Method for increasing nitidine chloride content in radix zanthoxyli tissue culture material and application

Abstract

The invention relates to a method for increasing the content of nitidine chloride in a radix zanthoxyli tissue culture material, and belongs to the technical field of biology. At present, the problems of long production period, unstable content and the like exist in the traditional method for extracting nitidine chloride by cultivating radix zanthoxyli. Therefore, the method provided by the invention comprises explant selection, bud induction culture, cluster bud culture, callus induction culture and directional regulation culture, in the directional regulation culture, cluster buds or calluses are transferred to a red light condition to be cultured for 1-2 months, the illumination intensity is 150-250 micromoles per day, the photoperiod is 10-14 hours per day, and the culture time is 2-3 days. The nitidine chloride synthesis pathway gene expression is activated, and the nitidine chloride content is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, the present invention relates to a method for increasing the content of nitidine chloride in tissue culture materials of Zanthoxylum nitidum and its application. Background Art

[0002] As a genuine medicinal material of "Ten Flavors of Guangxi" in Guangxi Zhuang Autonomous Region, Zanthoxylum nitidum is used as medicine with its root, having the effects of promoting blood circulation to remove blood stasis, promoting qi circulation to relieve pain, etc. Its index component nitidine chloride is a key component for quality control specified in the Pharmacopoeia of the People's Republic of China [1]. Affected by the sharp reduction of wild resources, the current production relies on artificial cultivation. However, the content difference of nitidine chloride in medicinal materials of different cultivation types and origins can reach 6 times (0.70 - 4.33 mg / g) [2], while Yang et al. detected the content of nitidine chloride in Zanthoxylum nitidum medicinal materials from 8 origins to be 0.50% - 0.91% [3], and Wu Luxiang et al. detected the content of nitidine chloride in Zanthoxylum nitidum medicinal materials from 10 origins to be 0.14% - 0.19% [4]. It can be seen that the content of nitidine chloride in Zanthoxylum nitidum medicinal materials fluctuates greatly, which brings challenges to the quality control of Zanthoxylum nitidum medicinal materials. And the traditional cultivation cycle is long, and the accumulation of secondary metabolites is significantly affected by the environment, resulting in difficult quality control.

[0003] Plant tissue culture technology has realized the efficient rapid propagation of Zanthoxylum nitidum. By optimizing the bud induction, subculture proliferation and rooting culture media, the induction rate of explants can reach 41.7% - 96.2%, and the annual proliferation rate reaches 3.612, and problems such as browning of tender buds are solved. However, the existing technology focuses on the optimization of rapid propagation technology, and there is insufficient research on the biosynthesis regulation of nitidine chloride in the tissue culture system. The synthesis pathway has not been directionally activated through environmental factors (such as light quality, culture medium components), and it cannot meet the requirements of stable and efficient accumulation of target components in large-scale production.

[0004] In view of the above situation, it is urgent to optimize tissue culture technology, break through the bottleneck of long traditional cultivation cycle and large content fluctuation, establish a method for directional regulation of nitidine chloride biosynthesis, and provide technical support for industrial production.

[0005] References: [1] Pharmacopoeia Commission of the People's Republic of China. Pharmacopoeia of the People's Republic of China (Part I). Beijing: China Medical Science and Technology Press, 2020; [2] Qin Yunrui, Jiang Zhen'ou, Lai Maoxiang, Huang Yunfeng, Wang Xinhong. Textual research on the original plants of Zanthoxylum nitidum and analysis of the content of its active ingredients. Guihaia, 2019, 39(04): 531 - 539; [3] Yang, Y., Li, Y., Amoroso, V., Acma, F., Guiang, M. M., & Wu, H. Comparison of production of bioactive components in Zanthoxylum nitidum taproots from different regions in southern China. Biomedical chromatography : BMC, 2023, 37(5): e5602; [4] Wu, L. X., Huang, R. G., Lan, X. D., Ma, E. Y., Chen, J. X., Yang, S. H., Liu, Q. Y., & Zhou, S. T. Quality evaluation of Zanthoxylum nitidum medicinal materials from different producing areas based on principal component analysis. Journal of Central South University of Forestry & Technology, 2023, 44(10): 23 - 26. Summary of the Invention

[0006] An object of the present invention is to solve at least the above - mentioned defects and provide at least the advantages described hereinafter.

[0007] The present invention provides a method for increasing the content of nitidine chloride in tissue - cultured materials of Zanthoxylum nitidum, aiming to optimize tissue - culture techniques to directionally regulate the biosynthesis of nitidine chloride in tissue - cultured materials of Zanthoxylum nitidum, increase the content of nitidine chloride, solve the problems such as the long production cycle required for extracting nitidine chloride from Zanthoxylum nitidum plants by cultivation techniques and the instability of the nitidine chloride content, and provide a new technology for large - scale production of nitidine chloride.

[0008] A method for increasing the content of nitidine chloride in tissue - cultured materials of Zanthoxylum nitidum provided by the present invention comprises the following steps: S1. Selection of explants; S2. Bud induction culture: After disinfecting the explants, inoculate them into a bud induction medium for culture; S3. Cluster - bud culture: Transfer the tender buds obtained in step S2 to a cluster - bud medium for culture; S4. Callus induction culture: Scratch the tender leaves obtained in step S2 and inoculate them into a callus induction medium for culture; S5. Directional regulation culture: Transfer the cluster - bud culture in step S3 or the callus induction culture in step S4 to be cultured under red - light conditions for 1 - 2 months, wherein the light intensity under red - light conditions is 150 - 250 μmol•m⁻²•s⁻¹, and the photoperiod is 10 - 14 hours per day.

