CN1759665A - Method for preparing polygonin through tissue culture and inducement of hairy roots of giant knotweed - Google Patents
Method for preparing polygonin through tissue culture and inducement of hairy roots of giant knotweed Download PDFInfo
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Abstract
A process for preparing the polygonoside from the trichiform root of rhizome polygoni cuspidati by tissue culture and inducing includes such steps as tissue culture of wild rhizome polygoni cuspidati, activating culture of Agrobacterium rhizogene, inducing the trichiform root of rhizome polygoni cuspidati by the T-DNA fragment on rooting plasmide of Agrobacterium rhizogene, optimalizing culture of said trichiform root, and extracting polygonoside from said trichiform root.
Description
Technical field
The present invention relates to a kind of tissue culture and inducement of hairy roots of giant knotweed and prepare the method for polygonin, belong to plant biotechnology field.
Background technology
Polygonin (polydatin) and resveratrol (resVeratrol) are the diphenylethylene polyphenol substance of natural generation in the plant corpus, it is monomeric compound with clear and definite chemical constitution and definite curative effect, human body is had cardiac stimulant expand blood vessel, suppress platelet aggregation, reducing blood lipid, effects such as antiviral and enhancing body immunity, it has what is more important lethality and does not influence normal cell at cancer cell specifically, be described as " 21st century is one of the most promising cancer therapy drug ", therefore be widely used in clinical treatment and healthcare industry, the domestic and international market is in great demand.
Polygonin and resveratrol all exist in natural a lot of plants, and for example grape, peanut, black false hellebore, giant knotweed, European spruce etc. are wherein abundant with the content in giant knotweed, considerably beyond other plant.Pharmaceuticals industry at present mainly relies on extracts polygonin and resveratrol from the wild giant knotweed root and rhizome of excavating in a large number, extract 10 tons of resveratrols according to statistics and need consume 5000 tons of giant knotweed raw materials.Polygonin and the resveratrol content in plant corpus is not only generally lower, but also the shadow that obviously is subjected to factors such as the place of production, collecting season and growing environment to, for example the content of polygonin is 3.24% in the giant knotweed rhizome in area, Yunnan, and Guangdong one band only is 0.64%; When gathering, the giant knotweed August in the same place of production contains resveratrol 0.30% in its rhizome, just reduce to 0.04g% when gathering to the October, so simple method of from plant material, extracting that relies on, not only difficult quality control, and the production cycle is long, cost is high, the product yield is low, even more serious is to excavate the extinction speed that also can accelerate wild resource with robbing formula for a long time, does not meet the strategic requirement of China's sustainable economic development.How under the condition of not destroying natural resources, producing resveratrol quality controllable, that purity is higher and glycoside thereof, is the hot issue of current research.
Biosynthesis (biosynthesis), promptly cultivate with optimization by screening to high-yield plant cell, tissue or organ, with plant corpus as one " medicine processing factory ", in specific bio-reactor, reach the efficient output of target component, significant for the production problem of sustainable use that solves Chinese herbal medicine resource and natural drug.At present, utilize biosynthesis pathway production polygonin or Study of Resveratrol mainly to concentrate on the cell culture aspect, for example patent 00113914.2 discloses the technical scheme of utilizing the grape cell line to produce resveratrol.But there are many deficiencies in the large-scale production that utilizes cell culture to carry out secondary metabolites, for example the general poor growth of cell and rely on that hormone is kept, content is low, when genetic stability difference and amplification culture to the shearing force sensitivity etc.
Summary of the invention
Technical problem to be solved by this invention is to utilize one section T-DNA on tissue culture technology and agrobacterium rhizogenes (the Agrobacter ium rhizogene) plasmid of taking root that the giant knotweed explant is carried out the method that hairy root that the genetic transformation inducing culture produced is produced polygonin.
