CN104726485A - Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2 - Google Patents
Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2 Download PDFInfo
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Abstract
The invention relates to a method for increasing the contents of tanshinone and salvianolic acid in a salvia miltiorrhiza hairy root by using a transgene AtMYC2, belonging to the technical field of gene engineering. The method comprises the steps of constructing a high-efficiency expression vector of a plant by using an arabidopsis transcription factor AtMYC2, and carrying out genetic transformation on salvia miltiorrhiza leaves to obtain a gene AtMYC2 overexpressed transgenetic salvia miltiorrhiza hairy root; analyzing the expression of AtMYC2 in the transgenetic salvia miltiorrhiza hairy root and related genes in biosynthetic pathways of tanshinone and salvianolic acid through qRT-PCR; measuring the contents of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a high-performance liquid chromatography (HPLC); and measuring the antioxidant activity of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a DPPH free radical scavenging method. The invention provides the method for simultaneously increasing the contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root and also provides a novel high-quality raw material for producing tanshinone and salvianolic acid with important clinic demands so as to have the positive promoting significance and application value for relieving the problem that the drug resources of tanshinone and salvianolic acid are short.
Description
Technical field
The present invention relates to a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza, belong to gene engineering technology field.
Background technology
Cardiovascular and cerebrovascular diseases threatens universe's health and life " number one killer " at present.According to statistics, cardiovascular and cerebrovascular diseases is died from the whole world every year nearly 1,700 ten thousand people, accounts for 1/3 of the total death toll in the whole world; China every year nearly 3,000,000 people dies from cardiovascular and cerebrovascular diseases.Therefore the clinical medicine of the treatment cardiovascular and cerebrovascular diseases of efficient, the low toxicity of active research and cheapness has very profound significance to raising level of human health.
The red sage root (Salvia miltiorrhiza Bunge) is Labiatae (Lamiaceae) Salvia (Salvia) per nnial herb, its root look red shape, like ginseng gain the name " red sage root ", is the conventional Chinese medicine of clinical treatment Cardial or cerebral vascular diseases.Effective component in red sage mainly comprises fat-soluble tanshinone component and water-soluble phenolic acrylic component, wherein TANSHINONES anti-oxidant, anti-inflammatory is antibacterial and antitumor etc. in there is significant curative effect, also there is anticoagulant, improve hypoxia-bearing capability, improve the pharmacological actions such as coronary artery blood supply; Salvianolic acid has the pharmacological actions such as anti-hepatic fibrosis, atherosclerosis, Improving memory dysfunction, and clinical application potentiality are very huge.Except for except pharmaceutical industries (FUFANG DANSHEN DIWAN, Radix Salviae Miltiorrhizae Injection, Compound Daushen Lozenge etc.), start to penetrate in the other fields such as health products trade (red rooted salvia etc.), cosmetic industry, have broad application prospects.And at present TANSHINONES, salvianolic acid depend on to extract from the red sage root and obtain.But due to red sage root cultivated since long quality deterioration serious; Active component content reduces; And growth cycle is long and restrict by the factor such as cultural area and environment.Therefore, in order to alleviate the sex chromosome mosaicism in short supply in TANSHINONES and the salvianolic acid medicine source with important clinical demand, a kind of novel method that simultaneously can improve TANSHINONES and salvianolic acid content is invented.
Prior art shows, in Arabidopis thaliana, bHLH class transcription factor MYC2 (AtMYC2) has vital role in the process such as to grow of regulating plant.But do not produce TANSHINONES and salvianolic acid class material in Arabidopis thaliana, whether AtMYC2 has promoter action to the synthesis of TANSHINONES and salvianolic acid and there is not yet open report.
The present invention utilizes genetic engineering means, Arabidopis thaliana transcription factor gene AtMYC2 genetic transformation red sage root blade is obtained the transgenosis Hairy Root Cultures of Salvia miltiorrhiza of AtMYC2 process LAN, raise the biosynthesizing of TANSHINONES and salvianolic acid simultaneously, obtain the Hairy Root Cultures of Salvia miltiorrhiza strain of TANSHINONES and the equal high yield of salvianolic acid, for commercially producing TANSHINONES and salvianolic acid provides novel high-quality medicine source.Not yet find the relevant report improving Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES and salvianolic acid content with the AtMYC2 gene overexpression strategy mentioned by present subject matter at present.Therefore, the problem that the present invention solves TANSHINONES and salvianolic acid medicine source property in short supply in reality has positive effect.