[0009] Preferably, the explant in step S1 is selected as the stem segment of Zanthoxylum nitidum seedling at the age of 1 to 6 months; the bud induction medium in step S2 is 1 / 2MS basal medium supplemented with 0.5 - 1.0 mg / L 6-BA, 0.2 - 0.4 mg / L IBA and 0.1 - 0.5 mg / L KT; the multiple shoot medium in step S3 is MS basal medium supplemented with 0.5 - 1.0 mg / L 6-BA, 0.2 - 0.4 mg / L IBA and 0.1 - 0.5 mg / L KT; the callus induction medium in step S4 is MS basal medium supplemented with 2 mg / L 6-BA, 0.1 - 0.2 mg / L KT, 0.2 - 1 mg / L 2,4-D and 1 - 1.5 mg / L NAA.

[0010] Preferably, the bud induction medium in step S2 is 1 / 2MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the multiple shoot medium in step S3 is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the callus induction medium in step S4 is MS basal medium supplemented with 2 mg / L 6-BA, 0.1 mg / L KT, 0.2 mg / L 2,4-D and 1 mg / L NAA; the light intensity of pure red light under red light conditions is 200 μmol•m⁻²•s⁻¹, and the photoperiod is 12 hours / day.

[0011] Preferably, the red light conditions activate the gene expression of nitidine chloride synthesis pathway, and the genes of nitidine chloride synthesis pathway include NCS, CYP450 and BBE.

[0012] Preferably, the culture under red light conditions for 1 - 2 months includes: The first stage, from the 1st to the 15th day under red light conditions, the light intensity is 150 - 180 μmol·m⁻²·s⁻¹, and the photoperiod is 10 hours / day; The second stage, from the 16th to the 30th day under red light conditions, the light intensity is increased to 200 - 220 μmol·m⁻²·s⁻¹, and the photoperiod is extended to 12 - 14 hours / day; The third stage, from the 31st day under red light conditions to the end of the culture, the light intensity is maintained at 200 μmol·m⁻²·s⁻¹, the photoperiod is 12 hours / day, and it is supplemented with 1 hour of blue light with an intensity of 50 μmol·m⁻²·s⁻¹ for intermittent irradiation every day; Among them, the wavelength of the red light is 620 - 660 nm, the wavelength of the blue light is 450 - 480 nm, and the intermittent irradiation is to insert 1 hour of blue light after every 6 hours of red light.

[0013] Preferably, in the cluster bud culture medium for culturing cluster buds in step S5 under red light conditions, 0.05 - 0.2 mg / L of SA and 5 - 10 μM of Trp are further added.

[0014] Preferably, the cluster bud culture medium in the first stage is MS basal medium supplemented with 0.6 mg / L of 6 - BA, 0.2 mg / L of IBA, 0.1 mg / L of KT, and 0.05 mg / L of SA; the cluster bud culture medium in the second stage is MS basal medium supplemented with 0.6 mg / L of 6 - BA, 0.2 mg / L of IBA, 0.1 mg / L of KT, and 0.15 mg / L of SA; the cluster bud culture medium in the third stage is MS basal medium supplemented with 0.6 mg / L of 6 - BA, 0.2 mg / L of IBA, 0.1 mg / L of KT, 0.1 mg / L of SA, and 6 μM of Trp.

[0015] Preferably, from the 1st day to the 15th day under red light conditions, the cluster buds are cultured using the cluster bud culture medium in the first stage; On the 16th day under red light conditions, the cluster buds are transferred to the pre - mixed cluster bud culture medium in the second stage for culturing. Among them, the replacement of the cluster bud culture medium in the second stage is synchronized with the stage of increasing red light intensity, and the red light intensity is increased from 150 μmol·m⁻²·s⁻¹ to 200 μmol·m⁻²·s⁻¹; On the 31st day under red light conditions, the cluster buds are transferred to the pre - mixed cluster bud culture medium in the third stage for culturing; among them, the cluster bud culture media in the first stage, second stage, and third stage are all pre - sterilized and encapsulated liquid media, which are directly injected into the culture container after opening or pre - encapsulated in the culture container.

[0016] The present invention also provides the application of the above - mentioned method in the production of nitidine chloride.

[0017] The present invention has at least the following beneficial effects: By optimizing the tissue culture technology to directionally regulate the biosynthesis of nitidine chloride in the tissue - cultured materials of Zanthoxylum nitidum, the content of nitidine chloride in the tissue - cultured materials of Zanthoxylum nitidum cultured under red light is increased by 2 - 10 times compared with the materials cultured under white light.

[0018] By directionally regulating the tissue culture technology, the expression of genes (such as NCS, CYP450, BBE, etc.) in the nitidine chloride synthesis pathway is activated under red light conditions, so that the content of the target component in the tissue - cultured materials (cluster buds or callus) is increased compared with that under white light culture. This method solves the problems of the long traditional cultivation cycle (3 - 5 years), large content fluctuations (the difference in different production areas can reach 6 times), and the insufficient regulation of secondary metabolites by the existing tissue culture technology. By controllable environmental factors, it directionally promotes the accumulation of nitidine chloride and provides stable technical support for industrial production.

[0019] The present invention activates gene expression by means of low-intensity red light with phased regulation, gradually enhances the promotion of metabolism accumulation by red light, and adopts the strategy of blue light cooperation to avoid photoreceptor inactivation. While reducing light stress, it continuously activates the synthesis pathway to synchronously optimize the content and stability of the target component, verifying the positive regulatory effect of the dynamic adjustment of light quality, light intensity and photoperiod on biosynthesis.

