Method for increasing glycyrrhizic acid content and liquiritin content in glycyrrhiza inflate roots through UV-B radiation

Abstract

The invention relates to the technical field of liquorice secondary metabolite accumulation and medicinal material quality improvement, in particular to a method for promoting efficient accumulation of triterpenoids (including glycyrrhizic acid) and flavonoids (including liquiritin) in a liquorice inflate root system through UV-B radiation. According to the invention, key genes (PAL1, CHS, CYP88D6 and CYP72A154) in phenylpropane metabolism and terpenoid synthesis pathways are activated through UV-B radiation treatment with specific intensity (107 [mu] W / cm < 2 >) and different durations (7d and 15d), so that the content of glycyrrhizic acid, liquiritin and other substances is remarkably increased. The content difference of glycyrrhizic acid, liquiritin and other substances in the glycyrrhiza inflate cultivated species (G) and the glycyrrhiza inflate wild species (W) is obvious. The invention provides an efficient and environment-friendly technical scheme for quality improvement and sustainable production of liquorice.

Description

Technical Field

[0001] The present invention belongs to the technical field of increasing the content of active substances in licorice, and specifically relates to a method for regulating the biosynthesis of triterpenes (including glycyrrhizic acid) and flavonoids (including liquiritin) in the roots of Glycyrrhiza inflata by UV-B radiation, so as to increase the content of glycyrrhizic acid and liquiritin. Background Art

[0002] Licorice is a perennial herbaceous plant of the genus Glycyrrhiza in the Leguminosae family. The 2020 edition of the Chinese Pharmacopoeia includes Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabral L. as medicinal herbs. Their main chemical components include triterpenoids (such as glycyrrhizic acid, glycyrrhetinic acid, etc.), flavonoids (such as liquiritin, glycyrrhizin, isoliquiritigenin, etc.), and polysaccharides. [1] More than 20 triterpenoids and more than 300 flavonoids have been isolated from licorice, and their main metabolic pathways have been identified. [2] Its dried roots and rhizomes are rich in triterpenoids and flavonoids, which have various pharmacological activities such as anti-inflammatory, antioxidant, and antiviral. [1,3 However, wild licorice is on the verge of extinction due to over-harvesting and habitat destruction, and artificially cultivated varieties generally have low levels of medicinal ingredients. [4-5] .

[0003] UV-B radiation, as an environmental stress factor, can increase the accumulation of active ingredients by activating plant secondary metabolic pathways. [6] UV-B-induced enzymes such as phenylalanine ammonia lyase (PAL) and the conversion of phenylpropanoid precursors (phenylalanine) to various other phenolic compounds play a key role in carbon transfer for the biosynthesis of secondary metabolites. [6] The study showed that UV-B radiation induced a shift from carbon assimilation to carbon accumulation in Astragalus mongolica [7] The molecular mechanism of UV-B radiation on the synthesis of triterpenes and flavonoids in licorice roots has not yet been clearly elucidated.

[0004] Problems with existing technologies: Although chemical induction or genetic engineering methods can increase the content of secondary metabolites, they are costly, complex, or have safety risks. Existing technologies do not yet have methods for improving drug quality and changing the ratio of active ingredients by precisely adjusting the UV-B radiation dose and treatment time. The present invention has found through experiments that using specific parameters of UV-B radiation (107 μW / cm 2, 4h daily, 9:00-11:00am, 15:00-17:00pm) treating Glycyrrhiza inflata seedlings can significantly improve the accumulation efficiency of active substances such as glycyrrhizic acid and liquiritin in their roots. Based on the joint analysis of transcriptome and metabolomics, the regulation rules of key genes and metabolic pathways in the roots of Glycyrrhiza inflata under UV-B radiation were revealed, providing an efficient and environmentally friendly method for improving the quality of licorice. Summary of the Invention

[0005] The present invention aims to provide a method for regulating the synthesis of triterpenes and flavonoids in the root system of Glycyrrhiza inflata by UV-B radiation, thereby providing valuable information for further developing licorice varieties with high medicinal components.

[0006] 1. A method for increasing the content of triterpenes and flavonoids in the roots of Glycyrrhiza inflata by UV-B radiation, the method comprising the following steps:

[0007] (1) Plant material cultivation

[0008] After germination of Glycyrrhiza inflata cultivar (G) or wild species (W), seedlings with uniform growth were selected and transplanted into a 5:3 mixture of nutrient soil and vermiculite. The seedlings were regularly watered with Hoagland nutrient solution and grown in a greenhouse at 25°C with a 12h / 12h photoperiod for 90 days.

[0009] (2) UV-B radiation treatment

[0010] The UV-B light source has a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0011] 2. Application of UV-B radiation to enhance the expression of key genes for the synthesis of flavonoids and triterpenes in the roots of Glycyrrhiza inflata. The transcript sequences of PAL1 and CHS, key genes in the flavonoid metabolic pathway, are shown in the sequence listings as SEQ No. 1 and SEQ No. 2; the transcript sequences of CYP88D6 and CYP72A154, key genes in the triterpenoid metabolic pathway, are shown in the sequence listings as SEQ No. 3 and SEQ No. 4. The expression levels of the above genes all reached peak values ​​after 7 days of UV-B radiation.

[0012] Preferably, the PAL1 gene CDS sequence is shown in the sequence listing as SEQ ID NO: 1, and the sequence ID published in the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / 1694854908) is MK341789; the CHS gene CDS sequence is shown in the sequence listing as SEQ ID NO: 2, and the sequence ID published in the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / KX260156.1) is KX260156; the CYP88D6 gene CDS sequence is shown in the sequence listing as SEQ ID NO: 3, and the sequence ID published in the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / MW071147.1) is MW071147; the CYP72A154 gene CDS sequence is shown in the sequence listing as SEQ ID As shown in NO: 4, the sequence ID published in the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / MK534533.1) is MK534533.

