CN104726485B - Turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza - Google Patents
Turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza Download PDFInfo
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Abstract
Turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza the present invention relates to a kind of, belong to gene engineering technology field.The present invention by arabidopsis transcription factor gene AtMYC2 by being built into high efficiency plant expression vector, genetic transformation red sage root blade obtains the transgenosis Hairy Root Cultures of Salvia miltiorrhiza of AtMYC2 gene overexpressions, in qRT PCR analysis transgenosis Hairy Root Cultures of Salvia miltiorrhiza in AtMYC2 and tanshinone and danshinolic acid biosynthesis pathway related gene expression, high performance liquid chromatography (HPLC) determines the content of tanshinone and danshinolic acid in transgenosis Hairy Root Cultures of Salvia miltiorrhiza, and DPPH removes the antioxidation activity that free radical method determines tanshinone and danshinolic acid in red sage root transgenic hairy root.The invention provides a kind of method that can improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza simultaneously, also a kind of novel high-quality raw material is provided to produce tanshinone and danshinolic acid with important clinical demand, there is positive promotion meaning and application value to the sex chromosome mosaicism in short supply for alleviating tanshinone and danshinolic acid medicine source.
Description
Technical field
Turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, category the present invention relates to a kind of
In gene engineering technology field.
Background technology
Cardiovascular and cerebrovascular disease is current threat whole mankind health and life " number one killer ".According to statistics, the whole world is annual
About 17,000,000 people die from cardiovascular and cerebrovascular disease, account for the 1/3 of global total death toll；Annual about 3,000,000 people of China
Die from cardiovascular and cerebrovascular disease.Therefore the clinical medicine of efficient, low toxicity and cheap treatment cardiovascular and cerebrovascular disease is actively studied to carrying
High level of human health has very profound significance.
The red sage root (Salvia miltiorrhiza Bunge) is that Labiatae (Lamiaceae) Salvia (Salvia) is more
Year raw herbaceous plant, its red shape of root color is gained the name " red sage root " like ginseng, is the conventional Chinese medicine of clinical treatment Cardial or cerebral vascular diseases.
Effective component in red sage mainly include fat-soluble tanshinone component and water-soluble phenolic acrylic component, wherein tanshinone it is anti-oxidant,
There is significant curative effect in terms of anti-inflammatory is antibacterial and antitumor, also with platelet aggregation is suppressed, improve hypoxia-bearing capability, improve hat
The pharmacological actions such as shape artery blood supply；Danshinolic acid has anti-hepatic fibrosis, antiatherosclerosis, improves the medicines such as memory dysfunction
Reason is acted on, and clinical practice potentiality are very huge.Except for pharmaceuticals industry, (compound danshen dripping pills, danshen injections, compound Danshen Root contain
Piece etc.) beyond, start to penetrate into the other fields such as health products trade (red rooted salvia etc.), cosmetic industry, should with wide
Use prospect.And tanshinone, danshinolic acid are depended on to extract from the red sage root and obtained at present.Yet with red sage root cultivated since long quality
Degenerate serious；Active component content is reduced；And growth cycle length and by the factors such as cultural area and environment restrict.Therefore, in order to
Alleviate the tanshinone with important clinical demand and the sex chromosome mosaicism in short supply of danshinolic acid medicine source, invention one kind can improve tanshinone simultaneously
With the new method of danshinolic acid content.
Prior art shows that bHLH classes transcription factor MYC2 (AtMYC2) is regulating and controlling growing for plant in arabidopsis
During play an important roll.But tanshinone and danshinolic acid class material are not produced in arabidopsis, whether AtMYC2 is to tanshinone
Synthesis with danshinolic acid has facilitation there is not yet open report.
The present invention utilizes genetic engineering means, and arabidopsis transcription factor gene AtMYC2 genetic transformation reds sage root blade is obtained
The transgenosis Hairy Root Cultures of Salvia miltiorrhiza that AtMYC2 is overexpressed, while raise the biosynthesis of tanshinone and danshinolic acid, obtain tanshinone and
The Hairy Root Cultures of Salvia miltiorrhiza strain of the equal high yield of danshinolic acid, novel high-quality medicine source is provided to commercially produce tanshinone and danshinolic acid.At present
Not yet find to improve Hairy Root Cultures of Salvia miltiorrhiza tanshinone and danshinolic acid with the AtMYC2 gene overexpressions strategy mentioned by present subject matter
The relevant report of content.Therefore, the present invention has positive on the problem of actually solving tanshinone and danshinolic acid medicine source property in short supply
Meaning.
The content of the invention
The purpose of the present invention is exactly the defect in order to overcome above-mentioned prior art presence and provides one kind and turn AtMYC2 genes
The method for improving tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza.
