CN104372034B - A kind of method that hairy roots of giant knotweed produces resveratrol and expansion culture - Google Patents
A kind of method that hairy roots of giant knotweed produces resveratrol and expansion culture Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, it is related to a kind of induction of hairy roots of giant knotweed and expands the method for culture, specially a kind of hairy roots of giant knotweed production resveratrol and the method for expanding culture using bioreactor.The present invention is using the wild stem with bud of polygonum cuspidate plant as explant, successfully induce polygonum cuspidate tissue-cultured seedling, and establish polygonum cuspidate tissue culturing system, and successfully induce hairy roots of giant knotweed system, and establish hairy roots of giant knotweed production resveratrol system, successfully passing airlift bioreactor makes hairy roots of giant knotweed produce resveratrol, and has carried out assay and extracted to separate to resveratrol.Resveratrol, which is produced, by hairy roots of giant knotweed has the characteristic of the fast-growth on no hormone culture-medium compared with production method now.Therefore this method is easy to operate, cost is relatively low, not the limitation of the natural conditions such as climate, soil the advantages that, for from now on progress industrialized production provide reliably source and material base.
Description
Technical field
The invention belongs to field of biotechnology, are related to the induction and utilization bioreactor expansion of a kind of hairy roots of giant knotweed
The method cultivated greatly, specially a kind of hairy roots of giant knotweed production resveratrol and the method for expanding culture.
Background technique
Resveratrol (Resveratrol, 3,4 ', 5-trihydroxy-trans-stilbene) i.e. trans- 3,4 ', 5- tri-
Hydroxyl Stilbene is diphenylethylene compounds, is present in polygonum cuspidate, grape, peanut etc. in the form of free state and two kinds of glucosides reference state
In various plants, wherein the abundantest with content in polygonum cuspidate.It is a kind of important phytoalexin.Resveratrol has a variety of medicines
Reason activity, such as antibacterial, anti-oxidant, anticancer, anti-platelet aggregation, anti-HIV activity, can repair atypical pneumonia prescription
Caused DNA Damage, especially to the formidable enemy of human health -- tumour and cardiopathic treatment and prevention have apparent effect.
Polygonum cuspidate be polygonaceae polygonum cuspidate category herbaceos perennial, be China's traditional Chinese medicine, be distributed widely in East China, it is Central-South and
The ground such as Shaanxi, Gansu, Sichuan, Gentrin Knotweed P.E have an anti-inflammatory, a variety of pharmacological activity of prevention cardiovascular disease etc., chief active at
It is divided into resveratrol and rheum emodin.According to statistics, polygonum cuspidate amount of regeneration is 9.8*106Kg/, sustainable excavation amount are 4.95*
106Kg/, market demand 6.0*106Kg/.Therefore, the wild resource of polygonum cuspidate has faced the situation excessively excavated.And according to
Statistics extracts 1 ton of resveratrol and needs to consume 500 tons of polygonum cuspidate raw materials, but resveratrol is generally lower in plant in-vivo content.
For the imbalance between supply and demand for solving medicinal plant, the method that people mostly use artificial cultivation expands medicine source.But in Planting
In the medicinal plant of training, some medicinal plant production cycles are very long, if breeding or nursery in conventional manner, when needing to spend longer
Between.There are also some medicinal plants because breeding coefficient is small, consumption kind amount is big, leads to that development speed is very slow and the production cost increases.In addition
Some medicinal plants have seriously affected yield and quality then because virus harm causes to degenerate.For guarantee resources of medicinal plant can
Sustainable utilization carries out the key gene gram of cell or tissue culture, effective component using the approach of bioengineering to precious resources
The technologies such as grand and conversion, it may be possible to solve the effective way of industrialization of Chinese medicine.Then, the actively regeneration of research resources of medicinal plant
Technology keeps limited resource mankind sustainable utilization extremely urgent.And hairy technology has fast growing, is not required to foreign aid plant
Hormone, synthesis secondary metabolites ability is strong and stablize, not by season and time restriction the advantages that.And hairy of Ri plasmid conversion
Growth is fast, is easy to cultivate, and effective component is high, has and expresses complete metabolic pathway, is the industrialization of medicinal plant metabolite
Production provides bright prospects.