Technical scheme of the present invention is achieved in that tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, and it is characterized in that: it comprises the steps:
A, the training of wild giant knotweed group: the young shoot of choosing well-developed wild giant knotweed plants stems section top, disinfect after differentiation culture, strong seedling culture, culture of rootage form grows complete seedling, and the explants such as stem section, blade, petiole and young shoot that cut aseptic tissue cultivating seedling are standby as infected material;
The activation culture of B, agrobacterium rhizogenes: take out agrobacterium rhizogene strain ATCC11325 equilibrium at room temperature 20～30min that refrigerator is preserved, drawing 500～800 μ l bacterium liquid on the superclean bench in the fresh YEB culture fluid of sterilized 100～150ml, then in 23～27 ℃, 120～160rmin
-1, dark shaken cultivation 20～24h gets final product;
The inducing culture of C, hairy roots of giant knotweed: scratch the acceptor material surface to form wound, the Agrobacterium bacterium liquid of putting into step B soaks 5sec～15min makes it abundant contact, take out infected material and dip in dried unnecessary bacterium liquid gently, place MS under 24～27 ℃, dark condition with aseptic blotting paper
0In carry out common cultivation.After 7～15 days in the germinating of the visible hairy root of wound location of infected material;
D, hairy roots of giant knotweed optimization are cultivated: adopt 1/2MS after Molecular Identification
0Liquid nutrient medium, additional 3% sucrose is 6.0～6.5 as carbon source in the pH value, 22～27 ℃ of temperature, shaking speed: 120～140rmin
-1, the hairy root strain that obtained a large amount of propagation under dark or the low light condition in 6～30 days is;
The extraction of polygonin in E, the hairy roots of giant knotweed: from culture fluid, take out hairy root, oven dry, grind into powder, (V: V) ethanol is handled in conjunction with 30～40min ultrasonic and is extracted with 50%, afterwards in the centrifugal 15～20min of 3000r/min, get the supernatant concentrating under reduced pressure, again through column chromatography for separation, concentrate, crystallization, freeze drying, can obtain the higher polygonin product of purity.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, sterilization method to wild material in the steps A is to adopt flowing water to rinse well to be placed on the superclean bench, 75% (V: V) alcohol-pickled 10～30sec, place 0.1% after the rinsed with sterile water three times again (W: V) mercuric chloride solution surface sterilization 3～8min, can inoculate after the abundant rinsing of sterile water; Induced bundle sprout the differentiation medium be meant MS+0.05mg/LTDZ+0.5mg/L NAA+0.1mg/L 6-BA; Strong sprout, the medium of growth was meant MS+0.1mg/LCPPU+1.0mg/L NAA, and root media is meant MS+1.0mg/L NAA+0.5% active carbon; Above-mentioned condition of culture is 27 ± 1 ℃ of daytimes, night 20 ± 1 ℃ of humidity 60～70%, light intensity 28 μ mol/m
2.s, light application time: 13～17h/d.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, and the method that improves the described hairy roots of giant knotweed inductivity of step G comprises:
A, agrobacterium rhizogenes and Agrobacterium tumefaciems double infection; The b supersonic oscillations are auxiliary to infect; C, the microwave radiation is auxiliary to be infected; D, choose the blade-section of climax leaves as infected material; E, infect blade without pre-when cultivating and cultivating altogether the blade epicuticle upwards place; Or induce the generation of root under the f, dark, room temperature environment.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, and the described Molecular Identification of step D is meant: cut the hairy root root segment that produces from infected material, be separated into and be forwarded to MS after single
0Grow in the solid culture medium, 10～15 days successive transfer culture once; The hairy root that the screening growth rapidly from a large amount of monoclonal feather shaped root systems, profile is sturdy, branch is more carries out the PCR Molecular Identification, confirms to have the hairy root of transmitting root agrobatcerium T-DNA character and enters next step operation.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, and the described cultivation cycle of step D is taked in 6～24 days to change culture fluid once in per 7 days.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, step D is obtained hairy roots of giant knotweed carry out the solid quality saving to be used for suitability for industrialized production.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, with growth rapidly, content is higher and the good hairy roots of giant knotweed monoclonal system of inheritance stability, be stored in to be added with 1% activated carbon and to increase sucrose content to 5% MS solid culture medium.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, enlarged culture is so that regularly gather in the crops hairy root under optimal conditions with hairy roots of giant knotweed that step D obtains, and inoculum concentration is controlled at 0.5～1.0g fresh weight hairy root during successive transfer culture: 100～150ml culture fluid.
