Sar1 gene of protostelium and application thereof

Abstract

This invention discloses a Sar1 transcription factor gene from *Stylosporium spp.* and its applications, belonging to the field of microbial genetic engineering technology. The nucleotide sequence of the Sar1 gene is shown in SEQ ID NO. 15, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO. 4. This gene is a key transcription factor for the synthesis of Altersolanol A in *Stylosporium spp.*, and overexpression in *Stylosporium spp.* can significantly increase the yield of Altersolanol A. Therefore, the discovery and cloning of the Sar1 gene in this invention provides a foundation for the artificial modification of microorganisms and the biosynthesis of Altersolanol A.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, and in particular to a Sar1 gene, a transcription factor from Crescentella asiatica, and its applications. Background Technology

[0002] Fungi are one of the most biodiverse groups of organisms and an important source of a range of small-molecule bioactive compounds. Their metabolites mainly consist of alkaloids, polyketides and their derivatives, terpenes, and polypeptides. Many of these compounds possess excellent biological activities, including antitumor, antiviral, antibacterial, anti-hyperlipidemia, and anti-inflammatory effects. For example, penicillins originate from the genus *Penicillium*, immunosuppressants like cyclosporine come from the genera *Trichoderma* and *Tolypocladium*, and lovastatin drugs, which have therapeutic effects on cardiovascular diseases, originate from the genus *Aspergillus*. In recent years, studies have found that over 50% of secondary metabolites with novel structures and activities originate from fungi.

[0003] Altersolanol A is a tetrahydroanthraquinone compound, first isolated from the metabolites of *Alternaria solani*, and subsequently reported in fungi such as *Stemphylium spp.* and *Phomopsis spp.*. Altersolanol A is a kinase inhibitor with strong inhibitory activity against various tumor cells, bacteria, fungi, and oomycetes, and its anticancer activity has attracted particular attention. Studies have shown that Altersolanol A exhibits cytotoxic activity against 34 types of human tumor cells, including bladder cancer, central nervous system cancer, colon cancer, gastric cancer, lung cancer, melanoma, breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, glioblastoma, kidney cancer, and uterine cancer, with an IC50 value of [missing information]. 50 The values ​​were between 0.005 μg / mL and 0.024 μg / mL, with an average IC50 value. 50 The concentration was 0.005 μg / mL. Studies have reported that Altersolanol A not only exhibits toxicity against cancer cells but also inhibits their proliferation and migration without affecting the survival of non-cancer cells. However, Altersolanol A is not yet a clinical drug, and research on its activity is insufficient, particularly regarding its mechanism of action. This is likely because the availability of Altersolanol A from natural sources is limited, thus failing to attract sufficient attention. Therefore, research on Altersolanol A from microbial sources has significant practical implications. Summary of the Invention

[0004] The purpose of this invention is to provide the Sar1 gene of the creeping mold transcription factor and its application, in order to solve the problems existing in the prior art. Overexpression of the Sar1 gene in creeping mold SvHN-02 can significantly increase the yield of Altersolanol A, providing a new direction for the development of microbial Altersolanol A.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a Sar1 gene, a transcription factor of Crested mold, whose nucleotide sequence is shown in SEQ ID NO.15. The gene sequence is not limited to the sequence shown in SEQ ID NO.15, but also includes gene sequences with more than 80% homology to the sequence shown in SEQ ID NO.15.

[0007] The present invention also provides a protein encoded by the Sar1 gene of the *Sarcandra glabra*, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides the application of the Sar1 gene or protein of *Strombyx mori* in promoting the production of Altersolanol A by *Strombyx mori*.

[0009] Furthermore, the production of Altersolanol A was increased by overexpressing the Sar1 gene in the *Pseudomonas* species.

[0010] The present invention also provides a method for producing Altersolanol A using the Sar1 gene of the *Pleurotus erythrorhizon* transcription factor, comprising the process of overexpressing the Sar1 gene of the *Pleurotus erythrorhizon* transcription factor in *Pleurotus erythrorhizon*.

[0011] Furthermore, the creeping molds include *Pseudomonas tomatoae* and *Pseudomonas sacchariformis*.

[0012] The present invention also provides the application of the Sar1 gene, a transcription factor of *Pseudomonas spp.*, or the protein therein, in the prevention and control of potato late blight.