At present it is reported that polygonum cuspidate successfully induces hairy, the content of resveratrol in the inducement of hairy roots of giant knotweed such as Li Zhuoxi
For 8.21mg/kg；The factor of the discussions such as Yu Shuhong influence hairy roots of giant knotweed high-frequency induction, the results showed that mature leaf is conversion ratio
Higher explant, infection 10d or so can form hairy, and average inductivity is 55.66%, and luring root density is 4.30；And seedling
Dawn swallow etc. studies hairy roots of giant knotweed DNA extraction method, the results showed that modified CTAB method solve polygonum cuspidate rich in polyphenol,
The substances such as polysaccharide are difficult to the problem of extracting high quality DNA, and the DNA mass of extraction is preferable, and purity is higher.It and is brave in setting macro wait
Cane hairy prepares polygonin and has further applied for patent, Patent No. 200510012809.7, but be merely resting on reality at present
It tests the stage, does not there is the research in terms of further progress industrialization also.Currently, having report using hairy roots of giant knotweed production resveratrol
Road, but had not been reported in terms of carrying out hairy roots of giant knotweed production resveratrol industrialization using bioreactor.Therefore, tiger is utilized
Hairy production resveratrol of cane carries out industrialization especially by bioreactor, produces to medicinal plant secondary metabolism is solved
The effective way that the industrialized production of object resveratrol provides is of great significance to polygonum cuspidate current resources situation is solved.
Summary of the invention
The purpose of the present invention is to provide a kind of hairy roots of giant knotweed production resveratrol and expand the method cultivated, and passes through
Polygonum cuspidate tissue culture technique, hairy culture technique and bioreactor carry out mass production resveratrol, to substitute wild money
Source provides an effective new way for the production of resveratrol.
In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:
A kind of method that hairy roots of giant knotweed produces resveratrol and expansion culture, method includes the following steps:
(1) disinfection of explant 1
Field polygonum cuspidate stem with bud tap water flowing water is taken to rinse 3h, alcohol impregnates 60s, sterile water punching in superclean bench
It washes 3-5 times, 0.1% mercuric chloride impregnates 5-8min (depending on material always tender degree), and sterile water rushes 5-6 times, with the filter to sterilize
Paper suck dry moisture, it is spare.
(2) induction of polygonum cuspidate tissue-cultured seedling and squamous subculture
Explant 1 selected in step (1) is cut into 1.5cm stem with bud and is inoculated in MS+6-BA2.5mg/L+
NAA0.5mg/L continues squamous subculture after belt length to 30d, selects MS+6-BA0.3mg/L+NAA0.5mg/L+TDZ0.05mg/L,
Condition of culture: 25 ± 2 DEG C of temperature, using fluorescent lamp, illumination 1500-2000lux, light application time 14-16h.Every 25-30 days subcultures
Once, wait grow, healthy and strong, well-developed complete polygonum cuspidate tissue-cultured seedling is spare after subculture 2-3 times.
(3) induction of hairy roots of giant knotweed
1) preculture
By the tissue culture seedling leaf obtained in step (2) (band petiole, 1.0cm*1.0cm) or stem (1.5cm), it is forwarded to MS sky
Dark culture 2 days in white culture medium, 25 ± 2 DEG C of cultivation temperature.
2) activation of agrobacterium rhizogenes
By agrobacterium rhizogenes ATCC15834 (being presented by Guangdong Academy of Forestry's biotechnology research institute) in YEB
It being activated in solid medium, picking single bacterium colony is inoculated in YEB fluid nutrient medium, at 28 DEG C, 120rmin-1With dark item
Shaken cultivation 20h under part reaches growth logarithmic phase, and to infect polygonum cuspidate explant 2, measuring its OD value is 0.2-0.5.
3) it infects and co-cultures
Explant 2 (blade or stem) Jing Guo preculture is transferred in activated Agrobacterium bacteria suspension, is disseminated
10min.Then the bacterium solution that 2 excess surface of explant is drawn with aseptic filter paper, is inoculated in MS blank cultures+acetyl cloves
Ketone (AS) 20-50mg/L, co-cultures 2d under 25 DEG C of dark conditions.
4) degerming and optimization culture
The explant 2 for co-culturing 2 days in step 3) is started into degerming and is transferred to MS+ cephalo thiophene with aseptic water washing 3 times
(bacterium culture medium is removed) in oxime sodium (cefotaxime) 500mg/L culture medium, 25 DEG C, 14hd-1It scatters and carries out degerming training under light
It supports.Every 5d switching is primary, subculture degerming repeatedly, until culture medium occurs without bacterial plaque.The hairy roots of giant knotweed induced is continued
Continue subculture in 1/2MS blank solution culture medium to rise in value 2-3 times.
(4) hairy roots of giant knotweed PCR is detected
The hairy roots of giant knotweed obtained in step 4) is subjected to DNA extraction, obtains and contains RolB and tms Gene response system,
The PCR product of acquisition is through agarose gel electrophoresis and ultraviolet detection.The amplified band size of RolB is 422bp, and the size of tms is
910bp。
(5) root system of hairy roots of giant knotweed preferably and bioreactor expansion culture
Hairy will detected through PCR, and select that degerming is thorough, and branch is more, it grows fast hairy and is cut into long 1-
3cm is added 1/2MS blank solution culture medium in 1L airlift bioreactor and 3% sucrose, acid hydrolyzed casein 0-- is added
5g/L, aspergillus niger 0--100mg/L, L-phenylalanine 0--50mg/L, inoculum concentration are 30g hairy of weight in wet base, it is desirable that whole nothings
Bacterium, PH5.8-6.5, temperature: 25 ± 2 DEG C, dark culture, ventilatory capacity 0.05vvm claims when growth of hair root reaches logarithmic phase
Take weight in wet base, dry weight.
