CN114982638A - Chemical inducer for astragalus adventitious root tissue culture and application of chemical inducer in astragalus adventitious root culture - Google Patents
Chemical inducer for astragalus adventitious root tissue culture and application of chemical inducer in astragalus adventitious root culture Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine astragalus adventitious root chemical induction, and provides a chemical inducer for astragalus adventitious root tissue culture and application thereof in astragalus adventitious root culture in order to solve the problems of green and sustainable development of astragalus resources at present; dissolving cis-3-hexenal in water to obtain 200mmol/L mother liquor, filtering and sterilizing with sterile filter membrane, and diluting with sterile water to obtain 50 or 12.5mmol/L mother liquor for direct use. Has feedback inhibition effect, promotes synthesis and accumulation of multiple triterpenoid saponins and isoflavone active ingredients of adventitious roots of astragalus in a dose-dependent manner, inhibits browning of the roots by inhibiting lipoxygenase activity, improves the growth state of the roots, and promotes synthesis and accumulation of astragaloside I, isoastragaloside I, astragaloside II, astragaloside III, formononetin, calycosin, glucoside thereof and the like. Is expected to be developed into a green additive, is applied to an astragalus mongholicus adventitious root tissue culture system, improves the growth state of a root culture, and improves the content of active substances of the root culture.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine astragalus adventitious root chemical induction, and particularly relates to a chemical inducer for astragalus adventitious root tissue culture and application thereof in astragalus adventitious root culture; the cis-3-hexenal is a configuration-specific feedback inhibitor of the astragalus adventitious root lipoxygenase and a chemical inducer accumulated by synthesizing various active ingredients. Cis-3-hexenal with different concentrations is added into a culture medium, so that not only is the lipoxygenase activity of the adventitious root tissue culture of the astragalus root inhibited, the growth state of the root tissue culture is changed, but also the content increase of astragaloside I, isoastragaloside I, astragaloside II, astragaloside III, formononetin, calycosin and glucoside in the culture is promoted in a concentration dependent mode.
Background
The green leaf volatile matter is a small molecular compound formed by hydrolyzing linoleic acid, linolenic acid or combined galactolipid released by cell membrane lipid with phospholipase under the condition that the plant is subjected to environmental stress such as mechanical injury, drought, fungal infection and the like or under the normal state, and sequentially acting with lipoxygenase and fatty acid hydroperoxide lyase, and mainly comprises C6 aldehyde, alcohol and ester thereof. Researchers at home and abroad find that the volatile matters of the green leaves play an important role in the phytochemistry defense process except for participating in the stress states of odor formation, drought characterization and the like. Such as trans-2-hexenal, has effects of inhibiting Aspergillus flavus spore germination, killing agricultural pest muscae laevigatae, etc [1, 2] (ii) a The cis-3-hexenyl acetate has effects of increasing sesquiterpene release amount of corn, killing larva of Spodoptera frugiperda, promoting synthesis of benzoxazolinone compounds, etc [3, 4] . Cis-3-hexenol presetting (priming) has effects of reducing tomato whitefly gnawing insect virus infection, increasing flavone synthesis, improving plant growth, etc [5] (ii) a The preset acetic acid cis-3-hexenyl ester has the functions of protecting or reducing abiotic stress damage and the like [6] . The cooperation of the central advanced innovation of molecular plants in Chinese academy of sciences and Mapu's chemical ecology in Germany, recently published in research results of science, found that cis-3-hexenal participates in the assembly of plant specific metabolite CPH (calcium acetylstrescine-green leaf calcium complex, m/z 347.19) to cope with the agricultural serious pest leafhopper [7] . In addition, the trans-2-hexenal has stronger effect of promoting the synthesis and accumulation of arabidopsis thaliana phytoalexin than cis-3-hexenal, the rhus macrostoma is more prone to predate tobacco hornworm larvae with high trans-2/cis-3-hexenal ratio and under the atmosphere of hexenol and acetic ester thereof, and female leafhoppers are more prone to lay eggs on tea tree leaves releasing cis-3-hexenol and acetic ester thereof [8-10] . These studies revealed that the role of green leaf volatiles in plant defense is related to the presence and configuration of their aldehydes, alcohols, esters.
The radix astragali is Mongolian radix astragali of LeguminosaeAstragalus membranaceus var. mongholicusOr Astragalus membranaceus (Fisch.) bgeAstragalus membranaceusThe dried root of (1) mainly contains active ingredients such as astragalus polysaccharide, triterpenoid saponin taking cycloastragenol as a framework and isoflavone represented by calycosin glucoside, etc. The triterpene saponin with cycloastragenol as skeleton mainly comprises astragaloside I, isoastragaloside I, astragaloside II, astragaloside III, astragaloside IV, etc.
Compared with the growth cycle of at least 2 years of field cultivation of the medicinal astragalus, the astragalus root culture has the advantages of short growth cycle, no pesticide and fertilizer pollution, no land resource occupation and the like, and is an important raw material for obtaining a large amount of medicinal astragalus roots and preparing the astragalus extract in a short time. Compared with the prior hairy root culture generated by the induction of the agrobacterium rhizogenes, which contains toxic opines and has higher preparation cost of later-stage extract, the adventitious root culture system is safer and more economic, and has great industrial development potential.
