CN105087599A - Application of panax japonicus transcription factor gene PjERF1 - Google Patents
Application of panax japonicus transcription factor gene PjERF1 Download PDFInfo
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- CN105087599A CN105087599A CN201510561815.1A CN201510561815A CN105087599A CN 105087599 A CN105087599 A CN 105087599A CN 201510561815 A CN201510561815 A CN 201510561815A CN 105087599 A CN105087599 A CN 105087599A
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- rhizome
- pjerf1
- transcription factor
- bipinnatifid ginseng
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Abstract
The invention discloses application of panax japonicus transcription factor gene PjERF1, namely, application in improving the expression quantity of key enzyme genes in the biological synthesis of panax japonicus saponins, and increasing the content of saponins in panax japonicus callus. The nucleotide sequence of the PjERF1 gene is shown as SEQ ID NO: 1, and AP2/ERF transcription factors are coded. Through the adoption of functional genomics and techniques related to metabolic engineering, a panax japonicus PjERF1 transcription factor is proved to have the effect of positively regulating the biological synthesis of panax japonicus saponins; when the panax japonicus PjERF1 transcription factor gene disclosed by the invention is established on a plant expression vector and transferred into the panax japonicus callus for over expression, the expression quantity of the key enzyme genes in the synthetic route of panax japonicus saponins is increased, and the yield of panax japonicus saponins is increased.
Description
Technical field
The present invention relates to molecular biology and genetically engineered field, especially a kind of Rhizome of Bipinnatifid Ginseng saponin(e biosynthesizing transcription factor gene
pjERF1application.
Background technology
Rhizome of Bipinnatifid Ginseng
panaxjaponicusc.A.Mey.var.
major(Burk.) C.Y.WuetK.M.Feng is Araliaceae Araliaceae Panax
panaxplant, because rhizome internode is very thin, joint enlarges into spherical beads shape and gains the name, and its rhizome is used as medicine.Rhizome of Bipinnatifid Ginseng, as medicinal material, beginning to be loaded in " the southern regions of the Yunnan Province book on Chinese herbal medicine ", recording kind for going through version " Chinese Pharmacopoeia ".Medicinal Rhizome of Bipinnatifid Ginseng main product is in Yunnan, and the expensive parts of generic medicinal plants of generic name is traditional medication of the ethnic minoritys such as the Yi nationality, distributed over Yunnan, Sichuan and Guizhou, Naxi, Bai nationality, Tibetan, Lisu.At present, wild Rhizome of Bipinnatifid Ginseng is mainly distributed in NW YUNNAN, Northeastern Yunnan, under being common in the sink-source dynamics and broad-leaf forest that height above sea level reaches 2500-4000m.Rhizome of Bipinnatifid Ginseng nature and flavor are bitter, sweet, be slightly cold, and return liver, lung, stomach warp, have tonifying lung yin-nourishing, stasis-dispelling and pain-killing, effect of hemostasis, is applied to deficiency of both qi and yin clinically, and dysphoria with smothery sensation is thirsty, and consumptive disease is coughed, wound, arthralgia, hemoptysis, spits blood, traumatic hemorrhage etc.
Rhizome of Bipinnatifid Ginseng saponin(e (
panaxjaponicussaponins, PJS) be the main active ingredient of Rhizome of Bipinnatifid Ginseng, comprise dammarane type, oleanane-type triterpene saponin.At present, from Rhizome of Bipinnatifid Ginseng rhizome and leaf, isolate more than 30 kind of saponin constituent, mainly comprise Rhizoma Panacis Japonici saponin IVa, Rhizoma Panacis Japonici saponin IV, Rhizoma Panacis Japonici saponin V, ginsenoside Ro, ginsenoside Re, Ginsenoside Rd, ginsenoside Rb1 etc.The representative species " ginseng " of Panax (
panaxginseng) main containing dammarane type saponin(e, oleanane glycoside only finds the ginsenoside Ro that content is atomic; The another kind of famous medicinal material of Panax " pseudo-ginseng " (
panaxnotoginseng), only containing dammarane type saponin(e, not containing oleanane glycoside.Saponin(e component contained by Rhizome of Bipinnatifid Ginseng with belong to together compared with " ginseng ", " pseudo-ginseng ", all there is notable difference in composition kind and each component concentration, because it contains a large amount of oleanane glycoside, cause special purpose clinically.
