A beauvericin synthesis regulatory gene CcWC-1 and its application

Abstract

The present invention belongs to the technical field of fungal molecular biology, and particularly relates to a beauvericin synthesis regulatory gene CcWC-1 and its application. The nucleotide sequence of the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.1. The present invention first provides the Cordyceps cicadae CcWC-1 gene related to the regulation of beauvericin synthesis, its encoded protein and application. By constructing a CcWC-1 gene knockout strain, the results show that compared with the wild-type strain, the beauvericin content of the knockout strain has changed significantly. Under light culture conditions, the beauvericin content of the CcWC-1 knockout strain is significantly higher than that of the wild type. The present invention reveals the regulatory mechanism of light on the synthesis of beauvericin in Cordyceps cicadae, provides new gene resources and production strategies for the artificial and efficient production of beauvericin, and helps to increase the yield of beauvericin.

Description

Technical Field

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

[0002] Cicada fungus is an important medicinal fungus with a long history of use in traditional Chinese medicine. Modern research has shown that cicada fungus contains a variety of bioactive components, including nucleosides, cordycepic acid, polysaccharides, ergosterol and its derivatives, and cyclic peptides. These active components contribute to its diverse pharmacological effects, including anti-fatigue, sedation and analgesia, blood pressure and blood sugar reduction, and anti-tumor properties. The Latin name for cicada fungus is Cordyceps chanhua.

[0003] Beauvericin is an important cyclic peptide active substance in cicada fungus. Beauvericin was originally isolated from Beauveria bassiana and was later discovered in various fungi. As a compound with multiple biological activities, beauvericin has attracted widespread attention from researchers. Studies have shown that beauvericin has multiple biological activities, including antibacterial, antiviral, anti-inflammatory, antioxidant, and anti-tumor activities. In terms of anti-tumor effects, beauvericin has shown growth inhibition effects on various 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 a key environmental factor influencing fungal growth and secondary metabolism. Studies have shown that light can regulate the growth, morphogenesis, and synthesis of secondary metabolites in a variety of fungi. For example, in Aspergillus flavus, light can affect the synthesis of aflatoxin, while in Cordyceps militaris, white and blue light can promote the synthesis of carotenoids. However, little research has examined the effects of light on the synthesis of beauvericin in Cicadae.

[0005] The light response of fungi involves a complex photosensitivity and signal transduction system. Among them, WC-1 (White Collar-1) encodes an important blue light receptor that participates in the regulation of 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 decrease in pigment synthesis. Therefore, in-depth research on the effects of light on the synthesis of beauvericin in Cicadae 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. At present, there is a lack of understanding of the key genes that regulate the synthesis of beauvericin in Cicadae. Summary of the Invention

[0006] To address the problem of the lack of understanding of key genes regulating the synthesis of beauvericin in the prior art, the present invention provides a beauvericin synthesis regulatory gene CcWC-1 and its application. To achieve the above objectives, the present invention adopts the following technical solutions:

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

[0008] The beauvericin synthesis regulatory gene CcWC-1 is a key gene that regulates beauvericin synthesis in cicada fungus. This present invention, through research on this gene and its mechanism of action, provides a beauvericin synthesis regulatory gene CcWC-1, addressing the prior art's lack of understanding of key genes regulating beauvericin synthesis. This present invention, through in-depth research on the regulatory mechanism of beauvericin synthesis in cicada fungus, offers the potential for increasing beauvericin production in cicada fungus through genetic engineering.

[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 shown in SEQ ID NO. 2 and having the function of the beauvericin synthesis regulatory gene CcWC-1.

[0012] The present invention also provides the use 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 1kb to 1.5kb homologous recombination arms of 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 enzyme sites.

[0018] Specifically, the fungal gene knockout vector selects 1kb to 1.5kb sequences from the upstream and downstream ORF regions of the beauvericin synthesis regulatory gene CcWC-1 as homologous recombination arms, respectively, and is recombined into the pDHt-SK-Bar plasmid using multi-fragment ligase through the restriction sites of PstI and Xba I. The final host cell structure constructed is: pDHt-SK-Bar skeleton-PstI-upstream homology arm-Bar gene-downstream homology 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 beauvericin content of Cicadae Convolvulus, which uses Agrobacterium-mediated genetic transformation to introduce the fungal gene knockout vector into Cicadae Convolvulus strains to obtain transgenic Cicadae Convolvulus 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 conker strain; the transformed cicada conker strain is screened on a selective culture medium; and the cicada conker transgenic strain with a high beauvericin content is regenerated to obtain the cicada conker 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 with the beauvericin synthesis regulatory gene CcWC-1 deleted, or the cicada fungus mutant strain with the beauvericin synthesis regulatory gene CcWC-1 knocked out.

