CN109295069A - The application of panax japonicus majoris transcription factor gene PjMYB1 - Google Patents
The application of panax japonicus majoris transcription factor gene PjMYB1 Download PDFInfo
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- CN109295069A CN109295069A CN201811092438.1A CN201811092438A CN109295069A CN 109295069 A CN109295069 A CN 109295069A CN 201811092438 A CN201811092438 A CN 201811092438A CN 109295069 A CN109295069 A CN 109295069A
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- Prior art keywords
- panax japonicus
- japonicus majoris
- pjmyb1
- saponin
- transcription factor
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- C—CHEMISTRY; METALLURGY
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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Abstract
The invention discloses a kind of panax japonicus majoris transcription factor genesPjMYB1Purposes, i.e., its improve panax japonicus majoris saponin(e biosynthesis key genePjASApplication in expression quantity and increase panax japonicus majoris callus in saponin content, the panax japonicus majoris transcription factor genePjMYB1Nucleotide sequence as shown in SEQ ID NO:1, encode MYB class transcription factor；The present invention proves that panax japonicus majoris PjMYB1 transcription factor has the function of just regulating and controlling panax japonicus majoris saponin(e biosynthesis using functional genomics and metabolic engineering the relevant technologies；By panax japonicus majoris of the present inventionPjMYB1Transcription factor gene is building up on plant expression vector, and is transferred in panax japonicus majoris callus and is made its overexpression, not only increases the expression quantity of panax japonicus majoris saponin formation pathway key enzyme gene, and improve the yield of panax japonicus majoris saponin(e.
Description
Technical field
The present invention relates to molecular biology and genetic engineering field, especially a kind of regulation panax japonicus majoris saponin(e biosynthesis
Transcription factor genePjMYB1Purposes.
Background technique
Panax japonicus majorisPanax japonicus C. A. Mey.var. major (Burk.) C. Y. Wu et K. M.
Feng is Araliaceae Araliaceae PanaxPanaxPlant, because rhizome internode is very thin, section enlarges into spherical beads shape and obtains
Name.Panax japonicus majoris records kind first recorded in " the southern regions of the Yunnan Province book on Chinese herbal medicine " as Chinese traditional herbs to go through version " Chinese Pharmacopoeia ".Medicinal panax japonicus majoris
It is used as medicine with its rhizome, main product is traditional medication of the ethnic groups such as the Yi nationality, distributed over Yunnan, Sichuan and Guizhou, Naxi, Bai nationality, Tibetan, Lisu in Yunnan.Mesh
Before, wild panax japonicus majoris is distributed mainly on NW YUNNAN, Northeastern Yunnan, is common in the Subalpine region needle that height above sea level reaches 2500-4000 m
Under woods and broad-leaf forest.Panax japonicus majoris is bitter in taste, sweet, slightly cold, returns liver, lung, stomach meridian, has tonifying lung yin-nourishing, stasis-dispelling and pain-killing, the function of hemostasis
Effect, is clinically applied to deficiency of both qi and yin, dysphoria and thirsty, tuberculosis cough, traumatic injury, arthralgia, and hemoptysis is spitted blood, and wound goes out
Blood etc..
Panax japonicus majoris saponin(e (Panax japonicusSaponins, PJS) be panax japonicus majoris main active, including
Dammarane type and oleanane-type triterpene saponin.Currently, more than 30 kinds of saponin constituents, generation have been isolated from panax japonicus majoris root, stem and leaf
Table ingredient is panax japonicus saponin IVa, panax japonicus saponin IV, panax japonicus saponin V, ginsenoside R0Equal oleanane glycosides saponin(e.
And the representative species " ginseng " of Panax (Panax ginseng) mainly containing dammarane saponins, oleanane glycoside is only sent out
The atomic ginsenoside R of existing content0；" Radix Notoginseng " (Panax notoginseng) containing only dammarane saponins, be free of oleanane
Type saponin(e.Saponin(e component contained by panax japonicus majoris is compared with belonging to " ginseng ", " Radix Notoginseng ", in ingredient type and each component content
There are notable differences, because it contains a large amount of oleanane glycosides, lead to the specific use of panax japonicus majoris clinically.Modern pharmacology
Learn studies have shown that panax japonicus majoris saponin(e have anti-inflammatory and antalgic, improve myocardial ischemia, increase cerebral blood flow (CBF), antitumor, immunological regulation with
And treatment leukopenia and other effects.
