A novel use of exopoid C, an isopentenylated indole alkaloid

Abstract

This invention discloses a novel use of isopentenylated indole alkaloid exopoid C, belonging to the field of marine microbial application technology. This invention is the first to discover and confirm that isopentenylated indole alkaloid exopoid C has significant growth-inhibiting activity against Arabidopsis thaliana. After culturing for 7 days in 1 / 2 MS medium containing 50 μg / mL exopoid C, the taproot elongation of Arabidopsis thaliana seedlings was significantly inhibited, with a taproot length reduction of more than 70% compared to the solvent control group. In the group containing 200 μg / mL exopoid C, the inhibition rate reached almost 100%, and Arabidopsis thaliana showed almost no growth. These results indicate that exopoid C has the potential to be developed as a plant growth inhibitor or a lead compound with herbicidal activity, opening up new directions for the application of indole alkaloid natural products in the pesticide field.

Description

Technical Field

[0001] This invention belongs to the field of marine microbial application technology, specifically relating to a new use of an isopentenylated indole alkaloid, exopoid C. Background Technology

[0002] Arabidopsis thaliana ( Arabidopsis thaliana Arabidopsis thaliana is one of the most important model plants in plant science, possessing advantages such as a short growth cycle, a small and fully sequenced genome, and a mature genetic transformation system. Screening for growth-inhibiting activities using Arabidopsis thaliana as an indicator plant is a crucial approach for discovering novel herbicidal compounds and studying herbicide targets and mechanisms of action. Compounds exhibiting growth-inhibiting activity in Arabidopsis thaliana often have targets that can be extended to various broadleaf weeds, thus possessing significant value for pesticide development. Summary of the Invention

[0003] The purpose of this invention is to provide a new use for the isopentenylated indole alkaloid exopoid C, specifically in its application as a plant growth inhibitor or herbicide, to overcome the shortcomings of the prior art.

[0004] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: An isopentenylated indole alkaloid, exopoid C, has the chemical formula shown in Formula I: .

[0005] The application of the compound exopoid C or its agrochemically acceptable salt in the preparation of products that inhibit Arabidopsis growth.

[0006] The use of the compound exopoid C or its agronomically acceptable salt in the preparation of plant growth inhibitors or herbicides.

[0007] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover and confirm that the isopentenylated indole alkaloid exopoid C exhibits significant growth-inhibiting activity against Arabidopsis thaliana. After culturing for 7 days in 1 / 2 MS medium containing 50 μg / mL exopoid C, the taproot elongation of Arabidopsis thaliana seedlings was significantly inhibited, with a taproot length reduction of over 70% compared to the solvent control group. In the group containing 200 μg / mL exopoid C, the inhibition rate reached almost 100%, with Arabidopsis thaliana showing almost no growth. These results indicate that exopoid C has the potential to be developed as a plant growth inhibitor or a lead compound with herbicidal activity, opening a new direction for the application of indole alkaloid natural products in the pesticide field. Attached Figure Description

[0008] Figure 1 The chemical structural formula of isopentenylated indole alkaloid exopoid C is given.

[0009] Figure 2 Photographs showing representative growth phenotypes of Arabidopsis seedlings after 7 days of treatment with different concentrations of exopoid C. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of protection of this invention.

[0011] The preliminary research for this invention involved: obtaining fungi from an intertidal soil sample from Antarctica. Penicillium sp The exopoid indole alkaloid biosynthesis gene cluster exo was discovered and identified in HDN14-431, and was detected by Aspergillus nidulans (…). Aspergillus nidulans A novel indole alkaloid, exopoid C, was successfully obtained using a heterologous expression system. The structure, biosynthetic gene cluster, and synthesis method of the above-mentioned compound have been disclosed in a prior application (application number: 2024115271334).

[0012] Among them, the marine-derived fungus is Penicillium HDN14-431. Penicillium sp. HDN14-431, accession number CCTCC NO: M 20242691, accession date: December 10, 2024, depositary institution: China Center for Type Culture Collection, address: School of Life Sciences, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0013] Example 1: Preparation of the test compound exopoid C exopoid C was prepared according to the biosynthetic method described in the prior application (application number: 2024115271334), as follows: Fungi originating from the polar intertidal zone Penicillium spUsing HDN14-431 genomic DNA as a template, exoA, exoB, and exoC were amplified by PCR using primers (specific primer sequences are shown in Table 1) pYTU-exoA-F / pYTU-exoA-R, pYTP-exoB-F / pYTP-exoB-R, and pYTR-exoC-F / pYTR-exoC-R. The PCR reaction mixture was subjected to agarose gel electrophoresis, and the exoA, exoB, and exoC fragments were purified using a gel extraction kit. Next, using a one-step cloning enzyme, the exoA, exoB, and exoC fragments were integrated into the NotI, BamHI, and BamHI-digested linearized vectors pYTU, pYTP, and pYTR, respectively, and then transformed into *E. coli*. In XL-1, positive clones were screened using ampicillin. The positive clones were selected for liquid fermentation, plasmids were extracted and sequenced for verification, and finally the correct expression vectors pYTU-exoA, pYTP-exoB, and pYTR-exoC were obtained.