[0013] Furthermore, the pathogens causing potato late blight include, but are not limited to, oomycetes.

[0014] The present invention also provides the application of the Sar1 gene of *Pseudomonas spp.* transcription factor or the protein thereon in the prevention and control of pepper blight.

[0015] Furthermore, the pathogens causing the pepper blight include, but are not limited to, *Phytophthora capsici*.

[0016] The present invention discloses the following technical effects:

[0017] This invention utilizes comparative transcriptome sequencing of *Stemphylium lycopersici* strains infected with the fungal virus *Stemphylium lycopersici* mycovirus 1 (SlMV1) to identify differentially expressed genes. Through analysis and screening, a novel *Stemphylium lycopersici* transcription factor, Sar1, was identified. This gene is a key transcription factor for Altersolanol A synthesis in *Stemphylium lycopersici*, and overexpression in *Stemphylium lycopersici* significantly increases Altersolanol A production. Therefore, the discovery and cloning of the Sar1 gene in this invention provides a foundation for the artificial modification of microorganisms for the biosynthesis of Altersolanol A. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The liquid chromatogram shows the change in Altersolanol A content before and after infection of S1MV1 by SvHN-02 strain;

[0020] Figure 2 This is a schematic diagram of the protein structure encoded by the differentially expressed transcription factor Sar1 gene;

[0021] Figure 3 The phenotypes of Sar1 gene knockout / reinforcement strains are as follows: SvHN-02 is a wild-type strain; KO-Sar1-3, KO-Sar1-22 and KO-Sar1-25 are gene knockout strains; and CO-Sar1-3, CO-Sar1-15 and CO-Sar1-16 are gene-reinforcement strains.

[0022] Figure 4 The content of Altersolanol A in the fermentation product of SvHN-02Sar1 gene knockout / complement strains is given. Among them, SvHN-02 is a wild-type strain; KO-Sar1-3, KO-Sar1-22 and KO-Sar1-25 are gene knockout strains; and CO-Sar1-3, CO-Sar1-15 and CO-Sar1-16 are gene complement strains.

[0023] Figure 5 To screen the three Sar1 gene overexpression transformants and wild-type *Sar1* phenotype, SvHN-02 was the wild-type strain; OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3 were overexpression strains.

[0024] Figure 6 The content of Altersolanol A in the fermentation product of strains overexpressing the Sar1 gene in strain SvHN-02 was determined. SvHN-02 is a wild-type strain; OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 are overexpressing strains.

[0025] Figure 7 The antibacterial effect of fermentation broths of OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 overexpression strains on potato late blight pathogens;

[0026] Figure 8 The antibacterial effects of fermentation broths from OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3 overexpression strains on *Phytophthora capsici* were investigated. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The strains involved in the embodiments of this invention, namely *Stachys cystis* strain SvHN-02 and *Stachys tomato* strain SlHN-10, were both isolated from lettuce leaves infected with leaf spot disease; the strains and their specific isolation methods are disclosed in patent document CN112280751B; the above strains are all preserved by Hunan Agricultural University and the Institute of Modern Agriculture of Peking University, and it is promised that all the above strains will be made available to the public within 20 years from the date of application of this invention.

[0033] Example 1: The synthesis of Altersolanol A in the *Stemphylium lycopersici* strain SvHN-02, infected with the fungal virus *Stemphylium lycopersici* mycovirus 1 (SlMV1), was significantly reduced.

[0034] 1. Preparation of fermentation broth for *Pseudomonas spp.*

[0035] (1) On a clean bench, four mycelial cakes were made by punching four mycelial cakes with a sterile 5mm puncher using the SvHN-02 strain of Cysticercus and the SvHN-02-V infected with SlMV1. The round mycelial cakes were picked up with a sterile inoculation needle and inoculated into 50mL of PS liquid medium. The mixture was then incubated in the dark at 28℃ for 12 days.

[0036] (2) Use a folded, sterilized gauze to separate the mycelium from the culture liquid after 12 days of static culture. Store the resulting fermentation broth at -20℃ for later use.