Panax japonicus majoris is perennial medicinal herb plant, need to grow for many years, can be used as medicine.Excessively adopting to wild resource for a long time
Digging causes panax japonicus majoris resource increasingly exhausted.In recent years, deepening continuously with panax japonicus majoris pharmacological research, medical value gradually by
To approving and paying attention to, society steps up the cognition degree of panax japonicus majoris medicinal efficacy, fast as the drug market of component using panax japonicus majoris
Speed expands, and panax japonicus majoris medicinal material demand rapidly increases, and imbalance between supply and demand is outstanding day by day.It is long in view of the panax japonicus majoris artificial cultivation time, it is medicinal
Components Chemical synthesis mechanism and not clear etc. enough drawbacks of route produce pearl using biotechnology and the method for gene regulation
Ginseng saponin(e is increasingly becoming research hotspot.
Transcription factor is the important regulation link of Secondary Metabolism of Plant process to the transcriptional activation of gene, exclusive with its
" multiple spot regulation " advantage, compensates in metabolic engineering operation that the single insufficient and multiple key genes of key gene effect may
The case where generating composition lethal expression.Transcription factor passes through the relevant enzyme base of function in activated plant secondary metabolism route of synthesis
The expression of cause, starting or closing secondary metabolism route of synthesis, to regulate and control the synthesis of secondary metabolites.
MYB class transcription factor is the important member of plant transcription factor family, since MYB discovery, has more and more been ground
Study carefully and show that it has a close relationship to the growth and development of plant and secondary metabolite, and discovered in recent years MYB transcription because
There are interactions between the transcription factor of sub and other regulation secondary metabolites.Such as rough gentian (Gentiana scabra)
Middle overexpression two kinds of R2R3-MYB type transcription factors GtMYBP3 and GtMYBP4 facilitate the biosynthesis and accumulation of flavones, lead to
Cross opening for the gene FNSII and F3H that transient expression discovery transcription factor GtMYBP3 and GtMYBP4 are raising flavones early stage synthesis
Promoter activity, to promote the accumulation of endogenous Flavonoids biosynthesis and flavones；AtMYBl23/TT2, TTl and R2R3-
MYB can to arabidopsis (Arabidopsis thaliana) kind skin procyanidine (PAs) biosynthesis rise coordinated regulation make
With.
With especially adjusting specific secondary generation to the deep parsing of Secondary Metabolism of Plant network and illustrating for regulatory mechanism
The separation and identification of the transcription factor of object synthesis are thanked, the genetic engineering based on transcription factor will be development and utilization Secondary Metabolism of Plant
Object provides significantly more efficient means.
Summary of the invention
The purpose of the present invention is the clear transcriptions that the controllable panax japonicus majoris saponin(e biosynthesis obtained is cloned from panax japonicus majoris
Factor genePjMYB1Purposes, i.e., its improve panax japonicus majoris saponin(e biosynthesis key geneFPS、DS、ASExpression quantity and
Increase the application in panax japonicus majoris callus in saponin content.
The present invention is based on the principles of homologous clone, and clone obtains the cDNA of MYB class transcription factor gene simultaneously from panax japonicus majoris
Albumen is encoded to it carries out Function Identification.This unnamed gene is by inventorPjMYB1, wherein the cDNA such as sequence table SEQ
Shown in ID NO:1, amino acid sequence albumen shown in SEQ ID NO:2 is encoded；Sequence analysis is carried out to the gene, is shownPjMYB1
CDNA size is 735 bp, is just open reading frame (Open reading frame, ORF), and coding contains 244 amino acid
Protein.It, will be of the invention by plant overexpression vector using agrobacterium-mediated transformationPjMYB1Transcription factor gene imports
In panax japonicus majoris cell, the expression quantity of panax japonicus majoris saponin formation pathway key enzyme gene can be improved, increase the yield of panax japonicus majoris saponin(e.