The purpose of the present invention can be achieved through the following technical solutions：
The present invention clones and isolates 1872bp AtMYC2 genes from arabidopsis, plant expression vector is built, with root of hair
Agrobacterium C58C1 is mediation, and genetic transformation red sage root blade obtains hairy root；PCR testing goal Gene As tMYC2 integration；
The expression of qRT-PCR analysis insertions target gene AtMYC2 and tanshinone and danshinolic acid biosynthesis related genes in hairy root
Situation；High performance liquid chromatography determines the content of tanshinone and danshinolic acid in transgenic hairy root；DPPH removes free radical experiment and surveyed
Determine the antioxidation activity of tanshinone in transgenosis Hairy Root Cultures of Salvia miltiorrhiza.
It is a kind of to turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, including following step
Suddenly：
(1) cloned using gene clone method from arabidopsis and obtain transcription factor gene AtMYC2, the AtMYC2 bases
Because sequence is SEQ ID:1 DNA sequence dna；
(2) AtMYC2 genes are operably connected in expression regulation sequence, form the plant expression of the gene containing AtMYC2
Carrier；
(3) by the plant expression vector transforming agrobacterium rhizogenes of the gene containing AtMYC2, obtain for converting containing for the red sage root
The agrobacterium rhizogene strain of AtMYC2 gene plant expression vectors；
(4) constructed agrobacterium rhizogene strain genetic transformation red sage root blade is utilized, turning through PCR test positive is obtained
Gene hairy root is cloned；
(5) quantitative RT-PCR determines AtMYC2 genes and tanshinone and danshinolic acid biosynthesis in red sage root transgenic hairy root
The relative expression quantity of related gene；
(6) the high effective liquid chromatography for measuring red sage root turns tanshinone and danshinolic acid content in AtMYC2 gene hairy roots, screening
The red sage root transgenic hairy root strain that tanshinone and danshinolic acid content are significantly improved；
(7) DPPH removes free radical method and determines the antioxidation activity that the red sage root turns tanshinone in AtMYC2 gene hairy roots.
Preferably, described agrobacterium rhizogenes is selected from bacterial strain C58C1.
PCR detection method described in step (4) is as follows：
(4.1) root of hair locus gene BrolB, kalamycin resistance gene kan specific PCR primers are separately designed；
(4.2) constitutive promoter for starting the tMYC2 expression of insertion Gene A is CaMV35S promoters；
(4.3) insertion gene internal and NOS terminator indoor design upstream and downstream specific primer, carry out DNA cloning；
(4.4) viewed under ultraviolet radiation purpose band strain, it is then the strain of positive transgenic Hairy Root Cultures of Salvia miltiorrhiza purpose band occur
System.
Step (5) described quantitative RT-PCR detecting method is as follows：
(5.1) the hairy root clone that the positive is accredited as to PCR carries out the extraction of total serum IgE, unified quantitative anti-to 0.5 μ g RNA
It is transcribed into the cDNA of 25 μ l systems；
(5.2) insertion target gene and house-keeping gene Actin primer are separately designed, is entered using same amount of cDNA as template
Row quantitative RT PCR analysis；
(5.3) the relative expression quantity situation of analysis Gene A tMYC2 and tanshinone and danshinolic acid biosynthesis related genes.
High performance liquid chromatography described in step (6) is as follows：
(6.1) tanshinone：Chromatographic column C-18 reverse phase silica gel posts, mobile phase is volume ratio 65：35 acetonitrile and water；Detect ripple
Long 220nm, 30 DEG C of column temperature, flow velocity 1ml/min, the μ l of sample size 20；
(6.2) danshinolic acid：Chromatographic column C-18 reverse phase silica gel posts, mobile phase is volume ratio 30：70 acetonitrile and water, and use phosphorus
Acid for adjusting pH is to 2.03；Detection wavelength 281nm, 35 DEG C of column temperature, flow velocity 1ml/min, the μ l of sample size 20.
In step (7), the assay method of the antioxidation activity of tanshinone and danshinolic acid is：
(7.1) red sage root turns to extract tanshinone and danshinolic acid in AtMYC2 gene hairy roots；
(7.2) sample obtained by step (7.1) is diluted to concentration gradient：0.25 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/
Ml, 4 μ g/ml each 1ml of methanol solution；
(7.3) the μ l of step (7.2) resulting solution 200 are taken, 800 μ l 0.1mM DPPH solution is separately added into, fully mixed；
(7.4) 30min is placed in darkroom at 25 DEG C of step (7.3) products therefrom；
(7.5) step (7.4) product is surveyed into absorption value under spectrophotometer at 517nm；
Wherein, DPPH free radical scavenging activities (%)=[(A0-A1)/A0× 100],
In formula, A0It is control sample (DPPH) absorbance；A1It is the absorbance of sample or standard items.