[0037] 2. Determination of Altersolanol A content by high performance liquid chromatography (HPLC)

[0038] (1) Instruments and chromatographic conditions

[0039] Analysis was performed using Shimadzu high-performance liquid chromatography (HPLC). A Waters ACQUITYUPLC BEH C18 column (100 mm x 2.1 mm, 1.7 μm) with an SPD-M20 APDA detector was used. The column temperature was 37 °C. The mobile phase consisted of 30% methanol and 70% water. The injection volume was 10 μL. The flow rate was 1 mL / min. The wavelength was 220 nm.

[0040] (2) Preparation of standard samples and preparation of standard curves

[0041] Altersolanol A standard was purchased from Sigama. The standard was accurately weighed using a precision balance, dissolved in methanol, and prepared into a standard solution with a concentration of 160 μg / mL. This solution was then serially diluted to concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL. A standard curve was constructed with different Altersolanol A concentrations plotted on the x-axis and the corresponding peak areas on the liquid chromatography phase plotted on the y-axis.

[0042] (3) Determination of Altersolanol A content

[0043] Transfer 10 mL of the filtered fermentation broth into a new 50 mL centrifuge tube, add 10 mL of methanol, mix well, and incubate overnight at 4°C. Then, add an equal volume of ethyl acetate, mix thoroughly, and let stand for 20 min. Collect the supernatant and dry it under nitrogen until orange-red crystals appear. Finally, add 5 mL of methanol, sonicate to dissolve, filter through a 0.22 μm bacterial filter, and then analyze.

[0044] The fermentation broths of *S. cytogenes* strain SvHN-02 and *S. cytogenes* strain SvHN-02-V infected with SlMV1 were analyzed by high-performance liquid chromatography (HPLC) with Altersolanol A standards. The results showed that Altersolanol A synthesis decreased significantly after infection with SlMV1 virus. Figure 1 ).

[0045] Example 2: Candidate and Validation of Key Transcription Factors Synthesized by Altersolanol A in Crested Yeast

[0046] The high-Altersolanol A-producing *Pseudomonas tomatoides* strains SlHN-10 and SvHN-02 were infected with SlMV1, resulting in strains SlHN-10-V and SvHN-02-V, respectively, where Altersolanol A synthesis was inhibited. Transcriptome sequencing was then performed on the two groups of strains before and after SlMV1 infection (SlHN-10 / SlHN-10-V and SvHN-02 / SvHN-02-V). Transcription factors related to the regulation of Altersolanol A synthesis were screened from differentially expressed genes. One homologous transcription factor, Sar1, was found among the two differentially expressed genes, encoding a protein with a sequence similarity of 98.3%. Furthermore, the expression level of Sar1 was significantly reduced in both wild-type strains SlHN-02 and SlHN-10 after SlMV1 infection, suggesting that this gene is involved in the regulation of Altersolanol A synthesis. Next, the function of the Sar1 gene was verified. Since this transcription factor was the first cloned transcription factor in *Stylosporium spp.* related to the regulation of Altersolanol A synthesis, it was named Sar1. The specific procedures are as follows:

[0047] Primers were designed based on transcriptome sequencing results: Sar1-F: 5'-GCCATCCGATCAGTCTCC-3' (SEQ ID NO.1); Sar1-R: 5'-AGCAACAACGCCATACGC-3' (SEQ ID NO.2).

[0048] The specific procedure for PCR amplification of the Sar1 gene is as follows:

[0049] 1. Extraction of fungal DNA and RNA

[0050] The experimental methods and procedures were performed in accordance with the instructions of the Rapid DNA Extraction and Detection Kit (KG203) and the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech (Beijing) Co., Ltd.

[0051] 2. Sar1 gene PCR amplification

[0052] PCR reaction system 50.0 μL: 10×PCR Buffer 5.0 μL, 2.5 mmol / L dNTP 4.0 μL, forward and reverse primers 2.0 μL each, DNA or cDNA template 2.0 μL, Pfu enzyme 1.0 μL, ddH2O to make up to 50.0 μL.

[0053] Amplification program: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, for 34 cycles; extension at 72℃ for 5 min. PCR products of DNA and cDNA were detected by 1% agarose gel electrophoresis and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0054] The results showed that the full-length DNA of the Sar1 gene was 3272 bp, and the nucleotide sequence was shown in SEQ ID NO.15.

