CN105602985A - Method for improving content of salvianolic acid in Salvia miltiorrhiza hairy root through transgenic SmMYB75 - Google Patents
Method for improving content of salvianolic acid in Salvia miltiorrhiza hairy root through transgenic SmMYB75 Download PDFInfo
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Abstract
The invention discloses a method for improving the content of salvianolic acid in Salvia miltiorrhiza hairy root through transgenic SmMYB75. The method comprises the following steps: cloning from Salvia miltiorrhiza through a gene cloning technology to obtain 645bp SmMYB75 gene, and constructing an SmMYB75 gene prokaryotic expression and subcellular positioning vector; and constructing a plant high-expression vector, genetically transforming Salvia miltiorrhiza blades to obtain SmMYB75 gene overexpression Salvia miltiorrhiza hairy root, and screening through a series of biotechnologies to obtain the transgenic SmMYB75 Salvia miltiorrhiza hairy root with obviously improved content of salvianolic acid. The method provides a novel high-quality raw material for production of salvianolic acid meeting important clinic demands, is of positive promotion significance to alleviate the shortage problem of salvianolic acid medicine sources, and has application values.
Description
Technical field
The present invention relates to technique for gene engineering, particularly one turns SmMYB75 gene and improves red phenol in Hairy Root Cultures of Salvia miltiorrhizaThe method of acid content.
Background technology
Cardiovascular and cerebrovascular disease is " the No.1 killer " who threatens at present whole mankind's health and life. According to statistics, the whole world is large every yearApproximately there are 1,700 ten thousand people to die from cardiovascular and cerebrovascular disease, account for 1/3 of the total death toll in the whole world, China annual nearly 3,000,000People dies from cardiovascular and cerebrovascular disease, and therefore, active research is efficient, the clinical medicine of low toxicity and cheap treatment cardiovascular and cerebrovascular diseaseThere is very profound significance to improving level of human health.
The red sage root (Salviamiltiorrhiza), is Labiatae (Labiatae) Salvia perennial herb plant, dry with itRoot or stem tuber be used as medicine, be widely used in treatment cardiovascular and cerebrovascular disease, be a kind of traditional Chinese herbal medicine plant. The red sage rootBioactive ingredients is mainly divided into two large classes: a class is water miscible phenolic acid compound, mainly comprises salviandic acid A, red phenolAcid B, salvianolic acid C, caffeic acid, Rosmarinic acid, danshensu etc.; Another kind of is fat-soluble tanshinone compound, mainComprise Tanshinone I, tanshinone IIA, Cryptotanshinone, dihydrotanshinone etc.
Danshinolic acid in the red sage root is playing an important role aspect prevention human diseases, and its BA mainly comprises antioxygenThe adjusting of change, anticoagulating active, renal function, the protection of liver, cardiovascular protection, anticancer, antibacterial, antiviral, anti-Inflammation etc. In addition, danshinolic acid also has the effect of anti-ischemia-reperfusion, antithrombotic, hypotensive and anti-fibrosis. ?China, the red sage root is made as medicament conventionally, as tablet, capsule, granule, injection, oral liquid, spraying, dripping pill etc.,There is high medicinal and economic worth. But, under traditional cultivation mode, when the red sage root is cultivated for a long time quality deterioration serious,Active component content reduces, growth cycle is long and be subject to the restriction such as cultural area and environment; In chemical synthesis process, pelletThe chemical synthesis process of phenolic acid is very loaded down with trivial details, and cost is higher and easily cause environmental pollution; Pass through thin in vitroThe cell that born of the same parents' cultural method obtains, its medicinal active ingredient accumulation is very low and stability is very poor, does not far reach businessThe requirement that industryization develops. Therefore, in order to alleviate the property problem in short supply in the danshinolic acid medicine source with important clinical demand,Urgently invent a kind of new method that improves danshinolic acid content, metabolic engineering develop rapidly with hair shape root culture technique dayBenefit is ripe for improving the content of danshinolic acid composition in Hairy Root Cultures of Salvia miltiorrhiza, and solving tanshinone medicine source property in short supply problem provides, new approaches.
There are some researches show, the secondary metabolism process of plant is arrived in the R2R3-MYB transcription factor wide participation in plant, because ofThe content that this R2R3-MYB transcription factor of excavating the red sage root self improves danshinolic acid is significant. Utilize geneEngineering means, transform red sage root blade acquisition SmMYB75 by red sage root transcription factor SmMYB75 gene genetic and cross turning of expressionGene Hairy Root Cultures of Salvia miltiorrhiza raises the biosynthesis of danshinolic acid simultaneously, obtains the Hairy Root Cultures of Salvia miltiorrhiza root system of danshinolic acid high yield, is businessIndustryization is produced danshinolic acid novel high-quality medicine source is provided. Not yet find at present to improve by SmMYB75 gene overexpression strategy redThe relevant report of danshinolic acid content in ginseng hair shape root.