Integrated application biology of the present invention and gene technology method such as vector construction, genetic transformation, Molecular Detection, quantitative
QRT-PCR analyses, tanshinone and the extraction of danshinolic acid and assay etc., have invented a kind of utilization arabidopsis AtMYC2 transcriptons
The method for improving two big active component tanshinones and danshinolic acid in the red sage root simultaneously.The overexpression AtMYC2 that the present invention is obtained turns base
Because total-tanshinone (dihydrotanshinone, Cryptotanshinone, Tanshinone I, tanshinone IIA) content is significantly improved in Hairy Root Cultures of Salvia miltiorrhiza, its
Middle tanshinone content highest strain is 14.06mg/g DW, is 6.45 times of control group (2.18mg/g DW)；The strain is red simultaneously
Phenolic content is 95.90mg/g DW, is 4.30 times of control group (22.32mg/g DW).The present invention is a large amount of productions of commercialization
Tanshinone and with danshinolic acid and reduction drug price provide may, be also a large amount of production tanshinones and danshinolic acid clinical medicine
Wilderness demand provides important sources.
Compared with prior art, the present invention has advantages below：
1st, the content of total-tanshinone and danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza can be significantly improved simultaneously.
2nd, inventive method effect is reliable.
3rd, the low cost of tanshinone and danshinolic acid is obtained.
4th, production process non-environmental-pollution.
Brief description of the drawings
Fig. 1 is pCAMBIA2300+::AtMYC2 vector construction schematic diagrames.
Fig. 2 is the PCR qualification result figures of transgenic hairy root.Detect that lower section is above Fig. 2 for the PCR of AtMYC2 genes
Hairy root rolB identified for genes figures.M,DL-2000 Marker(100–2,000bp)；PC, positive control (pCAMBIA2300+::
AtMYC2 plasmids are template)；NC, negative control (the hairy root that the empty Agrobacterium C58C1 genetic transformations red sage root is obtained).
Fig. 3-1 is expression analysis result figures of the AtMYC2 in transgenosis Hairy Root Cultures of Salvia miltiorrhiza；
Fig. 3-2 is the expression analysis result figure of tanshinone biosynthesis related genes；
Fig. 3-3 is the expression analysis result figure of danshinolic acid biosynthesis related genes；Empty vector are empty carriers
pCAMBIA2300+The hairy root that the genetic transformation red sage root is obtained.
Fig. 4-1 is the content detection result figure of tanshinone in AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza；Wherein DH-TI is dihydro
Tanshinone, CT is Cryptotanshinone, and T-I is Tanshinone I, and T-IIA is tanshinone IIA, and TT is total-tanshinone content.
Fig. 4-2 is the content detection result figure of danshinolic acid in AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza；Wherein CA is caffeic acid,
RA is Rosmarinic acid, and SA A are salviandic acid A, and SA B are tanshin polyphenolic acid B, and TS is total salvianolic acid, and Empty vector are empty carriers
pCAMBIA2300+The hairy root that the genetic transformation red sage root is obtained.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as molecular cloning
Condition described in (Sambrook etc.), or the incidental specification suggestion of reagent or kit provided according to manufacturer
Condition.
Embodiment 1
The clone of arabidopsis AtMYC2 genes
1.1. the extraction of arabidopsis total serum IgE
A small amount of arabidopsis young leaflet tablet is taken, after liquid nitrogen flash freezer, is ground with mortar rapidly, then according to TIANGEN companies
The RNAprep Pure Plant Kit operation instructions of offer extract total serum IgE.With the sugared gel electrophoresis (electrophoresis strip of plain agar
Part：Gum concentration 1.2%；0.5 × TBE electrophoretic buffers；150v, 15min) detection RNA integrality.Use Nano Drop
2000c ultramicrospectrophotometers detect its purity and concentration.
1.2. the clone of arabidopsis AtMYC2 genes
The 0.5 μ g arabidopsis total serum IgEs to be obtained carry out the first chain cDNA conjunction with reverse transcriptase XL (AMV) as initial amount
Into (instructions book that operating procedure is provided with reference to Promega companies).According to the coded sequence (SEQ of the AtMYC2 genes
ID NO.1), design amplifies the upstream and downstream primer of complete encoder block, and introduces restricted respectively on upstream and downstream primer
Restriction enzyme site (depending on this carrier visually selected), so as to construction of expression vector.Using the first described chain cDNA as template, warp
It is sequenced after PCR amplifications.Determined dna sequence is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing result
Show, the sequence cloned and AtMYC2 coded sequences (the SEQ ID announced on NCBI：1) it is completely the same.
Embodiment 2
The structure of the plant over-express vector of the genes of AtMYC2 containing arabidopsis
Vector construction ideograph is shown in Fig. 1.With pCAMBIA2300+For medial expression vector, with what is cloned from arabidopsis
AtMYC2 genes replace pCAMBIA2300+On gus genes.Specifically, Spe I/BstPI double digestions pMD18-T::AtMYC2
And pCAMBIA2300+；Reclaim AtMYC2 genes and pCAMBIA2300+Large fragment；Connection conversion, picking monoclonal bacterium colony PCR sieves
Select positive colony；Extract the further digestion verification of plasmid.As a result show, AtMYC2 genes have successfully been building up to plant expression load
Body pCAMBIA2300+In, so as to obtain the plant over-express vector pCAMBIA2300 of the gene containing AtMYC2+::AtMYC2。
Transcription factor AtMYC2 is operatively connectable to expression regulation sequence by the present embodiment, forms gene containing AtMYC2
Plant over-express vector pCAMBIA2300+::AtMYC2, the expression vector can be used for improving pellet by metabolic engineering strategies
The content of tanshinone and danshinolic acid in ginseng.