Described tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, can prepare resveratrol extract simultaneously.
Technological progress effect of the present invention shows:
(1) compares with the mode of production now of from wild giant knotweed root and rhizome, extracting polygonin and resveratrol, some active ingredient of the medicinal plant that utilizes hairy root to produce to be used as medicine based on root, have with short production cycle, quality and stable yield, advantage such as do not occupy cultivated land, and but the production of natural drug is to carry out under controlled and repeat condition, can not be subjected to the influence of factors such as damage by disease and insect and natural environment.
(2) compare with the method for chemosynthesis, polygonin and resveratrol utilize hairy root culture biosynthesis pathway except can be provided, can produce directly also that precursor and other have the pharmacologically active structure and chemical means is difficult to synthetic compound, in addition product can be infinitely, continuously, produce uniformly, separate, extract simple to operate, use manpower and material resources sparingly.
(3) compare with utilizing other tissue cultures (as natural root, callus, suspension cell etc.) mode of production, in the hairy root owing to imported T-DNA on the agrobacterium rhizogenes Ri plasmid, have the characteristic that does not rely on the autonomous quick growth of hormone, and other cultures must add costliness or the virulent exogenous hormone of human body is kept its growth.The high-quality hairy roots of giant knotweed strain system that screening obtains in the specific embodiment of the invention, the dry weight growth rate can reach 8.29 in the 30d culture period, is 192.8 times of the same terms low suspension cultured cell, is 8.4 times of nature root culture.In addition, the hairy root heritability is stable, and subculture is simple to operate, and growth does not need illumination, and is with low cost, is more suitable for batch production production.
(4) the present invention utilizes the mode of wild type agrobacterium rhizogenes and the two bacterium coinfections of Agrobacterium tumefaciems, not only can obviously go out root in advance, and effectively improved the inductivity of hairy root and lured root density, the average inductivity of hairy root can reach 97.1%, lure root density to reach 15.8, be higher than far away and belong to other plant together.In addition, different with bibliographical information in the past is, experience material in this technology and do not need pre-cultivation, directly be soaked in after the scuffing in the good bacterium liquid of activation, blade take root and hairy root successive transfer culture process in all do not need numerous and diverse degerming work, saved manpower and materials, also make hairy root induce and to cultivate operation more easy.
Description of drawings
Fig. 1 begins to occur hairy root from blade after infecting 7d
Fig. 2 forms raised growth feather shaped root system rapidly after infecting 20d
The fluid enlargement culture of Fig. 3 hairy roots of giant knotweed
The rolb gene primer is through the agarose electrophoresis collection of illustrative plates of pcr amplification afterproduct in Fig. 4 hairy roots of giant knotweed
Among the figure: be followed successively by from left to right: 1. blank (no template DNA); 2. Ri plasmid; 3. infect blade; 4. do not infect blade; 5. Marker; 6. hairy root; 7. natural root.
The tms gene primer is through the agarose electrophoresis collection of illustrative plates of pcr amplification afterproduct in Fig. 5 hairy roots of giant knotweed
Among the figure: be followed successively by from left to right: 1. Marker; 2. hairy root; 3. natural root; 4. blank (no template DNA).
