CN110066328B - Ginseng PgMYB2 transcription factor and application thereof in regulating and controlling synthesis of ginsenoside - Google Patents

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 application thereof in regulating and controlling synthesis of ginsenoside

Technical Field

The invention belongs to the technical field of biological gene engineering, relates to a regulation and control effect of a transcription factor on a target gene, and particularly relates to an expression effect of a ginseng PgMYB2 transcription factor promoting dammarenediol synthetase (DDS) gene. The invention aims to realize the improvement of saponin synthesis and accumulation level by disclosing the regulation mechanism of key enzyme in the synthetic pathway of ginsenoside, and has important application value.

Background

Ginseng (Panax ginseng c.a. meyer) is a perennial herbaceous plant and has a very high medicinal value. The main effective component of ginseng is ginsenoside, more than 50 ginsenoside monomers are separated from ginseng root at present, and most of the ginsenoside monomers have important medicinal values of resisting tumor, resisting aging, inhibiting apoptosis, enhancing immunity and the like, such as Rb₁And Rg₁Has anti-aging effect, Ra₁、Rg₃And Rh₂Has stronger anticancer activity and the like, and part of ginsenoside is widely applied to clinic. Although many ginsenoside monomers have the activities of resisting tumor and aging, inhibiting apoptosis, enhancing immunity and the like, most of the ginsenoside with strong activity has very low content in ginseng and can not meet the market demand, and the bottleneck is broken by depending on deep research on a regulation mechanism of ginsenoside biosynthesis.

The ginsenoside biosynthesis pathway comprises more than 20 continuous enzymatic reactions. With the research of the synthetic pathway of ginsenoside, more and more key genes of synthetase are cloned. Among the key enzymes are farnesyl diphosphate synthase (FPS), Squalene Synthase (SS), Squalene Epoxidase (SE), dammarenediol-II synthase (DDS), β -amyrin synthase (β -AS), cytochrome P450 (CYP 450), and Glycosyltransferase (GT). The first identified key enzyme in the ginsenoside synthesis pathway is dammarenediol-II synthetase (DDS), which catalyzes the cyclization of 2, 3-oxidosqualene to dammarenediol, which is the most important branch of the ginsenoside synthesis pathway and is a branch of the biosynthesis of triterpenoids and sterols in plants. The branch has the most amount and variety of ginsenoside, and the ginsenoside synthesized by the branch is called dammarane type ginsenoside. Many researchers at home and abroad explore the ginseng PgDDS gene, for example, after the ginseng PgDDS gene is transferred into yeast, the yeast can be detected to generate dammarenediol and hydroxydammarenone, the RNAi technology is utilized to silence the PgDDS gene in the ginseng callus and induce the gene into hairy roots, and the saponin content is found to be reduced to 84.5% [1] of the original saponin; transferring PgDDS into tobacco suspension cells, wherein the transgenic suspension cells can produce dammarendiol, and the content of the dammarendiol in dry weight reaches 573 mug/g <2 > after 3 weeks of culture; the ginseng PgDDS gene is transferred into tobacco, and the transgenic tobacco can generate marendiol and the resistance to tobacco mosaic virus is enhanced [3 ]; the ginseng PgDDS and other key enzyme genes are combined and transferred into saccharomyces cerevisiae to construct a 'ginseng yeast' strain, which can simultaneously generate sapogenin, oleanolic acid, protopanaxadiol (PPD) and protopanaxatriol (PPT), and the contents respectively reach 21.4mg/L, 17.2mg/L and 15.9mg/L [4-6 ]; no DDS exists in rice, dammarane type ginsenoside can not be synthesized, the ginseng PgDDS gene is transferred into the rice, and the transgenic rice can highly express dammarane diol synthetase and generate PPD and PPT [7 ]. These results indicate that DDS is critical for ginsenoside synthesis.

MYB protein is the largest transcription factor family in plants, is widely involved in growth and development of plants and metabolic regulation, such as regulation of secondary metabolism and response of biotic and abiotic stresses, and is becoming a hot spot of plant science research.

Reference to the literature

[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.

The contents of the above references are incorporated by reference into this document.

Disclosure of Invention

The invention aims to provide a MYB gene capable of indirectly regulating and controlling ginsenoside synthesis and application of transcription factor protein coded by the gene in regulating and controlling ginsenoside synthesis.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

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

The PgMYB2 gene sequence for coding the ginseng PgMYB2 transcription factor is shown as SEQ ID NO. 2. The total length of the polypeptide is 1401bp, wherein 370 to 1212 are sequences encoding SEQ ID NO. 1.

The sequences of the primer pair for amplifying the PgMYB2 gene are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The inventor discovers that a promoter of a ginseng PgDDS gene contains MYB binding sites through bioinformatics analysis, and speculates that the MYB transcription factors may participate in the synthesis and transport regulation of ginsenoside based on the phenomenon that the transcription factors can be combined with a specific sequence in a target gene promoter through a DNA binding domain. But whether MYB transcription factor can regulate the expression of dammarenediol synthetase and further indirectly regulate the biosynthesis of ginsenoside? The research on the problems is helpful for completely explaining the regulation and control mechanism of MYB on the synthesis and accumulation of ginsenoside, but no report about the regulation and control of MYB on the biosynthesis of ginsenoside exists in ginseng at present, so that the research on the regulation and control of MYB transcription factors of ginseng on the synthesis and accumulation of ginsenoside is particularly important.