With pCAMBIA2300 in the present embodiment+It is a kind of preferred embodiment for plant vector, in actual implementation carrier
During, other suitable carriers can be selected as the case may be, when from different carriers, introduce restriction enzyme position
Point is different.
Embodiment 3
The arabidopsis AtMYC2 gene genetics conversion red sage root of agrobacterium rhizogenes mediation obtains transgenosis Hairy Root Cultures of Salvia miltiorrhiza
3.1. the acquisition of the agrobacterium rhizogenes engineering bacteria containing plant expression vector
By the plant over-express vector pCAMBIA2300 of the gene containing AtMYC2 in embodiment 2+::AtMYC2 is transferred to root of hair agriculture
In bacillus C58C1, picking monoclonal bacterium colony enters performing PCR checking.As a result show, the plant over-express vector of the gene containing AtMYC2
pCAMBIA2300+::AtMYC2 is successfully building up in agrobacterium rhizogenes C58C1.
3.2. agrobacterium rhizogenes mediation AtMYC2 gene genetics convert the red sage root
3.2.1 the preculture of explant
Clip red sage root stalwartness tests for sterility (0.5cm2), it is inoculated on precultivation medium (1/2MS), 25 DEG C of light cultures
2 days.
3.2.2 the co-cultivation of Agrobacterium and explant
By the red sage root blade explant of above-mentioned preculture, 1/ containing the above-mentioned agrobacterium rhizogenes engineering bacteria activated is put into
Soaked in 2MS suspensions after 10 minutes (jiggle is that explant is fully contacted with bacterium solution), take out the red sage root blade after contaminating and use
Sterile blotting paper blots surface bacterium solution, goes in co-cultivation culture medium 1/2MS, light culture 2-3 days.
3.2.3 the induction of hairy root and squamous subculture
The above-mentioned red sage root explant for co-culturing 2-3 days is transferred to degerming solid medium (1/2MS+Cef500mg/L)
In, 25 DEG C of light cultures 2-3 weeks or so can grow hairy root from explant wound.The red sage root explant of root of hair is transferred
Onto degerming solid medium (1/2MS+Cef300mg/L), 25 DEG C of light cultures, 2 weeks clip lists when hairy root length is to more than 3cm
One hairy root continues to be inoculated in except light culture two weeks in bacterium culture medium (1/2MS+Cef100mg/L), directly as a clone
Overflowed to without Agrobacterium.
3.3. the PCR detections of transgenosis Hairy Root Cultures of Salvia miltiorrhiza
3.3.1 the extraction of transgenic hairy root genomic DNA
Transgenic hairy root genomic DNA is extracted using CTAB methods.The degerming transgenic hairy root finished in clip 3.2.3
5cm or so is put into 1.5mL centrifuge tubes, is added 600 μ L CTAB lysates (65 DEG C of preheatings, containing 1% beta -mercaptoethanol), is used group
Beveller is knitted to be fully ground.It is placed in 65 DEG C of water-baths 40-50 minutes, sample (secondary/15min) is repeatedly mixed therebetween, is cooled to
Isometric phenol/chloroform (1 is added after room temperature:1), gently overturn and mix emulsification 10min, 1200rpm centrifugation 15min, carefully
Supernatant is drawn in new EP pipes, isometric chloroform is added and mixes, 12000rpm centrifugations 15min, slow supernatant of drawing is in new EP
Guan Zhong, plus 2 times of volume precoolings absolute ethyl alcohol (putting 30min in -20 DEG C), separate out precipitation, 12000rpm centrifugation 15min, abandon
Clearly, add 75% ethanol to wash twice, suction out supernatant, room temperature is dried, 30-50 μ L water dissolves precipitation is added, after being handled with RNase
Frozen in -80 DEG C of ultra low temperature freezers, it is standby.