Above-mentioned transcription factor gene can be applied to the biosynthesis for just regulating and controlling panax japonicus majoris saponin(e, and concrete operations are as follows:
(1) it obtains target gene: extracting panax japonicus majoris total serum IgE, reverse transcription synthesizes the first chain of cDNA；It is amplified by RT-PCRPjMYB1Full length coding region, and be connected with pMD18-T easy carrier, the clone with target gene obtained through sequence verification；
(2) plant expression vector construction and genetic transformation: restriction enzyme is usedXbaI andSmaI digestion pMD18-T-PjMYB1
Plasmid and plant expression vector pCAMBIA2300S, glue recycle target gene fragment and carrier large fragment.Connect target gene piece
Section and pCAMBIA2300S carrier segments, construct plant overexpression vector pCAMBIA2300S-PjMYB1；Using frozen-thawed method
By pCAMBIA2300S-PjMYB1Plasmid utilizes the side of Agrobacterium-mediated genetic transformation after importing in agrobacterium strains EHA105
Overexpression vector containing target gene is imported in panax japonicus majoris cell and is expressed by method, is screened by antibiotic and qRT-PCR positive
Transgenic cell line；
(3) transgenic cell line saponin content detects: extracting the saponin(e in panax japonicus majoris transgenosis and Nontransgenic cells system, analysis
The difference of saponin content between transgenosis and Nontransgenic cells system.
The present invention provides a kind of new method to improve the content of saponin(e in panax japonicus majoris, utilizes biotechnology and gene
The method of regulation can higher efficiency synthesize panax japonicus majoris saponin(e, overcome artificial cultivation period long, chemical synthesis mechanism and route not
The disadvantages of enough clear；By transcription factorPjMYB1It is expressed in channel genes panax japonicus majoris cell, makes panax japonicus majoris saponin(e biosynthesis way
The expression quantity of diameter related keyword enzyme gene improves, and then increases the yield of panax japonicus majoris saponin(e, is large-scale industrialized production pearl
Son ginseng saponin(e provides theoretical reference and scientific basis.
Detailed description of the invention
Fig. 1 is panax japonicus majoris total serum IgE electrophorogram；
Fig. 2 isPjMYB1 RT-PCR testing result；Wherein Marker(M): DL2000 DNA Marker, by 2,000 bp, 1,
Six 000 bp, 750 bp, 500 bp, 250 bp and 100 bp DNA fragmentation compositions；1 is positive cell line；
The three-dimensional structure that Fig. 3 is PjMYB1 is predicted；
Fig. 4 is qRT-PCR result analysis chart, is indicated in panax japonicus majoris saponin formation approach by PjMYB1 regulationPjFPS、PjDS、 PjASExpression of the gene in wild type and transgenic cell line；C represents wild type (control), and 1,2,3,4 represent difference
It is overexpressedPjMYB1The cell line of gene；
Fig. 5 is the assay result of panax japonicus majoris saponin(e.
Specific embodiment
Below by drawings and examples, the present invention is further described, but the scope of the present invention is not limited in described
Hold.
Embodiment 1:PjMYB1The clone of gene and bioinformatic analysis
The total serum IgE (Fig. 1) for extracting panax japonicus majoris is taken using the guanidine isothiocyanate method of improvement, it is total with the panax japonicus majoris callus of extraction
RNA is template, referring to GoScriptTMReverse Transcriptase System kit specification synthesizes cDNA first
Chain, reaction system and operating process are as follows: take 5 μ g Total RNA, sequentially add 50 ng oligo (dT) 15、2 μL dNTP
(2.5 mM each), DEPC water to reaction volume are 13.5 μ L；It is mild to mix, exist rapidly after 70 DEG C of progress, 5min initial denaturation
Then 5 min of cooled on ice sequentially adds 4 μ L 5 × First-stand buffer, 0.5 μ L RNasin (200U), 1 μ
L M-MLV (200U) mixes simultaneously brief centrifugation, and 25 DEG C of annealing 5 min, 42 DEG C of 1.5 h of warm bath are finally placed in 70 DEG C of water-baths
Pot 10 min of heating, inactivate transcriptase to terminate reaction, synthesized the first chain of cDNA is placed in -20 DEG C and saves backup.