Rhizome of Bipinnatifid Ginseng is per nnial herb, need grow and can be used as medicine for more than 6 years.At present, Rhizome of Bipinnatifid Ginseng medicinal material mostly is wild product, and unordered excavating causes Rhizome of Bipinnatifid Ginseng resource day by day exhausted, and habitat is damaged.In recent years, be that the drug market of component expands rapidly, cause the demand growth of Rhizome of Bipinnatifid Ginseng medicinal material with Rhizome of Bipinnatifid Ginseng, imbalance between supply and demand is given prominence to.In view of the artificial culture time is long, the drawbacks such as chemosynthesis mechanism and route are clear not, utilize the method for biotechnology and gene regulating to become study hotspot gradually to produce Rhizome of Bipinnatifid Ginseng saponin(e.
The transcriptional activation of transcription factor to gene is the important regulation and control link of Secondary Metabolism of Plant process.Utilize transcription factor as the instrument of plant modification pathways metabolism, with " multiple spot regulation and control " advantage that it is exclusive, compensate for single key gene effect deficiency and multiple key gene in metabolic engineering operation and may produce the situation of the lethal expression of composition, become a kind of strategy newly.In secondary metabolism approach, multiple function involved enzyme gene being is often just is being regulated and controled or negative regulation by same transcription factor, carries out genetic modification obviously than the easier Breeding objectives secondary metabolism approach of polygene operation to transcription factor.
AP2/ERF (APETALA2/ETHYLENEResponsiveFactor) transcription factor is a class transcription factor specific to plant, and increasing research shows that such transcription factor has important regulating and controlling effect to Eukaryotic secondary metabolism process.Such as Vinca (
catharanthusroseus) AP2/ERF class transcription factor ORCA3 controllable participates in the expression of terpene indole alkaloid biosynthetic strictosidine synthase gene; Sweet wormwood (
artemisiaapiacea) AaERF1 with AaERF2 transcription factor can improve the expression amount of Artemisinin biosynthesizing relevant false indigo 4,11-diene synthase (ADS) and cytochrome P 450 monooxygenases (CYP71AV1) gene, just regulating and controlling the synthesis of Artemisinin; In addition, Ramulus et folium taxi cuspidatae (
taxuschinensis) expression of TcAP2 transcription factor controllable Taxol biosynthesis key gene.
Along with to the deep parsing of Secondary Metabolism of Plant network and illustrating of regulatory mechanism, the separation andpreconcentration of the transcription factor particularly regulating specific secondary metabolite to synthesize, the genetically engineered based on transcription factor will provide more effective means for developing Plant Secondary Metabolites.The present invention, clones for research object with the Rhizome of Bipinnatifid Ginseng callus of vitro culture
pjERF1transcription factor gene, and this transcription factor is analyzed and Function Identification, specify the importance and functions of PjERF1 transcription factor in Rhizome of Bipinnatifid Ginseng saponin(e biosynthetic process, for the foundation obtaining efficient, stable Rhizome of Bipinnatifid Ginseng saponin formation control technique and homology or allos efficient expression system provides theoretical reference and foundation.
Summary of the invention
The object of this invention is to provide a kind of Rhizome of Bipinnatifid Ginseng transcription factor gene
pjERF1purposes, i.e. Rhizome of Bipinnatifid Ginseng transcription factor gene
pjbHLH1improving key gene expression amount and the application increased in Rhizome of Bipinnatifid Ginseng callus in saponin content in Rhizome of Bipinnatifid Ginseng saponin formation pathways metabolism; The present invention clones and obtains the biosynthetic transcription factor gene of controllable Rhizome of Bipinnatifid Ginseng saponin(e from Rhizome of Bipinnatifid Ginseng
pjERF1and the application of clear and definite this transcription factor.
The present invention is based on the principle of homologous clone, from Rhizome of Bipinnatifid Ginseng, clone obtains the cDNA of AP2/ERF class transcription factor gene and carries out Function Identification to its proteins encoded.This unnamed gene is by contriver
pjERF1, gene accession number is KP890784, and wherein said cDNA is as shown in SEQIDNO ︰ 1.Sequential analysis is carried out to this gene, shows
pjERF1cDNA size is 808bp, has the open reading frame (Openreadingframe, ORF) of 801bp, and coding is containing 266 amino acid whose protein, and aminoacid sequence is as shown in SEQIDNO ︰ 2.Utilize plant expression vector, by agrobacterium-mediated transformation by of the present invention
pjERF1transcription factor gene imports in Rhizome of Bipinnatifid Ginseng callus, can improve the expression amount of Rhizome of Bipinnatifid Ginseng saponin formation pathway key enzyme gene, the output of Rhizome of Bipinnatifid Ginseng saponin(e is increased.
