CN110066328A

CN110066328A - Ginseng PgMYB2 transcription factor and application thereof in regulating and controlling synthesis of ginsenoside

Info

Publication number: CN110066328A
Application number: CN201910371237.3A
Authority: CN
Inventors: 罗志勇; 刘拓; 郭祥前; 罗眺; 李继佳
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2019-05-06
Filing date: 2019-05-06
Publication date: 2019-07-30
Anticipated expiration: 2039-05-06
Also published as: CN110066328B

Abstract

The invention discloses a transcription factor PgMYB2 capable of regulating and controlling a ginseng dammarenediol synthase gene (PgDDS). The protein sequence of the transcription factor is shown as SEQ ID NO. 1. The nucleotide sequence of the protein is SEQ ID NO. 2. The invention also discovers that the PgMYB2 transcription factor can be combined with a specific sequence in a PgDDS promoter to promote the expression of PgDDS and further promote the synthesis and accumulation of ginsenoside by researching the relation between the transcription factor and the expression of a madene diol synthase gene (PgDDS), and provides a new strategy for constructing transgenic ginseng cells, hairy roots and plants by utilizing genetic engineering and metabolic engineering in the future by researching the application of the PgMYB2 transcription factor in regulating and controlling the synthesis of the ginsenoside so as to improve the yield of the ginsenoside.

Description

Ginseng PgMYB2 transcription factor and its application in regulation ginsenoside synthesis

Technical field

The invention belongs to technical field of biological genetic engineering, are related to transcription factor to the regulating and controlling effect of target gene, specifically The expressional function of ginseng PgMYB2 transcription factor promotion dammarendiol synzyme (DDS) gene.The present invention is directed to pass through to disclose The regulatory mechanism of key enzyme in ginsenoside route of synthesis realizes the raising of saponin formation and accumulating level, has important answer With value.

Background technique

Ginseng (Panax ginseng C.A.Meyer) is a kind of herbaceos perennial, has high medical value. Main pharmacodynamics ingredient in ginseng is ginsenoside, isolates more than 50 ginsenoside monomers, big portion from ginseng at present The medical value for dividing ginsenoside monomer that there is antitumor, anti-aging, inhibit Apoptosis and strengthen immunity etc. important, such as Rb₁And Rg₁There are anti-aging, Ra₁、Rg₃And Rh₂There is stronger anticancer activity etc., part ginsenoside is widely used to It is clinical.Although many ginsenoside monomers have antitumor, anti-aging, inhibit Apoptosis and strengthen immunity isoreactivity, It is that the strong ginsenoside of wherein most activity content in ginseng is extremely low, is far from satisfying the market demand, to break this bottle Regulatory mechanism of the neck dependent on further investigation ginsenoside biosynthesis.

In ginsenoside biosynthesis approach, including more than 20 walk continuous enzymatic reaction.With ginsenoside route of synthesis Research is goed deep into, and more and more synzyme key genes are cloned out.Key enzyme therein has farnesyl pyrophosphate synthesis Enzyme (farnesyl diophosphate synthase, FPS), squalene synthetase (squalene synthase, SS), squalene Epoxidase (squalene epoxidase, SE), dammarendiol-II synzyme (darmmarenediol-II synthase, DDS), beta-amyrin synzyme (β-amyrin synthase, β-AS), Cytochrome P450 (cytochromeP450, ) and glycosyl transferase (glycosyltransferase, GT) etc. CYP450.First is accredited in ginsenoside route of synthesis Key enzyme out is dammarendiol-II synzyme (DDS), and catalysis 2,3- oxidosqualene cyclisation generates dammarendiol, this is Most important branch in ginsenoside route of synthesis is point of triterpene compound and phytosterin compound biosynthesis in plant Branch.Ginsenoside quantity, the most species of branch generation, the content highest in ginseng, the ginsenoside synthesized through the branch Referred to as dammarane type ginsenoside.Domestic and international many researchers explore ginseng PgDDS gene, such as by ginseng PgDDS Gene can detect that yeast generates dammarendiol and hydroxydammarenone Ⅱ after being transferred to yeast, be cured using RNAi technology silencing ginseng It PgDDS gene and is induced into root of hair in wound, wherein saponin content is reduced to 84.5% original [1] for discovery；PgDDS is transferred to cigarette Careless suspension cell, transgenic suspension cells can generate dammarendiol and Ma enediol content reaches in dry weight after culture 3 weeks 573μg/g[2]；Ginseng PgDDS gene is transferred to tobacco, discovery transgene tobacco can generate Ma enediol, and to tobacco The resistance of mosaic virus enhances [3]；It combines ginseng PgDDS with other key genes and is transferred to saccharomyces cerevisiae, construct " people Join yeast " bacterial strain, sapogenin, oleanolic acid, protopanoxadiol (PPD) and protopanaxatriol (PPT), content can be generated simultaneously Respectively reach 21.4mg/L, 17.2mg/L and 15.9mg/L [4-6]；There is no DDS in rice, dammarane type ginseng soap cannot be synthesized Ginseng PgDDS gene is transferred to rice by glycosides, transgenic paddy rice can high expression dammarendiol synzyme and generate PPD and PPT[7].These results illustrate that DDS is most important to the synthesis of ginsenoside.

MYB albumen is the transcription factor family of maximum kind in plant, the growth and development of wide participation plant and metabolic regulation, The regulation of such as cometabolism and the response of biology and abiotic stress, increasingly become the hot spot of plant science research.

Bibliography

[1]Han J Y,Kwon Y S,Yang D C,et al.Expression and RNA interference- induced silencing of the dammarenediol synthase gene in Panax ginseng[J] .Plant Cell Physiol,2006,47(12):1653-1662.

[2]Han J Y,Wang H Y,Choi Y E.Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco[J].Plant Cell Rep,2014,33(2):225-233.

[3]Lee M H,Han J Y,Kim H J,et al.Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase[J].Plant Cell Physiol,2012,53(1):173-182.

[4]Wang P,Wei Y,Fan Y,et al.Production of bioactive ginsenosides Rh2and Rg3by metabolically engineered yeasts[J].Metab Eng,2015,29:97-105.

[5]Dai Z,Wang B,Liu Y,et al.Producing aglycons of ginsenosides in bakers'yeast[J].Sci Rep,2014,4:3698.[6]Dai Z,Liu Y,Zhang X,et al.Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides[J] .Metab Eng,2013,20:146-156.

[7]Huang Z,Lin J,Cheng Z,et al.Production of dammarane-type sapogenins in rice by expressing the dammarenediol-II synthase gene from Panax ginseng C.A.Mey[J].Plant Sci,2015,239:106-114.

Content above with reference to document is integrated into present specification as background technique.

Summary of the invention

The present invention is intended to provide what a kind of myb gene for capableing of the synthesis of indirect adjustments and controls ginsenoside and the gene encoded Application of the transcription factor protein in regulation ginsenoside synthesis.

In order to achieve the above objectives, technical solution provided by the invention are as follows:

The sequence of the ginseng PgMYB2 transcription factor is as shown in SEQ ID NO.1.