Using the first chain cDNA of synthesis as template, according to MYB class relevant to regulation notoginsenoside biosynthesis in Radix Notoginseng
Transcription factor gene cDNA sequence designs specific primer, carries out high-fidelity PCR amplification, the primer sequence is respectively 5 '-
ATGGGGAGGAGCCCTTGCTGTGC-3 ' and 5 '-TCAAGACAGCCAATCTCCTCCGGAC -3 '.PCR reaction condition: 94
℃ 5 min；94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min, 32 cycles；72℃ 10 min.Reaction system (50 μ L)
For 4 the first chain of μ L cDNA, 5 μ L Ex Taq PCR Buffer (10 ×), 4 μ L dNTP Mixture (2.5 mM),
0.5 μ L forward primer (10 μM), 0.5 μ L reverse primer (10 μM), 0.5 μ L Ex Taq DNA polymerase (5
U/μL)、35.5 μL ddH2O.After PCR, take 5 μ L for agarose gel electrophoresis, detect the specificity of amplified production with
And size, remaining PCR product carry out glue recycling (Fig. 2).By target fragment glue recovery product carry out T-A clone, reaction system and
Operating process are as follows: take 3 μ L glue recovery products, sequentially add 1 μ L pMD18-T carrier, 5 μ L Solution I and 1 μ L
ddH2Brief centrifugation after O is mixed, 16 DEG C of 30 min of reaction.Connection product is transferred to by Escherichia coli using heat-shock transformed method
In Trans1-T1.With the LB solid medium screening positive clone for containing ampicillin (ampicillin, Amp).If selecting
Dry bacterium colony, uses amplification after shaking bacteriumPjMYB1Special primer identify multiple cloning sites insertionPjMYB1Clone, will identify
Clone be sequenced.
Finally obtainPjMYB1CDNA size be 735 bp, by NCBI ORF finder (http: //
Www.ncbi.nlm.nih.gov/gorf/gorf.html) analysis finds that it is just open reading frame (see sequence table).PjMYB1The molecular weight of coding albumen is about 27.7KD, isoelectric point 8.85, unstability index 68.98, predictionPjMYB1It compiles
The protein of code is unstable.Bioinformatics Prediction PjMYB1 does not include transmembrane region, is free of signal peptide, has a MYB transcription
Ratio characteristics conserved domain.Predict that PjMYB1 can be located at nucleus by online tool iPSORT.By SWISS-
MODEL tool, PjMYB1 carry out three dimensional joint element (Fig. 3) by template of 1a5j.1.A proto-oncogene protein, the results showed that
1a5j.1.A has the space structure of 43.93% sequence similarity.
Embodiment 2: plant expression vector construction
It is right using Primer Premier 5.0 according to the multiple cloning sites of plant expression vector pCAMBIA2300SPjMYB1Base
The selection of restriction enzyme site and the design of primer are carried out because of sequence.Using a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA (on
Hai Shenggong) extract insertionPjMYB1Escherichia coli plasmid pMD18-T-PjMYB1And plant expression vector pCAMBIA2300S
Plasmid, take 1 μ L for agarose gel electrophoresis to detect the integrality and concentration level of extracted plasmid.WithXbaI
(TaKaRa) andSmaI (TaKaRa) is respectively to plasmid pMD18-T-PjMYB1Double digestion (100 μ L are carried out with pCAMBIA2300S
System), reaction system and operating process are as follows: take 20 μ L pMD18-T-PjMYB1Or pCAMBIA2300S plasmid, sequentially add 10
μL 10×M buffer、5 μL Xba I、5 μL SmaI、60 μL ddH2O mixes, is placed in digestion 6 in 37 DEG C of water-baths
h.It is right using SanPrep pillar DNA plastic recovery kit (the raw work in Shanghai)PjMYB1Segment and pCAMBIA2300S large fragment point
It carry out not glue recycling.1 μ L recovery product is taken to detect the size and concentration of recycling segment by agarose gel electrophoresis, be placed in-
