Stemphylium transcription factor Sar1 gene and application thereof

Abstract

The invention discloses a Stemphylium transcription factor Sar1 gene and application thereof, and belongs to the technical field of microbial genetic engineering. The nucleotide sequence of the Stemphylium transcription factor Sar1 gene is as shown in SEQ ID NO.15, and the amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO.4. The gene is a key transcription factor for synthesis of the Altersolanol A in Stemphylium, and the yield of the Altersolanol A can be remarkably increased through overexpression in Stemphylium cystosum. Therefore, the discovery and cloning of the Sar1 gene disclosed by the invention provide a basis for artificially modifying microorganisms and biologically synthesizing AltersolanolA.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, in particular to a Pseudomonas stolonifer transcription factor Sar1 gene and its application. Background Art

[0002] Fungi are one of the most biodiverse groups and a significant source of a wide range of small-molecule active compounds. Their metabolites primarily include alkaloids, polyketides and their derivatives, terpenes, and peptides. Many of these compounds possess remarkable biological activities, including anti-tumor, antiviral, antibacterial, anti-hyperlipidemic, and anti-inflammatory properties. Examples include penicillins from the genus Penicillium, the immunosuppressant cyclosporine from the genera Trichoderma and Tolypocladium, and lovastatin, a drug with cardiovascular therapeutic potential, from the genus Aspergillus. In recent years, research has revealed that over 50% of secondary metabolites with novel structures and activities originate from fungi.

[0003] AltersolanolA is a tetrahydroanthraquinone compound that was first isolated from the metabolites of Alternaria solani and subsequently reported in fungi such as Stemphylium spp. and Phomopsis spp. AltersolanolA is a kinase inhibitor that has strong inhibitory activity against a variety of tumor cells, bacteria, fungi and oomycetes, especially its anti-cancer activity has attracted much attention. Studies have shown that AltersolanolA 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. Its IC 50 The values ​​ranged from 0.005 μg / mL to 0.024 μg / mL, and the average IC 50 The value is 0.005 μg / mL. Studies have reported that altersolanol A is not only toxic to cancer cells but also inhibits cancer cell proliferation and migration, without affecting the viability of non-cancerous cells. However, altersolanol A has not yet become a clinical drug, and research on its activity is insufficient, especially with limited literature reporting on its mechanism of action. This is presumably due to the limited availability of altersolanol A from natural sources, which has led to its lack of attention. Therefore, studying altersolanol A from microbial sources is of great practical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a transcription factor Sar1 gene of Stipes stolonifer and its application to solve the problems existing in the above-mentioned prior art. The overexpression of the transcription factor Sar1 gene of Stipes stolonifer in Stipes vesicularis SvHN-02 can significantly increase the production of Altersolanol A, providing a new direction for the development of Altersolanol A from microbial sources.

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

[0006] The present invention provides a transcription factor Sar1 gene of Sclerotium stoloniferum, 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 a homology of more than 80% to the sequence.

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

[0008] The present invention also provides the use of the Stipes stolonifer transcription factor Sar1 gene or the protein in promoting Stipes stolonifer to produce Altersolanol A.

[0009] Furthermore, the yield of the Altersolanol A is increased by overexpressing the Sar1 gene in the Pseudomonas stolonifer.

[0010] The present invention also provides a method for producing altersolanol A by utilizing the Stipes stolonifer transcription factor Sar1 gene, which comprises a process of overexpressing the Stipes stolonifer transcription factor Sar1 gene in Stipes stolonifer.

[0011] Furthermore, the Stenotrophomonas includes Stenotrophomonas lycopersicum and Stenotrophomonas cysticola.

[0012] The present invention also provides an application of the Sclerotium stolonifer transcription factor Sar1 gene or the protein in preventing and controlling potato late blight.

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

[0014] The present invention also provides an application of the Pseudomonas stolonifer transcription factor Sar1 gene or the protein in preventing and treating pepper blight.