[0055] SEQ ID NO.15:

[0056]

[0057] The RT-PCR amplification of the Sar1 gene CDS is performed as follows:

[0058] Reverse transcription of RNA was performed using the Quant cDNA first-strand synthesis kit (KR103) from Tiangen Biotech (Beijing) Co., Ltd. The PCR reaction system followed the Sar1 gene PCR amplification system. Amplification products were subjected to 1% agarose gel electrophoresis, and RT-PCR products were cloned into a T vector and sent to a sequencing company for verification. Results showed that the Sar1 gene CDS length was 3018 bp (SEQ ID NO. 3), and the amino acid sequence length of the encoded protein was 1005 A (SEQ ID NO. 4). Protein structure prediction indicated that the protein contains two zinc finger domains and one GAL4 domain, consistent with transcription factor characteristics. Figure 2 ).

[0059] SEQ ID NO.3:

[0060]

[0061] SEQ ID NO.4:

[0062]

[0063] 3. Verification of Sar1 gene function

[0064] Utilizing the principle of split-marker homologous recombination, the fusion product SY+HPH+XY, consisting of the upstream fragment SY of the target gene, the hygromycin gene fragment (HPH), and the downstream fragment XY of the target gene, was obtained by double-joint PCR amplification. Using specific primers, the split-marker fragments SY+HPH and HPH+XY for gene knockout were obtained. The split-marker fragments were introduced into *Sar1* protoplast cells via PEG-mediated protoplast transformation. Through three homologous recombinations, the hygromycin gene fragment replaced the target gene fragment, thereby achieving Sar1 gene knockout.

[0065] The Sar1 gene knockout vector pCX62 and the complementation vector KSTNP were used to construct knockout and complementation vectors, respectively. The specific procedures are as follows:

[0066] (1) Obtaining Sar1 knockout double fragments

[0067] The upstream and downstream flanking sequences of the Sar1 gene in the genome of *Stenocystis sylvestris* were obtained from the NCBI database. Primers Sar1-SY-F / R and Sar1-XY-F / R were designed using Primer 5.0. Using genomic DNA from *Stenocystis sylvestris* SvHN-02 as a template, the upstream fragment SY and the downstream fragment XY of Sar1 were amplified. Primers HPH-F / R were designed, and the HPH fragment was amplified using the plasmid vector pCX62 as a template. Amplification of the split-marker gene knockout fragment requires two steps. The first step is to ligate the upstream fragment SY, the downstream fragment XY, and the HPH fragment using double-joint PCR. The second step is to amplify the SY+HPH fragment using primers Sar1-SY-F and SPD-SY-R, and the HPH+XY fragment using primers SPD-XY-F and Sar1-XY-R. The relevant primers are as follows:

[0068] Sar1-SY-F: 5'-CGTGTTGTTGACTCGCTAGGTG-3' (SEQ ID NO.5),

[0069] Sar1-SY-R: 5'-TTGACCTCCACTAGCTCCAGCCAAGCC-3' (SEQ ID NO.6),

[0070] Sar1-XY-F: 5'-CGTCCGCAATGTGTTATTAAGTCGAC-3' (SEQ ID NO.7),

[0071] Sar1-XY-R: 5'-CACTGCTACTGGGCGATTGA-3' (SEQ ID NO.8),

[0072] HPH-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3' (SEQ ID NO.9),

[0073] HPH-R: 5'-GTCGACTTAATAACACATTGCGGACGT-3' (SEQ ID NO. 10),

[0074] SPD-SY-R: 5'-ACTTCGGGGCAGTCCTCG-3' (SEQ ID NO.11),

[0075] SPD-XY-F: 5'-GAACTCACCGCGACGTCTGT-3' (SEQ ID NO. 12).

[0076] (2) Obtaining the supplementary fragment

[0077] The method for constructing the complementation vector involved inserting the Sar1 gene and its upstream promoter sequence (approximately 1,500 bp) into the KSTNP vector. Primers were designed using Primer 5.0 based on the *Stenostomium cylindrica* genome information in the NCBI database. To facilitate gene cloning into the vector, a vector homologous sequence (in italics) was added before the primer sequences. Restriction enzyme sites (underlined) were also added to the primer sequences to facilitate verification of gene cloning into the vector. *Stenostomium cylindrica* complementation fragment amplification primers: Sar1-HB-F: GGTACCCGGG GGATCC GTTGCAGCGCGAACCTTG (SEQ ID NO. 13), Sar1-HB-R: AACGTTAAGT GGATC C ACCCCGCATCTACGAACA (SEQ ID NO.14), reaction system: 1 μL DNA template, 1 μL each of primers (10 μM), 12.5 μL Mix, 9.5 μL sterile ddH2O, and amplification was performed under the following conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for 30 cycles, with a final extension at 72℃ for 10 min. The target fragment containing the corresponding vector homologous arm sequence was obtained. PCR products were quality checked by 1% gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Fragments with correct sequences were recovered, digested with enzymes, and stored at -20℃ for later use.