(6) in hairy roots of giant knotweed resveratrol HPLC assay
Content through resveratrol in HPLC content detection hairy roots of giant knotweed is shown into resveratrol in hairy roots of giant knotweed
Content is 210--280 μ g/g, and polydatin is 1500-2340 μ g/g.
(7) extraction separation and purification of resveratrol
Hairy roots of giant knotweed in step (5) dry, pulverize, add distilled water (pH=10) stirring, filtering, filter residue is soaked again
It mentions, filters, merging filtrate, tune PH is faintly acid.Crude product is extracted with ethyl acetate, carries out silica gel column layer after extract liquor concentration
Analysis.After the ethanol in proper amount repeated crystallization of the chromatographed product containing resveratrol, 50 DEG C of vacuum drying.It is sealed in bottle, is protected from light
It saves.
Heretofore described culture medium and technical term:
(1) it includes a large amount of members that MS minimal medium, which is (unit mg/L): MS (Murashige and Skoog, 1962),
Plain (ammonium nitrate 1650, potassium nitrate 1900, calcium chloride dihydrate 440, epsom salt 370, biphosphate 170) can be made into 20 times of mothers
Liquid takes mother liquor 50ml/L；Microelement (manganese sulfate monohydrate 22.3, white vitriol 8.6, CoCL2 6H2O 0.025, anhydrous sulphur
Sour copper 0.025, boric acid 6.2, Sodium Molybdate Dihydrate 0.25, potassium iodide 0.83) 200 times of mother liquor are made into, take mother liquor 5ml/L；Molysite (seven
Aqueous ferrous sulfate 28.7, disodium ethylene diamine tetraacetate 37.3) 200 times of mother liquor are made into, take mother liquor 5ml/L；Organic (niacin 0.5, cigarette
Sour pyridoxol 0.5, thiamine hydrochloride 0.1, glycine 2.0) 200 times of mother liquor are made into, mother liquor 5ml/L is taken, inositol 0.1g, sugarcane are separately added
Sugared 30g, solid medium add agar powder 6.5g, and 1/2MS fluid nutrient medium is that a great number of elements halves, but ammonium nitrate contains in the present invention
Measure constant, remaining is constant, PH5.8-6.5.
(2) YEB culture medium prescription (unit g/L): beef extract 5.0, yeast extract 1.0, peptone 5.0, sucrose
5.0, epsom salt 0.5, solid medium add agar powder 8g, PH7.0.
(3) antibiotic is cefotaxime na concn (Cefotaxime), kanamycins (kan), carbenicillin sodium Cb
(Carbenicillin)
(4) heretofore described hormone is 6-BA (6- benzylamino adenine), NAA (methyl α-naphthyl acetate), TDZ (Thidiazuron)
(5) heretofore described explant 1 is wild polygonum cuspidate stem with bud.
(6) heretofore described explant 2 is the blade or stem sheared in polygonum cuspidate tissue-cultured seedling.
(5) heretofore described dry weight refer to hairy roots of giant knotweed in drying box 70 DEG C of dryings to constant weight.
(6) heretofore described weight in wet base, which refers to, takes hairy root culturd, draws hairy root surface excessive moisture with filter paper
It blots, claims to obtain weight in wet base.
(7) heretofore described 3% sucrose refers to addition 30g sucrose in 1L culture medium.
(8) heretofore described hairy root induction rate
The hairy hairy number of root explant 2 of root induction rate=generation/total explant 2 number × 100%.
(9) measuring method of 1 proliferation times of polygonum cuspidate explant
Proliferation times=(weight-inoculum concentration when harvest)/inoculum concentration
Growth rate=(finally harvesting dry weight-inoculation dry weight)/inoculation dry weight
(10) heretofore described resveratrol determination of yield method
Resveratrol yield=harvest dry weight * Resveratrol content
Heretofore described bioreactor is circulation fermentation pot (Nanjing Tian Hui AIF-S-XX) in 1L gas-lifting type, using logical
The upper up-flow for entering the filtrated air formation of reactor is supplied oxygen and is stirred, and by the control to each parameter, stablizes height to reach
It produces, reduces polygonum cuspidate raw materials consumption etc., each parameter includes: temperature, pH value, air mass flow, speed of agitator, dissolved oxygen speed, foam
Highly, pressure etc..By the design to these experimental datas, provided for the production that hairy roots of giant knotweed is cultivated in subsequent amplification important
Reference frame.