Above-mentioned transcription factor gene can be applicable to the biosynthesizing just regulating and controlling Rhizome of Bipinnatifid Ginseng saponin(e, and concrete operations are as follows:
(1) acquisition of gene: extract Rhizome of Bipinnatifid Ginseng total serum IgE, reverse transcription synthesis cDNA first chain.Amplified by RT-PCR
pjERF1full length coding region, be then connected on pGEM-Teasy carrier, obtain through sequence verification and there is the clone of goal gene;
(2) plant expression vector construction and genetic transformation: use restriction enzyme
smai and
xbai enzyme cuts pGEM-T-
pjERF1plasmid, is reclaimed by glue and obtains goal gene fragment.With same endonuclease digestion plant expression vector pCAMBIA2300S, glue reclaims and obtains carrier large fragment.Goal gene fragment is connected with pCAMBIA2300S carrier segments, builds plant overexpression vector pCAMBIA2300S-
pjERF1.By frozen-thawed method by pCAMBIA2300S-
pjERF1plasmid imports in agrobacterium strains EHA105.Utilize Agrobacterium-mediated genetic transformation method, will
pjERF1import in Rhizome of Bipinnatifid Ginseng callus and make its overexpression, screen positive transgenic clone by antibiotic-screening and qRT-PCR;
(3) transgenic cell line saponin content detects: extract the saponin(e in Rhizome of Bipinnatifid Ginseng transgenosis and Nontransgenic cells system, analyzes the difference of saponin content between transgenosis and Nontransgenic cells system.
The present invention is that the content improving saponin(e in Rhizome of Bipinnatifid Ginseng provides a kind of novel method, utilize the method for biotechnology and gene regulating can more high-level efficiency synthesis Rhizome of Bipinnatifid Ginseng saponin(e, the shortcoming such as overcome artificial culture cycle long, chemosynthesis mechanism and route is clear not.By transcription factor
pjERF1channel genes Rhizome of Bipinnatifid Ginseng cells, makes the expression amount of Rhizome of Bipinnatifid Ginseng saponin(e biosynthetic pathway key gene improve, adds the output of Rhizome of Bipinnatifid Ginseng saponin(e, for large-scale industrialized production Rhizome of Bipinnatifid Ginseng saponin(e provides theoretical reference and scientific basis.
Accompanying drawing explanation
Fig. 1 is Rhizome of Bipinnatifid Ginseng total serum IgE electrophoretogram in the present invention;
Fig. 2 is in the present invention
pjERF1rT-PCR detected result, wherein M is DL2000DNAMarker, and 1 is
pjERF1rT-PCR product;
Fig. 3 is the three-dimensional structure prediction of PjERF1 in the present invention;
Fig. 4 is qRT-PCR interpretation of result figure in the present invention, represents in Rhizome of Bipinnatifid Ginseng saponin formation approach and regulated and controled by PjERF1
fPSthe expression level of gene in wild-type and transgenic cell line, wherein C is wild-type cell system, and 1-3 is transgenic cell line;
Fig. 5 is the assay result of Rhizome of Bipinnatifid Ginseng saponin(e in the present invention, and wherein C is wild-type cell system, and 1-3 is transgenic cell line.
Embodiment
Below by drawings and Examples, the present invention is further described; but scope is not limited to described content; in embodiment, method is ordinary method if no special instructions, the reagent that the reagent of use is conventional commercial reagent if no special instructions or prepares according to a conventional method.