[0020] The present invention reduces light stress by regulating the plant hormone signal pathway with SA, improves the survival rate of cluster buds and enhances the expression of synthetic genes (such as BBE); Trp, as a precursor substance for alkaloid synthesis, directly supplements the substrates of the metabolic pathway to solve the limitation of insufficient precursors during single light quality regulation. The two act synergistically with phased light quality to form a multiple regulation mechanism, further optimizing the biosynthesis efficiency and stability of the target component, and providing a new technology for the large-scale production of nitidine chloride.

[0021] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and will also be understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0022] Figure 1 It is the morphology of Zanthoxylum nitidum cluster buds; Figure 2 It is the content of nitidine chloride in the tender shoots of Zanthoxylum nitidum cultured under different light qualities; Figure 3 It is the clustering heat map of genes related to the alkaloid synthesis pathway in the tender shoots of Zanthoxylum nitidum cultured under different light qualities; Figure 4 It is the morphology of Zanthoxylum nitidum callus; Figure 5 It is the content of nitidine chloride in the callus of Zanthoxylum nitidum cultured under different light qualities; Figure 6 It is the clustering heat map of genes related to the alkaloid synthesis pathway in the callus of Zanthoxylum nitidum cultured under different light qualities; Among them, berberine bridge enzyme (BBE); codeine 3-O-demethylase (DIOX); thebaine synthase (MLP31); caffeic acid 3-O-methyltransferase (COMT1); cytochrome P450 enzyme (cytochrome P450 CYP82D47, CYP82D6); cytochrome P450 enzyme (cytochrome P450 81E8, CYP81Q32); (R,S)-reticuline 7-O-methyltransferase (7OMT). Detailed implementation manners

[0023] The following further elaborates on the present invention in conjunction with embodiments, so that those skilled in the art can implement it with reference to the text of the specification.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation schemes are all conventional methods, and the reagents and materials, unless otherwise specified, can all be obtained from commercial channels; in the description of the present invention, it should be noted that, unless otherwise clearly defined and limited, the terms "installation", "connection", and "setting" should be understood in a broad sense. For example, they can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations. The orientation or positional relationship indicated by the terms "horizontal", "vertical", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation to the present invention.

[0025] The present invention provides a method for increasing the content of nitidine chloride in the tissue culture materials of Zanthoxylum nitidum, including the following steps: S1 Select an explant, and use the stem segment of a 1- to 6-month-old seedling as the explant; S2 Bud induction culture: After the explant is disinfected with 0.1% mercuric chloride, it is inoculated onto a bud induction medium (1 / 2MS + 0.5 - 1.0 mg / L 6-BA + 0.2 - 0.4 mg / L IBA + 0.1 - 0.5 mg / L KT). After 1 month of culture, the germination rate is over 90%, and the number of germinated buds on each explant is 2 - 5. S3 Multiple shoot culture: Transfer the shoots with a length of 2 - 3 cm obtained in step S2 to a multiple shoot medium (MS + 0.5 - 1.0 mg / L 6 - BA + 0.2 - 0.4 mg / L IBA + 0.1 - 0.5 mg / L KT). After 2 months of culture, each shoot differentiates into 3 - 10 shoots; S4 Callus induction culture: Slightly scratch the young leaves obtained in step S2 and transfer them to a callus induction medium (MS + 2 mg / L 6 - BA + 0.1 - 0.2 mg / L KT + 0.2 - 1 mg / L 2,4 - D + 1 - 1.5 mg / L NAA). After 2 months of culture, the callus induction rate is over 90%, and 0.2 - 0.1 g of callus grows from each young leaf; S5 Directional regulation of nitidine chloride biosynthesis culture: Transfer the multiple shoots or callus to be cultured under red light for 1 - 2 months, with a light intensity of 150 - 250 μmol•m⁻²•s⁻¹ and a photoperiod of 10 - 14 hours per day, to activate the expression of genes related to the nitidine chloride synthesis pathway in vivo (such as NCS, CYP450, BBE, etc.), so that the content of nitidine chloride is increased by 2 - 10 times or more compared with the materials cultured under white light.

[0026] The material is in the form of multiple shoots or callus.

[0027] Example 1 A method for increasing the content of nitidine chloride in tissue - cultured materials of Zanthoxylum nitidum, including the following steps: On September 20, 2023, 500 g of Zanthoxylum nitidum seeds were harvested and directly sown on a germination bed.

[0028] On October 20, 2023, the germinated seeds were selected and transferred to a seedling - raising plug tray, and nutrient solution was regularly irrigated to promote the growth of seedlings.

[0029] On December 23, 2023, the main stems of the seedlings were taken as explants for tissue - culture experiments. Each stem segment was cut into small segments with a length of 2 - 3 cm, washed clean with running water, disinfected in a 0.1% mercuric chloride solution for 5 min on a sterile operating table, and then rinsed 3 times with sterile water, and inoculated into a bud induction medium (1 / 2MS + 0.6 mg / L 6 - BA + 0.2 mg / L IBA + 0.1 mg / L KT). A total of 150 bottles were inoculated, and 1 material was inoculated in each bottle.

[0030] On January 8, 2024, it was recorded that 15 bottles were contaminated, and among the remaining 135 bottles, 96 bottles grew tender shoots.