The HPLC chromatogram of Fig. 6 polygonin reference substance
The HPLC chromatogram of Fig. 7 resveratrol reference substance
The HPLC chromatogram of Fig. 8 hairy roots of giant knotweed sample
Among the figure: polygonin reference substance 0.101mgmL
-1Resveratrol reference substance 0.0300mgmL
-1
1, polygonin 2, resveratrol
Embodiment
1, choose robust growth, physically well develop, the young shoot at the wild giant knotweed plants stems section top that no damage by disease and insect and mould are infected, routine disinfection are handled and to be placed on induced bundle and to sprout in the medium of differentiation: MS+0.05mg/LTDZ+0.5mg/L NAA+0.1mg/L 6-BA.Condition of culture is a day temperature: 27 ± 1 ℃, night temperature: 20 ± 1 ℃; Humidity: 60-65%, light intensity: 28 μ mol/m
2.s, light application time: 14h/d.The seedling of will growing thickly behind the 15d is separated into individual plant, and place the growth medium in strong sprout: MS+0.1mg/L CPPU+1.0mg/LNAA, condition of culture is the same.1 week back transplanting individual plant tissue cultivating seedling is in root media: MS+1.0mg/L NAA+0.5% active carbon, condition of culture is the same.With this indefinite bud organ proliferating way sustainable carry out numerous soon, the reproduction coefficient of every strain bud be 5～7,2 the week in can obtain well-developed whole plant.
2, it is standby as infected material to cut the leaf explant of the aseptic tissue cultivating seedling of giant knotweed.
3, wild type agrobacterium rhizogene strain ATCC11325 usually in the YEB liquid nutrient medium that is added with 20% glycerine-20 ℃ keep in Dark Place.From refrigerator, take out agrobacterium rhizogene strain ATCC11325 balance 30min, drawing 500 μ l bacterium liquid on the superclean bench in the fresh YEB culture fluid of sterilized 100ml, 25 ℃, 140rmin then
-1, dark shaken cultivation 22h can use.
4, carefully scratch blade surface to form wound with aseptic scalpel, good Agrobacterium bacterium liquid soaks 10min to put into activation, shakes gently and makes it abundant contact.Take out infected material, dip in gently with aseptic blotting paper and do unnecessary bacterium liquid, make the blade epicuticle be positioned over MS downwards
0In the medium, carry out common cultivation under 25 ℃, dark condition.Infect all left and right sides blade surface scuffing place and begin to occur intensive hairy root of growing thickly (Fig. 1), penetrate ramp in the medium downwards.Continue to cultivate altogether 20～30d and can form a large amount of abundant feather shaped root systems (Fig. 2).Do not occur the Agrobacterium growing state therebetween, therefore save the degerming link.
5, the Molecular Identification of hairy root: polymerase chain reaction technology is adopted in the evaluation of hairy root transgenosis character, and (polymerase chain reaction PCR), designs T in the agrobacterium rhizogenes Ri plasmid
L-DNA or T
R-DNA last with the relevant gene primer of taking root, extract the hairy root genomic DNA and increase, the product of amplification is carried out Analysis and Identification by agarose electrophoresis, determine the specific gene fragment that obtains.The rolb gene is agrobacterium rhizogenes Ri plasmid T
L-DNA (T-DNA left arm) goes up and takes root a closely-related gene, and the tms gene then is positioned at T
ROn-the DNA (T-DNA right arm), participate in the synthetic of coding growth hormone, if the hairy root of being induced has the root of forwarding agrobatcerium T-DNA character, then by design T
L-DNA or T
RThe last special gene primer of-DNA carries out pcr amplification with the hairy root genomic DNA that extracts.Concrete operations are with reference to volume " plant genetic engineering principle and technology " (Science Presses in 1998) such as Wang Guanlin.
From Fig. 4, Fig. 5 result displayed as seen, utilize the PCR primer of rolb and tms, can from metainfective giant knotweed blade and the total DNA of hairy root, increase the respectively 422bp of expectation and the DNA fragment specific about 910bp, and from unconverted natural root and the total DNA of blade that does not infect amplification less than any fragment.This explanation, the T of agrobacterium rhizogenes ATCC11325Ri plasmid
L-DNA and T
R-DNA part has obtained integrating in the hairy roots of giant knotweed genome and has expressed.
