CN1699566A - Improvement of Astragaloside content by endogenous gene overexpression technology - Google Patents
Improvement of Astragaloside content by endogenous gene overexpression technology Download PDFInfo
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Abstract
The invention discloses an improvement of Astragaloside content by endogenous gene overexpression technology, wherein the two metabolism reaction coupled key enzyme genes of the polysaccharide metabolic pathway, i.e., ugp gene (uridine diphosphate glucose phosphorylase gene) and gbss gene (adenosine diphosphate glucose glycoside transferase gene) are modified in vitro, so as to reinforce the enhanced expression regulation element and construct intermediate carrier, and electrical perforation method is employed to transfer the expression intermediate carrier pBI-UG into Agrobacterium rhizogenes LBA-9402, thus obtaining polysaccharide synthesize double gene Radix Astragali hairyroot.
Description
Technical field:
The present invention relates to technical field of traditional Chinese medicines.Be specifically related to a kind of method of native gene overexpression technology raising Astragaloside content.
Background technology:
The Radix Astragali (Astragalus membranacius or A.mongholicus) is one of important traditional Chinese medicinal materials assortment of China, has that help is shown admittedly, a diuresis toxin expelling, promoting tissue regeneration and ulcer healing, inducing diuresis to remove edema effect, is the main component of many Chinese medicine compound prescription, patent medicine and healthcare products.Along with the reinforcement of the growing health care idea of people, the demand of the Radix Astragali will increase year by year; But at present the wild resource of the Radix Astragali reduces year by year, and the Cultivar quality descends and faces the puzzlement of problems such as pesticidal contamination and heavy-metal residual, gives clinical and patent medicine processing, outlet bring certain difficulty.Thereby use the output that modern biotechnology improves the Radix Astragali, and improving its quality, the feasible way of exploration suitability for industrialized production has great importance to the production of the Radix Astragali.Cyclosiversioside F be Chinese Pharmacopoeia regulation contain the survey index, if can search out the artificial Radix Astragali product of high Astragaloside content, will have excellent development and application prospect.
Since the eighties, with whole protein peptide spectrum, isozymogram, amino acid sequence analysis, enzyme immunoassay and foranalysis of nucleic acids technology is the Protocols in Molecular Biology of main contents, particularly genetic engineering technique successfully is applied to Chinese medicine production, quality is identified in the improvement, for new vitality has been injected in Chinese medicine production.Induce the hairly root of generation to have growth rapidly with Agrobacterium rhizogenes (Agrobacterium rhizogenesis), do not need to add exogenous hormone, the pathways metabolism that has stock plant, inheritance stability, be easy to advantages such as enlarged culturing, if can increase its active constituent content again, will help realizing suitability for industrialized production.Because the superiority of Agrobacterium rhizogenes Ri plasmid vector system, the research of relevant this respect report is a lot of in recent years, but focus mostly in the importing of individual gene, as discovering of Hughes, the elicitor that to control a kind of glucocorticosteroid of green fluorescent protein (GFP) changes in Vinca (Catharanthus roseus) hairly root, and to be tangible dosage relevant for the content of glucocorticosteroid (dexamethasone) in this elicitor and the hairly root.Hashimoto etc. make carrier with Agrobacterium rhizogenes, the Semen Hyoscyami hydroxylase is imported be rich in the belladonna (Scopolia japonica) of Daturine, and scopolamine content is higher 5 times than wild-type (not containing the Semen Hyoscyami '-hydroxylase gene) hairly root in the hairly root of conversion.But polygenic importing report seldom, and mainly be relevant crop disease and pest resisting and crop, in these reports is directly foreign gene to be imported farm crop, and transgenic crop is grown big Tanaka again, so resulting transfer-gen plant still needs long growth cycle.
Summary of the invention:
Technical problem to be solved by this invention is that a hairly root technology combines with genetically engineered, obtains the synthetic dual-gene Hairy Root of Astragalus membranaceus of commentaries on classics polysaccharide that growth cycle is short, Astragaloside content is high.
The invention provides the method that native gene overexpression technology improves Astragaloside content.
Two key genes relevant that the present invention clones from the Radix Astragali---ugp gene (GenBank accession number: AF281081) with gbss gene (GenBank accession number: AF097922) with the polysaccharide metabolism, should be dual-gene through external modification, increase and strengthen the controlling element (the strong terminator sequence of CaMV 35s strong promoter and Nos-ter) of expressing, successfully construct and express intermediate carrier pBI-UG, to express intermediate carrier pBI-UG by electroporation and change Agrobacterium rhizogenes LBA-9402 over to, the inducing medical plant Radix Astragali obtains Hairy Root of Astragalus membranaceus.
The present invention is to having carried out a series of evaluations such as rolC characteristic primer PCR screening, kalamycin resistance screening, the synthetic dual nested PCR screening of dual-gene characteristic primer of commentaries on classics polysaccharide, Southern blot analysis, RT-PCR, the proteic detection of genetic expression by the Hairy Root of Astragalus membranaceus of aforesaid method acquisition, what prove acquisition is to change the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide.The HPLC-ELSD method is measured the content of Cyclosiversioside F, and the content of finding to change Cyclosiversioside F in the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide is than high nearly 6 times of transgenosis Hairy Root of Astragalus membranaceus not, and is higher 11 times than commodity medicinal material Radix Astragali.Carried out the detection of genetic stability and anabolite stability to changeing the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide, the result shows changes the character that the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide has genetic stability and anabolite stability.
Change the content that the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide can increase substantially Cyclosiversioside F, wherein strain is that 11 and 22 Astragaloside content is respectively 22.24mg/g and 22.13mg/g, being not 6.7 times of transgenosis Hairy Root of Astragalus membranaceus (Astragaloside content is 3.30mg/g) of wild-type, is 12.5 times that Radix Astragali (Astragaloside content is 1.77mg/g) is produced in Shanxi.Because the Astragaloside content of different places of production medicinal material is different, with reference to Chinese Pharmacopoeia
[1]Regulation: Cyclosiversioside F must not be less than 0.4mg/g, and the content of Cyclosiversioside F in the different sources commodity Radix Astragali that collect to measure of Chinese medicine stdn center, Shanghai City: 0.5mg/g～1.9mg/g, can infer that changeing the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide has the ability of good synthetic Cyclosiversioside F.
Cyclosiversioside F be Chinese Pharmacopoeia regulation contain the survey index
[1], have good cardiovascular and cerebrovascular pharmacology activity.Zhang Zhaocai etc.
