CN105087599B - A kind of application of panax japonicus majoris transcription factor gene PjERF1 - Google Patents
A kind of application of panax japonicus majoris transcription factor gene PjERF1 Download PDFInfo
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- CN105087599B CN105087599B CN201510561815.1A CN201510561815A CN105087599B CN 105087599 B CN105087599 B CN 105087599B CN 201510561815 A CN201510561815 A CN 201510561815A CN 105087599 B CN105087599 B CN 105087599B
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- panax japonicus
- japonicus majoris
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Abstract
The invention discloses a kind of panax japonicus majoris transcription factor genesPjERF1Purposes, i.e., improve panax japonicus majoris saponin(e biosynthesis key gene expression quantity and increase panax japonicus majoris callus in saponin content application,PjERF1Gene nucleotide series encode AP2/ERF class transcription factors as shown in SEQ ID NO ︰ 1；The present invention proves that panax japonicus majoris PjERF1 transcription factors have the function of just to regulate and control panax japonicus majoris saponin(e biosynthesis using functional genomics and metabolic engineering the relevant technologies.By panax japonicus majoris of the present inventionPjERF1Transcription factor gene, which is building up on plant expression vector and is transferred in panax japonicus majoris callus, makes its overexpression, enhances the expression quantity of panax japonicus majoris saponin formation pathway key enzyme gene, improves 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 panax japonicus majoris saponin(e biosynthesis transcription
Factor genePjERF1Application.
Background technology
Panax japonicus majorisPanax japonicus C. A. Mey.var. major (Burk.) C. Y. Wu et K. M.
Feng is Araliaceae Araliaceae PanaxesPanaxPlant, because rhizome internode is very thin, section enlarges into spherical beads shape and obtains
Name, rhizome are used as medicine.Panax japonicus majoris as medicinal material, first recorded in《The southern regions of the Yunnan Province book on Chinese herbal medicine》, to go through version《Chinese Pharmacopoeia》Record kind.Medicinal pearl
Son ginseng main product is in Yunnan, the expensive parts of generic medicinal plants of generic name, is the biography of the ethnic groups such as the Yi nationality, distributed over Yunnan, Sichuan and Guizhou, Naxi, Bai nationality, Tibetan, Lisu
System medication.At present, wild panax japonicus majoris is distributed mainly on NW YUNNAN, Northeastern Yunnan, is common in the Asia that height above sea level reaches 2500-4000 m
Under Alpine coniferous forests and broad-leaf forest.Panax japonicus majoris is bitter in taste, it is sweet, be slightly cold, Return liver, lung, stomach have tonifying lung yin-nourishing, stasis-dispelling and pain-killing,
The effect of hemostasis, clinically applied to deficiency of both qi and yin, dysphoria and thirsty, tuberculosis cough, traumatic injury, arthralgia, hemoptysis spits
Blood, traumatic hemorrhage etc..
Panax japonicus majoris saponin(e (Panax japonicusSaponins, PJS) be panax japonicus majoris main active, including
Dammarane type, oleanane-type triterpene saponin.At present, more than 30 kinds of saponin constituent has been isolated from panax japonicus majoris rhizome and leaf,
Mainly include panax japonicus saponin IVa, panax japonicus saponin IV, panax japonicus saponin V, ginsenoside Ro, ginsenoside Re, ginsenoside
Rd, ginsenoside Rb1 etc..Panax representative species " ginseng " (Panax ginseng) mainly containing dammarane saponins, neat pier
Fruit alkane type saponin(e only finds the atomic ginsenoside Ro of content；Another famous medicinal material of Panax " Radix Notoginseng " (Panax notoginseng), containing only dammarane saponins, without oleanane glycoside.Saponin(e component contained by panax japonicus majoris is with belonging to " people
Ginseng ", " Radix Notoginseng " are compared, there are notable difference in ingredient type and each component content, because it contains a large amount of oleananes
Type saponin(e leads to specific use clinically.