The PgMYB2 gene order of the ginseng PgMYB2 transcription factor is encoded as shown in SEQ ID NO.2.Its overall length is 1401bp, wherein the 370th to 1212 is the sequence for encoding SEQ ID NO.1.

The primer pair sequence of PgMYB2 gene is expanded as shown in SEQ ID NO.3 and SEQ ID NO.4.

The present inventor passes through bioinformatic analysis, finds to contain MYB binding site in ginseng PgDDS gene promoter, Based on transcription factor can by DNA binding domain in conjunction with particular sequence in target gene promoters this phenomenon, thus it is speculated that MYB transcription because Son may participate in the regulation of synthesis and the transhipment of ginsenoside.But whether myb transcription factor can regulate and control dammarendiol synzyme Expression so that indirect adjustments and controls ginsenoside biosynthesis? explore to this problem helps completely to illustrate MYB to people Join the regulatory mechanism of saponin formation and accumulation, but not yet ginsenoside biosynthesis is regulated and controled in relation to MYB in ginseng at present Report, so research ginseng myb transcription factor to ginsenoside synthesize and accumulate regulation be particularly important.

The present invention also provides the recombinant vectors for containing PgMYB2 gene as claimed in claim 2, specifically include: Clonal Carrier is named as recombinant vector pGEM-T Easy-PgMYB2, i.e., by PgMYB2 gene cloning to empty carrier pGEM-T Easy In, obtain recombinant vector pGEM-T Easy-PgMYB2；Plant transient expression vector is named as recombinant vector pCAMBIA1302- PgMYB2 obtains recombinant vector pCAMBIA1302- that is, by PgMYB2 gene cloning into empty carrier pCAMBIA1302 PgMYB2；Yeast one-hybrid prey vector is named as pGADT7-PgMYB2, i.e., by PgMYB2 gene cloning to empty carrier In pGADT7, recombinant vector pGADT7-PgMYB2 is obtained；Prokaryotic expression carrier, pCold/TF-PgMYB2, i.e., by PgMYB2 base Because being cloned into empty carrier pCold/TF, recombinant vector pCold/TF-PgMYB2 is obtained；Reporter gene effector plasmid, is named as PEGAD MYC-PgMYB2 obtains recombinant vector pEGAD MYC- that is, by PgMYB2 gene cloning into empty carrier pEGAD MYC PgMYB2。

By PgMYB2 genetic recombination into known carrier, and the those skilled in the art expressed in suitable system Well known technology.Therefore, in addition to the above-mentioned recombinant vector expressed, there can also be the recombinant vector of other genes containing PgMYB2.

By recombinant vector, ginseng suspension cell is transfected, makes to be overexpressed PgMYB2 gene order in ginseng gene.Pass through this Method can obtain Panax ginseng hairy or transgenic plant.

Thus the Panax ginseng hairy or transgenic plant that method obtains, the high expression with PgDDS.

How those skilled in the art are known obtains ginseng suspension cell, and known by recombinant vector, by PgMYB2 base Because being integrated into ginseng suspension cell, to obtain Panax ginseng hairy or transgenic plant.

Referring to Fig. 1 to Fig. 9, the present invention utilizes existing plant gene engineering technology, using the primer of specificity, passes through Round pcr obtains PgMYB2 gene order, and is determined channel genes onion endepidermis cell by agrobacterium rhizogenes mediated method The subcellular localization of PgMYB2.Find that PgMYB2 gene has expression in ginseng is respectively organized simultaneously, 100 μM of MeJA can Induce PgMYB2 gene expression up-regulation.The further experiments such as EMSA and luciferase reporter gene activity show PgMYB2 albumen Can be in conjunction with the site MBSII in PgDDS promoter region, and promote the expression of PgDDS gene.

Detailed description of the invention

Fig. 1 PCR amplification PgMYB2 product gel electrophoresis result, M are gene M arker；

Fig. 2 is that PgMYB2 albumen conserved domain (a), subcellular localization (b) and transmembrane domain predict (c)；

Subcellular Localization diagram of Fig. 3 PgMYB2 albumen in onion: figure a-d is control group expression and localization situation, E-h is GFP-PgMYB2 expressing fusion protein positioning scenarios；

Fig. 4 Real time-PCR detects PgMYB2 relative expression's situation in 4 years raw fresh ginseng different tissues；

Fig. 5 Real time-PCR detects PgMYB2 and PgDDS base after 100 μM of MeJA processing ginseng poultry's different times Because of relative expression's situation；

Fig. 6 yeast one-hybrid detects PgMYB2 and PgDDS promoter region interaction result: left figure is without containing AbA The growing state of each group yeast on SD/-Ura/-Leu solid medium, right figure are in the SD/- containing 200ng/mL AbA The growing state of each group yeast on Ura/-Leu solid medium；

Fig. 7 EMSA probes into the combination in the site MBSII in PgMYB2 albumen and PgDDS promoter region, each system at Divide as shown in table 2；Relative luciferase activity of Fig. 8 PgMYB2 in protoplasts of Arabidopsis thaliana broken by ultrasonic transient expression, each group profile such as table Shown in 3；

Fig. 9 Real time-PCR detects the relative expression levels of PgMYB2 and PgDDS in leaves of panax ginseng, and EV1-3 is note Penetrate the leaves of panax ginseng of pCAMBIA1302 empty plasmid: OV1-3 is the ginseng injected pCAMBIA1302-PgMYB2 and be overexpressed plasmid Blade.

Specific embodiment

Embodiment 1

The clone of PgMYB2 gene

The foundation of 1 ginseng poultry's cultivating system

The configuration of 1.1 1/2MS solid, liquid culture mediums:

1. the preparation of mother liquor

A.10 × a great number of elements mother liquor

Add ddH₂O dissolution, is finally settled to 1L, 4 DEG C of preservations after sterilizing；

B.200 × microelement mother liquor

Add ddH₂O dissolution is finally settled to 500mL, 4 DEG C of preservations；

C.100 × organic mother liquor

D.100 × mother liquid of iron salt

Na₂EDTA·2H₂O 3.725g

FeSO₄·7H₂O 2.785g

Add ddH₂O dissolution, is finally settled to 1L, is placed in 4 DEG C of brown bottle preservations.

2. 1/2MS culture medium prescription

3. the preparation of culture medium

Various mother liquors are first added by dosage in table, add weighed inositol and sucrose, with a certain amount of ddH₂O dissolution Afterwards, weighed agar powder is added, microwave stove heating is placed in, after agar is completely dissolved, supplies ddH₂O is settled to 1L, sufficiently It stirs and evenly mixs, pH value is adjusted to pH6.0 with NaOH solution.After training basigamy is good, it is dispensed into 100mL conical flask, every bottle of 30- 40mL, fluid nutrient medium is then not necessarily to plus agar powder, dispenses after direct constant volume.After sealing the sealing of bottle film, fastened with rope.

