATTACCCAGCATACGA-3'. The nucleotide sequence of the downstream homology arm primer-R is shown in SEQ ID NO.6: 5'-GGGAACAAAAGCTGGAGCTCGCGGACTATTGACCTTTGCA-3'. The homology arm amplification PCR system is shown in Table 1:

[0134] Table 1 Homologous arm amplification PCR system

[0135] Components Addition volume, 20 μL system Cicadae RCEF6887 DNA template 1μL Upstream / downstream homology arm primer F 0.5μL Upstream / downstream homology arm primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10μL <![CDATA[ddH 2 The]]> 8μL

[0136] The homology arm amplification PCR reaction program is shown in Table 2:

[0137] Table 2 Homologous arm amplification PCR reaction program

[0138]

[0139] The enzyme digestion system of Pst I and Xba I is shown in Table 3:

[0140] Table 3 Enzyme digestion system of Pst I and Xba I

[0141]

[0142]

[0143] After mixing the sample, pipette to mix well and place in a 37°C water bath for 30 min.

[0144] The multi-fragment recombination system is shown in Table 4:

[0145] Table 4 Multi-fragment recombination system

[0146] Reaction components Addition volume, 20μL system Multi-fragment recombinase 2μL Buffer 4μL Enzyme digestion products (length × 0.02) / concentration μL Upstream homology arm amplification product (length × 0.04) / concentration μL Downstream homology arm amplification product (length × 0.04) / concentration μL <![CDATA[ddH 2 The]]> Up to 20 μL

[0147] After mixing the sample, pipette to mix well, centrifuge briefly, and place in a 50°C water bath for 30 min.

[0148] The target gene length verification primer pair includes a length verification primer-F and a length verification primer-R. The nucleotide sequence of the length verification primer-F is shown in SEQ ID NO.7: 5'-TACCCGCCGAGAAATTATCC-3'.

[0149] The nucleotide sequence of the length verification primer-R is shown in SEQ ID NO.8: 5'-AGAAGAGGAACAAAGGCGTC-3'.

[0150] The target gene presence verification primer pair includes presence verification primer-F and presence verification primer-R. The nucleotide sequence of presence verification primer-F is shown in SEQ ID NO.9: 5'-TATCAGCAACACGACACAGC-3'.

[0151] The nucleotide sequence of the existence verification primer-R is shown in SEQ ID NO. 10: 5'-GTTTGTGTGCCAAGTATGGC-3'.

[0152] The verification PCR system is shown in Table 5:

[0153] Table 5 Verification of PCR system

[0154] Components Addition volume, 20 μL system Positive transformant DNA template 1μL Primer F 0.5μL Primer R 0.5μL 2×Taq Plus Master Mix (Dye) 10μL <![CDATA[ddH 2 The]]> 8μL

[0155] The verification PCR reaction program is shown in Table 6:

[0156] Table 6 Verification PCR reaction program

[0157]

[0158] 2.6 Transformation of E. coli Top10 strain

[0159] Take the E. coli Top10 strain and place it on ice for 15 minutes. Add 10 μL of the recombinant ligation product to every 100 μL of E. coli, mix well by pipetting, and place it on ice for 25 minutes. Heat shock at 42℃ for 45 seconds. After the heat shock, gently move it to ice and place it for 3 minutes. Add 800 μL of LB liquid culture medium without antibiotics, put it in a 37℃ shaker at 220 r / min for 1 hour, centrifuge at 6000 r / min for 1 minute to collect E. coli into the tube, discard 650 μL of supernatant, resuspend the bacterial liquid, and evenly spread it on a plate containing LB solid culture medium with kanamycin reagent. Incubate it in a 37℃ incubator for 16 hours. Pick the suspected positive transformants and place them in a centrifuge tube containing LB liquid culture medium with kanamycin reagent. After incubating it in a 37℃ shaker at 220 r / min for 6 hours, verify it by PCR, and select the positive transformants for sequencing.

[0160] The volume ratio of kanamycin reagent to LB solid culture medium is 100 μL / 100 mL.

[0161] The volume ratio of the kanamycin reagent to the LB liquid culture medium was 100 μL / 100 mL.