[0015] Furthermore, the pathogen of pepper blight includes but is not limited to Phytophthora capsici.

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

[0017] The present invention comparatively sequences transcriptomes of Stemphylium stolonifer strains infected with the fungal virus Stemphylium lycopersici mycovirus 1 (SlMV1) to obtain differentially expressed genes. Through analysis and screening, a new Stemphylium stolonifer transcription factor, the Sar1 gene, was identified. This gene is a key transcription factor for altersolanol A synthesis in Stemphylium stolonifer. Overexpression in Stemphylium stolonifer can significantly increase altersolanol A production. Therefore, the discovery and cloning of the Sar1 gene provides a foundation for artificially modifying microorganisms for the biosynthesis of altersolanol A. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a liquid chromatogram showing the changes in the content of Altersolananol A in the SvHN-02 strain before and after infection with S1MV1;

[0020] Figure 2 Schematic diagram of the protein structure encoded by the differentially expressed transcription factor Sar1 gene;

[0021] Figure 3 The phenotypes of Sar1 gene knockout / complementation strains are shown, among which SvHN-02 is a wild-type strain; KO-Sar1-3, KO-Sar1-22, and KO-Sar1-25 are gene knockout strains; CO-Sar1-3, CO-Sar1-15, and CO-Sar1-16 are gene complementation strains;

[0022] Figure 4 is the content of altersolanol A in the fermentation products of the SvHN-02Sar1 gene knockout / complementation strain, where SvHN-02 is the wild-type strain; KO-Sar1-3, KO-Sar1-22, and KO-Sar1-25 are gene knockout strains; CO-Sar1-3, CO-Sar1-15, and CO-Sar1-16 are gene complementation strains;

[0023] Figure 5 To screen the phenotypes of the three Sar1 gene overexpression transformants and the wild-type S. stolonifer, among which SvHN-02 is the wild-type strain; OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 are overexpression strains;

[0024] Figure 6 To determine the content of Altersolananol A in the fermentation products of the strain SvHN-02 overexpressing the Sar1 gene, wherein SvHN-02 is a wild-type strain; OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 are overexpression strains;

[0025] Figure 7 The antibacterial effect of the fermentation liquid of OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 overexpressing strains on potato late blight pathogen;

[0026] Figure 8 The antibacterial effect of the fermentation broth of OE-Sar1-1, OE-Sar1-2 and OE-Sar1-3 overexpressing strains on pepper blight pathogen. DETAILED DESCRIPTION

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

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

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

[0032] The strains involved in the embodiments of the present invention: the cystic sclerotinia strain SvHN-02 and the tomato sclerotinia 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 Peking University Modern Agriculture Research Institute, and it is promised that all the above strains will be open to the public and provided within 20 years from the date of application of this invention.

[0033] Example 1: Altersolanol A synthesis in S. vesicularis strain SvHN-02 infected with Stemphylium lycopersici mycovirus 1 (S1MV1) was significantly reduced

[0034] 1. Preparation of Sclerotium stolonifer fermentation broth

[0035] (1) Four bacterial cakes were punched out of the S. vesicularis strain SvHN-02 and SvHN-02-V infected with SlMV1 using a sterilized 5 mm puncher on a clean bench. The round bacterial cakes were picked up with a sterilized inoculation needle and inoculated into 50 mL of PS liquid culture medium. The culture was kept in a dark incubator at 28 °C for 12 days.

[0036] (2) Use sterilized gauze folded into four layers to separate the mycelium from the culture solution after 12 days of static culture. The resulting fermentation solution is stored at -20°C for later use.

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

[0038] (1) Instruments and chromatographic conditions

[0039] The analysis was performed using Shimadzu high performance liquid chromatography, a Waters ACQUITY UPLC BEH C18 column (100 mm2.1 mm, 1.7 μm), an SPD-M20APDA detector; the column temperature was 37°C; the mobile phase was 30% methanol and 70% water; the injection volume was 10 μL; the flow rate was 1 mL / min; and the wavelength was 220 nm.