However, the astragalus adventitious root culture has the defects of tissue culture browning, limited subculture times and the like. The addition of the inducer which has the functions of promoting the synthesis and accumulation of the active ingredients of the adventitious roots of the astragalus and inhibiting lipoxygenase contributes to the increase of the subculture times, the propagation expansion and the industrial development of the astragalus adventitious root culture.
As described above, cis-3-hexenal defense is related to its configuration, but studies have been mainly made in plants such as tobacco, Arabidopsis, and tea. The effect of cis-3-hexenal on feedback inhibition of lipoxygenase activity, a key enzyme in the synthetic route of the cis-3-hexenal, and the application of the cis-3-hexenal in the synthesis and accumulation of the non-volatile triterpene saponin and the isoflavone active ingredients of the medicinal plants are not reported at home and abroad.
Disclosure of Invention
The invention provides a chemical inducer for astragalus adventitious root tissue culture and application thereof in astragalus adventitious root culture, aiming at solving the problems of green and sustainable development of astragalus resources at present; the chemical inducer has the effects of feedback inhibition and promotion of synthesis and accumulation of active ingredients of astragaloside and isoflavone of adventitious roots in a dose-dependent manner, inhibits browning of roots by inhibiting lipoxygenase activity of the adventitious roots of the astragalus, further improves the growth state of the roots, and promotes synthesis and accumulation of astragaloside I, isoastragaloside I, astragaloside II, astragaloside III, formononetin, calycosin, glucoside thereof and the like in the roots.
The invention is realized by the following technical scheme: a chemical inducer for culturing adventitious root tissue of astragalus membranaceus is prepared by dissolving cis-3-hexenal in water, and the specific preparation method comprises the following steps: dissolving cis-3-hexenal in sterile water to prepare 200mmol/L mother liquor, filtering and sterilizing by using a sterile filter membrane, and diluting by using the sterile water according to the volume ratio of 1:4 or 1:16 to prepare 50mol/L or 12.5mol/L mother liquor for direct use.
The feedback inhibition and chemical inducer cis-3-hexenal is a 50% solution dissolved in triacetin obtained by a commercial purchase mode, and water for preparing mother liquor is sterilized deionized water.
The invention also provides application of the chemical inducer in inhibiting browning of the adventitious root of astragalus and improving the growth state of the adventitious root of astragalus.
Further, the chemical inducer completely inhibits the activity of lipoxygenase in the extracting solution of the astragalus root adventitious root source in vitro, and the working concentration of cis-3-hexenal is as follows: 3.20 nmol/. mu.L or 6.40 nmol/. mu.L.
The invention also provides a method for reducing the lipoxygenase activity of the astragalus adventitious root culture by using the chemical inducer, wherein the cis-3-hexenal is used for treating the astragalus adventitious root culture, the working concentration of the cis-3-hexenal is 12.5-200 mu mol/L, and the treatment lasts for 120 hours.
Further, the working concentration of cis-3-hexenal is: 12.5, 50 or 200 [ mu ] mol/L.
In addition, the method for promoting the synthesis and accumulation of active ingredients of formononetin, calycosin and glucoside in roots by the chemical inducer comprises the following specific steps: adding 0.1% v: v of 200mmol/L cis-3-hexenal mother liquor into 1/2 MS culture solution containing 1 mg/L indolebutyric acid and 30 g/L sucrose; inoculating the astragalus adventitious root culture according to the inoculation amount of 13% w: v, and performing shake culture for 168h at 25 ℃ under the condition of 100 rpm.
The method for promoting the synthesis and accumulation of active ingredients including astragaloside I, isoastragaloside I, astragaloside II, astragaloside III and cycloastragaloside II in roots by using the chemical inducer comprises the following specific steps: adding 0.1% v/v of 12.50 mmol/L and 50mmol/L cis-3-hexenal mother liquor into 1/2 MS liquid culture medium containing 1 mg/L indolebutyric acid and 30 g/L sucrose; inoculating the astragalus adventitious root culture according to the inoculation amount of 13% w: v, and performing shake culture for 168h at 25 ℃ under the condition of 100 rpm.
The chemical inducer is suitable for an adventitious root liquid culture system of Mongolian astragalus, and has the effects of reducing the activity of lipoxygenase of a root culture and improving the content of various active ingredients in roots under the condition of sterile shaking table culture.
The chemical elicitor related by the invention naturally exists in roots of Mongolian milkvetch roots of medicinal plants, has high content in genuine milkvetch roots, namely turbid source milkvetch roots, belongs to volatile organic compounds, is applied to an adventitious root culture system of milkvetch roots, and does not have the problem of secondary pollution; the elicitor cis-3-hexenal promotes the synthesis and accumulation of formononetin, calycosin and glucoside thereof in the adventitious roots of Mongolian milkvetch roots to be configuration specific; the characteristic of promoting the synthesis and accumulation of calycosin glucoside is more consistent with the result of treating astragalus membranaceus seedlings by lipoxygenase inhibitor-phenidone alone, and is presumed to be related to partial inhibition of lipoxygenase signal pathway [11] . Therefore, the cis-3-hexenal provides a natural environment-friendly chemical inducer for the liquid culture of the adventitious roots of the Mongolian astragalus.