Panax japonicus majoris is herbaceos perennial, and need to grow 6 years or more can be used as medicine.At present, panax japonicus majoris medicinal material is mostly wild
Product, unordered excavation lead to that panax japonicus majoris resource is increasingly exhausted, and habitat is destroyed.In recent years, using panax japonicus majoris as the drug city of component
Field expands rapidly, and leads to panax japonicus majoris medicinal material demand growth, and imbalance between supply and demand protrudes.In view of the artificial cultivation time is long, chemical synthesis machine
Reason and not clear etc. enough drawbacks of route, using biotechnology and the method for gene regulation come produce panax japonicus majoris saponin(e gradually into
For research hotspot.
Transcription factor is the important regulation and control link of Secondary Metabolism of Plant process to the transcriptional activation of gene.Utilize transcription factor
As the tool of plant modification metabolic pathway, with its exclusive " multiple spot regulation and control " advantage, compensate for single in metabolic engineering operation
Key gene effect is insufficient and multiple key genes are there may be the situation of the lethal expression of composition, becomes a kind of new plan
Slightly.In secondary metabolism approach multiple relevant enzyme genes of function often by same transcription factor just regulation and control or negative regulation, to transcription because
Son carries out genetic modification and is obviously easier Breeding objectives secondary metabolism approach than polygenes operation.
AP2/ERF (APETALA2/ETHYLENE Responsive Factor) transcription factor is one specific to plant
Class transcription factor, it is more and more research shows that such transcription factor has important regulating and controlling to Eukaryotic secondary metabolism process
Effect.Such as catharanthus roseus (Catharanthus roseus) the controllable participation terpene indoles of AP2/ERF class transcription factors ORCA3
The expression of the strictosidine synthase gene of alkaloid biosynthesis；Sweet wormwood (Artemisia apiacea) AaERF1 and
AaERF2 transcription factors can improve the relevant false indigo 4,11- diene synthase of qinghaosu biosynthesis（ADS）And cytochromes
P450 monooxygenases（CYP71AV1）The expression quantity of gene, the synthesis of positive regulation and control qinghaosu；In addition, Chinese yew (Taxus chinensis) the controllable Taxol biosynthesis key gene of TcAP2 transcription factors expression.
With to the deep parsing of Secondary Metabolism of Plant network and illustrating for regulatory mechanism, particularly adjusting specific secondary generation
Thank to the separation and identification of the transcription factor of object synthesis, the genetic engineering based on transcription factor will be to develop and use Secondary Metabolism of Plant
Object provides significantly more efficient means.The present invention is using the panax japonicus majoris callus of in vitro culture as research object, clonePjERF1Turn
Factor gene is recorded, and the transcription factor is analyzed and Function Identification, PjERF1 transcription factors is specified and is given birth in panax japonicus majoris saponin(e
Status and effect in object building-up process, for obtain efficiently, the panax japonicus majoris saponin formation control technique and homologous or heterologous stablized
The foundation of efficient expression system provides theoretical reference and foundation.
Invention content
The object of the present invention is to provide a kind of panax japonicus majoris transcription factor genesPjERF1Purposes, i.e. panax japonicus majoris transcription factor
GenePjbHLH1Key gene expression quantity and increase panax japonicus majoris callus in panax japonicus majoris saponin formation metabolic pathway is improved
Application in middle saponin content；The present invention clones the transcription factor for obtaining controllable panax japonicus majoris saponin(e biosynthesis from panax japonicus majoris
GenePjERF1And the clearly application of the transcription factor.
The present invention is based on the principles of homologous clone, and clone obtains AP2/ERF class transcription factor genes from panax japonicus majoris
CDNA simultaneously encodes it albumen progress Function Identification.This unnamed gene is by inventorPjERF1, gene accession number is
KP890784, wherein the cDNA is as shown in SEQ ID NO ︰ 1.Sequence analysis is carried out to the gene, is shownPjERF1CDNA is big
Small is 808 bp, has the open reading frame (Open reading frame, ORF) of 801 bp, and coding contains 266 amino acid
Protein, amino acid sequence is as shown in SEQ ID NO ︰ 2.Using plant expression vector, this is sent out by agrobacterium-mediated transformation
BrightPjERF1Transcription factor gene is imported in panax japonicus majoris callus, can improve panax japonicus majoris saponin formation pathway key enzyme gene
Expression quantity, make the yield of panax japonicus majoris saponin(e increase.
