CN120052257A - Method for improving content of nitidine chloride in tissue culture material of radix zanthoxyli and application - Google Patents

Abstract

The invention relates to a method for increasing the content of nitidine chloride in Zanthoxylum bungeanum tissue culture materials, and belongs to the field of biotechnology. At present, the traditional cultivation of Zanthoxylum bungeanum to extract nitidine chloride has the problems of long production cycle and unstable content. To this end, the method provided by the invention includes explant selection, bud induction culture, cluster bud culture, callus induction culture and directional regulation culture, wherein the directional regulation culture is to transfer the cluster buds or callus to red light conditions for 1 to 2 months, with a light intensity of 150 to 250 μmol・m⁻²・s⁻¹ and a photoperiod of 10 to 14 hours/day, so as to activate the expression of genes in the synthesis pathway of nitidine chloride and effectively increase the content of nitidine chloride.

Description

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving alkali content of a nitidine chloride in a nitidine tissue culture material and application thereof.

Background

Radix Zanthoxyli is used as a root of a Guangxi Zhuang nationality autonomous region ' Guishiwei ' genuine medicinal material, has the effects of promoting blood circulation, removing blood stasis, promoting qi circulation, relieving pain and the like, and the index component of the radix Zanthoxyli alkali chloride is a key quality control component [1] specified in the pharmacopoeia of the people's republic of China. Under the influence of sharp reduction of wild resources, the current production relies on artificial cultivation, but the difference of alkali content of the radix zanthoxyli in medicinal materials of different cultivation types and production places can reach 6 times (0.70-4.33 mg/g) [2], while Yang and the like detect 8 radix zanthoxyli medicinal materials of production places to obtain 0.50-0.91% alkali content of the radix zanthoxyli, wu Luxiang and the like detect 10 radix zanthoxyli medicinal materials of production places to obtain 0.14-0.19% alkali content of the radix zanthoxyli. Therefore, the content fluctuation of the chloridized nitidine in the radix zanthoxyli medicinal material is large, which brings challenges to the quality control of the radix zanthoxyli medicinal material. And the traditional cultivation period is long, secondary metabolites are accumulated obviously under the influence of the environment, and the quality control is difficult.

The plant tissue culture technology has realized the high-efficient rapid propagation of radix zanthoxyli, through optimizing bud induction, subculture multiplication and rooting culture medium, the induction rate of explants can reach 41.7% -96.2%, the annual multiplication rate reaches 3.612, and the problems of tender bud browning and the like are solved. However, the prior art focuses on the optimization of the rapid propagation process, has insufficient research on the biosynthesis regulation of the nitidine chloride in a tissue culture system, does not directionally activate the synthesis path of the nitidine chloride through environmental factors (such as light quality and culture medium components), and cannot meet the requirement of stably and efficiently accumulating target components in large-scale production.

Aiming at the current situation, the method for directionally regulating and controlling the biosynthesis of the nitidine chloride is established by optimizing the tissue culture technology to break through the bottleneck of long traditional cultivation period and large content fluctuation, and provides technical support for industrial production.

Reference is made to:

[1] the national formulary committee, pharmacopoeia of the people's republic of China (one part), beijing, chinese medical science and technology Press 2020;

[2] Qin Yunrui, jiang Zhenou, lai Maoxiang, huang Yunfeng, wang Xinhong. Zanthoxylum nitidum primary plant test card and analysis of active ingredient content. Guangxi Zhuang et al, 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 differentregions in southern China. Biomedical chromatography : BMC, 2023, 37(5): e5602;

[4] wu Luxiang, huang Xiang, lan Xiaodong, ma Enyao, chen Junxi, yang Shihui, liu Jiyue, zhou Shouting. Quality evaluation of Zanthoxylum nitidum medicinal materials in different places of origin based on principal component analysis. South and middle agricultural science and technology 2023,44 (10): 23-26.

Disclosure of Invention

It is an object of the present invention to address at least the above-mentioned drawbacks and to provide at least the advantages to be described later.

The invention provides a method for improving the content of nitidine chloride in a nitidine tissue culture material, which aims to directionally regulate and control the biosynthesis of nitidine chloride in the nitidine tissue culture material by optimizing a tissue culture technology, improve the content of nitidine chloride, solve the problems of long production period, unstable content of nitidine chloride and the like required by extracting nitidine chloride from nitidine plants by means of a cultivation technology at present, and provide a new technology for large-scale production of nitidine chloride.

The invention provides a method for improving the alkali content of a chlorinated radix zanthoxyli in a radix zanthoxyli tissue culture material, which comprises the following steps:

S1, selecting an explant;

s2, bud induction culture, namely sterilizing an explant and inoculating the explant into a bud induction culture medium for culture;

S3, cluster bud culture, namely transferring the tender buds obtained in the step S2 into a cluster bud culture medium for culture;

s4, callus induction culture, namely scratching tender leaves in the step S2, and inoculating the tender leaves to a callus induction culture medium for culture;

S5, directional regulation and control culture, namely transferring the cluster bud culture in the step S3 or the callus induction culture in the step S4 to a red light condition for culture for 1-2 months, wherein the illumination intensity of the red light condition is 150-250 mu mol.m-2.s < -1 >, and the photoperiod is 10-14 hours/day.