The invention also provides a recombinant vector containing the PgMYB2 gene in claim 2, which specifically comprises: cloning a carrier, namely a recombinant carrier pGEM-T Easy-PgMYB2, namely cloning a PgMYB2 gene into an empty carrier pGEM-T Easy to obtain a recombinant carrier pGEM-T Easy-PgMYB 2; plant transient expression vector named as recombinant vector pCAMBIA1302-PgMYB2, namely PgMYB2 gene is cloned into empty vector pCAMBIA1302 to obtain recombinant vector pCAMBIA1302-PgMYB 2; the yeast single hybrid prey vector is named as pGADT7-PgMYB2, namely PgMYB2 gene is cloned into an empty vector pGADT7 to obtain a recombinant vector pGADT7-PgMYB 2; a prokaryotic expression vector pCold/TF-PgMYB2, namely cloning a PgMYB2 gene into an empty vector pCold/TF to obtain a recombinant vector pCold/TF-PgMYB 2; the reporter gene effect vector is named as pEGAD MYC-PgMYB2, namely PgMYB2 gene is cloned into an empty vector pEGAD MYC to obtain a recombinant vector pEGAD MYC-PgMYB 2.

The PgMYB2 gene is recombined into a known vector and expressed in a suitable system by techniques well known to those skilled in the art. Therefore, in addition to the above-mentioned recombinant vector, there may be another recombinant vector containing the PgMYB2 gene.

The recombinant vector is used for transfecting the ginseng suspension cells, so that the PgMYB2 gene sequence is overexpressed in the ginseng gene. The method can obtain ginseng hairy root or transgenic plant.

The ginseng hairy root or the transgenic plant obtained by the method has high expression of PgDDS.

The skilled person is familiar with how to obtain ginseng suspension cells and is familiar with integrating the PgMYB2 gene into ginseng suspension cells by recombinant vectors to obtain ginseng hairy roots or transgenic plants.

Referring to fig. 1 to 9, the present invention utilizes the existing plant genetic engineering technology, utilizes specific primers, obtains the gene sequence of PgMYB2 by the PCR technology, and introduces the gene into onion epidermal cells by the agrobacterium rhizogenes mediated method, and determines the subcellular localization of PgMYB 2. Meanwhile, the PgMYB2 gene is expressed in each tissue of ginseng, and 100 mu M MeJA can induce the expression of the PgMYB2 gene to be up-regulated. Further EMSA and luciferase reporter gene activity and other experiments show that the PgMYB2 protein can be combined with the MBSII site in the PgDDS promoter region and promote the expression of the PgDDS gene.

Drawings

FIG. 1 shows the result of gel electrophoresis of the product PgMYB2 amplified by PCR, wherein M is a gene Marker;

FIG. 2 shows the conserved domain of the PgMYB2 protein (a), subcellular localization (b), and transmembrane domain prediction (c);

FIG. 3 schematic representation of subcellular localization analysis of PgMYB2 protein in onion: FIGS. a-d show the localization of expression in control, and e-h show the localization of expression of GFP-PgMYB2 fusion protein;

FIG. 4 Real time-PCR detection of PgMYB2 relative expression in different tissues of 4-year fresh ginseng;

FIG. 5 Real time-PCR detection of PgMYB2 and PgDDS gene relative expression after processing ginseng hairy roots with 100. mu.M MeJA for different time;

FIG. 6 results of yeast single-hybrid detection of interaction between PgMYB2 and PgDDS promoter region: the left panel shows the growth of the yeast groups on SD/-Ura/-Leu solid media without AbA, and the right panel shows the growth of the yeast groups on SD/-Ura/-Leu solid media with 200ng/mL AbA;

FIG. 7 EMSA explores the binding effect of the PgMYB2 protein and the MBSII site in the PgDDS promoter region, and the components of each system are shown in Table 2; FIG. 8 relative luciferase activity transiently expressed in Arabidopsis protoplasts by PgMYB2, the panel descriptions are shown in Table 3;

FIG. 9 relative time-PCR detection of PgMYB2 and PgDDS expression levels in ginseng leaf, EV1-3 is the ginseng leaf injected with pCAMBIA1302 no-load plasmid: OV1-3 is the ginseng leaf blade injected with pCAMBIA1302-PgMYB2 overexpression plasmid.

Detailed Description

Example 1

Cloning of the PgMYB2 Gene

1 establishment of Ginseng radix hairy root culture system

1.11/2 MS solid and liquid culture medium preparation:

preparation of mother liquor

a.10 x macroelement mother liquor

Add ddH₂Dissolving O, finally fixing the volume to 1L, sterilizing and storing at 4 ℃;

b.200X microelement mother liquor

Add ddH₂Dissolving O, finally fixing the volume to 500mL, and storing at 4 ℃;

c.100 Xorganic mother liquor

Add ddH₂Dissolving O, finally fixing the volume to 500mL, and storing at 4 ℃;

d.100 Xferric salt mother liquor

Na₂EDTA·2H₂O 3.725g

FeSO₄·7H₂O 2.785g

Add ddH₂Dissolving O, finally fixing the volume to 1L, and storing in a brown bottle at 4 ℃.

② 1/2MS culture medium formula

Preparation of culture medium

Adding various mother solutions according to the dosage in the table, adding weighed inositol and sucrose, and adding a certain amount of ddH₂Dissolving O, adding the weighed agar powder, heating in a microwave oven, and supplementing ddH after the agar is completely dissolved₂And (4) metering the volume of O to 1L, fully stirring and uniformly mixing, and adjusting the pH value to be 6.0 by using a NaOH solution. After the culture medium is prepared, subpackaging into 100mL conical bottles with 30-40mL each, and directly packaging after constant volume without adding agar powder into the liquid culture medium. Tying with rope after sealing with bottle sealing filmAnd (5) tightening.

