CN106148453B - A method of utilizing hairy production acteoside of glutinous rehmannia - Google Patents
A method of utilizing hairy production acteoside of glutinous rehmannia Download PDFInfo
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- CN106148453B CN106148453B CN201610554590.1A CN201610554590A CN106148453B CN 106148453 B CN106148453 B CN 106148453B CN 201610554590 A CN201610554590 A CN 201610554590A CN 106148453 B CN106148453 B CN 106148453B
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Abstract
The invention discloses a kind of methods for producing acteoside using glutinous rehmannia hairy, belong to technical field of biological culture.Method includes the following steps: being placed in Fiber differentiation in salicylated culture solution for glutinous rehmannia hairy.Method And Principle are as follows: hairy of the glutinous rehmannia handled through salicylic acid can improve the expression of acteoside biosynthesis key gene in short time (12~for 24 hours), such as phenylalanine lyase (PAL), 4- coumaric acyl-CoA ligase (4CL), cinnamic acid hydroxylase (C4H), coumaric acid hydroxylase (C3H), copper amine oxidase (CuAO) and tyrosine decarboxylase (TyDC), to complete the accumulation of acteoside, and further increase its yield, this method is easy to operate, cultivation cycle is short, it is convenient for industrialization, scale and Standardization Practice, it is of great significance to promoting acteoside to be mass produced.
Description
Technical field
The present invention relates to a kind of methods for producing acteoside using glutinous rehmannia hairy, belong to biological culture technigue neck
Domain.
Background technique
Glutinous rehmannia (Rehmannia glutinosa L.) is Scrophulariaceae herbaceos perennial, is used as medicine with root tuber, is China
One of famous " four-Huai medicine ", dosage is big, curative for effect, has important medical value and economic value, large to commonly use
Medicinal material is one of the big kind of the modern big Chinese medicine industrial chain formation keypoint recommendation research and utilization in China.
Acteoside is one of index components of glutinous rehmannia, has anti-oxidant, immunological regulation, anti-inflammatory, liver protection, enhancing memory
The bioactivity such as power, antitumor, toxic side effect are smaller.The content of acteoside is lower in glutinous rehmannia root tuber, " Chinese Pharmacopoeia " rule
The content for determining acteoside in radix rehmanniae recen must not be lower than 0.02%.And actual conditions are, verbascose between different glutinous rehmannia kinds
The content difference of glycosides is obvious, even same breed, in different regions, plantation also shows content difference, and range of variation compared with
Greatly, this results in the problems such as glutinous rehmannia quality is irregular, and acteoside extracts and application is limited.It is obtained using genetic engineering technology
The glutinous rehmannia kind for obtaining high acteoside content is a potential effective solution method, however so far, related glutinous rehmannia hair
The molecular genetic mechanism of stamen flower glucosides biosynthesis is unclear.
Studies have shown that glutinous rehmannia hairy can generate secondary metabolite identical with former plant, as can utilizing hairy
Acteoside is produced, a kind of new approach will be provided to obtain glutinous rehmannia acteoside.
Summary of the invention
The object of the present invention is to provide a kind of methods using hairy mass production acteoside of glutinous rehmannia.
In order to achieve the goal above, the technical scheme adopted by the invention is that:
A method of utilizing hairy production acteoside of glutinous rehmannia, comprising the following steps: be placed in glutinous rehmannia hairy
Fiber differentiation in salicylated culture solution.
Salicylic concentration is 10~40 μm of ol/L in the culture solution.Preferably, Determination of Salicylic Acid is 25 μm of ol/L.
The base fluid of the culture solution can be used in the MS culture medium of liquid, WPM culture medium, N6 culture medium, B5 medium etc.
Any one." the practical Plant Tissue Breeding skill that MS, WPM, N6, B5 fluid nutrient medium are edited using Cao Zi justice and Liu Guomin
Art study course " disclosed in be formulated.In addition, in culture solution can also containing the sucrose of 20~40g/L of mass concentration, as carbon source and
Energy substance.
The Fiber differentiation are as follows: 5~10d is cultivated under 100~150rpm of revolving speed, 24~26 DEG C of temperature, dark condition.It is excellent
Choosing, the dark culture 10d at revolving speed 130rpm, 26 DEG C of temperature.
The principle of the invention lies in: hairy of the glutinous rehmannia handled through salicylic acid can improve in short time (12~for 24 hours)
The expression of acteoside biosynthesis key gene, such as phenylalanine lyase (PAL), 4- coumaric acyl-CoA ligase
(4CL), cinnamic acid hydroxylase (C4H), coumaric acid hydroxylase (C3H), copper amine oxidase (CuAO) and tyrosine decarboxylase
(TyDC), to complete the accumulation of acteoside.