The explant in the step S1 is selected to be selected from stem segments of 1-6 month old Zanthoxylum nitidum seedlings as the explant, 0.5-1.0 mg/L6-BA, 0.2-0.4 mg/LIBA and 0.1-0.5 mg/L KT are added to the bud induction culture medium in the step S2, 0.5-1.0 mg/L6-BA, 0.2-0.4 mg/LIBA and 0.1-0.5 mg/L KT are added to the cluster bud culture medium in the step S3, and 2 mg/L6-BA, 0.1-0.2 mg/L KT, 0.2-1 mg/L2, 4-D and 1-1.5 mg/L NAA are added to the callus induction culture medium in the step S4.

Preferably, the bud induction medium in step S2 is 1/2MS basal medium supplemented with 0.6 mg/L6-BA, 0.2mg/LIBA and 0.1mg/L KT, the cluster bud medium in step S3 is MS basal medium supplemented with 0.6 mg/L6-BA, 0.2mg/LIBA and 0.1mg/L KT, the callus induction medium in step S4 is MS basal medium supplemented with 2 mg/L6-BA, 0.1mg/L KT, 0.2 mg/L2, 4-D and 1mg/L NAA, and the red light condition has a pure red light illumination intensity of 200 μmol.m-2.s-1 and a light cycle of 12 hours/day.

Preferably, the red light condition activates expression of a chlorinated nitidine synthesis pathway gene including NCS, CYP450 and BBE.

Preferably, the culture is carried out for 1-2 months under the condition of red light, and comprises the following steps:

In the first stage, the red light condition is 1 to 15 days, the illumination intensity is 150 to 180 mu mol m & lt-2 & gt s & lt-1 & gt, and the photoperiod is 10 hours/day;

In the second stage, the illumination intensity is increased to 200-220 mu mol m-2 s-1 under the condition of 16-30 days in red light, and the photoperiod is prolonged to 12-14 hours/day;

In the third stage, the illumination intensity is maintained to be 200 mu mol m-2 s-1 from the 31 st day of the red light condition to the end of the culture, the photoperiod is 12 hours/day, and the blue light intensity of 50 mu mol m-2 s-1 is added for 1 hour every day for intermittent irradiation;

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 blue light after every 6 hours red light.

Preferably, the clustered bud culture medium cultured under the red light condition in the step S5 is further added with SA of 0.05-0.2 mg/L and Trp of 5-10 mu M.

Preferably, the first stage of the cluster bud medium is added with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/LKT and 0.05mg/L SA to the MS basal medium, the second stage of the cluster bud medium is added with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/L KT and 0.15mg/L SA to the MS basal medium, and the third stage of the cluster bud medium is added with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/LKT, 0.1mg/L SA and 6 mu M Trp to the MS basal medium.

Preferably, the culture is carried out on days 1 to 15 under red light conditions by using the cluster bud culture medium of the first stage;

transferring the clustered shoots to the premixed clustered shoot culture medium of the second stage for culture on the 16 th day under the red light condition, wherein the replacement of the clustered shoot culture medium of the second stage is synchronous with the red light intensity increasing stage, and the red light intensity is increased from 150 mu mol.m-2.s-1 to 200 mu mol.m-2.s-1;

transferring the cluster buds to the premixed cluster bud culture medium of the third stage for culture on the 31 st day under the red light condition, wherein the cluster bud culture mediums of the first stage, the second stage and the third stage are all pre-sterilized packaging liquid culture mediums, and directly injecting the culture mediums into a culture container or pre-packaging the culture mediums after unsealing.

The invention also provides application of the method in the production of chloridized nitidine.

The invention at least comprises the following beneficial effects:

According to the invention, biosynthesis of the nitidine chloride in the nitidine tissue culture material is directionally regulated and controlled by optimizing a tissue culture technology, so that the content of the nitidine chloride in the nitidine tissue culture material cultured under red light is improved by 2-10 times compared with that of the material cultured under white light.

The invention activates the expression of the synthesis pathway genes (NCS, CYP450, BBE, etc.) of the chloridized nitidine under the red light condition by the directional regulation and control tissue culture technology, so that the content of target components in the tissue culture material (cluster buds or callus) is improved compared with that of white light culture. The method solves the problems of long period (3-5 years), large content fluctuation (the difference of different production areas can reach 6 times) and insufficient regulation and control of secondary metabolites in the conventional tissue culture technology, and provides stable technical support for industrial production by directionally promoting accumulation of nitidine chloride through controllable environmental factors.

According to the invention, through the strategy of starting gene expression by low-intensity red light regulated and controlled in stages, metabolism accumulation is promoted by the red light gradually, and light receptor passivation is avoided by blue light cooperation, the synthesis path is continuously activated while light stress is reduced, synchronous optimization of the content and stability of target components is realized, and forward regulation and control effects of dynamic regulation of light quality, light intensity and photoperiod on biosynthesis are verified.