Above-mentioned transcription factor gene can be applied to just regulate and control the biosynthesis of panax japonicus majoris saponin(e, and concrete operations are as follows：
（1）The acquisition of gene：Extract panax japonicus majoris total serum IgE, reverse transcription synthesis the first chains of cDNA.It is amplified by RT-PCRPjERF1Full length coding region, be subsequently attached on pGEM-T easy carriers, through sequence verification obtain have target gene
Clone；
（2）Plant expression vector construction and genetic transformation：Use restriction enzymeSmaI andXbaI digestions pGEM-T-PjERF1Plasmid is recycled by glue and obtains target gene fragment.With same endonuclease digestion plant expression vector
PCAMBIA2300S, glue recycling obtain carrier large fragment.Target gene fragment is connect with pCAMBIA2300S carrier segments, structure
Build plant overexpression vector pCAMBIA2300S-PjERF1.By frozen-thawed method by pCAMBIA2300S-PjERF1Plasmid is led
Enter in agrobacterium strains EHA105.It, will using Agrobacterium-mediated genetic transformation methodPjERF1It imports in panax japonicus majoris callus
Make its overexpression, positive transgenic cell line is screened by antibiotic-screening and qRT-PCR；
（3）Transgenic cell line saponin content detects：The saponin(e in panax japonicus majoris transgenosis and Nontransgenic cells system is extracted,
Analyze 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 and control can higher efficiency synthesis panax japonicus majoris saponin(e, overcome artificial cultivation period long, chemical synthesis mechanism and route not
The shortcomings of enough clear.By transcription factorPjERF1It is expressed in channel genes panax japonicus majoris cell, makes panax japonicus majoris saponin(e biosynthesis way
The expression quantity of diameter key gene improves, and increases the yield of panax japonicus majoris saponin(e, is large-scale industrialized production panax japonicus majoris saponin(e
Provide theoretical reference and scientific basis.
Description of the drawings
Fig. 1 is panax japonicus majoris total serum IgE electrophoresis pattern in the present invention；
Fig. 2 is in the present inventionPjERF1 RT-PCR testing results, wherein M are DL2000 DNA Marker, and 1 isPjERF1
RT-PCR products；
The three-dimensional structure prediction that Fig. 3 is PjERF1 in the present invention；
Fig. 4 is qRT-PCR interpretation of result figures in the present invention, is represented in panax japonicus majoris saponin formation approach by PjERF1 regulation and controlFPSExpression of the gene in wild type and transgenic cell line, wherein C are wild-type cell system, and 1-3 is transgenic cell
System；
Fig. 5 be the assay of panax japonicus majoris saponin(e in the present invention as a result, wherein C is wild-type cell system, 1-3 is transgenosis
Cell line.
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, method is conventional method unless otherwise specified in embodiment, and the reagent used is conventional commercial examination unless otherwise specified
Agent or the reagent prepared according to a conventional method.
Embodiment 1：PjERF1The clone of gene and bioinformatic analysis
Because containing more secondary metabolites in panax japonicus majoris, two-step method need to be used to carry out the extraction of total serum IgE.It first uses and changes
Good guanidine isothiocyanate method, which takes, slightly to be carried, and is then digested using DNA enzymatic and obtains purer pearl using chloroform
Join total serum IgE（Fig. 1）.Reverse transcriptase M-MLV (promega) is used using panax japonicus majoris total serum IgE as the first chains of templated synthesis cDNA, instead
System and the operating process is answered to be：5 μ g Total RNA are taken, sequentially add 50 ng oligo (dT) 15、2 μL dNTP (2.5
MM each), DEPC water to reaction volume be 13.5 μ L；After mixing, 5 are cooled down on ice rapidly after 70 DEG C of 5 min of heat denatured
Then min sequentially adds 4 μ L 5 × First-stand buffer, 0.5 μ L RNasin (200 U), 1 μ L M-MLV
(200 U), mixing and brief centrifugation, 42 DEG C of 1.5 h of warm bath, 70 DEG C of 10 min of heating, terminate reaction after taking-up.CDNA first
Chain synthesis is placed on -20 DEG C and saves backup.
