CN105602985B - Turn the method that SmMYB75 gene improves danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza - Google Patents
Turn the method that SmMYB75 gene improves danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza Download PDFInfo
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Abstract
The invention discloses a kind of methods for turning SmMYB75 gene and improving danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, it is cloned from Radix Salviae Miltiorrhizae by the method for gene cloning and has obtained the SmMYB75 gene of 645bp, construct the carrier of SmMYB75 gene prokaryotic and subcellular localization；Construct high efficiency plant expression vector, genetic transformation Radix Salviae Miltiorrhizae blade obtains the Hairy Root Cultures of Salvia miltiorrhiza of SmMYB75 gene overexpression, and screens to have obtained the Hairy Root Cultures of Salvia miltiorrhiza for turning SmMYB75 gene that danshinolic acid content significantly improves by the means of a series of biotechnology.Method of the invention to produce there is the danshinolic acid of important clinical demand to provide a kind of novel high-quality raw material, have positive promote meaning and application value to the property problem in short supply for alleviating danshinolic acid medicine source.
Description
Technical field
The present invention relates to technique for gene engineering, in particular to one kind turns red phenol in SmMYB75 gene raising Hairy Root Cultures of Salvia miltiorrhiza
The method of acid content.
Background technique
Cardiovascular and cerebrovascular disease is current threat whole mankind's health and life " number one killer ".According to statistics, the whole world is annual
About 17,000,000 people die of cardiovascular and cerebrovascular disease, account for about the 1/3 of global total death toll, China about 3,000,000 people every year
Cardiovascular and cerebrovascular disease is died of, therefore, actively research is efficiently, the clinical medicine of low toxicity and cheap treatment cardiovascular and cerebrovascular disease is to mentioning
High level of human health has very profound significance.
Radix Salviae Miltiorrhizae (Salvia miltiorrhiza) is Labiatae (Labiatae) Salvia perennial herb plant, with
Its root or tuber dried is used as medicine, and is widely used in treatment cardiovascular and cerebrovascular disease, is a kind of traditional Chinese herbal medicine plant.It is red
The bioactive ingredients of ginseng are broadly divided into two major classes: one kind is water-soluble phenolic acid compound, mainly includes salviandic acid A, pellet
Phenolic acid B, salvianolic acid C, caffeic acid, Rosmarinic acid, danshensu etc.；Another kind of is fat-soluble tanshinone compound, main to wrap
Include Tanshinone I, tanshinone IIA, Cryptotanshinone, dihydrotanshinone etc..
Danshinolic acid in Radix Salviae Miltiorrhizae plays an important role in terms of preventing human diseases, and biological activity mainly includes
It is anti-oxidant, anticoagulating active, the adjusting of renal function, the protection of liver, cardiovascular protection, anticancer, antibacterial, antiviral, anti-inflammatory
Disease etc..In addition to this, danshinolic acid also has the function of anti-ischemia-reperfusion, antithrombotic, blood pressure lowering and anti-fibrosis.In China,
Radix Salviae Miltiorrhizae is usually made as medicament, such as tablet, capsule, granule, injection, oral solution, spraying, dripping pill, has high
Medicinal and economic value.However, under traditional cultivation mode, when Radix Salviae Miltiorrhizae cultivated since long, quality deterioration is serious, active constituent contains
Amount reduces, growth cycle is long and restricts vulnerable to cultural area and environment etc.；During chemical synthesis, the chemical synthesis of danshinolic acid
Process is very complicated, higher cost and easily causes environmental pollution；In the cell that in vitro is obtained by cell culture processes,
Its medicinal active ingredient accumulation is very low and stability is very poor, and the requirement that commercial development utilizes far is not achieved.Therefore, in order to
Alleviate the property problem in short supply with the danshinolic acid medicine source of important clinical demand, it would be highly desirable to invent a kind of new side for improving danshinolic acid content
Method, the rapid development of metabolic engineering are to improve danshinolic acid ingredient in Hairy Root Cultures of Salvia miltiorrhiza with hairy the increasingly mature of culture technique
Content solves the problems, such as that tanshinone medicine source property in short supply provides a new approaches.
