CN106148453A - A kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside - Google Patents
A kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside Download PDFInfo
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- CN106148453A CN106148453A CN201610554590.1A CN201610554590A CN106148453A CN 106148453 A CN106148453 A CN 106148453A CN 201610554590 A CN201610554590 A CN 201610554590A CN 106148453 A CN106148453 A CN 106148453A
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Abstract
The invention discloses a kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside, belong to technical field of biological culture.The method comprises the following steps: will be placed in inducing culture in salicylated culture fluid by Radix Rehmanniae hairy root,.Method And Principle is: the Radix Rehmanniae hairy root processed through salicylic acid can improve the expression of verbascoside biosynthesis key gene within the short time (12～24h), such as PAL (PAL), 4 coumaroyl A ligase (4CL), cinnamic acid hydroxylase (C4H), coumaric acid hydroxylase (C3H), copper amino oxidase (CuAO) and tyrosine decarboxylase (TyDC), thus complete the accumulation of verbascoside, and improve its yield further, the method is easy and simple to handle, cultivation cycle is short, it is convenient for industrialization, scale and Standardization Practice, to advancing, verbascoside large-scale production is significant.
Description
Technical field
The present invention relates to a kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside, belong to biological culture technigue neck
Territory.
Background technology
Radix Rehmanniae (Rehmannia glutinosa L.) is Scrophulariaceae herbaceos perennial, is used as medicine with tuber, is China
One of famous " four-Huai medicine ", consumption is big, determined curative effect, has important medical value and economic worth, for conventional large
Medical material, is one of the big kind of the modern big Chinese medicine industrial chain formation keypoint recommendation research and utilization of China.
Verbascoside is one of index components of Radix Rehmanniae, have antioxidation, immunomodulating, antiinflammatory, protect the liver, hypermnesis
The biological activity such as power, antitumor, toxic and side effects is less.In Radix Rehmanniae tuber, the content of verbascoside is relatively low, and " Chinese Pharmacopoeia " advises
Determining the content of verbascoside in Radix Rehmanniae must not be less than 0.02%.And practical situation is, verbascose between different Radix Rehmanniae kinds
The content difference of glycosides is obvious, even same breed, in different regions, plantation also shows content difference, and range of variation is relatively
Greatly, it is uneven that this results in Radix Rehmanniae quality, and verbascoside extracts and applies the problems such as limited.Gene engineering is utilized to obtain
The Radix Rehmanniae kind obtaining high verbascoside content is a potential effective solution, but up to now, relevant Radix Rehmanniae hair
The stamen flower biosynthetic molecular genetic mechanism of glucosides is unclear.
Research shows, Radix Rehmanniae hairy root can produce the secondary metabolite identical with former plant, if utilizing hairy root
Produce verbascoside, by for obtaining Radix Rehmanniae verbascoside a kind of new approach of offer.
Summary of the invention
It is an object of the invention to provide a kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside in a large number.
In order to realize object above, the technical solution adopted in the present invention is:
A kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside, comprises the following steps: be placed in by Radix Rehmanniae hairy root
Inducing culture in salicylated culture fluid,.
In described culture fluid, salicylic concentration is 10～40 μm ol/L.Preferably, Determination of Salicylic Acid is 25 μm ol/L.
The base fluid of described culture fluid can use in the MS culture medium of liquid, WPM culture medium, N6 culture medium, B5 medium etc.
Any one.MS, WPM, N6, B5 fluid medium uses " the practical plant tissue culture skill of Cao Zi justice and Liu Guomin chief editor
Art study course " disclosed in formula.It addition, in culture fluid can also containing the sucrose of mass concentration 20～40g/L, as carbon source and
Energy substance.
Described inducing culture is: cultivate 5～10d under rotating speed 100～150rpm, temperature 24～26 DEG C, dark condition.Excellent
Choosing, light culture 10d at rotating speed 130rpm, temperature 26 DEG C.
The principle of the present invention is: the Radix Rehmanniae hairy root processed through salicylic acid can improve within the short time (12～24h)
The expression of verbascoside biosynthesis key gene, such as PAL (PAL), 4-coumaric acyl-CoA ligase
(4CL), cinnamic acid hydroxylase (C4H), coumaric acid hydroxylase (C3H), copper amino oxidase (CuAO) and tyrosine decarboxylase
(TyDC), thus complete the accumulation of verbascoside.
