Method for evaluating and regulating UV-B irradiation enriched grape secondary metabolites based on tubulin array

Abstract

The invention discloses a method for evaluating, regulating and controlling ultraviolet B-band (ultraviolet B, UV-B) irradiation enrichment of grape secondary metabolites on the basis of a microtubulin array. The material is used for evaluating a microtubulin array and preparing a sample solution for determining secondary metabolites by applying a high performance liquid chromatography, and by detecting the enrichment degrees of the cell metabolites of rutin, kaempferol, myricetin and resveratrol before and after irradiation, the microtubulin array can be used for evaluating the microtubulin array and preparing the sample solution for determining the secondary metabolites. The raw materials for preparing food and cosmetics with anti-inflammatory and anti-oxidation effects are obtained. The invention creates a complete method from treatment time and treatment steps of UV-B irradiation of the American sandy grape cell suspension, monitoring of dynamic changes of tubulin and cell morphology indexes, and detection of changes of cell metabolites such as rutin, kaempferol, myricetin and resveratrol before and after irradiation. And a new way is provided for applying the enriched grape natural secondary metabolites to production of food and cosmetics with anti-inflammatory and anti-oxidation effects.

Description

Technical Field

[0001] This invention belongs to the field of horticultural product nutrition technology, and relates to a method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array. Background Technology

[0002] Globally, grapes (Vitis vinifera L.) are among the most economically valuable fruit trees under sustainable agricultural development models. Plant suspension cell lines, characterized by material homogeneity, high cell count, rapid reproduction, and low variability, are ideal fine-scale model systems for enriching secondary metabolites. Ultraviolet B (UV-B 280-320nm) plays a significant role in the synthesis of secondary metabolites in grapes. Flavonoids are the most abundant category of phenolic compounds in grapes. Furthermore, flavonoids possess anti-inflammatory, anti-cancer, and antibacterial properties, making them ideal food and phytophagic components.

[0003] Rutin is a flavonol glycoside with anti-free radical, anti-lipid peroxidation, enzyme activity inhibition in inflammatory responses, and anti-inflammatory effects. Myricetin possesses multiple effects including free radical scavenging, anti-oxidation, anti-tumor, neurotoxicity reduction, influencing lymphocyte activation and proliferation, inhibiting platelet-activating factor, lowering blood sugar, relieving alcohol poisoning, and anti-inflammation. The US FDA has approved the widespread use of myricetin in pharmaceuticals, food, health products, and cosmetics. Resveratrol and its derivatives have antioxidant, antimicrobial, and anti-cancer cell proliferation activities and can be widely used in pharmaceuticals, health products, cosmetics, and food additives. Kaempferol participates in regulating glucose and lipid metabolism disorders and inhibits glycolysis, which can improve hepatitis and liver fibrosis induced by high-fat and high-glucose diets.

[0004] Existing data discloses three methods for producing metabolites. The first method involves using ultraviolet light to induce an increase in the types and amounts of secondary metabolites in the stems and leaves of the medicinal plant Clematis terniflora DC. [1] This method employs ultraviolet (UV) treatments of different wavelengths (UVA, UVB, or UVC) and combinations, with varying irradiation intensities, to increase the content of secondary metabolites. However, obtaining the target yield of secondary metabolites requires consuming large amounts of fresh plant material to acquire stem and leaf tissues. If this method is applied to suspension cell lines continuously propagating in a culture environment, UVA and UVC irradiation carries the potential risk of inducing phytotoxin production in the cells.

[0005] The second method involves immobilizing suspended cells by seeding them in a liquid culture medium containing an immobilization carrier, and then continuously culturing the immobilized cells in a liquid culture medium containing inducing factors to produce metabolites. [2]This method, due to the immobilization of cells during culture, results in a slower increase in cell expansion efficiency compared to suspension culture. This means that obtaining sufficient cells to achieve the target yield of secondary metabolites requires more time, increasing production costs.