[0013] 3. Application of UV-B treatment to alter root phenotype in Glycyrrhiza uralensis. Root fresh weight, dry weight, root length, and root diameter of cultivated (G) and wild-type (W) licorice showed significant increases under UV-B radiation; the UV-B treatment was performed with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0014] 4. Application of UV-B treatment to change the physiological and biochemical indicators of Glycyrrhiza inflata roots. The antioxidant enzyme activity and MDA content of the roots of cultivated (G) and wild (W) types irradiated with UV-B showed significant time-dependent differences and variety-specific responses. The antioxidant enzyme (CAT, SOD) activities of the roots of cultivated (G) were significantly higher than those of the wild type, and membrane lipid peroxidation damage (MDA) was reduced by 30%. The UV-B treatment was performed with a UV-B light source having a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0015] 5. Application of UV-B treatment to change the content of active substances in the roots of Glycyrrhiza inflata. The 15 key metabolites were significantly enriched under UV-B stress, covering amino acids, flavonoids, lipids and energy metabolites: L-glutamic acid, L-homoserine, L-histidine, L-cysteine, L-dendrobatidine, eriodictyol, naringenin-7-O-glucoside, piperine, sn-glycero-3-phosphocholine and 7-methoxyeriodictyol; the UV-B treatment was a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0016] 6. Application of UV-B treatment to alter the content of active substances in the roots of Glycyrrhiza inflata. The significantly enriched pathways are: secondary metabolite biosynthesis, flavonoid biosynthesis, flavonol biosynthesis, arginine and proline metabolism, L-glutamate metabolism, D-amino acid metabolism, and β-alanine metabolism; G and W roots are mainly enriched in flavonoid metabolism, amino acid metabolism, energy metabolism, and signaling and transport; the UV-B treatment is a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0017] 7. UV-B treatment activated the metabolic pathways of active substances in the roots of Glycyrrhiza inflata. The KEGG enrichment analysis showed that UV-B radiation significantly activated the secondary metabolite biosynthesis (ko01110), phenylpropanoid (ko00940), and terpenoid skeleton synthesis (ko00900) pathways. The UV-B treatment was performed using a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

[0018] Description of the accompanying tables and figures

[0019] Figure 1 :Phenotypic changes of Glycyrrhiza inflata under UV-B radiation, where: G: cultivated Glycyrrhiza inflata, W: wild Glycyrrhiza inflata;

[0020] Figure 2 : Glycyrrhizic acid and liquiritin contents of Glycyrrhiza inflata and its wild species under different radiation treatment times, where: G: cultivated species of Glycyrrhiza inflata, W: wild species of Glycyrrhiza inflata;

[0021] Figure 3: Bar graph of physiological responses of cultivated and wild species of Glycyrrhiza inflata to UV-B radiation; where: G: cultivated species of Glycyrrhiza inflata, W: wild species of Glycyrrhiza inflata;

[0022] Figure 4 : Principal component analysis (PCA) analysis of cultivated and wild Glycyrrhiza inflata species under different degrees of UV-B radiation, where: G: cultivated Glycyrrhiza inflata species, W: wild Glycyrrhiza inflata species;

[0023] Figure 5 :Statistical graph of the number of differentially expressed genes between different comparison groups;

[0024] Figure 6 : Venn diagram of DEGs between different comparison groups, where: G: cultivated species of Glycyrrhiza inflata, W: wild species of Glycyrrhiza inflata;

[0025] Figure 7 : GO annotation map of differentially expressed genes induced by UV-B radiation in the roots of cultivated and wild species of Glycyrrhiza inflata;

[0026] Figure 8 : KEGG annotation map of differentially expressed genes induced by UV-B radiation in the roots of cultivated and wild Glycyrrhiza inflata; the left image is of cultivated Glycyrrhiza inflata, and the right image is of wild Glycyrrhiza inflata;

[0027] Figure 9 : Heat map of differentially expressed genes in Glycyrrhiza inflata and its wild varieties under UV-B radiation;

[0028] Figure 10 : qRT-PCR validation analysis of differentially expressed genes in Glycyrrhiza inflata and its wild varieties under UV-B radiation;

[0029] Figure 11 : PCA analysis of samples of Glycyrrhiza inflata and its wild varieties under UV-B radiation;

[0030] Figure 12 : Classification of differential metabolites in the roots of Glycyrrhiza inflata and its wild varieties under UV-B radiation;

[0031] Figure 13 : Cluster analysis of differential metabolites in the roots of Glycyrrhiza inflata and its wild varieties under UV-B radiation;

[0032] Figure 14 : Diagram of the regulatory mechanism of UV-B radiation on the synthesis of triterpenes and flavonoids in the roots of cultivated and wild varieties of Glycyrrhiza uralensis, where: A: MVA pathway, B: MEP pathway, C: glycyrrhizic acid synthesis pathway, D: liquiritin synthesis pathway; from left to right: G-0, G-7, G-15, W-0, W-7, W-15.

[0033] Figure 15: Sequence map of PAL1, CHS, CYP88D6 and CYP72A154 gene transcripts in Glycyrrhiza inflata. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the invention clearer, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present invention shown in the accompanying drawings and described with reference to the accompanying drawings are merely exemplary, and the present invention is not limited to these embodiments. It should also be noted that in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution according to the present invention are shown in the accompanying drawings, and other details that are not closely related are omitted.