Using the first chain cDNA of synthesis as template, according in Radix Notoginseng with regulate and control the relevant AP2/ of notoginsenoside biosynthesis
ERF class transcription factor genes cDNA sequence designs specific primer, amplifying target genesPjERF1, the primer sequence is respectively
5 '-cccgggATGTGTGGAGGTGCAATCCTAGGTG-3 ' and 5 '-tctagaGTGCTCTTTAAA CGACATCGTCGAA-
3’.Using AdvantageTM2 PCR Enzyme (Clontech) amplify target gene.PCR reaction conditions：94 ℃ 5
min；94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 50 s, 32 cycles；72 ℃ 10 min.Reaction system (50 μ L) is
4 the first chains of μ L cDNA, 5 10 × Buffer of μ L, 1 μ L 50 × dNTP Mix, 0.5 μ L forward primers (10 μM), 0.5
μ L reverse primers (10 μM), 1 μ L AdvantageTM2 PCR Enzyme、38 μL PCR-Grade Water.PCR terminates
Afterwards, 5 μ L is taken to detect the specificity and size of amplified production for agarose gel electrophoresis（Fig. 2）, the progress of remaining PCR product
Glue recycles.Target fragment glue recovery product is subjected to TA clones, reaction system and operating process are：Take the recycling production of 3.6 μ L glue
Object sequentially adds 0.7 μ L pGEM-T easy vector (50 ng/ μ L), 5 μ 2 × Rapid of L Ligation
Buffer and 0.7 μ L T4 DNA Ligase mixings are placed in 8 DEG C of reaction overnights.Connection product is transferred to using heat-shock transformed method
In Escherichia coli Trans1-T1.With positive gram of the LB solid mediums screening containing ampicillin (ampicillin, Amp)
It is grand.Several white colonies are selected, amplification is used after shaking bacteriumPjERF1Special primer identify multiple cloning sites insertionPjERF1
Clone, the clone of identification is sequenced.
Finally obtainPjERF1CDNA sizes are 808 bp, pass through NCBI ORF finder (http://
Www.ncbi.nlm.nih.gov/gorf/gorf.html) analysis finds that it includes the ORF of 801 bp (see sequence table).PjERF1The molecular weight of coding albumen is about 30.1 KD, isoelectric point 5.40, unstability index 44.80, predictionPjERF1It compiles
The protein of code is unstable.Bioinformatics Prediction PjERF1 does not include transmembrane region, and without signal peptide, there are one AP2/ERF for tool
Class transcription factor feature conserved domain.Predict that PjERF1 can be located at cytoplasm or mitochondria by online tool iPSORT
Matrix.By SWISS-MODEL tools, PjERF1 carries out three dimensional joint element by template of arabidopsis ATERF1 transcription factors
（Fig. 3）, the results showed that PjERF1 and space structure of the ATERF1 transcription factors with 71.93% sequence similarity.
Embodiment 2：Plant expression vector construction
It is extracted and is inserted into using a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA (Shanghai life work)PjERF1Large intestine bar
Bacteria plasmid pGEM-T- PjERF1And the plasmid of plant expression vector pCAMBIA2300S, 1 μ L are taken for Ago-Gel electricity
The integrality and concentration level of swimming extraction plasmid to detect.WithXbaI (TaKaRa) andSmaI (TaKaRa) is respectively to plasmid
pGEM-T- PjERF1Double digestion (100 μ L systems) is carried out with pCAMBIA2300S, reaction system and operating process are：Take 20
μL pGEM-T- PjERF1Or pCAMBIA2300S plasmids, sequentially add 10 μ L 10 × T buffer, 10 μ L BSA, 5 μ LXba I、5 μL SmaI、50 μL ddH2O centrifuges after mixing, is placed in 37 DEG C of reaction overnights in short-term.By all digestion products points
Electrophoresis is carried out in Ago-Gel, it is then rightPjERF1Segment and pCAMBIA2300S large fragments carry out glue recycling respectively, whole
A process uses SanPrep pillar DNA plastic recovery kits (Shanghai life work).1 μ L recovery products is taken to pass through Ago-Gel electricity
The size and concentration of swimming detection recycling segment, are placed in -20 DEG C and save backup.