4. the sterilizing of culture medium

All packaged conical flasks are put into vertical pressure steam sterilizer and are sterilized, are set as 121 DEG C, 101kPa, 20min.After sterilizing, taking-up is shaken up, and culture bottle is placed on 2-3d in electro-heating standing-temperature cultivator after solidification is good, No fouled by microzyme rear to be confirmed can be used for the culture of ginseng poultry.

1.2 ginseng poultry's tissue cultures and hormone induction processing

Tweezers, culture medium and alcohol swab etc. are put into superclean bench, ultraviolet irradiation 20min.

1. solid culture

The preferable root of hair of appropriate growth conditions is taken with tweezers, is connected in 1/2MS solid training base, is dispersed as far as possible, then seal Bottleneck carries out dark culture, routine observation growth conditions and microbiological contamination situation in 24 DEG C of incubators.Every 30-40d subculture is primary.

2. Liquid Culture

The preferable root of hair of appropriate growth conditions is taken with tweezers, is connected in 1/2MS liquid training base, sealed membrane seals bottleneck.24 DEG C, dark culture in 110rpm shaken cultivation case, routine observation growth conditions and microbiological contamination situation, usual 10d carry out a subculture.

3. MeJA induction processing ginseng poultry

A. processing group: 46 μ L MeJA is taken to be dissolved in 454 μ L ethyl alcohol, then plus 500 μ L ddH₂O is made into final concentration of 200mM's MeJA mother liquor, with 0.22 μm of filter filtration sterilization, it is 100 μM that 5 μ L mother liquors, which are added, to concentration is used in every 10mL fluid nutrient medium；It is right According to group: 454 μ L ethyl alcohol being taken to be dissolved in 546 μ L ddH₂In O, with 0.22 μm of filter filtration sterilization, every 10mL fluid nutrient medium is added 10 μL。

B. the preferable ginseng poultry of solid medium growing way is inoculated in aforesaid liquid culture medium, 120rpm, 24 DEG C of dark trainings It supports, (0,1,3,6,12,24,36,48,72h) respectively takes out one bottle of processing group and control group in different time points, and root of hair is moved into In 1.5mL centrifuge tube, it is put into Liquid nitrogen storage, the extraction for subsequent RNA.

The extraction of 2 ginseng poultry's total serum IgEs

Tip, centrifuge tube, mortar, tweezers, spoon pipe support etc. need to impregnate in the DEPC water that concentration is 1 ‰ with preposition More than 12h, high-temperature sterilization is dried rear and be can be used.Illustrate to extract always with reference to the E.Z.N.A.Plant RNA Kit of OMEGA company RNA。

The synthesis of first chain of 3cDNA

Using the RevertAid of Thermo company^TMFirst Strand cDNA Synthesis Kit kit carries out The synthesis of first chain of cDNA.The first chain of cDNA of synthesis directly can carry out PCR as template.

The clone of 4 PgMYB2 genes

4.1 PCR amplification

Agents useful for same is the Ex Taq enzyme of TaKaRa company, and primer is the synthesis of Invitrogen company, and primer sequence is as follows:

Primer Sequence (' of 5 ' → 3)

PgMYB2-F CCACTCTCAACGCATTCTC(SEQ ID NO.3)

PgMYB2-R TAGGGGTAGGCACATTCTG(SEQ ID NO.4)

Reaction system:

PCR reaction condition:

The recycling of 4.2 PCR product glue

Used kit is Wizard SV Gel and PCR Clean-up system kit (Promega), with reference to saying Bright book carries out gel extraction.

The connection of 4.3 pGEM-T Easy carriers

The PCR product of glue recycling uses pGEM-T Easy Vector System (Promega, USA) connection, connector System:

It should be noted recycling segment when connection and pGEM-T Easy Vector molar ratio be 1:3-1:5, ratio connection effect Rate is higher.It is incubated for: 4 DEG C of overnight or room temperature 1h.

The preparation of 4.4 DH5 α competent cells

1. the DH5 α bacterial strain of -80 DEG C of picking preservations is crossed in LB solid plate and is activated, 37 DEG C of cultures；

2. picking single colonie is connected in 5mL LB liquid medium, and 37 DEG C, 200rpm, shaken cultivation 12-16h；

3. by the switching of 5mL bacterium solution in 500mL LB liquid medium, 37 DEG C, 200rpm, shaken cultivation 1-2h, to OD₆₀₀ Value is gone to rapidly on ice when being 0.4-0.6, ice bath 30min；

4. 500mL bacterium solution is gone in sterile centrifugation tube by the packing of 80mL portion, 4 DEG C, 4,000rpm, it is centrifuged 10min, is abandoned Supernatant；

5. the CaCl of the 0.1M of 20mL pre-cooling is added into centrifuge tube₂Solution, resuspended bacterium solution, after ice bath 30min, 4 DEG C, 4, 000rpm is centrifuged 10min, abandons supernatant；

6. the CaCl of the 0.1M of 4mL pre-cooling is added into centrifuge tube₂Solution (glycerol containing 15% volume), resuspended bacterium solution, Ice bath 10min is dispensed into sterile 1.5mL centrifuge tube, is put into -80 DEG C of refrigerators rapidly and is saved.

The conversion of 4.5 connection products and screening

1. taking out DH5 α competent cell from -80 DEG C of refrigerators, after thawing on ice, take 100 μ L that 5 μ L connection products are added, Flick mixing, ice bath 30min；

2. centrifuge tube is placed in 42 DEG C of water-baths, 60-90s is put on ice immediately after taking-up, places 3min；

3. the LB liquid medium of 500 μ L is added into centrifuge tube, 37 DEG C, 200rpm shaken cultivation 1h；

4. 100 μ L bacterium solutions is taken to be coated on the LB plate containing Amp, 37 DEG C are incubated overnight；

5. picking white single bacterium is fallen in LB liquid medium of the 5mL containing Amp, plasmid is extracted in 37 DEG C of shaken overnight cultures DNA identification.The extraction of 4.6 plasmids

Plasmid extraction kit is the Pure Plasmid Mini Kit of CWBIO company.Illustrate to extract referring to kit.

4.7 DNA sequencings and sequence bioinformatic analysis

1. DNA sequencing

Invitrogen company is sent to carry out DNA sequencing the plasmid containing target fragment.

2. amino acid sequence analysis a. conserved structure domain analysis: the website NCBI (http: // Www.ncbi.nlm.nih.gov/ Conserved Domain Search Service (CD Search) program)；B. albumen Matter analysis of physical and chemical property: the Protparam tool of the website ExPASy；C. transmembrane domains are analyzed: TMHMM SERVER 2.0；D. Protein secondary structure is analyzed: with the GOR tool of the website ExPASy；E. albumen tertiary structure analyze: the website Phyre2 and Swiss-Model on-line prediction software.