[0026] The co-culture conditions were as follows: incubation at 28°C in the dark for 48 hours.

[0027] The selective culture medium was prepared as follows: 10 g glucose, 3 g KNO3, 15 g agar powder, 62.5 mL M-100 salt solution, 0.16 g glufosinate ammonium, 0.27 g thiophanate-methyl, and pure water were added to make the volume 1 L.

[0028] The present invention constructed a cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 through gene knockout technology, and compared the beauvericin synthesis-related traits of the strain with those of a wild-type cicada fungus strain. The results showed that the beauvericin content of the cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 was significantly altered compared with the wild-type cicada fungus strain. Under light culture conditions, the beauvericin content of the cicada fungus mutant strain lacking the beauvericin synthesis regulatory gene CcWC-1 was significantly higher than that of the wild-type cicada fungus 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 the host cell's 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 a 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] The light culture conditions are 850 lx white light, blue light or red light, with alternating 12 hours of light and 12 hours of darkness, and the culture temperature is controlled at 25°C.

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

[0034] The primer pair includes an upstream homology arm primer pair and a downstream homology arm primer pair, and the upstream homology arm primer pair includes 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 a downstream homology arm primer-F and a 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 Cone CcWC-1 gene that regulates fungal metabolism, its encoded protein, and its application. The knockout vector of the beauvericin synthesis regulatory gene CcWC-1 was introduced into the wild-type Cicada Cone 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. This result provides a theoretical basis for studying how the fungal light sensing system regulates secondary metabolism, and also provides a theoretical basis and technical support for regulating the production of beauvericin and improving the medicinal value of Cicada Cone. By studying the gene and its mechanism of action, the present invention provides a beauvericin synthesis regulatory gene CcWC-1, which solves the problem of the lack of understanding of the key genes that regulate the synthesis of beauvericin in Cicada Cone in the prior art. The present invention conducts in-depth research on the regulatory mechanism of beauvericin synthesis in Cicada Cone, and provides the possibility of increasing the yield of beauvericin in Cicada Cone through genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0047] Figure 2 Screening of knockout strains of the beauvericin synthesis regulatory gene CcWC-1 in the present invention; wherein, Figure 2 Figure A: 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 Panel B shows the presence of the target gene by PCR. ΔCcWC-1: positive transformant for target gene knockout; C-ΔCcWC-1: positive transformant for target gene knockout complementation; WT: wild type.

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

[0050] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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 Cicadae confusa cell, in which the beauvericin synthesis regulatory gene CcWC-1 is knocked out and contains a Bar resistance marker gene. The Cicadae confusa cell is also called a Cicadae confusa 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 the 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 use of the CcWC-1 gene in regulating fungal metabolism, particularly 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 the upstream homologous arm of the CcWC-1 gene, the Bar resistance marker gene, and the downstream homologous arm of the CcWC-1 gene are sequentially connected in the multiple cloning site region.

[0079] The upstream and downstream homology arms were sequences of 1 to 1.5 kb 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 a downstream homology arm primer-F and a 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, which enables the cell to 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] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0096] The cicada fungus strain used in this example is RCEF5833, which was isolated from Platylomiapieli Kato collected from Jingting Mountain in Xuancheng, Anhui Province, China, and is deposited in the China Center for Microbiological Culture Collection with the accession number CGMCC NO. 40986. For details on the preservation and cultivation of the RCEF5833 strain, see "Zhao C, Bu H, Zhu J, et al. Integration of untargeted metabolomics with transcriptomics provides insights into beauvericin biosynthesis in Cordyceps chanhua under H2O2-induced oxidative stress[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 was pDHt-SK-Bar, which was obtained from the Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Escherichia coli Top10 and Agrobacterium tumefaciens AGL1 for DNA homologous 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 5.125g K2H2PO4, 3.625g KH2PO4, 1.250g MgSO4·7H2O, 0.375g NaCl, 0.165g CaCl2·H2O, 0.0062g FeSO4·7H2O, and 1.250g (NH4)2SO4 and place them in a beaker. Add ddH2O and stir evenly. Make up to 1L and store in a reagent bottle with a cap. Stir with a magnetic stirrer for 12h.