[8]Find that Cyclosiversioside F can improve myocarditis mouse survival rate, reduce the synthetic and apoptosis of cardiac muscle of collagen, safe and effective; Its anti-apoptosis effect is to delaying or the myocardial fibrosis of reversal of viral myocarditis plays an important role.(Chinese clinical neuroscience, 2000,8 (4): the Cyclosiversioside F that studies have shown that 280-281) can reduce the permeability of hemato encephalic barrier to Luo Yumin etc., is a kind of Protective agent of cerebral tissue.Li Mingya etc. (ACAD J GCP, 1997,13 (4): 219-221) studies show that Cyclosiversioside F is to KCl and CaCl
2Due to the isolated rabbit aortic annulus shrink and the spontaneous rhythm and pace of moving things of the guinea pig right atrium that exsomatizes had restraining effect.Wu greatly just waits that (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2001,26 (12): the cerebral microvascular permeability increase that is caused by histamine that studies show that 850-853) can be suppressed by Cyclosiversioside F.These have shown that all Cyclosiversioside F has good market application prospect, Astragaloside content is 12.5 times of commodity Milkvetch Root in the synthetic dual-gene Hairy Root of Astragalus membranaceus of the commentaries on classics polysaccharide that the present invention obtains, can be used as the extraction starting material of Cyclosiversioside F, have the excellent development prospect.
Description of drawings:
Fig. 1.: plasmid pBI-UG PCR identifies figure
Fig. 2: contain the synthetic dual-gene Agrobacterium rhizogenes LBA9402-UG three-wheel PCR of polysaccharide and identify
Fig. 3: rolC characteristic primer PCR identifies
Fig. 4: kalamycin resistance screening
The dual nested PCR of two pairs of characteristic primers of Fig. 5: ugp, gbss identifies
Fig. 6: change dual-gene Hairy Root of Astragalus membranaceus Southern and identify
Fig. 7: change dual-gene Hairy Root of Astragalus membranaceus RT-PCR and identify
Fig. 8: contain the synthetic dual-gene Hairy Root of Astragalus membranaceus Astragaloside content of polysaccharide and measure
Fig. 9: the HPLC-ELSD method is measured the content of Cyclosiversioside F
Fig. 9 .1: Cyclosiversioside F structure iron;
Fig. 9 .2: Cyclosiversioside F standard substance HPLC figure;
Fig. 9 .3: sample (changeing the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide) Cyclosiversioside F is measured HPLC figure;
Figure 10: change dual-gene Hairy Root of Astragalus membranaceus genetic stability and detect
Embodiment:
1) total RNA extracts (single stage method)
The preparation of reaction reagent: sex change liquid (26mM Trisodium Citrate pH 4.0,0.5% sarcosyls, 0.125M 2 mercapto ethanol, 4M guanidinium isothiocyanate); NaAc (2M, pH 4.7); Phenol: chloroform: primary isoamyl alcohol (25: 24: 1); 70% ethanol.All with the no RNase water preparation of 0.1%DEPC water treatment, Glass Containers all removes RNase to all reagent.
Schedule of operation: get the proper amount of fresh plant tissue and place ceramic mortar, add liquid nitrogen and be ground to fine powder fast, be transferred to mixing in the 1.5ml centrifuge tube that contains 650 μ l sex change liquid; Add 65 μ l 2M NaAc (pH 4.0), put upside down mixing repeatedly; Add 650 μ l phenol again: chloroform: primary isoamyl alcohol, mixing, ice bath 15min; 4 ℃, 12000rpm, centrifugal 20min; Supernatant is transferred in the new 1.5ml centrifuge tube, adds the equal-volume Virahol, puts 30min for-20 ℃ behind the mixing; 4 ℃, 12000rpm, centrifugal 10min precipitated rna; Abandon supernatant liquor, RNA is dissolved in the 0.5ml sex change liquid, 65 ℃ of heating are dissolved as early as possible; Add equal-volume phenol: chloroform: the extracting 1 time again of primary isoamyl alcohol, mixing solutions, promptly repeat preceding step; After centrifugal, abandon supernatant liquor, add 75% pre-cooled ethanol 1ml, the rinsing precipitation, centrifugal the same, abandon supernatant; After the dry air, add 20 μ l deionized water dissolving RNA, spectrophotometry and detected through gel electrophoresis concentration and purity, remaining part-20 ℃ preservation is standby.
2) reverse transcription cDNA
Oligo (dT) (37.5 μ M) 2 μ l, total RNA 10 μ g, ddH
2O 10.5 μ l, 70 ℃ of reaction 10min put on ice immediately, 5 * RT buffer, 4 μ l, RNasin (40U/ μ l) 0.5 μ l, 10mM dNTPs 1 μ l, M-MLV reversed transcriptive enzyme (200U/ μ l) 1 μ l, 42 ℃ of reaction 1.5h, 70 ℃ of reaction 10min deactivation reversed transcriptive enzymes.
3) design of primers
According to the cDNA sequences Design of UGPase that has delivered and GBSS following primer ugp P2:5 '-tcgagccttacaggtcctttgg-3 ', amplimer was to as follows when PxbaI:5 '-tttctaatggccaccgctactgc-3 ' gbss P20:5 '-cctctagatgttgctcttactgctctctc-3 ', P21:5 '-ccgagctctgtggctcaaaatccttctg-3 ' made up dual-gene carrier:
Psf：5’-cagaagtactattccagtatgacg-3’，Psr：5’-tttcatgatctagtaacatagtgacac-3’
4) pcr amplification
cDNA?1μl，Primer?1(5μM)2μl，Primer?2(5μM)2μl，10×Buffer?2μl，MgCl
2(25mM)1.6μl，dNTPs(10mM)0.5μl，Taq(3U/μl)0.3μl，ddH
2O
℃ extension 1.0min → 30 circulation of ℃ annealing 0.5min → 72, ℃ sex change 0.5min → 60,94 ℃ of sex change 5min → 94 is extended 7min for → 72 ℃.
5) connect the pGEM-T carrier
T4DNA damping fluid (10 *) 1 μ l, pGEM easy carrier (50ng/ μ l) 1 μ l, the PCR purified product is an amount of, T4 dna ligase (3U/ μ l) 1 μ l, ddH
2O is with 4 ℃ of placements behind the reaction solution mixing.