[0014] The primer sequences are shown in Table 1: Table 1 Primer Sequences

[0015] The fermentation strain was then transformed.

[0016] The strain of Aspergillus nidus was removed from the freezer at -80°C. Aspergillus nidulnas Spread the spores onto CD (supplemented with U, R, and P elements) solid plates and incubate at 37°C for 2-3 days. Collect the spores in sterile water using sterile cotton swabs and filter through a 40 μm cell filter to remove mycelium and other components. Add an appropriate amount of spores to 40 mL of CD (supplemented with U, R, and P elements) liquid medium and incubate at 37°C and 220 rpm for 8-9 h until most spores have germinated. Add 10 mL of protoplast enzymatic hydrolysate (0.02 g Yatalase™ and 0.03 g Lysing Enzymes from Trichoderma harzianum dissolved in 10 mL OM buffer, filtered through a 0.22 μm sterile filter membrane, and prepared fresh for each use) to resuspend the cells. Enzymatic digestion was performed overnight at 28℃ and 80 rpm. After complete digestion of protoplasts under microscopic examination, the protoplasts were resuspended in an appropriate amount of pre-cooled STC. Then, the correctly constructed recombinant plasmid was added to the protoplasts. Subsequently, 600 μl of PEG solution was added to the transformation system. After standing at room temperature for 25 min, the mixture was spread onto the CD regeneration medium of the corresponding defect type and cultured at 37℃ for two days. The transformed individuals were then transferred to the CD medium of the corresponding defect type for expansion culture. After 2 days, the correctly transfected strain AN-exoABC was obtained.

[0017] AN-exoABC transfected strain was fermented in 10L of CD-ST solid medium for 4 days. The fermentation medium was extracted with ethyl acetate, and the organic phase was evaporated to obtain 3.4g of crude extract. The crude extract was first separated by ODS (octadecylsilyl bonded silica gel) column. The target compound was found to be in a 55% methanol / water fraction. The fraction containing the target compound was separated by high performance liquid chromatography (HPLC) under the following conditions: MeOH / H2O (50:50), retention time 15.1 min, flow rate 3 ml / min. The pure compound was obtained, and its structure was analyzed by NMR and HRESIMS.

[0018] The Aspergillus nidulans auxotrophic CD medium consists of: 1% anhydrous glucose, 5% 20x Nitrate Salts, 0.1% Trace element, and U stock solution (1%), R stock solution (1‰) and P stock solution (1‰) added as needed before use.

[0019] The Aspergillus nidulans protoplast regeneration CDS solid medium consists of: 1% anhydrous glucose, 5% 20x Nitrate Salts, 0.1% Trace element, 21.86% D-sorbitol, and 2% agar. Before use, U stock solution (1%), R stock solution (1‰), and P stock solution (1‰) are added as needed.

[0020] The CD-ST culture medium for Aspergillus nidulans fermentation comprises: 2% soluble starch, 2% acid-hydrolyzed casein, 5% 20x Nitrate Salts, and 0.1% Trace element. Before use, U stock solution (1%), R stock solution (1‰), and P stock solution (1‰) are added as needed.

[0021] The composition of the U element mother liquor is: 0.56% Uracil, 24.442% Uridine; The composition of the R element mother liquor is: 0.0125% Riboflavin; The mother liquor containing phosphorus (P) consists of 0.05% Pyridoxal hydrochloride. The nitrogen source 20x Nitrate Salts consist of: 12% NaNO3, 1.04% KCl, 1.04% MgSO4·7H2O, and 3.04% KH2PO4.

[0022] The reagent Trace element consists of: 0.22% ZnSO4·7H2O, 0.11% H3BO3, 0.05% MnCl2·4H2O, 0.016% FeSO4·7H2O, 0.016% CoCl2·5H2O, 0.016% CuSO4·5H2O, 0.011% (NH4)6Mo7O24·4H2O, and 0.5% Na4EDTA.

[0023] This invention determined the structure of the compound exopoid C using high-resolution mass spectrometry and NMR, such as Figure 1 As shown.

[0024] Table 2 shows the NMR data of compound 1.

[0025] Example 2: Determination of the growth-inhibiting activity of exopoid C on Arabidopsis seedlings 2.1 Experimental Materials Test compound: exopoid C (prepared in Example 1, purity ≥95%), prepared into a stock solution of appropriate concentration with dimethyl sulfoxide (DMSO), and stored at -20°C in the dark for later use.