The astragalus medicinal material is mainly subjected to wild-simulated cultivation and flat land transplanting at present, the growth period of the wild-simulated cultivation astragalus is more than or equal to 4 years, the growth period of the flat land transplanting astragalus is more than or equal to 2 years, and the growth period is longer; spraying a chemical synthetic pesticide to prevent and control plant diseases and insect pests and applying a chemical fertilizer to roots to promote growth, so that pesticide residues of the astragalus medicinal material and the quality of the medicinal material are easily reduced, soil pollution, hardening, fertility reduction and the like can be caused, and the green development of limited land resources is not facilitated. A plant tissue culture method is applied, a new medicinal astragalus resource obtaining way is excavated, the method is an active, economic and efficient supply way for preparing the astragalus extract and ensuring green development and sustainable utilization of the astragalus industry, and the method can also be applied to breeding of high-quality astragalus varieties.
The chemical elicitor related to the present invention is obtained by screening in the following way. Firstly, respectively adding 0.1% v/v of cis-3-hexenal mother liquor of 200mmol/L, 50mmol/L and 12.5mmol/L into 1/2 MS liquid culture solution, shaking and culturing for 168h, and dynamically sampling. By analyzing the lipoxygenase activity and the contents of the astragalus saponin I, the isoastragalus saponin I, the astragalus saponin II, the astragalus saponin III, the formononetin, the calycosin and the glucoside in the treated and control samples and observing the root growth state and the root activity when the samples are cultured for 168 hours, the cis-3-hexenal is found to simultaneously have the functions of reducing the lipoxygenase activity of an adventitious root culture, changing the root growth state and promoting the synthesis and accumulation of the active ingredients in the root. The reduction of the activity of the adventitious root lipoxygenase and the synthesis accumulation promotion effect of dose-dependent triterpenoid saponin and isoflavone active ingredients are comprehensively considered, and the elicitor cis-3-hexenal is expected to be developed into a green additive to be applied to an adventitious root tissue culture system of astragalus mongholicus, so that the growth state of a root culture is changed, and the content of root active substances is increased.
Drawings
FIG. 1 shows the seedlings of Astragalus membranaceus and adventitious roots of Astragalus membranaceus induced by the seedlings and their liquid culture (A, B, C, D, E); in the figure, A is the apical bud (arrow mark) for inducing the tissue culture seedling and adventitious root of Astragalus membranaceus; b is the adventitious root early morphology induced (arrow-marked); c is adventitious root for liquid culture; d is astragalus adventitious root culture for subculture; e is an adventitious root culture of the astragalus root for treating cis-3-hexenal;
FIG. 2 is a graph of the optimization of the dose of positive control phenidone in vitro inhibition of lipoxygenase from Astragalus membranaceus;
FIG. 3 is a screen of green leaf volatiles with in vitro inhibition of lipoxygenase from Astragalus membranaceus; in the figure, different letters on the standard deviation of the histogram represent that the inhibition rate of the green leaf volatile substances has obvious difference, Mock is solvent control, Phenidine is positive control Phenidone, Hexanal is n-Hexanal, Hexanol is n-Hexanol, Hexyl acetate is Hexyl acetate,Cis-3-hexenol is cis-3-hexenal,Cis-3-hexenol is cis-3-hexenol, Leaf acetate is cis-3-hexenyl acetate,Trans-2-hexenol is trans-2-hexenal,Trans-2-hexenol is trans-2-hexenol;
FIG. 4 is a graph of the effect of treatment concentration of cis-3-hexenal (A) and low temperature storage (B) on inhibition of Astragalus lipoxygenase in vitro; in the figure, different letters on the histogram represent significant differences in inhibition rates of different concentrations of cis-3-hexenal, indicating that the inhibition rate of the cis-3-hexenal solution after 3 days of low temperature storage is significantly lower than that of the same concentration of fresh formulation;
FIG. 5 is a graph showing the inhibition of lipoxygenase of soybean (A) and mung bean (B) by cis-3-hexenal at different concentrations in vitro; in the graph, different letters on the standard deviation of the histogram represent the significant difference of the inhibition rates of different concentrations, Mock is a solvent control, and phenidone is a positive control phenidone;
FIG. 6 shows the lipoxygenase activity of adventitious roots cultures of Astragalus membranaceus treated with cis-3-hexenal at different concentrations for different periods of time; in the figure, different letters on the histogram standard deviation represent significant differences at the same time point between groups;
FIG. 7 is a graph showing the variation trend of the activity of lipoxygenase in adventitious root cultures of Astragalus membranaceus in different concentrations of cis-3-hexenal-treated groups;
FIG. 8 is an appearance trait of 168h Astragalus adventitious root culture treated with 200 μmol/L cis-3-hexenal (A, B); in the figure, A is a normal control, and B is a 200 [ mu ] mol/L cis-3-hexenal treatment group;
FIG. 9 shows the effect of different concentrations of cis-3-hexenal treatment on the growth status of radix astragali root cultures (A, B, C); in the figure, A is a normal control root culture for 120h growth, B is a root culture treated by 200 [ mu ] mol/L cis-3-hexenal for 120h, C is a root culture treated by 168h, Ca is a normal control, Cb is treated by 12.5 [ mu ] mol/L cis-3-hexenal, Cc is treated by 50 [ mu ] mol/L cis-3-hexenal, and Cd is treated by 200 [ mu ] mol/L cis-3-hexenal; the arrow points to the root growth point;
FIG. 10 shows the effect of treatment with different concentrations of cis-3-hexenal on the synthesis and accumulation of triterpenoid saponins in adventitious roots cultures of Astragalus (A, B, C, D); in the figure, A is astragaloside I, B is astragaloside II, C is astragaloside III, D is isoastragaloside I, Control is normal Control, and different letters on the standard deviation of a histogram represent the significant difference of the same time point among groups;
FIG. 11 shows the effect of different concentrations of cis-3-hexenal treatment on the accumulation of isoflavone synthesis in adventitious root cultures of Astragalus (A, B, C); in the figure, A is Calycosin glucoside (Calycosin-7-O-β-D-glucoside, CG), B is formononetin (Ononin), C is Calycosin (calcicin), Control is a normal Control, different letters on the histogram standard deviation represent significant differences at the same time point between groups.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize that equivalents to the specific embodiments described, as may be learned by routine experimentation, are intended to be encompassed by the present application.