6) preparation of competence bacterium
Take out bacterial classification DH5 α from-70 ℃ of cryogenic refrigerators, be inoculated in the LB flat board, 37 ℃ of overnight incubation activate.From activating picking list bacterium colony on the plate, insert 5ml and do not contain in the antibiotic LB liquid nutrient medium 37 ℃ of jolting overnight incubation (250rpm).Change in fresh LB liquid nutrient medium in 1% (V/V) ratio next day, and 37 ℃ of joltings are cultured to OD
600=0.3 (0.3～0.6 also can).Under aseptic condition, 50～100ml nutrient solution is changed in the aseptic centrifuge tube (Falcon 2070 pipes) of two precoolings, leave standstill 10min on ice.4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant.Nutrient solution is flow to end in inversion.The 0.1M CaCl that adds the 10ml precooling
2Solution, resuspension bacterium, ice bath 30min.4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant.The 0.1M CaCl that adds the 2ml precooling
2Solution, the resuspension bacterium is competent cell.
7) conversion and colony screening
In the competent cell that makes, add 4 μ l carriers and connect plasmid DNA, behind the ice bath 30min, in 42 ℃ of heat shock 1.5min, cooled on ice 1～2min; Add SOC substratum 800 μ l, in 37 ℃, the 180rpm shaking table is cultivated 1hr; Get culture 100 μ l shop LB flat board (adding corresponding screening microbiotic), add IPTG again, each 5 μ l of X-Gal, 37 ℃ of constant incubator incubated overnight are according to blue hickie screening positive clone.
8) respectively with primer to gbss characteristic primer P20-P21, ugp characteristic primer PxbaI-P2 the amplification gbss, ugpTemplate 1 μ l, Primer 1 (5 μ M) 2 μ l, Primer 2 (5 μ M) 2 μ l, 10 * Buffer, 2 μ l, MgCl
2(25mM) 1.6 μ l, dNTP (10mM) 0.5 μ l, Taq (3U/ μ l) 0.3 μ l, ddH
2O
→ 94 ℃ of sex change 0.5min → 55 of circulation, ℃ extension 1.0min → 3, ℃ annealing 0.5min → 72, ℃ sex change 0.5min → 45,94 ℃ of sex change 5min → 94 ℃ extension 1.0min → 27, ℃ annealing 0.5min → 72 → 72 ℃ of extensions of circulation 7min.
Order-checking.
9) double digestion (XbaI-SacI)
10 * double digestion damping fluid, 10 μ l, XbaI (10U/ μ l) 3 μ l, SacI (10U/ μ l) 3 μ l, DNA 10 μ g, ddH
2O
37 ℃ of water-bath 1hr take out 75 ℃ of deactivation restriction endonucleases, get 5 μ l electrophoresis and identify, rest part phenol: chloroform: behind primary isoamyl alcohol (25: 24: the 1) purifying, the 3M NaAc that adds 1/10 volume, 2 volume of ethanol deposit D NA, leave standstill 10min after, 12000rpm, 4 ℃, centrifugal 10min, precipitation is dried about 10min with after twice of 70% washing with alcohol, add the dissolving of an amount of sterilized water, carry out with carrier be connected or-20 ℃ of preservations stand-by.
10) extraction of plasmid pBI 121, double digestion
The preparation of reaction reagent: LB liquid nutrient medium; Solution 1 (50mM glucose Glucose, 25mM Tris-HCl, 10mMEDTA, pH 8.0); Solution 2 (0.2M NaOH, 1%SDS); Solution 3 (5M KAc 60ml, HAc 11.5ml, H
2O 28.5ml); Phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixed solution; RNA enzyme (20 μ g/ml); Dehydrated alcohol; 70% ethanol.
Schedule of operation: get 2ml and contain antibiotic LB liquid nutrient medium, add in the good test tube of the ventilation of 15ml, choose a single bacterium colony, 37 ℃, 300rpm, overnight incubation from transforming flat board then; Get the 1.5ml culture, 4 ℃, 12000rpm, centrifugal 0.5min removes supernatant liquor, and precipitation is dry as far as possible; Add 100 μ l solution 1 bacterium that suspends again, concuss; Add 200 μ l solution 2, cover tight lid, put upside down mixing fast, put on ice; Add the solution 3 of 150 μ l ice precooling, cover tight lid, put upside down mixing, put 3～5min on ice; 12000rpm, 4 ℃, centrifugal 5min, supernatant shifts; Add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, the vibration mixing, 4 ℃, 12000rpm, centrifugal 2min shifts supernatant liquor; Add 2 times of volume EtOH in supernatant, room temperature was placed 2 minutes; 12000rpm, 4 ℃, centrifugal 5min; Abandon supernatant, precipitation is dried; Add 1ml 70% washing with alcohol precipitation again, 4 ℃, 12000rpm, centrifugal 5min, precipitation dry air 10min; Add the ddH that 50 μ l contain Pancreatic RNase
2The O dissolving DNA, be stored in-20 ℃ stand-by.
Double digestion is with 9).
11) plasmid and segmental connection of insertion
10 * connect damping fluid 1 μ l, DNA behind the step 9) double digestion purifying, the molar ratio that carrier pBI 121 inserts fragment and carrier is about 2: 1, T4 dna ligase (5U/ μ l) 1 μ l, ddH
2O, 16 ℃ of water-baths are spent the night.
12) transform
In the competent cell that makes, add 4 μ l plasmids and connect product, behind the ice bath 30min, in 42 ℃ of heat shock 1.5min, cooled on ice 1～2min; Add SOC substratum 800 μ l, in 37 ℃, the 180rpm shaking table is cultivated 1hr; Get culture 100 μ l shop LB flat board (adding corresponding screening microbiotic), 37 ℃ of constant incubator incubated overnight, PCR method screening positive clone.
13) respectively with gbss, ugp characteristic primer pcr amplification screening positive clone, method is with 7).
14) extract plasmid pBI-GBSS, pBI-UGP respectively, connect acquisition pBI-UG.Wherein pBI-GBSS is carried out the ScaI enzyme and cut, reclaim endonuclease bamhi.To Psf-Psr, is template with plasmid pBI-UGP with primer, carries out pcr amplification.10 * reaction buffer, 2 μ l, MgCl
2(25mM) 1.6 μ l, dNTPs (10mM) 0.5 μ l, Taq enzyme (5U/ μ l) 0.2 μ l, Psf (5 μ M) 2 μ l, Psr (5 μ M) 2 μ l, Template (plasmid pBI-UGP) 0.5 μ l, ddH
2O.The PCR cycling condition is with 7).