Embodiment 1:
pjERF1the clone of gene and bioinformatic analysis
Because containing more secondary metabolites in Rhizome of Bipinnatifid Ginseng, two-step approach need be adopted to carry out the extraction of total serum IgE.First adopt the guanidine isothiocyanate method of improvement to get slightly to carry, then use DNA enzymatic carry out digesting and utilize chloroform to obtain purer Rhizome of Bipinnatifid Ginseng total serum IgE (Fig. 1).Adopt reversed transcriptive enzyme M-MLV (promega) with Rhizome of Bipinnatifid Ginseng total serum IgE for templated synthesis cDNA first chain, reaction system and operating process are: get 5 μ gTotalRNA, add 50ngoligo (dT) successively
15, 2 μ LdNTP (2.5mMeach), DEPC water to reaction volume be 13.5 μ L; After mixing, rapidly at cooled on ice 5min after 70 DEG C of heat denatured 5min, then 4 μ L5 × First-standbuffer, 0.5 μ LRNasin (200U), 1 μ LM-MLV (200U) is added successively, mixing is brief centrifugation also, 42 DEG C of temperature bath 1.5h, take out rear 70 DEG C of heating 10min, termination reaction.The synthesis of cDNA first chain is placed on-20 DEG C and saves backup.
With the first chain cDNA of synthesis for template, design Auele Specific Primer, amplifying target genes according to AP2/ERF class transcription factor gene cDNA sequence relevant to the biosynthesizing of regulation and control arasaponin in pseudo-ginseng
pjERF1, the primer sequence is respectively 5 '-cccgggATGTGTGGAGGTGCAATCCTAGGTG-3 ' and 5 '-tctagaGTGCTCTTTAAACGACATCGTCGAA-3 '.Adopt Advantage
tM2PCREnzyme (Clontech) amplifies goal gene.PCR reaction conditions: 94 DEG C of 5min; 94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 50s, 32cycles; 72 DEG C of 10min.Reaction system (50 μ L) is 4 μ L first chain cDNA, 5 μ L10 × Buffer, 1 μ L50 × dNTPMix, 0.5 μ L forward primer (10 μMs), 0.5 μ L reverse primer (10 μMs), 1 μ LAdvantage
tM2PCREnzyme, 38 μ LPCR-GradeWater.After PCR terminates, get 5 μ L for agarose gel electrophoresis, detect specificity and the size (Fig. 2) of amplified production, all the other PCR primer carry out glue recovery.Object fragment glue is reclaimed product and carries out TA clone, reaction system and operating process are: get 3.6 μ L glue and reclaim product, add 0.7 μ LpGEM-Teasyvector (50ng/ μ L), 5 μ L2 × RapidLigationBuffer and 0.7 μ LT4DNALigase mixing successively and are placed in 8 DEG C of reaction overnight.Heat-shock transformed method is adopted to be proceeded in intestinal bacteria Trans1-T1 by connection product.With the LB solid medium screening positive clone containing penbritin (ampicillin, Amp).Select several white colonies, with increasing after shaking bacterium
pjERF1special primer identify multiple clone site insert
pjERF1clone, the clone of qualification is checked order.
Final acquisition
pjERF1cDNA size is 808bp, is analyzed find that it comprises the ORF (see sequence table) of a 801bp by NCBIORFfinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).
pjERF1the molecular weight of proteins encoded is about 30.1KD, and iso-electric point is 5.40, and unstability index is 44.80, prediction
pjERF1the protein of coding is unstable.Bioinformatics Prediction PjERF1 does not comprise cross-film district, not containing signal peptide, has an AP2/ERF class transcription factor feature conserved domain.Predict that PjERF1 may be positioned tenuigenin or mitochondrial matrix by online tool iPSORT.By SWISS-MODEL instrument, PjERF1 is with Arabidopis thaliana ATERF1 transcription factor for template carries out three dimensional joint element (Fig. 3), and result shows that PjERF1 and ATERF1 transcription factor has the space structure of the sequence similarity of 71.93%.
Embodiment 2: plant expression vector construction
Adopt extraction agent box (the raw work in Shanghai) the extraction insertion in a small amount of SanPrep pillar plasmid DNA
pjERF1escherichia coli plasmid pGEM-T-
pjERF1and the plasmid of plant expression vector pCAMBIA2300S, get 1 μ L for agarose gel electrophoresis with detect the integrity of extraction plasmid and concentration level.With
xbai (TaKaRa) and
smai (TaKaRa) is respectively to plasmid pGEM-T-
pjERF1carry out double digestion (100 μ L system) with pCAMBIA2300S, reaction system and operating process are: get 20 μ LpGEM-T-
pjERF1or pCAMBIA2300S plasmid, add 10 μ L10 × Tbuffer, 10 μ LBSA, 5 μ L successively
xbai, 5 μ L
smai, 50 μ LddH
2o, centrifugal in short-term after mixing, be placed in 37 DEG C of reaction overnight.All digestion products points are carried out electrophoresis in sepharose, then right
pjERF1fragment and pCAMBIA2300S large fragment carry out glue recovery respectively, and whole process uses SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai).Get 1 μ L and reclaim product detects recovery fragment size and concentration by agarose gel electrophoresis, be placed in-20 DEG C and save backup.
