CN101121941B - Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system - Google Patents
Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system Download PDFInfo
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Abstract
The present invention discloses a method using Agrobacterium rhizogenes (Ar) gene to transform Rhodiola sachalinensis A.Bor to achieve a hairy root system for Salidroside production. By preparation of explant and cultivation in Ar bacteria liquid, Rhodiola sachalinensis A.Bor is induced to grow hairy roots by genetic transformation; the PCR testing proves that hairy roots are generated by transformation; a fermenter is used for massive production of hairy roots, and precursor substances and elicitors are added to improve the Salidroside content. The method is applied to produce Salidroside to substitute the drying-up Rhodiola sachalinensis A.Bor resources.
Description
Technical field
The invention belongs to gene engineering technology field.Specifically be that a kind of Agrobacterium rhizogenes genetic transformation Radix Rhodiolae that utilizes is set up the method that the hairly root culture system is produced rhodioloside.
Background technology
Radix Rhodiolae (Rhodiola sachalinensis A.Bor) has another name called storehouse page or leaf Root of Kirilow Rhodiola, is the perennial dicotyledonous herbaceous plant of Crassulaceae rhodiola (Rhodiola L.).Be one of rare medicinal plant, have the effect of " consolidate and set upright " in the traditional Chinese medical science, have the effect of " adaptogen ".Also have significantly function such as anti-hypoxia, cold resistance, antifatigue, resisting microwave radiation, and have the senility of humanbody of delaying, prevent effect such as geriatric disease.Because Radix Rhodiolae is in the exploitation of industrial aspect, wild plant is excavated in a large number, and resource is exhausted day by day.At present, local government has forbidden to excavate.From the initial stage eighties, just begin the research of artificial introducing culture, because the Radix Rhodiolae adaptation is high and cold, dry, the growing environment of high height above sea level, reasons such as non-refractory, humid climate and susceptible disease, commerial growing does not still succeed.The main medicinal ingredients of Radix Rhodiolae is a rhodioloside.Reports such as bright Haiquan utilize its glucoside unit alcohol to carry out glycosylation synthetic rhodioloside, but the reaction process complexity, yield is low.The Radix Rhodiolae cell cultures provides an approach for the production of rhodioloside, but output is very low, and the salidroside content in the culturing cell can only reach the level of wild plant at the most.Xu Jianfeng etc. utilize cell cultures, be used for Radix Rhodiolae secondary metabolite production research, dynamics research by external factor, precursor, elicitor and cell fermentation, can make the cell culture density maximum reach 18g DW/L, the rhodioloside output maximum in the culture reaches 1980mg/L.The content that is equivalent to rhodioloside in the wild Radix Rhodiolae, moreover, because the interpolation of hormone not only increases cost, and influence the quality of rhodioloside.
Because hairly root has fast growth, many, the weak geotropisms of branch, is in the organ level, hormone autotrophic, Physiology and biochemistry, hereditary feature are stablized, and stable secondary metabolite synthesis capability is arranged.A large amount of hairly root of cultivating substitute wild resource, promote modernization of Chinese medicine development.Therefore it is significant to set up a large amount of cultivations of Radix Rhodiolae hairly root culture systems.
Since Ackermann in 1997 at first transformed higher plant with Agrobacterium rhizogenes, existing so far 100 various plants had been set up culture systems.And utilize it to carry out secondary metabolite production.Middle pharmaceutically active ingredient is mostly at root.And the Ri plasmid induces the hairly root of generation to have the characteristic of root, and it is higher to have a fast growth, hormone autotrophic, differentiation degree, and advantage such as inherited character is relatively stable, therefore, more helps the mass production of secondary metabolite than cell cultures.
Summary of the invention
The objective of the invention is to by biotechnology means mass production Radix Rhodiolae hairly root, utilize hairly root to cultivate in a large number and produce the Radix Rhodiolae glucoside,, fill up the Chinese medicine breach in order to substitute wild resource; The present invention utilizes Agrobacterium rhizogenes genetic transformation Radix Rhodiolae to obtain hairly root, utilizes fermentor tank to cultivate hairly root in a large number in order to produce rhodioloside.