Existing research show the R2R3-MYB transcription factor wide participation in plant to the secondary metabolism process of plant, because
This R2R3-MYB transcription factor for excavating Radix Salviae Miltiorrhizae itself is of great significance to improve the content of danshinolic acid.Utilize genetic engineering
Radix Salviae Miltiorrhizae transcription factor SmMYB75 gene genetic conversion Radix Salviae Miltiorrhizae blade is obtained the transgenosis Radix Salviae Miltiorrhizae hair that SmMYB75 is overexpressed by means
Shape root, while the biosynthesis of danshinolic acid is raised, the Hairy Root Cultures of Salvia miltiorrhiza root system of danshinolic acid high yield is obtained, to commercially produce red phenol
Acid provides novel high-quality medicine source.Not yet discovery passes through red phenol in SmMYB75 gene overexpression strategy raising Hairy Root Cultures of Salvia miltiorrhiza at present
The relevant report of acid content.
Summary of the invention
The purpose of the present invention provides one kind and turns SmMYB75 gene exactly in order to overcome the problems of the above-mentioned prior art
Method to improve 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 is cloned from Radix Salviae Miltiorrhizae obtains the SmMYB75 gene of 645bp, the carrier of prokaryotic expression is constructed, in large intestine bar
SmMYB75 gene recombinant protein is expressed in bacterium；Its gene after different tissues and methyl jasmonic acid induction of quantitative PCR analysis
Expression quantity；The carrier of subcellular localization, instantaneous conversion tobacco leaf are constructed, confocal laser scanning microscope quantifies SmMYB75
Positioning scenarios of the gene in cell；Plant expression vector is constructed, agrobacterium rhizogenes C58C1 mediates genetic transformation Radix Salviae Miltiorrhizae blade to obtain
Obtain hairy of transgenosis；The integration of PCR testing goal gene SmMYB75；Quantitative PCR analysis is inserted into gene SmMYB75
And expression of the danshinolic acid biosynthesis related genes in hairy；High performance liquid chromatography measures red in transgenic hairy root
The content of phenolic acid.
A method of turning SmMYB75 gene and improve danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, comprising the following steps:
(1) it is cloned from Radix Salviae Miltiorrhizae using gene clone method and obtains gene SmMYB75, the SmMYB75 gene order
As shown in SEQ ID:1, sequence analysis is carried out to it.
(2) according to Radix Salviae Miltiorrhizae SmMYB75 gene order, SmMYB75 gene is operably implemented in the load of prokaryotic expression
Body is expressed in Escherichia coli, and recombinant protein carries out SDS-PAGE electrophoresis detection；The carrier is pET-30a (+),
Escherichia coli are bacterial strain Rosetta (DE3).
(3) according to Radix Salviae Miltiorrhizae SmMYB75 gene order, the primer of quantitative PCR is designed, to SmMYB75 at different groups of Radix Salviae Miltiorrhizae
Expression quantity in knitting and its inducing expression of methyl jasmonic acid regulation is analyzed.
(4) by the gene constructed carrier in subcellular localization of SmMYB75, agrobacterium tumefaciens is converted, tobacco leaf is carried out
Instantaneous conversion carries out Subcellular Localization to SmMYB75 gene；The carrier is pMON530, and agrobacterium tumefaciens are bacterial strain
Ase。
(5) SmMYB75 gene is operably implemented in expression regulation sequence, forms the plant of the gene containing SmMYB75
Expression vector；Plant expression vector is the pCAMBIA2300 obtained by transformation+Carrier includes CaMV35S promoter and termination
Son, multiple cloning sites, replication origin and kalamycin resistance site.
(6) it by the plant expression vector transforming agrobacterium rhizogenes C58C1 of step (5) resulting gene containing SmMYB75, obtains
For converting the agrobacterium rhizogene strain of the expression vector of gene plant containing SmMYB75 of Radix Salviae Miltiorrhizae.
(7) using agrobacterium rhizogene strain genetic transformation Radix Salviae Miltiorrhizae blade constructed by step (6), acquisition is detected as through PCR
Positive transgenic hairy root clone；The positive transgenic Hairy Root Cultures of Salvia miltiorrhiza through PCR detection refers to: design root of hair site
The upstream and downstream primer of gene BrolB and insertion gene SmMYB75 inside and NOS terminator interior design upstream and downstream it is special
Property primer, carry out DNA cloning, the root system of viewed under ultraviolet radiation to purpose band is positive transgenic Hairy Root Cultures of Salvia miltiorrhiza root system.