[0162] The nucleotide sequence of the PCR verification primer is the same as the upstream and downstream homology arm amplification primer sequences. The verification PCR system is shown in Table 7:

[0163] Table 7 Verification of PCR system

[0164] Components Addition volume, 20μL system Escherichia coli liquid 1μL Primer F 0.5μL Primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10μL <![CDATA[ddH 2 The]]> 8μL

[0165] The verification PCR reaction program is shown in Table 8:

[0166] Table 8 Verification of PCR reaction procedures

[0167]

[0168] 2.7 Transformation of Agrobacterium tumefaciens strain AGL1

[0169] Take the Agrobacterium AGL1 strain and put it in the palm of your hand to melt it. When it becomes an ice-water mixture, put it on ice to stand. Add 100ng of plasmid to every 100μL of Agrobacterium, mix it by blowing and let it stand for 5min, put it in liquid nitrogen for quick freezing for 5min, put it in a 37℃ water bath for water bath for 5min, and then put it on ice to stand for 5min; add 700μL of YEB liquid culture medium without antibiotics, put it in a shaker at 28℃ and 220r / min for recovery for 3h, centrifuge it at 8000r / min for 1min to collect Agrobacterium to the tube body, discard 650μL of supernatant, resuspend the bacterial liquid, evenly spread it on a plate of YEB solid culture medium containing the corresponding antibiotics, and culture it in a 28℃ incubator for 48h. Pick the suspected positive transformants in a centrifuge tube containing YEB liquid culture medium with the corresponding antibiotics, culture it in a shaker at 220r / min and 28℃ for 6h, verify it by PCR, and select the corresponding positive transformants for sequencing.

[0170] The PCR verification primer nucleotide sequence is the same as the upstream and downstream homology arm amplification primer sequence, and the verification PCR system is shown in Table 9:

[0171] Table 9 Verification of PCR system

[0172] Components Addition volume, 20 μL system Agrobacterium tumefaciens liquid 1μL Primer F 0.5μL Primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10μL <![CDATA[ddH 2 The]]> 8μL

[0173] The verification PCR reaction program is shown in Table 10:

[0174] Table 10 Verification of PCR reaction procedures

[0175]

[0176] 2.8 Agrobacterium-mediated genetic transformation of Cicadae

[0177] The Agrobacterium containing the recombinant plasmid was placed in 15 mL of YEB liquid medium containing kanamycin and cultured in a shaking incubator at 220 r / min and 28°C for 16 h. The Agrobacterium was then transferred to IM liquid medium containing AS and its OD was measured. 660 The value was between 0.15 and the culture was placed in a shaking incubator at 220 r / min and 28°C for 6 hours, so that the OD 660 The value is about 0.5. The spores of Cicadae confusa on the 12th day of culture were selected and placed in 1 mL of 0.5% Tween 80 solution by volume from the SDAY plate. The spores were quickly shaken for 5 min using a vortex shaker to evenly spread the spores in the solution. The mycelium and impurities were filtered out using a filter and the spore suspension was diluted to a concentration of 1×10 6 / mL. Take equal amounts of Agrobacterium bacterial suspension and Cicadae conidia suspension, mix them with a vortex shaker, and evenly apply them on the surface of IM solid culture medium containing AS, and culture them at 28℃ in the dark for 48h. Use M-100 solid culture medium containing thiosporin and the corresponding resistance reagent glufosinate ammonium of the carrier to cover the IM solid culture medium after 48h of culture, culture at 25℃ for 7d, pick the mycelium grown on the M-100 solid culture medium, and place it on the M-100 solid culture medium plate containing thiosporin and the corresponding resistance reagent glufosinate ammonium of the carrier for secondary resistance screening. The screened bacteria are verified by PCR, and finally positive transformants are obtained.

[0178] The volume ratio of kanamycin reagent to YEB liquid culture medium is 100 μL / 100 mL.

[0179] The volume ratio of AS to IM solid culture medium was 20 μL / 100 mL.

[0180] The volume ratio of thiophanate-methyl to M-100 solid culture medium is 90 μL / 100 mL, and the volume ratio of the carrier-corresponding resistance reagent glufosinate-ammonium to M-100 solid culture medium is 80 μL / 100 mL.

[0181] 2.9 Determination of the growth of cicada fungus mycelium

[0182] The concentration of each was 1×10 7 The CcWC-1 knockout strain and wild-type spore suspensions at 100 / mL were evenly spread on SDAY plates covered with cellophane, and the plates were placed under 850lx white light, blue light, and red light, respectively, and cultured for 6 days with 12h light and 12h dark alternation, and the culture temperature was controlled at 25°C. When the cicada fungus was cultured to the 6th day, all the cultures were collected, freeze-dried and weighed.