Hairy of the glutinous rehmannia can be used following steps and induce to obtain: taking the leaf tissue of bosom glutinous rehmannia aseptic seedling, is placed in use
It disseminates, is co-cultured after taking-up, then screening and culturing, until growing hairy in the agrobacterium rhizogenes bacterium solution of dip dyeing culture medium activation.
The common bacterial strains such as ACCC10060, ATCC15834, K599, C58C1 can be selected in the agrobacterium rhizogenes.
MS, WPM, N6 or B5 fluid nutrient medium containing 100 μm of ol/L acetosyringones can be used in the dip dyeing culture medium.Leaching
Dye condition are as follows: 5~15min is cultivated at 24~26 DEG C of Yu Wendu；It is preferred that are as follows: 8min is cultivated at 26 DEG C.
MS, WPM, N6 or B5 solid medium containing 100 μm of ol/L acetosyringones can be used in the co-cultivation.It co-cultures
Condition are as follows: 2~3d of dark culture at 24~26 DEG C of Yu Wendu；It is preferred that are as follows: dark culture 2d at 26 DEG C.
The screening and culturing uses MS, WPM, N6 or B5 of cephalosporin containing 500mg/L and 100 μm of ol/L acetosyringones
Solid medium.The condition of screening and culturing are as follows: dark culture at 24~26 DEG C of Yu Wendu, every 9~11d subculture 1 time；It is preferred that are as follows: 26
Dark culture at DEG C, every 10d subculture 1 time.
The first culture of hairy of the glutinous rehmannia are as follows: take glutinous rehmannia hairy (inducing) of 4~6 2~3cm of root long degree, connect
20~25d is cultivated after kind under 100~150rpm of revolving speed, 24~26 DEG C of temperature, dark condition.
The culture medium of the inoculation is MS, WPM, N6 or B5 fluid nutrient medium.Preferably, in revolving speed 130rpm, temperature 26
Dark culture 20d at DEG C.
Beneficial effects of the present invention:
The present invention utilizes hairy production acteoside of Induced by Salicylic Acid glutinous rehmannia, can significantly improve the production of acteoside
Amount, and it is easy to operate, cultivation cycle is short, be convenient for industrialization, scale and Standardization Practice, to promote acteoside it is big
Large-scale production is of great significance.Experiments have shown that feltwort can be made by adding 10 μm of ol/L, 25 μm of ol/L, 40 μm of ol/L salicylic acids
Glycosides Contents are increased to 0.899%, 1.166% and 0.643% from 0.511%, wherein to add 25 μm of salicylic effects of ol/L
Fruit is best, and hairy middle acteoside content is 2.28 times of blank control.
Detailed description of the invention
Induction, identification and the culture figure that suspends that Fig. 1 is glutinous rehmannia hairy；
Fig. 2 is the influence for adding salicylic acid to glutinous rehmannia hairy root biomass and acteoside content；
Fig. 3 is influence of the salicylic acid to the catalysis enzyme gene expression of induction hairy acteoside biosynthesis of glutinous rehmannia.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
A method of utilizing hairy production acteoside of glutinous rehmannia, comprising the following steps:
1) glutinous rehmannia hairy induction and identification
A. glutinous rehmannia hairy induction
The blade for cherishing glutinous rehmannia aseptic seedling is cut into about 1cm in superclean bench2Fritter, be placed in activated root of hair agriculture
In the dip dyeing culture medium (+100 μm of ol/L acetosyringones of MS fluid nutrient medium) of bacillus ACCC 10060, taken out after 10min, and
Bacterium solution is blotted on aseptic filter paper；Blade is inverted in and co-cultures solid medium (+100 μm of ol/L acetyl of MS solid medium
Syringone) in, 2d is co-cultured under 26 DEG C, dark condition；After aseptic water washing, then by blade inoculation in screening and culturing medium (MS
+ 100 μm of ol/L acetosyringones of solid medium+500mg/L cephalosporin) in, 26 DEG C of dark cultures, every 10d subculture 1 time, directly
To growing hairy (see Fig. 1 a, b).
Using not by bacterium solution disseminate blade as compare, 3 repetitions of every kind of processing.
B. glutinous rehmannia hairy PCR identification
The genomic DNA that the hairy stock system of 5 glutinous rehmannia randomly selected is extracted using CTAB method, utilizes specific primer
(being directed to rolB, rolC gene respectively) carries out standard PCR amplification, and amplified production is detected with 1% agarose gel electrophoresis, in gel
It observation analysis and takes pictures under imaging system.