Described Radix Rehmanniae hairy root can use following steps induction to obtain: takes the leaf tissue of bosom Radix Rehmanniae aseptic seedling, is placed in use
Contaminate in the Agrobacterium rhizogenes bacterium solution of culture medium activation and contaminate, co-culture after taking-up, then screening and culturing, until growing hairy root.
Described Agrobacterium rhizogenes can be selected for the conventional bacterial strain such as ACCC10060, ATCC15834, K599, C58C1.
Described dip-dye culture medium can use MS, WPM, N6 or B5 fluid medium containing 100 μm ol/L acetosyringones.Leaching
Dye condition is: cultivate 5～15min at temperature 24～26 DEG C；It is preferably: at 26 DEG C, cultivate 8min.
Described co-culture MS, WPM, N6 or B5 solid medium that can use containing 100 μm ol/L acetosyringones.Co-culture
Condition is: light culture 2～3d at temperature 24～26 DEG C；It is preferably: light culture 2d at 26 DEG C.
Described screening and culturing uses containing 500mg/L cephamycin and MS, WPM, N6 or B5 of 100 μm ol/L acetosyringones
Solid medium.The condition of screening and culturing is: light culture at temperature 24～26 DEG C, every 9～11d subculture 1 time；It is preferably: 26
Light culture at DEG C, every 10d subculture 1 time.
The first cultivation of described Radix Rehmanniae hairy root is: takes the Radix Rehmanniae hairy root (inducing) of 4～6 root length degree 2～3cm, connects
Under rotating speed 100～150rpm, temperature 24～26 DEG C, dark condition, 20～25d are cultivated after Zhong,.
The culture medium of described inoculation is MS, WPM, N6 or B5 fluid medium.Preferably, in rotating speed 130rpm, temperature 26
Light culture 20d at DEG C.
Beneficial effects of the present invention:
The present invention utilizes Induced by Salicylic Acid Radix Rehmanniae hairy root to produce verbascoside, can significantly improve the product of verbascoside
Amount, and easy and simple to handle, cultivation cycle is short, it is simple to carry out industrialization, scale and Standardization Practice, to advance verbascoside big
Large-scale production is significant.Test shows, adds 10 μm ol/L, 25 μm ol/L, 40 μm ol/L salicylic acid can make Herba Verbasci Thapsi
Glycosides Contents brings up to 0.899%, 1.166% and 0.643% from 0.511%, wherein to add the 25 salicylic effects of μm ol/L
Fruit is optimal, and in hairy root, verbascoside content is 2.28 times of blank.
Accompanying drawing explanation
Fig. 1 is the induction of Radix Rehmanniae hairy root, qualification and suspension culture figure；
Fig. 2 is to add salicylic acid to Radix Rehmanniae hairy root Biomass and the impact of verbascoside content；
Fig. 3 is the salicylic acid impact on induction Radix Rehmanniae hairy root verbascoside biosynthetic catalyzing enzyme gene expression.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
A kind of method utilizing Radix Rehmanniae hairy root to produce verbascoside, comprises the following steps:
1) induction of Radix Rehmanniae hairy root and qualification
A. the induction of Radix Rehmanniae hairy root
In superclean bench, the blade of bosom Radix Rehmanniae aseptic seedling is cut into about 1cm2Fritter, be placed in the root of hair agriculture activated
In the dip-dye culture medium (MS fluid medium+100 μm ol/L acetosyringone) of bacillus ACCC 10060, take out after 10min, and
Aseptic filter paper blots bacterium solution；Blade is inverted in and co-cultures solid medium (MS solid medium+100 μm ol/L acetyl
Syringone) in, in 26 DEG C, co-culture 2d under dark condition；After aseptic water washing, then by blade inoculation in screening culture medium (MS
Solid medium+500mg/L cephamycin+100 μm ol/L acetosyringone) in, 26 DEG C of light culture, every 10d subculture 1 time, directly
To growing hairy root (see Fig. 1 a, b).
Repeat for 3 times using the blade do not contaminated by bacterium solution as comparison, every kind of process.
B. the PCR of Radix Rehmanniae hairy root identifies
Use CTAB method to extract the genomic DNA of 5 the Radix Rehmanniae hairy root strains randomly selected, utilize specific primer
(being respectively directed to rolB, rolC gene) carries out standard PCR amplification, and amplified production detects with 1% agarose gel electrophoresis, at gel
Observation analysis taking pictures under imaging system.