Summary of the invention
Object of the present invention, the defect existing in order to overcome above-mentioned prior art exactly, provides one to turn SmMYB75 baseBecause improving the method for danshinolic 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 the SmMYB75 gene that obtains 645bp from the red sage root, builds the carrier of prokaryotic expression, at large intestine barIn bacterium, express SmMYB75 gene recombinant protein; Quantitative PCR analysis its after the induction of different tissues and methyl jasmonic acidThe expression of gene; Build the carrier of Subcellular Localization, instantaneous conversion tobacco leaf, confocal laser scanning microscope is fixedThe location situation of amount SmMYB75 gene in cell; Build plant expression vector, agrobacterium rhizogenes C58C1 mediates something lostPass and transform the genetically modified hair of red sage root blade acquisition shape root; The integration of PCR testing goal gene SmMYB75; QuantitativelyPcr analysis inserts gene SmMYB75 and the expression of danshinolic acid biosynthesis related genes in hair shape root; High-efficient liquidThe content of danshinolic acid in phase chromatographic determination transgenosis hair shape root.
Turn the method that SmMYB75 gene improves danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, comprise the following steps:
(1) adopt gene clone method to clone from the red sage root and obtain gene SmMYB75, described SmMYB75 gene orderRow, as shown in SEQID:1, carry out sequence analysis to it.
(2) according to red sage root SmMYB75 gene order, SmMYB75 gene is implemented in to prokaryotic expression operablyCarrier is expressed in Escherichia coli, and recombinant protein carries out SDS-PAGE electrophoresis detection; Described carrier isPET-30a (+), Escherichia coli are bacterial strain Rosetta (DE3).
(3) according to red sage root SmMYB75 gene order, the primer of design quantitative PCR, to SmMYB75 at the red sage rootExpression in different tissues and the abduction delivering that methyl jasmonic acid is regulated and controled thereof are analyzed.
(4) by gene constructed SmMYB75 in the carrier of Subcellular Localization, transform agrobacterium tumefaciens, carry out tobacco leafInstantaneous conversion, SmMYB75 gene is carried out to Subcellular Localization; Described carrier is pMON530, root nodule agricultureBacillus is strains A se.
(5) SmMYB75 gene is implemented in to expression regulation sequence operably, forms planting containing SmMYB75 geneThing expression vector; Plant expression vector is the pCAMBIA2300 obtaining through transformation+Carrier, comprises CaMV35SPromoter and terminator, MCS, replication origin and kalamycin resistance site.
(6) by the plant expression vector transforming agrobacterium rhizogenes C58C1 containing SmMYB75 gene of step (5) gained,Obtain the agrobacterium rhizogene strain containing SmMYB75 gene plant expression vector for transforming the red sage root.
(7) utilize the constructed agrobacterium rhizogene strain genetic transformation red sage root blade of step (6), obtain and detect through PCRPositive transgenosis hair shape root clone; The described positive transgenosis Hairy Root Cultures of Salvia miltiorrhiza detecting through PCR refers to: establishCount the upstream and downstream primer of root of hair locus gene BrolB and establish in insertion gene SmMYB75 inside and NOS terminator insideMeter upstream and downstream Auele Specific Primer, carry out DNA cloning, and the root system of observing object band under ultraviolet ray is sunProperty transgenosis Hairy Root Cultures of Salvia miltiorrhiza root system.
(8) SmMYB75 gene and danshinolic acid in the red sage root transgenosis hair shape root that quantitative PCR determination step (7) obtainsThe relative expression quantity of biosynthesis pathway related gene, and filtered out SmMYB75 in expression SmMYB75 gene root systemThe root system that the expression of gene improves; Concrete grammar is: PCR is accredited as to positive hair shape root clone and carries out total RNAExtraction, unified quantitatively to 0.5 μ gRNA, reverse transcription becomes the cDNA of 25 μ l systems, genes of interest is inserted in design respectivelyAnd the quantitative primer of house-keeping gene SmActin, carry out quantitative PCR analysis SmMYB75 taking the cDNA of same amount as templateAnd the relative expression quantity situation of danshinolic acid biosynthesis related genes.