6, the hairy root to screening is optimized cultivation, adopts 1/2MS
0Liquid nutrient medium does not add other plant growth regulator (as NAA, CPPU, TDZ or ethrel), and regulating the pH value is 6.0～6.5, and additional 3% sucrose is as carbon source.Temperature: 22～27 ℃, shaking speed: 120～140 rmin
-1, cultivate under the dark or the low light level.Inoculum concentration is controlled at 0.5～1.0g (fresh weight) hairy root during successive transfer culture: 100～150ml culture fluid is more suitable.Take the fluid infusion best cultivation at growth of hair root the fastest exponential phase (6～24 days), can farthest gather in the crops culture.Independently fast breeding such as Fig. 3 in the hairy root liquid medium within, daily growth amount average out to 0.214～0.275g dry weight/sky in the incubation, 30 days amount of growth (fresh weight) is 7～9 times of inoculum concentration, the dry weight growth rate can reach 8.29, being 192.8 times of the same terms low suspension cultured cell, is 8.4 times of nature root culture.
7, from culture fluid, take out hairy root, after low temperature (45 ℃) is dried to constant weight, be ground into fine powder with mortar, cross 60 mesh sieves, handle in conjunction with 30～40min ultrasonic with 50% (V: V, as follows) ethanol and extract, centrifugal 15～the 20min of 3000r/min afterwards, get the supernatant concentrating under reduced pressure, again through column chromatography for separation, concentrate, crystallization, freeze drying, can obtain higher polygonin of purity and resveratrol.
8, utilize high performance liquid chromatography (HPLC) to detect polygonin and Resveratrol content in the hairy root, compare discovery, contain polygonin and resveratrol composition in the hairy roots of giant knotweed as shown in Figure 8 with standard items Fig. 6, Fig. 7.With feather shaped root system provided by the invention and cultural method, hairy root daily growth amount average out to 0.214～0.275g dry weight/sky, polygonin content can reach 0.18～0.22% of culture dry weight, and Resveratrol content can reach 0.05～0.0g% of culture dry weight.
9, good hairy root strain system is transferred in being added with an amount of activated carbon,, and carries out the solid quality saving in the MS solid culture medium of increasing sucrose content as 5-10% as 1-2%, be used for suitability for industrialized production under optimal conditions enlarged culture so that regularly gather in the crops hairy root.
The stem section that cuts the giant knotweed aseptic seedling in step (2) is as infected material.In the step (3), add simultaneously Agrobacterium tumefaciems C58 bacterium liquid 500 μ l in 100ml YEB culture fluid and agrobacterium rhizogenes ATCC11325 at 25 ℃, 140rmin
-1, dark uses after cultivating 22h altogether.In the step (4), stem explants soaks 10min in two bacterium activating solutions, place MS under the dark condition
0In cultivate altogether.In the step (5), the stem section is induced the laggard performing PCR Molecular Identification of hairy root separation and Culture of acquisition; In the step (6), induce the hairy root of acquisition to be optimized cultivation, obtain the hairy root strain system of a large amount of propagation the stem section.In the step (7), induce extraction the hairy root of acquisition, separation, purifying polygonin and resveratrol product from the stem section.In the step (8), utilize the HPLC method stem section to be induced the content of polygonin and resveratrol detects in the hairy root of acquisition.In the step (9), directly carry out fluid enlargement culture after the stem section being induced the hairy root screening of acquisition, adopt 1/2MS
0Liquid nutrient medium is cultivated under optimized conditions, and a large amount of hairy root cultures that results obtain are used for extracting polygonin and resveratrol product, and other are with embodiment 1.