Basic operation condition: fermentor being connected after being sterilized, and uniformly inoculation is carried out successively in superclean bench,
The volume of culture solution is 1L in reactor, and inoculum concentration is the 30g of weight in wet base, and inoculation post-fermentation tank, which is put into constant temperature incubation room, to carry out secretly
Culture, pH value 5.8-6.0, temperature are 25 ± 2 DEG C, ventilatory capacity 0.05vvm.During the cultivation process, according to the water of evaporation
It is timed moisturizing by peristaltic pump, keeps total volume constant.
The positive effect of the present invention is embodied in:
(1) in the present invention, by the large-scale production of bioreactor, provide polygonum cuspidate production resveratrol industrialization
One effective way has great applicability.
(2) present invention carries out in laboratory conditions, by hairy mass production resveratrol, with wild and cultivation
Training polygonum cuspidate is compared, and be can get stable quality and yield, is not limited by natural conditions and time, be not take up cultivated area, this
Sample has been saved the plenty of time, manpower.
(3) by adding elicitor, precursor etc. in bioreactor, hairy roots of giant knotweed secondary metabolism is greatly improved
Productive biomass improves the content of resveratrol and its glycosides.In the present invention, hairy yield can reach dry weight
In 15.32-21.65g/L/ periods (30-40 days), wherein resveratrol maximum output is up to 269.48 μ g/g, resveratrol and its
The yield of glycosides is up to 210--280 μ g/g and 1500-2340 μ g/g, and hairy appreciation rate is up to 40-60 times.It is the same terms
3-4 times, 10 times of natural root of lower common hairy.
(4) by artificial regulatory optimum culture condition, hairy growth and the synthesis of secondary metabolite, pole can be improved
The yield of resveratrol is improved greatly.
(5) cultivating hairy culture medium in the present invention not add any hormone, dark culture.Therefore life can be substantially reduced
Cost is produced, production efficiency is improved.
(6) the hairy roots of giant knotweed production cycle is generally 30 days to 40 days, than field grown time (the general growth cycle of polygonum cuspidate
For 3-5) want it is short very much.The time has been saved in this way, has saved polygonum cuspidate wild resource, and certain work is also played to environmental protection
With.
(7) present invention process is easy to operate, reproducible, and large-scale production facilitates rapidly.
Specific embodiment
The present invention is described in further detail With reference to embodiment.But this should not be interpreted as to the present invention
The range of above-mentioned theme is only limitted to following embodiments, all that model of the invention is belonged to based on the technology that the content of present invention is realized
It encloses.Polygonum cuspidate plant used is from Liangshan State of Sichuan Province Ganluo County in embodiment.
Embodiment 1:
(1) explant 1 sterilizes
Polygonum cuspidate tender stem segments are chosen as explant 1, carry out disinfection processing, and steps are as follows: stem with bud tap water flowing water
3h is rinsed, alcohol impregnates 60s in superclean bench, and aseptic water washing 5 times, 0.1% mercuric chloride impregnates 5-8min, sterile water punching 6
Time, it is spare with the filter paper suck dry moisture to sterilize.
(2) induction of bud and subculture increment
The explant 1 for bacterium of having gone out in step (1) is forwarded to bud inducement cultivation base: MS solid minimal medium+6-
In BA2.5mg/L+NAA0.5mg/L+ sucrose 30g/L, starts to grow bud point after 7d, grow up to intact plant after 30d.Continue subculture
Culture, subculture medium: MS solid minimal medium+6-BA2.5mg/L+NAA0.5mg/L+TDZ0.05mg/L+ sucrose 30g/
L, it is spare after length to well-developed complete polygonum cuspidate tissue-cultured seedling.Growth conditions: 25 ± 2 DEG C of temperature, using fluorescent lamp, illumination
1500-2000lux, light application time 14-16h.
(3) induction of hairy roots of giant knotweed
Polygonum cuspidate tissue culture seedling leaf used in step (2) (band petiole part) is taken, dark culture 2 in MS blank cultures is forwarded to
It, 25 ± 2 DEG C of cultivation temperature.
(4) activation of strain
Picking is stored in the agrobacterium rhizogenes ATCC15834 on 4 DEG C of YEB solid plate culture mediums, picking single bacterium colony in
In YEB fluid nutrient medium, at 28 DEG C, 120rmin-1With shaken cultivation 20h under dark condition, growth logarithmic phase is reached,
To infect polygonum cuspidate blade.Measuring its OD value is 0.4.