[0040] (2) Preparation of standard samples and standard curve

[0041] Altersolanol A standards were purchased from Sigma. Accurately weigh the altersolanol A standard using a precision balance and dissolve it in methanol to prepare a 160 μg / mL standard solution. This solution was then serially diluted to 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL. A standard curve was constructed using the altersolanol A concentrations as the horizontal axis and the corresponding peak areas as the vertical axis.

[0042] (3) Determination of Altersolanol A content

[0043] Pipette 10 mL of filtered fermentation broth into a new 50 mL centrifuge tube, add 10 mL of methanol, and mix thoroughly. Incubate at 4°C overnight. Then, add an equal volume of ethyl acetate, mix thoroughly, and let stand for 20 minutes. Remove the supernatant and blow dry with nitrogen until orange-red crystals appear. Finally, add 5 mL of methanol, sonicate, and filter through a 0.22 μm bacterial filter before testing.

[0044] The fermentation broth of S. vesiculosus strain SvHN-02 and the fermentation broth of S. vesiculosus strain SvHN-02-V infected with SlMV1 were analyzed by high performance liquid chromatography with standard substances of altersolanol A. The results showed that the synthesis of altersolanol A decreased significantly after infection with SlMV1 virus ( Figure 1 ).

[0045] Example 2 Candidates and Verification of Key Transcription Factors for the Synthesis of Altersolanol A in Sclerotium

[0046] The high-altersolanol A-producing strains of S. stolonifer (SlHN-10) and S. cysticola (SvHN-02) were infected with SlMV1, resulting in strains SlHN-10-V and SvHN-02-V, respectively, with altersolanol A biosynthesis blocked. Transcriptome sequencing was then performed on the two groups of strains (SlHN-10 / SlHN-10-V and SvHN-02 / SvHN-02-V) before and after SlMV1 infection. The differentially expressed genes were then screened for transcription factors involved in the regulation of altersolanol A biosynthesis. A homologous transcription factor, Sar1, was found in the two differentially expressed gene groups. The protein sequence encoding the Sar1 gene shared a high similarity of 98.3%. Furthermore, the expression of this gene 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 biosynthesis. Next, the function of the Sar1 gene was verified. Since this transcription factor was the first cloned transcription factor in Sclerotium that was related to the regulation of Altersolanol A synthesis, it was named Sar1. The specific operation is as follows:

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

[0048] PCR amplification of the Sar1 gene, the specific operation process is as follows:

[0049] 1. Extraction of Fungal DNA and RNA

[0050] The experimental methods and steps were based on the instructions of the Tiangen Biochemical Technology (Beijing) Co., Ltd. Rapid DNA Extraction and Detection Kit (KG203) and RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441).

[0051] 2. PCR amplification of Sar1 gene

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

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

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

[0055] SEQ ID NO.15:

[0056]

[0057] RT-PCR amplification of Sar1 gene CDS, the specific operation is as follows:

[0058] The reverse transcription of RNA was performed with reference to the Quant cDNA First Strand Synthesis Kit (KR103) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The PCR reaction system was based on the Sar1 gene PCR amplification system. The amplified product was electrophoresed on a 1% agarose gel, and the RT-PCR product was cloned into a T vector and sent to a sequencing company for sequencing verification. The results showed that the Sar1 gene CDS length was 3018 bp (SEQ ID NO.3), and the amino acid sequence length of the protein it encoded was 1005 Aa as shown in (SEQ ID NO.4). Protein structure prediction showed that the protein contained two zinc finger domains and a GAL4 domain, which was consistent with the characteristics of a transcription factor ( 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, double-joint PCR amplification generated a fusion product, SY+HPH+XY, consisting of the upstream target gene fragment SY, the hygromycin gene fragment (HPH), and the downstream target gene fragment XY. Specific primers were then used to amplify the split-marker fragments SY+HPH and HPH+XY for gene knockout. The split-marker fragments were introduced into protoplasts of Sclerotium cystis through PEG-mediated protoplast transformation. Through three rounds of homologous recombination, the hygromycin gene fragment replaced the target gene fragment, achieving Sar1 gene knockout.