Embodiment 2

The subcellular localization and expression analysis of PgMYB2

1 mediated by agriculture bacillus PgMYB2 is tested in the subcellular localization of onion endepidermis

1.1 PCR amplification

In Invitrogen company synthesis both ends added with the primer of carrier homology arm, with the Taq in Kang Wei century MasterMix amplification:

Primer Sequence (' of 5 ' → 3)

1302-PgMYB2-F AGAACACGGGGGACTCTTGACCAAGAAGAAATTGACGACGATG(SEQ ID NO.5)

1302-PgMYB2-R GTGAAAAGTTCTTCTCCTTTACTATTCCTTTTCCCAACAGTCC(SEQ ID NO.6)

Reaction system:

PCR reaction condition:

The purifying of 1.2 products

1.3 digestion system

The homologous recombination of 1.4 purified products and expression vector

Connection uses ClonExpress^TMII one step cloning kit (Vazyme) carries out homologous recombination.37℃ It is incubated for 30min.

The configuration of 1.5 YEB solid, liquid culture mediums

The preparation of 1.6 Agrobacterium competent cells

1. the agrobacterium strains for taking -80 DEG C of refrigerators to save are crossed in YEB solid plate (50mg/L containing Rif) and are activated, and 28 DEG C Cultivate 2d；

2. picking single bacterium colony is connected in 5mL YEB fluid nutrient medium (50mg/L containing Rif), 28 DEG C, 220rpm, oscillation Culture is for 24 hours；

3. by 5mL bacterium solution switching 200mL YEB fluid nutrient medium (50mg/L containing Rif), 28 DEG C, 220rpm oscillation training 4-7h is supported, to OD₆₀₀Value is gone to rapidly on ice when being 0.4-0.6, ice bath 30min；

4. bacterium solution is gone in sterile centrifugation tube, 4 DEG C, 5,000rpm refrigerated centrifuge 5min, supernatant is abandoned；

5. the 0.1M CaCl of 20mL pre-cooling is added₂Cell is resuspended with liquid-transfering gun pressure-vaccum in solution, ice bath 30min, and 4 DEG C, 5, 000rpm is centrifuged 5min, abandons supernatant；

6. the 0.1M CaCl of 1mL pre-cooling is added₂Cell, ice is resuspended in the glycerol of solution and 150 μ L pre-cooling, liquid-transfering gun pressure-vaccum 10min is bathed, after the packing of 1.5mL centrifuge tube, -80 DEG C of refrigerators is put into rapidly and saves.

1.7 plant expression vectors convert Agrobacterium

It is EHA105 that the pCAMBIA1302-PgMYB2 built, which is converted Agrobacterium tumefaciems, using liquid nitrogen direct translation method.

Specific step is as follows:

1. taking out the 1.5mL centrifuge tube that EHA105 Agrobacterium competent cell is housed from -80 DEG C of refrigerators, thaw on ice, Recombinant plasmid 0.1-1 μ g (5-10 μ L) is added into 50 μ L Agrobacterium competent cells, flicks mixing, ice bath 30min；

2. centrifuge tube is put into liquid nitrogen, quenching processing 5min is transferred to rapidly 37 DEG C, water-bath 5min is put into immediately after taking-up On ice, 5min is placed；

3. the YEB fluid nutrient medium of 1mL is added into centrifuge tube, 28 DEG C, 200rpm, shaken cultivation 4h；

4. 100 μ L bacterium solutions is taken to be coated on YEB plate (containing 100 50 μ g/mL of μ g/mL, Kan of Rif), it is first just being placed in 28 DEG C In incubator, 48h is cultivated, picking single colonie is identified.

1.8 recombinational agrobacteriums infect onion endepidermis cell

A. take -80 DEG C of refrigerators save the agrobacterium strains containing recombinant vector, in YEB solid plate (50mg/L containing Rif, Kan 50mg/L) scribing line activation, 28 DEG C of cultures；

B. picking single bacterium colony is connected in 5mL YEB fluid nutrient medium (50mg/L containing Kan), 28 DEG C, 220rpm, oscillation Overnight incubation；

C. the bacterium solution for taking 1mL to be incubated overnight is connected in 10mL YEB fluid nutrient medium (50mg/L containing Kan), and 28 DEG C, 200rpm cultivates 18~22h (OD₆₀₀About 0.5)；

D. bacterium solution goes to sterile centrifugation tube, and 4,000rpm centrifugation 10min abandon supernatant, 5mL 1/2MS fluid nutrient medium is added Thallus is resuspended in (containing final concentration of 100 μM of AS), liquid-transfering gun piping and druming, and 28 DEG C, 200rpm cultivates 3~4h；

E. following table area 1.0cm is cut with scalpel²The onion tissues block of left and right, in 75% ethanol disinfection l min, 1/ 2MS fluid nutrient medium rinses once, and 2% liquor natrii hypochloritis sterilizes 2min, and 1/2MS fluid nutrient medium rinses 3~4 times, will disappear The onion tissues block that poison is handled well, which is put into 4M NaCl solution, handles 20min, and clear water rinses once, infects rapidly；

F. a few drop polysorbas20s are added into the Agrobacterium bacterium solution after culture 3-4h, onion block is then placed in suspension, is invaded Contaminate 90min；

G. the onion after infecting is taken out, and aseptic filter paper blots surface bacterium solution, isolates lower epidermis, is placed in not resistant On MS solid plate, dark culture 36-48h at 26 DEG C；

H. the onion lower epidermis for co-culturing 36-48h is taken out, sterile water wash twice, makes load, is added dropwise on glass slide The glycerol of several drops 10%, tile onion lower epidermis, covered, and fluorescence microscope is taken pictures.

2 Real-time PCR expression analysis

Reagent uses the Μ Ltra SYBR Mixture (With ROX) of CWBIO, with different groups of 4 years raw fresh ginsengs It knits and is template with the cDNA that the RNA reverse transcription extracted in the root of hair of 100 μM of MeJA processing different time obtains, with β-actin For internal reference, Real-time PCR amplification is carried out with primer in table 1, each sample is repeated 3 times.Confirmation amplification is bent after reaction Line and solubility curve, data result use 2^-△△CtMethod calculates the difference of gene expression dose, is analyzed using SPSS 15.0 soft Part is for statistical analysis.

1 Real-time PCR of table analyzes the primer

Reaction system:

Response procedures:

Embodiment 3

PgMYB2's and DDSpro mutually performs an analysis

1 PCR amplification

In the primer with restriction enzyme site of Beijing Bioisystech Co., Ltd, farsighted Boxing section synthesis, TransStart is used FastPfu DNAPolymerase is expanded.

A, promoter sequence expands:

Reaction system:

PCR response procedures:

B, PgMYB2 gene magnification:

Primer Sequence (' of 5 ' → 3)

pGADT7-PgMYB2-F CATATGGCCATGGAGGCCAGTATGATGGGACGTTCACCTTGC(SEQ ID NO.15)

pGADT7-PgMYB2-R ATCTGCAGCTCGAGCTCGATGTCTATTCCTTTTCCCAACAGTCC(SEQ ID NO.16)

Reaction system:

Response procedures:

The recycling of 2 PCR product glue

3 are cloned target gene in pGADT7 carrier using Gibson method, by DDS gene cloning into pAbAi carrier.Match 50 DEG C of placement 1h after recombination system well:

4 by the recombinant plasmid transformed in 3 into DH5 α.