Utilize T4DNALigase (TaKaRa), by what reclaim
pjERF1dNA fragmentation and pCAMBIA2300S carrier segments couple together, and reaction system (20 μ L) and operating process are: get 10 μ L
pjERF1dNA fragmentation adds 2 μ LpCAMBIA2300S carrier DNAs, 2 μ L10 × T4DNALigaseBuffer, 1 μ LT4DNALigase, 5 μ LddH successively
2o, centrifugal in short-term after mixing, be placed in 16 DEG C of water-bath reaction overnight.Then heat-shock transformed method is adopted to proceed in intestinal bacteria Trans1-T1 by connection product, with the solid medium screening positive clone containing 50mg/L kantlex (kanamycin, Km).Picking individual colonies shakes bacterium, with bacterium liquid for template amplification
pjERF1special primer carry out PCR, pick out
pjERF1the clone be successfully connected with pCAMBIA2300S, if the bacterial strain that detects is positive, adds 20% glycerine mixing and is placed on-80 DEG C and saves backup.
With the pCAMBIA2300S in test kit the extraction also above-mentioned intestinal bacteria of purifying
-PjERF1plasmid.Prepare the competent cell of Agrobacterium EHA105 bacterial strain and be sub-packed in 1.5mL centrifuge tube, often pipe 150 μ L, liquid nitrogen flash freezer is placed on-80 DEG C and saves backup.Adopt frozen-thawed method by the plant expression vector pCAMBIA2300S-of above-mentioned structure
pjERF1proceed in prepared Agrobacterium EHA105 competent cell.Operation steps is: get 3 μ gpCAMBIA2300S-
pjERF1plasmid adds in the centrifuge tube containing 150 μ L competent cells, and ice bath 30min after mixing, then proceeds to quick-frozen 5min in liquid nitrogen gently, then 37 DEG C of water-bath 5min are placed in rapidly, ice bath 2min immediately afterwards, adds 600 μ LLB liquid nutrient mediums, 28 DEG C of shaking culture 4h.Agrobacterium after activation is applied on the LB solid medium containing 50mg/LKm, 28 DEG C of quiescent culture.Picking individual colonies shakes bacterium, with amplification
pjERF1special primer carry out PCR, detect pCAMBIA2300S-
pjERF1whether proceed in Agrobacterium.For positive colony, add glycerine and be placed on-80 DEG C and save backup.
Embodiment 3: agriculture bacillus mediated Rhizome of Bipinnatifid Ginseng genetic transformation
That from-80 DEG C of refrigerators, takes out preservation contains pCAMBIA2300S
-PjERF1the Agrobacterium EHA105 bacterial classification of plasmid, be inoculated in 5mL and contain in the LB liquid nutrient medium of 50mg/LKm and 25mg/L Rifampin, 28 DEG C are cultured to muddiness.The bacterium liquid drawing 1mL muddiness, to containing on the LB solid medium of 50mg/LKm, cultivates 48h for 28 DEG C.Being scraped by Agrobacterium on LB solid medium is inoculated in MGL liquid nutrient medium in right amount, and the Syringylethanone of additional 40mg/L, 28 DEG C of shaking culture are to OD
600stop when being 0.6 shaking bacterium, gained bacterium liquid is used for infecting.
Good for growth conditions Rhizome of Bipinnatifid Ginseng callus is received on MS pre-culture medium (containing 35mg/L Syringylethanone) and carry out preculture 3 days.After preculture completes, callus is soaked in completely in above-mentioned Agrobacterium bacterium liquid and carries out shaking culture.Contaminate complete, removing bacterium liquid, draw the bacterium liquid on callus surface with aseptic filter paper, be finally inoculated into by callus in Dual culture base, carry out light culture in 25 DEG C, the time is three days.Callus after dip-dye is inoculated in MS Dual culture base (containing 35mg/L Syringylethanone), light culture 3 days, then with sterilized water, callus is washed, transfer in carrying out degerming cultivation containing on the MS substratum of 400mg/L cephamycin again, in 25 DEG C of light culture 15 days, prevent Agrobacteriuna overgrowth.Finally be transferred in screening culture medium by callus, every 45 days subcultures once.Through 5 screenings, finally isolate the pure clone that the value-added speed with Km resistance is fast, for subsequent detection.