[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 H₃BO₃, 0.07 g MnCl·4H₂O, 0.20 g ZnCl₂, 0.02 g Na₂MoO₄·2H₂O, 0.02 g FeCl₃·6H₂O, and 0.20 g CuSO₄·5H₂O into a beaker. Add ddH₂O and stir until the volume is 0.5 L. Store in a reagent bottle with a cap and stir with a magnetic stirrer for 12 h. The M-100 Trace Element Solution is used to prepare the M-100 salt solution.

[0103] (3) M-100 Salt Solution: Accurately weigh 16 g KH2PO4, 4 g Na2SO4, 8 g KCl, 2 g MgSO4·7H2O, and 1 g CaCl2 into a beaker. Add 8 mL M-100 trace element solution and ddH2O, mix well, and dilute to 1 L. Store in a reagent bottle with a cap and stir using 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 through an organic filter membrane, and store at -20°C. Acetosyringone is called Acetosyringone (AS). Dimethyl sulfoxide is called Dimethyl Sulphoxide (DMSO).

[0105] (5) Glufosinate reagent: Accurately weigh 0.3 g of glufosinate, add 1 mL of ddH2O, shake well, and filter using an organic filter membrane. The glufosinate reagent was stored at -20°C.

[0106] (6) Thiaprotinin reagent: Accurately weigh 0.3 g of thiaprotinin, add 1 mL of ddH2O, shake well, and filter through an organic filter membrane. Store the thiaprotinin reagent at -20°C.

[0107] (7) Kanamycin reagent: Accurately weigh 0.5 g of kanamycin, add 10 mL of ddH2O, shake well, and filter through an organic filter membrane. Store the kanamycin reagent at -20°C.

[0108] (8) Benomyl reagent: Accurately weigh 0.5 g of benomyl, add 10 mL of ddH2O, shake well, and filter through an organic filter membrane. Store the benomyl reagent at -20°C.

[0109] The culture medium required for this embodiment and its preparation method 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, add purified water to make up to 1 L, and autoclave at 121°C for 20 min.

[0111] Peptone medium, YEB medium: Weigh 10 g of tryptone, 1 g of yeast extract powder, 5 g of sucrose, 0.5 g of MgSO4·7H2O, and 15 g of agar powder respectively, add purified water to make up to 1 L, and autoclave at 121°C for 20 min.

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

[0113] Induction Medium, IM: Weigh 0.36 g glucose and 3.154 g MES respectively, add 80 mL 2.5× MM induction medium with salt solution, 1 mL 50% glycerol by volume, add purified 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% by volume glycerol, and add purified water to make up to 200 mL. Autoclave at 121°C for 20 min. When cooled to 45°C, add 40 μL 1 mol / L AS solution.

[0115] M-100 culture medium: Weigh 10 g glucose, 3 g KNO3, and 15 g agar powder respectively, add 62.5 mL M-100 salt solution, add pure water to make up to 1 L, and autoclave at 121°C for 20 min.

[0116] 2. Methods

[0117] 2.1 Cicada fungus strain culture

[0118] 100 μL of the cicada fungus strain RCEF5883 was drawn to a concentration of 1×10 7 A spore suspension of 1000 spores / mL was evenly spread on a SDAY plate covered with cellophane. The plate was placed under 850 lx white light and cultured for 6 days with alternating periods of 12 hours of light and 12 hours of darkness at 25°C. A control group was cultured in the dark under the same conditions.

[0119] 2.2 Transcriptome library construction and sequencing

[0120] Total RNA is processed using the mRNA enrichment method, fragmented using a shearing buffer, and reverse transcribed using random N6 primers. The cDNA second strand is then synthesized to form double-stranded DNA. The synthesized double-stranded DNA is blunt-ended and phosphorylated at the 5' end. A sticky end with an "A" protruding from the 3' end is then formed and ligated to a bubble-shaped adapter with a protruding "T" at the 3' end. The ligated product is amplified by PCR using specific primers. The PCR product is heat-denatured into single strands, and a bridge primer is then used to circularize the single-stranded DNA to generate a single-stranded circular DNA library, which is then sequenced.