Embodiment 3
In step (2), cut the young shoot of giant knotweed aseptic seedling as infected material.In the step (3), agrobacterium rhizogenes ATCC11325 is at 26 ℃, 150rmin
-1, dark uses after cultivating 24h altogether.In the step (4), the young shoot explant is soaked in the 100ml activation bacterium liquid, handles 5～10sec in ultrasonic oscillator, after unnecessary bacterium liquid is blotted in taking-up, places MS under the dark condition
0In cultivate altogether.In the step (5), young shoot is induced the laggard performing PCR Molecular Identification of hairy root separation and Culture of acquisition; In the step (6), induce the hairy root of acquisition to be optimized cultivation, obtain the hairy root strain system of a large amount of propagation young shoot.In the step (7), induce extraction the hairy root of acquisition, separation, purifying polygonin and resveratrol product from young shoot.In the step (8), utilize the HPLC method young shoot to be induced the content of polygonin and resveratrol detects in the hairy root of acquisition.In the step (9), directly carry out fluid enlargement culture after young shoot being induced the hairy root screening of acquisition, adopt 1/2MS
0Liquid nutrient medium is cultivated under optimized conditions, and a large amount of hairy root cultures that results obtain are used for extracting polygonin and resveratrol product, and other are with embodiment 1.
Embodiment 4
In step (2), cut the giant knotweed aseptic seedling mature leaf (leaf is long 〉=1.5cm) as infected material.In the step (3), agrobacterium rhizogenes ATCC11325 is at 27 ℃, 140rmin
-1, dark uses after cultivating 20h altogether.In the step (4), leaf explant is soaked in the 100ml activation bacterium liquid, and irradiation treatment 5～10sec in micro-wave oven after unnecessary bacterium liquid is blotted in taking-up, places MS under the dark condition
0In cultivate altogether.In the step (5), blade is induced the laggard performing PCR Molecular Identification of hairy root separation and Culture of acquisition; In the step (6), induce the hairy root of acquisition to be optimized cultivation, obtain the hairy root strain system of a large amount of propagation blade.In the step (7), induce extraction the hairy root of acquisition, separation, purifying polygonin and resveratrol product from blade.In the step (8), utilize the HPLC method blade to be induced the content of polygonin and resveratrol detects in the hairy root of acquisition.In the step (9), directly carry out fluid enlargement culture after blade being induced the hairy root screening of acquisition, adopt 1/2MS
0Liquid nutrient medium is cultivated under optimized conditions, and a large amount of hairy root cultures that results obtain are used for extracting polygonin and resveratrol product, and other are with embodiment 1.
Embodiment 5
The petiole part that cuts giant knotweed aseptic seedling blade in step (2) is as infected material.In the step (3), agrobacterium rhizogenes ATCC11325 is at 24 ℃, 130rmin
-1, dark uses after cultivating 23h altogether.In the step (4), petiole explant is soaked in 8min in the 100ml activation bacterium liquid, after unnecessary bacterium liquid is blotted in taking-up, places MS under the dark condition
0In cultivate altogether.In the step (5), petiole is induced the laggard performing PCR Molecular Identification of hairy root separation and Culture of acquisition; In the step (6), induce the hairy root of acquisition to be optimized cultivation, obtain the hairy root strain system of a large amount of propagation petiole.In the step (7), induce extraction the hairy root of acquisition, separation, purifying polygonin and resveratrol product from petiole.In the step (8), utilize the HPLC method petiole to be induced the content of polygonin and resveratrol detects in the hairy root of acquisition.In the step (9), directly carry out fluid enlargement culture after petiole being induced the hairy root screening of acquisition, adopt 1/2MS
0Night, the body medium was cultivated under optimized conditions, and a large amount of hairy root cultures that results obtain are used for extracting polygonin and resveratrol product, and other are with embodiment 1.
Embodiment 6
In step (2), cut the giant knotweed aseptic seedling mature leaf (leaf is long 〉=1.5cm) as infected material.In the step (3), agrobacterium rhizogenes ATCC11325 is at 23 ℃, 130rmin
-1, dark uses after cultivating 23h altogether.In the step (4), leaf explant is soaked in 15min in the 100ml activation bacterium liquid, after unnecessary bacterium liquid is blotted in taking-up, makes the blade epicuticle upwards be positioned over MS
0In the medium, carry out common cultivation under the low light condition.In the step (5), blade is induced the laggard performing PCR Molecular Identification of hairy root separation and Culture of acquisition; In the step (6), induce the hairy root of acquisition to be optimized cultivation, obtain the hairy root strain system of a large amount of propagation blade.In the step (7), induce extraction the hairy root of acquisition, separation, purifying polygonin and resveratrol product from blade.In the step (8), utilize the HPLC method blade to be induced the content of polygonin and resveratrol detects in the hairy root of acquisition.In the step (9), directly carry out fluid enlargement culture after blade being induced the hairy root screening of acquisition, adopt 1/2MS
0Liquid nutrient medium is cultivated under optimized conditions, and a large amount of hairy root cultures that results obtain are used for extracting polygonin and resveratrol product, and other are with embodiment 1.