The present invention is achieved by the following technical solutions:
(Rhodiola sachalinensis A.Bor) is explant with Radix Rhodiolae, utilize Agrobacterium rhizogenes (Agrobacterium rhizogenes, Ar) carry out genetic transformation and induce generation hairly root (hairy root), by culture condition (pH value, temperature, time of infection, incubation time and Syringylethanone interpolation altogether) optimization and improvement hairly root inductivity.Hairly root by the optimization of carbon source, nitrogenous source, illumination, temperature, pH value, elicitor, precursor substance and nutrient solution, improves the content of Radix Rhodiolae hairly root rhodioloside through fermentor cultivation mass production hairly root.By the separation and Extraction process of hairly root rhodioloside, produce rhodioloside.
The concrete steps of this method are as follows:
1, the preparation of explant: the aseptic seed of Radix Rhodiolae is put the 1/2MS substratum sprout, the green cotyledon (staying the cotyledon petiole about 2mm) of getting 3 days seedling ages places 25 ℃ of succeeding transfer culture on the MS+0.5mg/LNAA+3.0mg/L 6-BA substratum.With at MS+1.2mg/L 2, dark blade, stem section and the cotyledonary node of cultivating 2-4 days is explant in the 4-D+2.4mg/L 6-BA substratum;
2, Agrobacterium rhizogenes is cultivated: Agrobacterium rhizogenes R1000 or LBA9402 or R1601 or ATCC15834 or A4 (bacterial classification provides by French garden centre) are activated on the YEB solid medium, picking list bacterium colony, be seeded in the LB liquid nutrient medium, at 27 ℃, Agrobacterium rhizogenes bacterium liquid A is cultivated in the shaking table concussion under the 4000r/min dark
600Be to be used to infect explant at 0.45～0.60 o'clock.
3, infect, be total to cultivation, hairly root inducing culture, degerming and screening: explant is invaded Agrobacterium rhizogenes bacterium liquid respectively, infect 20～30min in the dark; The explant that will infect places (the medium pH value is 5.0～6.0) on MS+NAA (0.05mg/L)+Pro 700mg/L substratum, cultivates altogether 2～5 days in 18 ℃～20 ℃; Move on 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+50～100m mol/L Syringylethanone substratum, illumination cultivation 2～5 days, the low light level is cultivated and is induced hairly root; The hairly root of inducing generation is inserted antibacterial, screening culture medium 1/2MS+Cef (500mg/L)+Km (50mg/L) keep the low light level to cultivate, switching is once transferred after 3～5 times repeatedly weekly; Downcut the tip of a root of 2～5mm of anti-Km root system, insert the liquid nutrient medium enlarged culturing.
4, strain is screening and fluid enlargement culture: select the hairly root strain rapid, that branch is more of growth to be, cut long 2～4mm tip of a root partly, place the 1/2MS liquid nutrient medium, the 50ml nutrient solution is packed in the 150ml triangular flask under the low light level or the dark culture condition, 50～80rpm/min shaking culture on shaking table, 25 days subcultures once.Add elicitor and precursor substance in the optimization substratum and help the accumulation of Radix Rhodiolae secondary metabolite.Elicitor is glossy ganoderma or rainbow conk or aspergillus niger or Mucor, and concentration is 50mg/L～100mg/L; Precursor substance is tyrosol or tyrosine or phenylalanine, and concentration is 0.8～1.8mmol/L.Wherein elicitor with aspergillus niger for well, precursor with tyrosol for well.
5, salidroside content is measured in the hairly root: adopt high effective liquid chromatography for measuring.Its result is: the hairly root by Agrobacterium rhizogenes induces Radix Rhodiolae to obtain, can rise in value about 25 days 15～28 times.Salidroside content is 3.58% in the hairly root dry product, is 5 times of wild Changbai Mountain Radix Rhodiolae root approximately, 7 times of Tibet Radix Rhodiolae.Radix Rhodiolae growth of hair root amount maximum can reach 398.21g/L; The rhodioloside accumulation volume can reach 14.26g/L.Increase significantly than 0.59% of wild Changbai Mountain Radix Rhodiolae root.
6, the separation-extraction technology of rhodioloside in the Radix Rhodiolae hairly root: get Radix Rhodiolae hairly root culture in 60 ℃ of oven dry of air cycle moisture eliminator, dry powder is ground into 20 purpose meal with pulverizer, use 70% extraction using alcohol, or directly use 70% alcohol-pickled fresh hairly root culture, extract rhodioloside.Present method is 3.0%～3.50% of a hairly root dry weight by the content of the rhodioloside that obtains in the hairly root culture, and total secondary metabolite productivity is 14.06g/L.