Summary of the invention
Object of the present invention is exactly provide a kind of to overcome defect that above-mentioned prior art exists to turn the method that AtMYC2 gene improves TANSHINONES and salvianolic acid content in Hairy Root Cultures of Salvia miltiorrhiza.
Object of the present invention can be achieved through the following technical solutions:
The present invention clones and isolates the AtMYC2 gene of 1872bp from Arabidopis thaliana, builds plant expression vector, and with Agrobacterium rhizogenes C58C1 for mediation, genetic transformation red sage root blade obtains hairly root; The integration of PCR testing goal Gene A tMYC2; QRT-PCR analyzes and inserts goal gene AtMYC2 and TANSHINONES and the expression of salvianolic acid biosynthesis related genes in hairly root; The content of TANSHINONES and salvianolic acid in high-performance liquid chromatogram determination transgenic hairy root; The anti-oxidant activity of TANSHINONES in DPPH scavenging free radicals measuring transgenosis Hairy Root Cultures of Salvia miltiorrhiza.
Turn the method that AtMYC2 gene improves TANSHINONES and salvianolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, comprise the following steps:
(1) adopt gene clone method to clone from Arabidopis thaliana and obtain transcription factor gene AtMYC2, described AtMYC2 gene order is the DNA sequence dna of SEQ ID:1;
(2) AtMYC2 genes being operational is connected in expression regulation sequence, forms the plant expression vector containing AtMYC2 gene;
(3) by the plant expression vector transforming agrobacterium rhizogenes containing AtMYC2 gene, the agrobacterium rhizogene strain containing AtMYC2 gene plant expression vector for transforming the red sage root is obtained;
(4) the agrobacterium rhizogene strain genetic transformation red sage root blade constructed by utilization, the transgenic hairy root obtained through PCR test positive is cloned;
(5) quantitative RT-PCR measures the relative expression quantity of AtMYC2 gene and TANSHINONES and salvianolic acid biosynthesis related genes in red sage root transgenic hairy root;
(6) the high effective liquid chromatography for measuring red sage root turns TANSHINONES and salvianolic acid content in AtMYC2 gene hairly root, the red sage root transgenic hairy root strain that screening TANSHINONES and salvianolic acid content significantly improve;
(7) DPPH scavenging free radicals method measures the anti-oxidant activity that the red sage root turns TANSHINONES in AtMYC2 gene hairly root.
As preferably, described Agrobacterium rhizogenes is selected from bacterial strain C58C1.
PCR detection method described in step (4) is as follows:
(4.1) specific PCR primers of root of hair locus gene BrolB, kalamycin resistance gene kan is designed respectively;
(4.2) starting the constitutive promoter inserting Gene A tMYC2 expression is CaMV35S promotor;
(4.3) insert gene internal and NOS terminator indoor design upstream and downstream specific primer, carry out DNA cloning;
(4.4) viewed under ultraviolet radiation object band strain, occurs that object band is then positive transgenic Hairy Root Cultures of Salvia miltiorrhiza strain.
Step (5) described quantitative RT-PCR detecting method is as follows:
(5.1) extraction that positive hairly root clone carries out total serum IgE is accredited as to PCR, the unified cDNA quantitatively becoming 25 μ l systems to 0.5 μ g RNA reverse transcription;
(5.2) primer of goal gene and house-keeping gene Actin is inserted in design respectively, with the cDNA of identical amount for template carries out quantitative RT PCR analysis;
(5.3) the relative expression quantity situation of analyzing gene AtMYC2 and TANSHINONES and salvianolic acid biosynthesis related genes.
High performance liquid chromatography described in step (6) is as follows:
(6.1) TANSHINONES: chromatographic column C-18 reverse phase silica gel post, moving phase is acetonitrile and the water of volume ratio 65:35; Determined wavelength 220nm, column temperature 30 DEG C, flow velocity 1ml/min, sample size 20 μ l;
(6.2) salvianolic acid: chromatographic column C-18 reverse phase silica gel post, moving phase is acetonitrile and the water of volume ratio 30:70, and with phosphorus acid for adjusting pH to 2.03; Determined wavelength 281nm, column temperature 35 DEG C, flow velocity 1ml/min, sample size 20 μ l.
In step (7), the measuring method of the anti-oxidant activity of TANSHINONES and salvianolic acid is:
(7.1) red sage root turns in AtMYC2 gene hairly root and extracts TANSHINONES and salvianolic acid;
(7.2) be concentration gradient by step (7.1) gained diluted sample: 0.25 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, each 1ml of methanol solution of 2 μ g/ml, 4 μ g/ml;
(7.3) get step (7.2) gained solution 200 μ l, add the DPPH solution of 800 μ l 0.1mM respectively, fully mix;
(7.4) at step (7.3) products therefrom 25 DEG C, 30min is placed in darkroom;
(7.5) step (7.4) product 517nm place under spectrophotometer is surveyed absorption value;
Wherein, DPPH free radical scavenging activity (%)=[(A
0-A
1)/A
0× 100],
In formula, A
0it is the absorbance of control sample (DPPH); A
1it is the absorbance of sample or standard substance.