20 DEG C save backup.
The DNA fragmentation that recycling obtains is attached using T4 DNA Ligase, constructs recombinant vector
pCAMBIA2300S-PjMYB1.Reaction system (20 μ L) and operating process are as follows: take 10 μ LPjMYB1DNA fragmentation sequentially adds
2 μ L pCAMBIA2300S carrier DNAs, 2 μ L 10 × T4 DNA Ligase Buffer, 1 μ L T4 DNA Ligase, 5 μ
L ddH2O is mixed well, and carries out staying overnight connection reaction in 16 DEG C.Then connection product is transferred to by large intestine using heat-shock transformed method
In bacillus Trans1-T1, the bacterium solution after conversion is coated on the LB solid medium containing 50 mg/L kanamycins, 37 DEG C,
It is inverted overnight incubation, picking monoclonal shakes bacterium, using bacterium solution as template, usesPjMYB1Upstream and downstream primer carry out bacterium solution PCR inspection
It surveys, agarose gel electrophoresis detects PCR product, and screening contains recombinant plasmid pCAMBIA2300S- PjMYB1Escherichia coli it is single
Bacterium solution is cloned, addition 20% glycerol mixing is placed on -80 DEG C and saves backup.
It is extracted with plasmid extraction kit and purifies the pCAMBIA2300S in above-mentioned Escherichia coli-PjMYB1Plasmid.System
The competent cell of standby Agrobacterium EHA105 bacterial strain is simultaneously sub-packed in 1.5 mL centrifuge tubes, every 150 μ L of pipe, liquid nitrogen flash freezer postposition
It is saved backup in -80 DEG C.Using frozen-thawed method by the plant expression vector pCAMBIA2300S- of above-mentioned buildingPjMYB1It is transferred to
In prepared Agrobacterium EHA105 competent cell.Operating procedure are as follows: take 3 μ g pCAMBIA2300S-PjMYB1Plasmid adds
Enter in the centrifuge tube containing 150 μ L competent cells, mix gently rear 30 min of ice bath, is then transferred to quick-frozen 5 min in liquid nitrogen
After be immediately placed in 37 DEG C of 5 min of water-bath, immediately 2 min of ice bath.The Agrobacterium competent cell of conversion is moved into LB liquid
In culture medium (any antibiotic is not added), 28 DEG C, 200 rpm shaken cultivation 4h make cell recovery, are then coated on containing 50
On the LB solid medium of mg/L Km and 25 mg/L rifampins, in 28 DEG C of 48 h of culture carton upside down culture or so.Picking Dan Ke
It is grand, bacterium, bacterium solution PCR detection are shaken, screening contains recombinant vector pCAMBIA2300S-PjMYB1Agrobacterium EHA105 infect pearl
Join callus cell, and by strain glycerol be stored in -80 DEG C it is spare.
Embodiment 3: the panax japonicus majoris genetic transformation of mediated by agriculture bacillus
The panax japonicus majoris cell switching for choosing growth conditions good (cell grainless and presentation is faint yellow) is pre- in panax japonicus majoris cell
(contain 35 mg/L acetosyringones) on culture medium, confluent cultures primary surface, dark culture 3 days under the conditions of being placed in 25 DEG C.From -80
Taken out in DEG C refrigerator save contain pCAMBIA2300S- PjMYB1The Agrobacterium EHA105 strain of plasmid, with connecing for 0.1 %
Kind amount is inoculated in the LB liquid medium containing 50 mg/L kanamycins and 25 mg/L rifampins, and 28 DEG C, 200 rpm vibration
Culture is swung to OD600Value is 0.6 ~ 0.8, is then sub-packed in 50 mL centrifuge tubes, and 5000 rpm of room temperature is centrifuged 5 min and collects bacterium
Body.Thallus is moved in the MGL fluid nutrient medium containing 40 mg/L acetosyringones, 28 DEG C, 200 rpm shaken cultivations are extremely
OD600Value is 0.6 ~ 0.8；3 days panax japonicus majoris callus cells of above-mentioned preculture are totally submerged in bacterium solution, are shaken in 25 DEG C
Bed, 110 rpm shaken cultivation, 20 min, then with the bacterium solution of Buchner funnel and filter paper removal panax japonicus majoris callus surface.It will
Panax japonicus majoris cell is forwarded to surface and is covered on the co-cultivation base of aseptic filter paper, co-cultures 3 days under 25 DEG C of dark conditions；
After co-cultivation, panax japonicus majoris cell is transferred in sterilized beaker, with the nothing containing 400mg/L cephalosporin
Bacterium water cleans 5 ~ 6 times, sufficiently to remove Agrobacterium.After cleaning, reuses Buchner funnel and filter paper removal panax japonicus majoris is cured
The liquid of injured tissue cell surface.Panax japonicus majoris cell is forwarded to and removes dark culture on bacterium culture medium containing 400 mg/L cephalosporins
15 days, prevent Agrobacteriuna overgrowth.Finally callus is transferred in screening and culturing medium, every 45 days subcultures are primary.By 4
The transgenic cell line of Km resistance is provided in ~ 5 screenings, final separation, is used for subsequent detection.