[0035] Example 1

[0036] This embodiment provides a method for changing the growth and root quality of Glycyrrhiza inflata by UV-B radiation, as follows:

[0037] 1. Materials

[0038] Seeds of cultivated Glycyrrhiza uralensis (W) and wild species (Gi) with full grains were selected and treated with 98% concentrated sulfuric acid for 30 minutes to break seed dormancy. The seeds were stirred every 10 minutes and then rinsed with distilled water to remove residual sulfuric acid. The seeds were disinfected with 3% NaClO for 12 minutes, immersed in 75% ethanol for 2 minutes, and rinsed with distilled water (5-8 times). After the treated seeds were germinated on culture dishes for 5 days, the seedlings with consistent growth were transplanted into pots (15 cm × 12 cm × 14.5 cm) with nutrient soil: vermiculite = 5:3. During this period, the modified Hoagland nutrient solution was used for irrigation every 7 days. All seedlings were grown at a temperature of 25°C and a light intensity of 500 μmol (m 2 s) -1 In the greenhouse, the light / dark cycle was 12 / 12 h. When the seedlings grew to 90 days old, the plants with the same growth were selected and treated with UV-B (107 μW·cm -2 ) for 7 and 15 days, with irradiation for 4 hours daily, from 9:00 AM to 11:00 AM and from 3:00 PM to 5:00 PM. Licorice roots were rinsed with sterile water and used for biomass, physiological parameters, transcriptome sequencing, and metabolomics analysis.

[0039] 2. Metabolite Extraction and Non-targeted Metabolism Analysis

[0040] The powder was treated according to the method of Wang et al. [8]. Vacuum freeze drying: Accurately weigh 0.2 g of licorice root sample and treat it with vacuum freeze drying technology (cold trap temperature ≤ -50°C, vacuum degree <10 Pa) to obtain freeze-dried powder. Physical crushing treatment: Use a mortar to grind the freeze-dried sample. Active ingredient extraction: Solvent system construction, weigh 100 mg of powder, add 1.2 mL of 70% (v / v) methanol aqueous solution, and construct a low-polarity extraction system. Multi-stage vortex extraction: Initial vortex: vortex at 3000 rpm for 30 minutes at 4°C; step-by-step intensification: vortex again at an interval of 15 minutes, and complete 6 cycles in total (total extraction time 180 minutes); low-temperature standing treatment: The extraction mixture is transferred to a 4°C constant temperature incubator and left to stand overnight (12-16 hours) to promote the release of cell contents. Centrifugal purification: Use low-temperature high-speed centrifugation (4°C, 12000 rpm, 10 minutes), discard the precipitate and retain the supernatant. Membrane Filtration: Terminal filtration was performed using a 0.22 μm nylon microporous membrane to eliminate particulate matter interference. Sample Storage: The filtrate was transferred to an injection vial. An Agilent 1290 UPLC system coupled to a 6545 Q-TOF (equipped with an ESI Turbo ion source) was used, with full process control provided by a MassHunter workstation. The system included a two-dimensional liquid chromatography module (ZORBAX Eclipse Plus C18, 2.1 × 150 mm, 1.7 μm), a dual-piezoelectric electrospray ionization source (switching between positive and negative ion modes), and a high-precision time-of-flight mass analyzer (TOF mass accuracy <2 ppm). Chromatographic separation conditions: mobile phase system, phase A: 0.1% formic acid aqueous solution (containing 5 mM ammonium formate), phase B: 0.1% formic acid acetonitrile solution gradient program: 0-9 min: phase B 5%→95% (linear gradient), 9-10 min: phase B maintained at 95%, 10-11.1 min: phase B 95%→5% (gradient withdrawal), 11.1-14 min: phase B equilibrated to 5%, flow rate and injection, flow rate: 0.35 mL / min (column temperature 30°C), injection volume: 5 μL (autosampler temperature 4°C). Mass spectrometry analysis parameters: ionization system, electrospray voltage: 5500V (ESI+) / -4500V (ESI-), ion source temperature: 550°C, gas parameters: GSI 50psi / GSII60psi / CUR 25psi, mass analysis, mass range: m / z 100-1700 (full scan mode), resolution: Q-TOF mode 40,000FWHM (@m / z 200), secondary mass spectrometry: information-dependent acquisition (IDA), collision energy gradient optimization, calibration and tuning, tuning solution: 10 / 100μmol / L polypropylene glycol (PPG), calibration parameters: DP 60-120V, CE 10-40eV (MRM mode).Data acquisition strategy: MRM parameter optimization, 300 preset ion pairs (including Q1 / Q3 mass, DP, CE), dynamic background subtraction (DBS) window: ±50 Da, peak dwell time: 50 ms / ion pair. System validation: Linear range: 0.1-1000 ng / mL (R. 2 >0.999), sensitivity: LLOQ 0.05 ng / mL (S / N ≥ 10), reproducibility: retention time RSD < 0.8%, peak area RSD < 2.3%.

[0041] Mass spectrometry data processing method: Agilent MassHunter Profinder was used for multi-stage data processing: feature extraction algorithm, batch recursive feature extraction algorithm was enabled, and the following parameters were set: charge state range: 1-2+, isotope distribution model: common organic (excluding halogen interference), mass tolerance window: 10-15 ppm, peak intensity threshold: ≥5000 counts, cross-sample feature matching was performed based on retention time offset (±0.2 min) and mass deviation (±15 ppm), and an adaptive weight algorithm was used to optimize alignment accuracy.

[0042] Molecular formula inference method: accurate mass analysis, molecular weight is calculated by the accurate mass (m / z) of the primary mass spectrometry parent ion, combined with the following parameters: mass accuracy requirement: <5ppm, adduct ion identification: priority matching [M+H] + / [M+Na] + Typical adduct forms such as β-aminobenzoic acid and benzoic acid were selected for candidate molecular formula screening. A multidimensional verification strategy based on mass accuracy and adduct ion characteristics was used to extract candidate molecular formulas from databases such as HMDB and Massbank, and an elemental composition filtering algorithm (element deviation < 3%) was applied.