Integrated application biology of the present invention and gene engineering method as vector construction, genetic transformation, Molecular Detection, quantitatively qRT-PCR analyze, the extraction of TANSHINONES and salvianolic acid and assay etc., invented a kind of method utilizing Arabidopis thaliana AtMYC2 transcripton simultaneously to improve two large active component TANSHINONES and salvianolic acid in the red sage root.In the process LAN AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza that the present invention obtains, total tanshinone (dihydrotanshinone, Cryptotanshinone, Tanshinone I, Tanshinone II A) content significantly improves, wherein the highest strain of TANSHINONES content is 14.06mg/g DW, is 6.45 times of control group (2.18mg/g DW); This strain salvianolic acid content is 95.90mg/g DW simultaneously, is 4.30 times of control group (22.32mg/g DW).The present invention is a large amount of production TANSHINONES of commercialization and provides possibility, also for the heavy demand of a large amount of production TANSHINONES and salvianolic acid clinical medicine provides important sources with salvianolic acid and reduction drug price.
Compared with prior art, the present invention has the following advantages:
1, the content of total tanshinone and salvianolic acid in Hairy Root Cultures of Salvia miltiorrhiza can be significantly improved simultaneously.
2, inventive method is reliable for effect.
3, the cost obtaining TANSHINONES and salvianolic acid is low.
4, production process non-environmental-pollution.
Accompanying drawing explanation
Fig. 1 is pCAMBIA2300
+:: AtMYC2 vector construction schematic diagram.
Fig. 2 is the PCR qualification result figure of transgenic hairy root.Be the PCR detection of AtMYC2 gene above Fig. 2, below is hairly root rolB gene identification figure.M, DL-2000 Marker (100 – 2,000bp); PC, positive control (pCAMBIA2300
+:: AtMYC2 plasmid is template); NC, negative control (hairly root that the empty Agrobacterium C58C1 genetic transformation red sage root obtains).
Fig. 3-1 is the expression analysis result figure of AtMYC2 in transgenosis Hairy Root Cultures of Salvia miltiorrhiza;
Fig. 3-2 is the expression analysis result figure of TANSHINONES biosynthesis related genes;
Fig. 3-3 is the expression analysis result figure of salvianolic acid biosynthesis related genes; Empty vector is empty carrier pCAMBIA2300
+the hairly root that the genetic transformation red sage root obtains.
Fig. 4-1 is the content detection result figure of TANSHINONES in AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza; Wherein DH-TI is dihydrotanshinone, and CT is Cryptotanshinone, and T-I is Tanshinone I, and T-IIA is Tanshinone II A, and TT is total tanshinone content.
Fig. 4-2 is the content detection result figure of salvianolic acid in AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza; Wherein CA is coffic acid, and RA is rosmarinic acid, and SA A is salvianolic acid A, and SA B is salvianolic acid B, and TS is total salvianolic acid, and Empty vector is empty carrier pCAMBIA2300
+the hairly root that the genetic transformation red sage root obtains.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the such as condition described in molecular cloning (Sambrook etc.), or the condition of the reagent provided according to manufacturer or the incidental specification sheets suggestion of test kit.
Embodiment 1
The clone of Arabidopis thaliana AtMYC2 gene
1.1. the extraction of Arabidopis thaliana total serum IgE
Take a morsel Arabidopis thaliana young leaflet tablet, and after liquid nitrogen flash freezer, grind rapidly with mortar, the RNAprep Pure Plant Kit working instructions then provided according to TIANGEN company extract total serum IgE.With plain agar sugar gel electrophoresis (deposition condition: gum concentration 1.2%; 0.5 × TBE electrophoretic buffer; 150v, 15min) detect the integrity of RNA.Its purity and concentration is detected with Nano Drop 2000c ultramicrospectrophotometer.
1.2. the clone of Arabidopis thaliana AtMYC2 gene
With 0.5 obtained μ g Arabidopis thaliana total serum IgE for initial amount, carry out the synthesis (instructions book that operation steps provides with reference to Promega company) of the first chain cDNA with ThermoScript II XL (AMV).According to the encoding sequence (SEQ ID NO.1) of described AtMYC2 gene, design amplifies the upstream and downstream primer of complete encoder block, and on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the first described chain cDNA for template, check order after pcr amplification.Determined dna sequence is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing result shows, the sequence of cloning is completely the same with the AtMYC2 encoding sequence (SEQ ID:1) announced on NCBI.