The invention reduces light stress by regulating a plant hormone signal path through SA, improves cluster bud survival rate and enhances expression of a synthetic gene (such as BBE), takes Trp as an alkaloid synthesis precursor substance, directly supplements a metabolic path substrate, and solves the limitation of insufficient precursor during single light quality regulation. The two components and the staged light quality cooperate to form a multiple regulation mechanism, so that the biosynthesis efficiency and stability of the target component are further optimized, and a new technology is provided for large-scale production of the nitidine chloride.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows the form of a cluster bud of Zanthoxylum nitidum;

FIG. 2 shows the content of nitidine chloride in buds of nitidine cultivated under different light quality;

FIG. 3 is a cluster heat map of alkaloid synthesis pathway related genes in buds of radix Zanthoxyli cultured under different light quality;

FIG. 4 shows the morphology of Zanthoxylum nitidum;

FIG. 5 shows the content of nitidine chloride in the callus of radix Zanthoxyli cultured under different light quality;

FIG. 6 is a cluster heat map of alkaloid synthesis pathway-related genes in callus of radix Zanthoxyli cultured under different light quality;

Among them, berberine bridge enzyme (berberinebridgeenzyme, BBE), codeine3-O-demethylase (codeine 3-O-demethylase, DIOX), thebaine synthase (thebainesynthase, MLP 31), caffeic acid3-O-methyltransferase (caffeicacid 3-O-METHYLTRANSFERASE, COMT 1), cytochrome P450 enzyme (cytochromeP CYP82D47, CYP82D 6), cytochrome P450 enzyme (cytochromeP 45081E8, CYP81Q 32), 7-O-methyltransferase ((R, S) -reticuline7-O-METHYLTRANSFERASE,7 OMT).

Detailed Description

The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

In the description of the present invention, the terms "mounted," "connected," and "disposed" are to be construed broadly as being either fixedly connected, disposed, or detachably connected, disposed, or integrally connected, as described in the following embodiments, unless otherwise specifically indicated and as being otherwise readily available. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art. The terms "transverse," "longitudinal," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used for convenience in describing and simplifying the description of the present invention based on the orientation or positional relationship shown in the drawings, and do not denote or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the present invention.

The invention provides a method for improving the alkali content of a nitidine chloride in a nitidine tissue culture material, which comprises the following steps:

S1, selecting an explant, and taking a stem segment of a seedling with the age of 1-6 months as the explant;

S2, performing bud induction culture, sterilizing an explant by 0.1% mercuric chloride, inoculating the sterilized explant to a bud induction culture medium (1/2MS+0.5-1.0 mg/L6-BA+0.2-0.4 mg/LIBA+0.1-0.5 mg/L KT), and culturing for 1 month, wherein the germination rate is more than 90%, and the germination number on each explant is 2-5;

S3, cluster bud cultivation, namely transferring buds with the length of 2-3 cm obtained in the step S2 to a cluster bud culture medium (MS+0.5-1.0 mg/L6-BA+0.2-0.4 mg/LIBA+0.1-0.5 mg/L KT), and differentiating 3-10 buds from each bud after 2 months of cultivation;

S4, callus induction culture, namely slightly scratching tender leaves obtained in the step S2, transferring the slightly scratched tender leaves to a callus induction culture medium (MS+2mg/L6-BA+0.1-0.2 mg/L KT+0.2-1 mg/L2, 4-D+1-1.5 mg/LNAA), and culturing for 2 months, wherein the callus induction rate is more than 90%, and each tender leaf grows 0.2-0.1 g of callus;

S5, directionally regulating and controlling the biosynthesis and culture of the nitidine chloride, transferring cluster buds or calli to red light for culturing for 1-2 months, wherein the illumination intensity is 150-250 mu mol.m-2.s-1, the photoperiod is 10-14 hours/day, and activating the expression of related genes (NCS, CYP450, BBE and the like) of the nitidine chloride synthesis pathway in vivo, so that the nitidine chloride content is increased by more than 2-10 times compared with that of a material cultured under white light.

The material is in the form of cluster buds or calli.

Example 1

The method for improving the content of chloridized nitidine in the tissue culture material of the radix zanthoxyli comprises the following steps:

500g of zanthoxylum nitidum seeds are harvested in 2023, 9 and 20 days and directly sown on a germination bed.

And (3) selecting germinated seeds in 2023, 10 and 20 days, transferring the germinated seeds into seedling raising plug trays, and irrigating nutrient solution regularly to promote the growth of seedlings.

And taking the main stem of the seedling as an explant in 12 months and 23 days of 2023 to carry out a tissue culture experiment. Cutting each stem into small segments with the length of 2-3cm, washing with flowing water, sterilizing in 0.1% mercuric chloride solution for 5min on a sterile operation table, washing with sterile water for 3 times, inoculating into bud induction culture medium (1/2MS+0.6mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT), inoculating 150 bottles, and inoculating 1 material per bottle.

At day 1 and 8 of 2024, 15 bottles were contaminated and 96 of the remaining 135 bottles developed shoots.

The number of polluted bottles is recorded for 1 month and 23 days in 2024, 125 bottles of tender buds grow in the remaining 134 bottles (namely, after pollution is deducted, the total germination rate is 125/134=93.3%) and 10 bottles of materials are randomly counted to obtain 2.4 average buds/plant, wherein the minimum number of the tender buds grows out is 1 bud, and the maximum number of the tender buds grows out is 4 buds.

And cutting tender buds growing to a length of 2-3 cm on a bud induction culture medium on a sterile operation table for 5 days in 2024, transferring the tender buds to a cluster bud culture medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT), and inoculating 50 bottles altogether, wherein each bottle is inoculated with 3 buds.