(9) the middle red sage root of high effective liquid chromatography for measuring step (8) turns the content of danshinolic acid in SmMYB75 gene hair shape root,The red sage root transgenosis hair shape root root system that screening danshinolic acid content significantly improves; Described method is: the anti-phase silicon of chromatographic column C-18Glue post, mobile phase is acetonitrile and the water of volume ratio 30:70, and with phosphorus acid for adjusting pH to 2.03; Detect wavelength 281nm,35 DEG C of column temperatures, flow velocity 1ml/min, sample size 20 μ l.
Integrated application biology of the present invention and gene technology method are as vector construction, genetic transformation, Molecular Detection, quantitative PCRExtraction and the assay etc. of analysis, danshinolic acid, invented a kind of R2R3-MYB transcription factor of utilizing the red sage rootSmMYB75 gene improves the method for danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza. What the present invention obtained crosses expression SmMYB75 transgenosisIn Hairy Root Cultures of Salvia miltiorrhiza root system, high expressed amount is that total salvianolic acid content is 167.25mg/g dry weight, is control group (73.7mg/gDry weight) 2.26 times. The present invention produces in a large number danshinolic acid and reduces drug price for commercialization provides possibility, is also largeThe wilderness demand that amount is produced danshinolic acid clinical medicine provides important sources.
Compared with prior art, the present invention has the following advantages:
1, significantly improve the content of danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza.
2, inventive method is reliable for effect.
3, obtain the cost of danshinolic acid low.
4, production process non-environmental-pollution.
Brief description of the drawings
Fig. 1 is pCAMBIA2300+:: SmMYB75 vector construction schematic diagram.
Fig. 2 is that the SDS-PAGE of recombinant protein SmMYB75 analyzes.
Fig. 3 is the tissue expression analysis of red sage root SmMYB75.
Fig. 4 is the expression analysis of red sage root SmMYB75 under methyl jasmonic acid is processed. Wherein, Control is ethanol contrastGroup induction, MJ is methyl jasmonic acid induction.
Fig. 5 is the analysis result figure of the Subcellular Localization of SmMYB75. GFP, green fluorescent protein; Bright, light field;Merged, the merging figure of green fluorescent protein and light field; PMON530::GFP, empty carrier luciferase expression;PMON530::SmMYB75-GFP, the instantaneous luciferase expression of SmMYB75.
Fig. 6 is the expression analysis result figure of SmMYB75 in transgenosis Hairy Root Cultures of Salvia miltiorrhiza. Wherein, Control is C58C1Infect the hair shape root of acquisition.
Fig. 7 is the expression analysis result figure of danshinolic acid biosynthesis related genes. Wherein, Emptyvector is empty carrier.
Fig. 8 is the content detection result figure of danshinolic acid in SmMYB75 transgenosis Hairy Root Cultures of Salvia miltiorrhiza. Wherein, EmptyvectorIt is empty carrier.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail. Fig. 1 ispCAMBIA2300+:: SmMYB75 vector construction schematic diagram. Fig. 2 is that the SDS-PAGE of recombinant protein SmMYB75 dividesAnalyse.
The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example molecular cloningCondition described in (Sambrook etc.), or the reagent providing according to manufacturer or the incidental description of kitThe condition of suggestion.
Embodiment 1
The clone of red sage root SmMYB75 gene
1.1. the extraction of the total RNA of the red sage root
The red sage root young leaflet tablet that takes a morsel, with after liquid nitrogen flash freezer, grinds with mortar rapidly, then carries according to TIANGEN companyThe RNAprepPurePlantKit operation instructions of confession extract total RNA. With the gel electrophoresis of plain agar sugar (deposition condition:Gum concentration 1.2%; 0.5 × TBE electrophoretic buffer; 150v, 15min) detect the integrality of RNA. Use NanoDrop2000cUltramicrospectrophotometer detects its purity and concentration.
1.2. the clone of red sage root SmMYB75 gene
Taking the total RNA of the 0.5 μ g red sage root that obtained as initial amount, carry out the first chain cDNA with reverse transcriptase XL (AMV)Synthetic (instructions book that operating procedure provides with reference to Promega company). According to the volume of described SmMYB75 geneCode sequence (SEQIDNO.1), design amplifies the upstream and downstream primer of complete encoder block, and on upstream and downstream primerIntroduce respectively restriction endonuclease sites (this is determined by the carrier of selecting), so that carrier construction. With the first described chainCDNA is template, after pcr amplification, checks order. Determined dna sequence is had by Shanghai raw work biotechnology serviceLimit company completes. Sequencing result is shown in SEQNO.1.