[0078] (3) Activation of the vector and construction of the recombinant vector

[0079] The KSTNP vector, preserved in glycerol, was streaked on LB agar plates and incubated overnight at 37°C. Single colonies were picked and cultured in LB liquid medium at 180 rpm for 12-16 hours in a shaker at 37°C for plasmid extraction. Plasmid extraction was performed according to the high-purity plasmid miniprep kit (DP104) from Tiangen Biotech (Beijing) Co., Ltd. The vector was recovered after digestion with the appropriate enzymes, ligated to the target fragment, transformed into *E. coli*, and positive clones were selected by PCR and sent to Shanghai Sangon Biotech for sequencing verification.

[0080] (4) PEG-mediated protoplast transformation

[0081] 1) Take two pieces of SvHN-02 colony edge cakes that have grown for 4-5 days on a PDA plate, inoculate them into 50mL of sterile PD liquid medium, and incubate them in a constant temperature incubator at 28℃ with shaking for 2-3 days;

[0082] 2) Use a sterile 1mL pipette tip to draw up the short mycelial culture medium, add it to two layers of sterile lens paper for filtration, collect the tender mycelia, and wash them 3-4 times with sterile 0.7M NaCl solution;

[0083] 3) Collect mycelial clumps the size of mung beans and mix them thoroughly with the enzyme hydrolysate (note that the mycelium should not clump together at this time, but should be suspended as evenly as possible in the enzyme hydrolysate, and can be mixed by inverting and shaking). Incubate at 28℃ and 80r / min for 2.5 hours. Take 10μL of the enzyme hydrolysate for microscopic examination. If the enzyme hydrolysis is not complete, the enzyme hydrolysis time can be appropriately extended to about 3 hours.

[0084] 4) Filter with two layers of sterile lens paper, wash 8-10 times with 1 mL of sterile 0.7M NaCl solution each time (be careful not to puncture the lens paper with the pipette tip), and collect the filtrate;

[0085] 5) Centrifuge the collected filtrate at 5000 r / min at room temperature for 5 min, discard the supernatant (operate in a clean bench), collect the precipitate, enrich it into 1 tube, and suspend the protoplast precipitate in 200 μL of sterile STC (1M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl2).

[0086] 6) Take 10 μL of STC suspension for microscopic examination. Observe with a 10×40 magnification lens. More than 10 protoplasts per field of view can be used for PEG-mediated transformation.

[0087] 7) Add 10-15g of plasmid to 200μL of protoplasts, slowly add 400μL of sterile PTC (PEG8000 40g, add STC solution to 100mL, accelerate dissolution in a 65℃ water bath, filter sterilize with a 0.22μm bacterial filter), gently invert to mix, and incubate on ice for 10min.

[0088] 8) Add 700 μL PTC of sterile solution to the above liquid and incubate at room temperature for 10 min;

[0089] 9) Add all the liquid in the centrifuge tube to 6 mL of sterile RM liquid culture medium (antibiotic-free, 10 mL centrifuge tubes must be sterilized), incubate at 28°C and 80 rpm for 2-4 hours (until small white particles are visible to the naked eye).

[0090] 10) Add all the liquid to 100mL of sterile RM medium (containing the antibiotic corresponding to the transformed plasmid), mix well, and pour onto a sterilized petri dish. Incubate at 28℃. Transformants will grow in 2-3 days (approximately 10-20 colonies per dish). Then, cover the plate with antibiotic-containing medium three times (note that individual colonies must not grow together). Transfer any newly grown single colonies to fresh antibiotic-containing medium and cover three more times.

[0091] 11) DNA was extracted from transformants selected by antibiotic screening and tested by PCR to detect positive transformants.