[0183] 2.10 Sample processing of cicada fungus culture

[0184] When the cicada fungus is cultured to the 6th day, the culture on the cellophane is collected, quenched with liquid nitrogen, freeze-dried in a freeze dryer at -80℃ for 24h, ground into powder, weigh 100mg of mycelium in a 5mL centrifuge tube, add 3mL of 80% methanol by volume, ultrasonically clean for 30min, shake once every 10min, take out the centrifuge tube, and centrifuge at 12000rpm for 5min. After centrifugation, take 2mL of supernatant into a new centrifuge tube, place the opening of the centrifuge tube containing 2mL of supernatant in a centrifugal vacuum concentrator, and concentrate to dryness at 40℃. Take out the centrifuge tube, add 500μL of 90% methanol by volume for ultrasonic re-dissolution, centrifuge to take the supernatant, filter with a 0.22μm pore size filter membrane, obtain the sample extract, place it in an injection bottle, and prepare for HPLC use.

[0185] 2.11 Establishment of BEA standard curve and determination of BEA content

[0186] The sample extract was detected using an Agilent Eclipse XDB-C18 column. The mobile phase was a mixed solvent of distilled water and acetonitrile in a volume ratio of 30:70; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 30°C; detection wavelength: 215 nm; elution time: 30 min.

[0187] Among them, Agilent Eclipse XDB-C18 chromatographic column: 2.1×150mm, 5μm.

[0188] Accurately weigh 0.001 g of BEA standard, dissolve it in methanol to prepare a 300 μg / mL solution, and then dilute it sequentially into standard solutions with concentrations of 270 μg / mL, 240 μg / mL, 210 μg / mL, 180 μg / mL, 150 μg / mL, 120 μg / mL, 90 μg / mL, 60 μg / mL, and 30 μg / mL.

[0189] The above standard solutions of different concentrations are respectively injected into the chromatograph for chromatographic analysis according to the above detection conditions. After chromatographic analysis, a standard curve is obtained with concentration as the horizontal axis and peak area as the vertical axis.

[0190] The BEA content in the sample was obtained according to the standard curve and the peak area of ​​the test sample to prepare the standard curve: y = 44796x-148066, the correlation coefficient R 2 =0.9996, indicating that the standard curve is reliable in the range of 30-270 μg / mL.

[0191] 3. Experimental results

[0192] 3.1 Domain division of the protein encoded by the CcWC-1 gene

[0193] As attached Figure 1 As shown, CcWC-1 contains 3 PAS domains, 2 PAC domains and 1 GATA-type zinc finger domain. The PAS domain is a signal sensor domain that participates in a variety of signal proteins. The PAC motif appears at the C-terminus of a subset of all known PAS motifs. The PAS domain is usually associated with the PAC domain, and together they form a conserved 3D PAS fold. WC-1 and WC-2 are believed to be able to form heterodimers WCC through their respective PAS domains, thereby activating Frq periodic transcription; and the GATA-type zinc finger domain is a domain unique to GATA-type transcription factors, which participates in transcriptional regulation through sequence-specific DNA binding and zinc ion binding.

[0194] 3.2 Screening of CcWC-1 gene knockout strains

[0195] By constructing the vector plasmid pDHt-SK-Bar-CcWC-1, the CcWC-1 knockout positive transformant was obtained by Agrobacterium-mediated cicada fungus genetic transformation, and the PCR length verification was performed. The results are shown in the attached figure. Figure 2 As shown in Figure A, with WT and vector plasmid as controls, the target band is similar in size to the vector plasmid band and is significantly different from WT; the designed primers for CcWC-1 were used for PCR verification, with WT as the control, and the results are as follows Figure 2 As shown in Figure B, no bands similar to WT could be detected in the obtained positive transformants. Further screening was performed using glufosinate resistance, and all three obtained positive transformants were resistant to glufosinate. Therefore, it was confirmed that the obtained positive transformants were CcWC-1 knockout strains.