[0031] On January 23, 2024, the number of contaminated bottles was recorded as 1. Among the remaining 134 bottles, 125 bottles had germinated buds (i.e., after deducting the contamination, the overall germination rate was 125 / 134 = 93.3%). The number of buds on 10 randomly selected materials was counted, and the average number of germinations was found to be 2.4 per plant. The minimum number of buds was 1, and the maximum was 4.

[0032] On March 5, 2024, the buds that had grown to a length of 2 - 3 cm on the bud induction medium were cut under a sterile workbench and transferred to the cluster bud medium (MS + 0.6 mg / L 6 - BA + 0.2 mg / L IBA + 0.1 mg / L KT). A total of 50 bottles were inoculated, with 3 buds inoculated in each bottle.

[0033] On May 6, 2024, the growth status of the cluster buds was recorded. 3 bottles were randomly selected, with a total of 9 materials. It was calculated that the average number of buds per material was 5.6 (the minimum number of buds was 3, and the maximum was 18). The average length of the buds was 2.74 cm (the shortest bud length was 0.32 cm, and the longest was 5.62 cm) ( Figure 1 Morphology of Zanthoxylum nitidum cluster buds).

[0034] On June 5, 2024, the healthy buds on the bud induction medium of Zanthoxylum nitidum were selected, and single buds with a length greater than 2 cm were cut and transferred to the cluster bud medium (MS + 0.6 mg / L 6 - BA + 0.2 mg / L IBA + 0.1 mg / L KT). A total of 100 bottles were inoculated, with 3 buds inoculated in each bottle. They were cultured under pure red light (620 - 660 nm), pure blue light (450 - 480 nm), pure green light (500 - 570 nm), and white light (400 - 760 nm) respectively. The light intensity was 200 μmol•m⁻²•s⁻¹, and the photoperiod was 12 hours per day. 25 bottles were cultured under each light quality.

[0035] On August 6, 2024, the Zanthoxylum nitidum buds subcultured under different light qualities were taken out, and the nitidine chloride content was determined by UPLC - MS - MS, and transcriptome sequencing was carried out simultaneously. It was analyzed that the differences in the nitidine chloride content under the four light quality conditions were significant. Among them, the relative expression level of the nitidine chloride content under red light was the highest, which was 13.7 times that of white light ( Figure 2 Relative content of nitidine chloride in Zanthoxylum nitidum tender buds cultured under different light qualities). Based on the transcriptome data, it was found that the expressions of the genes (BBE, STOX, TNMT) related to the biosynthesis pathway of nitidine chloride under red light were significantly up - regulated ( Figure 3 Cluster heat map of genes related to the alkaloid synthesis pathway in Zanthoxylum nitidum tender buds cultured under different light qualities).

[0036] Among them, the nutrient solution regularly irrigated in the seedling-raising plug tray is the modified Hoagland's nutrient solution, and the specific formula is as follows: Macronutrients (mg / L): Calcium nitrate (Ca(NO 3 ) 2 ・4H 2 O) 1000, Potassium nitrate (KNO 3 ) 810, Potassium dihydrogen phosphate (KH 2 PO 4 ) 136, Magnesium sulfate (MgSO 4 ・7H 2 O) 493, Ammonium sulfate ((NH 4 ) 2 SO 4 ) 132; Micronutrients (μmol / L): Sodium ferric ethylenediaminetetraacetate (NaFe-EDTA) 100, Boric acid (H 3 BO 3 ) 20, Manganese sulfate (MnSO 4 ・H 2 O) 1, Zinc sulfate (ZnSO 4 ・7H 2 O) 0.2, Copper sulfate (CuSO 4 ・5H 2 O) 0.01, Sodium molybdate (Na 2 MoO 4 ・2H 2 O) 0.01; Preparation method: Dissolve macronutrients and micronutrients in distilled water respectively, adjust the pH to 5.8 - 6.2 after mixing, and irrigate once every 7 - 10 days.

[0037] I. UPLC-MS-MS detection method: 1. Sample pretreatment Take 0.1 g of fresh tissue culture material (buds or callus), add 1 mL of 80% methanol, grind in liquid nitrogen and extract by ultrasonic for 30 min (power 200 W, frequency 40 kHz), centrifuge at 12,000 rpm for 15 min, take the supernatant and filter through a 0.22 μm microporous filter membrane, and the filtrate is used for UPLC-MS-MS detection.

[0038] 2. Liquid chromatography (UPLC) conditions Chromatographic column: AgilentSB-C18 (1.8 μm, 2.1 mm × 100 mm) Mobile phase: Phase A is ultrapure water containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid Elution gradient: 0.00 min: 5% of phase B 0.01–9.00 min: Phase B linearly increases to 95% and is maintained for 1 min 10.00–11.10 min: Phase B decreases to 5% and is balanced to 14 min Flow rate: 0.35 mL / min Column temperature: 40 °C Injection volume: 2 μL 3. Mass spectrometry (MS-MS) conditions Ion source: Electrospray ionization (ESI+, positive ion mode) Ion source parameters: Temperature: 500 °C Spray voltage: 5500 V Curtain gas (CUR): 25 psi Nebulizing gas (GS1): 50 psi Auxiliary gas (GS2): 60 psi Scan mode: Multiple reaction monitoring (MRM) Target ion pairs: Precursor ion m / z 340.2, product ion m / z 192.1 (quantitative ion) Collision energy (CE): 35 eV Declustering potential (DP): 80 V 4. Content calculation Relative content: Peak area of the target group / Peak area of the white light group (the white light group is set to 1) Absolute content conversion: A standard curve is drawn using nitidine chloride standard (purity ≥ 98%) to calculate the absolute content of nitidine chloride in the sample (mg / g fresh sample).