3.3.2 design of primers and PCR detections
In pCAMBIA2300+::Separately designed on the insertion gene (AtMYC2) and NOS terminator of AtMYC2 carriers special
Property upstream and anti-sense primer (AtMYC2-F1080：5’-ACCAAGATCCGGCGAGATATTAAAC-3’,NOS-R:5’-
GCAAGACCGGCAACAGGATTC-3’).Specific upstream and downstream primer (rolB- is designed on root of hair locus gene BrolB simultaneously
F:5’-GCTCTTGCAGTGCTAGATTT-3’,rolB-R:5’-GAAGGTGCAAGCTACCTCTC-3’).Use PCR method simultaneously
Molecular Detection is carried out to the STb gene of above-mentioned hairy root, Fig. 2 is seen.As a result show, root of hair can be detected in all hairy stock systems
Locus gene, can be detected and positive control (pCAMBIA2300 in a part of transgenic hairy root+::AtMYC2 plasmids are
Template) sizable PCR primer.And the genomic DNA of hairy root obtained by the red sage root is infected using Agrobacterium C58C1 empty bacteriums as mould
During plate, any fragment is not amplified.As a result illustrate that AtMYC2 genes have been incorporated into red sage root genome.
Described plant expression vector transforming agrobacterium rhizogenes are obtained the plant for converting the red sage root and expressed by the present embodiment
The agrobacterium rhizogene strain C58C1 of carrier, using constructed agrobacterium rhizogene strain genetic transformation red sage root blade, is passed through
The transgenic hairy root of PCR test positive clone.The acquisition of transgenosis Hairy Root Cultures of Salvia miltiorrhiza is screening high yield tanshinone, danshinolic acid
Hairy root provide direct material.
It is a kind of preferred embodiment that agrobacterium rhizogenes C58C1 is selected in the present embodiment, in actual selection, root of hair agriculture bar
The bacterial strain of bacterium is not limited to C58C1, and other bacterial strains can be selected as the case may be.
Embodiment 4
The expression of related gene in qRT-PCR detection transgenosis Hairy Root Cultures of Salvia miltiorrhiza
4.1. hairy root Liquid Culture
The good hairy root of fast, branch is grown in selection example 3, clip 2-3cm is with sterile distillation on superclean bench
Water rinses out 1/2MS fluid nutrient medium subculture of the access equipped with 100mL after the agar on its surface and once, harvests, take after 60 days
Proper amount of fresh hairy root is blotted after surface moisture with blotting paper, to be packaged with masking foil and be stored in -80 after being freezed into liquid nitrogen
DEG C be used for RNA extract, remaining hairy root drying after be used for tanshinone danshinolic acid content extraction.
4.2.RNA extraction and the synthesis of the chains of cDNA first
Step 1.1 in method be the same as Example 1
The design and synthesis of 4.3 primers
Used respectively according to arabidopsis gene AtMYC2 and tanshinone and danshinolic acid biosynthesis related genes coded sequence
Primer5.0 design primers are used for the expression for detecting related gene in Hairy Root Cultures of Salvia miltiorrhiza, and house-keeping gene Actin is used as
Internal reference.The primer is synthesized by Shanghai Sheng Gong bio-engineering corporations.
The primer of quantitative PCR is as follows：
4.4. the QRT-PCR detections of transgenosis Hairy Root Cultures of Salvia miltiorrhiza
QRT-, as template, is carried out with the primer of above-mentioned design using the first chain of same amount of above-mentioned cDNA (10 times of dilution) respectively
PCR is expanded.Carried out with reference to the specification of the Biosystem StepOne instruments of Applied Biosystem companies of U.S. production
Quantitative PCR is operated, using the RT-PCR kit of Quan Shi King Companies.Quantitative PCR reaction system is as follows：
PCR reaction conditions：94 DEG C of 5min, 40 circulations (94 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extensions
30sec),72℃10min.Target gene and house-keeping gene respectively do three repetitions.
QRT-PCR results are shown (see Fig. 3-1, Fig. 3-2, Fig. 3-3)：Expression quantity of the AtMYC2 genes in strain is overexpressed
Significantly improve, do not expressed in empty carrier, but the expression quantity between different clones has certain difference, and expression quantity highest is
Minimum 21.69 times of expression quantity.In related gene in tanshinone biosynthesis pathway, SmDXS2, SmCPS, SmHMGR expression
Amount has obvious up-regulation, and with No. 3 strains, significantly (SmDXS2 expression quantity is 3.7 times of control group, and SmCPS expression quantity is control
6.1 times of group) SmHMGR up-regulated expressions are the most it is apparent that No. 5 strains, are 6.1 times of control group；Danshinolic acid biosynthesis is on the way
SmCYP 98A, SmRAS, Sm4CL1, SmTAT expression quantity has obvious up-regulation in related gene in footpath, is control group respectively
3.72 times (No. 33), 3.31 times (No. 33), 3.69 times (No. 43), 5.04 times (No. 3), the slight up-regulation of SmPAL1, SmC4H expression,
SmHPPD expression quantity is then remarkably decreased, and is only 0.13 times (No. 13) of control group.As a result prove AtMYC2 in Hairy Root Cultures of Salvia miltiorrhiza
After overexpression, the expression of most of tanshinone danshinolic acid biosynthesis related genes can be promoted, suppress SmHPPD expression, and
A key gene on SmHPPD exactly danshinolic acid synthesis branch roads.