[0043] Metabolite Identification System: Primary Identification Process: Primary Matching: Candidates are screened using dual thresholds of accurate mass and retention time. Secondary Verification: Spectral similarity comparison with standards (similarity > 80%). Metabolite Annotation: Integrate information from databases such as HMDB and KEGG to establish a compound annotation confidence scoring system. Secondary Identification Strategy: Fragment Ion Analysis: Extract the top 10 characteristic fragment ions, calculate the fragmentation pattern match, and analyze the deviation of isotope peak distribution from theoretical abundance.

[0044] Data analysis: Multi-dimensional statistical analysis was conducted based on the Maiwei Metabolism Cloud Platform: T-test: Calculate the significance of the difference in metabolite content between the two groups (P-value), fold analysis: Calculate the fold change, screen for significantly changed metabolites with |log2FC|≥2, multivariate statistical analysis: PCA analysis: Observe the sample clustering trend through the principal component score diagram, OPLS-DA analysis: Optimize orthogonal signal correction and improve the model explanatory power (R 2 Y>0.8,Q 2 >0.5). Key parameter calculation model evaluation indicators, OPLS-DA model: verification of model reliability through permutation test (Q 2 Intercept < 0.05), pathway annotation, KEGG enrichment analysis: Based on the mapping of differential metabolites to metabolic pathways, pathway enrichment was calculated (p < 0.05). Results screening criteria, a double significance threshold system was established: statistical significance: P < 0.05, biological significance: |log2FC| ≥ 2, and metabolites meeting either condition were identified as differential metabolites.

[0045] 8. RNA Extraction, RNA Sequencing, and Transcriptome Analysis

[0046] Sample Pretreatment: Cryogenically grind licorice root tissue (approximately 50 mg) in a pre-chilled mortar (frozen in liquid nitrogen), grind to a fine powder, and transfer to a 1.5 mL RNase-free centrifuge tube. Lysis and Extraction: Lysis system was established by adding 1 mL of Trizol reagent, vortexing thoroughly for 30 seconds, and incubating at room temperature (≥25°C) for 10 minutes. Organic Phase Separation: 200 μL of chloroform was added, vigorously shaken for 30 seconds, and centrifuged at 12,000 rpm for 10 minutes at 4°C. The upper aqueous phase was transferred to a fresh tube. For continuous purification, an equal volume of phenol:chloroform (25:24) was added, the centrifugation and shaking steps were repeated, and an equal volume of chloroform was added again, and the centrifugation was repeated. RNA Precipitation: Isopropanol precipitation was performed by adding an equal volume of pre-chilled isopropanol, incubating at -20°C for 1 hour, and centrifuging at 12,000 rpm for 10 minutes. Gradient Wash: Discard the supernatant, add 1 mL of 75% ethanol (prepared in DEPC water), centrifuge at 8,000 rpm for 5 minutes, and repeat the wash step once. RNA drying and dissolution: Vacuum dry, discard ethanol, briefly centrifuge, and vacuum dry for 2-4 minutes (avoid overdrying). Dissolution and reconstitution: Add 20-50 μL of RNase-free water, dissolve at room temperature for 10 minutes, and store at 4°C. Quality Assurance: Integrity assessment: RNA integrity index (RIN ≥ 7.0) is performed using an Agilent 2100 Bioanalyzer. Purity verification: An A260 / A280 ratio of 1.8-2.1 and an A260 / A230 ratio > 2.0 are acceptable.

[0047] mRNA is enriched using mRNACapture Beads magnetic beads. After purification, the mRNA is fragmented using high temperature. The fragmented mRNA is used as a template to synthesize the first strand of cDNA in a reverse transcriptase mixture. While synthesizing the second strand of cDNA, end repair and A tailing are completed. Then, the adapter is connected and the mRNA is synthesized using Hieff DNA Selection Beads magnetic beads were used to purify and select target fragments; PCR library amplification was then performed, and finally, Illumina Novasek X Plus was used for detection.

[0048] Quality control tool selection: fastp tool is used for full-process quality control analysis, which uses a sliding window algorithm to achieve dynamic identification of low-quality bases [9] Filtering strategy design, adapter contamination removal: automatically identify and remove 3' end adapter sequences, N base filtering: remove sequences containing N ratio ≥ 10%, homologous base filtering: remove all A sequences (A content = 100%), quality threshold control: discard sequences with low-quality base ratio > 50% (Q ≤ 20).

[0049] Quality distribution analysis, Phred quality value distribution: draw Q value histogram to evaluate the overall quality level, base uniformity detection: analyze the GC content distribution of each position (ideal value 45-55%), length distribution verification: count the effective sequence length (≥50bp).

[0050] mRNA enrichment strategy: Oligo (dT) magnetic beads are used to specifically capture poly-A tailed mRNA. This technology can capture eukaryotic mRNA with an efficiency of over 85%. Note: The magnetic beads must be pretreated with 0.1M NaOH to remove residual DNA before use, and the LiCl concentration in the binding buffer must be optimized to 500-1000mM to improve specificity. Alignment parameter settings: Use Bowtie2 for short sequence alignment

[10] .

[0051] HISAT 2 was used to align the sequences obtained from the paired-end sequencing to the reference genome of Glycyrrhiza uralensis, and the parameters were set to the default parameters.

[11] .