Embodiment 4:PjMYB1Gene is overexpressed to panax japonicus majoris saponin formation pathway key enzyme genePjFPS、PjDS、PjAS
The influence of expression quantity
Selection 25 days or so, the good panax japonicus majoris positive transgenic cell line of growth conditions and wild-type cell system, extract respectively
RNA, according to GoScriptTMReverse Transcriptase System kit by the RNA reverse transcription of extraction at cDNA, instead
Answer system and operating process are as follows: 5 μ g total serum IgEs, 1 μ L Random Primer and 1 μ L Oligo (dT) are added in centrifuge tube15,
It with Nuclease-free Water polishing to 10 μ L, mixes, carries out 70 DEG C, 5min initial denaturation, be immediately placed on ice on ice after the completion
Bathe 5min.The then of short duration centrifugation in centrifuge by centrifuge tube, makes reaction solution be collected in tube bottom, then 4 μ L are added thereto
GoScriptTM 5×Reaction Buffer、2µL MgCl2 (25 mM)、1µL PCR Nucleotide Mix(10 mM)、
0.5 μ L Recombinant RNasin Ribonuclease Inhibitor and 1 μ L GoScriptTM Reverse
Transcriptase is mixed, brief centrifugation, 25 DEG C of annealing 5 min, 42 DEG C of 1.5 h of extension, finally, placing in 70 DEG C of water-baths
15 min, inactivate reverse transcriptase, and reaction was completed, and of short duration centrifugation, -20 DEG C save backup.
The cDNA of reverse transcription synthesis is diluted 5 times, i.e., 20.0 μ L cDNA are diluted to 100.0 μ L.After dilution
CDNA is template, according to GoTaq®2-Step RT-qPCR System kit specification and panax japonicus majoris18S rRNAGene
(accession number: AB088018.1), farnesyl pyrophosphate synzyme (Farnesyl diphosphate synthase, FPS) base
Cause (accession number: KP684141),βAmyrin synzyme (β-amysyrin synthase, β AS) gene (accession number:
KP658156) and dammarane's synthase gene (dammarane synthase) design primer, quantitative fluorescent PCR is carried out.
The primer sequence isFPS F: 5 '-AGAATGAGCGATCTGAAGACGAG-3 ', FPS R: 5 '-ACAGACAACAAC
TTCCCCTCCAT-3'；βAS F: 5 '-GTATTCCCTGTAGAGCATCGCAT-3 ',βAS R: 5 '-
GGCACAGGCGTTGTTTTCAC-3'；DS F: 5 '-TGGGAGTTTCAGCCCGATG-3 ',DS R: 5 '-
GGGGAGGTGTATAAAGTAAAGAGCC-3'； 18S F: 5 '-GTTGTTGCAG TTAAAAAGCTCGTAG-3 ',18S R: 5 '-
ACCTCTGACTATGAAATACGAATGC-3’。
Specific reaction system and operating process are as follows: 20 ng cDNA, 25 μ L GoTaq are added in PCR pipe® qPCR
Master Mix (2 ×) and 0.2 μ L qPCR Primers (18S F /18S R,FPS F / FPS R,βAS F/ βAS R,DS F /DS R
10 mM), with Nuclease-Free Water polishing to 50 μ L.After reaction system whirlpool is mixed, centrifugation is collected into pipe
Bottom is subsequently placed in fluorescence quantitative PCR instrument and is reacted, and carries out quantitative fluorescent PCR using two-step method, response parameter is such as
Under: 95 DEG C of 2min of thermal starting；95 DEG C of 15s, 60 DEG C of 1min of annealing/extension are denaturalized, totally 45 circulations.Each sample is corresponding
Each Duplication detect 2 times.