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

[0122] Gene sequences derived from the cicada convolvulus transcriptome 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 convolvulus light-sensitive gene CcWC-1 was screened and verified by alignment with the NCBI website and mapped to the cicada convolvulus genome. Conserved functional domains of the proteins were analyzed using the SMART-PFAM software.

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

[0124] 2.4 Extraction of Cicada Fungus Genomic DNA

[0125] 0.30 g of Cicada fungus mycelium was placed in a 1.5 mL centrifuge tube. Two steel balls were added, and the tube was quickly frozen in liquid nitrogen for 60 seconds. The tube was then ground in a shaker at 60 Hz for 50 seconds, and repeated three times. 700 μL of CTAB extraction buffer was added, the tube was shaken to mix, and the tube was incubated at 65°C for 30 minutes. 800 μL of a mixture of phenol, chloroform, and isopropanol was added, the tube was inverted to mix, and the tube was centrifuged at 12,000 rpm for 10 minutes. After centrifugation, the aqueous phase was transferred to a new 1.5 mL centrifuge tube, an equal volume of a mixture of chloroform and isopropanol was added, the tube was inverted to mix, and the tube was centrifuged at 12,000 rpm for 10 minutes. After centrifugation, the supernatant was transferred to a new 1.5 mL centrifuge tube, an equal volume of isopropanol was added, the tube was inverted to mix, the tube was allowed to stand at -20°C for 20 minutes, and the tube was centrifuged at 13,000 rpm for 10 minutes. The supernatant was discarded. Add 1 mL of 75% ethanol by volume, mix thoroughly by inverting, centrifuge at 13,000 rpm for 1 minute, remove the supernatant, and repeat once. After evaporating any remaining ethanol in the tube, dissolve the DNA in 50 μL of ddH2O 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 target gene knockout vector

[0129] Two homology arms were designed at 1114bp and 1105bp in the ORF regions upstream and downstream of the target gene, respectively. Two pairs of primers were designed at both ends of the homology arms and homologous sequences of PstⅠ and XbaⅠ were added before the primers. The genomic DNA of cicada fungus was used as a template for PCR amplification. The products were recombined into the pDHt-SK-Bar plasmid using multi-fragment ligase through the restriction sites of Pst I and Xba I, 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 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 Homology arm amplification PCR system

[0135] Components Addition volume, 20 μL system Cicadae RCEF6887 DNA template 1 μL Upstream / downstream homology arm primer F 0.5μL Upstream / downstream homology arm primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10 μL <![CDATA[ddH2O]]> 8μL

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

[0137] Table 2 PCR reaction procedures for homology arm amplification

[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 thoroughly 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, 20 μL 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[ddH2O]]> Up to 20 μL

[0147] After mixing the sample, pipette to mix thoroughly, 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 presence 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 1 μL Primer F 0.5μL Primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10 μL <![CDATA[ddH2O]]> 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 thoroughly by pipetting, and let it stand for 25 minutes. Heat shock at 42°C for 45 seconds. After the heat shock, gently move it to ice and let it stand for 3 minutes. Add 800 μL of LB liquid medium without antibiotics and place it on a 37°C shaker at 220 r / min for 1 hour. Centrifuge at 6000 r / min for 1 minute to collect the E. coli into the tube. Discard 650 μL of the supernatant, resuspend the bacteria, and evenly spread it on a plate of LB solid medium containing kanamycin reagent. Incubate in a 37°C incubator for 16 hours. Pick suspected positive transformants and place them in a centrifuge tube of LB liquid medium containing kanamycin reagent. After incubation at 220 r / min and 37°C shaker for 6 hours, verify by PCR, and select 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 kanamycin reagent to LB liquid culture medium was 100 μL / 100 mL.