[0039] II. Transcriptome sequencing method: 1. RNA extraction and library construction Total RNA extraction: Using the PlantRNA Purification Reagent kit (Invitrogen), 50 mg of fresh tissue culture material is taken, ground in liquid nitrogen, and RNA is extracted according to the instructions. The purity is detected by NanoDrop 2000 (OD260 / 280 = 1.8 - 2.0), and the integrity is detected by Agilent 2100 Bioanalyzer (RIN ≥ 8.0).

[0040] cDNA library construction: Magnetic beads are used to enrich poly(A)+ mRNA, and fragmentation buffer is added to break it into 100 - 300 bp fragments; Random hexamer primers are used to synthesize the first strand of cDNA, and then the second strand of cDNA is synthesized; End repair, poly(A) tailing, ligation of sequencing adapters (including index), and magnetic bead screening of 200 - 400 bp fragments; PCR amplification for 12 cycles to construct a cDNA library.

[0041] 2. Sequencing and data analysis Sequencing platform: Illumina NovaSeq 6000, PE150 mode, single-sample sequencing data volume ≥ 6 Gb.

[0042] Data processing: The raw data was filtered by Fastp v0.23.2 (removing adapters and low-quality reads, Q ≥ 30) to obtain Clean Data; Reference genome: Zanthoxylum nitidum genome; Alignment tool: Hisat2 v2.2.1, mapping rate ≥ 85%; Differential gene analysis: DESeq2 v1.34.0, screening criteria: |log 2 (FoldChange)| ≥ 1 and FDR < 0.05, KEGG pathway enrichment analysis (p < 0.05).

[0043] III. Absolute content data of nitidine chloride: 1. Example 1 (bud material) Absolute content in the white light group: 0.52 mg / g fresh bud (the content of dry product can be converted according to the water content of 60% in fresh sample); Absolute content in the red light group: 6.85 mg / g fresh bud (relative content is 13.7 times, significantly higher than that in the white light group, and 1.58 times that of the highest value of 4.33 mg / g dry product in traditional cultivation); 2. Example 2 (callus) Absolute content in the white light group: 0.31 mg / g fresh callus; Absolute content in the red light group: 0.65 mg / g fresh callus (relative content is 2.1 times, higher than that in the white light group and the content of some medicinal materials from production areas); Explanation on the rationality of detecting fresh samples: Characteristics of tissue culture materials: During the tissue culture process, the samples need to maintain a fresh state to maintain metabolic activity. Drying treatment may lead to the degradation of secondary metabolites. Therefore, using fresh samples for detection can better reflect the real-time synthesis ability.

[0044] Conversion with dry product: The water content of fresh samples is about 60% - 70%. According to this conversion, the content of dry product of buds in the red light group is 17.13 - 22.83 mg / g (significantly higher than the pharmacopoeia standard of 0.13% and the highest value in traditional cultivation), meeting the requirements of industrial extraction.

[0045] Comparison with the prior art: In the adventitious buds treated with red light in the present invention, the absolute content of nitidine chloride is 6.85 mg / g of fresh sample. Calculated according to the water content of fresh Zanthoxylum nitidum tissues of about 60% - 70%, the content of its dried product can reach 17.13 - 22.83 mg / g (calculated based on the dried product).

[0046] Comparing with the content of dried Zanthoxylum nitidum (0.70 - 4.33 mg / g) reported by Qin Yunrui et al. (2019) for different cultivation types, the conversion value of the fresh adventitious buds in the present invention significantly exceeds the samples of existing medicinal materials; Compared with the medicinal materials from 10 producing areas detected by Wu Luxiang et al. (2023) (0.14% - 0.19%, i.e., 1.4 - 1.9 mg / g), the content in the present invention is significantly increased and significantly higher than the minimum standard specified in the Chinese Pharmacopoeia (0.13%, i.e., 1.3 mg / g).

[0047] More importantly, through the controllability of the tissue culture environment in the present invention, the content fluctuation range is narrowed, while the content difference in the existing cultivation technology can reach 6 times (Qin Yunrui et al., 2019), solving the core problem of "unstable content" in traditional production.

[0048] It can be seen that the present invention has the advantages of increasing the content of nitidine chloride in adventitious buds and improving the quality stability (in the determination of 20 bottles of materials randomly selected from each treatment group, the highest content of nitidine chloride in fresh buds in the red light treatment group is 6.89 mg / g, and the lowest is 5.91 mg / g; the highest content of nitidine chloride in fresh buds in the white light group is 1.79 mg / g, and the lowest is 0.27 mg / g). And it only takes 3 - 4 months from the inoculation of explants to the end of red light treatment, while traditional cultivation takes 3 - 5 years to harvest, so the present invention has an obvious cycle advantage.

[0049] Example 2 A method for increasing the content of nitidine chloride in tissue culture materials of Zanthoxylum nitidum, comprising the following steps: On May 6, 2024, take the leaves on the robust buds on the bud induction medium of Zanthoxylum nitidum, slightly scratch them with a scalpel, and transfer them to the callus induction medium (MS + 2 mg / L 6 - BA + 0.1 mg / L KT + 0.2 mg / L 2,4 - D + 1 mg / L NAA) on the sterile operating table. A total of 50 bottles are inoculated, and 3 leaf pieces are inoculated in each bottle.

