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 its application in regulating ginsenoside synthesis

technical field

The invention belongs to the technical field of biological genetic engineering, and relates to the regulation effect of transcription factors on target genes, in particular to the effect of ginseng PgMYB2 transcription factors to promote the expression of damalediol synthase (DDS) gene. The present invention aims to improve the synthesis and accumulation level of saponins by revealing the regulation mechanism of key enzymes in the synthesis pathway of ginsenosides, and has important application value.

Background technique

Ginseng (Panax ginseng CAMeyer) is a perennial herb with high medicinal value. The main medicinal components in ginseng are ginsenosides. At present, more than 50 kinds of ginsenoside monomers have been isolated from ginseng roots. Most ginsenoside monomers have important functions such as anti-tumor, anti-aging, inhibiting cell apoptosis and enhancing immunity. For example, Rb ₁ and Rg ₁ have anti-aging effects, and Ra ₁ , Rg ₃ and Rh ₂ have strong anti-cancer activities, etc. Some ginsenosides have been widely used in clinical practice. Although many ginsenoside monomers have anti-tumor, anti-aging, anti-apoptotic and immune-enhancing activities, most of the active ginsenosides have extremely low content in ginseng, which is far from meeting the market demand. The bottleneck relies on the in-depth study of the regulatory mechanisms of ginsenoside biosynthesis.

The ginsenoside biosynthesis pathway includes more than 20 consecutive enzymatic reactions. With the in-depth study of ginsenoside synthesis pathway, more and more key genes of synthase have been cloned. Among them, the key enzymes are farnesyl diophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarediol-II Synthetase (darmmarenediol-II synthase, DDS), β-amyrin synthase (β-amyrin synthase, β-AS), cytochrome P450 (cytochromeP450, CYP450) and glycosyltransferase (glycosyltransferase, GT) and so on. The first key enzyme identified in the ginsenoside synthesis pathway is dammarediol-II synthase (DDS), which catalyzes the cyclization of 2,3-oxysqualene to dammarediol, which is the synthesis of ginsenosides. The most important branch of the pathway is the branch of the biosynthesis of triterpenoids and sterols in plants. The number and types of ginsenosides generated by this branch are the largest, and the content is the highest in ginseng. The ginsenosides synthesized by this branch are called dammarane-type ginsenosides. Many researchers at home and abroad have explored the ginseng PgDDS gene. For example, after the ginseng PgDDS gene was transferred into yeast, it was detected that the yeast produced damarenediol and hydroxydamarenone, and the RNAi technology was used to silence the PgDDS gene in the ginseng callus and induce it. In hair roots, it was found that the saponin content was reduced to 84.5% of the original [1]; PgDDS was transferred into tobacco suspension cells, and the transgenic suspension cells were able to produce dammarediol and the dry weight of dammarediol content reached 573 μg/g after 3 weeks of culture. g[2]; The ginseng PgDDS gene was transferred into tobacco, and it was found that the transgenic tobacco could produce malediol and had enhanced resistance to tobacco mosaic virus[3]; The ginseng PgDDS and other key enzyme genes were combined into Saccharomyces cerevisiae , constructed a "ginseng yeast" strain, which can simultaneously produce sapogenin, oleanolic acid, protopanaxadiol (PPD) and protopanaxatriol (PPT), with the contents reaching 21.4mg/L, 17.2mg/L and 15.9mg/L, respectively. mg/L[4-6]; there is no DDS in rice, so dammarane-type ginsenosides cannot be synthesized. The ginseng PgDDS gene is transferred into rice, and the transgenic rice can express dammarediol synthase and produce PPD and PPT[7] ]. These results indicate that DDS is essential for the synthesis of ginsenosides.

MYB proteins are the largest family of transcription factors in plants. They are widely involved in plant growth, development and metabolic regulation, such as the regulation of secondary metabolism and the response to biotic and abiotic stresses. They are increasingly becoming the focus of plant research.

references

[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-IItriterpene in a cell suspension culture of transgenic tobacco[J].Plant CellRep,2014,33(2):225-233.