QRT-PCR is the results show that turnPjMYB1In gene panax japonicus majoris cellPjFPS、PjDS、PjASThe expression quantity of gene is wanted
(Fig. 4) higher than wild type illustrates PjMYB1 as transcription factor, can promote crucial in panax japonicus majoris saponin formation metabolic pathway
Enzyme genePjFPS、PjDS、PjASExpression；In figure, C indicates control group wild-type cell system, and 1,2,3 and 4 respectively indicate difference
Transgenic cell line experimental group.
Embodiment 5:PjMYB1Gene overexpresses the influence to panax japonicus majoris saponin formation amount
The transgenosis panax japonicus majoris cell and wild-type cell for collecting growth 35 days or so, are respectively placed in 55 DEG C of baking ovens and dry to constant weight,
It is sieved with 100 mesh sieve after being fully ground into powder.Weigh transgenosis and each 0.5g of wild type panax japonicus majoris cell powder, be respectively placed in containing
It is impregnated overnight in the clean triangular flask of 50mL methanol solution.In 60W, ultrasonic 4s, ultrasound 1.5 under the ultrasound condition of interval 2s ~
2.0h.4,000rpm is centrifuged 30min under normal temperature condition, collects supernatant, i.e. panax japonicus majoris total saposins solution, and protect in 4 DEG C of refrigerators
It deposits spare.
The above-mentioned corresponding total saposins solution of each panax japonicus majoris cell strain system prepared of 150 μ L of this sample is accurately drawn in band
In the test tube of plug (each sample is arranged 3 in parallel), 5% vanilla newly prepared is added in the evaporation of solvent under the conditions of 55 DEG C
200 μ L of aldehyde-glacial acetic acid solution, 800 μ L of perchloric acid, test tube plug beyond the Great Wall, 60 DEG C of 15 min of heating water bath after vortex mixes, every
5min sufficiently shakes once, promotes sufficiently reaction.Test tube is placed in cooling in ice immediately after reaction, then into reaction solution
5 mL glacial acetic acid are added, mixes well, is stored at room temperature 10min.Each reaction is measured at 550nm wavelength with ultraviolet specrophotometer
The light absorption value of liquid, reference standard curve calculate the content of total saposins in each transgenosis and wild type panax japonicus majoris cell strain system.Knot
Fruit shows, turnsPjMYB1Saponin content is apparently higher than saponin content in wild-type cell (Fig. 5), explanation in gene panax japonicus majoris cell
PjMYB1 transcription factor takes part in the anabolism regulation of panax japonicus majoris saponin(e, facilitates the raising of saponin(e yield.C indicates control group
Wild-type cell system, 1,2,3 and 4 respectively indicate different transgenic cell line experimental groups.