[0052] Screening of differentially expressed genes: The screening criteria were set, and a dual-threshold screening strategy was adopted: significance threshold: FDR-corrected p-value (FDR<0.05), difference fold threshold: |log2FC|>1 (corresponding to FC≥2 or ≤0.5), expression level normalization, and relative gene expression levels were calculated based on the FPKM (Fragments Per Kilobase Million) algorithm. The influence of library construction bias on quantitative results was effectively eliminated by transcript length normalization and sequencing depth correction.

[0053] Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to enrich the DEGs to obtain a detailed description of the DEGs in the roots of Glycyrrhiza inflata under UV-B radiation.

[0054] 9. Determination of glycyrrhizic acid and liquiritin content in the root of Glycyrrhiza uralensis

[0055] Preparation of standard products: Refer to the method of the 2020 edition of the Chinese Pharmacopoeia to obtain glycyrrhizic acid and liquiritin reference substances [1] , add 70% ethanol to prepare reference solutions of 20 μg / mL liquiritin and 0.2 mg / mL glycyrrhizic acid, respectively. Take 0.2 g of dried root sample powder passed through a No. 3 sieve and place it in a stoppered conical flask. Add 100 mL of 70% ethanol, stopper tightly, weigh, and ultrasonicate for 30 minutes. Cool, weigh, and make up the lost weight with 70% ethanol. Shake well and filter to obtain the sample solutions. Glycyrrhizic acid and liquiritin contents were determined using acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B. The detection wavelength was 237 nm, the column temperature was set at 30°C, and the injection volume was 10 μL.

[0056] Preparation of the sample to be tested: Accurately weigh 0.2g of sample powder and place it in a conical flask with a lid that has been pre-cooled to 4°C. Add 100mL of 70% (v / v) ethanol solution and immediately seal the bottle to ensure that there are no bubbles remaining in the system. Ultrasonic treatment is carried out for 30min at a frequency of 40kHz and a power of 250W, and the temperature does not exceed 25°C. Solvent compensation: After cooling to room temperature, weigh and calculate the amount of solvent volatilization, and fill it to the initial volume (100mL) with ethanol solution of the same concentration. Centrifugal separation: Transfer to a centrifuge tube, centrifuge at 5000rpm at 4°C for 10min, and take the supernatant for use. Membrane filtration treatment: Filter through a 0.22μm organic phase filter membrane to obtain the solution to be tested.

[0057] 10. Combined analysis of metabolism and transcriptome

[0058] DEGs and DAMs enriched in triterpenoid and flavonoid biosynthesis pathways were mapped using KEGG enrichment analysis. Differential gene expression and metabolite content were calculated using FPKM values.

[0059] 11. qRT-PCR Validation

[0060] The CDS sequences of candidate genes were found from the licorice transcriptome data, and primers were designed using the NCBI (National Center for Biotechnology Information (nih.gov) online website (Table 1). β-actin was selected as the internal reference gene. Total RNA was extracted using a plant tissue RNA rapid extraction kit (TIANGEN DP452), and then reverse transcribed into cDNA using a cDNA synthesis kit (TIANGEN KR118). qRT-PCR was performed, and each sample was assayed three times. Finally, 2 -ΔΔCt Algorithm calculation and analysis of the relative expression of candidate genes

[12] , and used Excel 2019 and Origin 2021 software to create graphs.

[0061] Table 1 qRT-PCR primer sequences

[0062]

[0063]

[0064] A standardized 20 μL reaction system was established according to the standard operating procedure of the Tiangen Biochemical qRT-PCR Kit. The initial denaturation phase was performed at 95°C for 15 minutes to completely melt the DNA double-strands and activate the Taq enzyme. The step-wise amplification phase involved 40 cycles of denaturation at 95°C for 10 seconds (to disrupt double-stranded DNA), annealing and extension at 60°C for 20 seconds (to allow primer binding and chain extension), and extension at 72°C for 30 seconds (to ensure complete amplification product formation). Melting curve analysis was performed with initial denaturation at 95°C for 15 seconds, annealing scan at 60°C for 1 minute (to collect fluorescence signals), and melting at 95°C for 1 second (to verify product specificity).

[0065] Example 2

[0066] This embodiment provides the experimental results of the above embodiment, which are as follows:

[0067] 1. Analysis of the growth and development of Glycyrrhiza uralensis roots and changes in physiological and biochemical indicators under UV-B radiation

[0068] like Figure 1 As shown, under UV-B radiation, the root fresh and dry weights of both G and W licorice showed a significant increase (P < 0.05). The root fresh weight of G increased from 0.35 g (day 0) to 0.39 g (day 15), an increase of 11.4%. The root fresh weight of W increased from 0.45 g (day 0) to 0.50 g (day 15), an increase of 11.1%. Furthermore, the root fresh weight of W was significantly higher than that of G at all treatment time points, with a difference of 0.11 g at day 15, indicating that wild-type Glycyrrhiza uralensis has greater resistance or adaptability to UV-B radiation.

[0069] like Figure 1 As shown in the figure, on the 7th day of UV-B radiation, the leaves of W were more severely curled than those of G, and withered and dried up. On the 15th day, almost all the leaves of W withered and withered, while the leaves of G changed more slowly than those of W. There were significant differences in plant height, root length and root diameter between G and W under UV-B radiation (P<0.05). Root length increased with the extension of radiation time. The root diameter of W was higher than that of G at all UV-B treatment time points. As the radiation time increased, the root diameter of G grew slower than that of W ( Figure 1 ).

[0070] like Figure 2 As shown, glycyrrhizic acid and liquiritin content in W roots were higher than in G at all treatment time points. Glycyrrhizic acid content reached its highest level in W roots on the 7th day of irradiation and decreased with increasing irradiation duration. While glycyrrhizic acid content in G roots reached its highest level on the 7th day, both the increase and decrease rates were relatively gradual. While liquiritin content in W roots increased with increasing irradiation duration, liquiritin content in G roots decreased on the 7th day, with the increase and decrease rates mirroring those of glycyrrhizic acid content.