The abbreviation term that relates among the present invention, substratum:
1. the 1.0cm that the aseptic seedling of explant---Radix Rhodiolae is cut into
2Blade, 1cm long shoot section and cotyledonary node.
2. Agrobacterium rhizogenes bacterial classification---R1000, R1601, ATCC15834, A
4, LBA9402 is by French gardening research centre (doctor Tepter is so kind as to give).
3. Agrobacterium rhizogenes substratum: LB, YEB
The LB liquid nutrient medium is: 10g/L peptone, 5g/L yeast extract, 10g/LNaCl, adjust pH to 7.2 back autoclaving.The LB solid medium adds the 15g/L agarose on above-mentioned basis.
The YEP substratum is: 10g/L peptone, 10g/L yeast extract, 5g/L beef extract, 15g/L agarose, adjust pH to 7.0 back autoclaving.
4. plant base basal culture medium: MS (Murashige and Skoog, 1962), 50mlMS macroelement (20
*), 5ml MS trace element (200
*), 5ml Fe salt (200
*), 10ml V VITAMIN (100
*) (V
B11000mg, V
B6100mg, nicotinic acid 100mg, glycine 200mg, inositol 10g, vitamin H 5mg.Be settled to 1000ml, be made into mother liquor), 0.2g casein, 0.2g asparagine, 0.5mg benzaminic acid, 0.25mg riboflavin.Folic acid 50mg is settled to 100ml with small amount of N aOH dissolving, and every liter of substratum is got 1ml (0.5mg/ml).6.5g/L agar, pH 5.8～6.0.
5. microbiotic: kantlex Km (Kanamycin), cephamycin C ef (cephamycin)
6. hormone: 2,4-D (2,4-Dichlorophenoxyacetic acid), NAA (Naphthaleneacetic acid), 6-BA (benzyladenine)
7. other reagent: Syringylethanone AS (3,5-mcthoxy-4-hydroxyacetophenone), proline(Pro) Pro (proline), rhodioloside (salidroside)
The effect that the present invention is useful is embodied in:
L, induce and culture technique solves Radix Rhodiolae shortage of resources problem by hairly root, for Chinese medicine Root of Kirilow Rhodiola source hews out an effective way.Radix Rhodiolae is the state guarantee plant, has important pharmaceutical use, utilizes a large amount of culture techniques of hairly root to produce rhodioloside, can protect limited wild resource, is fit to industrialized production, has practicality.
2, use the Radix Rhodiolae hairly root and cultivate in a large number and produce rhodioloside and compare with wild plant with cultivation, can obtain stable quality and output, be not subjected to the restriction of natural condition, the area that do not occupy cultivated land only need take limited factory building and culturing room.
3, optimize culture condition by artificial regulatory, can improve the growth of hairly root and synthesizing of secondary metabolite, improve the output of rhodioloside greatly.
4, this technology is not added any hormone in a large amount of culturing process of hairly root liquid, and therefore growth fast, can reduce cost greatly in dark culture environment.
Description of drawings
Fig. 1 is a Radix Rhodiolae aseptic seedling synoptic diagram.
Fig. 2 is the hairly root synoptic diagram that Agrobacterium rhizogenes is induced generation.
Fig. 3 is hairly root quick growth synoptic diagram on solid medium.
Fig. 4 is that the PCR of hairly root rol gene detects synoptic diagram.
Fig. 5 is the Radix Rhodiolae hairly root synoptic diagram of liquid culture.
Fig. 6 is a hairly root liquid nutrient medium growth curve synoptic diagram.
Embodiment
Embodiment
The acquisition of 1 aseptic seedling
Get the Radix Rhodiolae seed that picks up from 1700～2500 meters of Changbai Mountain height above sea level, clean with tap water flushing, in 75% alcohol, soak 1min after, again with the 15%NaClO 12min that sterilizes.Use aseptic water washing then 3～5 times, the empty dried 1/2MS+Pro700mg/L substratum (agar 0.6%) that is seeded in no hormone of water is gone up to sprout cultivated 10～18 days.25 ℃, light intensity 2000lux, illumination in 16 hours, 8 hours dark cultivations.Treat that 15 days aseptic seedling grow, the green cotyledon (staying the cotyledon petiole about 2mm) of getting 3 days seedling ages places on the MS+0.5mg/LNAA+3.0mg/L 6-BA substratum succeeding transfer culture as shown in Figure 1.