[0006] The third method involves collecting the extracellular secondary metabolites secreted by plant cells into the culture medium from the reactor and transferring them to a downstream storage tank for metabolite extraction. [3] This method continuously supplies culture medium containing the target secondary metabolites to the downstream storage tank by regulating the number of suspended cells and inducing factors in the upstream reactor. This method primarily targets the collection of secondary metabolites secreted extracellularly and cannot enrich intracellular secondary metabolites. Furthermore, this method only provides a means of collecting secondary metabolites and cannot precisely control the type and content of secondary metabolites produced by suspended cells at different culture time points. Summary of the Invention

[0007] Addressing the challenges of existing production methods, such as the inability of irradiated plant tissues to continuously produce secondary metabolites, the potential risk of plant toxin generation from broadband ultraviolet irradiation, the limitation of cell proliferation rates when immobilizing suspended cells, the inability to obtain intracellular metabolites from culture media collected in reactors, and the inability to precisely control the types and amounts of secondary metabolites at different culture time periods, this invention aims to establish a plant suspension cell line that can serve as an in vitro bioreactor for large-scale production of plant natural products, thereby reducing production costs. Furthermore, it provides a method based on microtubule arrays to assess cell morphology and physiological indicators, precisely controlling UV-B irradiation of the plant suspension cell line to achieve enrichment of specific secondary metabolite types and amounts at different culture time periods, providing a new approach for producing foods and cosmetics with anti-inflammatory and antioxidant effects.

[0008] To achieve the above-mentioned technical objectives, the present invention is specifically implemented through the following technical solutions:

[0009] A method for assessing the enrichment of grape secondary metabolites by regulating UV-B irradiation based on a microtubule array includes the following steps:

[0010] 1) Culture of cell suspension systems: Take plant tissue samples (such as those from vegetative organs or reproductive organs) to induce callus tissue. Take soft, light yellow callus tissue and place it into an Erlenmeyer flask containing liquid culture medium for suspension culture.

[0011] 2) UV-B irradiation of American sandy grape cell suspension: On the 3rd day of subculture of the suspension cell line, it was transferred into a self-made device for UV-B irradiation.

[0012] 3) After UV-B irradiation treatment for 20 min, cells were collected at 0 h, 12 h, 24 h and 72 h after treatment, filtered and aliquoted into centrifuge tubes;

[0013] 4) After UV-B irradiation treatment for 3 hours, cells were collected (counted as 0 hours after treatment) and the dynamic changes of tubulin were measured, including: the dispersion, density, and angle of periplasmic microtubules, and the dispersion and density of central microtubules.

[0014] 5) After UV-B irradiation treatment for 3 hours, cells were collected (counted as 0 hours after treatment) and morphological parameters were measured, including: cell death rate, mitotic index, and cell width-to-length ratio.

[0015] 6) Preparation of sample solution, blank solution, and standard solution: Weigh the grape cells from step 3), add 10 mL of methanol, extract by sonication for 10 min, then centrifuge to collect the supernatant. Repeat this step, combine the supernatants obtained from the two centrifugations, evaporate and dry, add 2 mL of acetonitrile to reconstitute, and filter through an organic filter membrane to obtain the sample solution. Prepare the blank solution in the same way. Weigh the cells and add acetonitrile to a brown volumetric flask to make up to 1 mg / mL. -1 Standard solutions are diluted to different concentrations before use.

[0016] 7) Screening chromatographic conditions and setting elution programs: Separate various substances, perform full-wavelength ultraviolet scanning to obtain the maximum absorption wavelength, and select the final determination wavelength channel based on the maximum absorption wavelength of each substance; the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, where A is 0.1% phosphoric acid aqueous solution and B is acetonitrile; column temperature: 25℃; injection volume: 10μL; flow rate: 0.7mL·min -1 Then, perform the elution process.

[0017] Further, in step 1), the callus induction method is as follows: Take plant tissue vegetative organs or reproductive organs, disinfect them, cut them into stem segments about 0.5 cm long, transfer them to MS solid medium, seal them, and place them in a dark incubator at 25°C and 50% humidity for culture.

[0018] Furthermore, the composition of the liquid culture medium in step 1) is as follows: MS medium powder 4.3 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 200 mg / L, inositol 100 mg / L, thiamine 1 mg / L, 2,4-D 0.2 mg / L, pH adjusted to 5.8 with KOH and HCl, and autoclaved at 121°C for 20 minutes.

[0019] Furthermore, in step 1), the suspension culture method involves taking an appropriate amount of soft, pale yellow callus tissue and transferring it to a 250ml conical flask (with a groove at the bottom) containing 40ml of liquid culture medium. The flask is then sealed with aluminum foil that has been burned by an alcohol lamp and placed in a shaker at 25°C, in the dark, with a rotation speed of 200rpm for oscillation culture.