Using T4 DNA Ligase (TaKaRa), by recyclingPjERF1DNA fragmentation and pCAMBIA2300S carrier-pellets
Section connects, and reaction system (20 μ L) and operating process are：Take 10 μ LPjERF1DNA 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 centrifuges after mixing, is placed in 16 DEG C of water-bath reaction overnights in short-term.Then connection product is transferred to greatly using heat-shock transformed method
In enterobacteria Trans1-T1, with positive gram of the solid medium screening containing 50 mg/L kanamycins (kanamycin, Km)
It is grand.Picking individual colonies shake bacterium, are expanded by template of bacterium solutionPjERF1Special primer carry out PCR, pick outPjERF1With
The clone that pCAMBIA2300S is successfully connected, the bacterial strain detected add in 20% glycerine mixing and are placed on -80 DEG C of guarantors if the positive
It deposits spare.
It is extracted with kit and purifies the pCAMBIA2300S in above-mentioned Escherichia coli-PjERF1Plasmid.Prepare Agrobacterium
The competent cell of EHA105 bacterial strains is simultaneously sub-packed in 1.5 mL centrifuge tubes, and often 150 μ L of pipe, liquid nitrogen flash freezer are placed on -80 DEG C
It saves backup.Using frozen-thawed method by the plant expression vector pCAMBIA2300S- of above-mentioned structurePjERF1It is transferred to prepared
Agrobacterium EHA105 competent cells in.Operating procedure is：Take 3 μ g pCAMBIA2300S-PjERF1Plasmid addition contains
In the centrifuge tube of 150 μ L competent cells, gently 30 min of ice bath after mixing, is then transferred to quick-frozen 5 min in liquid nitrogen, then
37 DEG C of 5 min of water-bath are immediately placed in, 2 min of ice bath, 600 μ L LB fluid nutrient mediums of addition, 28 DEG C of oscillations are trained immediately later
Support 4 h.Agrobacterium after activation is applied on the LB solid mediums containing 50 mg/L Km, 28 DEG C of quiescent cultures.Select list
Bacterium colony shakes bacterium, with amplificationPjERF1Special primer carry out PCR, detect pCAMBIA2300S-PjERF1Whether Agrobacterium is transferred to
In.For positive colony, addition glycerine is placed on -80 DEG C and saves backup.
Embodiment 3：Agriculture bacillus mediated panax japonicus majoris genetic transformation
That preservation is taken out from -80 DEG C of refrigerators contains pCAMBIA2300S-PjERF1The Agrobacterium EHA105 bacterium of plasmid
Kind, it is inoculated in the LB fluid nutrient mediums that 5 mL contain 50 mg/L Km and 25 mg/L rifampins, 28 DEG C of cultures to muddiness.It inhales
It takes on bacterium solution to the LB solid mediums containing 50 mg/L Km of 1 mL muddinesses, 28 DEG C of 48 h of culture.By LB solid mediums
On Agrobacterium scrape and be inoculated in MGL fluid nutrient mediums in right amount, add the acetosyringone of 40 mg/L, 28 DEG C of shaken cultivations
To OD600Stop shaking bacterium when being 0.6, gained bacterium solution is used to infect.
The preferable panax japonicus majoris callus of growth conditions is connected to MS pre-culture mediums（Containing 35 mg/L acetosyringones）It is enterprising
Row preculture 3 days.After the completion of preculture, callus is completely soaked and carries out shaken cultivation in above-mentioned Agrobacterium bacterium solution.Leaching
Dye finishes, and removes bacterium solution, and the bacterium solution on callus surface is drawn with sterile filter paper, callus finally is inoculated into co-cultivation
In base, light culture is carried out in 25 DEG C, the time is three days.Callus after dip dyeing is inoculated in MS and co-cultures base（Containing 35 mg/L
Acetosyringone）In, then light culture 3 days washs callus with sterile water, then transfer in containing 400 mg/L cephalos
Degerming culture is carried out on the MS culture mediums of mycin, in 25 DEG C of light cultures 15 days, prevents Agrobacteriuna overgrowth.Finally by callus
Tissue is transferred in screening and culturing medium, and every 45 days subcultures are primary.By 5 screenings, the increment of Km resistances is provided in final separation
Fireballing pure cell line, for subsequent detection.