The PCR product is cut glue and is reclaimed, and carries out enzyme with ScaI again and cuts, and reclaims to be connected with carrier pBI-GBSS after enzyme is cut product.Screening positive clone obtains pBI-UG.
Successfully construct double gene expression vector pBI-UG, added the strong terminator sequence of CaMV 35s strong promoter and Nos-ter respectively, the double PCR qualification result is seen Fig. 1.
1:pBI-UG ugp characteristic primer PCR result among the figure; 2:1kb DNA Marker; 3:pBI121 ugp characteristic primer PCR result; 4:pBI121 gbss characteristic primer PCR result; 5:pBI-UG gbss characteristic primer PCR result
15) electroporation transforming agrobacterium rhizogenes
Inoculation Agrobacterium rhizogenes LBA9402 is on the YMB solid medium that contains 100 μ g/ml Rifampins, and 28 ℃ are cultured to single bacterium colony and grow.Choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins, 28 ℃, 250rpm cultivates about 40hr.Inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, and 28 ℃, it is 0.5 that 250rpm is cultured to OD600.After putting on ice 5min, 5000rpm, 4 ℃ of centrifugal 10min.1mM HEPES (pH7.0) washing Agrobacterium precipitation 2～3 times is washed back 5000rpm respectively at every turn, and 4 ℃ of centrifugal 10min, 10% glycerine suspend and precipitate.Get the bacterium 1ml of suspension, add about conversion plasmid 200ng, behind the slight mixing, put 1min on ice.The bacterium liquid of getting the step on the 40 μ l places the electroporation cup of 2mm, add 2500V voltage, 25uF electric capacity, behind the electroporation 10mS, bacterium liquid is transferred in the 0.5ml YMB liquid nutrient medium, be laid on the YMB flat board that contains 50 μ g/ml kantlex behind 25 ℃ of cultivation 2～3hr, cultivate after 3～4 days for 25 ℃ and promptly see single bacterium colony.Choose single bacterium colony and carry out PCR evaluation (method is with 7), positive bacterium colony is inoculated in and contains 100 μ g/ml Rifampins, the YMB liquid nutrient medium of 50 μ g/ml kantlex, 28 ℃, 250rpm, overnight incubation, again bacterium liquid is carried out PCR and identify (the same 1.2.10 of method), determine positive bacteria LBA9402-UG.
1:1kb DNA marker among the figure; 2:GBSS cDNA ORF, about 1.8kb; 3:UGPase cDNA ORF, about 1.4kb; 4.rolC segment, about 570bp
16) change the synthetic dual-gene Agrobacterium rhizogenes of polysaccharide and infect Radix Astragali aseptic seedling
Inoculation Agrobacterium rhizogenes LBA9402-UG is on the YMB solid medium that contains 100 μ g/ml Rifampins and 50 μ g/ml kantlex, and 28 ℃ are cultured to single bacterium colony and grow.Choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins and 50 μ g/ml kantlex, 28 ℃, 250rpm cultivates about 40hr.Inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, and 28 ℃, the 250rpm overnight incubation.Add an amount of Syringylethanone to final concentration 50 μ M, cultivated again one day.Under the aseptic condition, cut the stem and the blade of Radix Astragali aseptic seedling, the synthetic dual-gene Agrobacterium rhizogenes LBA9402-UG bacterium liquid (OD600=0.5) of commentaries on classics polysaccharide in that incision inoculation suspension culture is spent the night is transferred to the MSOH solid medium then, and 25 ℃, dark condition were cultivated 2 days down.Behind the sterilized water washing explant 4～5 times, be transferred to the solid MSOH substratum that contains 500mg/L Claforan, 25 ℃, dark condition are cultivated down and are induced the generation hairly root.
17) hairly root rolC characteristic primer PCR screening
The extraction of hairly root DNA: the total DNA of Hairy Root of Astragalus membranaceus that CTAB method extraction step 16 induces.Get 500～600mg fresh material, the liquid nitrogen porphyrize changes it in the 1.5ml centrifuge tube rapidly to fine powdered then; Add 600 μ l 2%CTAB extracting solutions (60 ℃ of preheatings), mixing is placed 1hr in 60 ℃ of water-baths, and middle gentle mixing several times; The centrifugal 10min of 12000rpm gets supernatant liquor, adds isopyknic water-saturated phenol: chloroform: primary isoamyl alcohol (25: 24: 1), and mixing, 4 ℃ of centrifugal 10min of 12000rpm so repeat 2 times, do not have white precipitate between two liquid; Get supernatant liquor, add isopyknic chloroform: primary isoamyl alcohol (24: 1), mixing, 4 ℃ of centrifugal 10min of 12000rpm; Get supernatant liquor and add 1/10 NaAc and equal-volume Virahol, mixing is placed 1hr for-20 ℃; 4 ℃ of centrifugal 10min of 5000rpm abandon supernatant liquor, precipitate with 70% ethanol rinsing 2 times; Be deposited in the air after the seasoning, be dissolved among the 30 μ lTE.
PCR reaction: the primer of rolC gene: 5 '-GATATATGCCAAATTTACACTAG-3 '
5 '-GTTAACAAAGTAGGAAACAGG-3 ', expection fragment length 577bp.
Template?1μl，Primer?1(5μM)2μl，Primer?2(5μM)2μl，10×Buffer?2μl，MgCl
2(25?mM)1.6μl，dNTP(10mM)0.5μl，Taq(3U/μl)0.3μl，ddH
2O。℃ extension 0.75min → 30 circulation of ℃ annealing 0.5min → 72, ℃ sex change 0.5min → 45,94 ℃ of sex change 5min → 94 is extended 10min for → 72 ℃.
Among the figure 1: positive control (LBA9402-UG); 2: Radix Astragali aseptic seedling; 3: change the ugp Hairy Root of Astragalus membranaceus; 4: change the gbss Hairy Root of Astragalus membranaceus; 5: change dual-gene Hairy Root of Astragalus membranaceus; 6: plasmid pBI-UG; 7:100bp DNA Marker
18) resistance screening of Hairy Root of Astragalus membranaceus
To cultivate in containing the MSOH solid medium of 75mg/L kantlex by the Hairy Root of Astragalus membranaceus that step 17 obtains, the positive strain that screening has kalamycin resistance is.