Embodiment 2
The prokaryotic expression analysis of red sage root SmMYB75 gene
2.1.pET-30a (+):: the structure of SmMYB75 carrier
According to the ORF sequence of red sage root SmMYB75 of cloning acquisition, the primer of design Subcellular Localization vector construction,Build pET-30a (+):: SmMYB75, and by pET-30a (+):: SmMYB75 plasmid transformation escherichia coli Rosetta (DE3),Picking monoclonal bacterium colony carries out PCR checking. Result shows, the Subcellular Localization carrier that contains SmMYB75PET-30a (+):: SmMYB75 has successfully been transformed in Escherichia coli Rosetta (DE3), can be for follow-up tobacco winkTime express experiment.
2.2.SmMYB75 the SDS-PAGE of recombinant protein analyzes
Contain pET-30a (+) by what successfully construct:: the Escherichia coli Rosetta (DE3) of SmMYB75 be inoculated in contain card thatIn the LB culture medium of mycin (75mg/mL), cultivate 2-3 hour (OD600=0.4-0.5). Add 0.5 mMIPTG derivant abduction delivering, respectively at 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5Hour, within 6 hours, collect bacterium liquid, 12000 revs/min, centrifugal 1 minute results thalline, adds albumen sample-loading buffer,Boiling water boils 10 minutes, carries out SDS-PAGE electrophoretic analysis. Result demonstration, the size of expressing protein is about 30KD,Can see, after IPTG induction, compared with 30KD place is not when inducing, having obvious from SDS-PAGE electrophoretogramObject band, illustrates SmMYB75 successful expression in this expression system, and along with its induction of growth of induction time is shownThe amount of reaching obviously improves.
Embodiment 3
The tissue expression analysis of spectrum of red sage root SmMYB75
In order to study the tissue expression pattern of SmMYB75, extract respectively red sage root life in 2 years main root, fibrous root, stem,Total RNA of leaf, petiole, petal, calyx, eight tissues of filigree, and carry out respectively purity and concentration detection.Then reverse transcription becomes cDNA, and for the distribution expression pattern of quantitative PCR analysis SmMYB75, reaction system is usedSuperRealPreMix (SYBRGreen) kit that TIANGEN company provides, uses SmActin as interiorGinseng gene. Quantification PCR primer is:
Result shows, SmMYB75 detected institute in a organized way in, in stem, leaf and petiole, express stronger,Secondly in filigree and main root, expression is higher, expresses weak (seeing Fig. 3) in fibrous root, petal, calyx.
Embodiment 4
The response analysis of red sage root SmMYB75 to methyl jasmonic acid processing
In order to verify whether SmMYB75 responds the induction of methyl jasmonic acid, to cultivated 60 left and right in shaking flaskThe Hairy Root Cultures of Salvia miltiorrhiza of size is carried out methyl jasmonic acid processing, extracts the total of Hairy Root Cultures of Salvia miltiorrhiza after treated different timeRNA, and carry out respectively purity and concentration detection, then reverse transcription becomes cDNA, for quantitative PCR analysisThe distribution expression pattern of SmMYB75, quantitatively primer is with embodiment 3, and quantitative PCR result shows, when certainBetween in scope, along with the induction of methyl jasmonic acid, the expression of SmMYB75 obviously raises, and in the time of 4h, reachesHigh value, subsequently expression decline (seeing Fig. 4).
Embodiment 5
The Subcellular Localization of red sage root SmMYB75 gene
5.1.pMON530::MYB75 the structure of carrier
According to the ORF sequence of red sage root SmMYB75 of cloning acquisition, design Subcellular Localization vector constructionPrimer, build pMON530::SmMYB75, and by pMON530::SmMYB75 plasmid transform AgrobacteriumAse, picking monoclonal bacterium colony carries out PCR checking. Result shows, the Subcellular Localization carrier that contains SmMYB75PMON530::SmMYB75 has successfully been transformed in Agrobacterium Ase, can be for follow-up tobacco transient expressionExperiment.
5.2. the transient expression in tobacco
With asepsis injector draw build contain carrier pMON530::SmMYB75 and empty carrierThe engineering bacteria Ase of pMON530 carries out instantaneous conversion, illumination cultivation, 40-48 to well-grown tobacco leafAfter hour, get blade on slide, the back side upward, with distilled water infiltrate, with cover glass fixed blade (avoid productAngry bubble), under laser confocal microscope, observe. Result demonstration, SmMYB75 expresses in nucleus, withThey are consistent (seeing Fig. 5) as the function of transcription factor.