[0092] Then, using Altersolanol A as a standard, the fermentation broths of the wild-type strain, the gene knockout strain, and the complement strain were analyzed by high-performance liquid chromatography (see Example 1).

[0093] The results showed that AltersolanolA synthesis was inhibited in the Sar1 gene knockout strain, while it was restored in the complement strain. Figure 3 and Figure 4 This indicates that the Sar1 gene is a key transcription factor regulating the synthesis of AltersolanolA.

[0094] Example 3: Overexpression of the Sar1 gene in *Stylosporium sacchariformis* increases Altersolanol A production.

[0095] 1. Fungal RNA extraction is performed according to Example 2.

[0096] 2. Construction of Sar1 overexpression vector

[0097] The purified and recovered full-length Sar1 gene CDS was ligated to the KSTNP vector using Exnase III enzyme, and then transformed into E. coli DH5α by heat shock to construct the Sar1 gene overexpression vector.

[0098] 3. Genetic transformation and detection of Sar1 gene overexpression vector were performed as described in Example 2.

[0099] The Sar1 gene overexpression vector was transformed into the *Sarcandra glabra* strain SvHN-02, resulting in three Sar1 gene overexpression transformants (OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3). Observation of culture traits showed a significant increase in Altersolanol A synthesis (Altersolanol A was orange-red). Figure 5 The content of Altersolanol A in the fermentation broth of Sar1 gene overexpression transformants was significantly increased compared to SvHN-02, indicating a significantly enhanced ability of overexpression transformants to synthesize Altersolanol A. Figure 6 ).

[0100] Literature reports that Altersolanol A possesses multiple activities, including antibacterial and anticancer properties. The antibacterial effects of the fermentation broths of three overexpression transformants (OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3) against *Phytophthora indicum* and *Phytophthora capsici* were determined. The results showed that a 10-fold dilution of the fermentation broth from the wild-type *Phytophthora indicum* strain SvHN-02 exhibited good inhibitory effects against *Phytophthora indicum*, while a 40-fold dilution of the fermentation broths from the Sar1 gene overexpression strains OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3 still showed good inhibitory effects against both *Phytophthora indicum* and *Phytophthora capsici*. Figure 7 ; Figure 8 ).

[0101] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the Sar1 gene, a transcription factor of *Stenophyllus cystis*, or the protein encoded therein, in promoting the production of Altersolanol A by *Stenophyllus cystis*, characterized in that, The nucleotide sequence of the Sar1 gene transcription factor of *Stylosporium sacchariformis* is shown in SEQ ID NO. 15; The amino acid sequence of the protein is shown in SEQ ID NO.4; The production of Altersolanol A can be increased by overexpressing the Sar1 gene or increasing the expression level of the protein encoded by the Sar1 gene in the *Sarcandra glabra*.

2. A method for producing Altersolanol A using the Sar1 gene of the *Stachys syringae* transcription factor, characterized in that, The process includes overexpression of the Sar1 gene, a transcription factor of *Sarcandra glabra*, in *Sarcandra glabra*; the nucleotide sequence of the Sar1 gene is shown in SEQ ID NO.

15.

3. The application of the Sar1 gene, a transcription factor of *Stenophyllariae*, or its encoded protein, in enhancing the inhibitory effect of *Stenophyllariae* on *Phytophthora infestans*, characterized in that... The nucleotide sequence of the Sar1 gene transcription factor of *Pseudomonas stolonifer* is shown in SEQ ID NO.15; the amino acid sequence of the protein is shown in SEQ ID NO.4; by overexpressing the Sar1 gene or increasing the expression level of the protein encoded by the Sar1 gene in *Pseudomonas stolonifer*, the inhibitory effect of *Pseudomonas stolonifer* on potato late blight can be improved.

4. The application of the Sar1 gene, a transcription factor of *Phytophthora stolonifer*, or its encoded protein, in enhancing the inhibitory effect of *Phytophthora stolonifer* on *Phytophthora capsici*, characterized in that... The nucleotide sequence of the Sar1 gene transcription factor of *Phytophthora capsici* is shown in SEQ ID NO.15; the amino acid sequence of the protein is shown in SEQ ID NO.4; by overexpressing the Sar1 gene or increasing the expression level of the protein encoded by the Sar1 gene in *Phytophthora capsici*, the inhibitory effect of *Phytophthora capsici* on *Phytophthora capsici* can be improved.

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