The growth of clumped buds was recorded on day 5 and 6 of 2024, 3 bottles of the clumped buds were randomly removed, and an average of 5.6 buds per material (3 buds at least and 18 buds at most) were calculated (average length of buds was 2.74cm (shortest length of buds was 0.32cm and length of buds was 5.62 cm) (FIG. 1, clumped bud morphology).

And (3) taking strong buds on a needle bud induction medium in 2024 and 5 days, cutting single buds with the length of more than 2cm, continuously transferring the buds to a cluster bud medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT), inoculating 100 bottles, inoculating 3 buds in each bottle, respectively placing the buds 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 culture, wherein the illumination intensity is 200 mu mol.m-2.s-1, the photoperiod is 12 hours/day, and each light is cultured for 25 bottles.

And 2024, 8 and 6 days, taking out the sub-cultured zanthoxylum nitidum buds under different light quality, measuring the content of chloridized zanthoxylum nitidum by using UPLC-MS-MS, and simultaneously carrying out transcriptome sequencing. Analysis shows that the difference of the alkali content of the nitidine chloride under four light conditions is obvious, wherein the relative expression amount of the alkali content of the nitidine chloride under red light is the highest and is 13.7 times of white light (the relative content of the alkali content of the nitidine chloride in the nitidine buds cultured under different light conditions in FIG. 2). Based on transcriptome data, the expression of the related gene (BBE, STOX, TNMT) on the chloridized nitidine biosynthesis pathway under red light was significantly up-regulated (FIG. 3 cluster heat map of the related gene of the alkaloid biosynthesis pathway in the cultured nitidine buds under different light qualities).

Wherein, the nutrient solution which is irrigated in the fixed period of the seedling raising plug tray is an improved Hoagland nutrient solution, and the specific formula is as follows:

Macroelements (mg/L) calcium nitrate (Ca (NO ₃)₂・4H₂ O) 1000, potassium nitrate (KNO ₃) 810, monopotassium phosphate (KH ₂PO₄) 136, magnesium sulfate (MgSO ₄・7H₂ O) 493, ammonium sulfate ((NH ₄)₂SO₄) 132);

Microelements (mu mol/L) of sodium ferric ethylenediamine tetraacetate (NaFe-EDTA) 100, boric acid (H ₃BO₃) 20, manganese sulfate (MnSO ₄・H₂ O) 1, zinc sulfate (ZnSO ₄・7H₂ O) 0.2, copper sulfate (CuSO ₄・5H₂ O) 0.01, sodium molybdate (Na ₂MoO₄・2H₂ O) 0.01;

The preparation method comprises the steps of respectively dissolving major elements and trace elements in distilled water, mixing, adjusting the pH to 5.8-6.2, and irrigating once every 7-10 days.

1. UPLC-MS-MS detection method:

1. Sample pretreatment

Taking 0.1g of fresh tissue culture material (bud or callus), adding 1mL of 80% methanol, grinding with liquid nitrogen, performing ultrasonic extraction for 30min (power 200W, frequency 40 kHz), centrifuging at 12,000rpm for 15min, taking supernatant, passing through a 0.22 μm microporous filter membrane, and detecting filtrate by UPLC-MS-MS.

2. Liquid Chromatography (UPLC) conditions

AgilentSB-C18 column (1.8 μm,2.1 mm. Times.100 mm)

Mobile phase A is ultrapure water containing 0.1% formic acid, and B is acetonitrile containing 0.1% formic acid

Elution gradient:

0.00min phase B5%

The phase B line is raised to 95% in 0.01-9.00min, and maintained for 1min

Phase B is reduced to 5% in 10.00-11.10min, and balanced for 14min

Flow rate of 0.35mL/min

Column temperature of 40 DEG C

Sample injection amount 2. Mu.L

3. Mass Spectrometry (MS-MS) conditions

Ion Source electrospray ionization (ESI+, positive ion mode)

Ion source parameters:

Temperature 500 DEG C

Spray voltage of 5500V

Curtain gas (CUR) 25psi

Atomizing gas (GS 1) 50psi

Auxiliary gas (GS 2) 60psi

Scanning mode Multi-reaction monitoring (MRM)

Target ion pair:

parent ion m/z340.2, child ion m/z192.1 (quantitative ion)

Collision Energy (CE) 35eV

De-cluster voltage (DP) 80V

4. Content calculation

Relative content target group peak area/white light group peak area (white light group is set to 1)

And (3) converting the absolute content, namely drawing a standard curve through a standard product (the purity is more than or equal to 98%) of the nitidine chloride, and calculating the absolute content (mg/g fresh sample) of the nitidine chloride in the sample.

2. Transcriptome sequencing method:

RNA extraction and library construction

Total RNA extraction, namely, extracting RNA according to instructions after grinding 50mg of fresh tissue culture material by liquid nitrogen by adopting a PlantRNAPurificationReagent kit (Invitrogen), and detecting the purity by using a NanoDrop2000 (OD 260/280=1.8-2.0), wherein the integrity of the Agilent2100Bioanalyzer is detected (RIN is more than or equal to 8.0).