Embodiment 4：PjERF1Gene is overexpressed to panax japonicus majoris saponin formation pathway key enzyme geneFPSThe influence of expression quantity
Selection 25 days or so, the good panax japonicus majoris transgenic cell line of growth conditions and wild-type cell system, extract respectively
RNA, then according to GoTaq®2-Step RT-qPCR System kit specifications synthesize cDNA, reaction system and operate
Cheng Wei：4 μ g total serum IgEs and 1 μ L Oligo (dT) are added in centrifuge tube15, with Nuclease-free Water polishings to 10
Reaction system is put in 70 DEG C of 5 min of denaturation, is subsequently placed in 5 min on ice by μ L.It is then that centrifuge tube is of short duration in centrifuge
Centrifugation, makes reaction solution be collected in tube bottom, then add in 4 μ L GoScript theretoTM 5×Reaction Buffer、2 µL
MgCl2 (25 mM)、1 µL PCR Nucleotide Mix(10 mM)、0.5 µL Recombinant RNasin
Ribonuclease Inhibitor and 1 μ L GoScriptTMReverse Transcriptase, by entire reaction system whirlpool
Whirlpool mixing, is collected by centrifugation tube bottom, and reactant is placed in 42 DEG C of thermostat water baths and reacts 1 h, is tieed up in 70 DEG C of water-baths
15 min are held to terminate reaction, finally the cDNA of synthesis is placed in -20 DEG C of refrigerators and is saved backup.
Using the cDNA as template, according to panax japonicus majoris18S rRNAGene（Accession number：AB088018.1）With farnesyl coke phosphorus
Acid enzyme（Farnesyl diphosphate synthase, FPS）Gene（Accession number：KP684141）Primer is designed, according to
According to GoTaq®2-Step RT-qPCR System kit specifications carry out fluorescence semiquantitive PCR amplification panax japonicus majoris reference gene
WithFPSGene.The primer sequence is18S F：5 '-GTTGTTGCAG TTAAAAAGCTCGTAG-3 ',18S R：5’-
ACCTCTGACTATGAAATACGAATGC-3’；FPS F：5’-AGAATGAGCGATCTGAAGACG AG-3’；FPS B：5’-
ACAGACAACAACTTCCCCTCCAT-3’.Specific reaction system and operating process are：Added in PCR pipe 20 ng cDNA,
25 µL GoTaq®QPCR Master Mix (2 ×) and 0.2 μ L qPCR Primers (18S F /18S R, FPS B /FPS F, 10 mM), with Nuclease-Free Water polishings to 50 μ L.After reaction system whirlpool mixing, centrifuge and received
Collect tube bottom, be subsequently placed in fluorescence quantitative PCR instrument and reacted, quantitative fluorescent PCR, reaction ginseng are carried out using two-step method
Number is as follows：95 DEG C of 2 min of thermal starting；95 DEG C of 15 s, 60 DEG C of 1 min of annealing/extension are denaturalized, totally 45 cycles.Often
The corresponding each Duplication of a sample detects 2 times.
QRT-PCR is the results show that turnPjERF1In gene panax japonicus majoris cellFPSThe expression quantity of gene is higher than wild type
（Fig. 4）, illustrate that PjERF1 as transcription factor, can promote key gene in panax japonicus majoris saponin formation metabolic pathwayFPS's
Expression；In figure, C represents control group wild-type cell system, and 1,2 and 3 represent different transgenic cell line experimental groups respectively.
Embodiment 5：PjERF1Gene overexpresses the influence to panax japonicus majoris saponin formation amount
The transgenic cell line of growth 35 days or so and wild-type cell system are chosen, is respectively placed in 100 clean mL triangles
In bottle, after adding in the methanol solution soaked overnight of 20 mL, 1 h of room temperature ultrasonication.Filtering collects filtrate, filtrate is volatilized
Again with methanol dissolves afterwards, is settled to 25 mL, obtains crude extract.Residue at 50 DEG C is dried to constant weight, is weighed.Precision is drawn and is slightly carried
5 mL of liquid puts water bath method in 50 mL beakers.It is dissolved with the distilled water of 4 times of volumes after being evaporated, is fully filtered after dissolving by several times,
Filtrate is fully transferred in processed good Hsp100 macroporous resin columns, first slowly washes away carbohydrate with the distilled water of 2 column volumes
Wait impurity.Whether Molish reaction detection carbohydrate impurity removes totally, if be as a result positive, continues to be washed to the moon with distillation
Property, 2 column volumes then are eluted with 75% ethanol solution, collect alcohol liquid, water bath method, residue is dissolved with methanol solution, is settled to
25 mL。
It accurately draws in 150 μ L of this sample to the 10 mL test tubes with plug（If 3 repetitions）, solvent is volatilized, addition is newly matched
5% vanillic aldehyde-glacial acetic acid solution, 0.2 mL of system, 0.8 mL of perchloric acid, 60 DEG C of 15 min of heating water bath after mixing, use ice immediately
Water cooling adds in 5 mL glacial acetic acid, and 554 nm survey absorbance after static 10 min of mixing, and reference standard curve calculates PJS contents.
