CN103695462A - Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana - Google Patents
Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana Download PDFInfo
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- CN103695462A CN103695462A CN201310603423.8A CN201310603423A CN103695462A CN 103695462 A CN103695462 A CN 103695462A CN 201310603423 A CN201310603423 A CN 201310603423A CN 103695462 A CN103695462 A CN 103695462A
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Abstract
The invention belongs to the field of genetic engineering, specifically to a production method for chlorogenic acid and dicaffeoylquinic acid by establishing a hairy root culture system through genetic transformation of stevia rebaudiana with agrobacterium rhizogenes. The method comprises the following steps: preparation of an explant of stevia rebaudiana, activation and culture of agrobacterium rhizogenes, suspension culture and production of chlorogenic acid and dicaffeoylquinic acid. PCR detection results show that hairy roots of stevia rebaudiana are generated through transformation; HPLC detection results show that the hairy roots of stevia rebaudiana can produce chlorogenic acid and dicaffeoylquinic acid. Since the hairy roots used in the invention have the characteristic of rapid growth on a hormone-free medium, the method provided by the invention has the advantages of simple operation, low cost, no restriction by natural conditions like climate and land, etc. A stable and sustainable novel drug source and food function factors are provided for production of chlorogenic acid and dicaffeoylquinic acid through culture of the hairy roots, and a reliable source and a reliable substance base are provided for large scale production of chlorogenic acid and dicaffeoylquinic acid by using a bioreactor in the future.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of method that adopts Agrobacterium rhizogenes to infect sweet Stevia induction generation hairly root, and produce secondary metabolite chlorogenic acid and dicaffeoylquinic acid with this hairly root.
Background technology
Plant can produce natural medicinal ingredients, but also brings a large amount of situations that consume of plant resources thereupon.Utilize plant biological engineering to produce active components in medicinal plant, can effectively protect national resource, save soil, great for modern society's Significance of Sustainable Development.Genetic Transformation in Higher Plants Agrobacterium rhizogenes carrier system have heredity and biosynthetic stability and growth of hair root soon, be easy to the unique advantages such as cultivation, effective constituent height, for the suitability for industrialized production of Plant Secondary Metabolites provides wide prospect.
At present, although it is more to adopt Agrobacterium rhizogenes genetic transformation plant to obtain the correlative study of hairly root of secondary metabolite, genetic transformation sweet Stevia is obtained to yield of chlorogenic acid and the research of dicaffeoylquinic acid hairly root be still blank both at home and abroad.Sweet Stevia (Stevia rebaubiana), Dicotyledoneae, a kind of small-sized per nnial herb of genus chrysanthemum material Si Taiwei subgenus, has another name called sweet grass, stevia rebaudianum, sweet tea etc.Originate in Gran Chaco, mountain range is visitd by Oman that Paraguay and Brazil have a common boundary.China started successively from Japan, to introduce sweet Stevia by R&D institutions such as Zhong Shan Botanical gardens, Nanjing, the Chinese Academy of Agricultural Sciences and plants experimentally successfully from 1976.The beginning of the eighties, the sweet Stevia cured leaf main exit of production was Japanese to popularizing planting in all parts of the country.Stevioside plant body contains a variety of materials, comprises volatile oil, and two, triterpenes, coumarins, coffic acid and chlorogenic acid etc.Wherein, steviol glycoside (Stevioside, St) is the diterpene-kind compound in sweet Stevia leaf, is the sweet taste substance of natural high sugariness, and sugariness is 200～300 times of sucrose, receives people's concern always.In sweet Stevia, also contain the of great value composition chlorogenic acid of a class and dicaffeoylquinic acid, but content is lower, is not yet utilized effectively at present.Chlorogenic acid, has the effects such as anti-oxidant, antibacterial, antiviral, leukocyte increasing, immunomodulatory, antitumor, hypotensive, reducing blood-fat.Have result of study to show, chlorogenic acid is the lead compound of the anti AIDS virus (HIV) that gets a good chance of simultaneously.The dicaffeoylquinic acid phenolic acids natural component that to be a class formed by esterification condensation by quininic acid and number coffic acid not etc., extensively be present in vegitabilia as in the fruit trees such as the Chinese medicines such as Japanese Honeysuckle, Inula Flower and tealeaves, hawthorn, there is anti-hepatic fibrosis and lipid peroxidation.Along with improving constantly of biotechnology, utilize hairly root to carry out the quick production effective constituent of isolated culture, become one of effective way of resources of medicinal plant Sustainable development.