Embodiment 2
Containing the structure of the plant over-express vector of Arabidopis thaliana AtMYC2 gene
Vector construction mode chart is shown in Fig. 1.With pCAMBIA2300
+for medial expression vector, replace pCAMBIA2300 with the AtMYC2 gene of cloning from Arabidopis thaliana
+on gus gene.Particularly, Spe I/BstPI double digestion pMD18-T::AtMYC2 and pCAMBIA2300
+; Reclaim AtMYC2 gene and pCAMBIA2300
+large fragment; Connect and transform, picking mono-clonal bacterium colony PCR screening positive clone; Extract the further digestion verification of plasmid.Result shows, AtMYC2 gene is successfully building up to plant expression vector pCAMBIA2300
+in, thus obtain the plant over-express vector pCAMBIA2300 containing AtMYC2 gene
+:: AtMYC2.
Transcription factor AtMYC2 is connected to expression regulation sequence by the present embodiment operably, forms the plant over-express vector pCAMBIA2300 containing AtMYC2 gene
+:: AtMYC2, this expression vector can be used for the content being improved tanshinone in salvia miltiorrhiza bunge and salvianolic acid by metabolic engineering strategies.
With pCAMBIA2300 in the present embodiment
+for plant vector is a kind of preferred implementation, in the process of actual implementation carrier, other suitable carriers can being selected as the case may be, when selecting different carriers, introducing restriction endonuclease sites different.
Embodiment 3
The Arabidopis thaliana AtMYC2 gene genetic of Agrobacterium rhizogenes mediation transforms the red sage root and obtains transgenosis Hairy Root Cultures of Salvia miltiorrhiza
3.1. containing the acquisition of the Agrobacterium rhizogenes engineering bacteria of plant expression vector
By the plant over-express vector pCAMBIA2300 containing AtMYC2 gene in embodiment 2
+:: AtMYC2 proceeds in Agrobacterium rhizogenes C58C1, and picking mono-clonal bacterium colony carries out PCR checking.Result shows, containing the plant over-express vector pCAMBIA2300 of AtMYC2 gene
+:: AtMYC2 has successfully been building up in Agrobacterium rhizogenes C58C1.
3.2. Agrobacterium rhizogenes mediation AtMYC2 gene genetic transforms the red sage root
3.2.1 the preculture of explant
Healthy and strong tests for sterility (the 0.5cm of the clip red sage root
2), be inoculated on precultivation medium (1/2MS), 25 DEG C of light culture 2 days.
3.2.2 the Dual culture of Agrobacterium and explant
By above-mentioned pre-incubated red sage root leaf explant, after putting into 1/2MS suspension immersion 10 minutes (jiggle is that explant fully contacts with bacterium liquid) containing the above-mentioned Agrobacterium rhizogenes engineering bacteria activated, the red sage root blade taken out after contaminating blots surperficial bacterium liquid with aseptic thieving paper, forward in Dual culture substratum 1/2MS, light culture 2-3 days.
3.2.3 the induction of hairly root and succeeding transfer culture
The red sage root explant of above-mentioned Dual culture 2-3 days is transferred in degerming solid medium (1/2MS+Cef500mg/L), about 25 DEG C of light culture 2-3 weeks, hairly root can be grown from explant wound.The red sage root explant of root of hair is transferred on degerming solid medium (1/2MS+Cef300mg/L), 25 DEG C of light culture 2 weeks when hairly root grows to more than 3cm the single hairly root of clip as a clone, continue to be inoculated in except light culture in bacterium culture medium (1/2MS+Cef100mg/L) two weeks, until overflow without Agrobacterium.