The results show that turnPjERF1Saponin content is apparently higher than saponin content in wild-type cell in gene panax japonicus majoris cell（Fig. 5）, say
Bright PjERF1 transcription factors take part in the anabolism regulation and control of panax japonicus majoris saponin(e, contribute to the raising of saponin(e yield；C represents control
Group wild-type cell system, 1,2 and 3 represent different transgenic cell line experimental groups respectively.
Sequence table
<110>Kunming University of Science and Technology
<120>A kind of panax japonicus majoris transcription factor genePjERF1Application
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 808
<212> DNA
<213> Panax japonicus
<220>
<221> mRNA
<222> (1)..(808)
<220>
<221> CDS
<222> (1)..(801)
<220>
<221> 3'UTR
<222> (802)..(808)
<400> 1
atgtgtggag gtgcaatcct aggtgatctt accgctcgaa atttcaaccg ccgtgtctcc 60
gccgctgact tctggcccac atctctctcc gacaaactcg acaatttcca gtccgaattt 120
aatcatttcc ctccggagga gactcgaacc ctcaaaagag cgcagcctaa ttcaggtgga 180
gtacctctcg gaaagacaac aaagagacag aggaagaaca tgtacagagg aataaggcag 240
cgtccatggg ggaaatgggc agctgagatt agggatccga gaaaaggagt gagggtttgg 300
ctgggtactt tcaacacggc tgaggaggct gccagagcct acgacaaaga agctcgcaag 360
attagaggaa acaaagccaa agttaacttc ccaaatgagg actgctttaa tcaattcaat 420
gtcaaaaata tgaatcaatt tgggtcaaat tcttgttctg ggttttctgc attgaacagg 480
gaggaccaat cacctttgga ttttacctgt ctgaaaaatg gaagcgatgg acttagtatt 540
gctgagcagg aggagaaggt aaaagaagaa aaagaggaga aagaaaataa agagagtgtg 600
attgatcaag tggaggaaca gaacgaactg cagaagctct cggatgagct aatggcctac 660
gaatcttaca tgaaatttta tgaaattccg tatcttgatg gccagtcggc gacggcggca 720
gcagctccga cgagtgcggt gccggaaaac gtcgtagacg gtggtttgct aaatctttgg 780
agcttcgacg atgtcgttta aagagcac 808
<210> 2
<211> 266
<212> PRT
<213> Panax japonicus
<400> 2
Met Cys Gly Gly Ala Ile Leu Gly Asp Leu Thr Ala Arg Asn Phe Asn
1 5 10 15
Arg Arg Val Ser Ala Ala Asp Phe Trp Pro Thr Ser Leu Ser Asp Lys
20 25 30
Leu Asp Asn Phe Gln Ser Glu Phe Asn His Phe Pro Pro Glu Glu Thr
35 40 45
Arg Thr Leu Lys Arg Ala Gln Pro Asn Ser Gly Gly Val Pro Leu Gly
50 55 60
Lys Thr Thr Lys Arg Gln Arg Lys Asn Met Tyr Arg Gly Ile Arg Gln
65 70 75 80
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Arg Lys Gly
85 90 95
Val Arg Val Trp Leu Gly Thr Phe Asn Thr Ala Glu Glu Ala Ala Arg
100 105 110
Ala Tyr Asp Lys Glu Ala Arg Lys Ile Arg Gly Asn Lys Ala Lys Val
115 120 125
Asn Phe Pro Asn Glu Asp Cys Phe Asn Gln Phe Asn Val Lys Asn Met
130 135 140
Asn Gln Phe Gly Ser Asn Ser Cys Ser Gly Phe Ser Ala Leu Asn Arg
145 150 155 160
Glu Asp Gln Ser Pro Leu Asp Phe Thr Cys Leu Lys Asn Gly Ser Asp
165 170 175
Gly Leu Ser Ile Ala Glu Gln Glu Glu Lys Val Lys Glu Glu Lys Glu
180 185 190
Glu Lys Glu Asn Lys Glu Ser Val Ile Asp Gln Val Glu Glu Gln Asn
195 200 205
Glu Leu Gln Lys Leu Ser Asp Glu Leu Met Ala Tyr Glu Ser Tyr Met
210 215 220
Lys Phe Tyr Glu Ile Pro Tyr Leu Asp Gly Gln Ser Ala Thr Ala Ala
225 230 235 240
Ala Ala Pro Thr Ser Ala Val Pro Glu Asn Val Val Asp Gly Gly Leu
245 250 255
Leu Asn Leu Trp Ser Phe Asp Asp Val Val
260 265
<210> 3
<211> 31
<212> DNA
<213>Artificial sequence
<400> 3
cccgggatgt gtggaggtgc aatcctaggt g 31