(5) it infects and co-cultures
Blade surface (1.0cm*1.0cm) will be carefully scratched with sterile scalpel by the blade co-cultured in step (3),
Step (4) is then transferred in activated strain bacteria suspension, 10min is infected, then draws 2 table of explant with aseptic filter paper
The extra bacterium solution in face is inoculated in MS blank cultures+AS20mg/L, co-cultures 2d under 25 DEG C of dark conditions.
(6) degerming and optimization culture
It by the polygonum cuspidate blade co-cultured, with aseptic water washing 3 times, will be transferred to except bacterium culture medium MS is solid in step (5)
In body minimal medium+cef500mg/L, 25 DEG C, 14hd-1It scatters and carries out degerming culture under light.Every 5d switching is primary, repeatedly
Subculture degerming, until culture medium occurs without bacterial plaque.Degerming started to grow hairy after 5 days, started to grow after 20d a large amount of hairy
Root.Will induce to degerming completely after hairy roots of giant knotweed continue in 1/2MS blank solution culture medium continue subculture increment 2-3
It is secondary.Growth of hair root feature: root is elongated, multiple-limb, and apogetropism is radial.
(7) the PCR detection of hairy roots of giant knotweed
The rapid grind into powder of liquid nitrogen is added in the hairy roots of giant knotweed and blade (control) that will be obtained in step (6), 0.2g,
It is transferred in the 4ml centrifuge tube of CTAB extracting solution and 10 μ l mercaptoethanols that 800 μ l65 DEG C preheating has been added, firmly mixes.From
Heart pipe warm bath lh under 65 DEG C of water-baths was gently reversed every 10 minutes mix therebetween, continued 1min or so every time.It is added isometric
Chloroform/isoamyl alcohol is the mixed liquor of 24:1, is gently mixed by inversion 10min.At room temperature, 10000r/min is centrifuged 10min, in shifting
Clear liquid is into new pipe.Isometric isopropanol is added, flocculent deposit occurs.It is reverse to shake up, assemble flocculent deposit, and
5000rpm is centrifuged 5min and collects precipitating.The dehydrated alcohol rinse of 200 μ l is added twice in precipitating, and 5000rpm centrifugation 5min pours out nothing
Water-ethanol places a few minutes, and after ethyl alcohol volatilization to the greatest extent, 100 μ l dissolving DNA of TE solution is added.
The PeR primer of the PeR detection of hairy rolB gene, Ri plasmid rolB gene is synthesized by the raw work in Shanghai, and upstream is drawn
Object: 5 ' GCTCTTGCAGTGCTAGATTT-3 '；Downstream primer P25 '-GAA GGT GCA AGC TAC CTCTC-3 ', in 25 μ
In l1PCR reaction solution, Golden Easy PCR system (Tiangeng is biochemical) 12.5 μ l, upper and lower each 1 μ l (final concentration of primer
20pmol/L), 2 μ l of template DNA supplies 25 μ l with ultrapure water.94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 1min, 57 DEG C are annealed
1min, 72 DEG C of extension 2min, 35 circulations.
Amplified production is analyzed in 1.7% agarose gel electrophoresis and ultraviolet detection, the results showed that PCR reaction can be with
The specific fragment to expected 422bp and 910bp or so is expanded from hairy roots of giant knotweed, shows the rolB in ATCC15834
The part Ri TL-DNA and TR-DNA of gene and tms gene has been integrated into hairy roots of giant knotweed.And it is disseminated without ATCC15834
Blade specific fragment is not detected.
(8) root system of hairy roots of giant knotweed preferably and bioreactor expansion culture
Hairy will be detected by step (7), and select that degerming is thorough, and branch is more, and grown fast hairy and be cut into length
+ 3% sucrose of 1/2MS blank solution culture medium+black song of acid hydrolyzed casein 1g/L+ is added in 1-3cm in 1L airlift fermentor
Mould 10mg/L+L- phenylalanine 15mg/L+NH4NO3825mg/L, inoculum concentration are the 30g of weight in wet base, and whole process is in superclean bench
Middle progress, it is desirable that all sterile, pH value 5.8-6.5, temperature are 25 ± 2 DEG C, dark culture, ventilatory capacity 0.05vvm.It is cultivating
In the process, moisturizing is timed by peristaltic pump according to the water of evaporation, keeps total volume constant.
Embodiment 2:
(1) with embodiment 1 (1)
(2) induction of bud and subculture increment
The stem section for bacterium of having gone out in step (1) is forwarded to bud inducement cultivation base: MS solid minimal medium+6-
In BA2.0mg/L+NAA0.5mg/L+ sucrose 30g/L, starts to grow bud point after 7d, grow up to intact plant after 30d.Continue subculture
Culture, subculture medium: MS solid minimal medium+6-BA0.5mg/L+IAA0.5mg/L+TDZ0.05mg/L+ sucrose 30g/
L, it is spare after length to well-developed complete polygonum cuspidate tissue-cultured seedling.Growth conditions: 25 ± 2 DEG C of temperature, using fluorescent lamp, illumination
1500-2000lux, light application time 14-16h.