(8) SmMYB75 gene and danshinolic acid biology in the Radix Salviae Miltiorrhizae transgenic hairy root that quantitative PCR determination step (7) obtains
The relative expression quantity of route of synthesis related gene, and filter out the expression for being overexpressed SmMYB75 gene in SmMYB75 gene root system
Measure the root system improved；Method particularly includes: the extraction that hairy positive clone carries out total serum IgE is accredited as to PCR, it is unified quantitatively to arrive
0.5 μ g RNA, reverse transcription separately design insertion target gene and house-keeping gene SmActin are quantified at the cDNA of 25 μ l systems
Primer carries out the opposite of quantitative PCR analysis SmMYB75 and danshinolic acid biosynthesis related genes by template of same amount of cDNA
Expression quantity situation.
(9) Radix Salviae Miltiorrhizae turns the content of hairy middle danshinolic acid of SmMYB75 gene in high effective liquid chromatography for measuring step (8),
The Radix Salviae Miltiorrhizae transgenic hairy root root system that screening danshinolic acid content significantly improves；The method are as follows: chromatographic column C-18 reverse phase silica gel
Column, mobile phase are the acetonitrile and water of volume ratio 30:70, and with phosphorus acid for adjusting pH to 2.03；Detection wavelength 281nm, 35 DEG C of column temperature,
Flow velocity 1ml/min, 20 μ l of sample volume.
Integrated application biology of the present invention and gene technology method such as vector construction, Molecular Detection, quantify genetic transformation
PCR analysis, the extraction of danshinolic acid and assay etc., have invented a kind of R2R3-MYB transcription factor SmMYB75 using Radix Salviae Miltiorrhizae
The method that gene improves danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza.The overexpression SmMYB75 transgenosis Hairy Root Cultures of Salvia miltiorrhiza root that the present invention obtains
It is 167.25mg/g dry weight that highest expression quantity, which is total salvianolic acid content, in system, is 2.26 times of control group (73.7mg/g dry weight).
The present invention provides possibility for commercialization mass production danshinolic acid and reduction drug price, is also mass production danshinolic acid clinic medicine
The wilderness demand of object provides important sources.
Compared with prior art, the invention has the following advantages that
1, the content of danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza is significantly improved.
2, inventive method effect is reliable.
3, the cost for obtaining danshinolic acid is low.
4, production process non-environmental-pollution.
Detailed description of the invention
Fig. 1 is pCAMBIA2300+:: SmMYB75 vector construction schematic diagram.
The SDS-PAGE that Fig. 2 is recombinant protein SmMYB75 is analyzed.
Fig. 3 is the tissue expression analysis of Radix Salviae Miltiorrhizae SmMYB75.
Fig. 4 is expression analysis of the Radix Salviae Miltiorrhizae SmMYB75 under methyl jasmonic acid processing.Wherein, Control is ethanol control group
Induction, MJ are 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, green fluorescent protein merge figure with light field；PMON530::GFP, empty carrier luciferase expression；pMON530::
The prompt fluorescence of SmMYB75-GFP, SmMYB75 are expressed.
Fig. 6 is expression analysis result figure of the SmMYB75 in transgenosis Hairy Root Cultures of Salvia miltiorrhiza.Wherein, Control C58C1
Hairy for infecting acquisition.
Fig. 7 is the expression analysis result figure of danshinolic acid biosynthesis related genes.Wherein, Empty vector is unloaded
Body.
Fig. 8 is the content detection result figure of danshinolic acid in SmMYB75 transgenosis Hairy Root Cultures of Salvia miltiorrhiza.Wherein, Empty
Vector is empty carrier.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Fig. 1 is pCAMBIA2300+::
SmMYB75 vector construction schematic diagram.The SDS-PAGE that Fig. 2 is recombinant protein SmMYB75 is analyzed.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as molecular cloning
Condition described in (Sambrook etc.), or according to reagent provided by manufacturer or the incidental specification suggestion of kit
Condition.
Embodiment 1
The clone of Radix Salviae Miltiorrhizae SmMYB75 gene
1.1. the extraction of Radix Salviae Miltiorrhizae total serum IgE
It takes a small amount of Radix Salviae Miltiorrhizae young leaflet tablet to be ground with mortar rapidly after liquid nitrogen flash freezer, is then mentioned according to TIANGEN company
The RNAprep Pure Plant Kit operation instructions of confession extract total serum IgE.With the gel electrophoresis of plain agar sugar (deposition condition:
Gum concentration 1.2%；0.5 × TBE electrophoretic buffer；150v, 15min) detection RNA integrality.It is super with Nano Drop 2000c
Micro-spectrophotometer detects its purity and concentration.