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

[0066] (1) Obtaining Sar1 knockout double fragments

[0067] The upstream and downstream flanking sequences of the Sar1 gene in the S. cysticola genome 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 S. cysticola SvHN-02 as a template, the upstream SY and downstream XY fragments of Sar1 were amplified. Primers HPH-F / R were designed and, using plasmid vector pCX62 as a template, the HPH fragment was amplified. Amplification of the split-marker gene knockout fragment requires two steps. The first step is to connect the upstream SY, downstream XY, and HPH fragments 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) Acquisition of backfill fragments

[0077] The method for constructing the complementation vector is to insert the Sar1 gene and its promoter sequence approximately 1,500 bp upstream into the KSTNP vector. Primers were designed using Primer 5.0 based on the genome information of the Pseudomonas serrata included in the NCBI database. To facilitate gene cloning into the vector, vector homologous sequences (in italics) were added before the primer sequences. To facilitate verification of gene cloning into the vector, restriction enzyme sites (underlined) were added to the primer sequences. Primers for amplifying the complementation fragment of Pseudomonas serrata: 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 primer (10 μM), 12.5 μL Mix, 9.5 μL sterile ddH2O, and amplification under the following conditions: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 90 seconds, 30 cycles, and a final extension at 72°C for 10 minutes. The target fragment with the corresponding vector homology arm sequence was obtained. The PCR product was tested for quality by 1% gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The fragments with the correct sequence were recovered and digested at -20°C for later use.

[0078] (3) Vector activation and construction of recombinant vector

[0079] KSTNP vectors stored in glycerol were streaked onto LB plates and cultured overnight at 37°C. Single colonies were picked and transferred to LB liquid medium. Incubated in a shaker at 180 rpm and 37°C for 12-16 hours before plasmid extraction. Plasmid extraction procedures were similar to the high-purity plasmid miniprep kit (DP104) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The vectors were digested with the appropriate enzymes, recovered, and ligated with the target fragment. E. coli was then transformed, and positive clones were selected by PCR and sent to Shanghai Biotechnology for sequencing verification.

[0080] (4) PEG-mediated protoplast transformation

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

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

[0083] 3) Collect mung bean-sized mycelial clumps and mix thoroughly with the enzymatic hydrolysate (note that the mycelia should not clump at this point and must be suspended as evenly as possible in the enzymatic hydrolysate. Mix thoroughly by inverting and shaking). Incubate at 28°C, 80 rpm, and shake for 2.5 hours. Take 10 μL of the enzymatic hydrolysate for microscopic examination. If the enzymatic hydrolysis is not complete, extend the enzymatic hydrolysis time to about 3 hours.

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

[0085] 5) The collected filtrate was centrifuged at 5000 rpm for 5 min at room temperature, the supernatant was discarded (hygrometer operation), the precipitate was collected and enriched into one tube, and the protoplast pellet was suspended in 200 μL of sterile STC (1 M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl2);

[0086] 6) Take 10 μL of STC suspension and examine under a microscope using a 10×40 magnification lens. At least 10 protoplasts per field of view can be used for PEG-mediated transformation.

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

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

[0089] 9) Add all the liquid in the centrifuge tube to 6 mL of sterile RM liquid medium (without antibiotics, the 10 mL centrifuge tube must be sterilized) and incubate at 28°C, 80 rpm, for 2-4 hours (until small white particles are visible to the naked eye);

[0090] 10) Add all the liquid to 100 mL of sterile RM medium (containing the antibiotic corresponding to the transformed plasmid), mix well, and pour into sterile culture plates. Incubate at 28°C for 2-3 days to grow transformants (approximately 10-20 colonies per plate). Then, overlay the plate with the transformants with the medium containing antibiotics three times (note that individual colonies must not grow together). Pick out the individual colonies and place them on fresh medium containing antibiotics, overlaying them three more times.