5. picking white single colonie does bacterium colony PCR identification.

The bacterium colony for the result that is positive in 5 pickings 4 in LB liquid medium of the 5mL added with Amp, 37 DEG C be incubated overnight after mention Take plasmid.

6 will construct successful pAbAi-DDSpro carrier (constructs that is, DDS promoter gene is cloned into pAbAi carrier Carrier) use BbsI linearization for enzyme restriction.

Digestion system is placed in 65 DEG C of 2h after preparing:

Product after digestion in 6 is carried out glue recycling by 7.Recycling step is the same as 2 in example 4.

8 convert the product recycled in 7 into yeast Y1HGold, and steps are as follows for yeast conversion:

The preparation of A competent yeast

1. picking from the plate Y1HGold into suitable YPDA culture medium, 28 DEG C of 180rpm are incubated overnight

2. shaking good yeast liquid to be transferred in 1.5mL EP pipe, 5000g is centrifuged 5min at room temperature.

3. removing supernatant, precipitated with 1mL sterile water washing thalline, 5000g is centrifuged 5min at room temperature.

4. removing supernatant, thallus is resuspended with 100 1 × TE/LiAc of μ L.

B yeast conversion

1. being added to Pignus pignoris grain (single-turn is greater than 200ng, and double turns are greater than 300ng) and 10 μ L Carrier DNA (salmon essence DNA)。

2. 600 μ 1 × PEG4000/LiAc/TE of L are added, turning upside down mixes well and (acutely mixes, can use vortex).

3. 30 DEG C, 200rpm, 30min.

4. 70 μ L DMSO, mixing of turning upside down rapidly is added.

5. 42 DEG C of water-bath 15min.

6. placing 1min (ice bath overlong time can make PEG that presentation white precipitate be precipitated) on ice, brief centrifugation to maximum turns 5s is stopped after speed.

7. removing supernatant, 300 1 × TE of μ L is added to be resuspended.

8. 100 μ L or so is taken to apply SD/-Ura plate (gradient applies plate, applies plate amount and depends on the circumstances), 30 DEG C of inversion trainings It supports 2-4 days.

Single colonie in 9 pickings 8 does bacterium colony PCR identification

1. taking medium sized yeast bacterial plaque half, it is dissolved in 30 μ L 0.2%SDS solution, vibrates 15s；

2. 90 DEG C of metal bath 4min；

3. being centrifuged 1min, maximum (top) speed is sucted and is emptied in clean new pipe, (can be stored in -20 as crude DNA ℃)。

PCR system are as follows:

Response procedures:

4. the agarose gel electrophoresis with 1.5% detects.

10 will identify that the yeast being positive is incubated overnight with 28 DEG C of 180rpm of SD/-Ura culture medium in 8 and 9.

11ABA Resistance detecting

1. taking 1-2 positive colony after identifying positive colony, being resuspended in the NaCl solution of 1mL 0.9%；

2. doing negative control with ghost, measurement space-time culture medium adjusts OD as empty map₆₀₀(about to 0.002 2000cells/100μL)；

3. being coated with 150 μ L bacterium solutions to ready 4 kinds of culture plates, 30 DEG C are cultivated 2-3 days

12 observe in 11 as a result, determining optimal AbA concentration.

13 convert pGADT7-PgMYB2 plasmid in the yeast into 9.Step of converting is same as above, but the plate finally applied is SD/-Ura/-Leu and added with the AbA of concentration 200ng/mL.

14 observation 13 in as a result, simultaneously picking single colonie is crossed again into the culture medium of SD/-Ura/-Leu, culture is tested Card.

Embodiment 4

The combination of EMSA analyzed in vitro PgMYB2 transcription factor protein and the site MBSII

The building of 1 prokaryotic expression plasmid pCold/TF-PgMYB2

A, according to ClonExpress^TMThe design of II one step cloning kit specification has prokaryotic expression carrier The primer of pCold/TF homology arm is synthesized with Invitrogen company, and sequence is as follows:

Primer Sequence (' of 5 ' → 3)

pCold/TF-PgMYB2-F ATGGAGCTCGGTACCCTCGAGATGGGACGTTCACCTTGC(SEQ ID NO.17)

pCold/TF-PgMYB2-R AGACTGCAGGTCGACAAGCTTATGTTTTCCCAACAGATGA(SEQ ID NO.18)

PCR system are as follows:

Response procedures:

PCR product detects by 1% agarose gel electrophoresis and carries out glue recovery purifying, and step is same as above.

B, pCold/TF plasmid double digestion

1 μ g of pCold/TF plasmid

Xhol-Re-Mix 2μL

Hind III-HF 1μL

After enzyme reaction system is placed in 37 DEG C of water-bath 1-2h, enzyme reaction product is recycled, recycling step is same as above.

C, by PCR product and pCold/TF digested plasmid homologous recombination, and by homologous recombination product convert BL21 (DE3), Screening and identification, step is the same as example 1.

2 PgMYB2 transcription factor protein inducing expressions

(1) picking positive bacteria falls within 3mL LB liquid medium, and 37 DEG C, 180rpm, shaken cultivation is overnight；

(2) it is drawn 1mL bacterium solution by 1:100 and is transferred in 100mL liquid LB and expand culture, be placed in 37 DEG C, 150rpm, oscillation It cultivates to OD₆₀₀=0.4-0.6；

(3) the addition IPTG to final concentration of 1mM into bacterium solution, 37 DEG C, 150rpm, successive induction 4h；

(4) bacterium solution is dispensed into two 50mL centrifuge tubes, and 4 DEG C, 4,800rpm centrifugation 15min collect thallus；

(5) supernatant is abandoned, thallus is resuspended in the PBS that 2mL pre-cooling is added, and 4 DEG C, 4,800rpm are centrifuged 15min；

(6) supernatant is abandoned, 2-5mL lysate, resuspended bacterium solution is added in every g weight in wet base；Take the 500 μ L of the full bacterium solution Yu Yixin of 10 μ L from In heart pipe, isometric 2 × SDS sample-loading buffer is added and mixes；

(7) lysozyme (1mg/mL) and nuclease (3U/mL) is added, is incubated for 30min on ice；

(8) Ultrasonic Cell Disruptor is used, sample places progress ultrasound, ultrasound 6 times, each 10s, interval 10s, power 200- on ice 300W；4 DEG C, 10,000 × g is centrifuged 20min；

3 PgMYB2 albumen large-scale purifications

(1) take 1ml Ni-NTA suspension into 15mL centrifuge tube, 4 DEG C, brief centrifugation is carefully sopped up with micropipettor 2mL Lysis buffer is added into resin, is mixed by inversion for supernatant.