[0071] like Figure 3 As shown, under UV-B radiation stress, antioxidant enzyme activities and MDA content in the roots of G and W exhibited significant time-dependent and cultivar-specific responses. POD activity in W roots decreased rapidly with increasing irradiation duration, by as much as 75%. In contrast, POD activity in G roots gradually decreased, but remained largely unchanged at 15 and 7 days. At the same irradiation time point, POD activity in W roots was consistently higher than that in G. Superoxide dismutase (SOD) activity in G roots decreased at 7 days and increased at 15 days. SOD activity in W roots remained largely unchanged at 7 days but increased at 15 days. SOD activity in G roots was significantly higher than that in W throughout the irradiation period. CAT activity in G roots decreased at 7 days but remained stable at 15 days, while that in W roots peaked at 7 days and declined at 15 days. At the same time point, CAT activity in G roots was significantly higher than that in W roots at both 0 and 15 days, but higher than that in W roots at 7 days. The MDA content of both materials reached its lowest on the 7th day, with the MDA content of G roots decreasing significantly, while that of W roots changed slowly. As the irradiation time prolonged, the MDA content of W roots increased significantly less than that of G.

[0072] 2. Transcriptomic Analysis of Glycyrrhiza uralensis Roots Under UV-B Radiation

[0073] In order to clarify the response of licorice root system to UV-B radiation, the RPKM value, PCA analysis and correlation analysis ( Figure 4A, B, C, D), the situation of obtaining differentially expressed genes in different comparison groups is as follows Figure 5 As shown in the figure, this study was divided into nine comparison groups to display the number of up- and down-regulated DEGs. In the G-7 vs. G-15 comparison of G roots, the number of up-regulated DEGs was 2169, 3165, and 1942, respectively, while the number of down-regulated genes was 4347, 7585, and 4429, respectively. This indicates that negative regulation was dominant on the 7th day of radiation, and a stronger inhibitory response was triggered on the 15th day. In the W-7 vs. W-15 comparison of W, the number of up-regulated DEGs was 1620, 4214, and 2905, respectively, while the number of down-regulated genes was 1993, 4450, and 3335, respectively. This indicates that positive regulation dominated in W roots after exposure to UV-B radiation, and that the roots gradually adapted to the radiation with increasing radiation time. In the G-0 vs W-0, G-7 vs W-7, and G-15 vs W-15 comparison groups, the numbers of upregulated DEGs were 2602, 2174, and 3804, respectively, and the numbers of downregulated genes were 5301, 2953, and 2466, respectively.

[0074] The DEGs of different comparison groups were analyzed separately. Figure 6 As shown, there are 3276 genes shared by G-0vs G-7 and G-0vs G-15, 722 genes shared by G-0vs G-7 and G-7vs G-15, 3028 genes shared by G-0vs G-15 and G-7vs G-15, 1504 genes shared by W-0vs W-7 and W-0vs W-15, 521 genes shared by W-0vs W-7 and W-7vs W-15, 3813 genes shared by W-0vs W-15 and W-7vs W-15, 1134 genes shared by G-0vs W-0 and G-7vs W-7, 1220 genes shared by G-0vs W-0 and G-15vs W-15, and 726 genes shared by G-7vs W-7 and G-15vs W-15 has a total of 945 genes.

[0075] In this study, we conducted a Gene Ontology (GO) functional annotation analysis on the DEGs in the roots of Glycyrrhiza uralensis (G) and its wild type (W) treated with UV-B radiation for 7 days and 15 days. Functional classification mainly includes: biological process (BP): analyzing the life activities such as metabolic regulation and stress response in which genes participate; cellular component (CC): locating the distribution characteristics of gene products in organelles or subcellular structures; molecular function (MF): revealing the molecular mechanism of action such as the enzyme activity or binding characteristics of gene-encoded products. Figure 7As shown in the figure, the top ten functional categories significantly enriched in biological process (BP) include: GO:0009987 (cellular process), GO:0008152 (metabolic process), GO:0065007 (biological regulation), GO:0050896 (stimulus response), GO:0050789 (biological process regulation), GO:0051179 (localization), GO:0032502 (developmental process), GO:0032501 (multicellular organism process), GO:0023052 (signal transduction), and GO:0000003 (reproduction). Three core terms significantly enriched in cellular component (CC) include: GO:0110165 (cell morphology entity), GO:0032991 (protein complex), and GO:0044423 (viral component). The top ten functional categories with significant enrichment of molecular function (MF) included: GO:0005488 (binding activity), GO:0003824 (catalytic activity), GO:0005215 (transport activity), GO:0140657 (ATP-dependent activity), GO:0140110 (transcriptional regulatory element activity), GO:0005198 (structural molecule activity), GO:0098772 (molecular function regulatory element), GO:0016209 (antioxidant activity), GO:0060089 (molecular signaling activity), and GO:0045182 (translation regulatory element activity).

[0076] KEGG analysis was performed on the DEGs of G and W root systems at different treatment time points, and the top 20 pathways were extracted and ranked according to the degree of differential gene enrichment, such as Figure 8The top 10 activities of G roots are as follows: metabolic pathways (ko01100), biosynthesis of secondary metabolites (ko01110), carbon metabolism (ko01200), plant hormone signal transduction (ko04075), biosynthesis of amino acids (ko01230), plant-pathogen interaction (ko04626), starch and sucrose metabolism (ko00500), phenolic propanoid biosynthesis (ko00940), glycolysis / gluconeogenesis (ko00010), and amino sugar and nucleotide sugar metabolism (ko00520).