Construction of cDNA library:

enriching poly (A) +mRNA by using magnetic beads, and adding fragmentationbuffer to break into fragments of 100-300 bp;

synthesizing a first strand cDNA by using a six-base random primer, and then synthesizing a second strand cDNA;

Repairing the tail end, adding an A tail, connecting a sequencing joint (containing index), and screening a fragment of 200-400 bp by using magnetic beads;

PCR was performed for 12 cycles to construct a cDNA library.

2. Sequencing and data analysis

IlluminaNovaSeq6000, 6000 and PE150 modes, and the single sample sequencing data volume is more than or equal to 6Gb.

And (3) data processing:

filtering off-machine data by Fastpvc 0.23.2 (removing joints and low-quality reads, Q is more than or equal to 30) to obtain CLEANDATA;

reference genome, nitidine genome;

the comparison tool is Hisat2v2.2.1, and the Mapped percent is more than or equal to 85 percent;

Differential Gene analysis DESeq2v1.34.0, screening conditions were |log ₂ (FoldChange) |gtoreq.1 and FDR <0.05, KEGG pathway enrichment analysis (p < 0.05).

3. Absolute content data of nitidine chloride:

1. example 1 (bud Material)

The absolute content of the white light component is 0.52mg/g of fresh buds (the content of a dried product can be converted according to the water content of 60% of a fresh sample);

6.85mg/g fresh buds (the relative content is 13.7 times, obviously higher than that of a white light set and is 1.58 times of the highest value of 4.33mg/g dry product of traditional cultivation);

2. example 2 (callus)

The absolute content of the white light group is 0.31mg/g fresh callus;

The absolute content of the red light group is 0.65mg/g of fresh callus (the relative content is 2.1 times higher than that of the white light group and partial medicinal materials in the production place);

Rationality description for fresh sample detection:

the tissue culture material has the characteristics that the samples need to be kept in a fresh state in the tissue culture process to maintain metabolic activity, and secondary metabolites can be degraded by drying treatment, so that the real-time synthesis capability can be reflected by adopting fresh sample detection.

The water content of the fresh sample is about 60% -70% in terms of the dry product, the content of the red light group bud dry product is 17.13-22.83 mg/g (obviously higher than the pharmacopoeia standard 0.13% and the traditional cultivation maximum value) in terms of the fresh sample, and the industrial extraction requirement is met.

Compared with the prior art:

In the cluster buds treated by the red light, the absolute content of the nitidine chloride in the cluster buds is 6.85mg/g of fresh sample. The content of the dry product of the radix zanthoxyli can reach 17.13-22.83 mg/g (calculated by dry product) according to the conversion of the water content of the fresh tissue of the radix zanthoxyli of about 60-70%.

Comparing Qin Yunrui and 2019, wherein the content (0.70-4.33 mg/g) of the dried radix zanthoxyli products of different cultivation types is reported, and the conversion value of the fresh cluster buds is obviously higher than that of a sample of the existing medicinal material;

Compared with 10 medicinal materials (0.14% -0.19%, namely 1.4-1.9 mg/g) in origin detected by Wu Luxiang and the like (2023), the content of the medicinal composition is obviously improved and is obviously higher than the minimum standard (0.13%, namely 1.3 mg/g) specified in Chinese pharmacopoeia.

More importantly, the invention reduces the fluctuation range of the content by the controllability of the tissue culture environment, and the content difference in the existing cultivation technology can reach 6 times (Qin Yunrui and the like, 2019), thereby solving the core problem of unstable content in the traditional production.

Therefore, the method has the advantages of improving the alkali content of the chlorinated nitidine of the cluster buds, improving the quality stability (in the measurement of extracting 20 bottles of materials from each treatment group, the alkali content of the chlorinated nitidine of the fresh buds of the red light treatment group is the highest of 6.89mg/g and the lowest of 5.91mg/g, the alkali content of the chlorinated nitidine of the fresh buds of the white light treatment group is the highest of 1.79mg/g and the lowest of 0.27 mg/g), and the period from the inoculation of the explants to the end of the red light treatment can be shortened by 3-4 months, and the traditional cultivation can be completed by 3-5 years.

Example 2

The method for improving the content of chloridized nitidine in the tissue culture material of the radix zanthoxyli comprises the following steps:

And 4, at 5 months and 6 days of 2024, taking leaves on strong buds on a double-sided needle bud induction culture medium, slightly scratching the leaves by using a scalpel, transferring the leaves onto a callus induction culture medium (MS+2mg/L6-BA+0.1 mg/L KT+0.2 mg/L2, 4-D+1mg/L NAA) on a sterile operation table, inoculating 50 bottles altogether, and inoculating 3 leaves per bottle.

Recording growth of callus on day 7 and 6 of 2024, randomly taking out 10 bottles of the callus, calculating 30 materials to obtain callus induction rate of 100%, randomly taking out 2 bottles of the callus induction rate, and weighing to obtain average weight of 0.74g (form of double needle callus in FIG. 4).

Transferring the light green callus on a new callus induction culture medium (MS+2mg/L6-BA+0.1 mg/L KT+0.2 mg/L2, 4-D+1mg/L NAA) at the time of 7 months and 6 days in 2024, inoculating 80 bottles, respectively placing 3 materials in each bottle 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 culture, wherein the illumination intensity is 200 mu mol.m-2.s-1, the photoperiod is 12 hours/day, and each culture is 20 bottles under each light.