[0050] On July 6, 2024, record the growth of callus. Randomly take out 10 bottles, a total of 30 materials, and calculate that the callus induction rate is 100%; then randomly take out 2 bottles and weigh to obtain the average weight of the materials as 0.74 g ( Figure 4 Morphology of Zanthoxylum nitidum callus).

[0051] On July 6, 2024, the light green callus was transferred to a new callus induction medium (MS + 2 mg / L 6-BA + 0.1 mg / L KT + 0.2 mg / L 2,4-D + 1 mg / L NAA). A total of 80 bottles were inoculated, with 3 materials in each bottle, and they were placed under pure red light (620 - 660 nm), pure blue light (450 - 480 nm), pure green light (500 - 570 nm), and white light (400 - 760 nm) for cultivation. The light intensity was 200 μmol•m⁻²•s⁻¹, the photoperiod was 12 hours per day, and 20 bottles were cultivated under each light quality.

[0052] On August 6, 2024, the Zanthoxylum nitidum callus cultivated under different light qualities was taken out, and the content of nitidine chloride was determined by UPLC-MS-MS. At the same time, transcriptome sequencing was carried out (the determination method was the same as in Example 1). It was analyzed that the content differences of nitidine chloride under the four light quality conditions were significant. Among them, the relative expression level of nitidine chloride content under red light was the highest, which was 2.1 times that of white light ( Figure 5 Relative content of nitidine chloride in the Zanthoxylum nitidum callus cultivated under different light qualities). Based on the transcriptome data, it was found that the expression of genes related to the biosynthesis pathway of nitidine chloride (NCS, CYP450, BBE, etc.) was significantly up-regulated under red light ( Figure 6 Cluster heat map of genes related to the alkaloid biosynthesis pathway in the Zanthoxylum nitidum callus cultivated under different light qualities).

[0053] Example 3 The method for increasing the content of nitidine chloride in the tissue culture materials of Zanthoxylum nitidum was basically the same as in Example 1, except that the red light condition in this example adopted stage-by-stage regulation, specifically as follows: On June 5, 2024, healthy buds on the Zanthoxylum nitidum bud induction medium were taken, and single buds with a length greater than 2 cm were cut and transferred to a cluster bud medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT). A total of 25 bottles were inoculated, with 3 buds in each bottle.

[0054] In the first stage, from the 1st to the 15th day under the red light condition, the light intensity was 150 μmol•m⁻²•s⁻¹, and the photoperiod was 10 hours per day; In the second stage, from the 16th to the 30th day under the red light condition, the light intensity was increased to 200 μmol•m⁻²•s⁻¹, and the photoperiod was extended to 13 hours per day; In the third stage, from the 31st day to the end of the cultivation under the red light condition, the light intensity was maintained at 200 μmol•m⁻²•s⁻¹, the photoperiod was 12 hours per day, and it was supplemented with 1 hour of blue light with an intensity of 50 μmol•m⁻²•s⁻¹ for intermittent irradiation every day; Among them, the wavelength of the red light is 620 - 660 nm, the wavelength of the blue light is 450 - 480 nm, and the intermittent irradiation is that 1 hour of blue light is inserted after every 6 hours of red light. Such intermittent irradiation avoids the passivation of photoreceptors and enhances the continuous activation of metabolic pathways.

[0055] The staged regulation of the present invention starts the induction of gene expression by using red light with a lower intensity (150 μmol•m⁻²•s⁻¹) in the first stage to reduce light stress, then gradually increases the intensity of the red light and extends the light irradiation time to promote the accumulation of secondary metabolites, and finally combines with short-term blue light (450 - 480 nm) to increase the content of nitidine chloride through the synergistic effect of light quality.

[0056] On August 6, 2024, the roots of Zanthoxylum nitidum subcultured under different light qualities were taken out, and the content of nitidine chloride was measured by UPLC-MS-MS (the measurement method was the same as that in Example 1). Among the randomly selected 20 bottles of materials for measurement, the average content of nitidine chloride in the cluster buds was 7.02 mg / g (fresh sample), the highest content was 7.21 mg / g, and the lowest was 6.38 mg / g. From the measurement results, it can be seen that the content of nitidine chloride in the cluster buds with staged regulation was significantly increased, and there was a tendency to narrow the content fluctuation.

[0057] The staged regulation of the present invention adopts the strategy of "starting gene expression with low-intensity red light, gradually enhancing red light to promote metabolic accumulation, and blue light synergistically avoiding photoreceptor passivation", continuously activates the synthesis pathway while reducing light stress, realizes the synchronous optimization of the content and stability of the target component, and verifies the positive regulatory effect of the dynamic adjustment of light quality, light intensity and photoperiod on biosynthesis.

[0058] Example 4 The method for increasing the content of nitidine chloride in the tissue culture materials of Zanthoxylum nitidum is basically the same as that in Example 1. The difference is that in this example, the red light conditions adopt staged regulation and synchronous gradient addition of L-tryptophan (Trp) and salicylic acid (SA). Specifically: On June 5, 2024, the robust buds on the induction medium of Zanthoxylum nitidum were taken, and the single buds with a length greater than 2 cm were cut and transferred to the cluster bud medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.05 mg / L SA) in the first stage. A total of 25 bottles were inoculated, and 3 buds were inoculated in each bottle.