[0020] Furthermore, in step 2), the homemade device is a quartz dish covered with an ultraviolet filter.

[0021] Furthermore, in step 2), the irradiation conditions are ultraviolet irradiation at a wavelength of 305–315 nm and a distance of 30 cm.

[0022] Furthermore, in step 4), the methods for measuring the dispersion, density, and angle of periplasmic microtubules, as well as the dispersion and density of central microtubules, are as follows: Prepare a temporary slide using 20 μl of cell suspension after 3 hours of UV-B irradiation. Image the periplasmic and central microtubules of the cells under an Olympus inverted fluorescence microscope (GFP channel). Using the FIJI plugin (http: / / hasezawa.ib.ku-tokyo.ac.jp / zp / Kbi / HigStomata), the dispersion and density of periplasmic and central microtubules are statistically analyzed. The dispersion calculation formula is:

[0023]

[0024] The total number of cytoskeleton pixels is N, expressed in units of a given pixel intensity. Average pixel intensity

[0025] Density calculation formula:

[0026] Where nMT: number of pixels in the cytoskeleton; nCell: total number of pixels in the measurement area.

[0027] The FIJI angle measurement tool measures the angle between the periplasmic microtubules and the long axis of the cell center.

[0028] Further, in step 5), the cell death rate determination method is as follows: after ultraviolet irradiation, 200 μL of suspended cells are transferred to a 1.5 mL sterile centrifuge tube, and 10 mL of prepared 2.5% (w / v) Evans Blue is added to the centrifuge tube. The tube is stained for 4 min, and then filtered with water using a homemade filter to remove the culture medium. Then, 30 μL is transferred to a glass slide for observation and recording under a microscope. Cells that are stained are considered dead cells, and cells that are not stained are considered live cells.

[0029] Furthermore, in step 5), the mitotic index was determined on day 3 of subculture and used as a growth indicator. 500 μL of suspended cells was transferred from the culture flask to a 1.5 mL sterile centrifuge tube, and 10 ng / mL of the solution was added. -1 Stain with Hoechst 33258 for 2 min and add one drop of 10% (v / v) Triton X-100. Observe under the DAPI light field of an Olympus inverted fluorescence microscope and determine the mitotic index.

[0030] Further, step 5) cell width-to-length ratio determination uses the length-to-width measurement method of Imag J FIJI software.

[0031] Furthermore, in step 6), the centrifugation conditions are: 6000 r / min for 5 min.

[0032] Furthermore, in step 6), filtration is performed using a 0.22μm microporous organic filter membrane.

[0033] Furthermore, in step 7), the final measurement wavelength channel is 270 nm.

[0034] Furthermore, in step 7), the detailed settings for B in the elution program are as follows: 0–20 min, B is 10%–15% acetonitrile; 20–40 min, B is 15%–25% acetonitrile; 40–60 min, B is 25%–30% acetonitrile; 60–80 min, B is 30%–10% acetonitrile.

[0035] The beneficial effects of this invention are as follows:

[0036] 1) Material selection: Compared with plant tissue, suspension cell lines can achieve homogeneity of production materials. The suspension cell line induced by callus tissue was created as a homogeneous cell population, which is a stable and efficient system for producing secondary metabolites, providing a new method for the large-scale production of target secondary metabolites.

[0037] 2) Cell status assessment: cytoskeleton and physiological indicators. Cell status is assessed by measuring cytoskeleton indicators such as the dispersion and density of periplasmic microtubules and central microtubules; further physiological indicators such as cell death rate, cell aspect ratio, and mitotic index are measured to ensure that cells used for the synthesis of secondary metabolites are in optimal condition.

[0038] 3) Irradiation time control: After UV-B irradiation, the secondary metabolites are transferred back to the conical flask for continued cultivation. The changes in the types and enrichment of secondary metabolites at different time periods are used to precisely control the processing time according to production needs, so as to obtain the corresponding secondary metabolites and improve production efficiency.

[0039] 4) Potential value in food, cosmetics and other fields: The secondary metabolites enriched by this cell line after UV-B irradiation have anti-inflammatory, antioxidant, anti-cancer and antibacterial effects, and have potential value in cosmetics, health products, functional beverages and other fields.