3.3. the PCR of transgenosis Hairy Root Cultures of Salvia miltiorrhiza detects
3.3.1 the extraction of transgenic hairy root genomic dna
CTAB method is adopted to extract transgenic hairy root genomic dna.In clip 3.2.3, degerming complete about transgenic hairy root 5cm puts into 1.5mL centrifuge tube, adds 600 μ L CTAB lysates (65 DEG C of preheatings, containing 1% beta-mercaptoethanol), fully grinds with tissue grinder instrument.Be placed in 65 DEG C of water-bath 40-50 minute, repeatedly mix sample (secondary/15min) therebetween, isopyknic phenol/chloroform (1:1) is added after being cooled to room temperature, put upside down mixing emulsification 10min gently, the centrifugal 15min of 1200rpm, careful absorption supernatant is in new EP pipe, add the mixing of isopyknic chloroform, the centrifugal 15min of 12000rpm, slow absorption supernatant is in new EP pipe, add the dehydrated alcohol (putting 30min in-20 DEG C) of 2 times of volume precoolings, separate out precipitation, the centrifugal 15min of 12000rpm, abandon supernatant, add 75% washing with alcohol twice, sucking-off supernatant, room temperature is dried, add 30-50 μ L water dissolution precipitation, with frozen in-80 DEG C of Ultralow Temperature Freezers after RNA ferment treatment, for subsequent use.
3.3.2 design of primers and PCR detect
At pCAMBIA2300
+:: the insertion gene (AtMYC2) of AtMYC2 carrier and NOS terminator design respectively specific upstream and downstream primer (AtMYC2-F1080:5 '-ACCAAGATCCGGCGAGATATTAAAC-3 ', NOS-R:5 '-GCAAGACCGGCAACAGGATTC-3 ').On root of hair locus gene BrolB, design specificity upstream and downstream primer (rolB-F:5 '-GCTCTTGCAGTGCTAGATTT-3 ', rolB-R:5 '-GAAGGTGCAAGCTACCTCTC-3 ') simultaneously.By PCR method, Molecular Detection is carried out to the STb gene of above-mentioned hairly root simultaneously, see Fig. 2.Result shows, all root of hair locus gene can be detected in all hairly root strains, can detect and positive control (pCAMBIA2300 in a part of transgenic hairy root
+:: AtMYC2 plasmid is template) sizable PCR primer.And the genomic dna infecting the hairly root of red sage root gained with the empty bacterium of Agrobacterium C58C1 for template time, do not amplify any fragment.Result illustrates that AtMYC2 gene has been incorporated in red sage root genome.
The present embodiment is by described plant expression vector transforming agrobacterium rhizogenes, obtain the agrobacterium rhizogene strain C58C1 of the plant expression vector for transforming the red sage root, agrobacterium rhizogene strain genetic transformation red sage root blade constructed by utilization, obtains the transgenic hairy root through PCR test positive clone.The acquisition of transgenosis Hairy Root Cultures of Salvia miltiorrhiza is screening high yield TANSHINONES, the hairly root of salvianolic acid provides direct material.
Select Agrobacterium rhizogenes C58C1 to be a kind of preferred implementation in the present embodiment, in actual selection, the bacterial strain of Agrobacterium rhizogenes is not limited to C58C1, can select other bacterial strains as the case may be.
Embodiment 4
QRT-PCR detects the expression of genes involved in transgenosis Hairy Root Cultures of Salvia miltiorrhiza
4.1. hairly root liquid culture
Select the hairly root that in embodiment 3, growth is fast, branch is good, after the agar that clip 2-3cm sterile distilled water rinses out on its surface on Bechtop, access is equipped with the 1/2MS liquid nutrient medium subculture of 100mL once, gather in the crops after 60 days, get after proper amount of fresh hairly root thieving paper blots surface-moisture, with masking foil package enter freezing in liquid nitrogen after be stored in-80 DEG C and extract for RNA, all the other hairly root dry after for the extraction of TANSHINONES salvianolic acid content.
The synthesis of extraction 4.2.RNA and cDNA first chain
Method is with the step 1.1 in embodiment 1
The design of 4.3 primers and synthesis
Encoding sequence according to arabidopsis gene AtMYC2 and TANSHINONES and salvianolic acid biosynthesis related genes designs primer for detecting the expression of genes involved in Hairy Root Cultures of Salvia miltiorrhiza with Primer5.0 respectively, and house-keeping gene Actin is used as internal reference.The primer is synthesized by Shanghai Sheng Gong bio-engineering corporation.
The primer of quantitative PCR is as follows:
4.4. the QRT-PCR of transgenosis Hairy Root Cultures of Salvia miltiorrhiza detects
For template, QRT-PCR amplification is carried out with the primer of above-mentioned design respectively with the above-mentioned cDNA of identical amount (diluting 10 times) the first chain.Specification sheets with reference to the Biosystem StepOne instrument of Applied Biosystem company of U.S. production carries out quantitative PCR operation, adopts the RT-PCR kit of Quan Shi King Company.Quantitative PCR reaction system is as follows:
PCR reaction conditions: 94 DEG C of 5min, 40 circulations (72 DEG C extend 30sec for 94 DEG C of sex change 30sec, 60 DEG C of annealing 30sec), 72 DEG C of 10min.Goal gene and house-keeping gene respectively do three repetitions.