Summary of the invention
The object of the invention is to, provide a kind of transgenic technology genetic transformation sweet Stevia to obtain the method for the hairly root that produces medicinal ingredients chlorogenic acid and dicaffeoylquinic acid, the method imports the T-DNA of Agrobacterium rhizogenes in sweet Stevia, obtain sweet Stevia hairly root, for effectively utilizing new way of sweet Stevia Resource Supply.
The present invention is achieved by the following technical solutions:
1, Stevia seed is placed in the culture medium culturing such as MS, B5, WPM;
2, the T-DNA of the Ri plasmid of employing transgenic method transfer Agrobacterium rhizogenes, to sweet Stevia blade, comprises the following steps:
A. Agrobacterium rhizogenes is seeded in the activation of YEB substratum;
B. with Agrobacterium rhizogenes, infect sweet Stevia aseptic explant;
C. Agrobacterium rhizogenes and sweet Stevia explant are cultivated altogether;
D. explant is cultivated in microbiotic degerming such as carbenicillin sodium, cefotaxime sodiums.
3, sweet Stevia hairly root obtains and succeeding transfer culture;
4, the Molecular Detection of sweet Stevia hairly root, method is as follows:
A. extract sweet Stevia hairly root DNA;
B. obtain the PCR reaction system that contains rolB and rolC primer;
The c.PCR reaction amplification peculiar gene rolB of hairly root and rolC;
D. electrophoresis detection target stripe.
5, the content detection of sweet Stevia hairly root Content of Chlorogenic Acid.
6, sweet Stevia chlorogenic acid and dicaffeoylquinic acid are extracted and purifying
The abbreviation term relating in the present invention, substratum:
1. explant---stem section and cotyledonary node that sweet Stevia aseptic seedling is cut into;
2. Agrobacterium rhizogenes substratum: YEB, LB;
3. plant base basal culture medium: MS (Murashige and Skoog, 1962), B5, WPM, White;
4. the microbiotic such as microbiotic: Rifampin Rif (Rifampicin), kantlex (kan), cefotaxime sodium Cef (Cefotaxime), carbenicillin sodium Cb (Carbenicillin).
The useful effect of the inventive method is embodied in::
1, in the present invention, utilize transgenic technology, the T-DNA of Agrobacterium rhizogenes is imported to sweet Stevia cell and obtain hairly root, by screening choiceness, obtain chlorogenic acid and the relatively high and stable hairly root clone of dicaffeoylquinic acid content, for the production of sweet Stevia provides a kind of sustainable medicine source and food function factor of novel high-quality;
2, compare with field cultivation sweet Stevia, sweet Stevia hairly root can obtain stabilised quality and output, is not subject to the restriction of natural condition, and the area that do not occupy cultivated land can be produced continuously in industrialization;
3, by artificial regulatory optimum culture condition, can significantly improve the growth of hairly root and the accumulation of secondary metabolite, there is controllability;
4, this technology, in hairly root culturing process, is not added any hormone, therefore, can greatly reduce costs.
Accompanying drawing explanation
Accompanying drawing 1 is sweet Stevia aseptic seedling photo.
The hairly root photo that accompanying drawing 2 produces for Agrobacterium rhizogenes induction.
Accompanying drawing 3 is hairly root Fast Growth photo on solid medium.
Accompanying drawing 4 is that the PCR of hairly root rol gene detects photo.
The sweet Stevia hairly root photo that accompanying drawing 5 is liquid culture.
Accompanying drawing 6 is sweet Stevia hairly root Content of Chlorogenic Acid.
Embodiment
Following examples are only for setting forth the present invention, and protection scope of the present invention is not only confined to following examples.Person of an ordinary skill in the technical field, according to above content disclosed by the invention and scope that each parameter is got, all can realize object of the present invention.
The acquisition of embodiment 1 sweet Stevia aseptic explant
Pappus on artificial removal's Stevia seed is incubated 1-28 hour in 20-40 ℃ of water-bath, and filter paper blots, by 70% alcohol disinfecting 20-60S for Stevia seed, with aseptic water washing 2-3 time, then with 0.1% mercury solution sterilization 2-20min, with aseptic water washing 5-6 time.Be seeded on solid medium, this culture medium prescription is: MS minimum medium, and 10～60g/L sucrose, 0.01～1mg/LNAA and 0～1mg/L BA, then add the agar powder of 7-8g/L.Under 25 ± 2 ℃ of conditions of temperature, cultivate, dark culturing 10-20d, then moves to light chamber, and intensity of illumination is 2000lx, and light application time is 12h/d, obtains aseptic seedling.