[0040] 5) A method for plant tissue sampling, callus induction, and suspension cell line establishment was invented. This method can be extended to the application of medicinal plants, crops, horticultural plants, and the protection and application of endangered plants.

[0041] 6) Experimental setup for UV-B irradiation: When using Philips UV-B lamps (290–315 nm) for irradiation, there are low-band UVB rays that negatively impact plant metabolism. Using a UV filter can filter out UV-B wavelengths shorter than 305 nm, precisely controlling the UV-B wavelength within the 305–315 nm range. This filter is placed over a quartz dish containing suspended cells for suspension culture. Attached Figure Description

[0042] Figure 1 : Schematic diagram of the process of this invention.

[0043] Figure 2 Cell mortality rate of American sand grapes under UV-B irradiation. A: Microscopic observation of cell mortality rate of American sand grapes (DIC light field); B: Statistical analysis of mortality rate at 0h, 24h, and 48h after 20min of UV-B irradiation. Data are from the mean ± standard error of three biological replicates. * indicates a significant difference in t-test (**p < 0.01).

[0044] Figure 3 Detection of mitotic index in American sandy grape cells under UV-B irradiation. A: Microscopic observation of mitotic index in American sandy grape cells (DAPI light field); B: Statistical analysis of mitotic index at 0h, 24h, and 48h after 20min of UV-B irradiation. Data are from the mean ± standard error of three biological replicates. * indicates a significant difference in t-test (**p < 0.01).

[0045] Figure 4 Statistical analysis of microtubule structure and arrangement in American sandy grape cells under UV-B irradiation. A: Microscopic observation of central microtubules, periplasmic microtubules, and microtubule aggregation in the nucleus in the UV-B treated group and the control group; scale bar = 20 μm; B: Periplasmic microtubule occupancy rate after UV-B stress compared to the control, expressed as skewness; C: Degree of periplasmic microtubule polymerization; D: Angle of periplasmic microtubule arrangement; E: Degree of central microtubule polymerization; F: Central microtubule occupancy rate; G: Number of microtubule aggregation points in the nucleus. Data are from the mean ± standard error of three biological replicates. * indicates a significant difference in t-test (**p < 0.01).

[0046] Figure 5 Chromatograms of four standards detected in the 270nm channel. A: Rutin; B: Kaempferol; C: Trans-resveratrol; D: Myricetin.

[0047] Figure 6 Chromatogram of American sandy grape cells detected in the 270nm UV absorption channel after 20 min of UV-B irradiation. A: 0h; B: 24h; C: 48h.

[0048] Figure 7 Content of four substances after 20 min of UV-B irradiation. A: Comparison of content between irradiated and unirradiated samples after 0 h of incubation; B: Comparison of content after irradiation at 0 h, 24 h, and 48 h of incubation. Data are from the mean ± standard error of three biological replicates. * indicates a significant difference in t-test (**p < 0.01, ***p < 0.001). Detailed Implementation

[0049] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] Example 1:

[0051] 1. Test materials

[0052] Plant material: The plant material used in this experiment was a transgenic grape cell suspension line (V. rupestris expressing GFP-AtTUB6) expressing Arabidopsis β-tubulin 6 and fused with jellyfish green fluorescent protein.

[0053] Experimental reagents: 2.5% Evans Blue (SIGMA), 10% (v / v) Triton X-100, 10 ng / mL - 1 Hoechst 33258, chromatographically pure methanol, chromatographically pure acetonitrile (Fisher Scientific, Santa Clara, CA), acetic acid, anhydrous ethanol, ethyl acetate, methanol, and phosphoric acid, etc.

[0054] Instruments and equipment: UV-B lamp (120cm long, Philips TL40W / 12RS), quartz petri dishes, ultraviolet filter (305nm, 90% transmittance, 16cm side length, Nantong Huijie Optical Components Technology Co., Ltd.), high performance liquid chromatograph (Waters e2695), photodiode array detector (Waters 2998), TG-16 high-speed centrifuge (Sichuan Shuke Instrument Co., Ltd.), ZLNS-1 vacuum centrifuge concentrator (Hongqi Instrument Co., Ltd.), KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0055] 2. Test methods