[3] Lee M H, Han J Y, Kim H J, et al. Dammarenediol-II production confersTMV 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 ginsenosidesRh2and Rg3by metabolically engineered yeasts[J].Metab Eng,2015,29:97-105.

[5]Dai Z,Wang B,Liu Y,et al.Producing aglycons of ginsenosides inbakers'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-typesapogenins in rice by expressing the dammarenediol-II synthase gene fromPanax ginseng C.A.Mey[J].Plant Sci,2015,239:106-114.

The contents of the above references are incorporated into the present application as background art.

SUMMARY OF THE INVENTION

The present invention aims to provide a MYB gene capable of indirectly regulating the synthesis of ginsenosides, and the application of the transcription factor protein encoded by the gene in regulating the synthesis of ginsenosides.

In order to achieve the above object, the technical scheme provided by the invention is:

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

The PgMYB2 gene sequence encoding the ginseng PgMYB2 transcription factor is shown in SEQ ID NO.2. Its full length is 1401bp, and the 370th to 1212th are the sequences encoding SEQ ID NO.1.

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

Through bioinformatics analysis, the inventors found that the ginseng PgDDS gene promoter contains a MYB binding site. Based on the phenomenon that the transcription factor can bind to a specific sequence in the target gene promoter through the DNA binding domain, it is speculated that the MYB transcription factor may be involved in ginseng Regulation of the synthesis and transport of saponins. But can the MYB transcription factor regulate the expression of dammarediol synthase and thus indirectly regulate the biosynthesis of ginsenosides? Exploring this issue will help to fully explain the regulatory mechanism of MYB on ginsenoside synthesis and accumulation. However, there is no report on MYB's regulation of ginsenoside biosynthesis in ginseng. control is particularly important.

The present invention also provides a recombinant vector containing the PgMYB2 gene of claim 2, specifically including: a cloning vector, named as a recombinant vector pGEM-T Easy-PgMYB2, that is, cloning the PgMYB2 gene into an empty vector pGEM-T Easy to obtain Recombinant vector pGEM-T Easy-PgMYB2; plant transient expression vector, named as recombinant vector pCAMBIA1302-PgMYB2, clone PgMYB2 gene into empty vector pCAMBIA1302 to obtain recombinant vector pCAMBIA1302-PgMYB2; yeast one-hybrid prey vector, named pGADT7-PgMYB2 , that is, clone the PgMYB2 gene into the empty vector pGADT7 to obtain the recombinant vector pGADT7-PgMYB2; the prokaryotic expression vector, pCold/TF-PgMYB2, clone the PgMYB2 gene into the empty vector pCold/TF to obtain the recombinant vector pCold/TF-PgMYB2; report The gene effect vector, named pEGAD MYC-PgMYB2, cloned the PgMYB2 gene into the empty vector pEGAD MYC to obtain the recombinant vector pEGAD MYC-PgMYB2.

Techniques well known to those skilled in the art for recombination of the PgMYB2 gene into known vectors and expression in suitable systems. Therefore, in addition to the above-mentioned recombinant vectors, there may be other recombinant vectors containing the PgMYB2 gene.

The ginseng suspension cells were transfected with the recombinant vector to overexpress the PgMYB2 gene sequence in the ginseng gene. Ginseng hairy roots or transgenic plants can be obtained by this method.

The ginseng hairy roots or transgenic plants obtained by this method have high expression of PgDDS.

Those skilled in the art are familiar with how to obtain ginseng suspension cells, and are familiar with integrating the PgMYB2 gene into ginseng suspension cells through recombinant vectors, thereby obtaining ginseng hairy roots or transgenic plants.

Referring to Fig. 1 to Fig. 9, the present invention utilizes the existing plant genetic engineering technology, utilizes specific primers, obtains the PgMYB2 gene sequence through PCR technology, and introduces the gene into onion inner epidermal cells through the Agrobacterium rhizogenes-mediated method, and determines Subcellular localization of PgMYB2. At the same time, it was found that PgMYB2 gene was expressed in all tissues of ginseng, and 100μM MeJA could induce the up-regulation of PgMYB2 gene expression. Further experiments such as EMSA and luciferase reporter gene activity showed that PgMYB2 protein could bind to the MBSII site in the promoter region of PgDDS and promote the expression of PgDDS gene.