[0196] 3.3 Phenotypic analysis of CcWC-1 gene knockout strains

[0197] Figure 3 Figure A shows the dry weight of mycelium of the CcWC-1 gene knockout strain ΔCcWC-1 and the wild type WT on the 6th day of growth under different conditions. Among them, the dry weight of mycelium of ΔCcWC-1 was significantly higher than that of WT under white light culture conditions (P<0.01), blue light culture conditions (P<0.05), and red light culture conditions (P<0.01), indicating that the loss of CcWC-1 promoted the mycelium growth of Cicada fungus under light culture.

[0198] Figure 3 Figure B shows the BEA content in freeze-dried cultures of the CcWC-1 knockout strain ΔCcWC-1 and the wild-type WT on the 6th day of growth under different conditions. The BEA content in ΔCcWC-1 was significantly higher than that in WT under white light, blue light, and red light illumination conditions (P<0.01), indicating that the loss of CcWC-1 promoted the BEA synthesis of cicada fungus under light cultivation. Among them, BEA is the English name of beauvericin.

[0199] From the above experimental results, it can be seen that the CcWC-1 gene can act as a negative regulatory factor in cicada fungus, inhibiting hyphae growth and BEA synthesis. The loss of this gene leads to accelerated hyphae growth and increased BEA content, and this effect exists under different light conditions. This discovery reveals the important role of the CcWC-1 gene in the light response and metabolic regulation of cicada fungus, and provides a potential strategy for increasing the growth rate of cicada fungus and the production of secondary metabolites such as BEA through genetic engineering. Among them, different light refers to white light, blue light, and red light.

[0200] The present invention provides a beauvericin synthesis regulatory gene CcWC-1 by studying the gene and its mechanism of action, solving the problem of the lack of knowledge of key genes regulating the synthesis of beauvericin in the prior art. The present invention conducts an in-depth study on the synthesis regulatory mechanism of beauvericin in cicada flower, and provides the possibility of increasing the yield of beauvericin in cicada flower by genetic engineering means.

[0201] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0202] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A beauvericin synthesis regulatory gene CcWC-1, It is characterized in that The nucleotide sequence of the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.

1.

2. The beauvericin synthesis regulatory gene CcWC-1 according to claim 1, It is characterized in that The amino acid sequence of the protein expressed by the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.

2.

3. Use of the beauvericin synthesis regulatory gene CcWC-1 according to claim 1 in regulating fungal metabolism.

4. The use according to claim 3, It is characterized in that By using genetic engineering means, the beauvericin synthesis regulatory gene CcWC-1 is knocked out, or the expression level of the beauvericin synthesis regulatory gene CcWC-1 is reduced, and a cicada fungus mutant with a high beauvericin content is obtained, thereby regulating fungal metabolism.

5. A fungal gene knockout vector, It is characterized in that The fungal gene knockout vector is a knockout vector targeting the beauvericin synthesis regulatory gene CcWC-1 as claimed in claim 1; The fungal gene knockout vector comprises homologous recombination arms of 1 kb to 1.5 kb in the upstream and downstream ORF regions of the beauvericin synthesis regulatory gene CcWC-1, and a resistance screening marker gene Bar.

6. A fungal gene knockout vector according to claim 5, It is characterized in that The fungal gene knockout vector is based on the pDHt-SK-Bar plasmid and is constructed by recombination of Pst I and Xba I restriction sites.

7. A method for increasing the content of beauvericin in Cicadae convolvulus, It is characterized in that The fungal gene knockout vector as claimed in claim 5 is introduced into the Cicadae Constricta strain by using Agrobacterium-mediated genetic transformation to obtain a Cicadae Constricta transgenic strain with a high content of beauvericin.

8. The method according to claim 7, It is characterized in that A T-DNA vector containing a knockout sequence of the beauvericin synthesis regulatory gene CcWC-1 is constructed; the constructed T-DNA vector is transformed into Agrobacterium; the Agrobacterium carrying the T-DNA vector is co-cultured with a cicada confusa strain, and then the cicada confusa transgenic strain with a high beauvericin content is obtained through selection, screening and regeneration.

9. A genetically engineered host cell, It is characterized in that The beauvericin synthesis regulatory gene CcWC-1 as claimed in claim 1 is knocked out in the genome of the host cell.

10. Use of the host cell according to claim 9 in increasing the yield of beauvericin, It is characterized in that The host cells are cultured under light conditions to increase the beauvericin production of the host cells.

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