The experimental procedures in the following examples are all conventional ones unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1: inducing adventitious roots of Mongolian astragalus and subculturing
Taking a Mongolian astragalus mongholicus tissue culture seedling which is propagated in a long period (> 5 years) in a laboratory and is in a vigorous growth period as a starting material, selecting a terminal bud with good growth vigor and is 1-2 cm in length as shown in an attached drawing 1, aseptically shearing the terminal bud, transferring the terminal bud into an MS solid culture medium containing 0.7% of agar, 30 g/L of sucrose and 1 mg/L of naphthylacetic acid, culturing the terminal bud for 8 weeks under the conditions of 25 ℃ and 16 hours (light)/8 hours (dark), and inducing early morphological characteristics of grown adventitious roots to be shown as B in the attached drawing 1, wherein the morphological characteristics of the adventitious roots used for liquid culture are shown as C in the attached drawing 1.
Then, after aseptic shearing and washing adventitious roots with MS liquid culture medium, transferring and culturing to 30 mL 1/2 MS liquid culture medium (100 mL triangular flask) containing 30 g/L sucrose and 1 mg/L indolebutyric acid, and shake culturing at 25 ℃ and 100rpm for 4 weeks to obtain an adventitious root seed culture, as shown in D in figure 1; dividing the culture into two parts, inoculating the root seed culture into a 250 mL triangular flask containing 100 mL of the same culture medium, and performing shake culture for 4 weeks under the same condition for adventitious root subculture; dividing into two parts, transferring into 500 mL triangular flask containing 200 mL identical culture medium, and shake culturing under the same condition for 4 weeks to obtain induced radix astragali adventitious root culture, as shown in E of figure 1. Since adventitious roots of Astragalus membranaceus are formed by induced differentiation of apical buds of tissue culture seedlings of Astragalus membranaceus, and belong to sterile cultures, bacterial culture based on a tissue grinding method (7 days at 27 ℃ C., formation of sterile colonies) also confirmed that the adventitious root cultures are sterile materials.
Example 2: lipoxygenase extraction, activity determination and optimization of phenidone in vitro incubation dose
Randomly weighing 1.00g of astragalus adventitious root with good growth vigor, grinding the astragalus adventitious root into powder by using liquid nitrogen, adding 0.05 mol/L borate buffer solution with the pH value of 9.0 according to the material ratio of 1:10 (w: v), and fully mixingHomogenizing, leaching in ice water bath for 2 hr, centrifuging at 4 deg.C and 8000 rpm for 10 min, transferring supernatant to new centrifuge tube to obtain crude lipoxygenase extractive solution, and storing at 4 deg.C. Transferring 30 mu L of the lipoxygenase crude extract into a new centrifuge tube under the low temperature condition, adding 1000 mu L of 0.435 mmol/L linoleic acid solution which is preheated to 25 ℃ and has pH9.0, fully mixing uniformly, incubating at 25 ℃ for 10 min, and adding 970 mu L of absolute ethyl alcohol to terminate the reaction; simultaneously, a reaction system with absolute ethyl alcohol added firstly and substrate solution added later is used as a blank control to measure OD 235 And obtaining the activity data of the lipoxygenase in the root culture.
Selecting the same lipoxygenase crude extract, sucking 120 μ L into a new centrifuge tube, adding 0.01 mmol/L, 0.1 mmol/L, 1.0 mmol/L, 10 mmol/L phenidone solution and methanol (solvent control) according to a volume ratio of 30:1, mixing well, incubating at 25 deg.C for 10 min, determining OD according to the above method 235。 Using the obtained OD 235 The inhibition ratio is calculated according to the following formula:
inhibition rate = [ (OD) 235, Normal control －OD 235, phenidone ）/OD 235, Normal control ]×100%。
As shown in FIG. 2, the inhibition ratios were 19.52%, 36.65%, 100% and 100% at the concentrations of 0.01, 0.1, 1.0 and 10.0 mM, respectively, so that the minimum concentration of 1.0 mM with the inhibition ratio of 100% was selected, and the working concentration was 0.032 nmol/. mu.L, and the subsequent experiments were carried out.