Sterilization of culture Medium

And putting all the sealed conical flasks into a vertical pressure steam sterilizer for sterilization, wherein the temperature is set to be 121 ℃, and the pressure is 101kPa for 20 min. After sterilization, taking out and shaking up, placing the culture bottle in an electric heating constant temperature incubator for 2-3 days after solidification, and culturing ginseng hairy roots after confirming that the aseptic body is polluted.

1.2 tissue culture and hormone induction of Ginseng radix hairy root

Placing tweezers, culture medium and alcohol cotton into a clean bench, and ultraviolet irradiating for 20 min.

Solid culture

Taking appropriate amount of hairy root with good growth state with tweezers, inoculating into 1/2MS solid culture medium, dispersing as much as possible, sealing the bottle, dark culturing in 24 deg.C incubator, and periodically observing growth state and contamination condition. Subculturing every 30-40 d.

② liquid culture

Taking appropriate amount of hairy root with good growth state with tweezers, inoculating into 1/2MS liquid culture medium, sealing with sealing film, and sealing the bottle. The culture was carried out at 24 ℃ in a shaking incubator at 110rpm in the dark, and the growth state and contamination were observed periodically, usually for 10 days for one subculture.

③ MeJA induction treatment of ginseng hairy root

a. Treatment group: dissolving 46 μ L MeJA in 454 μ L ethanol, and adding 500 μ L ddH₂O, preparing MeJA mother liquor with the final concentration of 200mM, filtering and sterilizing by using a 0.22 mu M filter, and adding 5 mu L of mother liquor into each 10mL of liquid culture medium until the use concentration is 100 mu M; control group: 454 μ L ethanol was dissolved in 546 μ L ddH₂O, filter-sterilized with a 0.22 μm filter, and 10. mu.L of each 10mL of the liquid medium was added.

b. Inoculating Ginseng radix hairy root with good growth vigor in the solid culture medium, culturing at 120rpm and 24 deg.C in dark, taking out one bottle of the treated group and one bottle of the control group at different time points (0, 1, 3, 6, 12, 24, 36, 48, 72h), transferring hairy root into 1.5mL centrifuge tube, and storing in liquid nitrogen for subsequent RNA extraction.

2 extraction of Total RNA from Ginseng radix hairy root

The Tip head, the centrifuge tube, the mortar, the tweezers, the spoon tube frame and the like are required to be soaked in DEPC water with the concentration of 1 per mill for more than 12 hours before use, and can be used after high-temperature sterilization and drying. Total RNA extraction was described with reference to the E.Z.N.A.plant RNA Kit from OMEGA.

3 Synthesis of first Strand of cDNA

RevertAId from Thermo corporation was used^TMFirst Strand cDNA Synthesis Kit, for First Strand cDNA Synthesis. The first strand of the synthesized cDNA can be used directly as a template for PCR.

Cloning of the 4 PgMYB2 Gene

4.1 PCR amplification

The reagent used was Ex Taq enzyme from TaKaRa, the primers were synthesized by Invitrogen, and the sequences of the primers were as follows:

Primer Sequence(5＇→3＇)

PgMYB2-F CCACTCTCAACGCATTCTC(SEQ ID NO.3)

PgMYB2-R TAGGGGTAGGCACATTCTG(SEQ ID NO.4)

reaction system:

and (3) PCR reaction conditions:

4.2 PCR product gel recovery

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the Gel cutting recovery was performed according to the instructions.

4.3 pGEM-T Easy vector ligation

PCR products recovered from the gel were ligated using pGEM-T Easy Vector System (Promega, USA) in a ligation System:

when in connection, the molar ratio of the recovered fragment to pGEM-T Easy Vector is 1:3-1:5, and the connection efficiency is higher. And (3) incubation: 4 ℃ overnight, or room temperature for 1 h.

4.4 preparation of DH5 alpha competent cells

Selecting DH5 alpha strain stored at-80 ℃ to be streaked and activated on an LB solid plate, and culturing at 37 ℃;

picking single colony, inoculating the single colony in 5mL LB liquid culture medium, performing shaking culture at 37 ℃ and 200rpm for 12-16 h;

③ transferring 5mL of bacterial liquid into 500mL of LB liquid culture medium, culturing at 37 ℃ and 200rpm for 1-2h with shaking until OD is reached₆₀₀Rapidly transferring to ice when the value is 0.4-0.6, and ice-cooling for 30 min;

subpackaging 80mL of 500mL of bacterial liquid into a sterile centrifuge tube, centrifuging at 4 ℃ and 4,000rpm for 10min, and discarding the supernatant;

fifthly, adding 20mL of precooled 0.1M CaCl into the centrifuge tube₂The solution is subjected to ice bath for 30min, then centrifuged for 10min at 4 ℃ and 4,000rpm, and the supernatant is discarded;

sixthly, 4mL of precooled 0.1M CaCl is added into the centrifuge tube₂The solution (containing 15% volume of glycerol), the heavy suspension of the bacteria solution, ice bath for 10min, subpackaging into sterile 1.5mL centrifuge tubes, and rapidly placing into a refrigerator at-80 ℃ for storage.

4.5 ligation product transformation and screening

Taking out DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, unfreezing the cells on ice, adding 5 mu L of the ligation product into 100 mu L of the cells, flicking and uniformly mixing the cells, and carrying out ice bath for 30 min;

placing the centrifugal tube in a water bath kettle at 42 ℃ for 60-90s, taking out, immediately placing on ice, and placing for 3 min;

③ adding 500 mu L of LB liquid culture medium into a centrifuge tube, and carrying out shaking culture at 37 ℃ and 200rpm for 1 h;

fourthly, 100 mu L of bacterial liquid is taken and coated on an LB plate containing Amp, and the culture is carried out overnight at 37 ℃;

fifthly, picking white single colony to be cultured in 5mL LB liquid culture medium containing Amp, shaking at 37 ℃ overnight to extract plasmid DNA for identification. 4.6 extraction of plasmid

The Plasmid extraction Kit is Pure Plasmid Mini Kit of CWBIO company. Extraction was described with reference to the kit.