(2) 4 DEG C, brief centrifugation carefully sops up supernatant with micropipettor, and the supernatant after ultrasound in 2 is added in resin, 60min is incubated in 4 DEG C of jogs；

(3) said mixture is transferred in purification column, opening bottom cap oozes supernatant and collect efflux, uses It is detected in SDS-PAGE；

(4) the Wash buffer that 2.5mL (5 × bed volume) is slowly added into column bed is washed 2 times, washes away foreign protein, is received Collect efflux to detect for SDS-PAGE；

(5) it is slowly added to 1mL Elution buffer into column bed to wash 4 times, collects efflux and is examined in 4 centrifuge tubes Survey eluting peak.

4BCA method surveys protein concentration

(1) according to A liquid: B liquid=50:1 configures BCA working solution, mixes well；

(2) protein standard substance (2mg/mL) is diluted to 1,0.5,0.25,0.125,0.0625mg/mL step by step；

(3) protein standard substance of every kind of concentration takes 20 μ L to be placed in 96 orifice plates, three multiple holes of each sample, will be to test sample Product dilute 10 times, take 20 μ L to be placed in 96 orifice plates, three multiple holes are arranged；

(4) 200 μ L BCA working solutions are added in each reacting hole, 96 orifice plates are placed in 37 DEG C of incubation 15-30min；

(5) OD is detected with microplate reader₅₇₀Value, and standard curve is drawn according to measured value, calculate protein sample concentration.

5 gel shift retardation experiments (EMSA)

(1) EMSA agents useful for same formula

1. 5 × TBE:

2. 6.5%PAGE glue formula:

(2) EMSA experimental group and control group design are as follows:

2 EMSA experimental group of table and control group design component table (volume unit is μ L)

X represents the reference protein of pCold/TF empty carrier expression, and Y represents PgMYB2:TF fusion protein.In order to avoid non-spy Total system, should be reacted at room temperature 5min before the probe that biotin labeling is added, whole process avoids being vortexed by opposite sex reaction.Add Reaction 20min is stood at room temperature after entering probe.

(3) electrophoresis

1. prerunning: on ice with 0.5 × TBE being pre-chilled, 100V prerunning 30min；

2. using ddH before loading₂O and electrophoretic buffer are washed well 3-5 times respectively；

3. electrophoresis: Loading buffer being added after reaction and mixes rear electrophoresis, as the 2/3- of bromophenol blue electrophoresis to glue Stop electrophoresis (100V, 60min) when 3/4 position, the specific time can adjust with gum concentration and probe size, the probe being not bound with Follow bromophenol blue closely.

(4) transferring film

1. before transferring film, film needs impregnate at least 10min in 0.5 × TBE；

2. a small angle of very clean tweezers, powder-free gloves folder film need to be used when transferring film；

3. transferring film must use clean sponge, used sponge in WB cannot be used；

4. transferring film voltage 100V, 30min.

(5) it is crosslinked

Gel imager UV crosslinking 15min, face down.

(6) film is washed

1. reagent volume below can be appropriate if film used is bigger than normal or less than normal suitable for the film of 10 × 10cm Adjust reagent volume；Film is placed in clean glass dish；

2. confining liquid and 4 × Wash Buffer are placed in 37-50 DEG C of water-bath preheating in advance, until all components are sufficiently molten Solution；16mL confining liquid is added into glass dish, shakes closing 15min slowly (time can extend)；

3. preparing in conjunction with liquid/confining liquid: taking the Stabilizde Streptavidin-Horseradish of 50 μ L Peroxidase Conjugate is added in the confining liquid of 16mL (dilution of 1:300)；

4. outwelling confining liquid in 2., combination/confining liquid in 3. is then added, shaking incubation 15min slowly, (time can extend to 45min)；

5. preparing 1 × Wash solution: adding the ultrapure water of 4 × Wash Buffer to 120mL of 40mL, by film transfer Into new container, 1 × Wash solution that 20mL is added shakes rinsing 5min slowly；

6. repeating the 5. step 3 times (rinsing 4 times in total)；

7. the Substrate Equilibration of 30mL is added by film transfer into another new container Buffer shakes be incubated for 5min slowly；

(7) develop

1. preparing chemiluminescence reaction liquid: 6mL Luminol/Enhancer solution is added to 6mL Stable (daylight or any intense light irradiation can all damage luminous working solution to Peroxide solution, preferably be stored in luminous working solution In brown bottle, and avoid prolonged illumination.Short time, which is exposed under standard laboratory fluorescent lamp, to have working solution It influences)；

2. taking the film out, carefully blot, film is laid in clean Plastic film surface, chemical luminescence for liquid is added to film table Face (makes solution that film surface be completely covered), static incubation 5min；

3. taking the film out, and residual buffer is blotted, but film cannot be allowed dry, with the wet film of preservative film package, avoided Generate bubble and fold.With gel imager, 2-5min is exposed under X-ray.

Embodiment 5

PgMYB2 analyzes the regulating and controlling effect of PgDDS in protoplasts of Arabidopsis thaliana broken by ultrasonic

1PCR amplification

A, DDS promoter sequence expands

DDS promoter overall length is expanded with 5.0 software Design primers of Primer Premier, forward primer has Spe digestion Site, reverse primer have NcoI restriction enzyme site, are synthesized with Invitrogen company, and sequence is as follows:

Primer Sequence (' of 5 ' → 3)

LUC-DDSpro-F GACTAGTTTCTTCCAATACTTGTAG(SEQ ID NO.19)

LUC-DDSpro-R CATGCCATGGCATTCTTAAGTCTACTAC(SEQ ID NO.20)

Reaction system:

PCR reaction condition:

B, PgMYB2 sequence amplification

PgMYB2 overall length is expanded with 5.0 software Design primers of Primer Premier, forward primer has EcoRI digestion Site, reverse primer have BamHI restriction enzyme site, are synthesized with Invitrogen company, and sequence is as follows:

Primer Sequence (' of 5 ' → 3)

pEGAD-PgMYB2-F CCGGAATTCATGGGACGTTCACCTTGC(SEQ ID NO.21)

pEGAD-PgMYB2-R CGCGGATCCACAATATCTGTAAAACCCA(SEQ ID NO.22)

Reaction system:

PCR reaction condition:

The recycling of 2PCR product glue

Used kit is Wizard SV Gel and PCR Clean-up system kit (Promega), with reference to saying Bright book carries out gel extraction.3 vector plasmid digestions

PGreenII 0800-LUC plasmid carries out digestion, pEGAD MYC plasmid EcoRI and BamHI with Spe and NcoI Digestion is carried out, enzyme reaction solution is placed in 37 DEG C of water-bath 2h, and system is as follows:

4 PCR purified products are connect with carrier double enzyme digestion product

The conversion of 5 connection products, screening, upgrading grain

Such as step 4.5-4.7 in embodiment 1.