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

[0163] Table 7 Verification PCR system

[0164] Components Addition volume, 20μL system Escherichia coli liquid 1 μL Primer F 0.5μL Primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10 μL <![CDATA[ddH2O]]> 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 tumefaciens AGL1 strain and thaw it in the palm of your hand. Once it becomes an ice-water mixture, place it on ice. Add 100 ng of plasmid to every 100 μL of Agrobacterium, mix thoroughly by pipetting, and let it stand for 5 minutes. Then, quickly freeze it in liquid nitrogen for 5 minutes, place it in a 37°C water bath for 5 minutes, and then let it stand on ice for another 5 minutes. Add 700 μL of antibiotic-free YEB liquid medium, and resuscitate it in a shaker at 220 rpm at 28°C for 3 hours. Centrifuge it at 8000 rpm for 1 minute to collect the Agrobacterium in the tube. Discard 650 μL of supernatant, resuspend the bacteria, and evenly spread it on a plate containing YEB solid medium containing the corresponding antibiotic. Incubate it in a 28°C incubator for 48 hours. Pick suspected positive transformants and place them in a centrifuge tube containing YEB liquid medium containing the corresponding antibiotic. After incubation at 220 rpm at 28°C for 6 hours, verify them by PCR, and select the corresponding positive transformants for sequencing.

[0170] The nucleotide sequence of the PCR verification primers was the same as that of the upstream and downstream homology arm amplification primers. The verification PCR system is shown in Table 9:

[0171] Table 9 Verification of PCR system

[0172] Components Addition volume, 20μL system Agrobacterium culture solution 1 μL Primer F 0.5μL Primer R 0.5μL 2×TaqPlusMasterMix(Dye) 10 μL <![CDATA[ddH2O]]> 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 h, 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. A vortex shaker was used to quickly shake the spores for 5 minutes to evenly distribute 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 Equal amounts of Agrobacterium tumefaciens and Cicadae conidia suspension were mixed using a vortex shaker and evenly spread on the surface of IM solid medium containing AS. The mixture was incubated at 28°C in the dark for 48 hours. M-100 solid medium containing thiosporin and glufosinate, a resistance reagent corresponding to the carrier, was used to overlay the IM solid medium after 48 hours of incubation. After incubation at 25°C for 7 days, mycelium grown on the M-100 solid medium was selected and plated on M-100 solid medium plates containing thiosporin and glufosinate, a resistance reagent corresponding to the carrier, for secondary resistance screening. The screened cells were verified by PCR to obtain positive transformants.

[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 medium was 20 μL / 100 mL.

[0180] The volume ratio of thiosporin to the M-100 solid culture medium is 90 μL / 100 mL, and the volume ratio of the carrier-corresponding resistance reagent glufosinate-ammonium to the 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 1×10 7 Spore suspensions of the CcWC-1 knockout strain and wild-type strain were evenly spread on SDAY plates covered with cellophane. The plates were then incubated under 850 lx white, blue, and red light, alternating between 12 hours of light and 12 hours of darkness for 6 days at 25°C. On the sixth day of culture, the entire culture was harvested, freeze-dried, and weighed.

[0183] 2.10 Processing of Cicadae culture samples

[0184] On the sixth day of culture, harvest the culture on the cellophane paper, quench with liquid nitrogen, and freeze-dry in a freeze dryer at -80°C for 24 hours. Grind into a powder, weigh 100 mg of mycelium into a 5 mL centrifuge tube, add 3 mL of 80% methanol by volume, and sonicate for 30 minutes in an ultrasonic cleaner, shaking every 10 minutes. Remove the tube and centrifuge at 12,000 rpm for 5 minutes. After centrifugation, remove 2 mL of the supernatant and transfer it to a new tube. Place the tube containing the 2 mL supernatant in a centrifugal vacuum concentrator and concentrate to dryness at 40°C. Remove the tube, add 500 μL of 90% methanol by volume for sonication, and centrifuge to obtain the supernatant. Filter through a 0.22 μm pore size filter to obtain the sample extract, which is then placed in an injection vial for HPLC use.

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

[0186] The sample extracts were analyzed using an Agilent Eclipse XDB-C18 column. The mobile phase consisted of a mixture of distilled water and acetonitrile in a 30:70 volume ratio; the flow rate was 0.3 mL / min; the injection volume was 10 μL; the column temperature was 30°C; the detection wavelength was 215 nm; and the elution time was 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 to 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 were injected into the chromatograph according to the above detection conditions for chromatographic analysis. After chromatographic analysis, a standard curve was 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 a standard curve: y = 44796x-148066, with a 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 three PAS domains, two PAC domains and one GATA-type zinc finger domain. The PAS domain is a signal sensor domain that participates in a variety of signaling 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, together forming a conserved 3D PAS fold. WC-1 and WC-2 are believed to be able to form a heterodimer WCC through their respective PAS domains, thereby activating the periodic transcription of Frq; 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 transformants were obtained by Agrobacterium-mediated cicada fungus genetic transformation, and the length was verified by PCR. 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. PCR verification of the designed primers for CcWC-1 was performed, with WT as the control. The results are shown in Figure 2 As shown in Figure B, no bands similar to WT were detected in the positive transformants obtained. Further screening was performed using glufosinate resistance, and all three positive transformants obtained were resistant to glufosinate. Therefore, the positive transformants obtained were confirmed to be CcWC-1 knockout strains.