Embodiment 4:
pjERF1gene overexpression is to Rhizome of Bipinnatifid Ginseng saponin formation pathway key enzyme gene
fPSthe impact of expression amount
Choose about 25 days, the good Rhizome of Bipinnatifid Ginseng transgenic cell line of growth conditions and wild-type cell system, extract RNA respectively, then according to GoTaq
?2-StepRT-qPCRSystem test kit specification sheets synthesis cDNA, reaction system and operating process are: in centrifuge tube, add 4 μ g total serum IgE and 1 μ LOligo (dT)
15, with Nuclease-freeWater polishing to 10 μ L, reaction system is put in 70 DEG C of sex change 5min, is then placed in 5min on ice.Subsequently that centrifuge tube is of short duration centrifugal in centrifuges, reaction solution is collected at the bottom of pipe, then adds 4 μ LGoScript wherein
tM5 × ReactionBuffer, 2 μ LMgCl
2(25mM), 1 μ LPCRNucleotideMix (10mM), 0.5 μ LRecombinantRNasinRibonucleaseInhibitor and 1 μ LGoScript
tMreverseTranscriptase, by whole reaction system whirlpool mixing, at the bottom of collected by centrifugation to pipe, reactant is placed in 42 DEG C of thermostat water baths and reacts 1h, in 70 DEG C of water-baths, maintain 15min with termination reaction again, finally the cDNA of synthesis is placed in-20 DEG C of refrigerators and saves backup.
With this cDNA for template, according to Rhizome of Bipinnatifid Ginseng
18SrRNAgene (accession number: AB088018.1) and farnesyl pyrophosphate synthetic enzyme (Farnesyldiphosphatesynthase, FPS) gene (accession number: KP684141) design primer, according to GoTaq
?2-StepRT-qPCRSystem test kit specification sheets carry out fluorescence semiquantitive PCR amplification Rhizome of Bipinnatifid Ginseng reference gene and
fPSgene.The primer sequence is
18S f: 5 '-GTTGTTGCAGTTAAAAAGCTCGTAG-3 ',
18S r: 5 '-ACCTCTGACTATGAAATACGAATGC-3 ';
fPS f: 5 '-AGAATGAGCGATCTGAAGACGAG-3 ';
fPS b: 5 '-ACAGACAACAACTTCCCCTCCAT-3 '.Concrete reaction system and operating process are: in PCR pipe, add 20ngcDNA, 25 μ LGoTaq
?qPCRMasterMix (2 ×) and 0.2 μ LqPCRPrimers (
18S f/
18S r,
fPS b/
fPS f, 10mM), with Nuclease-FreeWater polishing to 50 μ L.After being mixed by reaction system whirlpool, centrifugally collected at the bottom of pipe, be placed in quantitative real time PCR Instrument subsequently and react, adopt two-step approach to carry out quantitative fluorescent PCR, reaction parameter is as follows: warm start 95 DEG C of 2min; Sex change 95 DEG C of 15s, annealing/extend 60 DEG C of 1min, totally 45 circulations.Each gene redundancy that each sample is corresponding detects 2 times.
QRT-PCR result shows, and turns
pjERF1in gene Rhizome of Bipinnatifid Ginseng cell
fPSthe expression amount of gene, than the height (Fig. 4) of wild-type, illustrates that PjERF1 is as transcription factor, can promote key gene in Rhizome of Bipinnatifid Ginseng saponin formation pathways metabolism
fPSexpression; In figure, C represents control group wild-type cell system, and 1,2 and 3 represent different transgenic cell line experimental group respectively.