1.2. the clone of Radix Salviae Miltiorrhizae SmMYB75 gene
Using the 0.5 μ g Radix Salviae Miltiorrhizae total serum IgE obtained as initial amount, the synthesis of the first chain cDNA is carried out with reverse transcriptase XL (AMV)
(instructions book that operating procedure is provided referring to Promega company).According to the coded sequence (SEQ of the SmMYB75 gene
ID NO.1), design amplifies the upstream and downstream primer of complete encoder block, and introduces respectively on upstream and downstream primer restricted
Restriction enzyme site (this can be depending on the carrier of selection), so as to carrier construction.Using the first chain cDNA as template, through PCR
It is sequenced after amplification.Determined dna sequence is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing result is shown in
SEQ NO.1。
Embodiment 2
The prokaryotic expression of Radix Salviae Miltiorrhizae SmMYB75 gene is analyzed
2.1.pET-30a (+):: the building of SmMYB75 carrier
According to the ORF sequence for the Radix Salviae Miltiorrhizae SmMYB75 for having cloned acquisition, the primer of subcellular localization vector construction is designed,
It constructs pET-30a (+):: SmMYB75, and by pET-30a (+):: SmMYB75 plasmid converts Escherichia coli Rosetta (DE3),
Picking monoclonal colonies carry out PCR verifying.The result shows that the subcellular localization carrier pET-30a (+) containing SmMYB75::
Successful conversion can be used for subsequent tobacco transient expression experiment in Escherichia coli Rosetta (DE3) to SmMYB75.
2.2.SmMYB75 the SDS-PAGE analysis of recombinant protein
Building is successfully contained into pET-30a (+):: the Escherichia coli Rosetta (DE3) of SmMYB75 is inoculated in containing card
In the LB culture medium of that mycin (75mg/mL), 2-3 hours (OD are cultivated600=0.4-0.5).0.5 mM of IPTG is added to lure
Agent inducing expression is led, respectively in 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours collection bacterium solutions,
12000 revs/min, it is centrifuged 1 minute harvest thallus, albumen sample-loading buffer is added, boiling water boils 10 minutes, carries out SDS-PAGE
Electrophoretic analysis.The results show that the size of expression albumen is about 30KD, it can be seen that, induced from SDS-PAGE electrophoresis in IPTG
Afterwards, there is apparent purpose band compared with when not inducing at 30KD, illustrate SmMYB75 success table in this expression system
It reaches, and its inducing expression amount significantly improves with the growth of induction time.
Embodiment 3
The tissue expression spectrum analysis of Radix Salviae Miltiorrhizae SmMYB75
In order to study the tissue expression pattern of SmMYB75, the biennial main root of Radix Salviae Miltiorrhizae, fibrous root, stem, leaf, leaf are extracted respectively
Handle, petal, calyx, filigree eight total serum IgEs organized, and purity and Concentration Testing are carried out respectively.Then reverse transcription is used at cDNA
In the SuperReal PreMix that the distribution expression pattern of quantitative PCR analysis SmMYB75, reaction system TIANGEN company provide
(SYBR Green) kit, uses SmActin as reference gene.Quantification PCR primer are as follows:
The results show that SmMYB75 it is detected it is organized in, expressed in stem, leaf and petiole it is relatively strong, secondly in flower
It expresses higher in silk and main root, weaker (see Fig. 3) is expressed in fibrous root, petal, calyx.
Embodiment 4
The response analysis that Radix Salviae Miltiorrhizae SmMYB75 handles methyl jasmonic acid
In order to verify the induction whether Sm MYB75 responds methyl jasmonic acid, to having cultivated 60 or so sizes in shaking flask
Hairy Root Cultures of Salvia miltiorrhiza carries out methyl jasmonic acid processing, extracts the total serum IgE of Hairy Root Cultures of Salvia miltiorrhiza after handling different time, and respectively into
Row purity and Concentration Testing, then reverse transcription is at cDNA, for the distribution expression pattern of quantitative PCR analysis SmMYB75, quantitative primer
With embodiment 3, quantitative PCR is the results show that within the regular hour, with the induction of methyl jasmonic acid, the table of SmMYB75
Up to the obvious up-regulation of amount, peak is reached in 4h, subsequent expression quantity declines (see Fig. 4).