2 cultivate in advance
Get cotyledonary node, stem section and the blade of the aseptic seedling of succeeding transfer culture, the cotyledon dorsal ventral side of blade and band petiole rows dry with cutter, the stem section is cut into the long explant of doing of 1cm, be seeded in MS+1.2mg/L2,4-D+2.4mg/L pre-the cultivation 2～3 days on the 6-BA+Pro 700mg/L substratum treats that the edge slightly is used to infect during the callus phenomenon.
3 Agrobacterium rhizogenes are cultivated
Agrobacterium rhizogenes ATCC15834 is seeded on the YEB solid medium streak culture, in 27 ℃ of down activation 3 times, single bacterium colony on the picking flat board is seeded in 25ml LB liquid nutrient medium.At 27 ℃, shaking table concussion is cultivated under the 4000r/min dark, behind the subculture 3 times, and Agrobacterium rhizogenes bacterium liquid A
600Be to be used to infect explant at 0.45 o'clock.
The infecting of 4 Agrobacterium rhizogenes, cultivation, degerming and screening altogether
Above-mentioned pre-incubated explant immersed respectively be in logarithmic phase bacterium liquid A
600Be in 0.45 the Agrobacterium rhizogenes ATCC15834 bacterium liquid, infect 24min in the dark, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, place cultivate 2 days altogether on MS+NAA (0.05mg/L)+Pro 700mg/L substratum after, move on the 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+60m mol/L Syringylethanone substratum, in 20 ℃, the scattering low light level is cultivated according to degerming.Changeed a subculture in per 5 days.And be contrast with Radix Rhodiolae cotyledon, stem section and the cotyledonary node that infects without Agrobacterium rhizogenes.Produce hairly root as shown in Figure 2 about 15 days.Cut and move on on the new substratum 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L infecting the hairly root that grows on the explant of back, transfer weekly 1 time, after degerming is finished, move on to not contain on the antibiotic 1/2MS substratum and cultivate as shown in Figure 3.
The screening of 5 hairly root high-quality strains system
Select the hairly root that degerming is thorough, process PCR detects, branch is many, the fast hairly root of growth is cut about long 1～3cm, in the immigration 1/2MS+Pro 700mg/L nutrient solution.In the 150ml Erlenmeyer flask, every bottled liquid 50ml inserts a hairly root.Observe growing state after 25 days, select eugonic be used for subculture and a large amount of the cultivation.
The detection of 6 hairly root
The genomic dna trace extracts
1) get 0.5g～1.0g and under liquid nitrogen, grind through the hairly root that screens, and the 50ml centrifuge tube of packing into adding 20ml Extraction Buffer (100mM Tris-HCl, PH 8.0; 0.35mMSorbitol; 5mM EDTA, PH 8.0; 1%2-Mercaptoethanol (use before add)) and place on ice.
2) 4 ℃, centrifugal 10 minutes of 10000r/min removes supernatant, with twice of 20ml ExtractionBuffer dissolution precipitation and centrifugal.
3) go supernatant also to suspend and precipitate with 5ml Extraction Buffer, and adding 3.5mlHigh-Salt CTAB Buffer (50mM Tris-HCl, PH 8.0; 4M NaCl; 1.8%CTAB; 25mM EDTA, PH 8.0) and 0.3mlSarkosyl (concentration is 30% the aqueous solution), 55 ℃ of incubations 60～90 minutes.
4) add isopyknic chloroform: primary isoamyl alcohol (24: 1), centrifugal 10 minutes of 10000r/min.
5) go supernatant to add the Virahol that is mixed with 1/10 volumes of acetic acid sodium of 2/3 volume precooling.4 ℃, centrifugal 20 minutes of 10000r/min.
6) remove supernatant, washing with alcohol precipitation with cold 75%.Remove ethanol, at air drying DNA, and with 200 μ lTE Buffer (10mM Tris, PH 8.0; 1mM EDTA, PH8.0) dissolving.