QRT-PCR result display (see Fig. 3-1, Fig. 3-2, Fig. 3-3): the expression amount of AtMYC2 gene in process LAN strain significantly improves, do not express in empty carrier, but the expression amount between different clone has certain difference, 21.69 times that what expression amount was the highest is expression amount is minimum.In genes involved in TANSHINONES biosynthetic pathway, SmDXS2, SmCPS, SmHMGR expression amount has obvious rise, with No. 3 strains, significantly (SmDXS2 expression amount is 3.7 times of control group, SmCPS expression amount is 6.1 times of control group,) SmHMGR up-regulated is apparent that No. 5 strains the most, is 6.1 times of control group; In genes involved in salvianolic acid biosynthetic pathway, SmCYP 98A, SmRAS, Sm4CL1, SmTAT expression amount has obvious rise, 3.72 times (No. 33), 3.31 times (No. 33), 3.69 times (No. 43) of control group, 5.04 times (No. 3) respectively, SmPAL1, SmC4H express slight rise, SmHPPD expression amount then significantly declines, and is only 0.13 times (No. 13) of control group.Result proves that AtMYC2 after process LAN, can promote the expression of most of TANSHINONES salvianolic acid biosynthesis related genes in Hairy Root Cultures of Salvia miltiorrhiza, the expression of suppression SmHPPD, and SmHPPD just salvianolic acid synthesize a key gene on branch road.
According to the literature, MYC2 is by regulating and controlling the expression of downstream target gene in conjunction with the G-box in downstream target gene promotor.Key gene promotor in TANSHINONES, salvianolic acid route of synthesis is analyzed, find on the gene promoter by AtMYC2 response containing G-box, therefore infer that AtMYC2 is by regulating and controlling the expression of key gene in conjunction with G-box, thus the synthesis of regulation and control TANSHINONES and salvianolic acid.
Embodiment 5
HPLC is utilized to measure TANSHINONES, salvianolic acid content in transgenosis Hairy Root Cultures of Salvia miltiorrhiza
5.1. the extraction of TANSHINONES content in hairly root
Hairly root embodiment 4 gathered in the crops is dried to constant weight, and grind into powder, accurately takes 0.2g hairly root powder in 50mL centrifuge tube, add 16mL methyl alcohol: methylene dichloride (3:1, v/v), ultrasonic 60min, room temperature, lucifuge, overnight stand, 12000rpm, centrifugal 10min, draw the 60 DEG C of vacuum-dryings in Rotary Evaporators of supernatant extraction liquid, resistates uses the methyl alcohol (analytical pure) of 2mL to dissolve, by sample with to be measured after the membrane filtration of 0.22 μm again.
5.2. in hairly root, the HPLC of TANSHINONES content measures
Dihydrotanshinone (DH-TI), Cryptotanshinone (CT), Tanshinone I (T-I) and Tanshinone II A (T-IIA) standard substance are configured to respectively the concentration of 1M with analytical pure methyl alcohol, be stored in-20 DEG C for subsequent use.
Chromatographic condition is: C-18 reverse phase silica gel post (Symmetry Shield TM C18,5 μm, 250 × 4.6mm, Waters); Acetonitrile: water (65:35) is moving phase; Column temperature is 30 DEG C; Flow velocity is 1mL/min; Wavelength is 270nm.
Above-mentioned standard substance stock solution is got 5 μ l respectively, 10 μ l, 20 μ l, 30 μ l, 40 μ l sample introduction under corresponding chromatographic condition, four kinds of compositions are separated completely, and peak type is good, record collection of illustrative plates and chromatographic parameter, carry out regression analysis with peak area (Y) to standard concentration (X, mg/mL) respectively.
TANSHINONES crude extract after above-mentioned 0.22 μm of membrane filtration respectively gets 20 μ L, injects high performance liquid chromatograph.Record the peak area of each TANSHINONES component, after substituting into equation of linear regression, calculate and obtain sample TANSHINONES content.
5.3. the extraction of salvianolic acid content in hairly root
Hairly root embodiment 4 gathered in the crops is dried to constant weight, grind into powder, accurately take 0.1g hairly root powder in 50mL centrifuge tube, add 10mL ethanol: water (4:1, v/v), ultrasonic 20min, 8000rpm, centrifugal 10min, draw the 70 DEG C of vacuum-dryings in Rotary Evaporators of supernatant extraction liquid, resistates dissolves with the distilled water of 2mL again, by sample with to be measured after the membrane filtration of 0.22 μm.