At 2024, 8 and 6 days, the callus of Zanthoxylum nitidum cultured under different light quality was taken out, and the content of nitidine chloride was measured by UPLC-MS-MS and transcriptome sequencing was performed (measurement method is the same as in example 1). Analysis shows that the difference of the alkali content of the chlorinated nitidine under four light conditions is obvious, wherein the relative expression amount of the alkali content of the chlorinated nitidine under red light is 2.1 times that of white light (the relative content of the alkali content of the chlorinated nitidine in the callus of the nitidine cultured under different light conditions in FIG. 5). Based on transcriptome data, expression of related genes (NCS, CYP450, BBE and the like) on the biosynthesis pathway of the nitidine chloride under red light is obtained to be significantly up-regulated (FIG. 6 is a cluster thermal diagram of related genes of the biosynthesis pathway of the alkaloids in the cultured nitidine calli under different light qualities).

Example 3

The method for increasing the alkali content of the nitidine chloride in the tissue culture material of the radix zanthoxyli is basically the same as that of the embodiment 1, except that the red light condition of the embodiment adopts staged regulation and control, and specifically comprises the following steps:

And 2024, 6 months and 5 days, taking strong buds on a nitidissima induction culture medium, cutting single buds with the length of more than 2cm, transferring the single buds to a cluster bud culture medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT), inoculating 25 bottles in total, and inoculating 3 buds per bottle.

In the first stage, the red light condition is 1 to 15 days, the illumination intensity is 150 mu mol.m to 2.s < -1 >, and the photoperiod is 10 hours/day;

In the second stage, the illumination intensity is increased to 200 mu mol.m-2.s-1 under the condition of red light for 16-30 days, and the photoperiod is prolonged to 13 hours/day;

in the third stage, the illumination intensity is maintained to be 200 mu mol.m-2.s-1 from the 31 st day under the red light condition to the end of the culture, the photoperiod is 12 hours/day, and the blue light intensity is assisted by 1 hour per day to be 50 mu mol.m-2.s-1, and the intermittent irradiation is carried out;

Wherein, the wavelength of red light is 620-660 nm, the wavelength of blue light is 450-480 nm, and the intermittent irradiation is that 1 hour blue light is inserted after every 6 hours of red light. Such intermittent irradiation avoids photoreceptor passivation, enhancing sustained activation of metabolic pathways.

According to the invention, the staged regulation and control are carried out, the gene expression is induced and started by adopting low-intensity red light (150 mu mol. M-2. S-1) in the first stage, the light stress is reduced, then the red light intensity is gradually enhanced, the illumination time is prolonged, the accumulation of secondary metabolites is promoted, and finally the alkali content of Gao Lvhua radix zanthoxyli is improved by combining short-time blue light (450-480 nm) and through a light quality synergistic effect.

And 2024, 8 and 6 days, taking out the subcultured nitidine buds under different light quality, and measuring the content of chloridized nitidine by using UPLC-MS-MS (the measuring method is the same as that of example 1). In the measurement of randomly extracting 20 bottles of materials, the average content of the nitidine chloride in the cluster buds is 7.02mg/g (fresh sample), the highest content is 7.21mg/g, the lowest content is 6.38mg/g, and the measurement result shows that the content of the nitidine chloride in the cluster buds regulated and controlled in stages is obviously improved, and the content fluctuation trend is reduced.

The invention regulates and controls the gene expression by stages through low-intensity red light, gradually enhances red light to promote metabolism accumulation, and blue light cooperates with a strategy of avoiding light receptor passivation, continuously activates a synthesis passage while reducing light stress, realizes synchronous optimization of the content and stability of target components, and verifies the forward regulation and control effect of dynamic regulation of light quality, light intensity and photoperiod on biosynthesis.

Example 4

The method for increasing the alkali content of the nitidine chloride in the tissue culture material of the radix zanthoxyli is basically the same as that of the embodiment 1, except that the red light condition of the embodiment adopts staged regulation and control and synchronous gradient addition with L-tryptophan (Trp) and Salicylic Acid (SA), and specifically comprises the following steps:

and 2024, 6 months and 5 days, taking strong buds on a nitidissima induction culture medium, cutting single buds with the length of more than 2cm, transferring to a first-stage cluster bud culture medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT+0.05mg/L SA), inoculating 25 bottles altogether, and inoculating 3 buds per bottle.

In the first stage, the red light condition is 1 to 15 days, the illumination intensity is 150 mu mol.m to 2.s < -1 >, and the photoperiod is 10 hours/day;

A second stage, namely, transferring the cluster buds from the 16 th day to a premixed cluster bud culture medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT+0.15mg/L SA) of the second stage under the red light condition for 16-30 th day, wherein the replacement of the cluster bud culture medium of the second stage is synchronous with the red light intensity lifting stage, the red light intensity is lifted to 200 mu mol.m-2.s-1, and the photoperiod is prolonged to 13 hours/day;

a third stage, namely, transferring cluster buds to a premixed cluster bud culture medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT+0.1mg/L SA+6 mu M Trp) in the third stage on 31 th day from 31 th day to the end of the culture under the red light condition, maintaining the illumination intensity to be 200 mu mol.m-2.s-1, and performing intermittent irradiation by assisting 1 hour blue light intensity to be 50 mu mol.m-2.s-1 every day;

the culture mediums of the cluster buds in the first stage, the second stage and the third stage are all pre-sterilized packaging liquid culture mediums, and are directly injected into a culture container after being unsealed.