Example 3: in vitro screening of green leaf volatiles with lipoxygenase activity inhibiting effect
Selecting the same batch of lipoxygenase crude extract, sucking 120 mu L of lipoxygenase crude extract into a new centrifuge tube, and adding 50mmol/L of the following green leaf volatile substance solution according to the volume ratio of 30: 1: hexyl hexanol, hexyl acetate, cis-3-hexenal, cis-3-hexenol, trans-2-hexenal, trans-2-hexenol, cis-3-hexenyl acetate, along with normal control, solvent control (adding methanol at the same volume ratio) and positive control (obtained by screening in example 2), mixing well, incubating at 25 deg.C for 10 min, and determining OD according to the method in example 2 235 Meter for measuringCalculating the inhibition rate; number of repetitions of each process: 3.
the result is shown in figure 3, and it can be seen from the figure that cis-3-hexenal inhibition rate is the highest in the detected green leaf volatile, and is at the same level with the inhibition rate of the positive control phenidone; the inhibition rate of other volatile matters is obviously lower than that of cis-3-hexenal and phenidone, and the volatile matters and the solvent control are at the same level, which indicates that the volatile matters in green leaves do not have the function of obviously inhibiting the lipoxygenase of the adventitious root of the astragalus.
Example 4: characterization of effect of cis-3-hexenal on inhibition of astragalus lipoxygenase crude enzyme solution and influence of low-temperature storage on inhibition effect
Three batches of crude enzyme solutions of Astragalus adventitious root lipoxygenase were prepared as in example 1, 120. mu.L was pipetted into a new centrifuge tube, 0.1 mmol/L, 1mmol/L, 10 mmol/L, 25 mmol/L, 50mmol/L, 100 mmol/L, 200mmol/L cis-3-hexenal was added at a ratio of 1:30 (v: v), mixed well, incubated at 25 ℃ for 10 min, and OD was measured as in example 2 235 Calculating the inhibition rate; blank control and solvent control were set simultaneously, biological replicates: 3. the results are shown in figure 4A, from which it can be seen that cis-3-hexenal inhibition in vitro is related to its concentration, and when the working concentration is between 0.0032-0.032 nmol/. mu.L, the inhibition effect is the weakest, and the inhibition rate is the lowest<5% of the same order of magnitude as the solvent control; when the working concentration is 0.32 nmol/mu L, the inhibition rate is 47.85 +/-2.28 percent; the inhibition rate is 72.72 +/-1.36% when the concentration is 0.80 nmol/mu L; when the working concentration is 1.60 nmol/mu L, the inhibition rate is 90.91 +/-0.48 percent; when the working concentration is 3.20 and 6.40 nmol/. mu.L, the inhibition rate is more than 99 percent, the lipoxygenase activity is almost completely inhibited, and the inhibition rate is equivalent to that of the phenidone with the working concentration of 0.032 nmol/. mu.L.
Trans-2-hexenal is a cis-3-hexenal isomerization product and is stable in configuration, but the results in example 3 show that trans-2 configuration does not have obvious effect of inhibiting the activity of lipoxygenase in vitro, which indicates that cis-3-hexenal inhibits the lipoxygenase of astragalus in vitro specifically. Considering that cis-3-hexenal is unstable in configuration and easily isomerized into trans-2-hexenal without inhibitory effect, and using freshly prepared cis-3-hexenal with the same concentration as a control, we further examined the inhibitory effect of 10, 25, 50mM cis-3-hexenal solution stored at low temperature, i.e. 4 ℃ for 3 days, on three batches of crude extract of astragalus lipoxygenase, and the results are shown in fig. 4B. As can be seen from the figure, the inhibition rate of 10 mM cis-3-hexenal stored for 3 days at low temperature is significantly lower than that of the fresh formulation, suggesting that under the low temperature storage condition, part of cis-3-hexenal in the solution has been isomerized into trans-2 configuration or converted into other substances, resulting in a decrease in the inhibition rate thereof; it can also be seen from the figure that there was no significant difference between the inhibition rates of 25, 50mM cis-3-hexenal stored for 3 days at low temperature and the freshly prepared solution of the same concentration, suggesting that the isomerization or conversion degree of cis-3-hexenal was very weak in these two solutions stored for 3 days at low temperature. In other words, the isomerization of cis-3-hexenal in an aqueous solution upon storage at low temperature is concentration dependent, high concentration and low degree of isomerization.
Example 5: research on effect of cis-3-hexenal on inhibiting lipoxygenase from other plant sources in vitro
Lipoxygenase is a key enzyme in the synthesis path of the green leaf volatile matter, and feedback inhibition of the synthesis path is prompted by the action of cis-3-hexenal for inhibiting the lipoxygenase of the astragalus in vitro; this feedback inhibition is present only in astragalus membranaceus, is also ubiquitous in the plant kingdom, or is present in certain types of plants; to clarify the existence range of the inhibition, using the lipoxygenase extraction and activity assay method of example 2, we investigated the inhibition of cis-3-hexenal on representative other plant lipoxygenase, specifically including: (1) the medicinal plants containing trans-2-hexenal, such as radix bupleuri, platycodon grandiflorum and radix codonopsis, are selected from fresh roots; (2) model plants of arabidopsis thaliana and tobacco are selected as the whole plant; (3) bean sprouts are selected as the materials of beans such as soybeans and mung beans. As a result, the lipoxygenase activity of soybean and mung bean sprouts is higher, and the lipoxygenase activity of other materials is very low, so that the lipoxygenase is extracted from the soybean sprouts and mung bean sprouts to study the inhibition effect of cis-3-hexenal, and the results are shown in figures 5A and 5B.