[0059] In the first stage, from the 1st to the 15th day under the red light condition, the light intensity was 150 μmol•m⁻²•s⁻¹, and the photoperiod was 10 hours / day; In the second stage, from the 16th to the 30th day under red light conditions, the cluster buds are transferred to the pre-mixed cluster bud medium for the second stage (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.15 mg / L SA) for cultivation on the 16th day. Among them, the replacement of the cluster bud medium for the second stage is carried out synchronously with the stage of increasing red light intensity. The red light intensity is increased to 200 μmol•m⁻²•s⁻¹, and the photoperiod is extended to 13 hours per day; In the third stage, from the 31st day to the end of cultivation under red light conditions, the cluster buds are transferred to the pre-mixed cluster bud medium for the third stage (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.1 mg / L SA + 6 μM Trp) for cultivation on the 31st day. The light intensity is maintained at 200 μmol•m⁻²•s⁻¹, the photoperiod is 12 hours per day, and it is supplemented with 1-hour intermittent irradiation of blue light with an intensity of 50 μmol•m⁻²•s⁻¹ every day; The cluster bud media for the first stage, the second stage, and the third stage are all pre-sterilized and packaged liquid media, which are directly injected into the culture containers after opening.

[0060] Among them, the red light wavelength is 620 - 660 nm, the blue light wavelength is 450 - 480 nm, and the intermittent irradiation is to insert 1 hour of blue light after every 6 hours of red light. Such intermittent irradiation avoids photoreceptor inactivation and enhances the continuous activation of metabolic pathways.

[0061] In the present invention, low-concentration SA in the first stage reduces light stress and improves the survival rate of cluster buds. Then, high-concentration SA in the second stage synergizes with red light to amplify the expression of the BBE gene, and Trp, as a precursor for alkaloid synthesis, directly increases the metabolic flux, solving the problem of content fluctuation caused by insufficient precursors when red light is regulated alone.

[0062] On August 6, 2024, the subcultured nitidin buds under different light qualities were taken out, and the nitidin chloride content was determined by UPLC-MS-MS (the determination method was the same as in Example 1). 20 bottles of materials were randomly selected for determination, and the average content of nitidin chloride in the clustered buds was 7.87 mg / g (fresh sample), with the highest content being 7.91 mg / g and the lowest being 7.73 mg / g. It can be seen from the determination results that the nitidin chloride content of the clustered buds with staged regulation and synchronous gradient addition of L-tryptophan (Trp) and salicylic acid (SA) was further increased, and the content fluctuation was significantly reduced, significantly improving stability. The SA of the present invention reduces light stress by regulating the plant hormone signaling pathway, improves the survival rate of clustered buds and enhances the expression of synthetic genes (such as BBE); Trp, as a precursor substance for alkaloid synthesis, directly supplements the metabolic pathway substrate to solve the limitation of insufficient precursors during single light quality regulation. The two work synergistically with the staged light quality to form a multiple regulatory mechanism of "environmental factors activate gene expression, enhance the supply of precursor substances, and increase the flux of metabolic pathways", further optimizing the biosynthetic efficiency and stability of the target components, and verifying the technical advantages of combining culture medium composition with light quality regulation.

[0063] Comparative Example 1 The method for increasing the content of nitidine chloride in the tissue culture material of Zanthoxylum bungeanum is basically the same as that in Example 4, except that the red light condition in this example is not regulated in stages, and L-tryptophan (Trp) and salicylic acid (SA) are added at one time, specifically: On June 5, 2024, take the robust buds on the Zanthoxylum bud induction medium, cut single buds longer than 2 cm, and transfer them to the cluster bud medium (MS+0.6mg / L 6-BA+0.2mg / LIBA+0.1mg / L KT+0.1mg / L SA+6μMTrp), inoculate a total of 25 bottles, with 3 buds in each bottle, and place them under red light intensity of 200μmol•m⁻²•s⁻¹, photoperiod of 12 hours / day, supplemented with 1 hour of blue light intensity of 50μmol•m⁻²•s⁻¹ per day, with intermittent irradiation; Among them, the wavelength of red light is 620~660nm, the wavelength of blue light is 450~480nm, and the intermittent irradiation is 1 hour of blue light inserted after every 6 hours of red light.

[0064] On August 6, 2024, the buds of Zanthoxylum bungeanum subcultured under different light qualities were taken out, and the content of nitidine chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). 20 bottles of materials were randomly selected for determination, and the average content of nitidine chloride in clustered buds was 5.96 mg / g (fresh sample), with the highest content being 6.14 mg / g and the lowest being 5.02 mg / g.

[0065] Comparative Example 2 The method for increasing the content of nitidine chloride in the Zanthoxylum bungeanum tissue culture material is basically the same as that in Example 4, except that the red light condition in this example is regulated in stages, but L-tryptophan (Trp) and salicylic acid (SA) are added at one time, and no blue light is added, specifically: On June 5, 2024, take the robust buds on the Zanthoxylum bud induction medium, cut single buds longer than 2 cm, and transfer them to the cluster bud medium (MS+0.6mg / L 6-BA+0.2mg / LIBA+0.1mg / L KT+0.1mg / L SA+6μMTrp), inoculate 25 bottles in total, and inoculate 3 buds in each bottle. In the first stage, the red light condition is from the 1st to the 15th day, the light intensity is 150μmol•m⁻²•s⁻¹, and the photoperiod is 10 hours / day; In the second stage, from the 16th to the 30th day of red light conditions, the red light intensity was increased to 200 μmol•m⁻²•s⁻¹, and the photoperiod was extended to 13 hours / day; The third stage, red light conditions from the 31st day to the end of culture, maintained light intensity at 200 μmol•m⁻²•s⁻¹, photoperiod of 12 h / day; Among them, the wavelength of red light is 620~660nm.