According to the literature, MYC2 can regulate and control downstream target gene by combining the G-box in downstream target gene promoter
Expression.Key gene promoter in tanshinone, danshinolic acid route of synthesis is analyzed, the base responded by AtMYC2 is found
Because containing G-box in promoter, therefore speculate AtMYC2 by combining G-box to regulate and control the expression of key gene, so as to adjust
Control the synthesis of tanshinone and danshinolic acid.
Embodiment 5
Tanshinone, danshinolic acid content in transgenosis Hairy Root Cultures of Salvia miltiorrhiza are determined using HPLC
5.1. in hairy root tanshinone content extraction
The hairy root that embodiment 4 is harvested is dried to constant weight, grind into powder, accurately weigh 0.2g hairy root powder in
In 50mL centrifuge tubes, 16mL methanol is added：Dichloromethane (3：1, v/v), ultrasonic 60min, room temperature, lucifuge, overnight stand,
12000rpm, centrifuges 10min, draws the 60 DEG C of vacuum drying in Rotary Evaporators of supernatant extract, residue is again with 2mL's
Methanol (analysis pure) dissolving, by sample with to be measured after 0.22 μm of membrane filtration.
5.2. the HPLC of tanshinone content is determined in hairy root
By dihydrotanshinone (DH-TI), Cryptotanshinone (CT), Tanshinone I (T-I) and tanshinone IIA (T-IIA) standard items
Respectively with the concentration for analyzing pure methanol and being configured to 1M, be stored in -20 DEG C it is standby.
Chromatographic condition is：C-18 reverse phase silica gels post (Symmetry Shield TM C18,5 μm, 250 × 4.6mm,
Waters)；Acetonitrile：Water (65：35) it is mobile phase；Column temperature is 30 DEG C；Flow velocity is 1mL/min；Wavelength is 270nm.
Above-mentioned standard product stock solution is taken into 5 μ l respectively, 10 μ l, 20 μ l, 30 μ l, 40 μ the l sample introductions under corresponding chromatographic condition,
Four kinds of compositions are kept completely separate, and peak type is good, record collection of illustrative plates and chromatographic parameter, respectively with peak area (Y) to standard concentration (X,
Mg/mL regression analysis) is carried out.
Tanshinone crude extract after above-mentioned 0.22 μm of membrane filtration respectively takes 20 μ L, injects high performance liquid chromatograph.Record is each
The peak area of tanshinone component, is substituted into after equation of linear regression, and calculating produces sample tanshinone content.
5.3. in hairy root danshinolic acid content extraction
The hairy root that embodiment 4 is harvested is dried to constant weight, grind into powder, accurately weigh 0.1g hairy root powder in
In 50mL centrifuge tubes, 10mL ethanol is added：Water (4：1, v/v), ultrasonic 20min, 8000rpm, centrifuge 10min, draw supernatant extraction
Liquid 70 DEG C of vacuum drying in Rotary Evaporators are taken, residue is dissolved with 2mL distilled water again, by sample with 0.22 μm of filter
It is to be measured after membrane filtration.
5.4. the HPLC of danshinolic acid content is determined in hairy root
Precision, which weighs salviandic acid A, tanshin polyphenolic acid B, Rosmarinic acid and caffeic acid standard items and is each configured to concentration with methanol, is
1M standard items stock solution, be stored in -20 DEG C it is standby.
Chromatographic condition：Chromatographic column is C-18 reverse phase silica gel posts, and mobile phase is acetonitrile：Water (30：70), water adjusts PH with phosphoric acid
For 2.03, Detection wavelength 281nm, 35 DEG C of column temperature, flow velocity 1mL/min.
Above-mentioned standard product stock solution is taken into 5 μ l respectively, 10 μ l, 20 μ l, 30 μ l, 40 μ the l sample introductions under corresponding chromatographic condition,
Four kinds of water soluble ingredients are kept completely separate, and peak type is good, record collection of illustrative plates and chromatographic parameter, respectively with peak area (Y) to standard items
Concentration (X, mg/mL) carries out regression analysis.
Danshinolic acid crude extract after above-mentioned 0.22 μm of membrane filtration respectively takes 20 μ L, is detected with high performance liquid chromatograph, record
Each component peak area, substitutes into equation of linear regression, calculates the content for producing sample water soluble ingredient.
In the present invention, be overexpressed AtMYC2 Hairy Root Cultures of Salvia miltiorrhiza strains in detected 4 kinds of tanshinones (dihydrotanshinone,
Cryptotanshinone, salvia miltiorrhiza bge I, tanshinone IIA) content is significantly improved compared to control group, wherein No. 3 strain tanshinone total amounts reach
14.06mg/g DW, are 6.45 times of control group；Dihydrotanshinone, Cryptotanshinone, Tanshinone I content are improved more significantly, red
Ginseng ketone IIA is not changed much then (see Fig. 4-1).4 kinds of danshinolic acid (caffeic acid, Rosmarinic acid, salviandic acid A, tanshin polyphenolic acid B) contents
Also significantly improved relative to control group, No. 3 strain danshinolic acid total amounts reach 95.90mg/g DW, be 4.30 times of control group；Coffee
Coffee acid and content of danshinolic acid B are significantly improved, and Rosmarinic acid and salviandic acid A content are then without significant change (see Fig. 4-2).