4.7 DNA sequencing and sequence bioinformatics analysis

(ii) DNA sequencing

The plasmid containing the target fragment was sent to Invitrogen corporation for DNA sequencing.

Amino acid sequence analysis a. conserved domain analysis: the served Domain Search Service (CD Search) program of the NCBI website (http:// www.ncbi.nlm.nih.gov /); b. protein physicochemical property analysis: the Protparam tool of the ExPASy website; c. protein transmembrane domain analysis: TMHMM SERVER 2.0.0; d. protein secondary structure analysis: GOR tool with ExPASy website; e. analyzing the tertiary structure of the protein: the Phyre2 website and the Swiss-Model online prediction software.

Example 2

Subcellular localization and expression analysis of PgMYB2

1 Agrobacterium mediated subcellular localization of PgMYB2 in onion inner epidermis

1.1 PCR amplification

Primers with vector homology arms added to both ends were synthesized by Invitrogen and amplified using Taq MasterMix in the conway century:

Primer Sequence(5＇→3＇)

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

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

reaction system:

and (3) PCR reaction conditions:

1.2 purification of the product

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the Gel cutting recovery was performed according to the instructions.

1.3 enzyme digestion System

1.4 homologous recombination of the purified product with an expression vector

The connection adopts Clon express^TMII one step cloning kit (Vazyme) was subjected to homologous recombination. Incubate at 37 ℃ for 30 min.

1.5 preparation of YEB solid and liquid culture Medium

1.6 Agrobacterium competent cell preparation

Taking agrobacterium strains stored in a refrigerator at the temperature of minus 80 ℃ to a YEB solid plate (containing Rif 50mg/L), marking and activating, and culturing for 2d at the temperature of 28 ℃;

selecting a single colony, inoculating the single colony in 5mL YEB liquid culture medium (containing Rif 50mg/L), performing shake culture at 28 ℃ and 220rpm for 24 hours;

thirdly, transferring 5mL of the bacterial liquid into 200mL of YEB liquid culture medium (containing Rif 50mg/L), and carrying out shaking culture at 28 ℃ and 220rpm for 4-7h until OD is reached₆₀₀Rapidly transferring to ice when the value is 0.4-0.6, and ice-cooling for 30 min;

transferring the bacterial liquid into a sterile centrifuge tube, freezing and centrifuging for 5min at 4 ℃ and 5,000rpm, and discarding the supernatant;

fifthly, adding 20mL precooled 0.1M CaCl₂The solution is blown and sucked by a pipette gun to resuspend the cells, ice-bathed for 30min, the temperature is 4 ℃, the rpm is 5,000, the centrifugation is carried out for 5min, and the supernatant is discarded;

sixthly, 1mL of precooled 0 is added.1M CaCl₂The solution and 150 mu L of precooled glycerol are used for blowing and sucking the resuspended cells by a pipette gun, the cells are cooled in ice for 10min, and the cells are quickly placed into a refrigerator at minus 80 ℃ for storage after being subpackaged by a 1.5mL centrifuge tube.

1.7 plant expression vectors for transformation of Agrobacterium

The constructed pCAMBIA1302-PgMYB2 is transformed into Agrobacterium tumefaciens EHA105 by a liquid nitrogen direct transformation method.

The method comprises the following specific steps:

taking out a 1.5mL centrifuge tube filled with EHA105 agrobacterium-infected cells from a refrigerator at the temperature of-80 ℃, unfreezing the centrifuge tube on ice, adding 0.1-1 mu g (5-10 mu L) of recombinant plasmid into 50 mu L of agrobacterium-infected cells, mixing the mixture by flicking, and carrying out ice bath for 30 min;

secondly, putting the centrifugal tube into liquid nitrogen, carrying out shock cooling treatment for 5min, rapidly turning to 37 ℃, carrying out water bath for 5min, immediately putting the centrifugal tube on ice after taking out, and standing for 5 min;

thirdly, adding 1mL of YEB liquid culture medium into the centrifuge tube, performing shaking culture at 28 ℃ and 200rpm for 4 hours;

and fourthly, coating 100 mu L of bacterial liquid on a YEB plate (containing Rif 100 mu g/mL and Kan 50 mu g/mL), putting the YEB plate in an incubator at 28 ℃ for culturing for 48h, and picking out a single colony for identification.

1.8 infection of onion inner epidermal cells by recombinant Agrobacterium

a. Taking the agrobacterium strain which is preserved in a refrigerator at the temperature of minus 80 ℃ and contains the recombinant vector, marking and activating on a YEB solid plate (containing Rif 50mg/L and Kan 50mg/L), and culturing at the temperature of 28 ℃;

b. picking a single colony, inoculating the single colony in 5mL YEB liquid culture medium (containing Kan 50mg/L), culturing at 28 ℃ and 220rpm under shaking overnight;

c. inoculating 1mL of overnight-cultured bacterial liquid into 10mL of YEB liquid medium (containing Kan 50mg/L), culturing at 28 deg.C and 200rpm for 18-22 h (OD)₆₀₀About 0.5);