The left side is for having the positive Hairy Root of Astragalus membranaceus strain system of kalamycin resistance among the figure, can grow on the substratum of kantlex adding; The right side is not for having the negative Hairy Root of Astragalus membranaceus strain system of kalamycin resistance, can not grow adding on the substratum of kantlex
19) change the dual nested PCR screening of the synthetic dual-gene characteristic primer of polysaccharide
The positive strain that step 18 is filtered out is that 17 method is extracted total DNA set by step; Primer carries out the PCR reaction to being respectively ugp characteristic primer PxbaI-P2 and gbss characteristic primer P20-P21.
Among the figure 1,8: positive control (LBA9402-UG); 2,9: Radix Astragali aseptic seedling; 3,10: transgenosis Hairy Root of Astragalus membranaceus not; 4,11: change dual-gene Hairy Root of Astragalus membranaceus; 5,12: change the gbss Hairy Root of Astragalus membranaceus; 6,13: change the ugp Hairy Root of Astragalus membranaceus; 7,14:1kb DNA Marker1--6:ugp characteristic primer PCR result, the about 1.4kb of purpose band; 8--13:gbss characteristic primer PCR result, the about 1.8kb of purpose band
20) the Southern blot that changes the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide analyzes
It is DNA that the CTAB method is extracted positive strain, gets after 15 μ g DNA of plants cut and spend the night with SacI and XbaI (5～10 times excessive) enzyme, and 0.8% sepharose 70V voltage electrophoresis changes film and fixing, molecular hybridization and signal detection.
Among the figure 1,7: change the ugp Hairy Root of Astragalus membranaceus; 2,8; Change dual-gene Hairy Root of Astragalus membranaceus; 3,9: change the gbss Hairy Root of Astragalus membranaceus; 4,10: Radix Astragali aseptic seedling; 5,11: transgenosis Hairy Root of Astragalus membranaceus not; 6,12: positive control (LBA9402-UG)
1--6:gbss feature probe Southern results of hybridization; 7--12:ugp feature probe Southern results of hybridization
21) change the RT-PCR that polysaccharide synthesizes dual-gene Hairy Root of Astragalus membranaceus
The total RNA of plant extracts set by step that l carries out, and synthetic cDNA first chain of reverse transcription 2 carries out set by step, and total RNA application of sample amount is 3 μ g, and PCR reacts with step 5.
Among the figure 1.: transgenosis Hairy Root of Astragalus membranaceus not; 2.: change the gbss Hairy Root of Astragalus membranaceus; 3.: change the ugp Hairy Root of Astragalus membranaceus; 4.: change dual-gene Hairy Root of Astragalus membranaceus; 5.: positive control (LBA9402-UG); 6.: Radix Astragali aseptic seedling
22) the proteic detection of genetic expression
With reference to Elling-Kula
[6]The coupled method of UDPG desaturase that adopts is carried out extraction and the determination of activity of UGPase, with reference to the method for Fujita etc.
[7]Carry out extraction and the GBSS enzyme assay of GBSS.The result shows, the dual-gene strain of 57.6% commentaries on classics is the UGPase activity more than or equal to transgenic line not, and the dual-gene strain of 45.5% commentaries on classics is the GBSS activity more than or equal to transgenic line not; Wherein 33.3% strain ties up on these two indexs all increases to some extent.This presentation of results, part change in the dual-gene strain system, and dual-gene the enhancing on translation skill expressed, and improved the activity of target product (UGPase and GBSS).
Through the evaluation and the detection of step 17～step 22, can determine to obtain to change the Hairy Root of Astragalus membranaceus of polysaccharide synthetic dual-gene (ugp gene and gbss gene).
23) the HPLC-ELSD method is measured the content of Cyclosiversioside F
The extraction of sample and preparation
Sample is pulverized, and crosses 80 mesh sieves, and every kind of sample takes by weighing 3 parts, and every part of 0.300g adds 80% methyl alcohol 10ml, after the soaked overnight, and 80 ℃ of supersound extraction 3 times, each 30min, suction filtration, merging filtrate.Evaporate to dryness filtrate, residue add ddH while hot
2O 5ml dissolving is transferred in the separating funnel, and 5ml water-saturated n-butanol extraction 3 times merges propyl carbinol liquid, 0.15%NaOH washing 1 time is collected propyl carbinol liquid, the evaporate to dryness propyl carbinol, residue is settled to 2ml with dissolve with methanol, shakes up back 0.45 μ m filtering with microporous membrane, promptly gets test liquid.
Chromatographic condition
Chromatographic column: Inertsil ODS-3 (4.6*250mm, 5 μ m); Mobile phase of acetonitrile and water (35: 65); Flow velocity: 1.0ml/min; Column temperature: room temperature; ELSD parameter: vaporization temperature: 100 ℃, atomization temperature: 80 ℃, temperature out: 60 ℃; Flow rate of carrier gas: 1.2ml/min.
The drafting of typical curve
Precision takes by weighing Cyclosiversioside F reference substance 2.31mg, with dissolve with methanol and be settled to 2ml, 0.45 μ m filtering with microporous membrane.Draw filtrate 1,2,5,10,15,20 μ l successively respectively by above-mentioned chromatographic condition analysis, measure peak area.Logarithmic value with reference substance content (μ g) is X-coordinate (X), and the logarithmic value of reference substance peak area (A) is ordinate zou (Y), the drawing standard curve.And regression equation, relation conefficient and linearity range are: Y=1.5856X+0.5769, r=0.9995,1.155～23.100 μ g.
The precision experiment
The accurate Cyclosiversioside F reference substance solution 10 μ l that draw repeat 5 sample introductions and measure, as a result Cyclosiversioside F RSD=1.114% (n=5).
Stability experiment
Same sample is respectively 0,2,4,6,8,10,12,14,16,18,20,22, and the 24h sample introduction calculates content, measures day internal stability, a RSD=0.79% as a result.
Repeated experiment
Sample of the same race takes by weighing 5 parts, and every part of 0.300g presses method extraction and determination Astragaloside content under the 3.2.3 item, calculates the average content 17.352mg/g of Cyclosiversioside F, and RSD is 2.30%.
Rate of recovery experiment
Precision takes by weighing not transgenosis Hairy Root of Astragalus membranaceus sample of 5 parts of known Astragaloside contents, every part of 0.300g, and the accurate respectively Cyclosiversioside F standard substance that add, with method extraction and determination Astragaloside content, recording average recovery rate is 96.72%, RSD=2.04%.