The present embodiment determines the content of transgenosis Hairy Root Cultures of Salvia miltiorrhiza tanshinone and danshinolic acid using HPLC methods.As a result show
The Hairy Root Cultures of Salvia miltiorrhiza total-tanshinone and danshinolic acid content ratio control group for being overexpressed AtMYC2 genes are significantly improved, wherein No. 3 strains
It is the most obvious that content is improved.
Embodiment 6
DPPH removes the antioxidation activity that free radical method determines tanshinone danshinolic acid in transgenic hairy root
6.1. prepared by sample solution：
Tanshinone in embodiment 5 and danshinolic acid crude extract are diluted to following gradient concentration：
0.25 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 4 μ g/ml each 1ml of methanol solution, takes 200 μ l, Ran Houjia
Enter 800 μ l 0.1mM 1,1- diphenyl picryl phenylhydrazine solution D PPH, fully mix, and dark place is incubated 30min at 25 DEG C.
6.2. the conversion of the absorbance measurement of sample and DPPH free radical scavenging activities：
By the sample in step (1), the 517nm under spectrophotometer is determined and is recorded its absorption value respectively.According to following
Absorbance is converted into DPPH free radical scavenging activities by formula：
DPPH free radical scavenging activities (%)==[(A0-A1)/A0 × 100]
A0 is control sample absorbance,
A1 is sample or standard items absorbance.
6.3.DPPH the comparison of free radical scavenging activity ability：
Method (Metabolic engineering, 2011) by delivering before us carries out DPPH radicals scavenging work
Property compare, as a result show, AtMYC2 transgenosis Hairy Root Cultures of Salvia miltiorrhiza strain M3 is compared with control group, the tanshinone of same substance amount and
Danshinolic acid crude extract shows similar radical scavenging activity.And sample M3 total-tanshinone and danshinolic acid content is respectively pair
According to 6.45 and 4.3 times, illustrate that the total antioxidant activity of sample M3 tanshinones and danshinolic acid is significantly higher than control group.
This example demonstrated the pellet for the same units amount being overexpressed in the Hairy Root Cultures of Salvia miltiorrhiza that AtMYC2 genetic methods are obtained
The oxidation resistance and control of ginseng ketone and danshinolic acid are suitable, therefore the tanshinone and danshinolic acid that are obtained by the present invention are while high yield
Hairy Root Cultures of Salvia miltiorrhiza method be efficiently it is feasible, with good application prospect.
The present invention obtains the pellet of high yield tanshinone and danshinolic acid simultaneously using the metabolic engineering strategies for turning AtMYC2 genes
Join transgenic hairy root strain, be a large amount of production tanshinones, danshinolic acid, alleviate tanshinone medicine source problem in short supply there is provided a kind of new
Type effective ways.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without passing through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel are according to the announcement of the present invention, and not departing from improvement and modification that scope made all should be the present invention's
Within protection domain.
Claims (6)
1. a kind of turn the method that AtMYC2 genes improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, it is characterised in that bag
Include following steps：
(1) cloned using gene clone method from arabidopsis and obtain transcription factor gene AtMYC2, the AtMYC2 genes sequence
It is classified as SEQ ID:1 DNA sequence dna；
(2) AtMYC2 genes are operably connected in expression regulation sequence, form the plant expression vector of the gene containing AtMYC2；
(3) by the plant expression vector transforming agrobacterium rhizogenes of the gene containing AtMYC2, obtain for convert the red sage root contain AtMYC2
The agrobacterium rhizogene strain of gene plant expression vector；
(4) constructed agrobacterium rhizogene strain genetic transformation red sage root blade is utilized, the transgenosis through PCR test positive is obtained
Hairy root is cloned；
(5) AtMYC2 genes and tanshinone are related to danshinolic acid biosynthesis in quantitative RT-PCR measure red sage root transgenic hairy root
The relative expression quantity of gene；
(6) the high effective liquid chromatography for measuring red sage root turns tanshinone and danshinolic acid content in AtMYC2 gene hairy roots, screens the red sage root
The red sage root transgenic hairy root strain that ketone and danshinolic acid content are significantly improved；
(7) DPPH removes free radical method and determines the antioxidation activity that the red sage root turns tanshinone in AtMYC2 gene hairy roots；
Described tanshinone is dihydrotanshinone, Cryptotanshinone, Tanshinone I or tanshinone IIA；
Described red sage root acid is caffeic acid, Rosmarinic acid, salviandic acid A or tanshin polyphenolic acid B.