[0056] 2.1 Cell morphology determination

[0057] 2.1.1 UV-B irradiation of American sandy grape cell suspension

[0058] In a clean bench, 40 mL of suspension cells, passaged for 3 days, was transferred into a quartz dish and covered with a UV filter. The dish was irradiated for 20 min in a UV lamp (wavelength 305–315 nm, distance 30 cm). Afterward, 600 μL of suspension cells was transferred to a 1.5 mL centrifuge tube to determine cell death rate, 2 mL to a 2 mL sterile centrifuge tube to determine mitotic index, and 10 mL to a 50 mL centrifuge tube to determine extracellular alkalization. These were recorded as cells at 0 h post-treatment. The irradiated suspension cells were then transferred to a 250 mL Erlenmeyer flask and cultured in the dark at 200 rpm for 24 h. Cells were then collected using the method described above.

[0059] 2.1.2 Determination of Mortality Rate

[0060] After UV irradiation, 200 μL of suspended cells were transferred to a 1.5 mL sterile centrifuge tube. 10 mL of prepared 2.5% (w / v) Evans Blue was added to the tube, and staining was performed for 4 min. The solution was then filtered with sterile water using a custom-made filter to remove the culture medium. 30 μL of the solution was then transferred to a glass slide for observation and recording under a microscope. Stained cells were identified as dead cells, while unstained cells were identified as live cells.

[0061] 2.1.3 Determination of the mitotic index

[0062] Since mitosis is most vigorous on day 3 of cell subculture, measuring the mitotic index can serve as an important indicator of cell growth status. Take 500 μL of sample from the culture flask into a 1.5 mL sterile centrifuge tube, and add 10 ng / mL... - 1 The cells were stained with Hoechst 33258 for two minutes and then one drop of 10% (v / v) Triton X-100 was added to the suspension cells. The cells were then observed and the mitotic index was determined under the DAPI light field of an Olympus inverted fluorescence microscope.

[0063] 2.2 Assessment of dynamic changes in grape microtubule protein

[0064] Suspension cells from both the UV-B irradiated American sandy grape treatment group and the control group were resuspended and prepared into temporary slides. Using an Olympus inverted fluorescence microscope (GFP channel), the central microtubules, periplasmic microtubules, and microtubule layers showing the aggregation of tubulin fluorescent dots in the cell nucleus were recorded in 50 cells. The degree of aggregation and occupancy of central and periplasmic microtubules were statistically analyzed using the FIJI plugin (http: / / hasezawa.ib.ku-tokyo.ac.jp / zp / Kbi / HigStomata). [4] The FIJI angle measurement tool was used to measure the angle between the periplasmic microtubules and the long axis of the cell center, and the number of microtubule-protein fluorescent spots in the cell nucleus was counted. [5] .

[0065] 2.3 Determination of secondary metabolites

[0066] 2.3.1 Extraction of flavonoids and resveratrol

[0067] Flavonoid extraction: Weigh 0.2500 g (accurate to 0.0001 g) of cells from 2.1.1 into a centrifuge tube, add 10 mL of methanol, and extract using a KQ-500DE CNC ultrasonic cleaner for 10 min. Then centrifuge at 6000 r / min for 5 min using a TG-16 high-speed centrifuge. Transfer the supernatant to a 5 mL centrifuge tube, add another 10 mL of methanol to the original centrifuge tube, repeat the ultrasonic extraction for 10 min, and centrifuge again. Combine the supernatants obtained from the two centrifugations and evaporate to near dryness at 2000 r / min and 67 °C using a ZLNS-1 vacuum centrifuge concentrator. Add 2 mL of 5% (w / v) NaHCO3 and 5 mL of ethyl acetate, and completely dry the mixed ethyl acetate aqueous phase. Redissolve the ethyl acetate phase in 2 mL of acetonitrile, filter through a 0.22 μm microporous organic filter membrane, and prepare a blank solution using the same method for analysis.

[0068] 2.3.2 Preparation of Standard Solutions

[0069] Weigh 1.0 mg of each of the four standards and dilute to volume with acetonitrile in a brown volumetric flask to prepare a 1.0 mg / mL stock solution. Store at -20°C in the dark for later use. When needed, take 0.1 mL of each stock solution and dilute to volume with 0.9 mL of acetonitrile. Then dilute to prepare a gradient concentration solution and filter through a 0.22 μm microporous organic filter membrane for instrumental analysis.