1, the activation of Agrobacterium rhizogenes
Agrobacterium rhizogenes is inoculated in containing antibiotic YEB solid mediums such as Rifampins, picking list bacterium colony, be seeded in containing in the microbiotic YEB liquid nutrient mediums such as Rifampin, 28～30 ℃ of temperature, rotating speed 50～400r/min, concussion incubated overnight, then the bacterium liquid 10-500 μ L getting after activation joins 10～50mL containing in antibiotic YEB (LB) liquid nutrient mediums such as Rif, 28 ℃ of temperature, 50～400rpm concussion is secretly cultured to OD value for 0.5-1.0.
2, the resuspension of Agrobacterium rhizogenes
The centrifugal 2-20min of room temperature 10000～5000rpm, abandons supernatant, isopyknic 1/2MS resuspension for thalline, and at 25-35 ℃, 50-200rpm concussion is dark cultivates half an hour, makes bacterial concentration reach OD600=0.2-1.5 left and right, for infecting.
3, the preculture of explant
Clip is cultivated sweet Stevia aseptic seedling blade and the stem explants on MS solid medium, is inoculated in the MS substratum that contains AS 25 ℃ of dark 2d that cultivate.
4, Agrobacterium rhizogenes is cultivated altogether in sweet Stevia
By above-mentioned through pre-incubated sweet Stevia blade explant (as young leaflet tablet), the 1/2MS suspension of putting into containing the above-mentioned Agrobacterium rhizogenes engineering bacteria having activated soaks after 10min (jiggle explant is fully contacted with bacterium liquid), the blade taking out after contaminating blots surperficial bacterium liquid with aseptic thieving paper, forward in common culture medium 1/2MS, secretly cultivate 2-6d.
5, the degerming of sweet Stevia is cultivated
Sweet Stevia blade after common cultivation 3d is blotted to blade surface moisture with aseptic thieving paper with aseptic water washing for several times, be placed in the upper degerming cultivation except bacterium culture medium 1/2MS+100-500mg/LCb, after 7-10 days, be transferred on 1/2MS+400～600mg/L cephalo thiophene palate sodium (Cef), during this period, will there is successively white hairly root in the wound of sweet Stevia blade; 1-2 is transferred to after week on 1/2MS+100～300mg/L cephalo thiophene palate sodium (Cef), 1-2 Zhou Houzai forwards on 1/2MS+100～300mg/L cephalo thiophene palate sodium (Cef), after two weeks, after basic degerming, be transferred to not containing on antibiotic 1/2MS substratum.Culture condition is 25 ± 2 ℃, dark culturing.
6, the screening of hairly root high-quality strain
Select degerming hairly root thorough, that detect through PCR, branch is many, the hairly root fast of growing is transferred in 1/2MS nutrient solution, carries out succeeding transfer culture.
The Molecular Detection of embodiment 3 sweet Stevia hairly root
1, the extraction of sweet Stevia hairly root genomic dna, method is as follows:
1) get young tender sweet Stevia hairly root 100-200mg, add liquid nitrogen to clay into power;
2) the sweet Stevia hairly root of claying into power is packed in the centrifuge tube of 1.5mlEppendorf, adds 600 μ l through 2 * CTAB of 60 ℃ of preheatings and the mercaptoethanol of 10 μ l, mix and be placed on 40-50min in 60 ℃ of water-baths, during frequently put upside down and mix;
3) after lysate is cooled to room temperature, add equal-volume phenol (pH8.0), soft upset mixes, standing 10min;
4) 12000rpm, draws oyster white supernatant to new pipe after centrifugal 20min, write down volume during absorption;
5) add equal-volume phenol: chloroform (1:1), soft upset mixes, standing 10min;
6) 12000rpm, draws oyster white supernatant to new pipe after centrifugal 20min, write down volume during absorption;
7) add equal-volume chloroform, soft upset mixes, standing 10min;
8) 12000rpm, draws oyster white supernatant to new pipe after centrifugal 20min, write down volume during absorption;
9) add two volumes to meet cold dehydrated alcohol, separate out flocks;
10) with suction nozzle, white flocks is chosen in EP pipe, 75% ethanol cleans twice, dries;
11) add again Rnase to be placed in 37 ℃ of water-bath 30min after adding sterilized water fully to dissolve, be placed in-20 ℃ standby.