[0066] On August 6, 2024, the buds of Zanthoxylum bungeanum subcultured under different light qualities were taken out, and the content of nitidine chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). 20 bottles of materials were randomly selected for determination, and the average content of nitidine chloride in clustered buds was 6.13 mg / g (fresh sample), with the highest content being 6.57 mg / g and the lowest being 5.20 mg / g.

[0067] It can be seen from the results of comparative examples 1 and 2 that staged light regulation is crucial for sustained activation of gene expression, and one-time addition of SA / Trp may lead to premature consumption of precursors or accumulation of light stress. Intermittent irradiation with blue light may affect the passivation of photoreceptors and the activation effect of metabolic pathways, which can illustrate that blue light is an important influencing factor in the staged coordinated regulation of red light.

[0068] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized.

Claims

1. A method for increasing the content of nitidine chloride in a Zanthoxylum bungeanum tissue culture material, characterized in that: The steps include: S1. Explant selection; S2. Bud induction culture: sterilize the explants and inoculate them into bud induction medium for culture; S3. Cluster bud culture: The buds obtained in step S2 are transferred to cluster bud culture medium for culture; S4. Callus induction culture: Take the tender leaves from step S2 and scratch them, then inoculate them into callus induction medium for culture; S5. Directed regulation culture: transfer the cluster bud culture of step S3 or the callus induction culture of step S4 to red light conditions for 1 to 2 months, wherein the light intensity of the red light conditions is 150 to 250 μmol•m⁻²•s⁻¹ and the photoperiod is 10 to 14 hours / day.

2. The method according to claim 1, characterized in that The explant selection in step S1 is to select the stem segments of 1-6 month-old Zanthoxylum bungeanum seedlings as the explants; the bud induction medium in step S2 is 1 / 2MS basic medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA and 0.1-0.5 mg / L KT; the cluster bud medium in step S3 is MS basic medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA and 0.1-0.5 mg / L KT; the callus induction medium in step S4 is MS basic medium supplemented with 2 mg / L 6-BA, 0.1-0.2 mg / L KT, 0.2-1 mg / L 2,4-D and 1-1.5 mg / L NAA.

3. The method according to claim 1, characterized in that The bud induction medium described in step S2 is 1 / 2MS basic medium supplemented with 0.6mg / L 6-BA, 0.2mg / L IBA and 0.1mg / L KT; the cluster bud medium described in step S3 is MS basic medium supplemented with 0.6mg / L 6-BA, 0.2mg / L IBA and 0.1mg / L KT; the callus induction medium described in step S4 is MS basic medium supplemented with 2mg / L 6-BA, 0.1mg / L KT, 0.2mg / L 2,4-D and 1mg / L NAA; the pure red light intensity of the red light condition is 200μmol•m⁻²•s⁻¹, and the photoperiod is 12 hours / day.

4. The method according to claim 1, characterized in that: The red light condition activates the expression of genes in the nitidine chloride synthesis pathway, and the genes in the nitidine chloride synthesis pathway include NCS, CYP450 and BBE.

5. The method according to claim 1, characterized in that The culturing under the red light condition for 1 to 2 months comprises: In the first stage, from the 1st to the 15th day under red light conditions, the light intensity was 150-180 μmol·m⁻²·s⁻¹, and the photoperiod was 10 h / day; In the second stage, from the 16th to the 30th day under red light conditions, the light intensity was increased to 200-220 μmol·m⁻²·s⁻¹, and the photoperiod was extended to 12-14 hours / day; The third stage, red light conditions from the 31st day to the end of culture, maintained light intensity of 200 μmol·m⁻²·s⁻¹, light cycle of 12 hours / day, supplemented with 1 hour of blue light intensity of 50 μmol·m⁻²·s⁻¹ per day, intermittent irradiation; The wavelength of the red light is 620-660 nm, the wavelength of the blue light is 450-480 nm, and the intermittent irradiation is to insert 1 hour of blue light after every 6 hours of red light.

6. The method according to claim 5, characterized in that In step S5, the bud cluster culture medium cultured under red light conditions is further supplemented with 0.05-0.2 mg / L SA and 5-10 μM Trp.

7. The method according to claim 6, characterized in that The cluster bud culture medium of the first stage is MS basal culture medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / LIBA, 0.1 mg / L KT and 0.05 mg / L SA, the cluster bud culture medium of the second stage is MS basal culture medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / LIBA, 0.1 mg / L KT and 0.15 mg / L SA, and the cluster bud culture medium of the third stage is MS basal culture medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / LIBA, 0.1 mg / L KT, 0.1 mg / L SA and 6 μM Trp.

8. The method according to claim 7, characterized in that From the 1st day to the 15th day under the red light condition, the clustered shoot medium of the first stage is used for cultivation; On the 16th day under the red light condition, the clustered buds were transferred to the premixed second-stage clustered bud culture medium for cultivation, wherein the replacement of the second-stage clustered bud culture medium was carried out simultaneously with the red light intensity increase stage, and the red light intensity was increased from 150 μmol·m⁻²·s⁻¹ to 200 μmol·m⁻²·s⁻¹; On the 31st day under red light conditions, the cluster buds are transferred to the premixed third-stage cluster bud culture medium for cultivation; wherein the first-stage, second-stage and third-stage cluster bud culture media are all pre-sterilized packaged liquid culture media, which are directly injected into the culture container or pre-packaged in the culture container after opening.

9. Use of the method according to any one of claims 1 to 8 in the production of nitidine chloride.

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