(3) induction of hairy roots of giant knotweed
Polygonum cuspidate tissue-cultured seedling stem used in step (2) is taken, dark culture 2 days in MS blank cultures, cultivation temperature 25 ± 2 are forwarded to
℃。
(4) with embodiment 1 (4)
(5) it infects and co-cultures
Stem section (1.0cm*1.0cm) will be scratched by the stem sterile scalpel co-cultured in step (3), is then transferred to
Step (4) in activated strain bacteria suspension, infects 10min, and the bacterium solution of stem excess surface is then drawn with aseptic filter paper, will
It is inoculated in MS blank cultures+AS20mg/L, co-cultures 2d under 25 DEG C of dark conditions.
(6) degerming and optimization culture
It will be transferred to aseptic water washing 3 times except bacterium culture medium MS solid in step (5) by the polygonum cuspidate stem co-cultured
In minimal medium+kan500mg/L, 25 DEG C, 14hd-1It scatters and carries out degerming culture under light.Every 5d switching is primary, repeatedly after
For degerming, until culture medium occurs without bacterial plaque.Degerming started to grow hairy after 5 days, started to grow after 20d hairy a large amount of.
Continuation subculture increment 2-3 times in 1/2MS blank solution culture medium to hairy roots of giant knotweed continuation after degerming completely that will be induced.
Growth of hair root feature: root is elongated, multiple-limb, and apogetropism is radial.
(7) with embodiment 1 (7)
(8) root system of hairy roots of giant knotweed preferably and bioreactor expansion culture
Hairy will be detected by step (7), and select that degerming is thorough, and branch is more, and grown fast hairy and be cut into length
+ 3% sucrose of 1/2MS blank solution culture medium+black song of acid hydrolyzed casein 3g/L+ is added in 1-3cm in 1L airlift fermentor
Mould 30mg/L+NH4NO3825mg/L, inoculum concentration are 30g (weight in wet base), and whole process carries out in superclean bench, it is desirable that whole nothings
Bacterium, pH value 5.8-6., temperature are 25 ± 2 DEG C, dark culture, ventilatory capacity 0.05vvm.During the cultivation process, according to evaporation
Water is timed moisturizing by peristaltic pump, keeps total volume constant.
Embodiment 3:
(1) with embodiment 1 (1)
(2) induction of bud and subculture increment
By bacterium of having gone out in step (1)+be forwarded to bud inducement cultivation base: MS solid minimal medium+6-BA2.0mg/L
In+NAA0.5mg/L+ sucrose 30g/L, starts bud point occur after 7d, grow up to intact plant after 30d.Continue squamous subculture, subculture
Culture medium: MS solid minimal medium+6-BA2.0mg/L+NAA0.5mg/L+ sucrose 30g/L, to long to well-developed complete
It is spare after polygonum cuspidate tissue-cultured seedling.Growth conditions: 25 ± 2 DEG C of temperature, using fluorescent lamp, illumination 1500-2000lux, light application time
14-16h。
(3) (4) (5) are the same as 1 step of embodiment (3) (4) (5)
(6) degerming and optimization culture
Degerming culture will be transferred to aseptic water washing 3 times by the polygonum cuspidate tissue culture seedling leaf co-cultured in step (5)
In base MS solid minimal medium+cb300mg/L, 25 DEG C, 14hd-1It scatters and carries out degerming culture under light.Every 5d switching is primary,
Subculture degerming repeatedly, until culture medium occurs without bacterial plaque.Degerming started to grow hairy after 5 days, started to grow a large amount of hairs after 20d
Shape root.The complete hairy roots of giant knotweed of degerming is continued to continue subculture increment 2-3 times in 1/2MS blank solution culture medium.It is hairy
Root growth feature: root is elongated, multiple-limb, and apogetropism is radial.
(7) with the step (7) in embodiment 1
(8) root system of hairy roots of giant knotweed preferably and bioreactor expansion culture
Hairy will be detected by step (7), and select that degerming is thorough, and branch is more, and grown fast hairy and be cut into length
+ 3% sucrose of 1/2MS blank solution culture medium+black song of acid hydrolyzed casein 1g/L+ is added in 1-3cm in 1L airlift fermentor
Mould 30mg/L- phenylalanine 15mg/L+NH4NO3825mg/L, inoculum concentration are 30g (weight in wet base), and whole process is in superclean bench
It carries out, it is desirable that all sterile, PH5.8-6.5, temperature: 25 ± 2 DEG C, dark culture, ventilatory capacity 0.05vvm.In incubation
In, moisturizing is timed by peristaltic pump according to the water of evaporation, keeps total volume constant.