Description of drawings

Fig. 1 Gel electrophoresis results of PCR-amplified PgMYB2 products, M is gene Marker;

Figure 2 shows the conserved domain (a), subcellular localization (b) and predicted transmembrane domain (c) of PgMYB2 protein;

Figure 3. Subcellular localization analysis of PgMYB2 protein in onion: Figures a-d are the expression and localization of the control group, and e-h are the expression and localization of the GFP-PgMYB2 fusion protein;

Figure 4 Real time-PCR detection of the relative expression of PgMYB2 in different tissues of 4-year-old fresh ginseng;

Figure 5 Real time-PCR detection of the relative expression of PgMYB2 and PgDDS genes after 100μM MeJA treatment of ginseng hairy roots for different times;

Figure 6. The results of yeast one-hybrid detection of the interaction between PgMYB2 and PgDDS promoter region: the left picture shows the growth of each group of yeast on SD/-Ura/-Leu solid medium without AbA, and the right picture shows the growth of yeast in each group containing 200ng/mL Growth of each group of yeast on SD/-Ura/-Leu solid medium of AbA;

Figure 7 EMSA to explore the binding effect of PgMYB2 protein and MBSII site in the PgDDS promoter region, the components of each system are shown in Table 2; Figure 8 The relative luciferase activity of PgMYB2 transiently expressed in Arabidopsis protoplasts, the description of each group is as follows as shown in Table 3;

Figure 9 Real time-PCR detection of the relative expression levels of PgMYB2 and PgDDS in ginseng leaves. EV1-3 are ginseng leaves injected with pCAMBIA1302 empty plasmid; OV1-3 are ginseng leaves injected with pCAMBIA1302-PgMYB2 overexpression plasmid.

Detailed ways

Example 1

Cloning of PgMYB2 gene

1 Establishment of ginseng hair root culture system

1.1 Configuration of 1/2MS solid and liquid medium:

①Preparation of mother liquor

a.10×major element mother liquor

Add ddH ₂ O to dissolve, make the final volume to 1L, and store at 4°C after sterilization;

b.200×trace element mother liquor

Add ddH ₂ O to dissolve, make up the final volume to 500mL, and store at 4°C;

c.100×organic mother liquor

Add ddH ₂ O to dissolve, make up the final volume to 500mL, and store at 4°C;

d.100×iron salt mother liquor

Na ₂ EDTA·2H ₂ O 3.725g

FeSO ₄ ·7H ₂ O 2.785g

Add ddH ₂ O to dissolve, make up the final volume to 1L, and store in a brown bottle at 4°C.

②1/2MS medium formula

③ Preparation of culture medium

According to the dosage in the table, first add various mother solutions, then add the weighed inositol and sucrose, dissolve with a certain amount of ddH ₂ O, then add the weighed agar powder, heat it in a microwave oven, and make up ddH after the agar is completely dissolved. Dilute to 1 L with ₂ O, stir and mix well, and adjust the pH to pH 6.0 with NaOH solution. After the medium is prepared, it is divided into 100mL conical flasks, each bottle is 30-40mL, and the liquid medium does not need to add agar powder. After the bottle is sealed, fasten it with a string.

④ Sterilization of culture medium

Put all the sealed conical flasks into a vertical pressure steam sterilizer for sterilization, set to 121°C, 101kPa, 20min. After sterilization, take out and shake well. After solidification, place the culture bottle in an electric heating constant temperature incubator for 2-3 days. After confirming the aseptic body contamination, it can be used for the cultivation of ginseng hairy roots.

1.2 Ginseng root tissue culture and hormone induction treatment

Put tweezers, culture medium and alcohol cotton into the ultra-clean workbench, and irradiate with UV light for 20min.

①Solid culture

Use tweezers to take an appropriate amount of hair roots with good growth conditions, connect them to 1/2 MS solid medium, disperse them as much as possible, then seal the bottle mouth, and culture in the dark in a 24°C incubator. Regularly observe the growth state and bacterial infection. Subculture every 30-40d.