Embodiment 6
Contain the structure of the plant over-express vector of red sage root SmMYB75 gene
Vector construction ideograph is shown in Fig. 1. With pCAMBIA2300+For expression vector, the SmMYB75 base that clone is obtainedBecause replacing pCAMBIA2300+On gus gene. Particularly, SpeI/BstPI double digestion pMD18-T::SmMYB75 andpCAMBIA2300+; Reclaim SmMYB75 gene and pCAMBIA2300+Large fragment; Connect and transform, picking monoclonalBacterium colony PCR screening positive clone; Extract the further enzyme of plasmid and cut checking. Result shows, SmMYB75 gene is by successBe building up to plant expression vector pCAMBIA2300+In, thereby obtain the plant over-express vector containing SmMYB75 genepCAMBIA2300+::SmMYB75。
Transcription factor SmMYB75 gene is connected in operably expression regulation sequence by the present embodiment, and formation containsThe plant over-express vector pCAMBIA2300 of SmMYB75 gene+:: SmMYB75, this expression vector can be used for by generationThank to engineering strategy and improve the content of danshinolic acid in the red sage root.
In the present embodiment with pCAMBIA2300+For medial expression vector is a kind of preferred embodiment, at actual implementation carrierProcess in, can select as the case may be other suitable carriers, in the time selecting different carriers, introduce restriction enzymeEnzyme site difference.
Embodiment 7
The red sage root SmMYB75 gene genetic of agrobacterium rhizogenes mediation transforms the red sage root and obtains transgenosis Hairy Root Cultures of Salvia miltiorrhiza
7.1. contain the acquisition of the agrobacterium rhizogenes engineering bacteria of plant expression vector
By the plant over-express vector pCAMBIA2300 containing SmMYB75 gene in embodiment 2+:: SmMYB75 proceeds to and sends outIn root Agrobacterium C58C1, picking monoclonal bacterium colony carries out PCR checking. Result shows, containing planting of SmMYB75 geneThing over-express vector pCAMBIA2300+:: SmMYB75 has successfully been transformed in agrobacterium rhizogenes C58C1.
7.2. agrobacterium rhizogenes mediation SmMYB75 gene genetic transforms the red sage root
7.2.1 the preculture of explant
The healthy and strong aseptic seedling blade of the clip red sage root (0.5cm2), be inoculated into preculture culture medium (1/2MS) upper, 25 DEG C of dark trainingsSupport 2 days.
7.2.2 the common cultivation of Agrobacterium and explant
By above-mentioned pre-incubated red sage root leaf explant, put into the 1/2MS containing the above-mentioned agrobacterium rhizogenes engineering bacteria having activatedIn suspension, soak after 10 minutes (jiggle is that explant fully contacts with bacterium liquid), the red sage root blade taking out after contaminating is usedAseptic blotting paper blots surperficial bacterium liquid, forwards in common culture medium 1/2MS, secretly cultivates 2-3 days.
7.2.3 the induction of hair shape root and subculture are cultivated
The above-mentioned common red sage root explant of cultivating 2-3 days is transferred in degerming solid medium (1/2MS+Cb300mg/L),25 DEG C of dark cultivations about 2-3 week, can grow from explant wound a mao shape root. The red sage root explant of root of hair is transferred toDegerming solid medium (1/2MS+Cb200mg/L) is upper, and 25 DEG C dark cultivates and within 2 weeks, grow to 3cm until a mao shape root and cut when aboveGet monoclonal hair shape root as a clone, continue to be inoculated in except secretly training in bacterium culture medium (1/2MS+Cb100mg/L)Support two weeks, until without the bacterium phenomenon of overflowing. Monoclonal hair shape root is transferred to and on the 1/2MS culture medium of antibiotic-free, continues dark trainingSupport.