[0091] 11) DNA was extracted from the transformants screened with antibiotics and positive transformants were detected by PCR.

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

[0093] The results showed that the synthesis of altersolanol A was inhibited in the Sar1 gene knockout strain, while the synthesis was restored in the complementation strain ( Figure 3 and Figure 4 ), indicating that Sar1 gene is a key transcription factor in the regulation of AltersolanolA synthesis.

[0094] Example 3 Overexpression of the Sar1 gene in Sclerotium saccateum to increase the yield of Altersolanol A

[0095] 1. Extraction of fungal RNA was performed as described in Example 2.

[0096] 2. Construction of Sar1 overexpression vector

[0097] The purified and recovered full-length CDS of Sar1 gene was ligated with KSTNP vector using ExnaseⅢ enzyme, and then heat-shocked and transformed into E.coli DH5ɑ to construct Sar1 gene overexpression vector.

[0098] 3. Genetic transformation and detection of Sar1 gene overexpression vector refer to Example 2

[0099] The Sar1 gene overexpression vector was transformed into the S. vesicularis strain SvHN-02, and three Sar1 gene overexpression transformants (OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3) were obtained. The culture characteristics showed that the synthesis of altersolanol A was significantly increased (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 with SvHN-02 ( Figure 6 ).

[0100] Literature reports that altersolanol A has multiple activities, including antibacterial and anticancer activities. Three overexpression transformants, OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3, were selected to determine the antibacterial effects of their fermentation broths on the oomycetes potato late blight and pepper Phytophthora. The results showed that the fermentation broth of the wild-type S. vesicularis strain SvHN-02 had a good inhibitory effect on potato late blight when diluted 10 times, while the fermentation broths of the Sar1 gene overexpression strains OE-Sar1-1, OE-Sar1-2, and OE-Sar1-3 still had a good inhibitory effect on late blight and pepper Phytophthora when diluted 40 times. Figure 7 ; Figure 8 ).

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

Claims

1. A transcription factor Sar1 gene of Strigospora stolonifera, characterized in that: The nucleotide sequence of the Pseudomonas aeruginosa transcription factor Sar1 gene is shown in SEQ ID NO.

15.

2. The protein encoded by the transcription factor Sar1 gene of Stipes stolonifer according to claim 1, wherein The amino acid sequence of the protein is shown in SEQ ID NO.

4.

3. Use of the transcription factor Sar1 gene of Stipes stolonifer according to claim 1 or the protein of claim 2 in promoting the production of Altersolananol A by Stipes stolonifer.

4. The use according to claim 3, characterized in that The production of the Altersolanol A is increased by overexpressing the Sar1 gene in the Pseudomonas stolonifer.

5. A method for producing Altersolananol A using the transcription factor Sar1 gene of Stipes stolonifer according to claim 1, characterized in that: The method comprises the process of over-expressing the Stipes stolonifer transcription factor Sar1 gene in Stipes stolonifer.

6. The method according to claim 5, characterized in that The described Stenotrophomonas include Stenotrophomonas lycopersicum and Stenotrophomonas cystis.

7. Use of the transcription factor Sar1 gene of Styptoptilospora as claimed in claim 1 or the Altersolanol A synthesized under the regulation of the protein as claimed in claim 2 in preventing and controlling the oomycete disease potato late blight.

8. The use according to claim 7, characterized in that The oomycete diseases include, but are not limited to, potato late blight.

9. Use of the transcription factor Sar1 gene of Stipes stolonifer according to claim 1 or the Altersolanol A synthesized under the regulation of the protein according to claim 2 in preventing and controlling pepper blight.

10. The use according to claim 9, characterized in that The pathogen of pepper blight includes but is not limited to Phytophthora capsici.

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