[0070] 2.3.3 Screening of chromatographic conditions

[0071] A full-wavelength ultraviolet scan was performed using a photodiode array detector (Waters 2998) to obtain the maximum absorption wavelength. An InertSustain C18 column (250 mm × 4.6 mm, 5 μm) was used, and the separation of various substances was achieved by varying the concentration of acetonitrile-0.1% phosphoric acid aqueous solution. The selection of the detection wavelength for flavonoid HPLC analysis was based on the maximum absorption wavelength of each substance, with 270 nm chosen as the determination wavelength channel.

[0072] 2.3.4 Setting the elution program

[0073] An InertSustain C18 column (250 mm × 4.6 mm, 5 μm) with a photodiode array detector (Waters 2998) was used. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution. A was 0.1% phosphoric acid aqueous solution, and B was acetonitrile. Column temperature: 25℃, injection volume: 10 μL, flow rate: 0.7 mL / min. The optimized elution program was set as follows: 0–20 min, B: 10%–15% acetonitrile; 20–40 min, B: 15%–25% acetonitrile. * For 40–60 min, B is 25%–30% acetonitrile; for 60–80 min, B is 10%–30% acetonitrile. The four standards were injected according to the optimized elution program.

[0074] 3. Results and Analysis

[0075] 3.1 Mortality of cell suspension systems of American sandy grapes after UV-B irradiation

[0076] The cell membrane of a living cell is a selectively permeable membrane composed of a phospholipid bilayer, acting as a protective barrier for the cell and allowing only selective passage of substances. After cell death, the cell membrane is damaged, increasing its permeability. Evans Blue is a non-cell membrane permeable dye. When the plasma membrane is damaged, the dye can enter the cytoplasm and nucleus, thus staining the cell blue, which can be used to detect cell viability. After 20 minutes of UV-B irradiation, cells cultured for 0h, 24h, and 48h were collected, and the cell death rate was statistically analyzed under a DIC light field using an inverted fluorescence microscope. Figure 2 ).

[0077] 3.2 Mitotic index of American sandy grape cell suspensions after UV-B irradiation

[0078] The mitotic index (MI) is the percentage of cells in the mitotic phase out of the total cell count, often used to indicate the degree of cell proliferation. In plant cells, tubulin forms an early prophase zone in the cytoplasm during prophase of mitosis. This zone precisely regulates cell division by limiting spindle rotation and determining the position of the new cell wall. Therefore, measuring the mitotic index can reflect the impact of cytoskeleton rearrangement on mitosis. After 20 min of UV-B irradiation, cells cultured for 0 h, 24 h, and 48 h were collected, and the mitotic index was statistically analyzed under a DAPI light field using an inverted fluorescence microscope. Figure 3 ).

[0079] 3.3 Determination of microtubule protein in American sandy grape cell suspension system after UV-B irradiation

[0080] Plant microtubules are involved in early signal sensing and transduction of endogenous immunity. Previous studies have shown that structural rearrangement, changes in polymerization degree, and angle alterations of grape microtubules and microfilaments are among the most important defense responses against biotic / abiotic stresses. Therefore, measuring the dynamic changes of microtubules can assess the physiological state of cells. After 3 hours of UV-B irradiation treatment, cells were collected to measure the dynamic changes of microtubules, specifically including: the dispersion, density, and angle of periplasmic microtubules, and the dispersion, density, and number of aggregation points in the nucleus of central microtubules. Figure 4 ).

[0081] 3.4 Determination of secondary metabolites

[0082] 3.4.1 HPLC analysis of standards

[0083] Four standards were prepared into gradient concentrations: 1.000000 μg / L, 0.500000 μg / L, 0.250000 μg / L, 0.125000 μg / L, 0.062500 μg / L, 0.031250 μg / L, and 0.015625 μg / L. These standards were injected separately under the same chromatographic conditions to determine their retention times. A standard curve was then plotted, and the data were recorded. Regression analysis was performed with peak area (AU*min) on the x-axis and concentration (mg / L) on the y-axis to plot the standard curve. Figure 5 ).

[0084] 3.4.2 Analysis of Metabolite Types

[0085] After 20 min of UV-B irradiation, cells were cultured for 0 h, 24 h, and 48 h, respectively. Cells were then collected, extracted with methanol using ultrasound, and loaded onto the control group. Chromatograms of the UV-B treated cells and the control group were obtained. Figure 6After irradiating American sandy grape cells with UV-B, the composition of metabolites was analyzed, and the peaks appearing in the chromatogram were numbered, with 1-8 representing the eight secondary metabolites that appeared.