Listed examples of the present invention is intended to further illustrate the method that this tissue culture and inducement of hairy roots of giant knotweed prepares polygonin, and scope of the present invention is not constituted any restriction, the product that obtains with the embodiment of the invention and prepare polygonin and resveratrol product via the described tissue culture and inducement of hairy roots of giant knotweed that all can obtain of claims of the present invention.Need to prove that resveratrol and glycosides thereof can transform mutually in plant corpus, therefore its content is needed comprehensive evaluation.
The abbreviation term that relates among the embodiment, medium and statistical method explanation:
1, bacterial classification: agrobacterium rhizogenes ATCC11325 and Agrobacterium tumefaciems C58 preserve the center available from Institute of Microorganism, Academia Sinica's bacterial classification.
2, Agrobacterium activation medium: YEB (yeast e * tract bacto) medium is the medium of gene engineering field cultivation Agrobacterium commonly used, and its prescription can obtain in engineered experiment instruction handbook.
3, plant culture: MS (Murashige ﹠amp; Skoog, 1962) medium, be Plant Tissue Breeding field minimal medium commonly used, all can obtain its prescription in textbook aspect tissue culture or the experiment instruction handbook.MS
0Refer to not add the MS minimal medium of any growth regulator; 1/2MS
0Refer to the MS that the macroelement composition reduces by half
0Medium.
4, standard items: resveratrol (resveratrol, purity 99%) is available from Sigma company; Polygonin (polydatin, purity 98%) is available from Tianjin spike natural products research and development Co., Ltd.
6, dry weight growth rate=(results dry weight-inoculation dry weight)/inoculation dry weight; Hairy root inductivity=generation hairy root explant number/total explant number * 100%; Bar number/explant the number of taking root that lures root density=hairy root to produce.Active constituent content (%)=active ingredient quality (g)/100g culture dry weight.
Claims (9)
1, tissue culture and inducement of hairy roots of giant knotweed prepares the method for polygonin, it is characterized in that: it comprises the steps:
A, the training of wild giant knotweed group: the young shoot of choosing well-developed wild giant knotweed plants stems section top, disinfect after differentiation culture, strong seedling culture, culture of rootage form grows complete seedling, and the explants such as stem section, blade, petiole and young shoot that cut aseptic tissue cultivating seedling are standby as infected material;
The activation culture of B, agrobacterium rhizogenes: take out agrobacterium rhizogene strain ATCC11325 balance 20～30min that refrigerator is preserved, drawing 500～800 μ l bacterium liquid on the superclean bench in the fresh YEB culture fluid of sterilized 100～150ml, then in 23～27 ℃, 120～160rmin
-1, dark shaken cultivation 20～24h gets final product;
The inducing culture of C, hairy roots of giant knotweed: scratch the acceptor material surface to form wound, the Agrobacterium bacterium liquid of putting into step B soaks 5sec～15min makes it abundant contact, take out infected material and dip in dried unnecessary bacterium liquid gently, place MS0 to carry out common cultivation germinating at the visible hairy root of wound location of infected material after 7～15 days under 24～27 ℃, dark condition with aseptic blotting paper;
D, hairy roots of giant knotweed optimization are cultivated: adopt the 1/2MS0 liquid nutrient medium after Molecular Identification, additional 3% sucrose is 6.0～6.5 as carbon source in the pH value, 22～27 ℃ of temperature, shaking speed: 120～140rmin
-1, a large amount of value-added hairy root strains of acquisition in 6～30 days under dark or low light condition;
The extraction of polygonin in E, the hairy roots of giant knotweed: from culture fluid, take out hairy root, oven dry, grind into powder, (V: V) ethanol is handled in conjunction with 30～40min ultrasonic and is extracted with 50%, afterwards in the centrifugal 15～20min of 3000r/min, get the supernatant concentrating under reduced pressure, again through column chromatography for separation, concentrate, crystallization, freeze drying, can obtain the higher polygonin product of purity.