6 recombinant plasmid corotation protoplasts of Arabidopsis thaliana broken by ultrasonic simultaneously detect Dual-Luciferase activity

Corotation, the operation of all pairs of protoplasts are carried out by following combination, it is necessary to pipette tips are cut into tip and keep smooth, To reduce injury of the generated shearing force to protoplast when piping and druming mixes；Steps are as follows:

(1) plasmid of corrresponding quality is taken to be tested according to the form below grouping:

3 luciferase reporter gene experimental group of table and control group design component table

(2) 0.2g celluLase R-10 (solarbio) and 0.08g macerozyme R-10 (solarbio) are weighed It is added in 20mL enzyme liquid storage and dissolves, in 55 DEG C of water-bath 10min, 200 μ L 1M CaCl are added after cooling₂With 0.02g BSA (0.1%)；

(3) Arabidopsis leaf is won, after selecting 4 weeks, before bolting, health, the good peak green plumpness leaf of growth conditions Piece (each conversion about 10), cuts off leaf leading edge and petiole, blade is cut into 1mm wide slice with blade, is placed in enzymolysis liquid, keeps away Light, 23 DEG C, 40-50rpm digests 2.5-3h；

(4) enzymolysis liquid 100-200 mesh sieve is filtered in 50mL centrifuge tube, 4 DEG C, 70 × g, brake=4, centrifugation 5min；

(5) supernatant is abandoned, the W5 solution for precipitating 4mL ice bath is softly dispelled into washing, 4 DEG C, 70 × g, brake=4, is centrifuged 5min；

(6) supernatant is abandoned, precipitating is softly dispelled with the W5 solution of 4mL ice bath again, on ice avoid light place 30min；

(7) 23 DEG C/room temperature, 70 × g, brake=4, it is centrifuged 5min, abandons supernatant, it is molten by each conversion plus 100 μ L MMG Precipitating is softly resuspended liquid with MMG solution；

(8) it takes the plasmid of corrresponding quality in 1.5mL centrifuge tube by the component in table 3, the original in 100 μ L steps 6 is added Raw plastid is soft to mix；

(9) 110 μ L PEG-Ca solution are added, it is soft rapidly to mix, it prevents protoplast from agglomerating, is placed at room temperature for 20- 30min；

(10) 440 μ L W5 solution are added into centrifuge tube, it is soft to mix, 23 DEG C/room temperature, 70 × g, brake=4, centrifugation 5min；

(11) supernatant to be abandoned, 500 μ L W5 solution are added, soft mix is washed, and 23 DEG C/room temperature, 70 × g, brake=4, from Heart 5min；

(12) supernatant is abandoned, 500 μ L W5 solution are added, soft to mix, immigration cleans up and with the 12 of W5 solution rinse In orifice plate, with W5 solution polishing to 1mL, slightly mix；

(13) 23 DEG C (as in arabidopsis incubator), it is protected from light culture 6-18h；

(14) it usesReporter Assay System (Promega) detects Dual-Luciferase activity.

Embodiment 6

The function analysis of transient expression PgMYB2 in leaves of panax ginseng

The EHA105 bacterium colony for being overexpressed plasmid containing pCAMBIA1302-PgMYB2 contains accordingly in 1mL in 1 picking embodiment 2 It in the LB liquid medium of antibiotic, is placed in 28 DEG C of constant-temperature tables, 250rpm is cultivated for 24 hours, picking plasmid containing pCAMBIA1302 EHA105 as a control group, processing mode is all the same；

2 take the MES (pH=5.7) that 100 μ L concentration are 0.5M and the acetosyringone solution that 2 μ L concentration are 100mM to add Into the LB of 5mL corresponding antibiotic, the bacterium solution cultivated in 50 μ L steps 1 is inoculated, 28 DEG C, 250rpm expands about 16h to OD₆₀₀ =1.0 or so；

3 23 DEG C, 4,000 × g is centrifuged 10min, abandons supernatant, bacterial sediment is collected, with 10mM MgCl₂Thallus is resuspended in solution It is precipitated to OD₆₀₀=1.0, according to the volume of re-suspension liquid, appropriate 100mM acetyl cloves is added in the ratio of every milliliter of 2 μ L of re-suspension liquid Ketone mixes gently, and stands 3h or more at room temperature；

4 take two months or so, the leaves of panax ginseng in growth animated period, draw Agrobacterium with 1mL asepsis injector and suspend Liquid gently supports face of blade with finger, and bacterium solution is entered from the injection of blade reverse side, can be with sterile razor blade in blade for convenience of injecting On mark a trail wound, then bacterium solution is injected along wound, every kind of bacterium solution at least takes three pieces blade to be injected；

Ginseng plant after injection is placed in 25 DEG C of incubators and cultivates 2d by 5, and 16h illumination and 8h dark treatment is given once daily； Each group leaves of panax ginseng is collected, RNA is extracted, utilizes the expression of RT-PCR and Real time-PCR detection related gene, step With embodiment 2.

SEQUENCE LISTING

<110>Central South University

<120>ginseng PgMYB2 transcription factor and its application in regulation ginsenoside synthesis

<130> 11

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 280

<212> PRT

<213>artificial synthesized

<400> 1

Met Gly Arg Ser Pro Cys Cys Glu Lys Ala His Thr Asn Lys Gly Ala

1 5 10 15

Trp Thr Lys Glu Glu Asp Gln Arg Leu Ile Asn Tyr Ile Arg Leu His

20 25 30

Gly Glu Gly Cys Trp Arg Ser Leu Pro Lys Ser Ala Gly Leu Leu Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp

50 55 60

Leu Lys Arg Gly Asn Phe Thr Glu Glu Glu Asp Glu Leu Ile Ile Lys

65 70 75 80

Leu His Ser Leu Leu Gly Asn Lys 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 His Ile