Embodiment 5
The Subcellular Localization of Radix Salviae Miltiorrhizae SmMYB75 gene
5.1.pMON530::MYB75 the building of carrier
According to the ORF sequence for the Radix Salviae Miltiorrhizae SmMYB75 for having cloned acquisition, the primer of subcellular localization vector construction is designed,
PMON530::SmMYB75 is constructed, and pMON530::SmMYB75 plasmid is converted into Agrobacterium Ase, picking monoclonal colonies carry out
PCR verifying.The result shows that the subcellular localization carrier pMON530::SmMYB75 containing SmMYB75 successful conversion to agriculture
In bacillus Ase, it can be used for subsequent tobacco transient expression experiment.
5.2. the transient expression in tobacco
The work of the pMON530 containing carrier pMON530::SmMYB75 and empty carrier built is drawn with asepsis injector
Journey bacterium Ase carries out instantaneous conversion to well-grown tobacco leaf, and illumination cultivation takes blade on glass slide after 40-48 hours,
The back side upward, is infiltrated with distilled water, with coverslip fixed blade (avoiding generating bubble), is seen under laser confocal microscope
It examines.The results show that SmMYB75 is expressed in nucleus, it is consistent as the function of transcription factor (see Fig. 5) with them.
Embodiment 6
The building of the plant over-express vector of the gene of SmMYB75 containing Radix Salviae Miltiorrhizae
Vector construction ideograph is shown in Fig. 1.With pCAMBIA2300+For expression vector, the SmMYB75 gene that clone is obtained
Replace pCAMBIA2300+On gus gene.Specifically, Spe I/BstPI double digestion pMD18-T::SmMYB75 and
pCAMBIA2300+；Recycle SmMYB75 gene and pCAMBIA2300+Large fragment；Connection conversion, picking monoclonal colonies PCR sieve
Select positive colony；Extract the further digestion verification of plasmid.The result shows that SmMYB75 gene has successfully been building up to plant expression
Carrier pCAMBIA2300+In, to obtain the plant over-express vector pCAMBIA2300 of the gene containing SmMYB75+::SmMYB75。
Transcription factor SmMYB75 gene is operatively connectable to expression regulation sequence by the present embodiment, and formation contains
The plant over-express vector pCAMBIA2300 of SmMYB75 gene+:: SmMYB75, the expression vector can be used for passing through metabolic engineering
Strategy improves the content of danshinolic acid in Radix Salviae Miltiorrhizae.
With pCAMBIA2300 in the present embodiment+It is a kind of preferred embodiment for medial expression vector, is carried in actual implementation
During body, other suitable carriers can be selected as the case may be, when selecting different carriers, introduce restriction enzyme
Enzyme site is different.
Embodiment 7
The Radix Salviae Miltiorrhizae SmMYB75 gene genetic conversion Radix Salviae Miltiorrhizae that agrobacterium rhizogenes mediates obtains transgenosis Hairy Root Cultures of Salvia miltiorrhiza
7.1. the acquisition of the agrobacterium rhizogenes engineering bacteria containing plant expression vector
By the plant over-express vector pCAMBIA2300 of the gene containing SmMYB75 in embodiment 2+:: SmMYB75 is transferred to root of hair
In Agrobacterium C58C1, picking monoclonal colonies carry out PCR verifying.It is carried the result shows that the plant of the gene containing SmMYB75 is overexpressed
Body pCAMBIA2300+:: SmMYB75 successful conversion into agrobacterium rhizogenes C58C1.
7.2. agrobacterium rhizogenes mediates SmMYB75 gene genetic to convert Radix Salviae Miltiorrhizae
7.2.1 the preculture of explant
Clip Radix Salviae Miltiorrhizae stalwartness tests for sterility (0.5cm2), it is inoculated on precultivation medium (1/2MS), 25 DEG C of dark cultures
2 days.
7.2.2 the co-cultivation of Agrobacterium and explant
By the Radix Salviae Miltiorrhizae blade explant of above-mentioned preculture, it is put into 1/ containing activated above-mentioned agrobacterium rhizogenes engineering bacteria
After impregnating 10 minutes (jiggle is that explant and bacterium solution come into full contact with) in 2MS suspension, the Radix Salviae Miltiorrhizae blade after taking out dip dyeing is used
Sterile blotting paper blots surface bacterium solution, goes in co-culture medium 1/2MS, and dark culture 2-3 days.