As can be seen from the attached figure 5A, when the cis-3-hexenal working concentration is 0.80, 1.60, 3.20 nmol/muL, the inhibition rate is greater than 99%, the inhibition rate is close to the positive control phenidone and is at the same level; when the working concentration is 0.032 and 0.32 nmol/mu L, the inhibition rates are 59.72 +/-3.95 percent and 86.80 +/-2.56 percent respectively; compared with the results of example 4, cis-3-hexenal has stronger effect of inhibiting soybean lipoxygenase in vitro than astragalus lipoxygenase in vitro. Compared with astragalus and soybean lipoxygenase, the activity of the mung bean lipoxygenase is higher, the concentration of the positive control phenidone obtained by re-screening under the same condition is 10 mmol/L (the working concentration is 0.32 nmol/muL), and is 9 times higher than the phenidone concentration of the first two plants with the same inhibition rate. As can be seen from the attached figure 5B, when the concentration of cis-3-hexenal mother liquor is increased to 250 mM and 500 mM, namely the working concentration is respectively 8.0 nmol/muL and 16.0 nmol/muL, the inhibition rates are respectively 74.42 +/-11.84% and 73.54 +/-5.96%, and are still remarkably lower than the inhibition rate of positive control phenidone, which indicates that cis-3-hexenal has weak effect of inhibiting mung bean lipoxygenase. In summary, the cis-3-hexenal feedback inhibition is obviously present in Astragalus mongholicus, soybeans and mung beans of Leguminosae.
Example 6: influence of different concentrations of cis-3-hexenal treatment on lipoxygenase activity of radix astragali adventitious root culture and synthesis and accumulation of triterpenoid saponin and isoflavone
In order to further verify that cis-3-hexenal inhibits lipoxygenase and explore the induction effect of lipoxygenase, an astragalus adventitious root culture system is utilized, and the effect of the lipoxygenase in astragalus root culture is further characterized. Firstly, 200mmol/L cis-3-hexenal mother liquor is prepared by sterile water, and is divided into 3 parts after filtration sterilization, wherein 2 parts are respectively diluted by 16 times and 4 times by sterile water to prepare 12.5mmol/L and 50mmol/L cis-3-hexenal mother liquor which is immediately used. The specific treatment method comprises the following steps: adding the mother solution into 1/2 MS liquid culture medium containing 1 mg/L indolebutyric acid and 30 g/L sucrose according to the volume ratio of 0.1%; inoculating radix astragali adventitious root culture according to the inoculation amount of 13% (w: v), and shake-culturing at 25 deg.C and 100rpm for 168 hr; a normal culture control was also set. Samples were taken 4, 8, 12, 24, 48, 72, 120, 168h after treatment, respectively, with a biological repetition number of 3 per sample point per set. During sampling, firstly transferring root culture onto fresh filter paper, sucking culture solution, dividing into 2 parts, transferring one part into a nut tube, quickly freezing by liquid nitrogen, transferring into a refrigerator at minus 80 ℃ for lipoxygenase activity determination; the other part was placed in an oven at 60 ℃ and dried to constant weight for chemical analysis. And dividing the 120h sample and the 168h sample into 3 parts, respectively using for lipoxygenase activity determination, crude protein content determination and chemical analysis, directly photographing the third part, and immediately placing the third part in TTC dye liquor for microscopic observation of root system activity.
The method of example 2 was used to prepare the crude lipoxygenase extract and to determine its activity; simultaneous determination of OD of crude extract 280 And drawing a working curve by taking bovine serum albumin as standard protein, and calculating to obtain the protein content of the lipoxygenase crude extract. The lipoxygenase activity unit is defined as: using linoleic acid as substrate, in a reaction system with pH9.0, 25 deg.C and 2 mL, per minute per microgram lipoxygenase crude extract OD 235 The increase of 0.001 is one activity unit.
The preparation method of the test solution for chemical analysis comprises the following steps: grinding root culture with mortar into powder, adding 70% ethanol (pH6.0) according to a material ratio of 1:30 (g: v), ultrasonic extracting for 1 h, centrifuging for 10 min at 8,000 g, and collecting supernatant; extracting once again, mixing extractive solutions, concentrating to dry with nitrogen blowing instrument, dissolving in 2 mL of chromatographic pure methanol containing 0.1% formic acid by vortex shaking, filtering with 0.22 μm filter membrane, transferring filtrate into sample vial, and measuring contents of astragaloside I, isoastragaloside I, astragaloside II, astragaloside III, formononetin, calycosin and glucoside thereof by UHPLC-MS method. The specific chromatographic and mass spectrum conditions are as follows: ultimate 3000 HPLC chromatography-Orbitrap Q active Plus mass spectrometer (Thermo Fischer), scan mode: FULL MS-DD MS 2; sample introduction amount: 2 mu L of the solution; a chromatographic column for separation: syncronis C18 column (2.1 mm. times.100 mm, 1.7 μm; Thermo Fischer). Chromatographic conditions are as follows: flow rate 0.2 mL/min, column temperature: 40 ℃; mobile phase: acetonitrile (a) -0.1% formic acid (a), gradient elution conditions: 0-2 min, 20% A; 2-4 min, 20% A → 40% A; 4-7 min, 40% A → 45% A; 7-10 min, 45% A; 10-14 min, 45% A → 80% A; 14-16 min, 80% A → 95% A; 16-18.5 min, 95% A; 18.5-19 min, 95% A → 20% A; 19-22 min, 20% A. The ion information for mass spectrometric qualitative identification and quantitative analysis is shown in table 1.