100 105 110

Lys Arg Lys Leu Ile Ser Arg Gly Leu Asp Pro Gln Thr His Arg Pro

115 120 125

Leu Asn Ala Thr Ala Thr Ala Ala Thr Ala Ile Thr Ala Thr Ser Leu

130 135 140

Asp Phe Arg Asn Thr Val Pro Ser Asn Ile Ile Pro Thr Glu Asn Asn

145 150 155 160

Ile Tyr Lys Leu Lys Thr Glu Ser Leu Glu Asp Gly Asn Cys Ser Ser

165 170 175

Ser Thr Thr Glu Glu Thr Gln Gln His Gln Gln Tyr Phe Ala Lys Phe

180 185 190

Gln Asn Ser Gln Val Leu Asp Leu Glu Leu Ser Ile Gly Leu Pro Ser

195 200 205

Ser Arg Thr Gln Thr Asn Asp Ser Ser Leu Ser Val Asn Ser Ile Glu

210 215 220

Ser Asn Val Arg Arg Gln Phe Met Met Val Ala Pro Pro Leu Pro Val

225 230 235 240

Leu Ser Thr Thr Val Ala Pro Arg Met Cys Leu Cys Trp Lys Leu Gly

245 250 255

Phe Gln Lys Gly Gly Gln Gln Gln Gln Leu Cys Ser Asn Cys Lys Ser

260 265 270

Thr Ser Gly Phe Tyr Arg Tyr Cys

275 280

<210> 2

<211> 1401

<212> DNA

<213>artificial synthesized

<400> 2

gaaagagaaa agaaaaatag atgatcgcga gtctattggc tacactccct gtttctccct 60

tatttctcct ccctccactc tcaacgcatt ctctctcctc ctttttacct tccccctctc 120

cacatacttc cttcttatat ctgtctctct ttcttcccac ttctttcaat aaaactctga 180

cctctctaaa aactctcttc tctctctctc cttcctcagc tttctttcag tcttatctct 240

atatcatcaa caatctacaa cagtacacat aaggcccctt atccatttac tctctctata 300

tatctctctt cctttaagct gtatatatat tacagagaaa aattaaggag ctagaagaaa 360

ttgacgacga tgggtcgttc accttgctgc gagaaagctc ataccaacaa aggcgcctgg 420

accaaagaag aagatcaacg cctcatcaac tatatccggc ttcacggcga aggctgctgg 480

cgttccctcc ccaagtccgc cggattattg agatgcggga agagttgcag attacggtgg 540

ataaactacc tccggccaga cctaaagaga gggaatttca cagaagaaga agatgagcta 600

attatcaagc ttcacagttt gctgggaaac aaatggtctt tgatagctgg aagattaccc 660

ggaaggactg ataatgaaat caagaactac tggaacaccc acatcaaacg gaaactcatc 720

agccgtggac tcgacccgca aactcaccgg ccgctaaacg ccactgccac ggctgccaca 780

gctatcaccg ccacgtctct agacttcaga aacactgttc catcaaatat tatacccacc 840

gaaaacaaca tatacaagct caaaacggag tccctggaag atggaaactg cagtagcagc 900

acaactgaag aaacacagca acatcaacaa tatttcgcca aattccaaaa cagtcaagtt 960

ctagacctcg agttatcgat aggactcccg agttcacgga ctcagactaa tgattcctcg 1020

ttatccgtaa actcgatcga gtctaatgtt cggcgccagt tcatgatggt ggctccgccg 1080

ttgccagttc tgtcaacgac ggtggcccca cggatgtgtt tgtgttggaa gttagggttt 1140

cagaaaggag gtcagcagca gcagttgtgt agtaattgca aaagcacaag tgggttttac 1200

agatattgtt gactgttggg aaaatgaata gaactgtact ctagttgtta ggttttagat 1260

atttaatgtt tttattactt actattactc ctaccatcag aatgtgccta cccctaccac 1320

caaatgtaat caatccattt gcacaccaat atttactaga caaattataa atagagtggt 1380

gaaactaatt tatcttgtga c 1401

<210> 3

<211> 19

<212> DNA

<213>artificial synthesized

<400> 3

ccactctcaa cgcattctc 19

<210> 4

<211> 19

<212> DNA

<213>artificial synthesized

<400> 4

taggggtagg cacattctg 19

<210> 5

<211> 43

<212> DNA

<213>artificial synthesized

<400> 5

agaacacggg ggactcttga ccaagaagaa attgacgacg atg 43

<210> 6

<211> 43

<212> DNA

<213>artificial synthesized

<400> 6

gtgaaaagtt cttctccttt actattcctt ttcccaacag tcc 43

<210> 7

<211> 18

<212> DNA

<213>artificial synthesized

<400> 7

cggattattg agatgcgg 18

<210> 8

<211> 23

<212> DNA

<213>artificial synthesized

<400> 8

tgatgtgggt gttccagtag ttc 23

<210> 9

<211> 21

<212> DNA

<213>artificial synthesized

<400> 9

tgagattaga tgaaaacgaa c 21

<210> 10

<211> 21

<212> DNA

<213>artificial synthesized

<400> 10

ggcaatgata aggggaggtg t 21

<210> 11

<211> 18

<212> DNA

<213>artificial synthesized

<400> 11

gcggttgagg tggtgggt 18

<210> 12

<211> 20

<212> DNA

<213>artificial synthesized

<400> 12

gtcctactaa caaggcagag 20

<210> 13

<211> 42

<212> DNA

<213>artificial synthesized

<400> 13

aattcgagct cggtacccgg gcttgtagtt ttgtgatttt cc 42

<210> 14

<211> 42

<212> DNA

<213>artificial synthesized

<400> 14

atacagagca catgcctcga gacttgttgg tatgtggtgt aa 42

<210> 15

<211> 42

<212> DNA

<213>artificial synthesized

<400> 15

catatggcca tggaggccag tatgatggga cgttcacctt gc 42

<210> 16

<211> 44

<212> DNA

<213>artificial synthesized

<400> 16

atctgcagct cgagctcgat gtctattcct tttcccaaca gtcc 44

<210> 17

<211> 39

<212> DNA

<213>artificial synthesized

<400> 17

atggagctcg gtaccctcga gatgggacgt tcaccttgc 39

<210> 18

<211> 40

<212> DNA

<213>artificial synthesized

<400> 18

agactgcagg tcgacaagct tatgttttcc caacagatga 40

<210> 19

<211> 25

<212> DNA

<213>artificial synthesized

<400> 19

gactagtttc ttccaatact tgtag 25

<210> 20

<211> 28

<212> DNA

<213>artificial synthesized

<400> 20

catgccatgg cattcttaag tctactac 28

<210> 21

<211> 27

<212> DNA

<213>artificial synthesized

<400> 21

ccggaattca tgggacgttc accttgc 27

<210> 22

<211> 28

<212> DNA

<213>artificial synthesized

<400> 22

cgcggatcca caatatctgt aaaaccca 28

Claims

1. a kind of ginseng PgMYB2 transcription factor for promoting the expression of dammarendiol synthase gene, which is characterized in that the people Join the sequence of PgMYB2 transcription factor as shown in SEQ ID NO.1.

2. encoding ginseng PgMYB2 transcription factor described in claim 1PgMYB2Gene, which is characterized in that describedPgMYB2Base Because sequence is as shown in SEQ ID NO.2.

3. one kind is for expanding described in claim 2PgMYB2The primer pair of gene, which is characterized in that the primer pair sequence is such as Shown in SEQ ID NO.3 and SEQ ID NO.4.

4. a kind of recombinant vector, which is characterized in that include empty carrier and as claimed in claim 2 in the recombinant vectorPgMYB2 Gene.

5. recombinant vector according to claim 4, which is characterized in that the empty carrier be selected from pGEM-T Easy, pCAMBIA1302、pCold/TF、pGADT7、pEGAD MYC。

6. ginseng according to claim 2PgMYB2Application of the gene in regulation ginsenoside synthesis.

7. application according to claim 6, which is characterized in that the application is that ginseng PgMYB2 transcription factor passes through regulation GinsengPgDDSGene expression regulates and controls the synthesis of ginsenoside.

8. application according to claim 6, it is characterised in that the application is by havingPgMYB2The overexpression of gene Plasmid is transfected into ginseng suspension cell.

9. a kind of Panax ginseng hairy is by being overexpressed in ginseng genomePgMYB2It is obtained after gene order.

10. a kind of ginseng transgenic plant is by being overexpressed in ginseng genomePgMYB2It is obtained after gene order.