The mensuration of sample
Take by weighing the Shanxi business men taste with discrimination the Radix Astragali medicinal material, the transgenosis Hairy Root of Astragalus membranaceus, change the synthetic dual-gene Hairy Root of Astragalus membranaceus powder of polysaccharide, 3 parts every kind, every part of 0.300g is equipped with test liquid with legal system, by the content of Cyclosiversioside F in the identical chromatographic conditions mensuration test liquid, external standard method is calculated peak area.
Measurement result
The result shows that the synthetic dual-gene Hairy Root of Astragalus membranaceus Astragaloside content of commentaries on classics polysaccharide is generally very high, as strain is that 11 and 22 Astragaloside content is respectively 22.24mg/g and 22.13mg/g, produces Radix Astragali medicinal material (Astragaloside content is 1.77mg/g) apparently higher than non-transgenic Hairy Root of Astragalus membranaceus (Astragaloside content is 3.30mg/g) and Shanxi.
Fig. 9 .1: Cyclosiversioside F structure iron; Fig. 9 .2: Cyclosiversioside F standard substance HPLC figure; Fig. 9 .3: sample (changeing the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide) Cyclosiversioside F is measured HPLC figure; ASI is the abbreviation of Cyclosiversioside F (Astragaloside IV) among the figure
24) detection of the stability of genetic stability and anabolite
Stable growth 15 days (promptly transforming the present age), the synthetic dual-gene Hairy Root of Astragalus membranaceus extraction DNA of the commentaries on classics polysaccharide in 1 year of succeeding transfer culture carry out ugp and gbss characteristic primer PCR after getting conversion respectively; The result show change over to dual-gene after 1 year still stable existence in the transgenic line genome, show that transgenic line has genetic stability.Get respectively and transform the present age and the synthetic dual-gene Hairy Root of Astragalus membranaceus extraction and determination Cyclosiversioside F of the commentaries on classics polysaccharide in 1 year of succeeding transfer culture; Presentation of results transforms the Astragaloside content no significant difference in the present age and the synthetic dual-gene Hairy Root of Astragalus membranaceus of the commentaries on classics polysaccharide of subculture after 1 year, and this result shows, changes the synthetic dual-gene Hairy Root of Astragalus membranaceus of polysaccharide and has the ability of the synthetic Cyclosiversioside F of inheritance stability.
Among the figure 1,9: the dual-gene Hairy Root of Astragalus membranaceus of annual commentaries on classics; 2,10: transgenosis Hairy Root of Astragalus membranaceus not; 3,11: transforming changes dual-gene Hairy Root of Astragalus membranaceus the present age; 4,12: Radix Astragali aseptic seedling; 5,8: positive control (LBA9402-UG); 6,7:1kb DNA Marker.
Claims (1)
1, a kind of native gene overexpression technology improves the method for Astragaloside content, it is characterized in that: comprise the following steps:
1) total Yeast Nucleic Acid extracts:
The preparation of reaction reagent: sex change liquid 26mM Trisodium Citrate pH4.0,0.5% sarcosyl, 0.125M 2 mercapto ethanol, 4M guanidinium isothiocyanate; NaAc2M, pH4.7; Phenol: chloroform: primary isoamyl alcohol 25: 24: 1; 70% ethanol; All with the deoxyribonuclease water preparation of 0.1% diethylpyrocarbonate water treatment, Glass Containers is the stoning ribonuclease T. all for all reagent;
Schedule of operation: get the proper amount of fresh plant tissue and place ceramic mortar, add liquid nitrogen and be ground to fine powder fast, be transferred to mixing in the 1.5ml centrifuge tube that contains 650 μ l sex change liquid; Add 65 μ l 2M NaAc pH4.0, put upside down mixing repeatedly; Add 650 μ l phenol again: chloroform: primary isoamyl alcohol 25: 24: 1, mixing, ice bath 15min; 4 ℃, 12000rpm, centrifugal 20min; Supernatant is transferred in the new 1.5ml centrifuge tube, adds the equal-volume Virahol, puts 30min for-20 ℃ behind the mixing; 4 ℃, 12000rpm, centrifugal 10min precipitation Yeast Nucleic Acid; Abandon supernatant liquor, Yeast Nucleic Acid is dissolved in the 0.5ml sex change liquid, 65 ℃ of heating are dissolved as early as possible; Add equal-volume phenol: chloroform: the extracting 1 time again of primary isoamyl alcohol, mixing solutions, promptly repeat preceding step; After centrifugal, abandon supernatant liquor, add 75% pre-cooled ethanol 1ml, the rinsing precipitation, centrifugal the same, abandon supernatant; After the dry air, add the aseptic two ionized water dissolving Yeast Nucleic Acid that boil off of 20 μ l, spectrophotometry and detected through gel electrophoresis concentration and purity, remaining part-20 ℃ preservation is standby;
2) reverse transcription complementary DNA (cDNA)
Oligomerization deoxythymidylic acid 37.5 μ M 2 μ l, total Yeast Nucleic Acid 10 μ g, aseptic two ionized water 10.5 μ l that boil off, 70 ℃ of reaction 10min put on ice immediately, 5 * reverse transcription damping fluid, 4 μ l, ribonuclease inhibitor 40U/ μ l 0.5 μ l, 10mM picodna ribonucleoside triphosphote 1 μ l, reversed transcriptive enzyme 200U/ μ l 1 μ l, 42 ℃ of reaction 1.5h, 70 ℃ of reaction 10min deactivation reversed transcriptive enzymes;
3) design of primers
Complementary DNA sequence design primer according to UDPglucose pyrophosphorylase gene and adenosine diphosphate glucose glycosides transferase gene
P2：5’-tcgagccttacaggtcctttgg-3’，PxbaI：5’-tttctaatggccaccgctactgc-3’
P20：5’-cctctagatgttgctcttactgctctctc-3’，P21：5’-ccgagctctgtggctcaaaatccttctg-3’
Amplimer is when making up dual-gene carrier:
Psf：5’-cagaagtactattccagtatgacg-3’，Psr：5’-tttcatgatctagtaacatagtgacac-3’
4) polymerase chain reaction (PCR) amplification
Complementary DNA (cDNA) 1 μ l, primer 15 μ M 2 μ l, primer 25 μ M 2 μ l, 10 * damping fluid, 2 μ l, MgCl
225mM1.6 μ l, picodna ribonucleoside triphosphote 10mM 0.5 μ l, deoxyribonucleic acid polymerase 3U/ μ l 0.3 μ l, aseptic two ionized waters that boil off
℃ extension 1.0min → 30 circulation of ℃ annealing 0.5min → 72, ℃ sex change 0.5min → 60,94 ℃ of sex change 5min → 94 is extended 7min for → 72 ℃;
5) connect the pGEM-T carrier:
T4 picodna damping fluid 10 * 1 μ l, pGEM easy carrier 50ng/ μ l 1 μ l, the polymerase chain reaction purified product is an amount of, T4 picodna ligase enzyme 3U/ μ l 1 μ l, aseptic pair boils off ionized water, with 4 ℃ of placements behind the reaction solution mixing;