[0077] In order to verify the reliability of the transcriptome data, 14 differentially expressed genes (flavonoid metabolism-related genes: PAL1, 4CL3, CHS1, CHS, CHI, FLS, triterpenoids: CYP88D6, CYP72A154, SQE1, UGT1, b-AS1, other genes: NADP-ME4, ALDC, FDX3) were selected and the expression patterns of DEGs were analyzed by FPKM values ​​and qRT-PCR. Figure 9 As shown in Figure 2, the expression levels of CHS1, FDX3, and NADP-ME4 continued to increase in G roots, while FDX3 first decreased and then increased in W roots; the expression levels of PAL1, 4CL3, CHS, FLS, CYP88D6, and CYP72A154 genes reached their peak values ​​in G and W roots 7 days after irradiation ( Figure 10), indicating that flavonoids and triterpenes accumulated most on the 7th day; while the FPKM of UGT1 and β-AS1 decreased first and then increased in both materials. SQE1, UGT1, and b-AS1 showed a trend of decreasing first and then increasing in both G and W roots. qRT-PCR results showed that SQE1 and UGT1 in G showed the lowest relative expression levels on the 7th day of irradiation and then increased with prolonged irradiation, while UGT1 in W roots reached its highest level on the 7th day and then decreased on the 15th day. The FPKM values ​​of NADP-ME4 and ALDC in W roots initially increased and then decreased. The expression trend of NADP-ME4 in G roots mirrored that of W, while ALDC continued to increase in G roots with prolonged irradiation. qRT-PCR verification confirmed that these FPKM values ​​were consistent with the transcriptome FPKM trend, indicating that the transcriptome data were reliable.

[0078] 3. Metabolomics analysis of Glycyrrhiza uralensis roots under UV-B radiation

[0079] Principal component analysis (PCA) was used to systematically evaluate the metabolome data of licorice roots treated with UV-B radiation: a two-dimensional score plot (PC1 = 60.21%, PC2 = 8.85%) was constructed, showing that G and W samples exhibited significant discrete distribution characteristics, and the metabolic profiles of each group exhibited dose-dependent shifts. To address the intra-group variation interference problem of PCA, orthogonal partial least squares discriminant analysis (OPLS-DA) was used for in-depth analysis: the internal aggregation degree of each group of samples was more aggregated than that of the PCA method, and the groups were more open ( Figure 11 ).

[0080] The threshold variable importance (VIP) value (VIP ≥ 1.0) in the OPLS-DA model was used to screen the differential metabolites and classify the differential metabolites into Figure 12 As shown, flavonoids (Flavonoids) 46.45%, alkaloids (Alkaloids) 3.87%, nitrogen compounds (Nitrogen Compounds) 7.74%, nucleoside and its derivatives (Nucleotide and derivatives) 1.29%, organic acids (Organic Acids) 12.90%, organic compounds (Organic Compounds) 3.87%, phenylpropanoids (Phenylpropanoids) 3.87%, phospholipids (Phospholipids) 6.45%, polyphenols (Polypheno) 11.94%, quinones (Quinones) 2.58%, terpenes (Terpene) 5.16%, and others (Others) 3.87%.

[0081] Cluster analysis of differential metabolites of flavonoids and triterpenoids in licorice roots irradiated with UV-B at different times Figure 13 As shown in the figure, the upregulated metabolites in the W root system at 7 days after irradiation were kaempferol-3-O-rhamnoside-7-O-rhamnoside, apigenin-7-rutinoside, rutin, trihydroxy isoflavone glucoside, quercetin-7-O-maleylhexoside, C-pentosyl-apigenin-O-salicyloylhexoside, narcissin, luteolin-O-hexoside, apigenin, eriodictyol, 7,4'-dihydroxyflavone, trihydroxyphenol acetone, glycyrrhizic acid, glycyrrhizin, and galangin. VI, 7-methoxyeriodictyol, pinoresin, C-hexosyl-hypericin-O-hexoside, C-hexosyl-luteolin-O-hexoside, licorice chalcone E, dehydroliquiritigenin C, 3',4'5,7-tetrahydroxyflavone-3-O-uronic acid, luteolin C-hexoside, 8-isoprenyl-biochanin, dehydroliquiritigenin, 18α-glycyrrhizic acid, apigenin-8-C-pentososide, naringenin-7-O-glucoside and swellin I. The upregulated metabolites in G roots at 7 days after irradiation were liquiritigenin D, licorice chalcone C, emodin, 3-hydroxyflavone, kaempferol, inflatoside V, proanthocyanidin B3, isoliquiritigenin A, ursolin, coumarin (dihydrokaempferol), genistein (4',5,7-trihydroxyisoflavone), pinosylvin, hydrogenated glycyrrhizin E, hypericin O-hexoside-O-hexoside methyl ether, quercetin O-hexoside and licorice chalcone A.

[0082] 4. Combined analysis of transcriptome and metabolome of Glycyrrhiza uralensis roots under UV-B radiation

[0083] The biosynthesis of the triterpenoid compound glycyrrhizic acid is regulated by multiple enzymes. The precursor, isopentenyl pyrophosphate (IPP), is synthesized via two distinct pathways: the mevalonate pathway (MVA) in the cytoplasm and the methylisopropyl phosphate pathway (MEP) in the plastids. The synthesis of glycyrrhizic acid from the precursor IPP is catalyzed by multiple key synthases.