Specific primer for rolB gene is:
F:5 '-GCTCTTGCAGTGCTAGATTT-3 ',
R:5 '-GAAGGTGCAAGCTACCTCTC-3 '；
Specific primer for rolC gene is:
F:5 '-CTCCTGACATCAAACTCGTC-3 ',
R:5 '-TGCTTCGAGTTATGGGTACA-3 '.
Fig. 1 c shows, 5 hairy root strains (1～5#) all can amplify purpose fragment, the T-DNA district of Ri plasmid has been described
Being incorporated into Radix Rehmanniae genome, hairy root is the positive.
2) the first cultivation of Radix Rehmanniae hairy root
The Radix Rehmanniae hairy root of degerming 5 clean root length degree about 2.5cm is inoculated in 50mL MS fluid medium, is turning
Speed 130rpm, temperature 26 DEG C, dark condition low suspension cultivate 20d, and the Radix Rehmanniae hairy root after cultivation is shown in Fig. 1 d.
3) Induced by Salicylic Acid is utilized to produce verbascoside
Adding salicylic acid in culture fluid makes its concentration be 25 μm ol/L, gathers in the crops (see Fig. 1 e, f) after continuing to cultivate 10d.
Using without salicylic hairy root as comparison, increase Determination of Salicylic Acid 10 μm ol/L, 40 μm ol/L simultaneously
Process group, measures the content of verbascoside in the hairy root dry weight and hairy root gathered in the crops, and result is shown in Fig. 2.
In other embodiments of the invention, can use in WPM culture medium, N6 culture medium, B5 medium alternative embodiment 1
MS culture medium.
In Radix Rehmanniae hairy root, the assay method of verbascoside content is:
A. the preparation of reference substance solution
Weigh verbascoside reference substance 0.25mg, add 25mL acetonitrile-0.1% acetum (16:84)), constant volume, stand
Standby.
B. the mensuration of verbascoside
Precision weighs hairy root that 0.4g dries to constant weight in tool plug conical flask, and the accurate methanol 25mL that adds is weighed heavy
Amount, in 60 DEG C of heating and refluxing extraction 1.5h, lets cool, more weighed weight, supplies the weight of less loss with methanol, shakes up, and filters, accurate
Measuring subsequent filtrate 10mL in evaporating dish, 60 DEG C are concentrated near dry, and residue acetonitrile-0.1% acetum (16:84) dissolves,
It is transferred in 5mL measuring bottle, and is diluted to scale with acetonitrile-0.1% acetum (16:84), shake up, with 22 μm membrane filtrations,
Stand for standby use.Employing high performance liquid chromatography detects, and with octadecylsilane chemically bonded silica as filler, acetonitrile-0.1% acetic acid is molten
Liquid (16:84) is flowing phase, detects wavelength 334nm.The peak area of record verbascoside, substitutes into equation of linear regression and (i.e. marks
Directrix curve) in, it is calculated the content of verbascoside.
Result shows, adds 10 μm ol/L, 25 μm ol/L salicylic hairy root dry weight has increase trend, but the most aobvious
Work level, and add 40 μm ol/L salicylic hairy root dry weight and significantly reduce.Meanwhile, add 10 μm ol/L, 25 μm ol/L, 40
In the salicylic hairy root of μm ol/L, the content of verbascoside all dramatically increases, content be respectively comparison 175.9%,
228.2% and 125.8%, the best results wherein processed with 25 μm ol/L salicylic acid.
MS fluid medium, the formula of solid medium see table 1.
Table 1 MS culture medium prescription
Test example
The expression analysis of the catalyzing enzyme gene of participation verbascoside synthesis in Radix Rehmanniae hairy root:
A. the liquid culture of Radix Rehmanniae hairy root
Take the Radix Rehmanniae hairy root (with embodiment 1) of suspension culture 20d, add salicylic acid extremely final concentration of 25 μm ol/L, point
Not gathering in the crops hairy root at 0h, 3h, 9h, 12h and the 24h cultivated, fresh sample quick-freezing in liquid nitrogen of hairy root is placed on-80 DEG C of refrigerators
Middle preservation, for the extraction of RNA.