For the specific primer of rolB gene are as follows:
F:5 '-GCTCTTGCAGTGCTAGATTT-3 ',
R:5 '-GAAGGTGCAAGCTACCTCTC-3 '；
For the specific primer of rolC gene are as follows:
F:5 '-CTCCTGACATCAAACTCGTC-3 ',
R:5 '-TGCTTCGAGTTATGGGTACA-3 '.
Fig. 1 c shows that 5 hairy stock systems (1~5#) can amplify target fragment, has illustrated the area T-DNA of Ri plasmid
It is integrated into glutinous rehmannia genome, hairy is the positive.
2) glutinous rehmannia hairy first culture
Glutinous rehmannia hairy of 5 clean root long degree about 2.5cm of degerming is inoculated in 50mL MS fluid nutrient medium, is being turned
Suspend culture 20d under fast 130rpm, 26 DEG C of temperature, dark condition, and glutinous rehmannia hairy after culture is seen Fig. 1 d.
3) acteoside is produced using Induced by Salicylic Acid
Salicylic acid is added in culture solution makes 25 μm of ol/L of its concentration, continues to harvest (see Fig. 1 e, f) after cultivating 10d.
Not add salicylic hairy as control, while increasing by 10 μm of ol/L of Determination of Salicylic Acid, 40 μm of ol/L
Processing group measures the hairy root dry weight of harvest and the content of hairy middle acteoside, as a result sees Fig. 2.
In other embodiments of the invention, WPM culture medium, N6 culture medium can be used, in B5 medium alternative embodiment 1
MS culture medium.
The measuring method of glutinous rehmannia hairy middle acteoside content are as follows:
A. the preparation of reference substance solution
Acteoside reference substance 0.25mg is weighed, -0.1% acetum of 25mL acetonitrile (16:84) is added), constant volume is stood
It is spare.
B. the measurement of acteoside
Precision weigh 0.4g drying to constant weight hairy in stuffed conical flask, methanol 25mL is added in precision, weighed heavy
Amount, in 60 DEG C of heating and refluxing extraction 1.5h, lets cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, and filters, accurate
Subsequent filtrate 10mL is measured in evaporating dish, 60 DEG C are concentrated into close dry, residue -0.1% acetum of acetonitrile (16:84) dissolution,
It is transferred in 5mL measuring bottle, and is diluted to scale with -0.1% acetum of acetonitrile (16:84), shake up, with 22 μm of membrane filtrations,
Stand for standby use.Using high performance liquid chromatography detection, using octadecylsilane chemically bonded silica as filler, -0.1% acetic acid of acetonitrile is molten
Liquid (16:84) is mobile phase, Detection wavelength 334nm.The peak area of acteoside is recorded, equation of linear regression is substituted into and (marks
Directrix curve) in, the content of acteoside is calculated.
The result shows that adding 10 μm of ol/L, the salicylic hairy root dry weight of 25 μm of ol/L has increase trend, but not up to aobvious
Level is write, and adding the salicylic hairy root dry weight of 40 μm of ol/L significantly reduces.Meanwhile adding 10 μm of ol/L, 25 μm of ol/L, 40
The content of μm salicylic hairy middle acteoside of ol/L dramatically increases, content be respectively 175.9% compareed,
228.2% and 125.8%, wherein best with the effect that 25 μm of ol/L salicylic acids are handled.
MS fluid nutrient medium, solid medium formula see the table below 1.
1 MS culture medium prescription of table
Test example
The expression analysis of the catalysis enzyme gene of acteoside synthesis is participated in glutinous rehmannia hairy:
A. glutinous rehmannia hairy Liquid Culture
Glutinous rehmannia hairy (with embodiment 1) of the culture 20d that suspends is taken, addition salicylic acid to final concentration of 25 μm of ol/L divides
Hairy is not harvested in 0h, 3h, 9h, 12h of culture and for 24 hours, hairy fresh sample is quick-frozen in liquid nitrogen to be placed on -80 DEG C of refrigerators
Middle preservation, the extraction for RNA.
B. the qRT-PCR detection of enzyme gene is catalyzed in hairy
Base area verbascum thapsiformis Schraeder glucosides biosynthesis pathway is catalyzed enzyme gene (PAL, 4CL, C4H, C3H, CuAO, TyDC's)
Coded sequence designs special primer (see the table below 2), carries out qRT-PCR detection by internal reference of TIP41 gene.
The primer of 2 qRT-PCR of table
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 of ol/L) 1 μ L,
Reverse Primer (10 μm of ol/L) 1 μ L, cDNA template 2.0 μ L, ddH28.5 μ L of O amounts to 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.