Wherein, the wavelength of red light is 620-660 nm, the wavelength of blue light is 450-480 nm, and the intermittent irradiation is that 1 hour blue light is inserted after every 6 hours of red light. Such intermittent irradiation avoids photoreceptor passivation, enhancing sustained activation of metabolic pathways.

According to the invention, the light stress is relieved through the low-concentration SA in the first stage, the survival rate of the cluster buds is improved, then the high-concentration SA in the second stage is cooperated with red light, BBE gene expression is amplified, trp is used as an alkaloid synthesis precursor, the metabolic flux is directly increased, and the problem of content fluctuation caused by insufficient precursors during independent regulation and control of the red light is solved.

And 2024, 8 and 6 days, taking out the subcultured nitidine buds under different light quality, and measuring the content of chloridized nitidine by using UPLC-MS-MS (the measuring method is the same as that of example 1). 20 bottles of material were randomly withdrawn and assayed, the average content of the cluster bud chlorinated nitidine was 7.87mg/g (fresh sample), with the highest content being 7.91mg/g and the lowest content being 7.73mg/g. The determination result shows that the content of the nitidine chloride in the cluster buds, which is synchronously and gradiently added with the L-tryptophan (Trp) and the Salicylic Acid (SA), is further improved by stage regulation, the content fluctuation is obviously reduced, and the stability is obviously improved. The SA of the invention reduces light stress by regulating a plant hormone signal path, improves cluster bud survival rate and enhances expression of a synthetic gene (such as BBE), takes Trp as an alkaloid synthesis precursor substance, directly supplements a metabolic path substrate, and solves the limitation of insufficient precursor during single light quality regulation. The two are cooperated with the staged light quality to form a multiple regulation mechanism of 'environmental factor activating gene expression, precursor substance supply strengthening and metabolic channel flux improving', the biosynthesis efficiency and stability of the target component are further optimized, and the technical advantage of combining the culture medium component with the light quality regulation is verified.

Comparative example 1

The method for increasing the alkali content of the chlorinated radix zanthoxyli in the radix zanthoxyli tissue culture material is basically the same as that of the embodiment 4, except that the red light condition of the embodiment does not adopt staged regulation and control, and the L-tryptophan (Trp) and the Salicylic Acid (SA) are added at one time, specifically:

Taking strong buds on a needle bud induction medium in 2024, cutting single buds with the length of more than 2cm, transferring the buds to a cluster bud medium (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT+0.1mg/L SA+6 mu M Trp), co-inoculating 25 bottles, inoculating 3 buds per bottle, placing the buds in a red light illumination intensity of 200 mu mol.m-2.s-1, and intermittently irradiating for 12 hours/day with blue light intensity of 50 mu mol.m-2.s-1 for 1 hour per day;

wherein, the wavelength of red light is 620-660 nm, the wavelength of blue light is 450-480 nm, and the intermittent irradiation is that 1 hour blue light is inserted after every 6 hours of red light.

And 2024, 8 and 6 days, taking out the subcultured nitidine buds under different light quality, and measuring the content of chloridized nitidine by using UPLC-MS-MS (the measuring method is the same as that of example 1). 20 bottles of material are randomly extracted for measurement, and the average content of the cluster bud chloridized nitidine is 5.96mg/g (fresh sample), and the content is 6.14mg/g at the highest and 5.02mg/g at the lowest.

Comparative example 2

The method for increasing the alkali content of the chlorinated nitidine in the tissue culture material of radix zanthoxyli is basically the same as that of the embodiment 4, except that the red light condition of the embodiment adopts staged regulation and control, but L-tryptophan (Trp) and Salicylic Acid (SA) are added at one time, blue light is not added, and the method specifically comprises the following steps:

Taking strong buds on a culture medium for inducing the buds of the radix zanthoxyli in 2024 and 5 days, cutting single buds with the length of more than 2cm, transferring the single buds to a culture medium for clustered buds (MS+0.6 mg/L6-BA+0.2 mg/LIBA+0.1mg/L KT+0.1mg/L SA+6 mu M Trp), co-inoculating 25 bottles, inoculating 3 buds to each bottle, and in the first stage, carrying out red light conditions for 1-15 days, wherein the illumination intensity is 150 mu mol.m-2.s-1, and the photoperiod is 10 hours/day;

in the second stage, the red light intensity is improved to 200 mu mol.m-2.s-1 under the condition of 16-30 days, and the photoperiod is prolonged to 13 hours/day;

in the third stage, the light intensity is maintained to be 200 mu mol.m-2.s-1 from the 31 st day under the red light condition to the end of the culture, and the photoperiod is 12 hours/day;

Wherein the wavelength of the red light is 620-660 nm.

And 2024, 8 and 6 days, taking out the subcultured nitidine buds under different light quality, and measuring the content of chloridized nitidine by using UPLC-MS-MS (the measuring method is the same as that of example 1). 20 bottles of material were randomly withdrawn for measurement, and the average content of the cluster bud chloridized nitidine was 6.13mg/g (fresh sample), with the highest content being 6.57mg/g and the lowest content being 5.20mg/g.