d. transferring the bacterial liquid to a sterile centrifuge tube, centrifuging for 10min at 4,000rpm, discarding the supernatant, adding 5mL 1/2MS liquid culture medium (containing 100 mu M of AS), blowing and beating the resuspended thalli by a pipette gun, and culturing for 3-4h at 28 ℃ and 200 rpm;

e. cut with a dissector to remove a surface area of 1.0cm²The left and right onion tissue blocks were sterilized with 75% ethanolmin, washing 1/2MS liquid culture medium once, disinfecting with 2% sodium hypochlorite solution for 2min, washing 1/2MS liquid culture medium for 3-4 times, treating the disinfected onion tissue blocks in 4M NaCl solution for 20min, washing with clear water once, and rapidly infecting;

f. adding a few drops of Tween 20 into the Agrobacterium liquid after 3-4h culture, placing onion blocks into the suspension, and infecting for 90 min;

g. taking out the infected onion, sucking dry surface bacteria liquid with sterile filter paper, separating out lower epidermis, placing on MS solid plate without resistance, and culturing in dark at 26 deg.C for 36-48 h;

h. taking out the lower epidermis of the onion which is co-cultured for 36-48h, cleaning twice with sterile water, preparing a mounting piece, dripping a few drops of 10% glycerol on a glass slide, laying the lower epidermis of the onion flat, covering a cover glass, observing by a fluorescence microscope, and taking a picture.

2 Real-time PCR expression analysis

The reagent adopts mu Ltra SYBR mix (With ROX) of CWBIO, takes cDNA obtained by reverse transcription of RNA extracted from different tissues of 4-year fresh ginseng and hairy roots treated by 100 mu M MeJA for different time as a template, takes beta-actin as an internal reference, and takes the primers in the table 1 to carry out Real-time PCR amplification, and each sample is repeated for 3 times. After the reaction, the amplification curve and the dissolution curve were confirmed, and the data obtained was 2^-△△CtThe method calculates the difference of gene expression level, and statistical analysis is carried out by SPSS 15.0 analysis software.

TABLE 1 primers used for Real-time PCR analysis

Reaction system:

reaction procedure:

example 3

Interaction analysis of PgMYB2 with DDSpro

1PCR amplification

The primers with restriction sites synthesized by Biotech Limited of Boxing, Rui, Beijing were amplified with TransStart Fastpfu DNApolymerase.

A. And (3) amplifying a promoter sequence:

reaction system:

PCR reaction procedure:

B. PgMYB2 gene amplification:

Primer Sequence(5＇→3＇)

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

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

reaction system:

reaction procedure:

2PCR product gel recovery

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the Gel cutting recovery was performed according to the instructions.

3 cloning the target gene into pGADT7 vector by Gibson method, and cloning the DDS gene into pAbAi vector. After a recombination system is prepared, placing for 1h at 50 ℃:

4 the recombinant plasmid in 3 was transformed into DH5 alpha.

Taking out DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, unfreezing the cells on ice, adding 5 mu L of the ligation product into 100 mu L of the cells, flicking the cells, uniformly mixing the cells, and carrying out ice bath for 30 min;

placing the centrifugal tube in a water bath kettle at 42 ℃ for 60-90s, taking out, immediately placing on ice, and placing for 3 min;

③ adding 500 mu L of LB liquid culture medium into a centrifuge tube, and carrying out shaking culture at 37 ℃ and 200rpm for 1 h;

fourthly, 100 mu L of bacterial liquid is taken and coated on an LB plate containing Amp, and the culture is carried out overnight at 37 ℃;

fifthly, selecting the white single colony for colony PCR identification.

5 the colonies showing positive results in 4 were picked up and cultured overnight at 37 ℃ in 5mL of liquid LB medium containing Amp, and plasmids were extracted.

6 the successfully constructed pAbAi-DDSpro vector (i.e., the vector constructed by cloning the DDS promoter gene into the pAbAi vector) is linearized by digestion with BbsI.

After the enzyme digestion system is prepared, the mixture is placed at 65 ℃ for 2 h:

7, performing gel recovery on the product obtained after the enzyme digestion in the step 6. The recovery procedure was the same as 2 in example 4.

8 transforming the product recovered in 7 into the yeast Y1HGold, the yeast transformation procedure is as follows:

competent preparation of A Yeast

Selecting Y1HGold from the plate to a proper amount of YPDA medium, and culturing overnight at 28 ℃ and 180rpm

② the shaken yeast liquid is transferred to a 1.5mL EP tube and centrifuged for 5min at 5000g at room temperature.

③ removing the supernatant, washing the bacterial pellet with 1mL of sterile water, and centrifuging for 5min at room temperature at 5000 g.

The supernatant was removed and the cells were resuspended in 100. mu.L of 1 XTE/LiAc.

B Yeast transformation

Adding plasmid to be transferred (one rotation is more than 200ng, two rotations are more than 300ng) and 10 mu L of Carrier DNA (salmon sperm DNA).

② adding 600 μ L of 1 × PEG4000/LiAc/TE, reversing up and mixing well (mixing vigorously, or using vortex).

③30℃，200rpm，30min。

Fourthly, 70 mu L DMSO is added, and the mixture is quickly inverted and evenly mixed.

Fifthly, carrying out water bath at 42 ℃ for 15 min.

Sixthly, the mixture is placed on ice for 1min (the PEG is separated out to form white precipitate after the ice bath time is too long), and the mixture is instantly centrifuged to the maximum rotating speed and then stays for 5 s.

Seventhly, removing supernatant, and adding 300 mu L of 1 × TE for resuspension.

Taking 100 mu L of the mixture, coating SD/-Ura plates (gradient coating plates, the amount of the coated plates is determined according to the situation), and carrying out inverted culture at the temperature of 30 ℃ for 2-4 days.