②Liquid culture

Use tweezers to take an appropriate amount of hair roots with good growth conditions, connect them to 1/2 MS liquid medium, and seal the bottle mouth with parafilm. Incubate in dark at 24°C, 110rpm shaking incubator, observe the growth state and bacterial infection regularly, usually subculture once every 10d.

③MeJA induced treatment of ginseng hairy roots

a. Treatment group: Dissolve 46 μL of MeJA in 454 μL of ethanol, add 500 μL of ddH ₂ O to prepare a MeJA stock solution with a final concentration of 200 mM, filter and sterilize with a 0.22 μm filter, and add 5 μL of the stock solution per 10 mL of liquid medium to a concentration of 100 μM; control group: dissolve 454 μL of ethanol in 546 μL of ddH ₂ O, filter and sterilize with a 0.22 μm filter, and add 10 μL per 10 mL of liquid medium.

b. Inoculate the ginseng hairy roots with good growth in the solid medium in the above liquid medium, 120rpm, 24°C dark culture, at different time points (0, 1, 3, 6, 12, 24, 36, 48, 72h) One bottle of the treatment group and the control group was taken out, and the hair roots were transferred into a 1.5 mL centrifuge tube and stored in liquid nitrogen for subsequent RNA extraction.

2 Extraction of total RNA from hairy roots of ginseng

Tip tip, centrifuge tube, mortar, tweezers, spoon tube holder, etc. should be soaked in DEPC water with a concentration of 1‰ for more than 12 hours before use, and can be used after high temperature sterilization and drying. Refer to OMEGA's E.Z.N.A. Plant RNA Kit instructions to extract total RNA.

3. Synthesis of the first strand of cDNA

The first strand of cDNA was synthesized using the RevertAid ^™ First Strand cDNA Synthesis Kit from Thermo Company. The first strand of the synthesized cDNA can be used directly as a template for PCR.

4 Cloning of PgMYB2 gene

4.1 PCR amplification

The reagent used is the Ex Taq enzyme of TaKaRa company, and the primer is synthesized by Invitrogen company, and the primer sequence is as follows:

Primer Sequence (5'→3')

PgMYB2-F CCACTCTCAACGCATTCTC (SEQ ID NO. 3)

PgMYB2-R TAGGGGTAGGCACATTCTG (SEQ ID NO. 4)

reaction system:

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 was cut and recovered according to the instructions.

4.3 pGEM-T Easy vector ligation

The PCR products recovered from the gel were connected using pGEM-T Easy Vector System (Promega, USA). The connection system:

When ligating, it should be noted that the molar ratio of the recovered fragment to the pGEM-T Easy Vector is 1:3-1:5, which has a higher ligation efficiency. Incubation: overnight at 4°C, or 1h at room temperature.

4.4 Preparation of DH5α competent cells

①Pick the DH5α strain stored at -80°C, streak and activate it on LB solid plate, and cultivate at 37°C;

②Pick a single colony, connect it to 5mL LB liquid medium, 37℃, 200rpm, shake culture for 12-16h;

③ Transfer 5mL of bacterial liquid to 500mL of LB liquid medium, 37°C, 200rpm, shake culture for 1-2h, quickly transfer to ice when the OD ₆₀₀ value is 0.4-0.6, and ice bath for 30min;

④ Transfer 500 mL of bacterial liquid into 80 mL portions into sterile centrifuge tubes, centrifuge at 4,000 rpm for 10 min at 4°C, and discard the supernatant;

⑤ Add 20 mL of pre-cooled 0.1 M CaCl ₂ solution to the centrifuge tube, resuspend the bacterial solution, ice bath for 30 min, centrifuge at 4°C, 4,000 rpm for 10 min, and discard the supernatant;

⑥ Add 4mL of pre-cooled 0.1M CaCl ₂ solution (containing 15% volume of glycerol) to the centrifuge tube, resuspend the bacterial solution, take an ice bath for 10min, dispense into sterile 1.5mL centrifuge tubes, and quickly put - Store in a refrigerator at 80°C.