2. a kind of AtMYC2 genes that turn according to claim 1 improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Method, it is characterised in that the agrobacterium rhizogenes described in step (3) is selected from bacterial strain C58C1.
3. a kind of AtMYC2 genes that turn according to claim 1 improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Method, it is characterised in that the PCR detection method described in step (4) is as follows：
(4.1) root of hair locus gene BrolB, kalamycin resistance gene kan specific PCR primers are separately designed；
(4.2) constitutive promoter for starting the tMYC2 expression of insertion Gene A is CaMV35S promoters；
(4.3) insertion gene internal and NOS terminator indoor design upstream and downstream specific primer, carry out DNA cloning；
(4.4) viewed under ultraviolet radiation purpose band strain, it is then positive transgenic Hairy Root Cultures of Salvia miltiorrhiza strain purpose band occur.
4. a kind of AtMYC2 genes that turn according to claim 1 improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Method, it is characterised in that step (5) described quantitative RT-PCR detecting method is as follows：
(5.1) the hairy root clone that the positive is accredited as to PCR carries out the extraction of total serum IgE, unified quantitative to 0.5 μ g RNA reverse transcriptions
Into the cDNA of 25 μ L systems；
(5.2) insertion target gene and house-keeping gene Actin primer are separately designed, is determined using same amount of cDNA as template
Measure RT-PCR analyses；
(5.3) the relative expression quantity situation of analysis Gene A tMYC2 and tanshinone and danshinolic acid biosynthesis related genes.
5. a kind of AtMYC2 genes that turn according to claim 1 improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Method, it is characterised in that the high performance liquid chromatography described in step (6) is as follows：
(6.1) tanshinone：Chromatographic column C-18 reverse phase silica gel posts, mobile phase is volume ratio 65：35 acetonitrile and water；Detection wavelength
220nm, 30 DEG C of column temperature, flow velocity 1mL/min, the μ L of sample size 20；
(6.2) danshinolic acid：Chromatographic column C-18 reverse phase silica gel posts, mobile phase is volume ratio 30：70 acetonitrile and water, and adjusted with phosphoric acid
Save pH to 2.03；Detection wavelength 281nm, 35 DEG C of column temperature, flow velocity 1mL/min, the μ L of sample size 20.
6. a kind of AtMYC2 genes that turn according to claim 1 improve tanshinone and danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Method, it is characterised in that in step (7), the assay method of the antioxidation activity of tanshinone and danshinolic acid is：
(7.1) red sage root turns to extract tanshinone and danshinolic acid in AtMYC2 gene hairy roots；
(7.2) sample obtained by step (7.1) is diluted to concentration gradient：0.25 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 4
μ g/mL each 1mL of methanol solution；
(7.3) the μ L of step (7.2) resulting solution 200 are taken, 800 μ L 0.1mM DPPH solution is separately added into, fully mixed；
(7.4) 30min is placed in darkroom at 25 DEG C of step (7.3) products therefrom；
(7.5) step (7.4) product is surveyed into absorption value under spectrophotometer at 517nm；
Wherein, DPPH free radical scavenging activities (%)=[(A0-A1)/A0× 100],
In formula, A0It is control sample (DPPH) absorbance；A1It is the absorbance of sample or standard items.
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| CN105602985B (en) * | 2015-10-28 | 2019-07-26 | 上海师范大学 | Turn the method that SmMYB75 gene improves danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza |
| CN106636142A (en) * | 2017-02-25 | 2017-05-10 | 中国医学科学院药用植物研究所 | Clone identification and application of 2-oxoglutarate-dependent dioxygenase (2OGD-5) gene participating in tanshinone synthesis |
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| CN112501177B (en) * | 2019-09-16 | 2022-09-06 | 上海长征医院 | Method for promoting development of lateral root of salvia miltiorrhiza and improving content of tanshinone in salvia miltiorrhiza root, salvia miltiorrhiza gene sequence and overexpression vector |
| CN110590923B (en) * | 2019-10-18 | 2020-12-29 | 中国药科大学 | Application of Salvia miltiorrhiza NAC1 transcription factor and coding gene thereof in regulating and controlling content of salvianolic acid compounds in Salvia miltiorrhiza |
| CN110819636B (en) * | 2019-11-08 | 2021-09-17 | 浙江中医药大学 | SmbZIP1 gene and application thereof in increasing content of salvianolic acid in salvia miltiorrhiza |
| CN112695043B (en) * | 2021-01-18 | 2023-02-14 | 浙江中医药大学 | Application of SmbZIP2 gene in improving content of salvianolic acid in salvia miltiorrhiza |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN114410656B (en) * | 2021-06-03 | 2023-12-01 | 浙江农林大学 | Application of BrMYC3-2 gene overexpression in plant growth |
| CN114645061B (en) * | 2022-04-11 | 2023-10-20 | 浙江中医药大学 | SmMYB76 gene and application thereof in improving salvianolic acid content in salvia miltiorrhiza bunge |
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