5.4. in hairly root, the HPLC of salvianolic acid content measures
Precision takes salvianolic acid A, salvianolic acid B, rosmarinic acid and coffic acid standard substance methyl alcohol and is configured to the standard substance stock solution that concentration is 1M respectively, be stored in-20 DEG C for subsequent use.
Chromatographic condition: chromatographic column is C-18 reverse phase silica gel post, and moving phase is acetonitrile: water (30:70), water phosphoric acid regulates PH to be 2.03, determined wavelength 281nm, column temperature 35 DEG C, flow velocity 1mL/min.
Above-mentioned standard substance stock solution is got 5 μ l respectively, 10 μ l, 20 μ l, 30 μ l, 40 μ l sample introduction under corresponding chromatographic condition, four kinds of water soluble components are separated completely, and peak type is good, record collection of illustrative plates and chromatographic parameter, carry out regression analysis with peak area (Y) to standard concentration (X, mg/mL) respectively.
Salvianolic acid crude extract after above-mentioned 0.22 μm of membrane filtration respectively gets 20 μ L, detects, record each component peaks area with high performance liquid chromatograph, substitutes into equation of linear regression, calculates and obtain the content of sample water soluble components.
In the present invention, 4 kinds of TANSHINONES (dihydrotanshinone, Cryptotanshinone, tanshinone Ⅰ, the tanshinone IIA) content detected in process LAN AtMYC2 Hairy Root Cultures of Salvia miltiorrhiza strain significantly improves compared to control group, wherein No. 3 strain TANSHINONES total amounts reach 14.06mg/g DW, are 6.45 times of control group; Dihydrotanshinone, Cryptotanshinone, Tanshinone I content improve comparatively remarkable, and Tanshinone II A does not then have too large change (see Fig. 4-1).4 kinds of salvianolic acid (coffic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B) content also significantly improve relative to control group, and No. 3 strain salvianolic acid total amounts reach 95.90mg/g DW, are 4.30 times of control group; Coffic acid and content of danshinolic acid B significantly improve, and rosmarinic acid and salvianolic acid A content then do not have considerable change (see Fig. 4-2).
The present embodiment adopts HPLC method to determine the content of transgenosis Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES and salvianolic acid.Result shows that the Hairy Root Cultures of Salvia miltiorrhiza total tanshinone of process LAN AtMYC2 gene and salvianolic acid content significantly improve than control group, and wherein No. 3 strain content raisings is the most obvious.
Embodiment 6
DPPH scavenging free radicals method measures the anti-oxidant activity of TANSHINONES salvianolic acid in transgenic hairy root
6.1. sample solution preparation:
TANSHINONES in embodiment 5 and salvianolic acid crude extract are diluted to following gradient concentration:
The each 1ml of methanol solution of 0.25 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 4 μ g/ml, gets 200 μ l, then adds 1, the 1-phenylbenzene picryl phenylhydrazine solution D PPH of 800 μ l 0.1mM, fully mixes, and dark place is incubated 30min at 25 DEG C.
6.2. the absorbance measurement of sample and the conversion of DPPH free radical scavenging activity:
By the sample in step (1) respectively under spectrophotometer 517nm measure and record its absorption value.Absorbance is converted to DPPH free radical scavenging activity according to formula below:
DPPH free radical scavenging activity (%)==[(A0-A1)/A0 × 100]
A0 is control sample absorbance,
A1 is sample or standard substance absorbance.
6.3.DPPH the comparison of free radical scavenging activity ability:
By method (the Metabolic engineering delivered before us, 2011) carry out DPPH free radical scavenging activity to compare, result shows, AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza strain M3 compares with control group, and the TANSHINONES of same substance amount shows similar radical scavenging activity with salvianolic acid crude extract.And the total tanshinone of sample M3 and salvianolic acid content are 6.45 and 4.3 times of contrast respectively, the total antioxidant activity of interpret sample M3 TANSHINONES and salvianolic acid is significantly higher than control group.
This example demonstrated the TANSHINONES of the same units amount in the Hairy Root Cultures of Salvia miltiorrhiza of process LAN AtMYC2 genetic method acquisition suitable with contrast with the resistance of oxidation of salvianolic acid, the method of the TANSHINONES therefore obtained by the present invention and the salvianolic acid Hairy Root Cultures of Salvia miltiorrhiza of high yield is simultaneously efficiently feasible, has good application prospect.