4.5 Conversion and screening of ligation products

① Take out the DH5α competent cells from the -80°C refrigerator, thaw on ice, add 5 μL of the ligation product to 100 μL, mix by flicking, and take an ice bath for 30 minutes;

②Place the centrifuge tube in a 42°C water bath for 60-90s, and immediately place it on ice for 3min;

③ Add 500 μL of LB liquid medium to the centrifuge tube, and incubate with shaking at 200 rpm for 1 h at 37 °C;

④ Take 100 μL of bacterial solution and spread it on the LB plate containing Amp, and cultivate at 37°C overnight;

⑤Pick white single colonies in 5 mL of LB liquid medium containing Amp, and shake overnight at 37°C to extract plasmid DNA for identification. 4.6 Extraction of plasmids

The plasmid extraction kit was Pure Plasmid Mini Kit from CWBIO Company. Extract according to the kit instructions.

4.7 DNA sequencing and sequence bioinformatics analysis

①DNA sequencing

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

②Amino acid sequence analysis a. Conserved domain analysis: Conserved Domain Search Service (CD Search) program of NCBI website (http://www.ncbi.nlm.nih.gov/); b. Protein physicochemical property analysis: ExPASy website Protparam tool; c. Protein transmembrane domain analysis: TMHMM SERVER2.0; d. Protein secondary structure analysis: GOR tool using ExPASy website; e. Protein tertiary structure analysis: Phyre2 website and Swiss-Model online prediction software.

Example 2

Subcellular localization and expression analysis of PgMYB2

1. Subcellular localization experiment of Agrobacterium-mediated PgMYB2 in the inner epidermis of onion

1.1 PCR amplification

Synthesize primers with vector homology arms at both ends in Invitrogen, and use TaqMasterMix of Converse to amplify:

Primer Sequence (5'→3')

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

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

reaction system:

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 was recovered by referring to the instructions.

1.3 Enzyme digestion system

1.4 Homologous recombination of purified product and expression vector

Ligation was performed by homologous recombination using the ClonExpress ^™ II one step cloning kit (Vazyme). Incubate at 37°C for 30min.

1.5 Configuration of YEB solid and liquid medium

1.6 Preparation of Agrobacterium competent cells

① Take the Agrobacterium strains stored in the refrigerator at -80°C, streak and activate them on YEB solid plate (containing Rif 50mg/L), and cultivate at 28°C for 2 days;

②Pick a single colony, connect it to 5mL YEB liquid medium (containing Rif 50mg/L), 28°C, 220rpm, shake culture for 24h;

③ Transfer 5mL of bacterial liquid to 200mL of YEB liquid medium (containing Rif 50mg/L), 28°C, 220rpm for shaking culture for 4-7h, when the OD ₆₀₀ value is 0.4-0.6, quickly transfer to ice, and ice bath for 30min;

④ Transfer the bacterial liquid to a sterile centrifuge tube, freeze and centrifuge at 5,000 rpm at 4°C for 5 min, and discard the supernatant;

⑤Add 20mL of pre-cooled 0.1M CaCl ₂ solution, resuspend the cells by pipetting, ice bath for 30min, 4℃, 5,000rpm, centrifuge for 5min, discard the supernatant;

⑥ Add 1 mL of pre-cooled 0.1M CaCl ₂ solution and 150 μL of pre-cooled glycerol, resuspend the cells by pipetting, ice bath for 10 min, divide into 1.5 mL centrifuge tubes, and quickly store them in a -80°C refrigerator.

1.7 Transformation of Agrobacterium with plant expression vector

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

Specific steps are as follows:

①Take out a 1.5mL centrifuge tube containing EHA105 Agrobacterium competent cells from the -80°C refrigerator, thaw on ice, add 0.1-1μg (5-10μL) of recombinant plasmid to 50μL Agrobacterium competent cells, and mix by flicking , ice bath for 30min;

②Put the centrifuge tube into liquid nitrogen, quench it for 5 minutes, quickly transfer it to 37°C, take a water bath for 5 minutes, put it on ice immediately after taking it out, and leave it for 5 minutes;

③ Add 1 mL of YEB liquid medium to the centrifuge tube, 28 ° C, 200 rpm, shaking culture for 4 hours;

④ Take 100 μL of bacterial solution and apply it to YEB plate (containing Rif 100 μg/mL, Kan 50 μg/mL), first place it in a 28°C incubator, cultivate for 48 hours, pick a single colony for identification.