7.2.3 hairy induction and squamous subculture
The above-mentioned Radix Salviae Miltiorrhizae explant for co-culturing 2-3 days is transferred to degerming solid medium (1/2MS+Cb300mg/L)
In, 25 DEG C dark culture 2-3 weeks or so, hairy can be grown from explant wound.The Radix Salviae Miltiorrhizae explant of root of hair is transferred
To on degerming solid medium (1/2MS+Cb200mg/L), clip list when hairy root long is to 3cm or more of 25 DEG C of dark cultures 2 weeks
It clones hairy and is used as a clone, continue to be inoculated in except dark culture two weeks in bacterium culture medium (1/2MS+Cb100mg/L), directly
To without bacterium phenomenon of overflowing.Monoclonal hairy is transferred on the 1/2MS culture medium of antibiotic-free and continues dark culture.
7.3. the PCR detection of transgenosis Hairy Root Cultures of Salvia miltiorrhiza
7.3.1 the extraction of transgenic hairy root genomic DNA
Transgenic hairy root genomic DNA is extracted using CTAB method.The transgenic hairy root that degerming finishes in clip 3.2.3
5cm or so is put into 1.5mL centrifuge tube, and 600 μ L CTAB lysates (65 DEG C of preheatings, contain 1% beta -mercaptoethanol) are added, use group
Beveller is knitted to be fully ground.It is placed in 65 DEG C of water-baths 40-50 minutes, repeatedly mixes sample (secondary/15min) therebetween, be cooled to
Isometric phenol/chloroform (1:1) is added after room temperature, is gently mixed by inversion emulsification 10min, 1200rpm is centrifuged 15min, carefully
Supernatant is drawn in new EP pipe, isometric chloroform is added and mixes, 12000rpm is centrifuged 15min, slowly draws supernatant in new EP
Guan Zhong, the dehydrated alcohol (putting 30min in -20 DEG C) for adding 2 times of volumes to be pre-chilled are precipitated and precipitate, 12000rpm centrifugation 15min, in abandoning
Clearly, 75% ethanol washing is added twice, supernatant is sucked out, room temperature is dried, and 30-50 μ L water dissolution precipitating is added, after being handled with RNA enzyme
It is frozen in -80 DEG C of ultra low temperature freezers, it is spare.
7.3.2 design of primers and PCR detection
In pCAMBIA2300+:: spy is separately designed on the insertion gene (SmMYB75) and NOS terminator of SmMYB75 carrier
Anisotropic upstream and downstream primer, while specific upstream and downstream primer is designed on root of hair locus gene BrolB.Simultaneously with the side PCR
Method carries out Molecular Detection to above-mentioned hairy total DNA.The result shows that being detected in transgenic hairy root root system and positive control
(pCAMBIA2300+:: SmMYB75 plasmid is template) sizable PCR product；And pellet is infected with Agrobacterium C58C1 empty bacterium
Join resulting hairy genomic DNA be template when, do not amplify any segment, as a result illustrated SmMYB75 gene
It is integrated into Radix Salviae Miltiorrhizae genome.
The plant expression vector transforming agrobacterium rhizogenes are obtained the plant for converting Radix Salviae Miltiorrhizae and expressed by the present embodiment
The agrobacterium rhizogene strain C58C1 of carrier is passed through using constructed agrobacterium rhizogene strain genetic transformation Radix Salviae Miltiorrhizae blade
The transgenic hairy root of PCR test positive clone.The acquisition of transgenosis Hairy Root Cultures of Salvia miltiorrhiza is the hairy of screening high yield danshinolic acid
Root provides 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, can select other bacterial strains as the case may be.
Embodiment 8
The expression of related gene in quantitative PCR detection transgenosis Hairy Root Cultures of Salvia miltiorrhiza
8.1. hairy Liquid Culture
Good hairy of fast, branch, the sterile distillation of clip 2-3cm on superclean bench are grown in selection example 7
1/2MS fluid nutrient medium subculture of the access equipped with 100mL is primary after water rinses out the agar on its surface, harvests, takes after 60 days
After proper amount of fresh hairy is blotted surface moisture with blotting paper, -80 are stored in after being packaged with masking foil into freezing in liquid nitrogen
DEG C for RNA extract, remaining hairy drying after be used for tanshinone danshinolic acid content extraction.