7.3. the PCR of transgenosis Hairy Root Cultures of Salvia miltiorrhiza detects
7.3.1 the extraction of transgenosis hair shape kan gene group DNA
Adopt CTAB method to extract transgenosis hair shape kan gene group DNA. The complete transgenosis hair shape root of degerming in clip 3.2.35cm puts into 1.5mL centrifuge tube in left and right, adds 600 μ LCTAB lysates (65 DEG C of preheatings, containing 1% beta-mercaptoethanol),With organizing beveller fully to grind. Be placed in 65 DEG C of water-bath 40-50 minute, repeatedly mix sample (inferior/15min) therebetween,After being cooled to room temperature, add isopyknic phenol/chloroform (1:1), put upside down gently and mix emulsification 10min, 1200rpm is centrifugal15min, carefully draws supernatant in new EP pipe, adds isopyknic chloroform to mix, and the centrifugal 15min of 12000rpm is slowThe slow supernatant of drawing, in new EP pipe, adds the absolute ethyl alcohol (putting 30min in-20 DEG C) of 2 times of volume precoolings, separates out precipitation,The centrifugal 15min of 12000rpm, abandons supernatant, adds 75% ethanol washed twice, sucking-off supernatant, and room temperature is dried, and adds 30-50 μ LWater dissolution precipitation, frozen, for subsequent use in-80 DEG C of ultra low temperature freezers after processing with RNA enzyme.
7.3.2 design of primers and PCR detect
At pCAMBIA2300+:: on the insertion gene (SmMYB75) of SmMYB75 carrier and NOS terminator, establish respectivelyMeter specificity upstream and downstream primer design specificity upstream and downstream primer simultaneously on root of hair locus gene BrolB. Use simultaneouslyPCR method carries out Molecular Detection to total DNA of above-mentioned hair shape root. Result shows, in transgenosis hair shape root root system, detectsAnd positive control (pCAMBIA2300+:: SmMYB75 plasmid is template) sizable PCR product; And with agriculture barWhen the genomic DNA that the empty bacterium of bacterium C58C1 is infected the hair shape root of red sage root gained is template, do not amplify any fragment, knotFruit illustrates that SmMYB75 gene has been incorporated in red sage root genome.
The present embodiment, by described plant expression vector transforming agrobacterium rhizogenes, obtains the plant expression vector for transforming the red sage rootAgrobacterium rhizogene strain C58C1, utilize constructed agrobacterium rhizogene strain genetic transformation red sage root blade, obtain through PCRTest positive clone's transgenosis hair shape root. The acquisition of transgenosis Hairy Root Cultures of Salvia miltiorrhiza is that the hair shape root of screening high yield danshinolic acid is carriedSupply direct material.
In the present embodiment, selecting agrobacterium rhizogenes C58C1 is a kind of preferred embodiment, in actual selection, and root of hair agriculture barThe bacterial strain of bacterium is not limited to C58C1, can select as the case may be other bacterial strains.
Embodiment 8
The expression of related gene in quantitative PCR detection transgenosis Hairy Root Cultures of Salvia miltiorrhiza
8.1. hair shape root Liquid Culture
Select the hair shape root that in embodiment 7, growth is fast, branch is good, the aseptic distillation of clip 2-3cm on superclean benchWater rinses out 1/2MS fluid nutrient medium subculture that access after its lip-deep agar is equipped with 100mL once, 60 days results afterwards,Get proper amount of fresh hair shape root blotting paper and blot after surface moisture, package to enter after freezing in liquid nitrogen with masking foil and be stored in-80 DEG C are extracted for RNA, all the other mao of shape root dry after for the extraction of tanshinone danshinolic acid content.
8.2.RNA synthesizing of extraction and cDNA the first chain
Method is with the step 1.1 in embodiment 1
The design of 8.3 primers and synthetic
Use respectively according to the coded sequence of red sage root gene SmMYB75 and tanshinone and danshinolic acid biosynthesis related genesPrimer5.0 design primer is for detection of the expression of related gene in Hairy Root Cultures of Salvia miltiorrhiza, and house-keeping gene Actin is used asInternal reference. The primer is synthetic by Shanghai Sheng Gong bio-engineering corporation.
The primer of quantitative PCR is as follows:
8.4. the quantitative PCR detection of transgenosis Hairy Root Cultures of Salvia miltiorrhiza
Above-mentioned cDNA (diluting 10 times) the first chain taking same amount, as template, is determined with the primer of above-mentioned design respectivelyAmount pcr amplification. The description of the BiosystemStepOne instrument of producing with reference to AppliedBiosystem company of the U.S. entersThe operation of row quantitative PCR, the quantitative PCR kit of employing Quan Shi King Company. Quantitative PCR reaction system is as follows:
PCR reaction condition: 94 DEG C 5 minutes, 40 circulations (94 DEG C of sex change 30 seconds, 60 DEG C of annealing 30 seconds, 72 DEG C of extensions30 seconds), 72 DEG C 10 minutes. Genes of interest and house-keeping gene respectively do three repetitions.