1.8 Recombinant Agrobacterium infects onion inner epidermal cells

a. Take the Agrobacterium strain containing the recombinant vector stored in the refrigerator at -80°C, streak and activate it on YEB solid plate (containing Rif 50mg/L, Kan 50mg/L), and cultivate at 28°C;

b. Pick a single colony, connect it to 5mL YEB liquid medium (containing Kan 50mg/L), 28°C, 220rpm, shake and culture overnight;

c. Take 1 mL of overnight cultured bacterial solution and connect it to 10 mL of YEB liquid medium (containing Kan 50 mg/L), and culture at 28°C and 200 rpm for 18-22 hours (OD ₆₀₀ is about 0.5);

d. Transfer the bacterial liquid to a sterile centrifuge tube, centrifuge at 4,000 rpm for 10 min, discard the supernatant, add 5 mL of 1/2 MS liquid medium (containing AS with a final concentration of 100 μM), and resuspend the bacterial cells by pipetting at 28°C. Culture at 200rpm for 3-4h;

e. Use a scalpel to cut off the onion tissue pieces with a surface area of about 1.0 cm ² , sterilize in 75% ethanol for 1 min, rinse once with 1/2 MS liquid medium, sterilize with 2% sodium hypochlorite solution for 2 min, and rinse with 1/2 MS liquid medium for 3~ 4 times, put the sterilized onion tissue pieces into 4M NaCl solution for 20min, rinse with clean water once, and quickly infect;

f. Add a few drops of Tween 20 to the Agrobacterium liquid after culturing for 3-4h, then put the onion pieces into the suspension, infect for 90min;

g. Take out the infected onion, dry the surface bacterial liquid with sterile filter paper, separate the lower epidermis, place it on the MS solid plate without resistance, and cultivate it in the dark at 26°C for 36-48h;

h. Take out the lower epidermis of the onion that has been co-cultured for 36-48 hours, wash it twice with sterile water, make a slide, add a few drops of 10% glycerol on the glass slide, flatten the lower epidermis of the onion, cover with a cover glass, fluorescence Microscope observation and photographing.

2 Real-time PCR expression analysis

The reagents were MLtra SYBR Mixture (With ROX) from CWBIO, and the cDNAs obtained by reverse transcription of RNA extracted from different tissues of 4-year-old fresh ginseng and hair roots treated with 100 μM MeJA at different times were used as templates, and β-actin was used as an internal reference. The primers in Table 1 were amplified by Real-time PCR, and each sample was repeated 3 times. After the reaction, the amplification curve and the dissolution curve were confirmed, and the difference in gene expression levels was calculated by the 2- ^△△Ct method, and the SPSS 15.0 analysis software was used for statistical analysis.

Table 1 Primers used in Real-time PCR analysis

reaction system:

Reaction program:

Example 3

Interaction analysis of PgMYB2 and DDSpro

1 PCR amplification

The primers with restriction sites synthesized in Beijing Ruibo Xingke Biotechnology Co., Ltd. were amplified with TransStartFastPfu DNAPolymerase.

A. Promoter sequence amplification:

reaction system:

PCR reaction program:

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 program:

2 PCR product gel recovery

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the gel was cut and recovered according to the instructions.