Table 1: ion information table for qualitative identification and quantitative analysis of mass spectrum
The results of the lipoxygenase activity analysis of the adventitious root culture of Astragalus membranaceus in the group treated with cis-3-hexenal at different concentrations are shown in the attached figure 6. As can be seen from the figure, the enzyme activity unit of the treatment group of 200. mu. mol/L is significantly lower than that of the treatment group of 12.5. mu. mol/L when cis-3-hexenal is treated for 4, 48 and 72 hours; after treatment for 8-24 h and 120h, the lipoxygenase activity of all cis-3-hexenal treatment groups is lower than that of a control group; after 168 hours of treatment, the lipoxygenase activity of the cis-3-hexenal treatment group and the control group has no obvious difference. In short, in addition to the in vitro inhibitory effect, cis-3-hexenal treatment resulted in a decrease in lipoxygenase activity of the astragalus root culture, suggesting that it has the effect of inhibiting the lipoxygenase of the root culture in vivo, the inhibition being related to the treatment dose and treatment time; the optimum processing time is 120h in terms of the processing time currently involved.
The activity trend of lipoxygenase in the culture of adventitious roots of Astragalus membranaceus treated with cis-3-hexenal at different concentrations is shown in figure 7. As can be seen from the figure, the activity of lipoxygenase showed a trend of "first rising and then falling" in the control group and the cis-3-hexenal-treated adventitious root culture of Astragalus membranaceus, and particularly the activity of the enzyme in the blank control group was the highest 12 h after inoculation. The existence of the high-activity lipoxygenase is not beneficial to the growth of adventitious roots, the aging process of plants is accelerated, the addition of cis-3-hexenal obviously delays the rise of the activity of the lipoxygenase, and the activity of the lipoxygenase is reduced to a certain extent; the treatment group with the concentration of 200 mumol/L is most remarkable, and the activity of the lipoxygenase is always maintained at a lower level. The growth states of the adventitious roots of the astragalus mongholicus in the cis-3-hexenal treatment group and the blank control group at the sampling time of 200 mu mol/L on the 7 th day are compared, and an assistant evidence can be provided for the inference. As shown in figure 8, the root culture of the 200 mu mol/L treatment group has more white growing points, white root, increased root amount and obviously better growth state than the control; the cis-3-hexenal is suggested to promote the growth of the adventitious roots of the astragalus and possibly relate to the inhibition of the lipoxygenase activity of the astragalus.
TTC, triphenyltetrazolium Chloride (2,3, 5-triphenyl Tetrazolium Chloride), is a redox species. When TTC in the colorless and transparent aqueous solution is reduced by dehydrogenase in root system cells, the product is red tritylhydrazone which is insoluble in water and not easily oxidized by oxygen existing in the air. The reduction capacity of TTC is utilized to reflect the vitality of the plant root system to a certain extent: the higher the respiratory rate of the root system is, the stronger the activity of the root system is, and the more the amount of TTC reduction in the root system is; as the respiration rate of the root system becomes weaker, the amount of reduced TTC is relatively reduced. By utilizing the principle, the root culture is placed in a centrifuge tube containing 1.8 mL of 0.4% TTC solution, incubated for 1 h under the dark condition at 25 ℃, added with 0.2 mL of 1mol/L sulfuric acid solution, stopped to react, and placed in an LEICA M205 FA fluorescence microscope for observation, and the root culture of a normal control group and a 12.5 mu mol/L treatment group is found to have closer growth characteristics, more branches and short branches and blunt-rounded top ends, and representative results are shown in figures 9A, 9 Ca and 9 Cb; in addition, the root cultures of the 50 and 200. mu. mol/L treated groups had closer growth characteristics, fewer branches, and longer branches, with the tips of the growing points being sharp-rounded and darker red, and the representative results are shown in FIGS. 9B, 9Cc, and 9 Cd. As can be seen by comparing FIGS. 9A, 9B and 9C, the root culture growth spots were lighter in color in 120h of shake culture than in 168h of culture, indicating that the root culture growth status was better in 168h of culture. In short, cis-3-hexenal affects the growth state of adventitious root cultures of astragalus in a dose-dependent manner, the 12.5 mu mol/L treatment has small influence on the differentiation and growth of roots, the growth characteristics are closer to those of a control, the 50 and 200 mu mol/L cis-3-hexenal treatment has large influence on the differentiation and growth of roots, and the morphology and TTC staining results are summarized to play roles in inhibiting the differentiation and promoting the elongation and growth of roots.