Sequence table
<110>Kunming University of Science and Technology
<120>application of panax japonicus majoris transcription factor gene PjMYB1
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 735
<212> DNA
<213> Panax japonicus
<400> 1
atggggagga gcccttgctg tgcaaaggta gggctaaaca aaggtgcatg gacttctgtt 60
gaggacaaga ttctcagaga ctacattaag ctccatggtg aaggcagatg gagaagcctc 120
cccaaaagag caggtttaaa gagatgtggg aagagttgca ggcttagatg gttgaattat 180
ctgagaccag acatcaagag aggcaacatc tcccctgatg aagaagaact catcatccgc 240
cttcacaagc tcttgggaaa tagatggtct ctaatagctg ggaggcttcc ggggcgaaca 300
gacaatgaaa taaagaacta ctggaataca accttgagaa aaaaggttca tgacaatcac 360
tcctcctctg cttcagcttc agcttcagct ccagctccag ctcctcccaa gaaagactgg 420
cagcagcagc agcatttggt tcggagcaga tcggcgcatg agtgggataa taaaattgat 480
gattccagtt caaaggcaga agaaaaatat gtagcaaaag aatgtattag tccagctgca 540
ttggatttgg tgcatccgga tgtggaattt tgcttaacgg agcttttaaa ctcggatttc 600
tggggtttat ttgagagccc tctgcagccg gaggctaatt ggaatgtcgg tgattttgtt 660
caaccaaatg tggcttccac tcatcattat tttgctaata atatttctga gtccggagga 720
gattggctgt cttga 735
<210> 2
<211> 244
<212> PRT
<213> Panax japonicus
<400> 2
Met Gly Arg Ser Pro Cys Cys Ala Lys Val Gly Leu Asn Lys Gly Ala
1 5 10 15
Trp Thr Ser Val Glu Asp Lys Ile Leu Arg Asp Tyr Ile Lys Leu His
20 25 30
Gly Glu Gly Arg Trp Arg Ser Leu Pro Lys Arg Ala Gly Leu Lys Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Asn Ile Ser Pro Asp Glu Glu Glu Leu Ile Ile Arg
65 70 75 80
Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Thr Leu
100 105 110
Arg Lys Lys Val His Asp Asn His Ser Ser Ser Ala Ser Ala Ser Ala
115 120 125
Ser Ala Pro Ala Pro Ala Pro Pro Lys Lys Asp Trp Gln Gln Gln Gln
130 135 140
His Leu Val Arg Ser Arg Ser Ala His Glu Trp Asp Asn Lys Ile Asp
145 150 155 160
Asp Ser Ser Ser Lys Ala Glu Glu Lys Tyr Val Ala Lys Glu Cys Ile
165 170 175
Ser Pro Ala Ala Leu Asp Leu Val His Pro Asp Val Glu Phe Cys Leu
180 185 190
Thr Glu Leu Leu Asn Ser Asp Phe Trp Gly Leu Phe Glu Ser Pro Leu
195 200 205
Gln Pro Glu Ala Asn Trp Asn Val Gly Asp Phe Val Gln Pro Asn Val
210 215 220
Ala Ser Thr His His Tyr Phe Ala Asn Asn Ile Ser Glu Ser Gly Gly
225 230 235 240
Asp Trp Leu Ser
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
atggggagga gcccttgctg tgc 23
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
tcaagacagc caatctcctc cggac 25
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
agaatgagcg atctgaagac gag 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 6
acagacaaca acttcccctc cat 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial)
<400> 7
gtattccctg tagagcatcg cat 23
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 8
ggcacaggcg ttgttttcac 20
<210> 9
<211> 19
<212> DNA
<213>artificial sequence (Artificial)
<400> 9
tgggagtttc agcccgatg 19
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 10
ggggaggtgt ataaagtaaa gagcc 25
<210> 11
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 11
gttgttgcag ttaaaaagct cgtag 25
<210> 12
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 12
acctctgact atgaaatacg aatgc 25
Claims (1)
1. a kind of panax japonicus majoris transcription factor genePjMYB1Improving panax japonicus majoris saponin(e biosynthesis key genePjASExpression
Application in amount and increase panax japonicus majoris callus in saponin content, it is characterised in that: the panax japonicus majoris transcription factor genePjMYB1Nucleotide sequence as shown in SEQ ID NO:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846106A (en) * | 2021-10-13 | 2021-12-28 | 杭州三叶青农业科技有限公司 | Gene PnDCD and application thereof in regulating and controlling saponin synthesis |
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Cited By (2)
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| CN113846106A (en) * | 2021-10-13 | 2021-12-28 | 杭州三叶青农业科技有限公司 | Gene PnDCD and application thereof in regulating and controlling saponin synthesis |
| CN113846106B (en) * | 2021-10-13 | 2023-08-29 | 杭州三叶青农业科技有限公司 | Gene PnDCD and application thereof in regulating and controlling saponin synthesis |
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