3. The target gene was cloned into pGADT7 vector by Gibson method, and the DDS gene was cloned into pAbAi vector. After the recombination system is prepared, place it at 50°C for 1h:

4 Transform the recombinant plasmid from 3 into DH5α.

① Take out the DH5α competent cells from the -80°C refrigerator, thaw on ice, add 5 μL of the ligation product to 100 μL, mix by flicking, and take an ice bath for 30 minutes;

②Place the centrifuge tube in a 42°C water bath for 60-90s, and immediately place it on ice for 3min;

③ Add 500 μL of LB liquid medium to the centrifuge tube, and incubate with shaking at 200 rpm for 1 h at 37 °C;

④ Take 100 μL of bacterial solution and spread it on the LB plate containing Amp, and cultivate at 37°C overnight;

⑤Pick white single colonies for colony PCR identification.

5. Pick the positive colonies in 4 and put them in 5 mL of liquid LB medium supplemented with Amp, and culture them at 37°C overnight to extract plasmids.

6. The successfully constructed pAbAi-DDSpro vector (ie, the vector constructed by cloning the DDS promoter gene into the pAbAi vector) was cut and linearized with BbsI.

After the enzyme digestion system is prepared, place it at 65°C for 2h:

7. Gel recovery of the digested product in 6. The recovery step is the same as 2 in Example 4.

8. The product recovered in 7 is transformed into yeast Y1HGold, and the yeast transformation steps are as follows:

A yeast competent preparation

①Pick Y1HGold from the plate into an appropriate amount of YPDA medium, and incubate overnight at 28°C and 180rpm

② Transfer the shaken yeast solution to a 1.5mL EP tube and centrifuge at 5000g for 5min at room temperature.

③ Remove the supernatant, wash the cell pellet with 1 mL of sterile water, and centrifuge at 5000 g for 5 min at room temperature.

④ Remove the supernatant and resuspend the cells with 100 μL of 1×TE/LiAc.

B yeast transformation

①Add the plasmid to be transfected (more than 200ng for single-transformation, more than 300ng for double-transformation) and 10 μL Carrier DNA (salmon sperm DNA).

②Add 600 μL of 1×PEG4000/LiAc/TE, invert upside down and mix thoroughly (mix vigorously, vortex is available).

③30℃, 200rpm, 30min.

④Add 70μL DMSO and mix by inverting quickly.

⑤ 42 ℃ water bath for 15 minutes.

⑥Place on ice for 1min (the ice bath time is too long, PEG will precipitate and appear as white precipitate), centrifuge instantaneously to the maximum speed and then stay for 5s.

⑦ Remove the supernatant and add 300 μL of 1×TE to resuspend.

⑧ Take about 100 μL of SD/-Ura plate (gradient coating, the amount of coating depends on the situation), and invert at 30°C for 2-4 days.

9 Pick a single colony in 8 for colony PCR identification

① Take half of the medium-sized yeast plaque, dissolve it in 30μL of 0.2% SDS solution, and shake for 15s;

②90℃ metal bath for 4min;

③ Centrifuge for 1 min at maximum speed, aspirate the supernatant and pour it into a clean new tube as crude DNA (can be stored at -20°C).

The PCR system is:

Reaction program:

④ Detected by 1.5% agarose gel electrophoresis.

10. Yeasts identified in 8 and 9 were cultured overnight in SD/-Ura medium at 28°C and 180rpm.

11ABA resistance test

① After identifying positive clones, take 1-2 positive clones and resuspend them in 1 mL of 0.9% NaCl solution;

②Use empty cells as a negative control, measure the space-time medium as an empty control, and adjust the OD ₆₀₀ to 0.002 (about 2000cells/100μL);

③ Spread 150 μL of bacterial solution onto the prepared 4 kinds of culture plates, and culture at 30°C for 2-3 days

12 Observe the results in 11 to determine the optimal AbA concentration.

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

14 Observe the results in 13, and pick a single colony and re-streaked it into the medium of SD/-Ura/-Leu for verification by culture.

Example 4

In vitro analysis of the binding effect of PgMYB2 transcription factor protein and MBSII site by EMSA

1 Construction of prokaryotic expression plasmid pCold/TF-PgMYB2

A. Design primers with homology arms of prokaryotic expression vector pCold/TF according to the instructions of ClonExpress ^TM II one step cloning kit, and synthesize them with Invitrogen company, the sequence is 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:

Reaction program:

PCR products were detected by 1% agarose gel electrophoresis, and the gel was recovered and purified, and the steps were the same as above.