7) add 10 μ lRnase (1mg/ml) 37 ℃ of following incubations 40 minutes, add isopyknic phenol chloroform, protein is removed in extracting.High speed centrifugation 10 minutes (preferably more than the 13000r/min) under the room temperature.
8) get 100% the chloroform that supernatant adds the equal-volume precooling, precipitation is removed protein.High speed centrifugation is 10 minutes under the room temperature.
9) get the cold dehydrated alcohol (sodium acetate that mixes 1/10 volume) that supernatant adds two volumes, deposit D NA then it is put in-20 ℃ following 30 minutes.
10) use maximum speed of revolution deposit D NA 15 minutes.Cold washing with alcohol DNA precipitation with 75%, and at air drying.With 50～00 μ l TE Buffer dissolving DNAs.
The PCR of hairly root detects
The genomic dna of hairly root 100ng of screening of learning from else's experience is a template, with the test-tube plantlet normal root as negative control, the positive contrast of Ri plasmid.The primer sequence of rolC gene (precious biosynthesizing):
Primer I sequence 5`-GATATATGCCAAATTTACACTAG-3`
Primer I I sequence 5`-GTTAACAAAGTAGGAAACAGG-3`
The PCR reaction totally is 50ul
Taq Mix (available from precious biological) 25ul
Primer I 2 (20pmol) ul
Primer I I 2 (20pmol) ul
Template 5 (100ng) ul
Deionization sterilized water 16ul
(UNOII reacts in Biometra), and response procedures is 94 ℃, 45s at the PCR instrument; 45 ℃, 30s; 72 ℃, 45s is totally 30 circulations, and 72 ℃ prolong 10min, as shown in Figure 4.
The mensuration of 7 growth of hair root curves:
Selecting eugonic hairly root strain is to insert in the 1/2MS nutrient solution.In the 150ml Erlenmeyer flask, every bottled liquid 50ml inserts hairly root fresh weight 0.390g (dry weight 0.041g), totally 30 bottles.Fresh weight of per 5 days mensuration and dry weight are that index is drawn growth curve with the weight in average.Fresh weight system is mensuration during to anhydrous dripping with the B suction filtration, and dry weight is to record as shown in Figure 6 after 60 ℃ of oven dry.
The liquid nutrient medium of 8 hairly root is cultivated in a large number:
Select eugonic hairly root strain system, insert the 150ml triangle and cultivate in the 1/2MS+Pro 700mg/L liquid nutrient medium of bottled liquid 50ml as shown in Figure 5.Wherein carbon source is sucrose or fructose or glucose (30g/L); NH
4 +And NO
3 -Be nitrogenous source, total nitrogen concentration is 80mmol/L; Illumination is the 2000xl scattered light; Temperature is 18 ℃～20 ℃; The pH value is 6.5～7.2; Elicitor is glossy ganoderma or rainbow conk or aspergillus niger or Mucor, and concentration is 50mg/L～100mg/L; Precursor substance is tyrosol or tyrosine or phenylalanine, and concentration is 0.8～1.8mmol/L.Wherein elicitor with aspergillus niger for well, precursor with tyrosol for well.
The separation and Extraction of 9 hairly root rhodiolosides
Chromatographic condition: C
18Chromatographic column (250mm * 4.6mm, 5 μ m), moving phase is methanol-water (20: 80), flow velocity 1ml/min detects the mensuration that wavelength 274nm is used for rhodioloside.
The preparation of reference substance and need testing solution:
The rhodioloside standard substance are available from Chinese biological goods calibrating institute.
Precision takes by weighing rhodioloside reference substance 5.08mg and puts in the 10ml measuring bottle, adds methyl alcohol to scale, shakes up, and gets rhodioloside reference substance stock solution; Precision is measured this stock solution 1ml and is put in the 10ml measuring bottle, adds methyl alcohol to scale, shakes up and promptly gets rhodioloside reference substance solution (50.8 μ g/ml).Get the about 0.4g of hairly root sample meal, the accurate title, decide, and puts in the Erlenmeyer flask, add methyl alcohol 90ml, supersound extraction 30min behind the immersion jolting 60min is put cold, filter, with a little methanol wash container, filter, filtrate is incorporated the 100ml measuring bottle into, add methyl alcohol to scale, shake up, filter with the 0.45Lm millipore filtration, as need testing solution.