8.2.RNA the synthesis of the first chain of extraction and cDNA
Method is the same as the step 1.1 in embodiment 1
The design and synthesis of 8.3 primers
It is used respectively according to the coded sequence of Radix Salviae Miltiorrhizae gene SmMYB75 and tanshinone and danshinolic acid biosynthesis related genes
Primer5.0 design primer is used to detect the expression of 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 corporation.
The primer of quantitative PCR is as follows:
8.4. the quantitative PCR detection of transgenosis Hairy Root Cultures of Salvia miltiorrhiza
It as template, is quantified respectively with the primer of above-mentioned design using the first chain of same amount of above-mentioned cDNA (10 times of dilution)
PCR amplification.It is carried out referring to the specification of the Biosystem StepOne instrument of U.S. Applied Biosystem company production
Quantitative PCR operation, using the quantitative PCR kit of Quan Shi King Company.Quantitative PCR reaction system is as follows:
PCR reaction condition: 94 DEG C 5 minutes, 40 circulation (94 DEG C be denaturalized 30 seconds, 60 DEG C anneal 30 seconds, 72 DEG C extend 30
Second), 72 DEG C 10 minutes.Target gene and house-keeping gene are respectively done to be repeated three times.
Quantitative PCR is as the result is shown: comparing empty carrier root system, SmMYB75 gene is obvious in the expression quantity being overexpressed in root system
It improves, but the expression quantity between different clones has certain difference, it is clone 9,14,18,19, No. 23 that wherein expression quantity is higher
(see Fig. 6).At the same time, after SmMYB75 is overexpressed in Hairy Root Cultures of Salvia miltiorrhiza, key gene in danshinolic acid metabolic pathway
Expression quantity significantly improves, and illustrates that SmMYB75 can promote the expression of related gene in danshinolic acid biosynthesis pathway (see Fig. 7).
According to the literature, myb transcription factor can be by adjusting in conjunction with the cis-acting elements in downstream target gene promoter
Control the expression of downstream target gene.Key gene promoter in danshinolic acid route of synthesis is analyzed, is found by SmMYB75
Containing the cis-acting elements of MYB on the gene promoter of response, therefore speculate that SmMYB75 is cis- in promoter by combining
Functional element regulates and controls the expression of key gene, to regulate and control the synthesis of danshinolic acid.
Embodiment 9
Utilize danshinolic acid content in HPLC measurement transgenosis Hairy Root Cultures of Salvia miltiorrhiza
9.1. the extraction of hairy middle danshinolic acid content
Drying to constant weight by hairy that embodiment 4 is harvested, grind into powder, accurately weigh hairy powder of 0.1g in
In 50mL centrifuge tube, 10mL ethyl alcohol: water (4:1, v/v) is added, ultrasonic 20min, 8000rpm are centrifuged 10min, draw supernatant extraction
Liquid 70 DEG C of vacuum drying in Rotary Evaporators are taken, residue uses the distilled water of 2mL to dissolve again, by 0.22 μm of filter of sample
It is to be measured after film filtering.
9.2. the HPLC measurement of hairy middle danshinolic acid content
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
The standard items stock solution of 1mM, be stored in -20 DEG C it is spare.
Chromatographic condition: chromatographic column is C-18 reverse phase silica gel column, and mobile phase is acetonitrile: water (30:70), and water adjusts PH with phosphoric acid
It is 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, map and chromatographic parameter are recorded, 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 10 μ L, is detected with high performance liquid chromatograph, record
Peak area of each component substitutes into equation of linear regression, calculates the content up to sample water soluble ingredient.
In the present invention, in addition to caffeinic content is without apparent upper downward relationship, relative to control group, Rosmarinic acid,
Salviandic acid A, three kinds of danshinolic acids of tanshin polyphenolic acid B content obviously raise, the content of total salvianolic acid also significantly improves.Wherein, expression quantity
The content (167.25) of danshinolic acid is 2.26 times (see Fig. 8) of control group (73.7) in highest transgenic hairy root.