Quantitative PCR result shows: compare empty carrier root system, to express expression in root system obvious crossing for SmMYB75 geneImprove, but expression between different clone has certain difference, wherein expression higher for clone 9,14,18,19,No. 23 (seeing Fig. 6). Meanwhile, after SmMYB75 crosses and expresses in Hairy Root Cultures of Salvia miltiorrhiza, the pass in danshinolic acid metabolic pathwayThe expression of key enzyme gene all obviously improves, and illustrates that SmMYB75 can promote related gene in danshinolic acid biosynthesis pathwayExpress (seeing Fig. 7).
According to the literature, myb transcription factor can be by regulating and controlling in conjunction with the cis-acting elements in the target gene promoters of downstreamThe expression of downstream target gene. Key gene promoter in danshinolic acid route of synthesis is analyzed, found to be subject to SmMYB75The cis-acting elements that contains MYB on the gene promoter of response, therefore infers that SmMYB75 passes through in conjunction with in promoterCis-acting elements regulate and control the expression of key gene, thereby regulation and control danshinolic acid is synthetic.
Embodiment 9
Utilize HPLC to measure danshinolic acid content in transgenosis Hairy Root Cultures of Salvia miltiorrhiza
9.1. the extraction of danshinolic acid content in hair shape root
The hair shape root that embodiment 4 is gathered in the crops is dried to constant weight, and grind into powder, accurately takes 0.1g hair shape root powder in 50mLIn centrifuge tube, add 10mL ethanol: water (4:1, v/v), ultrasonic 20min, 8000rpm, centrifugal 10min, inhalesGet the 70 DEG C of vacuum drying in Rotary Evaporators of supernatant extract, residue dissolves with the distilled water of 2mL again, by sampleTo be measured after membrane filtration with 0.22 μ m.
9.2. in hair shape root, the HPLC of danshinolic acid content measures
Precision takes salviandic acid A, tanshin polyphenolic acid B, Rosmarinic acid and caffeic acid standard items methyl alcohol and is configured to respectively concentration and isThe standard items stock solution of 1mM, be stored in-20 DEG C for subsequent use.
Chromatographic condition: chromatographic column is C-18 reverse phase silica gel post, and mobile phase is acetonitrile: water (30:70), water regulates with phosphoric acidPH is 2.03, detects wavelength 281nm, 35 DEG C of column temperatures, flow velocity 1mL/min.
Above-mentioned standard items stock solution is got respectively to 5 μ l, 10 μ l, 20 μ l, 30 μ l, 40 μ l sample introduction under corresponding chromatographic condition,Four kinds of water soluble ingredients separate completely, and peak type is good, record collection of illustrative plates and chromatographic parameter, respectively with peak area (Y) to markAccurate product concentration (X, mg/mL) is carried out regression analysis.
Danshinolic acid crude extract after above-mentioned 0.22 μ m membrane filtration is respectively got 10 μ L, and with high performance liquid chromatograph detection, record is eachComponent peaks area, substitution equation of linear regression, calculates and obtains the content of sample water soluble components.
In the present invention, except caffeinic content is without significantly upper downward relation, with respect to control group, Rosmarinic acid, pelletThe content of phenolic acid A, three kinds of danshinolic acids of tanshin polyphenolic acid B obviously raises, and the content of total salvianolic acid also obviously improves. Wherein, table2.26 times (seeing Fig. 8) that in the highest transgenosis hair shape root of the amount of reaching, the content (167.25) of danshinolic acid is control group (73.7).
The present invention adopts the metabolic engineering strategy that turns SmMYB75 gene to obtain the red sage root transgenosis hair shape root of high yield danshinolic acidRoot system, for a large amount of production danshinolic acids, alleviation danshinolic acid medicine source problem in short supply provide a kind of novel effective ways.
The above-mentioned description to embodiment is can understand and use invention for ease of those skilled in the art. Be familiar withThe personnel of art technology obviously can easily make various amendments to these embodiment, and General Principle described hereinBe applied in other embodiment and needn't pass through performing creative labour. Therefore, the invention is not restricted to above-described embodiment, this areaTechnical staff is according to announcement of the present invention, and not departing from improvement and the amendment that category of the present invention makes all should be guarantor of the present inventionWithin protecting scope.