The analysis results of the content of astragaloside in the cis-3-hexenal-treated root cultures with different concentrations are shown in figure 10. As can be seen from FIG. 10A, after treatment for 4 and 168 hours, the content of astragaloside I in all the treatment groups is significantly higher than that in the control group, and the content of astragaloside I in the treatment group is the highest at 12.5. mu. mol/L; the obvious increase of the content of astragaloside I also exists in the following groups: the root cultures were treated at 12.5 and 50. mu. mol/L for 8 and 48 h, respectively. As can be seen from FIGS. 10B, 10C and 10D, cis-3-hexenal promotes the synthesis and accumulation of astragaloside II, astragaloside III and isoastragaloside I in the adventitious root culture of Astragalus membranaceus, and the induction and promotion effects of 12.5. mu. mol/L and 50. mu. mol/L are similar, especially the treatment group of 12.5. mu. mol/L.
The results of the analysis of isoflavone content in the root cultures treated with cis-3-hexenal at different concentrations are shown in FIG. 11. As can be seen from the attached figures 11A and 11B, when cis-3-hexenal of 200. mu. mol/L is treated for 4-24 hours, the content of calycosin glucoside and formononetin in the root culture is the highest and is obviously higher than that of the control and low-concentration treatment groups; over time, the promoting effect of this treatment on both diminishes. As can be seen from FIG. 11C, 200. mu. mol/L cis-3-hexenal promoted the best accumulation of calycosin synthesis, followed by 12.5. mu. mol/L cis-3-hexenal treatment group, with the worst effect of intermediate dose treatment.
The data of the content of triterpenoid saponin and isoflavone in the astragalus mongholicus in the cis-3-hexenal treatment groups with different concentrations are summarized, the lowest concentration, namely 12.5 mu mol/L cis-3-hexenal treatment group, has the highest quantity of the components for promoting synthesis and accumulation, and the highest concentration, namely 200 mu mol/L cis-3-hexenal treatment group, has the strongest effect of promoting synthesis and accumulation of calycosin glucoside and formononetin.
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finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. A chemical inducer for culturing adventitious root tissue of astragalus is characterized in that: the chemical inducer is prepared by dissolving cis-3-hexenal in water, and the specific preparation method comprises the following steps: dissolving cis-3-hexenal in sterile water to prepare 200mmol/L mother liquor, filtering and sterilizing by using a sterile filter membrane, and diluting by using sterile water according to the volume ratio of 1:4 or 1:16 to prepare 50mol/L or 12.5mol/L mother liquor for direct use.
2. The use of the chemical inducer for culturing astragalus adventitious root tissue of claim 1 in culturing astragalus adventitious roots, characterized in that: the chemical inducer is applied to inhibiting browning of adventitious roots of astragalus and improving the growth state of the adventitious roots of the astragalus.
3. Use according to claim 2, characterized in that: the chemical inducer completely inhibits the activity of lipoxygenase in an extracting solution of an astragalus adventitious root source in vitro, and the working concentration of cis-3-hexenal is as follows: 3.20 nmol/. mu.L or 6.40 nmol/. mu.L.
4. Use according to claim 2, characterized in that: the chemical inducer reduces the activity of lipoxygenase of the astragalus adventitious root culture, inhibits browning of the astragalus adventitious root and improves the growth state of the astragalus adventitious root, the cis-3-hexenal treats the astragalus adventitious root culture, and the working concentration of the cis-3-hexenal is 12.5 mu mol/L-200 mu mol/L; and processing for 120 h.
5. Use according to claim 4, characterized in that: the cis-3-hexenal working concentration is as follows: 12.5, 50 or 200 [ mu ] mol/L.
6. The use of the chemical inducer for culturing adventitious roots of astragalus membranaceus according to claim 1 in culturing adventitious roots of astragalus membranaceus, wherein the chemical inducer comprises: the method for promoting the synthesis and accumulation of active ingredients of formononetin, calycosin and glucoside in roots by the chemical inducer comprises the following steps: adding 0.1% v: v of 200mmol/L cis-3-hexenal mother liquor into 1/2 MS culture solution containing 1 mg/L indolebutyric acid and 30 g/L sucrose; inoculating astragalus adventitious root culture according to the inoculation amount of 13% w: v, and performing shaking culture on a shaking table for 168 hours at the temperature of 25 ℃ and the speed of 100 rpm.
7. The use of the chemical inducer for culturing adventitious roots of astragalus membranaceus according to claim 1 in culturing adventitious roots of astragalus membranaceus, wherein the chemical inducer comprises: the method for promoting the synthesis and accumulation of active ingredients including astragaloside I, isoastragaloside I, astragaloside II, astragaloside III and cycloastragaloside II in roots by using the chemical inducer comprises the following specific steps: adding 0.1% v/v of 12.5mmol/L or 50mmol/L cis-3-hexenal mother liquor into 1/2 MS liquid culture solution containing 1 mg/L indolebutyric acid and 30 g/L sucrose; inoculating the astragalus adventitious root culture according to the inoculation amount of 13% w: v, and performing shake culture for 168h at 25 ℃ under the condition of 100 rpm.
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