The present invention is hairy using the Radix Salviae Miltiorrhizae transgenosis that the metabolic engineering strategies for turning SmMYB75 gene obtain high yield danshinolic acid
Root root system provides a kind of novel effective ways for mass production danshinolic acid, alleviation danshinolic acid medicine source problem in short supply.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (4)
1. a kind of method for turning SmMYB75 gene and improving danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, which is characterized in that including following step
It is rapid:
(1) it is cloned from Radix Salviae Miltiorrhizae using the method for gene cloning and obtains SmMYB75 gene, the SmMYB75 gene order such as SEQ
Shown in ID NO:1；
(2) SmMYB75 gene is operably implemented in expression regulation sequence, forms the plant expression of the gene containing SmMYB75
Carrier；
(3) it by the plant expression vector transforming agrobacterium rhizogenes of step (2) resulting gene containing SmMYB75, obtains for converting
The agrobacterium rhizogene strain of the expression vector of gene plant containing SmMYB75 of Radix Salviae Miltiorrhizae；
(4) it using agrobacterium rhizogene strain genetic transformation Radix Salviae Miltiorrhizae blade constructed by step (3), obtains through PCR test positive
Radix Salviae Miltiorrhizae transgenic hairy root clone；
(5) the SmMYB75 gene in the Radix Salviae Miltiorrhizae transgenic hairy root that quantitative PCR determination step (4) obtains and danshinolic acid biology close
At the relative expression quantity of key gene in approach, and filters out gene expression amount in the root system for being overexpressed SmMYB75 gene and mention
High root system；
(6) Radix Salviae Miltiorrhizae turns the content of hairy middle danshinolic acid of SmMYB75 gene in high effective liquid chromatography for measuring step (5), screening
The transgenosis Hairy Root Cultures of Salvia miltiorrhiza root system that danshinolic acid content significantly improves；
Plant expression vector described in step (2) is the pCAMBIA2300 obtained by transformation+Carrier starts comprising CaMV35S
Son and NOS terminator, multiple cloning sites, replication origin and kalamycin resistance site；
Agrobacterium rhizogenes described in step (3) is selected from bacterial strain C58C1；
The danshinolic acid is Rosmarinic acid, salviandic acid A or tanshin polyphenolic acid B.
2. the method according to claim 1 for turning SmMYB75 gene and improving danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, feature
It is, PCR detection method described in step (4) is as follows:
(1) specific PCR primers for designing root of hair locus gene rolB, carry out PCR amplification；
(2) inside insertion gene SmMYB75 and NOS terminator interior design upstream and downstream specific primer, carry out DNA expansion
Increase；
(3) viewed under ultraviolet radiation purpose band, purpose band occur then is positive transgenic Hairy Root Cultures of Salvia miltiorrhiza root system.
3. the method according to claim 1 for turning SmMYB75 gene and improving danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, feature
It is, quantitative PCR measuring method described in step (5) is as follows:
(1) extraction that hairy positive clone carries out total serum IgE is accredited as to PCR, it is unified it is quantitative to 0.5 μ g, RNA reverse transcription at
The cDNA of 25 μ l systems；
(2) separately design insertion target gene and house-keeping gene SmActin quantitative primer, using same amount of cDNA as template into
Row quantitative PCR analysis；
(3) the relative expression quantity situation of SmMYB75 and danshinolic acid biosynthesis related genes is analyzed.
4. the method according to claim 1 for turning SmMYB75 gene and improving danshinolic acid content in Hairy Root Cultures of Salvia miltiorrhiza, feature
It is, high performance liquid chromatography described in step (6) is as follows:
Chromatographic column C-18 reverse phase silica gel column, mobile phase are the acetonitrile and water of volume ratio 30:70, and with phosphorus acid for adjusting pH to 2.03；
Detection wavelength 281nm, 35 DEG C of column temperature, flow velocity 1ml/min, 20 μ l of sample volume.
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| CN106497939B (en) * | 2016-10-14 | 2019-06-11 | 昆明理工大学 | A kind of Radix Notoginseng transcription factor gene PnMYB1 and its application |
| CN106706381B (en) * | 2016-11-09 | 2019-04-12 | 桂林理工大学 | The separating and extracting process of total soluble phenolics in plant sub-cellular |
| CN109136235A (en) * | 2018-09-10 | 2019-01-04 | 浙江中医药大学 | A method of turning SmMYB2 gene while improving danshinolic acid and anthocyanidin content in Radix Salviae Miltiorrhizae |
| 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 |
| 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 |
| CN114807177B (en) * | 2022-06-23 | 2022-09-02 | 中国中药有限公司 | Wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof |
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