2, tissue culture and inducement of hairy roots of giant knotweed according to claim 1 prepares the method for polygonin, it is characterized in that: the sterilization method to wild material in the steps A is to adopt flowing water to rinse well to be placed on the superclean bench, 75% (V: V) alcohol-pickled 10～30sec, place 0.1% after the rinsed with sterile water three times again (W: V) mercuric chloride solution surface sterilization 3～8min, can inoculate after the abundant rinsing of sterile water; Induced bundle sprout the differentiation medium be meant MS+0.05mg/L TDZ+0.5mg/L NAA+0.1mg/L 6-BA; Strong sprout, the medium of growth was meant MS+0.1mg/L CPPU+1.0mg/L NAA, and root media is meant MS+1.0mg/L NAA+0.5% active carbon; Above-mentioned condition of culture is 27 ± 1 ℃ of daytimes, 20 ± 1 ℃ of nights; Humidity: 60～70%, light intensity: 28 μ mol/m
2.s, light application time: 13～17h/d.
3, tissue culture and inducement of hairy roots of giant knotweed according to claim 1 prepares the method for polygonin, it is characterized in that the method that improves the described hairy roots of giant knotweed inductivity of step C comprises:
A, agrobacterium rhizogenes and Agrobacterium tumefaciems double infection; The b supersonic oscillations are auxiliary to infect; C, the microwave radiation is auxiliary to be infected; D, choose the blade-section of climax leaves as infected material; E, infect blade without pre-when cultivating and cultivating altogether the blade epicuticle upwards place; Or induce the generation of root under the f, dark, room temperature environment.
4, tissue culture and inducement of hairy roots of giant knotweed according to claim 1 prepares the method for polygonin, it is characterized in that: the described Molecular Identification of step D is meant: cut the hairy root root segment that produces from infected material, be separated into to be forwarded in the MS0 solid culture medium after single and grow, 10～15 days successive transfer culture once; The hairy root that the screening growth rapidly from a large amount of monoclonal feather shaped root systems, profile is sturdy, branch is more carries out the PCR Molecular Identification, confirms to have the hairy root of transmitting root agrobatcerium T-DNA character and enters next step operation.
5, tissue culture and inducement of hairy roots of giant knotweed according to claim 1 prepares the method for polygonin, it is characterized in that: the described cultivation cycle of step D is taked in 6～24 days to change culture fluid once in per 7 days.
6, the method for preparing polygonin according to the described tissue culture and inducement of hairy roots of giant knotweed of claim 1 is characterized in that step D is obtained hairy roots of giant knotweed carries out the solid quality saving to be used for suitability for industrialized production.
7, the method for preparing polygonin according to the described tissue culture and inducement of hairy roots of giant knotweed of claim 6, it is characterized in that growth is rapid, content is higher and the good hairy roots of giant knotweed monoclonal of inheritance stability is to be stored in the MS solid culture medium that is added with 1% activated carbon and increases by 5% cane sugar content.
8, the method for preparing polygonin according to the described tissue culture and inducement of hairy roots of giant knotweed of claim 6, enlarged culture is so that regularly gather in the crops hairy root under optimal conditions to it is characterized in that step D is obtained hairy roots of giant knotweed, and inoculum concentration is controlled at 0.5～1.0g fresh weight hairy root during successive transfer culture: 100～150ml culture fluid.
9, the method for preparing polygonin according to the described tissue culture and inducement of hairy roots of giant knotweed of claim 1-8 is characterized in that said method can prepare resveratrol extract simultaneously.
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