Typical curve: accurate respectively rhodioloside reference substance solution 0.5,1,2,5, the 10ml of drawing, be diluted to methyl alcohol and contain rhodioloside 1.56,4.68,7.8,10.92,15.6 μ g/ml5 kind solution, the drawing standard curve, its regression equation is: Y=34663.4X-2445.1, r=0.9997 (n=5).Rhodioloside is good linear relationship in 0.20～8.00 μ g/ml scope.
Analyzing salidroside content as calculated is 3.58% of hairly root dry weight, is 5 times of wild Changbai Mountain Radix Rhodiolae root approximately, 7 times of Tibet Radix Rhodiolae.Radix Rhodiolae growth of hair root amount maximum can reach 398.21g/L; The rhodioloside accumulation volume can reach 14.26g/L.
The separation-extraction technology of rhodioloside in the 10 Radix Rhodiolae hairly root
Get Radix Rhodiolae hairly root culture in 60 ℃ of oven dry of air cycle moisture eliminator, dry powder is ground into 20 purpose meal with pulverizer, uses 70% extraction using alcohol, or directly uses 70% alcohol-pickled fresh hairly root culture, extracts rhodioloside.Present method is 3.0%～3.50% of a hairly root dry weight by the content of the rhodioloside that obtains in the hairly root culture, and total secondary metabolite productivity is 14.06g/L.
1,2,5,6,7,8,9,10 steps among the following embodiment 2,3,4,5 and embodiment 1 identical omission.
Embodiment 2
3, Agrobacterium rhizogenes is cultivated
With 5 kinds of Agrobacterium rhizogenes LBA9402, be seeded on the YEB solid medium streak culturely, in 27 ℃ of down activation 3 times, single bacterium colony on the picking flat board is seeded in 25ml LB liquid nutrient medium.At 27 ℃, shaking table concussion is cultivated under the 4000r/min dark, behind the subculture 3 times, and Agrobacterium rhizogenes bacterium liquid A
600Be to be used to infect explant at 0.55 o'clock.
4, the infecting of Agrobacterium rhizogenes, cultivation, degerming and screening altogether
Above-mentioned pre-incubated explant immersed respectively be in logarithmic phase bacterium liquid A
600Be in 0.45～0.60 the Agrobacterium rhizogenes LBA9402 bacterium liquid, infect 25min in the dark, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, place cultivate 2.5 days altogether on MS+NAA (0.05mg/L)+Pro 700mg/L substratum after, move on the 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+80m mol/L Syringylethanone substratum, in 20 ℃, the scattering low light level is cultivated according to degerming.Changeed a subculture in per 5 days.And be contrast with Radix Rhodiolae cotyledon, stem section and the cotyledonary node that infects without Agrobacterium rhizogenes.Cut and move on on the new substratum 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L infecting the hairly root that grows on the explant of back, transfer weekly 1 time, after degerming is finished, move on to not contain on the antibiotic 1/2MS substratum and cultivate.
Embodiment 3
3, Agrobacterium rhizogenes is cultivated
5 kinds of Agrobacterium rhizogenes A4 are seeded on the YEB solid medium streak culture, in 27 ℃ of down activation 3 times, single bacterium colony on the picking flat board is seeded in 25ml LB liquid nutrient medium.At 27 ℃, shaking table concussion is cultivated under the 4000r/min dark, behind the subculture 3 times, and Agrobacterium rhizogenes bacterium liquid A
600Be to be used to infect explant at 0.50 o'clock.
4, the infecting of Agrobacterium rhizogenes, cultivation, degerming and screening altogether
Above-mentioned pre-incubated explant immersed respectively be in logarithmic phase bacterium liquid A
600Be in 0.50 the Agrobacterium rhizogenes A4 bacterium liquid, infect 24min in the dark, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, place cultivate 2 days altogether on MS+NAA (0.05mg/L)+Pro 700mg/L substratum after, move on the 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+90m mol/L Syringylethanone substratum, in 25 ℃, the scattering low light level is cultivated according to degerming.Changeed a subculture in per 5 days.And be contrast with Radix Rhodiolae cotyledon, stem section and the cotyledonary node that infects without Agrobacterium rhizogenes.Cut and move on on the new substratum 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L infecting the hairly root that grows on the explant of back, transfer weekly 1 time, after degerming is finished, move on to not contain on the antibiotic 1/2MS substratum and cultivate.