Claims (8)
1. turn the method that SmMYB75 gene improves danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, it is characterized in that, compriseFollowing steps:
(1) method of employing Gene cloning is cloned and is obtained SmMYB75 gene, described SmMYB75 gene order from the red sage rootRow are as shown in SEQID:1;
(2) according to red sage root SmMYB75 gene order, SmMYB75 gene is implemented in to prokaryotic expression operablyCarrier is expressed in Escherichia coli;
(3) expression of SmMYB75 in the different tissues of the red sage root measured and analyzed to quantitative PCR and to methyl jasmineThe abduction delivering amount of acid regulation and control;
(4) by gene constructed SmMYB75 in the carrier of Subcellular Localization, transform agrobacterium tumefaciens, carry out tobacco leafInstantaneous conversion, SmMYB75 is carried out to Subcellular Localization;
(5) SmMYB75 gene is implemented in to expression regulation sequence operably, forms planting containing SmMYB75 geneThing expression vector;
(6), by the plant expression vector transforming agrobacterium rhizogenes containing SmMYB75 gene of step (5) gained, obtain and useIn the agrobacterium rhizogene strain containing SmMYB75 gene plant expression vector that transforms the red sage root;
(7) utilize the constructed agrobacterium rhizogene strain genetic transformation red sage root blade of step (6), obtain and detect through PCRPositive red sage root transgenosis hair shape root clone;
(8) SmMYB75 gene and red phenol in the red sage root transgenosis hair shape root that quantitative PCR determination step (7) obtainsThe relative expression quantity of key gene in acid biosynthesis pathway, and filtered out base in the root system of expressing SmMYB75 geneBecause of the root system of expression raising;
(9) the middle red sage root of high effective liquid chromatography for measuring step (8) turns the content of danshinolic acid in SmMYB75 gene hair shape root,The transgenosis Hairy Root Cultures of Salvia miltiorrhiza root system that screening danshinolic acid content significantly improves.
2. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, itsBe characterised in that, the carrier of the described prokaryotic expression of step (2) is pET-30a (+), and Escherichia coli are bacterial strain Rosetta (DE3),The method of prokaryotic expression is as follows:
(1) primer of design SmMYB75 gene prokaryotic, the carrier of the connection prokaryotic expression of operability, and turnChange Escherichia coli;
(2) the recombination bacillus coli Rosetta (DE3) successfully constructing is added to IPTG derivant abduction delivering, carry outSDS-PAGE electrophoretic analysis.
3. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, itsBe characterised in that, the carrier of the described Subcellular Localization of step (4) is pMON530, and agrobacterium tumefaciens is strains A se, AsiaThe method of celluar localization is as follows:
(1) carrier of the Subcellular Localization that contains SmMYB75 gene is transformed to agrobacterium tumefaciens;
(2) draw Agrobacterium bacterium liquid with asepsis injector well-grown tobacco leaf is carried out to instantaneous conversion;
(3) illumination cultivation, got blade on slide after 40-48 hour, and lid, with distilled water infiltration, is used upward in the back sideSlide fixed blade is observed under laser confocal microscope.
4. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1, itsBe characterised in that, the described plant expression vector of step (5) is the pCAMBIA2300 obtaining through transformation+Carrier, comprisesCaMV35S promoter and NOS terminator, MCS, replication origin and kalamycin resistance site.
5. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1,It is characterized in that, the described agrobacterium rhizogenes of step (6) is selected from bacterial strain C58C1.
6. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1,It is characterized in that, the described PCR detection method of step (7) is as follows:
(1) the specific PCR primer of design root of hair locus gene BrolB, carries out pcr amplification;
(2) insert gene SmMYB75 inside and NOS terminator indoor design upstream and downstream Auele Specific Primer, carry outDNA cloning;
(3) ultraviolet ray is lower observes object band, occurs positive transgenosis Hairy Root Cultures of Salvia miltiorrhiza root system of object band.
7. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1,It is characterized in that, described in step (3) and step (8), quantitative PCR assay method is as follows:
(1) PCR is accredited as to positive hair shape root clone and carries out the extraction of total RNA, unified quantitatively to 0.5 μ g, RNAReverse transcription becomes the cDNA of 25 μ l systems;
(2) the quantitative primer of genes of interest and house-keeping gene SmActin is inserted in design respectively, taking the cDNA of same amount asTemplate is carried out quantitative PCR analysis;
(3) the relative expression quantity situation of analysis SmMYB75 and danshinolic acid biosynthesis related genes.
8. the method that turns danshinolic acid content in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza according to claim 1,It is characterized in that, the described high performance liquid chromatography of step (9) is as follows:
Chromatographic column C-18 reverse phase silica gel post, mobile phase is acetonitrile and the water of volume ratio 30:70, and with phosphorus acid for adjusting pH extremely2.03; Detect wavelength 281nm, 35 DEG C of column temperatures, flow velocity 1ml/min, sample size 20 μ l.
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