Embodiment 4
Resveratrol content measurement (HPLC measurement) in hairy roots of giant knotweed
(1) drafting of standard curve
Weighing resveratrol and its glycosides 2.5mg respectively, (reference substance comes from Beijing Century AudioCodes Bioisystech Co., Ltd, contains
98% or more amount), it is placed in 25ml volumetric flask, methanol solution dissolution is configured to compare containing resveratrol and its glycosides 0.10mg/ml
Product solution, it is accurate respectively to draw reference substance solution 1.0,2.0,3.0,4.0,5.0ml, it is placed in 10ml volumetric flask, methanol dilution
To scale, the reference substance solution of different solubility is made.
(2) preparation of test solution
Hairy obtained in any one of Example 1-3 embodiment, 70 DEG C of drying in electric drying oven with forced convection,
It is ground into a powder, takes hairy powder 0.1g, accurately weighed, Diluted Alcohol 25ml is added in precision, and weighed weight is heated to reflux 30min,
It is cooled to room temperature, then weighed weight, the weight of less loss is supplied with Diluted Alcohol, is shaken up, take supernatant, filter, take subsequent filtrate, i.e.,
?.
(3) chromatographic condition
Chromatographic column: Japanese Shimadzu CLC-ODS column (150mm*6mm, i.d.5 μm)；Mobile phase is acetonitrile: water=41:59, stream
Speed: 1.0ml/min, column temperature: 30 DEG C；Sample volume: 10 μ L, Detection wavelength: 303nm.Theoretical cam curve is not less than 3000, separating degree
Greater than 1.5.Respectively with the solution of the above different solubility, each 10 μ L of sample volume measures each peak area according to above-mentioned chromatographic condition.
Linear regression is carried out with peak area and content respectively.Integrating peak areas value Y is ordinate, and reference substance sample volume X draws for abscissa
Standard curve processed carries out linear regression.
(4) measuring method
It is close respectively to draw reference substance solution and each 10 μ l of test solution, inject in HPLC, measurement to get.Root
Obtaining Resveratrol content in hairy roots of giant knotweed according to testing result is 210--280 μ g/g, and polydatin is 1500-2340 μ g/
g。
Embodiment 5
The extraction separation and purification of resveratrol:
Hairy roots of giant knotweed in any one of embodiment 1-3 embodiment step (8) is dry, and it is crushed to certain particle size,
And the alkaline aqueous solution for adjusting pH value=10 in advance is added, it at the uniform velocity stirs and heats and boil 1h, filter while hot；In the same way
It extracts again primary.And filtrate is adjusted into PH=3 with 1mol/LHCL, it stands.Unchanged to filtrate PH, then precipitation of resveratrol is complete
Entirely, by precipitation and centrifugal separation, vacuum drying.
It will be extracted after the dissolution of above-mentioned crude product with ethyl acetate, after extract liquor concentration, sample played the part of with the silica gel of certain mesh number, on
Column carries out silica gel column chromatography, is the ethyl acetate-light petrol isocratic elution of 2:1 with volume ratio, quantitatively receives eluent, be used in combination
Ultraviolet specrophotometer monitors its light absorption value in 307nm, using TLC tracing detection (solvent is chloroform: ethyl acetate: formic acid=
5:4:1), wait elute completely, merge identical eluent, vacuum is spin-dried for get the higher resveratrol product of purity.
Comparative example 1:
Hairy roots of giant knotweed produces resveratrol and expands the method and step of culture with the step and culture item in embodiment 1
Part only changes explant 2, and the influence that measurement explant 2 induces hairy roots of giant knotweed, influencing result see the table below:
Remarks: data are five duplicate averages in table.
As seen from the above table: the different selections of explant 2 have a certain impact to hairy induction, and different explants 2 are right
The inductivity of hairy roots of giant knotweed is different.As can be seen from the above data, the induction infected than stem section of the blade to hairy roots of giant knotweed
Rate is high by 16%.
Comparative example 2:
Hairy roots of giant knotweed produces resveratrol and expands the method and step of culture with the step and culture item in embodiment 1
Part only changes the substance added in bioreactor, measures addition different material in bioreactor and induces hairy roots of giant knotweed
Influence, measurement result see the table below:
Data are three duplicate averages in table.
As seen from the above table: to yield carry out SPSS variance analysis, as the result is shown: the 14th group in the level of signifiance (p <
0.05), remaining display is not significant.Its yield increases 2.73 times compared with the control.