9 picking out single colony in 8 for colony PCR identification

Dissolving half of yeast bacterial plaque with medium size in 30 mu L of 0.2% SDS solution, and oscillating for 15 s;

② metal bath for 4min at 90 ℃;

thirdly, centrifuging for 1min, rotating at the maximum speed, sucking the supernatant, pouring the supernatant into a new clean tube, and using the tube as the crop DNA (which can be stored at-20 ℃).

The PCR system is as follows:

reaction procedure:

fourthly, detecting by using 1.5 percent agarose gel electrophoresis.

10 Yeast identified as positive in 8 and 9 were grown overnight in SD/-Ura medium at 28 ℃ and 180 rpm.

11ABA resistance assay

After positive clones are identified, taking 1-2 positive clones, and suspending in 1mL of 0.9% NaCl solution;

② taking the empty cell as negative control, measuring the space-time culture medium as empty control, adjusting OD₆₀₀To 0.002 (about 2000cells/100 μ L);

③ applying 150. mu.L of bacteria solution to 4 prepared culture plates, and culturing at 30 ℃ for 2-3 days

12 observe the results in 11 and determine the optimal concentration of AbA.

13 pGADT7-PgMYB2 plasmid was transformed into yeast 9. The transformation procedure was as above, but the final coated plate was SD/-Ura/-Leu and was loaded with AbA at a concentration of 200 ng/mL.

14 the results in 13 were observed and single colonies were picked and restreaked to SD/-Ura/-Leu medium for culture validation.

Example 4

EMSA in vitro analysis of binding effect of PgMYB2 transcription factor protein and MBSII site

1 prokaryotic expression plasmid pCold/TF-PgMYB2 construction

A. According to Clonexpress^TMII one step cloning kit Specification primers with the homology arm of prokaryotic expression vector pCold/TF were designed and synthesized by Invitrogen, and the sequences were as follows:

Primer Sequence(5＇→3＇)

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

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

the PCR system is as follows:

reaction procedure:

the PCR product was detected by 1% agarose gel electrophoresis and gel recovery and purification were performed as described above.

B. pCold/TF plasmid double enzyme digestion

pCold/TF plasmid 1. mu.g

Xhol-Re-Mix 2μL

Hind III-HF 1μL

And (3) placing the enzyme reaction system in water bath at 37 ℃ for 1-2h, and then recovering the enzyme reaction product, wherein the recovery steps are the same as the above.

C. The PCR product and pCold/TF enzyme cutting plasmid are subjected to homologous recombination, and the homologous recombination product is transformed into BL21(DE3) and screened and identified, and the steps are the same as example 1.

2 PgMYB2 transcription factor protein induction expression

(1) Selecting positive colonies in 3mL liquid LB culture medium, shaking and culturing at 37 deg.C and 180rpm overnight;

(2) sucking 1mL of bacterial liquid according to a ratio of 1:100, transferring the bacterial liquid into 100mL of liquid LB for amplification culture, placing the liquid at 37 ℃ and 150rpm, and performing shaking culture until the liquid is OD₆₀₀＝0.4-0.6；

(3) Adding IPTG into the bacterial liquid until the final concentration is 1mM, 37 ℃, and continuously inducing for 4h at 150 rpm;

(4) subpackaging the bacterial liquid into two 50mL centrifuge tubes, centrifuging at 4 ℃, 4,800rpm for 15min and collecting thalli;

(5) discarding the supernatant, adding 2mL of precooled PBS to resuspend the thalli, and centrifuging at 4 ℃, 4,800rpm for 15 min;

(6) discarding the supernatant, adding 2-5mL of lysate per g of wet weight, and re-suspending the bacterial liquid; putting 10 μ L of whole bacteria liquid into a new 500 μ L centrifuge tube, adding equal volume of 2 xSDS loading buffer liquid, and mixing;

(7) adding lysozyme (1mg/mL) and nuclease (3U/mL), and incubating on ice for 30 min;

(8) placing the sample on ice by using an ultrasonic crusher for ultrasonic treatment for 6 times, wherein the ultrasonic treatment is carried out for 10s each time and the interval is 10s, and the power is 200-; centrifuging at 10,000 Xg for 20min at 4 deg.C;

large-scale purification of 3 PgMYB2 protein

(1) 1mL of the Ni-NTA suspension was put into a 15mL centrifuge tube, centrifuged instantaneously at 4 ℃, the supernatant was carefully aspirated off with a micropipette, 2mL lysine buffer was added to the resin, and the mixture was mixed by inversion.

(2) Centrifuging at 4 deg.C, sucking off the supernatant carefully with a micropipette, adding the supernatant obtained by ultrasonic treatment in step 2 into resin, and incubating at 4 deg.C for 60min with gentle shaking;

(3) transferring the mixture to a purification column, opening the bottom cap to allow the supernatant to flow out slowly, and collecting the effluent for SDS-PAGE detection;

(4) slowly adding 2.5mL (5 times of the volume of the column bed) of Wash buffer into the column bed for washing for 2 times, washing away impure proteins, and collecting effluent for SDS-PAGE detection;

(5) 1mL of Elution buffer was slowly added to the column bed for 4 washes, and the effluent was collected and the Elution peak detected in 4 centrifuge tubes.