Embodiment 5:
pjERF1gene overexpression is on the impact of Rhizome of Bipinnatifid Ginseng saponin formation amount
Choose the growth transgenic cell line of about 35 days and wild-type cell system, be placed in clean 100mL triangular flask respectively, after adding the methanol solution soaked overnight of 20mL, normal temperature ultrasonication 1h.Filter, collect filtrate, use dissolve with methanol again after filtrate being volatilized, be settled to 25mL, obtain crude extract.Residue is dried to constant weight at 50 DEG C, weighs.Accurate absorption crude extract 5mL, puts water bath method in 50mL beaker.Dissolve with the distilled water gradation of 4 times of volumes after evaporate to dryness, filter after fully dissolving, filtrate is all transferred in processed good Hsp100 macroporous resin column, first slowly washes away the impurity such as carbohydrate with the distilled water of 2 column volumes.Whether Molish reaction detection carbohydrate impurity is removed totally, if result is positive, continuation distilled water is washed till feminine gender, then uses 75% ethanolic soln wash-out, 2 column volumes, and collect alcohol liquid, water bath method, residue dissolve with methanol solution, is settled to 25mL.
In this sample 150 of accurate absorption μ L to the 10mL test tube of band plug (establishing 3 repetitions), volatilize solvent, add the 5% Vanillin-glacial acetic acid solution 0.2mL of new preparation, perchloric acid 0.8mL, mix rear 60 DEG C of heating in water bath 15min, immediately with frozen water cooling, add 5mL Glacial acetic acid, after mixing static 10min, 554nm surveys absorbancy, reference standard curve calculation PJS content.Result shows, and turns
pjERF1in gene Rhizome of Bipinnatifid Ginseng cell, saponin content is apparently higher than saponin content in wild-type cell (Fig. 5), illustrates that PjERF1 transcription factor take part in the anabolism regulation and control of Rhizome of Bipinnatifid Ginseng saponin(e, contributes to the raising of saponin(e output; C represents control group wild-type cell system, and 1,2 and 3 represent different transgenic cell line experimental group respectively.
sequence table
<110> Kunming University of Science and Technology
<120> Rhizome of Bipinnatifid Ginseng transcription factor gene
pjERF1application
<160>8
<170>PatentInversion3.5
<210>1
<211>808
<212>DNA
<213>Panaxjaponicus
<220>
<221>mRNA
<222>(1)..(808)
<220>
<221>CDS
<222>(1)..(801)
<220>
<221>3'UTR
<222>(802)..(808)
<400>1
atgtgtggaggtgcaatcctaggtgatcttaccgctcgaaatttcaaccgccgtgtctcc60
gccgctgacttctggcccacatctctctccgacaaactcgacaatttccagtccgaattt120
aatcatttccctccggaggagactcgaaccctcaaaagagcgcagcctaattcaggtgga180
gtacctctcggaaagacaacaaagagacagaggaagaacatgtacagaggaataaggcag240
cgtccatgggggaaatgggcagctgagattagggatccgagaaaaggagtgagggtttgg300
ctgggtactttcaacacggctgaggaggctgccagagcctacgacaaagaagctcgcaag360
attagaggaaacaaagccaaagttaacttcccaaatgaggactgctttaatcaattcaat420
gtcaaaaatatgaatcaatttgggtcaaattcttgttctgggttttctgcattgaacagg480
gaggaccaatcacctttggattttacctgtctgaaaaatggaagcgatggacttagtatt540
gctgagcaggaggagaaggtaaaagaagaaaaagaggagaaagaaaataaagagagtgtg600
attgatcaagtggaggaacagaacgaactgcagaagctctcggatgagctaatggcctac660
gaatcttacatgaaattttatgaaattccgtatcttgatggccagtcggcgacggcggca720
gcagctccgacgagtgcggtgccggaaaacgtcgtagacggtggtttgctaaatctttgg780
agcttcgacgatgtcgtttaaagagcac808
<210>2
<211>266
<212>PRT
<213>Panaxjaponicus
<400>2
MetCysGlyGlyAlaIleLeuGlyAspLeuThrAlaArgAsnPheAsn
151015
ArgArgValSerAlaAlaAspPheTrpProThrSerLeuSerAspLys
202530
LeuAspAsnPheGlnSerGluPheAsnHisPheProProGluGluThr
354045
ArgThrLeuLysArgAlaGlnProAsnSerGlyGlyValProLeuGly
505560
LysThrThrLysArgGlnArgLysAsnMetTyrArgGlyIleArgGln
65707580
ArgProTrpGlyLysTrpAlaAlaGluIleArgAspProArgLysGly
859095
ValArgValTrpLeuGlyThrPheAsnThrAlaGluGluAlaAlaArg
100105110
AlaTyrAspLysGluAlaArgLysIleArgGlyAsnLysAlaLysVal