Claims (4)
1. a kind of hairy roots of giant knotweed production resveratrol and the method for expanding culture, it is characterised in that this method includes following step
It is rapid:
(1) selection and processing of vegetable material
It chooses wild polygonum cuspidate stem with bud and is cut into the segment of 2-4cm as explant 1, and the processing that carries out disinfection to it；
(2) induction of Plant tissue culture seedling
Explant 1 in step (1) is inoculated in induced medium, temperature is 25 ± 2 DEG C, intensity of illumination 1500-2000lux,
Its polygonum cuspidate induced bud is carried out squamous subculture after 25 days by light application time 14-16h；The induced medium is MS+6-
BA2.5mg/L+NAA0.5mg/L；
(3) tissue-cultured seedling squamous subculture
The induced bud obtained in step (2) is inoculated in subculture medium, squamous subculture, the same step of condition of culture (2) are continued；
It is opened completely to blade, the culture of hairy roots of giant knotweed is used for after length to mature tissue-cultured seedling；The subculture medium is MS+6-
BA0.3mg/L+NAA0.5mg/L+TDZ0.05mg/L, pH value 5.8-6.0；
(4) induction of hairy roots of giant knotweed and squamous subculture
It is induced using the mature tissue culture seedling leaf obtained in step (3) or stem as explant 2 for hairy roots of giant knotweed；Specific steps
It is as follows:
A, the preculture of explant 2: by blade or stem in MS blank cultures dark culture two days；
B, actication of culture: agrobacterium rhizogenes strain ATCC15834 is activated in YEB culture medium；
C, it co-cultures: after the agrobacterium rhizogenes after activation is infected 10-30min with polygonum cuspidate blade, stem, placing MS blank cultures
In plus AS20mg/L, dark culture two days；
D, degerming culture: by after co-cultivation blade or stem in the MS containing Cefotaxime Sodium or kanamycins or carbenicillin sodium
Degerming culture in blank cultures, until degerming is complete, the cefotaxime na concn is 10-500mg/L, kanamycins 10-
300mg/L, carbenicillin sodium 10-300mg/L；
Condition of culture: temperature is 25 ± 2 DEG C, and dark culture, every degerming in 3-6 days is primary, until degerming is complete；
E, squamous subculture: the hairy 0.1-0.5g that degerming is grown completely, which is inoculated in 1/2MS blank solution culture medium, to carry out
Squamous subculture 2-5 times, every 20 days squamous subcultures are primary, cultivate under conditions of 25 ± 2 DEG C；
(5) hairy roots of giant knotweed PCR is detected
DNA is extracted: extracting hairy roots of giant knotweed DNA；
PCR system: the PCR system containing rolB and tms primer is obtained；
Target stripe is obtained using agarose gel electrophoresis；
(6) bioreactor large-scale culture hairy roots of giant knotweed
1/2MS blank solution culture medium is added in circulation fermentation pot in 1L gas-lifting type, it is another that 3% sucrose, acid hydrolyzed casein is added
1-5g/L、NH4NO3 825mg/L, aspergillus niger 10-100mg/L, L-phenylalanine 15-50mg/L, inoculum concentration are the 30g hair of weight in wet base
Shape root, it is desirable that all it is sterile, pH value be 5.8-6.5, temperature: 25 ± 2 DEG C, dark culture, ventilatory capacity be 0.05vvm under the conditions of into
Row culture is timed moisturizing by peristaltic pump according to the water of evaporation, keeps total volume constant during the cultivation process；
(7) the extraction separation of hairy roots of giant knotweed；
(8) using the content of resveratrol in HPLC measurement hairy roots of giant knotweed；The 1/2MS fluid nutrient medium refers to a large amount of members
Element halves, but ammonium nitrate content is constant, remaining is constant, PH 5.8-6.5.
2. hairy roots of giant knotweed production resveratrol according to claim 1 and the method for expanding culture, it is characterised in that:
It is 1.0cm*1.0cm, stem length 1.5cm that step (4), which requires 2 leaf blade size of explant,.
3. hairy roots of giant knotweed production resveratrol according to claim 1 and the method for expanding culture, it is characterised in that:
In the step (4) by carry out bacteria removing after hairy squamous subculture be temperature be 25 ± 2 DEG C, shake in dark culture
Culture is swung, used medium is 1/2MS blank cultures, without any hormone, shaking speed 120r/min.
4. hairy roots of giant knotweed production resveratrol according to claim 1 and the method for expanding culture, it is characterised in that:
The gene rolB and tms of hairy of the induction from Ri plasmid are incorporated in the step (5) in hairy roots of giant knotweed.
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| CN105766653B (en) * | 2016-04-22 | 2018-05-29 | 宜春学院 | The method containing resveratrol root system is prepared using giant knotweed leaf culture |
| CN106265963A (en) * | 2016-08-26 | 2017-01-04 | 天津市尖峰天然产物研究开发有限公司 | A kind of produce that low agriculture is residual, the method for the Rhizoma Polygoni Cuspidati extract of low emodin content |
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