6) preparation of competence bacterium:
Take out bacterial classification DH5 α from-70 ℃ of cryogenic refrigerators, be inoculated in the LB flat board, 37 ℃ of overnight incubation activate, and from activating picking list bacterium colony on the plate, insert 5ml and do not contain in the antibiotic LB liquid nutrient medium, 37 ℃ of jolting overnight incubation 250rpm, change in fresh LB liquid nutrient medium in the 1%V/V ratio next day, 37 ℃ of joltings be cultured to OD600=0.3～0.6 also can, under aseptic condition, 50～100ml nutrient solution is changed in the aseptic centrifuge tube of two precoolings, leave standstill 10min on ice.4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant.Nutrient solution is flow to end in inversion.The 0.1M CaCl that adds the 10ml precooling
2Solution, the resuspension bacterium, ice bath 30min, 4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant adds the 0.1M CaCl of 2ml precooling
2Solution, the resuspension bacterium is competent cell,
7) conversion and colony screening
In the competent cell that makes, add 4 μ l carriers and connect the plasmid picodna, behind the ice bath 30min, in 42 ℃ of heat shock 1.5min, cooled on ice 1～2min; Add SOC substratum 800 μ l, in 37 ℃, the 180rpm shaking table is cultivated 1hr; Get culture 100 μ l shop LB flat board and add corresponding screening microbiotic, add isopropylthio-again, each 5 μ l of 5-bromo-4-chloro-3-indoles-β-D-galactoside, 37 ℃ of constant incubator incubated overnight are according to blue hickie screening positive clone;
8) respectively with primer to adenosine diphosphate glucose glycosides transferase gene characteristic primer P20-P21, UDPglucose pyrophosphorylase gene expression characteristics primer PxbaI-P2 amplification adenosine diphosphate glucose glycosides transferase gene and UDPglucose pyrophosphorylase gene
Template 1 μ l, primer 15 μ M 2 μ l, primer 5 μ M 2 μ l, 10 * damping fluid, 2 μ l, MgCl
225mM 1.6 μ l, picodna ribonucleoside triphosphote 10mM 0.5 μ l, deoxyribonucleic acid polymerase 3U/ μ l 0.3 μ l, aseptic two ionized waters that boil off
→ 94 ℃ of sex change 0.5min → 55 of circulation, ℃ extension 1.0min → 3, ℃ annealing 0.5min → 72, ℃ sex change 0.5min → 45,94 ℃ of sex change 5min → 94 ℃ extension 1.0min → 27, ℃ annealing 0.5min → 72 → 72 ℃ of extensions of circulation 7min, order-checking;
9) double digestion:
10 * double digestion damping fluid, 10 μ l, restriction endonuclease XbaI 10U/ μ l 3 μ l, restriction endonuclease SacI 10U/ μ l 3 μ l, thymus nucleic acid 10 μ g, aseptic two ionized waters that boil off
37 ℃ of water-bath 1hr take out 75 ℃ of deactivation restriction endonucleases, get 5 μ l electrophoresis and identify, rest part phenol: chloroform: behind 25: 24: 1 purifying of primary isoamyl alcohol, the 3M NaAc that adds 1/10 volume, 2 volume of ethanol bulk deoxidation Yeast Nucleic Acid, leave standstill 10min after, 12000rpm, 4 ℃, centrifugal 10min, precipitation is dried about 10min with after twice of 70% washing with alcohol, add the dissolving of an amount of sterilized water, carry out with carrier be connected or-20 ℃ of preservations stand-by;
10) extraction of plasmid pBI121, double digestion
The preparation of reaction reagent: LB liquid nutrient medium; Solution 150mM glucose, 25mM Tris-HCl, 10mM ethylenediamine tetraacetic acid (EDTA), pH8.0; Solution 20.2M NaOH, 1% sodium lauryl sulphate; Solution 35M KAc 60ml, HAc 11.5ml, aseptic two ionized water 28.5ml that boil off; Phenol: chloroform: 25: 24: 1 mixed solutions of primary isoamyl alcohol; Rnase 20 μ g/ml; Dehydrated alcohol; 70% ethanol;
Schedule of operation: get 2ml and contain antibiotic LB liquid nutrient medium, add in the good test tube of the ventilation of 15ml, choose a single bacterium colony, 37 ℃, 300rpm, overnight incubation from transforming flat board then; Get the 1.5ml culture, 4 ℃, 12000rpm, centrifugal 0.5min removes supernatant liquor, and precipitation is dry as far as possible; Add 100 μ l solution 1 bacterium that suspends again, concuss; Add 200 μ l solution 2, cover tight lid, put upside down mixing fast, put on ice; Add the solution 3 of 150 μ l ice precooling, cover tight lid, put upside down mixing, put 3～5min on ice; 12000rpm, 4 ℃, centrifugal 5min, supernatant shifts; Add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, the vibration mixing, 4 ℃, 12000rpm, centrifugal 2min shifts supernatant liquor; Add 2 times of volume ethanol in supernatant, room temperature was placed 2 minutes; 12000rpm, 4 ℃, centrifugal 5min; Abandon supernatant, precipitation is dried; Add 1ml 70% washing with alcohol precipitation again, 4 ℃, 12000rpm, centrifugal 5min, precipitation dry air 10min; The aseptic two ionized waters dissolving thymus nucleic acids that boil off that add 50 μ l qiagen rnase enzymes, be stored in-20 ℃ stand-by;
Again set by step 9) method double digestion;
11) plasmid and segmental connection of insertion
10 * connect damping fluid 1 μ l, thymus nucleic acid behind the step 9) double digestion purifying, the molar ratio that carrier pBI121 inserts fragment and carrier is about 2: 1, T4 deoxyribonucleic acid ligase 5U/ μ l 1 μ l, aseptic pair boils off ionized water, and 16 ℃ of water-baths are spent the night;
12) transform
In the competent cell that makes, add 4 μ l plasmids and connect product, behind the ice bath 30min, in 42 ℃ of heat shock 1.5min, cooled on ice 1～2min; Add SOC substratum 800 μ l, in 37 ℃, the 180rpm shaking table is cultivated 1hr; Get culture 100 μ l shop LB flat board and add corresponding screening microbiotic, 37 ℃ of constant incubator incubated overnight, PCR method screening positive clone;
13) respectively with adenosine diphosphate glucose glycosides transferase gene characteristic primer and UDPglucose pyrophosphorylase gene expression characteristics primer-oligomerization PCR amplification screening positive clone, set by step 7) method is carried out;
14) extract plasmid pBI-GBSS, pBI-UGP respectively, connect acquisition pBI-UG.Wherein pBI-GBSS is carried out restriction endonuclease ScaI enzyme and cut, reclaim endonuclease bamhi.To Psf-Psr, is template with plasmid pBI-UGP with primer, carries out polymerase chain reaction (PCR) amplification, 10 * reaction buffer, 2 μ l, MgCl
225mM 1.6 μ l, picodna ribonucleoside triphosphote 10mM 0.5 μ l, deoxyribonucleic acid polymerase 5U/ μ l 0.2 μ l, primer 1 Psf 5 μ M 2 μ l, primer 2 Psr 5 μ M 2 μ l, plasmid pBI-UGP0.5 μ l, aseptic pair boils off ionized water, and the polymerase chain reaction cycling condition is identical with step 7).