4BCA method for measuring protein concentration

(1) According to the formula of solution A: solution B is 50: 1, preparing a BCA working solution, and fully and uniformly mixing;

(2) gradually diluting the protein standard substance (2mg/mL) to 1, 0.5, 0.25, 0.125 and 0.0625 mg/mL;

(3) respectively taking 20 mu L of protein standard substance with each concentration, placing the protein standard substance in a 96-well plate, placing each sample in three multiple wells, diluting the sample to be detected by 10 times, placing the 20 mu L of protein standard substance in the 96-well plate, and setting the three multiple wells;

(4) adding 200 mu L of BCA working solution into each reaction well, and incubating a 96-well plate at 37 ℃ for 15-30 min;

(5) detection of OD Using microplate reader₅₇₀And drawing a standard curve according to the measured value, and calculating the concentration of the protein sample.

5 gel migration retardation assay (EMSA)

(1) Reagent formula for EMSA

①5×TBE：

(6.5%) PAGE gel formula:

(2) the EMSA experimental group and the control group are designed as the following table:

TABLE 2 EMSA experimental and control groups component tables (volume units are. mu.L)

X represents the control protein expressed by pCold/TF empty vector, and Y represents PgMYB2: TF fusion protein. To avoid non-specific reactions, the total system should be reacted at room temperature for 5min before adding the biotin-labeled probe, avoiding vortexing throughout. After the probe is added, the mixture is kept stand and reacted for 20min at room temperature.

(3) Electrophoresis

Pre-electrophoresis: pre-electrophoresed with pre-chilled 0.5 × TBE on ice at 100V for 30 min;

② use ddH before loading₂Washing the sample adding holes for 3-5 times respectively with an electrophoresis buffer solution;

③ electrophoresis: and adding a Loading buffer after the reaction is finished, uniformly mixing, performing electrophoresis, stopping electrophoresis (100V, 60min) when the bromophenol blue electrophoresis reaches the 2/3-3/4 position of the gel, wherein the specific time can be adjusted according to the gel concentration and the size of the probe, and the unbound probe immediately follows the bromophenol blue.

(4) Rotary film

Firstly, before membrane conversion, the membrane needs to be soaked in 0.5 times TBE for at least 10 min;

secondly, when the film is rotated, a very clean forceps and a powder-free glove are needed to clamp a small corner of the film;

thirdly, the transfer film must use clean sponge, and can not use the sponge used in WB;

fourthly, the membrane voltage is changed to 100V for 30 min.

(5) Cross-linking

The gel imager was UV cross-linked for 15min with the front facing down.

(6) Washing membrane

The reagent volume used below is suitable for a membrane of 10X 10cm, and if the membrane used is too large or too small, the reagent volume can be appropriately adjusted; placing the membrane in a clean glass dish;

placing the confining liquid and 4 multiplied by Wash Buffer in water bath at 37-50 ℃ for preheating in advance until all components are fully dissolved; adding 16mL of sealing solution into a glass dish, and sealing for 15min by slow shaking (the time can be prolonged);

preparing binding liquid/sealing liquid: 50 μ L of Stabilizde Streptavidin-Horseradish Peroxidase Conjugate was added to 16mL of blocking solution (1:300 dilution);

pouring off the middle sealing liquid, adding the binding/sealing liquid in the third step, and incubating for 15min (the time can be prolonged to 45min) with slow shaking;

preparing 1 × Wash solution: adding 40mL of 4 xWash Buffer to 120mL of ultrapure water, transferring the membrane into a new container, adding 20mL of 1 xWash solution, and rinsing for 5min with slow shaking;

sixthly, repeating the fifth step for 3 times (rinsing for 4 times in total);

seventhly, transferring the membrane to another new container, adding 30mL of Substrate emulsification Buffer, and incubating for 5min with slow shaking;

(7) development

Preparing a chemiluminescent reaction solution: adding 6mL of lumineol/Enhancer solution to 6mL of Stable Peroxide solution (sunlight or any strong light can damage the luminous working solution, preferably the luminous working solution is stored in a brown bottle, and long-time light is avoided; short-time exposure under a standard laboratory fluorescent lamp can not affect the working solution);

taking out the membrane, carefully sucking the membrane, flatly paving the membrane on the surface of a clean plastic film, adding chemiluminescent liquid to the surface of the membrane (so that the solution completely covers the surface of the membrane), and standing and incubating for 5 min;

taking out the film, sucking the residual buffer, but not drying the film, and wrapping the wet film with a preservative film to avoid generating bubbles and wrinkles. Exposing with gel imager under X-ray for 2-5 min.

Example 5

Analysis of PgMYB2 regulation and control effects on PgDDS in Arabidopsis protoplasts

1PCR amplification

A. Amplification of DDS promoter sequence

Primers were designed using Primer Premier 5.0 software to amplify the entire length of the DDS promoter, the forward Primer carrying a Spe cleavage site and the reverse Primer carrying a NcoI cleavage site, and synthesized by Invitrogen, having the following sequences:

Primer Sequence(5＇→3＇)

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

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

reaction system:

and (3) PCR reaction conditions:

B. PgMYB2 sequence amplification

The PgMYB2 full length was amplified using Primer Premier 5.0 software design primers, forward with an EcoRI cleavage site and reverse with a BamHI cleavage site, synthesized by Invitrogen, with the following sequences:

Primer Sequence(5＇→3＇)

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

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

reaction system:

and (3) PCR reaction conditions:

2PCR product gel recovery

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the Gel cutting recovery was performed according to the instructions. 3 vector plasmid cleavage

pGreenII 0800-LUC plasmid was digested with Spe and NcoI, pEGAD MYC plasmid was digested with EcoRI and BamHI, and the enzyme reaction mixture was placed in a 37 ℃ water bath for 2h as follows:

4 connecting the PCR purified product with the vector double enzyme digestion product

5 connected product conversion, screening and quality improvement particle

As in example 1, steps 4.5-4.7.