Embodiment 4
3, Agrobacterium rhizogenes is cultivated
5 kinds of Agrobacterium rhizogenes R1601 are seeded on the YEB solid medium streak culture, in 27 ℃ of down activation 3 times, single bacterium colony on the picking flat board is seeded in 25ml LB liquid nutrient medium.At 27 ℃, shaking table concussion is cultivated under the 4000r/min dark, behind the subculture 3 times, and Agrobacterium rhizogenes bacterium liquid A
600Be to be used to infect explant at 0.60 o'clock.
4, the infecting of Agrobacterium rhizogenes, cultivation, degerming and screening altogether
Above-mentioned pre-incubated explant immersed respectively be in logarithmic phase bacterium liquid A
600Be in 0.60 the Agrobacterium rhizogenes R1601 bacterium liquid, infect 25min in the dark, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, place cultivate 3 days altogether on MS+NAA (0.05mg/L)+Pro 700mg/L substratum after, move on the 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+70mmol/L Syringylethanone substratum, in 25 ℃, the scattering low light level is cultivated according to degerming.Changeed a subculture in per 5 days.And be contrast with Radix Rhodiolae cotyledon, stem section and the cotyledonary node that infects without Agrobacterium rhizogenes.Cut and move on on the new substratum 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L infecting the hairly root that grows on the explant of back, transfer weekly 1 time, after degerming is finished, move on to not contain on the antibiotic 1/2MS substratum and cultivate.
Embodiment 5
3, Agrobacterium rhizogenes is cultivated
With 5 kinds of Agrobacterium rhizogenes R1000, be seeded on the YEB solid medium streak culturely, in 27 ℃ of down activation 3 times, single bacterium colony on the picking flat board is seeded in 25ml LB liquid nutrient medium.At 27 ℃, shaking table concussion is cultivated under the 4000r/min dark, behind the subculture 3 times, and Agrobacterium rhizogenes bacterium liquid A
600Be to be used to infect explant at 0.45～0.60 o'clock.
4, the infecting of Agrobacterium rhizogenes, cultivation, degerming and screening altogether
Above-mentioned pre-incubated explant immersed respectively be in logarithmic phase bacterium liquid A
600Be in 0.55 the Agrobacterium rhizogenes R1000 bacterium liquid, infect 30min in the dark, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, place cultivate 3 days altogether on MS+NAA (0.05mg/L)+Pro 700mg/L substratum after, move on the 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L+90mmol/L Syringylethanone substratum, in 25 ℃, the scattering low light level is cultivated according to degerming.Changeed a subculture in per 5 days.And be contrast with Radix Rhodiolae cotyledon, stem section and the cotyledonary node that infects without Agrobacterium rhizogenes.Cut and move on on the new substratum 1/2MS+ kantlex 50mg/L+ cephamycin 450mg/L infecting the hairly root that grows on the explant of back, transfer weekly 1 time, after degerming is finished, move on to not contain on the antibiotic 1/2MS substratum and cultivate.
Claims (1)
1. one kind is utilized Agrobacterium rhizogenes genetic transformation Radix Rhodiolae to set up the method that the hairly root culture system is produced rhodioloside, this method is an explant with Radix Rhodiolae (Rhodiola sachalinensis A.Bor), utilize Agrobacterium rhizogenes (Agrobacterium rhizogenes, Ar) carry out genetic transformation and induce generation hairly root (hairy root), by culture condition optimization and improvement hairly root inductivity; Hairly root by the optimization of carbon source, nitrogenous source, illumination, temperature, pH value, elicitor, precursor substance and nutrient solution, improves the content of Radix Rhodiolae hairly root rhodioloside through fermentor cultivation mass production hairly root; By the separation and Extraction process of hairly root rhodioloside, produce rhodioloside, it is characterized in that: elicitor is any one in glossy ganoderma or rainbow conk or aspergillus niger or the Mucor in the described nutrient solution, and the interpolation concentration of elicitor is 50mg/L～100mg/L; Precursor substance is any one in tyrosol or tyrosine or the phenylalanine, and the interpolation concentration of precursor substance is 0.8～1.8mmol/L.
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