The present invention adopts the metabolic engineering strategies turning AtMYC2 gene to obtain the red sage root transgenic hairy root strain of high yield TANSHINONES and salvianolic acid simultaneously, is a large amount of production TANSHINONES, salvianolic acid, alleviates TANSHINONES medicine source problem in short supply and provides a kind of novel effective ways.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (6)
1. turn the method that AtMYC2 gene improves TANSHINONES and salvianolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, it is characterized in that, comprise the following steps:
(1) adopt gene clone method to clone from Arabidopis thaliana and obtain transcription factor gene AtMYC2, described AtMYC2 gene order is the DNA sequence dna of SEQ ID:1;
(2) AtMYC2 genes being operational is connected in expression regulation sequence, forms the plant expression vector containing AtMYC2 gene;
(3) by the plant expression vector transforming agrobacterium rhizogenes containing AtMYC2 gene, the agrobacterium rhizogene strain containing AtMYC2 gene plant expression vector for transforming the red sage root is obtained;
(4) the agrobacterium rhizogene strain genetic transformation red sage root blade constructed by utilization, the transgenic hairy root obtained through PCR test positive is cloned;
(5) quantitative RT-PCR measures the relative expression quantity of AtMYC2 gene and TANSHINONES and salvianolic acid biosynthesis related genes in red sage root transgenic hairy root;
(6) the high effective liquid chromatography for measuring red sage root turns TANSHINONES and salvianolic acid content in AtMYC2 gene hairly root, the red sage root transgenic hairy root strain that screening TANSHINONES and salvianolic acid content significantly improve;
(7) DPPH scavenging free radicals method measures the anti-oxidant activity that the red sage root turns TANSHINONES in AtMYC2 gene hairly root.
2. a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, it is characterized in that, the Agrobacterium rhizogenes described in step (3) is selected from bacterial strain C58C1.
3. a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, it is characterized in that, the PCR detection method described in step (4) is as follows:
(4.1) specific PCR primers of root of hair locus gene BrolB, kalamycin resistance gene kan is designed respectively;
(4.2) starting the constitutive promoter inserting Gene A tMYC2 expression is CaMV35S promotor;
(4.3) insert gene internal and NOS terminator indoor design upstream and downstream specific primer, carry out DNA cloning;
(4.4) viewed under ultraviolet radiation object band strain, occurs that object band is then positive transgenic Hairy Root Cultures of Salvia miltiorrhiza strain.
4. a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, it is characterized in that, step (5) described quantitative RT-PCR detecting method is as follows:
(5.1) extraction that positive hairly root clone carries out total serum IgE is accredited as to PCR, the unified cDNA quantitatively becoming 25 μ l systems to 0.5 μ g RNA reverse transcription;
(5.2) primer of goal gene and house-keeping gene Actin is inserted in design respectively, with the cDNA of identical amount for template carries out quantitative RT PCR analysis;
(5.3) the relative expression quantity situation of analyzing gene AtMYC2 and TANSHINONES and salvianolic acid biosynthesis related genes.
5. a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, it is characterized in that, the high performance liquid chromatography described in step (6) is as follows:
(6.1) TANSHINONES: chromatographic column C-18 reverse phase silica gel post, moving phase is acetonitrile and the water of volume ratio 65:35; Determined wavelength 220nm, column temperature 30 DEG C, flow velocity 1ml/min, sample size 20 μ l;
(6.2) salvianolic acid: chromatographic column C-18 reverse phase silica gel post, moving phase is acetonitrile and the water of volume ratio 30:70, and with phosphorus acid for adjusting pH to 2.03; Determined wavelength 281nm, column temperature 35 DEG C, flow velocity 1ml/min, sample size 20 μ l.
6. a kind of method turning TANSHINONES and salvianolic acid content in AtMYC2 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, it is characterized in that, in step (7), the measuring method of the anti-oxidant activity of TANSHINONES and salvianolic acid is:
(7.1) red sage root turns in AtMYC2 gene hairly root and extracts TANSHINONES and salvianolic acid;
(7.2) be concentration gradient by step (7.1) gained diluted sample: 0.25 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, each 1ml of methanol solution of 2 μ g/ml, 4 μ g/ml;
(7.3) get step (7.2) gained solution 200 μ l, add the DPPH solution of 800 μ l 0.1mM respectively, fully mix;
(7.4) at step (7.3) products therefrom 25 DEG C, 30min is placed in darkroom;
(7.5) step (7.4) product 517nm place under spectrophotometer is surveyed absorption value;
Wherein, DPPH free radical scavenging activity (%)=[(A
0-A
1)/A
0× 100],
In formula, A
0it is the absorbance of control sample (DPPH); A
1it is the absorbance of sample or standard substance.
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