[0026] Test plant: seeds of Arabidopsis thaliana, Columbia ecotype (Col-0).

[0027] Culture medium: 1 / 2 Murashige & Skoog (MS) medium, with the following formula: 2.2 g / L Murashige & Skoog powder, 10.0 g / L sucrose, 20.0 g / L agar powder, pH 5.8, autoclaved at 121℃ for 20 min.

[0028] 2.2 Preparation of drug-containing culture medium plates Cool the sterilized 1 / 2 MS medium to about 50°C. Add exopoid C stock solution of different concentrations prepared with DMSO in a clean bench, mix thoroughly, and pour into square petri dishes (10 cm × 10 cm), about 40 mL of medium per dish. Let it cool and solidify before use.

[0029] Treatment groups were set up: 10, 50, and 200 μg / mL exopoid C, and a solvent control group was set up, with two replicates for each group.

[0030] 2.3 Surface disinfection and inoculation of Arabidopsis thaliana seeds Arabidopsis seeds should be surface sterilized according to the following steps: (1) Rinse the seeds twice with sterile double-distilled water (ddH2O); (2) Add 75% ethanol for 2 min, then rinse repeatedly with sterile ddH2O 4 times to fully remove residual ethanol; (3) Add 5% sodium hypochlorite (NaClO) solution and treat for 4 min, then rinse with sterile ddH2O 3-4 times to fully remove residual NaClO.

[0031] Evenly sow 24 seeds per half MS medium plate onto the surface of the sterilized seeds. Seal the petri dishes with sealing film after sowing.

[0032] 2.4 Culture conditions and observation records After inoculation, the culture dishes were placed in the dark at 4°C for vernalization for 2 days. After vernalization, the culture dishes were transferred to an artificial climate incubator and cultured vertically for 7 days.

[0033] The cultivation conditions were as follows: light intensity 15000 lx, photoperiod 16 h light / 8 h dark; temperature 22℃ and humidity 70% during the light period; temperature 20℃ and humidity 70% during the dark period.

[0034] 2.5 Growth index determination This experiment used the vertical plate culture method, where Arabidopsis thaliana seeds were sown on 1 / 2 MS solid medium supplemented with different concentrations of the target compound. The following treatment groups were set up: Negative control group: only an equal volume of solvent was added, and the target compound was not present; Low concentration treatment group: 10 μg / mL target compound, 2 parallel plates were set up; Medium concentration treatment group: 50 μg / mL target compound, 2 parallel plates were set up; High concentration treatment group: 200 μg / mL target compound, 2 parallel plates were set up.

[0035] All plates were vertically cultured for 7 days at 22 ℃ under 16 h light / 8 h darkness conditions. The length of the taproot was measured using a background grid of 10×10 cm (approximately 1.67 cm per grid). The mean taproot length, standard deviation, and root length inhibition rate were calculated.

[0036] 2.6 Experimental Results The experimental results showed that Arabidopsis seedlings in the solvent control group grew normally, indicating that this concentration of DMSO had no significant effect on Arabidopsis growth.

[0037] Compared with the solvent control group, when the exopoid C concentration was 50 μg / mL, the elongation of the taproot of Arabidopsis seedlings was significantly inhibited, while at 200 μg / mL, the seedlings showed no growth at all. Figure 2 The statistical results are shown in Table 3.

[0038] Table 3 Effect of exopoid C on taproot length of Arabidopsis thaliana seedlings (50 μg / mL, cultured for 7 days)

[0039] In the 50 μg / mL treatment, one parallel plate experienced separation of the culture medium from the petri dish wall, causing the seedling roots to detach from the medium, making it impossible to obtain effective taproot length data. Therefore, only data from one valid parallel plate in this group were retained as representative results. This result meets the verification requirements of this invention regarding the growth-inhibiting activity of Arabidopsis thaliana, and forms a complete dose-response relationship with the complete inhibition effect of the high-concentration group (200 μg / mL) and the lack of significant inhibition effect of the low-concentration group (10 μg / mL), without affecting the core conclusions of this invention.

[0040] 2.7 Conclusion The above results indicate that exopoid C at concentrations above 50 μg / mL significantly inhibits the elongation of the taproot of Arabidopsis thaliana seedlings. Therefore, exopoid C can be effectively used to inhibit the growth of Arabidopsis thaliana and has the potential to be used as a plant growth inhibitor.

[0041] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an isopentenylated indole alkaloid, exopoid C, or a pesticide-acceptable salt thereof, in the preparation of plant growth inhibitors or herbicides; characterized in that, The chemical structure of the compound exopoid C is shown in Formula I: 。 2. The application of an isopentenylated indole alkaloid, exopoid C, or a pesticide-acceptable salt thereof, in the preparation of products inhibiting Arabidopsis growth; characterized in that, The chemical structure of the compound exopoid C is shown in Formula I: 。

Images