[0084] In the MVA and MEP pathways ( Figure 14 A, B), HMGS and HMGR2 were upregulated in G and W roots at 7 days after UV-B irradiation, and downregulated with increasing irradiation time at 7 and 15 days. The expression levels of W roots were higher than those of G at all treatment time points. DXS, a member of the MEP pathway, was lowly expressed in both G and W roots. HDR1 had the highest expression level in G roots at 7 days after irradiation, while it reached the highest expression level in W roots at 15 days after irradiation.

[0085] As the irradiation time prolonged, the expression of FPS1 and GGPS genes in G and W roots was down-regulated at 7d and 15d irradiation treatments, while the expression of SQE1 and bAS1 was up-regulated at 7d and 15d irradiation treatments in G and W roots. Figure 14 C).

[0086] Cytochrome P450 reductase is essential for the oxidation of C-11 and C-30, balancing the cellular redox environment and promoting the synthesis of glycyrrhetinic acid. Finally, glycyrrhizinyltransferase (UGT) catalyzes the glucuronidation of the C-3 hydroxyl group of glycyrrhetinic acid, ultimately producing glycyrrhizic acid. During this phase, CYP88D6 and CYP72A154 expression levels peaked on the 7th day of irradiation in both G and W roots, with an initial upregulation followed by a downregulation during the irradiation period. The relative content trends of differentially expressed metabolites showed that inflatoside I reached its highest level on the 7th day of irradiation in both G and W. Inflatoside VI, with increasing irradiation duration, decreased in G at 7 and 15 days, while in W, it initially decreased and then increased. Inflatoside V reached its highest level on the 15th day of irradiation in W. The relative content of glycyrrhizic acid reached its highest level on the 7th day in both G and W. The biosynthesis of the flavonoid compound liquiritin begins with phenylalanine, which is produced through the shikimate pathway and then enters the phenylpropanoid metabolic pathway ( Figure 14 D) In ​​this study, CHS reached its highest expression level on the 7th day after irradiation in G and W roots. CHS and CHS1 jointly regulate the production of naringenin chalcone and isoliquiritigenin, with CHS1 expression continuously increasing in G roots. Isoliquiritigenin is further catalyzed by CHI to liquiritigenin. CHI reached its highest expression level on the 7th day after irradiation in G and W roots. The final step in the liquiritin biosynthesis pathway is glycosylation, with UGT1 catalyzing the 4'-O-glycosylation of liquiritigenin to form the final product, liquiritin.

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Claims

1. A method for increasing the content of triterpenes and flavonoids in the roots of Glycyrrhiza inflata by UV-B radiation, characterized in that The method comprises the following steps: (1) Plant material planting: After the seeds of Glycyrrhiza inflata cultivated species (G) or wild species (W) germinated, seedlings with uniform growth were selected and transplanted into a 5:3 mixture of nutrient soil and vermiculite. They were regularly watered with Hoagland nutrient solution and grown in a greenhouse environment at 25°C with a photoperiod of 12 h / 12 ​​h for 90 days. (2) UV-B radiation treatment: using a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

2. The application of UV-B radiation to enhance the expression of key genes for the synthesis of flavonoids and triterpenoids in the roots of Glycyrrhiza inflata, characterized in that The transcript sequences of the key genes PAL1 and CHS for flavonoid synthesis are shown in the sequence listings SEQ No. 1 and SEQ No. 2; the transcript sequences of the key genes CYP88D6 and CYP72A154 for triterpenoid synthesis are shown in the sequence listings SEQ No. 3 and SEQ No.

4. The expression levels of the above genes all reach peak values ​​after 7 days of UV-B radiation.

3. The application of UV-B treatment to change the root phenotype of Glycyrrhiza inflata is characterized by The root fresh weight, dry weight, root length and root diameter of cultivated (G) and wild (W) licorice showed significant increases under UV-B radiation; the UV-B treatment was a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

4. The application of UV-B treatment to change the physiological and biochemical indices of Glycyrrhiza inflata root system is characterized by The antioxidant enzyme activity and MDA content of the roots of the cultivated type (G) and the wild type (W) irradiated by UV-B showed significant time-dependent differences and variety-specific responses. The antioxidant enzyme CAT and SOD activities of the roots of the cultivated type (G) were significantly higher than those of the wild type, and the membrane lipid peroxidation damage MDA was reduced by 30%. The UV-B treatment was a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

5. The application of UV-B treatment to change the content of active substances in the roots of Glycyrrhiza inflata is characterized in that The root active substances are significantly enriched under UV-B stress, including L-glutamic acid, L-homoserine, L-histidine, L-cysteine, L-dendropicrin, eriodictyol, naringenin-7-O-glucoside, piperine, sn-glycero-3-phosphocholine and 7-methoxyeriodictyol; the UV-B treatment is a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

6. Application of UV-B treatment to change the content of active substances in the roots of Glycyrrhiza inflata, characterized in that The pathways of the active substances are: secondary metabolite biosynthesis, flavonoid biosynthesis, flavonol biosynthesis, arginine and proline metabolism, L-glutamate metabolism, D-amino acid metabolism and β-alanine metabolism; G and W roots are mainly enriched in flavonoid metabolism, amino acid metabolism, energy metabolism and signal and transport; the UV-B treatment is a UV-B light source with a wavelength of 280-320nm and an intensity of 107μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

7. UV-B treatment activates the metabolic pathway of active substances in the roots of Glycyrrhiza inflata, which is characterized by The KEGG enrichment analysis showed that UV-B radiation significantly activated the secondary metabolite biosynthesis ko01110, phenylpropanoid ko00940, and terpenoid skeleton synthesis ko00900 pathways; the UV-B treatment was a UV-B light source with a wavelength of 280-320 nm and an intensity of 107 μW / cm 2 , irradiated for 2 hours each day at 9:00-11:00 and 15:00-17:00, for 7 days and 15 days respectively.

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