The polymerase chain reaction product is cut glue and is reclaimed, and carries out enzyme with restriction endonuclease ScaI again and cuts, and reclaims to be connected with carrier pBI-GBSS after enzyme is cut product, and screening positive clone obtains pBI-UG;
Successfully construct double gene expression vector pBI-UG, added the strong terminator sequence of CaMV 35s strong promoter and Nos-ter respectively;
15) electroporation transforming agrobacterium rhizogenes
Inoculation Agrobacterium rhizogenes LBA9402 is on the YMB solid medium that contains 100 μ g/ml Rifampins, 28 ℃ are cultured to single bacterium colony and grow, choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins, 28 ℃, 250rpm cultivates about 40hr, and the inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, 28 ℃, it is 0.5 that 250rpm is cultured to OD600.After putting on ice 5min, 5000rpm, 4 ℃ of centrifugal 10min, 1mM N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid, pH7.0 washing Agrobacterium precipitation 2～3 times, each washing back is 5000rpm respectively, 4 ℃ of centrifugal 10min, 10% glycerine suspends and precipitates, and gets the bacterium 1ml of suspension, adds to transform about plasmid 200ng, behind the slight mixing, put 1min on ice.The bacterium liquid of getting the step on the 40 μ l places the electroporation cup of 2mm, add 2500V voltage, 25uF electric capacity, behind the electroporation 10mS, bacterium liquid is transferred in the 0.5ml YMB liquid nutrient medium, be laid on the YMB flat board that contains 50 μ g/ml kantlex behind 25 ℃ of cultivation 2～3hr, cultivate after 3～4 days for 25 ℃ and promptly see single bacterium colony, choose single bacterium colony and carry out the polymerase chain reaction and identify set by step 7) method carries out, and positive bacterium colony is inoculated in and contains 100 μ g/ml Rifampins, the YMB liquid nutrient medium of 50 μ g/ml kantlex, 28 ℃, 250rpm, overnight incubation, again bacterium liquid is carried out the polymerase chain reaction and identify, determine positive bacteria LBA9402-UG.
16) change the synthetic dual-gene Agrobacterium rhizogenes of polysaccharide and infect Radix Astragali aseptic seedling
Inoculation Agrobacterium rhizogenes LBA9402-UG is on the YMB solid medium that contains 100 μ g/ml Rifampins and 50 μ g/ml kantlex, 28 ℃ are cultured to single bacterium colony and grow, choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins and 50 μ g/ml kantlex, 28 ℃, 250rpm, cultivate about 40hr, inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, 28 ℃, the 250rpm overnight incubation, add an amount of Syringylethanone to final concentration 50 μ M, cultivated again one day, and under the aseptic condition, cut the stem and the blade of Radix Astragali aseptic seedling, the commentaries on classics polysaccharide that spends the night in incision inoculation suspension culture synthesizes dual-gene Agrobacterium rhizogenes LBA9402-UG bacterium liquid OD600=0.5, be transferred to the MSOH solid medium then, 25 ℃, dark condition was cultivated 2 days down, behind the sterilized water washing explant 4～5 times, be transferred to the solid MSOH substratum that contains the 500mg/L Cefotaxime, 25 ℃, dark condition is cultivated down and is induced the generation hairly root.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717782B (en) * | 2009-11-03 | 2012-01-25 | 华南农业大学 | Method for improving biological output and polysaccharide content of lucid ganoderma |
| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN106222163A (en) * | 2016-07-27 | 2016-12-14 | 郑州点石生物技术有限公司 | Solution for extraction of rna and extracting method |
| CN110012836A (en) * | 2019-04-29 | 2019-07-16 | 江苏灵福嘉叶生物科技有限公司 | A method of large-scale production Hairy Root of Astragalus membranaceus |
| CN110100739A (en) * | 2019-06-24 | 2019-08-09 | 延边大学 | A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100367969C (en) * | 2003-07-29 | 2008-02-13 | 上海博泰医药科技有限公司 | Injection formulation of astragalus root saponin and its preparation mehod |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717782B (en) * | 2009-11-03 | 2012-01-25 | 华南农业大学 | Method for improving biological output and polysaccharide content of lucid ganoderma |
| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN106222163A (en) * | 2016-07-27 | 2016-12-14 | 郑州点石生物技术有限公司 | Solution for extraction of rna and extracting method |
| CN110012836A (en) * | 2019-04-29 | 2019-07-16 | 江苏灵福嘉叶生物科技有限公司 | A method of large-scale production Hairy Root of Astragalus membranaceus |
| CN110100739A (en) * | 2019-06-24 | 2019-08-09 | 延边大学 | A method of promoting astragalus root of Radix Astragali Its Isoflavonoid Accumulation |
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