<210> 4
<211> 31
<212> DNA
<213>Artificial sequence
<400> 4
tctagagtgc tctttaaacg acatcgtcga a 31
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
gttgttgcag ttaaaaagct cgtag 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
acctctgact atgaaatacg aatgc 25
<210> 7
<211> 23
<212> DNA
<213>Artificial sequence
<400> 7
agaatgagcg atctgaagac gag 23
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence
<400> 8
acagacaaca acttcccctc cat 23
Claims (2)
1. a kind of panax japonicus majoris transcription factor genePjERF1The key gene in panax japonicus majoris saponin formation metabolic pathway is improvedFPS
Expression quantity and the application for increasing saponin content in panax japonicus majoris callus, it is characterised in that：The panax japonicus majoris transcription factor genePjERF1Nucleotide sequence as shown in SEQ ID NO ︰ 1.
2. panax japonicus majoris transcription factor gene according to claim 1PjERF1In panax japonicus majoris saponin formation metabolic pathway is improved
Key geneFPSExpression quantity and the application for increasing saponin content in panax japonicus majoris callus, it is characterised in that concrete operations are such as
Under：
（1）By panax japonicus majoris transcription factor genePjERF1It is connect with plant overexpression vector pCAMBIA2300S, structure plant surpasses
Expression vector；
（2）The expression vector of above-mentioned structure is transferred to by agriculture bacillus mediated in panax japonicus majoris callus；
（3）By antibiotic-screening panax japonicus majoris transgenic cell line, panax japonicus majoris saponin(e closes in qRT-PCR detection transgenic cell lines
Into pathway key enzyme gene expression amount；
（4）The saponin(e in panax japonicus majoris transgenosis and Nontransgenic cells system is extracted, is analyzed between transgenosis and Nontransgenic cells system
The difference of saponin content finally filters out the positive transgenic cell line that saponin content is improved.
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| CN105441462B (en) * | 2016-01-06 | 2018-10-23 | 昆明理工大学 | A kind of Radix Notoginseng transcription factor gene PnERF1 and its application |
| CN108866229A (en) * | 2018-07-27 | 2018-11-23 | 中国林业科学研究院林业研究所 | Primer combination, kit and method suitable for the detection TbAP2a gene expression of qRT-PCR method |
| CN109295080B (en) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | Application of rhizoma panacis majoris beta-balsamol synthetase gene Pj beta-AS |
| CN109295069B (en) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | Application of rhizoma panacis majoris transcription factor gene PjMYB1 |
| CN110484562B (en) * | 2019-09-25 | 2020-09-25 | 福建农林大学 | Method for improving sterol content of rice by using ginseng transcription factor |
| CN115725620B (en) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | Method for synthesizing panax japonicus saponins in pseudo-ginseng cells |
| CN116287148B (en) * | 2023-05-24 | 2023-08-15 | 云南珩柯生物科技有限公司 | Method for identifying Panax schinseng, primer, probe and application thereof |
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