As a result (Fig. 3) is shown, 6 catalysis enzyme genes expression quantity after salicylic acid processing is significantly increased, and in salicylic acid
Reach highest after culture 12h.Illustrate that salicylic acid passes through the catalysis enzyme gene of induction participation acteoside biosynthesis pathway
Expression promotes the accumulation of acteoside.
Claims (8)
1. a kind of method for producing acteoside using glutinous rehmannia hairy, it is characterised in that: the following steps are included: by glutinous rehmannia hair
Shape root is placed in Fiber differentiation in salicylated culture solution；
Salicylic concentration is 10 ~ 40 μm of ol/L in the culture solution；
The Fiber differentiation are as follows: 5 ~ 10d is cultivated under 100 ~ 150rpm of revolving speed, 24 ~ 26 DEG C of temperature, dark condition.
2. according to the method described in claim 1, it is characterized by: the base fluid of the culture solution uses MS, WPM, N6 or B5 liquid
Body culture medium.
3. according to the method described in claim 2, it is characterized by: containing the sugarcane of 20 ~ 40g/L of mass concentration in the culture solution
Sugar.
4. according to the method described in claim 1, it is characterized by: the glutinous rehmannia hairy induces to obtain using following steps:
The leaf tissue for taking bosom glutinous rehmannia aseptic seedling is placed in and is disseminated in the agrobacterium rhizogenes bacterium solution of dip dyeing culture medium activation, after taking-up altogether
Culture, then screening and culturing, until growing hairy.
5. according to the method described in claim 4, it is characterized by: the dip dyeing culture medium, co-cultivation are respectively adopted containing 100 μ
MS, WPM, N6 or B5 solid medium of mol/L acetosyringone.
6. according to the method described in claim 5, it is characterized by: the screening and culturing using cephalosporin containing 500mg/L with
MS, WPM, N6 or B5 solid medium of 100 μm of ol/L acetosyringones.
7. according to the method described in claim 6, it is characterized by: the condition of the screening and culturing are as follows: at 24 ~ 26 DEG C of Yu Wendu
Dark culture, every 9 ~ 11d subculture 1 time.
8. according to the method described in claim 7, being inoculated with it is characterized by: take the glutinous rehmannia induced hairy of 2 ~ 3cm of length
20 ~ 25d is cultivated under 100 ~ 150rpm of revolving speed, 24 ~ 26 DEG C of temperature, dark condition afterwards, Induced by Salicylic Acid culture can be carried out.
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| CN109486806B (en) * | 2018-12-27 | 2022-03-01 | 三明学院 | Phenylalanine ammonia lyase of anoectochilus formosanus, and coding gene, recombinant vector, recombinant engineering bacterium and application thereof |
| CN109679991B (en) * | 2019-01-18 | 2020-11-03 | 马鞍山师范高等专科学校 | Transgenic plant with increased phenylethanoid glycoside content and production method thereof |
| CN112315831B (en) * | 2020-11-18 | 2021-09-10 | 广州市万千粉丝化妆品有限公司 | Anti-radiation whitening and moisturizing face cream and preparation method 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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| WO2002072110A1 (en) * | 2001-03-09 | 2002-09-19 | Aventis Pharma Deutschland Gmbh | Caloporoside derivatives, methods for the production thereof and their use |
| CN1709835A (en) * | 2005-05-03 | 2005-12-21 | 曲沃县万乡红肥业有限公司 | Glutinous rehmannia anti-continous-cropping yield-increasing agent and its preparing method |
| CN101182542A (en) * | 2007-11-27 | 2008-05-21 | 西南大学 | Method for genetic transforming scutellaria viscidula to obtain hairy roots producing baicalin by agrobacteriitm rhizogenes |
| CN104380954A (en) * | 2014-10-23 | 2015-03-04 | 周琪 | Seedling raising method of rehmannia chingii |
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| WO2002072110A1 (en) * | 2001-03-09 | 2002-09-19 | Aventis Pharma Deutschland Gmbh | Caloporoside derivatives, methods for the production thereof and their use |
| CN1709835A (en) * | 2005-05-03 | 2005-12-21 | 曲沃县万乡红肥业有限公司 | Glutinous rehmannia anti-continous-cropping yield-increasing agent and its preparing method |
| CN101182542A (en) * | 2007-11-27 | 2008-05-21 | 西南大学 | Method for genetic transforming scutellaria viscidula to obtain hairy roots producing baicalin by agrobacteriitm rhizogenes |
| CN104380954A (en) * | 2014-10-23 | 2015-03-04 | 周琪 | Seedling raising method of rehmannia chingii |
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