6 recombinant plasmid cotransformation of Arabidopsis protoplast and detection of dual-luciferase activity

Carrying out co-rotation according to the following combination, wherein tip ends of gun heads are required to be removed and kept smooth for all operations on the protoplast, so that the damage of shearing force generated during blowing and uniformly mixing to the protoplast is reduced; the method comprises the following steps:

(1) the plasmids with corresponding mass were taken out for experiments according to the following groups:

TABLE 3 Dual-luciferase reporter gene experimental group and control group design group component table

(2) 0.2g of celluLase R-10(solarbio) and 0.08g of mecerozyme R-10(solarbio) are weighed and added into 20mL of enzyme stock solution to be dissolved, water bath is carried out at 55 ℃ for 10min, 200 mu L of 1M CaCl is added after cooling₂And 0.02g BSA (0.1%);

(3) picking leaves of Arabidopsis thaliana, selecting tender green and thick leaves (each converted about 10 leaves) which are healthy and have good growth state after 4 weeks and before bolting, cutting off the front edge and the petiole of the leaves, cutting the leaves into thin strips with the width of 1mm by a blade, placing the thin strips in enzymolysis liquid, keeping out of the sun, and carrying out enzymolysis at 23 ℃ and 40-50rpm for 2.5-3 hours;

(4) filtering the enzymolysis solution with 100-sand 200-mesh sieve in a 50mL centrifuge tube, centrifuging for 5min at 4 ℃ under 70 Xg and brake ═ 4;

(5) discarding the supernatant, gently blowing and washing the precipitate with 4mL of iced W5 solution, centrifuging for 5min at 4 ℃ at 70 Xg and brake ═ 4;

(6) discarding the supernatant, gently blowing off the precipitate with 4mL of iced W5 solution, and standing on ice away from light for 30 min;

(7) centrifugation at 23 deg.C/room temperature for 5min with a shake of 4, discarding the supernatant, adding 100. mu.L of MMG solution for each transformation, and gently resuspending the pellet with MMG solution;

(8) taking plasmids with corresponding mass according to the components in the table 3, putting the plasmids into a 1.5mL centrifuge tube, adding 100 mu L of the protoplast obtained in the step 6, and gently and uniformly mixing;

(9) adding 110 μ L PEG-Ca solution, mixing quickly and gently to prevent protoplast agglomeration, standing at room temperature for 20-30 min;

(10) adding 440 mu L W5 solution into a centrifuge tube, gently mixing uniformly, centrifuging for 5min at 23 ℃/room temperature with 70 Xg brake ═ 4;

(11) discarding the supernatant, adding 500 mu L W5 solution, mixing and washing gently, at 23 ℃/room temperature, 70 Xg, brake 4, centrifuging for 5 min;

(12) discarding the supernatant, adding 500 μ L W5 solution, mixing gently, transferring into 12-well plate cleaned and rinsed with W5 solution, replenishing to 1mL with W5 solution, and mixing slightly;

(13)23 ℃ (in an arabidopsis culture box), and culturing for 6-18h in a dark place;

(14) by using

The Reporter Assay System (Promega) detects dual luciferase activity.

Example 6

Effect analysis of transient expression PgMYB2 in ginseng leaf

1 selecting EHA105 colony containing pCAMBIA1302-PgMYB2 overexpression plasmid in example 2 to be placed in 1mL of liquid LB culture medium containing corresponding antibiotics, placing the liquid LB culture medium in a constant temperature shaking table at 28 ℃, culturing for 24h at 250rpm, selecting EHA105 containing pCAMBIA1302 plasmid as a control group, and treating in the same way;

2 mu.L of MES (pH 5.7) with a concentration of 0.5M and 2. mu.L of acetosyringone solution with a concentration of 100mM were added to 5mL of LB corresponding to the antibiotic, and 50. mu.L of the bacterial solution cultured in step 1 was inoculated, expanded at 28 ℃ and 250rpm for about 16 hours to OD₆₀₀About 1.0;

centrifuging at 323 deg.C for 10min at 4,000 Xg, discarding supernatant, collecting thallus precipitate, and adding 10mM MgCl₂Resuspending the bacterial pellet to OD₆₀₀Adding a proper amount of 100mM acetosyringone according to the volume of the resuspension and the proportion of 2 mu L per ml resuspension, gently mixing, and standing for more than 3h at room temperature;

4, taking ginseng leaves which are about two months and in a vigorous growth period, sucking the agrobacterium suspension by using a 1mL sterile injector, lightly pressing the front sides of the leaves by using fingers, injecting the bacterial liquid from the back sides of the leaves, marking a small wound on the leaves by using a sterile blade for convenient injection, then injecting the bacterial liquid along the wound, and taking at least three leaves of each bacterial liquid for injection;

5 placing the injected ginseng plant into an incubator at 25 ℃ for 2d, and giving 16h of illumination and 8h of dark treatment every day; collecting each group of ginseng leaves, extracting RNA, and detecting the expression level of related genes by using RT-PCR and Real time-PCR, wherein the steps are the same as the example 2.

SEQUENCE LISTING

<110> university of south-middle school

<120> ginseng PgMYB2 transcription factor and application thereof in regulation and control of ginsenoside synthesis

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<160> 22

<170> PatentIn version 3.5

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Claims (2)

1. The application of the ginseng PgMYB2 gene in promoting expression of the ginseng dammarenediol synthetase gene is characterized in that the sequence of the PgMYB2 gene is shown as SEQ ID No. 2.

2. The use according to claim 1, wherein the use is by transfection of an overexpression plasmid with the PgMYB2 gene into ginseng suspension cells.

Images