[0086] 3.4.3 Metabolite content analysis

[0087] By substituting the peak areas corresponding to different substances into the abscissa of the relevant regression equation, the contents of the four substances after 20 min of UV-B irradiation were compared with those after 0 h. Simultaneously, the contents of secondary metabolites in cells cultured for 0 h, 24 h, and 48 h after UV-B irradiation were compared. Figure 7 ).

[0088] References

[0089] [1] Zhejiang University. A method for inducing an increase in the types and contents of secondary metabolites in the medicinal plant Clematis paniculata [P]. Chinese Patent: CN101732434A, 2010-06-16

[0090] [2] Tsinghua University. Solid-liquid two-step culture method for producing useful metabolites from plant cells [P]. Chinese Patent: CN1556201A, 2004-12-22

[0091] [3] Zhejiang Mideyou Biotechnology Co., Ltd. A method for promoting the release of secondary metabolites, total flavonoids from licorice cells into a suspension culture medium [P]. Chinese Patent: CN116574668A, 2023-08-11

[0092] [4] Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S. Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsisguard cells. Plant J. 2010; 61: 156-165.

[0093] [5]Guan X,Buchholz G,Nick P.Tubulin marker line of grapevinesuspension cells as a tool to follow early stress responses.J PlantPhysiol.2015;176:118-128。

Claims

1. A method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule arrays, characterized in that, Includes the following steps: 1) Culture of cell liquid suspension: Take plant tissue samples (such as those from vegetative organs or reproductive organs) to induce callus, and select soft, light yellow callus tissue to be transferred into conical flasks containing liquid culture medium for suspension culture. 2) UV-B (ultraviolet B band) irradiation of American sandy grape cell suspension: On the 3rd day of subculture of the suspension cell line, it was transferred into a self-made device for UV-B irradiation. 3) After UV-B irradiation treatment for 20 min, the cells were transferred back to the conical flask for suspension culture. Cells were collected at 0 h, 12 h, 24 h and 72 h after treatment, filtered and aliquoted into centrifuge tubes. 4) After UV-B irradiation treatment for 3 hours, cells were collected to measure the dynamic changes of tubulin, specifically including the dispersion, density, and angle of periplasmic microtubules, and the dispersion and density of central microtubules. 5) After 3 hours of UV-B irradiation treatment, cells were collected to determine morphological parameters, including: cell death rate, mitotic index, and cell width-to-length ratio. 6) Preparation of sample solution, blank solution, and standard solution: Weigh the grape cells from step 3), add 10 mL of methanol, sonicate for 10 min, then centrifuge to collect the supernatant. Repeat this step, combine the supernatants obtained from the two centrifugations, evaporate to dryness, add 2 mL of acetonitrile to reconstitute, and filter through a filter membrane to obtain the sample solution. Prepare the blank solution in the same way. Separately, weigh the cells and add acetonitrile to a brown volumetric flask to prepare a solution of 1 mg / mL. -1 Standard solutions should be diluted to prepare samples with gradient concentrations before use. 7) Screening chromatographic conditions and setting elution programs: Separate various substances, perform full-wavelength ultraviolet scanning to obtain the maximum absorption wavelength, and set the measurement wavelength channels according to the maximum absorption wavelength of each substance to screen chromatographic conditions; the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, where A is 0.1% phosphoric acid aqueous solution and B is acetonitrile; column temperature: 25℃; injection volume: 10μL; flow rate: 0.7mL·min -1 The elution process was optimized.

2. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, Step 1) Callus induction method: Take tissues from plant vegetative organs (roots, stems, leaves) or reproductive organs (flowers, fruits, seeds), sterilize them in a clean bench, cut them into tissues with an area of ​​about 0.7cm*0.7cm, transfer them to MS solid medium, seal them, and place them in a dark incubator at 25℃ and 70% humidity for culture.

3. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, Step 1) The liquid culture medium consists of: MS (Murashige & Skoog) medium powder 4.3 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 200 mg / L, inositol 100 mg / L, thiamine 1 mg / L, 2,4-D 0.2 mg / L, pH adjusted to 5.8 with potassium hydroxide and hydrochloric acid, and autoclaved at 121°C for 20 min.

4. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 1), the cell suspension culture method involves taking an appropriate amount of soft, pale yellow callus tissue and placing it in a 250ml conical flask (with a groove at the bottom) containing 40ml of liquid culture medium. The flask is then sealed with flame-sterilized aluminum foil and placed in a shaker at 25°C, in the dark, with a rotation speed of 200rpm for culture.

5. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 2), the UV-B irradiation time for the suspended cells is the 3rd day after subculturing, because the cell mitotic index is most active at this time.

6. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, Step 2) involves a homemade irradiation apparatus using a 120cm long UV-B lamp (Philips TLA0W / 12RS), an 8cm diameter quartz petri dish, and a 305nm UV filter (90% transmittance, 16cm side length, Nantong Huijie Optical Components Technology Co., Ltd.). The lamp is placed 30cm above the petri dish, and the dish with the filter is placed in an incubator for the irradiation experiment. The UV filter can be used to filter out the UV-B band with wavelengths less than 305nm, retaining the UV-B band from 305nm to 315nm.

7. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 4), the dispersion, density, and angle of periplasmic microtubules, as well as the dispersion and density of central microtubules, were measured by preparing a temporary slide using 20 μL of cell suspension after 3 hours of UV-B irradiation. Periplasmic and central microtubules were then imaged under an Olympus inverted fluorescence microscope (GFP channel). The dispersion and density of periplasmic and central microtubules were calculated using the ImageJ FIJI software plugin (http: / / hasezawa.ib.ku-tokyo.ac.jp / zp / Kbi / HigStomata). The dispersion was calculated using the following formula: The total number of cytoskeleton pixels is N, with pixel intensity as the unit of measurement. Average pixel intensity; Density calculation formula: Where, n MT : Number of pixels in the cytoskeleton; n cell : Total number of pixels in the measurement area; The angle between the periplasmic microtubules and the long axis of the cell center was measured using the angle measurement tool in Image J FIJI software.

8. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 5), the cell death rate determination method is as follows: After UV-B irradiation, 200 μL of suspended cells are transferred to a 1.5 mL sterile centrifuge tube, Evans Blue is added to a concentration of 2.5% (w / v), and staining is performed for 4 min. Then, a self-made cell sieve is used: a 3 cm section of nylon mesh (Millipore, USA) with a 30 μm pore size is cut from a 2 mL centrifuge tube to make a cell sieve, which is then washed three times with sterile ddH2O. 30 μL of suspended cells is then placed on a glass slide, observed and recorded under a microscope. Cells that are stained are dead cells, and cells that are not stained are live cells.

9. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, Step 5) involves determining the mitotic index by taking 500 μL of grape cell suspension from the culture flask and placing it into a 1.5 mL sterile centrifuge tube, then adding 10 ng / mL... -1 Stain with Hoechst 33258 for 2 min, then add one drop of 10% (v / v) Triton X-100 to the suspended cells. Take 20 μL of the suspended cells and place them on a glass slide. Observe them under the DAPI channel of an Olympus inverted fluorescence microscope and count the mitotic index.

10. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on a microtubule array according to claim 1, characterized in that, In step 5), the cell length-to-width ratio is determined by taking 500 μL of the sample from the culture flask and placing it in a 1.5 mL sterile centrifuge tube, adding one drop of 10% (v / v) Triton X-100, taking 20 μL of suspended cells on a glass slide, observing it in bright field under an Olympus inverted fluorescence microscope, and measuring the length and width using the length-to-width measurement tool in Image J FIJI software.

11. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on a microtubule array according to claim 1, characterized in that, The centrifugation conditions in step 6) are: centrifuge at 6000 r / min for 5 min.

12. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 6), filtration is performed using a 0.22μm microporous organic filter membrane.

13. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, The optimal measurement wavelength in step 7) is 270 nm.

14. The method for evaluating and regulating the enrichment of grape secondary metabolites by UV-B irradiation based on microtubule array according to claim 1, characterized in that, In step 7), the elution program is set to: 0–20 min, B is 10%–15% acetonitrile; 20–40 min, B is 15%–25% acetonitrile; For 40–60 min, B is 25%–30% acetonitrile; for 60–80 min, B is 30%–10% acetonitrile.

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