As can be seen from the results of the assays of comparative examples 1 and 2, staged light modulation is critical for sustained activation of gene expression, and the addition of SA/Trp at once may lead to premature precursor consumption or light stress accumulation. The intermittent irradiation of blue light can influence the passivation of the light receptor, influence the activation effect of the metabolic pathway, and can indicate important influencing factors of blue light in the staged cooperative regulation of red light.

Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art.

Claims (9)

1. The method for improving the content of chloridized nitidine in the tissue culture material of radix zanthoxyli is characterized by comprising the following steps:

S1, selecting an explant;

s2, bud induction culture, namely sterilizing an explant and inoculating the explant into a bud induction culture medium for culture;

S3, cluster bud culture, namely transferring the tender buds obtained in the step S2 into a cluster bud culture medium for culture;

s4, callus induction culture, namely scratching tender leaves in the step S2, and inoculating the tender leaves to a callus induction culture medium for culture;

S5, directional regulation and control culture, namely transferring the cluster bud culture in the step S3 or the callus induction culture in the step S4 to a red light condition for culture for 1-2 months, wherein the illumination intensity of the red light condition is 150-250 mu mol.m-2.s < -1 >, and the photoperiod is 10-14 hours/day.

2. The method according to claim 1, wherein the explant of step S1 is selected from stem segments of 1-6 month old Zanthoxylum nitidum seedlings as the explant, the bud induction medium of step S2 is a 1/2MS basal medium supplemented with 0.5-1.0 mg/L6-BA, 0.2-0.4 mg/L IBA and 0.1-0.5 mg/L KT, the clustered bud medium of step S3 is a MS basal medium supplemented with 0.5-1.0 mg/L6-BA, 0.2-0.4 mg/L IBA and 0.1-0.5 mg/L KT, and the callus induction medium of step S4 is a MS basal medium supplemented with 2 mg/L6-BA, 0.1-0.2 mg/L KT, 0.2-1 mg/L2, 4-D and 1-1.5 mg/L NAA.

3. The method of claim 1, wherein the shoot induction medium is added with 0.6 mg/L6-BA, 0.2mg/L IBA and 0.1mg/L KT to 1/2MS basal medium in step S2, wherein the clustered shoot medium is added with 0.6 mg/L6-BA, 0.2mg/L IBA and 0.1mg/L KT to MS basal medium in step S3, wherein the callus induction medium is added with 2 mg/L6-BA, 0.1mg/L KT, 0.2 mg/L2, 4-D and 1mg/L NAA to MS basal medium in step S4, and wherein the red light condition has a pure red light illumination intensity of 200 μmol.m-2.s-1 and a light cycle of 12 hours/day.

4. The method of claim 1, wherein the red light conditions activate expression of a chlorinated nitidine synthesis pathway gene comprising NCS, CYP450, and BBE.

5. The method of claim 1, wherein the culturing under red light conditions for 1-2 months comprises:

In the first stage, the red light condition is 1 to 15 days, the illumination intensity is 150 to 180 mu mol m & lt-2 & gt s & lt-1 & gt, and the photoperiod is 10 hours/day;

In the second stage, the illumination intensity is increased to 200-220 mu mol m-2 s-1 under the condition of 16-30 days in red light, and the photoperiod is prolonged to 12-14 hours/day;

In the third stage, the illumination intensity is maintained to be 200 mu mol m-2 s-1 from the 31 st day of the red light condition to the end of the culture, the photoperiod is 12 hours/day, and the blue light intensity of 50 mu mol m-2 s-1 is added for 1 hour every day for intermittent irradiation;

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 blue light after every 6 hours red light.

6. The method according to claim 5, wherein the clustered shoot culture medium cultured under the red light condition in step S5 is further added with 0.05-0.2 mg/L SA, 5-10. Mu.M Trp.

7. The method of claim 6, wherein the first stage of the rosette culture medium is supplemented with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/L KT and 0.05mg/L SA for the MS basal medium, the second stage of the rosette culture medium is supplemented with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/L KT and 0.15mg/L SA for the MS basal medium, and the third stage of the rosette culture medium is supplemented with 0.6 mg/L6-BA, 0.2mg/LIBA, 0.1mg/L KT, 0.1mg/L SA and 6 μM Trp for the MS basal medium.

8. The method of claim 7, wherein the step of determining the position of the probe is performed,

Culturing on days 1 to 15 under red light conditions using the first stage of clustered shoot medium;

transferring the clustered shoots to the premixed clustered shoot culture medium of the second stage for culture on the 16 th day under the red light condition, wherein the replacement of the clustered shoot culture medium of the second stage is synchronous with the red light intensity increasing stage, and the red light intensity is increased from 150 mu mol.m-2.s-1 to 200 mu mol.m-2.s-1;

transferring the cluster buds to the premixed cluster bud culture medium of the third stage for culture on the 31 st day under the red light condition, wherein the cluster bud culture mediums of the first stage, the second stage and the third stage are all pre-sterilized packaging liquid culture mediums, and directly injecting the culture mediums into a culture container or pre-packaging the culture mediums after unsealing.

9. Use of a process according to any one of claims 1-8 for the production of chlorinated nitidine.