The formula of 2 * CTAB extracting solution (1000ml) is as follows:
2, in sweet Stevia hairly root, the PCR of rolB and rolC gene detects
RolB gene amplification primer is
frolb(5’-GCTCTTGCAGTGCTAGATTT-3’)
And rrolb (5 '-GAAGGTGCAAGCTACCTCTC-3 ')
RolC gene amplification primer is
frolc(5’-CTCCTGACATCAAACTCGTC-3’)
And rrolc (5 '-TGCTTCGAGTTATGGGTACA-3 ')
Reaction system is (50 μ L): ddH2O16 μ L, Template DNA5 μ L, Forward Primer (10 μ M) 2 μ L, Reverse Primer (10 μ M) 2 μ L, 2 * EasyTaq PCR SuperMix25 μ L.
PCR reaction conditions is: 94 ℃ of 5min, 35 circulations (94 ℃ of 60sec, 55 ℃ of 60sec, 72 ℃ of 90sec), 72 ℃ of 10min.
Positive control is corresponding engineering bacteria, the negative contrast of sweet Stevia blade.PCR product is through agarose gel electrophoresis and ultraviolet detection.The amplified band size of rolB is 423bp, and rolC is 626bp.
The mensuration of main effective constituent in embodiment 4 sweet Stevia hairly root
1, the extraction of main effective constituent in sweet Stevia hairly root
Results sweet Stevia hairly root, 60 ℃ of oven dry, grinding powder.Take 0.2g left and right powder, add 5mL60% ethanol, ultrasonic assisted extraction 30min, collects supernatant liquor, and distilled water is settled to 10mL, makes sweet Stevia hairly root extracting solution.
2, the making of reference substance typical curve
Accurately take chlorogenic acid standard substance 3.8mg, 3,5-diCQA 3.7mg, Isochlorogenic acid C 3.9mg, 1mL methyl alcohol ultrasonic dissolution, make 3.8mg/mL chlorogenic acid, 3.7mg/mL3,5-dicaffeoylquinic acid, 3.9mg/mL4,5-dicaffeoylquinic acid standardized solution, draw a certain amount of standardized solution, with distilled water diluting, obtain 0.095mg/mL, 0.0925mg/mL and 0.0975mg/mL standardized solution, 0.22 μ m filtering with microporous membrane, HPLC analyzes.Chromatographic condition: high performance liquid chromatograph (Agilent1260), chromatographic column is Waters C
18post (4.6mm * 250mm, 5um); Moving phase is methyl alcohol-acetic acid water (gradient elution), and elution requirement is as follows:
0 min methyl alcohol: water (containing 0.2% acetic acid)=30:70
10 min methyl alcohol: water (containing 0.2% acetic acid)=30:70
15 min methyl alcohol: water (containing 0.2% acetic acid)=55:45
25 min methyl alcohol: water (containing 0.2% acetic acid)=55:45
30 min methyl alcohol: water (containing 0.2% acetic acid)=30:70
Flow velocity: 1.0mL/min; Column temperature: 40 ℃; Detect wavelength: 327nm.With same concentrations different volumes sample introduction, obtain color atlas and chromatographic parameter, with peak area and content (μ g), carry out linear regression respectively, take content as X-coordinate, reference substance peak area is ordinate zou, drawing standard curve.
3, the mensuration of sweet Stevia hairly root Content of Chlorogenic Acid, 3,5-diCQA and Isochlorogenic acid C content
HPLC assay result: the content of sweet Stevia hairly root Content of Chlorogenic Acid, up to 35.2mg/g, higher than normal root (0.61mg/g) and blade (8.25mg/g), is respectively 57 times and 4.27 times of normal blade of normal root; The content of 3,5-diCQA, up to 55.69mg/g, is respectively 16.67 times (3.34mg/g) of normal root and 1.93 times (28.8mg/g) of blade; The content of Isochlorogenic acid C is 4.9mg/g, is 4 times (1.22mg/g) of normal root, 0.21 times (23.08mg/g) of blade.Sweet Stevia growth of hair root speed is fast, and chlorogenic acid content is high, can become a kind of effectively mode of suitability for industrialized production chlorogenic acid.