115120125
AsnPheProAsnGluAspCysPheAsnGlnPheAsnValLysAsnMet
130135140
AsnGlnPheGlySerAsnSerCysSerGlyPheSerAlaLeuAsnArg
145150155160
GluAspGlnSerProLeuAspPheThrCysLeuLysAsnGlySerAsp
165170175
GlyLeuSerIleAlaGluGlnGluGluLysValLysGluGluLysGlu
180185190
GluLysGluAsnLysGluSerValIleAspGlnValGluGluGlnAsn
195200205
GluLeuGlnLysLeuSerAspGluLeuMetAlaTyrGluSerTyrMet
210215220
LysPheTyrGluIleProTyrLeuAspGlyGlnSerAlaThrAlaAla
225230235240
AlaAlaProThrSerAlaValProGluAsnValValAspGlyGlyLeu
245250255
LeuAsnLeuTrpSerPheAspAspValVal
260265
<210>3
<211>31
<212>DNA
<213> artificial sequence
<400>3
cccgggatgtgtggaggtgcaatcctaggtg31
<210>4
<211>31
<212>DNA
<213> artificial sequence
<400>4
tctagagtgctctttaaacgacatcgtcgaa31
<210>5
<211>25
<212>DNA
<213> artificial sequence
<400>5
gttgttgcagttaaaaagctcgtag25
<210>6
<211>25
<212>DNA
<213> artificial sequence
<400>6
acctctgactatgaaatacgaatgc25
<210>7
<211>23
<212>DNA
<213> artificial sequence
<400>7
agaatgagcgatctgaagacgag23
<210>8
<211>23
<212>DNA
<213> artificial sequence
<400>8
acagacaacaacttcccctccat23
Claims (2)
1. a Rhizome of Bipinnatifid Ginseng transcription factor gene
pjERF1improving key gene expression amount and the application increasing saponin content in Rhizome of Bipinnatifid Ginseng callus in Rhizome of Bipinnatifid Ginseng saponin formation pathways metabolism, it is characterized in that: described Rhizome of Bipinnatifid Ginseng transcription factor gene
pjERF1nucleotide sequence as shown in SEQIDNO ︰ 1.
2. Rhizome of Bipinnatifid Ginseng transcription factor gene according to claim 1
pjERF1application, is characterized in that concrete operations are as follows:
(1) by Rhizome of Bipinnatifid Ginseng transcription factor gene
pjERF1be connected with plant overexpression vector pCAMBIA2300S, build plant overexpression vector;
(2) expression vector of above-mentioned structure is proceeded in Rhizome of Bipinnatifid Ginseng callus by agriculture bacillus mediated;
(3) by antibiotic-screening Rhizome of Bipinnatifid Ginseng transgenic cell line, qRT-PCR detects Rhizome of Bipinnatifid Ginseng saponin formation pathway key enzyme gene expression amount in transgenic cell line;
(4) extract the saponin(e in Rhizome of Bipinnatifid Ginseng transgenosis and Nontransgenic cells system, analyze the difference of saponin content between transgenosis and Nontransgenic cells system, finally filter out the positive transgenic clone that saponin content is improved.
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| CN110484562A (en) * | 2019-09-25 | 2019-11-22 | 福建农林大学 | A method of rice sterol content is improved using ginseng transcription factor |
| CN115725620A (en) * | 2022-09-12 | 2023-03-03 | 昆明理工大学 | Method for synthesizing panax japonicus saponin in panax notoginseng cells |
| CN116287148A (en) * | 2023-05-24 | 2023-06-23 | 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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| CN109295069A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | The application of panax japonicus majoris transcription factor gene PjMYB1 |
| CN109295080A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes |
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| CN110484562A (en) * | 2019-09-25 | 2019-11-22 | 福建农林大学 | A method of rice sterol content is improved using ginseng transcription factor |
| CN115725620A (en) * | 2022-09-12 | 2023-03-03 | 昆明理工大学 | Method for synthesizing panax japonicus saponin in panax notoginseng cells |
| CN115725620B (en) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | Method for synthesizing panax japonicus saponins in pseudo-ginseng cells |
| CN116287148A (en) * | 2023-05-24 | 2023-06-23 | 云南珩柯生物科技有限公司 | Method for identifying Panax schinseng, primer, probe and application thereof |
| 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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