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

[0197] Figure 3 Panel A shows the dry weight of mycelia on day 6 of the CcWC-1 knockout strain ΔCcWC-1 and the wild-type WT under different conditions. ΔCcWC-1 exhibited significantly higher mycelial dry weight than WT under white light (P<0.01), blue light (P<0.05), and red light (P<0.01), indicating that CcWC-1 deficiency promotes mycelial growth in light-treated Cicada fungus.

[0198] Figure 3Panel B shows the BEA content in freeze-dried cultures of the CcWC-1 knockout strain ΔCcWC-1 and the wild-type WT on day 6 of growth under different conditions. BEA content in ΔCcWC-1 was significantly higher than in WT under white, blue, and red light illumination (P<0.01), indicating that CcWC-1 deficiency promotes BEA production in Cicada fungus under light-induced culture. BEA is the English name for beauvericin.

[0199] These experimental results indicate that the CcWC-1 gene acts as a negative regulator in cicada fungi, inhibiting hyphal growth and BEA synthesis. Deletion of this gene results in accelerated hyphal growth and increased BEA content, an effect that persists across different light conditions. This discovery reveals the crucial role of the CcWC-1 gene in regulating light response and metabolism in cicada fungi, providing a potential strategy for increasing growth and the production of secondary metabolites, such as BEA, through genetic engineering. The different light conditions involved are white, blue, and red light.

[0200] By studying this gene and its mechanism of action, the present invention provides a gene regulating beauvericin synthesis, CcWC-1, addressing the prior art's lack of understanding of key genes regulating beauvericin synthesis in cicada flowers. This in-depth study of the regulatory mechanism of beauvericin synthesis in cicada flowers provides the potential for increasing beauvericin production through genetic engineering.

[0201] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. 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 the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of beauvericin synthetic regulatory gene CcWC-1 in increasing the content of cicada flower beauvericin, characterized in that, The nucleotide sequence of the beauvericin synthesis regulatory gene CcWC-1 is shown in SEQ ID NO.1; By means of genetic engineering, knocking out the beauvericin synthesis regulatory gene CcWC-1 or reducing the expression level of the beauvericin synthesis regulatory gene CcWC-1 to obtain a Cordyceps cicadae mutant with a high beauvericin content, thereby regulating fungal metabolism.

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

2.

3. The application according to claim 2, wherein Culturing the host cell with the beauvericin synthesis regulatory gene CcWC-1 knocked out according to claim 1 under light conditions to increase the beauvericin production of the host cell; The host cell is Cordyceps cicadae.

4. A method for increasing the content of cicada flower beauvericin, characterized in that, Using the Agrobacterium-mediated genetic transformation method to introduce the knockout vector targeting the beauvericin synthesis regulatory gene CcWC-1 as claimed in claim 1 into the Cordyceps cicadae strain to obtain a Cordyceps cicadae transgenic strain with a high beauvericin content.

5. The method according to claim 4, wherein Constructing a T-DNA vector containing the knockout sequence of the beauvericin synthesis regulatory gene CcWC-1; transforming the constructed T-DNA vector into Agrobacterium; co-culturing the Agrobacterium carrying the T-DNA vector with the Cordyceps cicadae strain, and then through selection, screening and regeneration to obtain the Cordyceps cicadae transgenic strain with a high beauvericin content.

6. The method according to claim 4, characterized in that The knockout vector targeting the beauvericin synthesis regulatory gene CcWC-1 as claimed in claim 1 is called a fungal gene knockout vector; The fungal gene knockout vector contains 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 the resistance screening marker gene Bar.

7. The method according to claim 6, characterized in that, The fungal gene knockout vector is recombinantly constructed based on the pDHt-SK-Bar plasmid through the restriction enzyme digestion sites of Pst I and Xba I.

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