Embodiment 4 sweet Stevia chlorogenic acids and dicaffeoylquinic acid are extracted and purifying example
After sweet Stevia hairly root drying is processed, pulverization process, adds 1-100 water doubly, boiling is extracted 2～3 times, and united extraction liquid filters, 55-80 ℃ of concentrating under reduced pressure, is adjusted to pH2-4, macroporous resin adsorption, upper prop adsorbs flow velocity 1-6BV/h, uses afterwards the ethanol of 10-80%, pH6-9, eluent flow rate 1-4BV/h, carries out wash-out, and elution volume is 3-8BV, concentrating under reduced pressure reclaims, and obtains product.
Claims (4)
1. a method that obtains the sweet Stevia hairly root of yield of chlorogenic acid and dicaffeoylquinic acid, is characterised in that employing transgenic technology, with Agrobacterium rhizogenes, infects sweet Stevia explant, comprises the steps:
(1) acquisition of sweet Stevia aseptic seedling;
(2) T-DNA of the Ri plasmid of employing transgenic method transfer Agrobacterium rhizogenes, to sweet Stevia blade, comprises the following steps:
A. Agrobacterium rhizogenes is seeded in the activation of YEB substratum;
B. with Agrobacterium rhizogenes, infect sweet Stevia aseptic explant;
C. Agrobacterium rhizogenes and sweet Stevia explant are cultivated altogether;
D. explant is cultivated in carbenicillin sodium, cefotaxime sodium degerming.
(3) sweet Stevia hairly root obtains and succeeding transfer culture;
(4) Molecular Detection of sweet Stevia hairly root, method is as follows:
E. extract sweet Stevia hairly root DNA;
F. obtain the PCR reaction system that contains rolB and rolC primer;
The g.PCR reaction amplification peculiar gene rolB of hairly root and rolC;
H. electrophoresis detection target stripe.
(5) content detection of sweet Stevia hairly root Content of Chlorogenic Acid and dicaffeoylquinic acid;
(6) extraction of sweet Stevia hairly root Content of Chlorogenic Acid and dicaffeoylquinic acid and purifying.
2. method according to claim 1, is characterized in that the explant in step b, is the blade being obtained through sterile culture by Stevia seed, and blade is cut into 0.1～10cm
2fritter.
3. method according to claim 1, is characterized in that carbenicillin sodium, the cefotaxime na concn in steps d is respectively 0～500mg/L and 0～1000mg/L.
4. method according to claim 1, utilizes that sweet Stevia hairly root extracts, purifying obtains chlorogenic acid and dicaffeoylquinic acid, it is characterized in that production process do not add hormone, extracts and purge process is easy, cost is low.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109293712A (en) * | 2018-09-30 | 2019-02-01 | 晨光生物科技集团股份有限公司 | The industrialized utilization method and its steviol glycoside and chlorogenic acid of a kind of STEVIA REBAUDIANA |
| CN109511553A (en) * | 2019-01-24 | 2019-03-26 | 赵静 | A kind of coptis hairy highly effective revulsion induction method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
-
2013
- 2013-11-15 CN CN201310603423.8A patent/CN103695462B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| ANNA STOJAKOWSKA等: "Caffeic acid derivatives from a hairy root culture of Lactuca virosa", 《ACTA PHYSIOL PLANT》 * |
| HANDE KARAKöSE等: "Characterization and Quantification of Hydroxycinnamate Derivatives in Stevia rebaudiana Leaves by LC-MS", 《J. AGRIC. FOOD CHEM.》 * |
| KRÓLICKA A等: "Establishment of hairy root cultures of Ammi majus", 《PLANT SCI.》 * |
| TAKESHI YAMAZAKI等: "EXAMINATION OF STEVIOL GLUCOSIDES PRODUCTION BY HAIRY ROOT AND SHOOT CULTURES OF STEVIA REBAUDIANA", 《JOURNAL OF NATURAL PRODUCTS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109293712A (en) * | 2018-09-30 | 2019-02-01 | 晨光生物科技集团股份有限公司 | The industrialized utilization method and its steviol glycoside and chlorogenic acid of a kind of STEVIA REBAUDIANA |
| CN109511553A (en) * | 2019-01-24 | 2019-03-26 | 赵静 | A kind of coptis hairy highly effective revulsion induction method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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