B. the qRT-PCR detection of catalyzing enzyme gene in hairy root
Base area verbascum thapsiformis Schraeder glucosides biosynthesis pathway catalyzing enzyme gene (PAL, 4CL, C4H, C3H, CuAO, TyDC)
Coded sequence design special primer (see table 2), carries out qRT-PCR detection with TIP41 gene for internal reference.
The primer of table 2 qRT-PCR
QRT-PCR assay kit isPremix Ex TaqTMII (Tli RNaseH Plus) (Takara, greatly
Even).PCR amplification system:Premix Ex Taq 12.5 μ l, Forward Primer (10 μm ol/L) 1 μ L,
Reverse Primer (10 μm ol/L) 1 μ L, cDNA template 2.0 μ L, ddH2O 8.5 μ L, altogether 25 μ L.PCR reaction condition: 95
℃30s；95 DEG C of 5s, 60 DEG C of 30s, 40 circulations.With 2-ΔΔCtCalculate the relative expression quantity of gene.
Result (Fig. 3) shows, 6 catalyzing enzyme gene expressions after salicylic acid processes are significantly increased, and at salicylic acid
Reach the highest after cultivating 12h.Illustrate that salicylic acid participates in the catalyzing enzyme gene of verbascoside biosynthesis pathway by induction
Express the accumulation promoting verbascoside.
Claims (10)
1. one kind utilizes the method that Radix Rehmanniae hairy root produces verbascoside, it is characterised in that: comprise the following steps: by Radix Rehmanniae hair
Shape root is placed in inducing culture in salicylated culture fluid,.
Method the most according to claim 1, it is characterised in that: in described culture fluid, salicylic concentration is 10～40 μ
mol/L。
Method the most according to claim 2, it is characterised in that: the base fluid of described culture fluid uses MS, WPM, N6 or B5 liquid
Body culture medium.
Method the most according to claim 3, it is characterised in that: containing mass concentration 20～40g/L in described culture fluid
Sucrose.
Method the most according to claim 4, it is characterised in that: described inducing culture is: in rotating speed 100～150rpm, temperature
Spend 24～26 DEG C, cultivate 5～10d under dark condition.
Method the most according to claim 1, it is characterised in that: described Radix Rehmanniae hairy root uses following steps induction to obtain:
Take the leaf tissue of bosom Radix Rehmanniae aseptic seedling, be placed in contaminating dip-dye in the Agrobacterium rhizogenes bacterium solution that culture medium activates, after taking-up altogether
Cultivate, then screening and culturing, until growing hairy root.
Method the most according to claim 6, it is characterised in that: described dip-dye culture medium, co-culture and be respectively adopted containing 100 μ
MS, WPM, N6 or B5 solid medium of mol/L acetosyringone.
Method the most according to claim 7, it is characterised in that: described screening and culturing use containing 500mg/L cephamycin and
MS, WPM, N6 or B5 solid medium of 100 μm ol/L acetosyringones.
Method the most according to claim 8, it is characterised in that: the condition of described screening and culturing is: in temperature 24～26 DEG C
Lower light culture, every 9～11d subculture 1 time.
Method the most according to claim 9, it is characterised in that: take the Radix Rehmanniae hairy root induced of length 2～3cm, connect
Under rotating speed 100～150rpm, temperature 24～26 DEG C, dark condition, cultivate 20～25d after Zhong, Induced by Salicylic Acid training can be carried out
Support.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486806A (en) * | 2018-12-27 | 2019-03-19 | 三明学院 | A kind of phenylalanine lyase and its encoding gene, recombinant vector, recombination engineering and application of anoectochilus formosanus |
| CN109679991A (en) * | 2019-01-18 | 2019-04-26 | 马鞍山师范高等专科学校 | A kind of transgenic plant and production method that benzyl carbinol glycosides content improves |
| CN112315831A (en) * | 2020-11-18 | 2021-02-05 | 熊林 | Anti-radiation whitening and moisturizing face cream and preparation method thereof |
| CN113388621A (en) * | 2021-07-09 | 2021-09-14 | 河南农业大学 | Rehmannia WRKY transcription factor RgWRKY37 gene and application thereof |
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Cited By (8)
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| CN113388621A (en) * | 2021-07-09 | 2021-09-14 | 河南农业大学 | Rehmannia WRKY transcription factor RgWRKY37 gene and application thereof |
| CN113388621B (en) * | 2021-07-09 | 2023-06-16 | 河南农业大学 | Rehmannia WRKY transcription factor RgWRKY37 gene and application thereof |
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