B. Double digestion of pCold/TF plasmid

pCold/TF plasmid 1μg

Xhol-Re-Mix 2μL

Hind III-HF 1 μL

After placing the enzyme reaction system in a 37° C. water bath for 1-2 hours, the enzyme reaction product was recovered, and the recovery steps were the same as above.

C. Homologously recombine the PCR product with the pCold/TF digested plasmid, transform the homologous recombination product into BL21 (DE3), screen and identify, the steps are the same as in Example 1.

2 Induced expression of PgMYB2 transcription factor protein

(1) Pick positive colonies in 3mL liquid LB medium, 37°C, 180rpm, shake and culture overnight;

(2) Transfer 1 mL of bacterial liquid to 100 mL of liquid LB at 1:100 to expand the culture, place at 37°C, 150 rpm, and shake to culture to OD ₆₀₀ =0.4-0.6;

(3) Add IPTG to the bacterial solution to a final concentration of 1 mM, 37°C, 150 rpm, and continue to induce for 4 hours;

(4) The bacterial liquid was divided into two 50mL centrifuge tubes, and the bacteria were collected by centrifugation at 4,800rpm for 15min at 4°C;

(5) Discard the supernatant, add 2 mL of pre-cooled PBS to resuspend the cells, and centrifuge at 4,800 rpm for 15 min at 4°C;

(6) Discard the supernatant, add 2-5 mL of lysate per gram of wet weight, and resuspend the bacterial solution; take 10 μL of the whole bacterial solution into a new 500 μL centrifuge tube, add an equal volume of 2×SDS loading buffer and mix well;

(7) Add lysozyme (1mg/mL) and nuclease (3U/mL) and incubate on ice for 30min;

(8) Using a sonicator, place the sample on ice for sonication, sonication 6 times, 10s each time, 10s intermittently, power 200-300W; 4°C, 10,000×g centrifugation for 20min;

3 Mass purification of PgMYB2 protein

(1) Take 1ml of Ni-NTA suspension into a 15mL centrifuge tube, centrifuge briefly at 4°C, carefully remove the supernatant with a micropipette, add 2mL of Lysis buffer to the resin, and mix by inversion.

(2) Centrifuge briefly at 4°C, carefully remove the supernatant with a micropipette, add the supernatant after sonication in 2 to the resin, and incubate at 4°C for 60 min with gentle shaking;

(3) Transfer the above mixture to the purification column, open the bottom cap to let the supernatant slowly flow out and collect the effluent for SDS-PAGE detection;

(4) Slowly add 2.5mL (5×column bed volume) of Wash buffer to the column bed to wash twice to remove impurities, and collect the effluent for SDS-PAGE detection;

(5) Slowly add 1 mL of Elution buffer to the column bed to wash 4 times, collect the effluent and detect the elution peaks in 4 centrifuge tubes.

4BCA method to measure protein concentration

(1) Prepare BCA working solution according to A solution: B solution = 50:1, and mix well;

(2) Dilute the protein standard (2mg/mL) stepwise to 1, 0.5, 0.25, 0.125, 0.0625mg/mL;

(3) Take 20 μL of protein standards of each concentration and place them in a 96-well plate, with three replicate wells for each sample, dilute the sample to be tested 10 times, take 20 μL and place it in a 96-well plate, and set up three replicate wells ;

(4) Add 200μL of BCA working solution to each reaction well, and incubate the 96-well plate at 37°C for 15-30min;

(5) Detect the OD ₅₇₀ value with a microplate reader, draw a standard curve according to the measured value, and calculate the protein sample concentration.

5 Gel Migration Arrest Assay (EMSA)

(1) Reagent formula used in EMSA

①5×TBE:

②6.5% PAGE gel formula:

(2) The design of the EMSA experimental group and the control group is as follows:

Table 2 EMSA experimental group and control group design component table (volume unit is μL)

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

(3) Electrophoresis

①Pre-electrophoresis: pre-electrophoresis with pre-cooled 0.5×TBE on ice, 100V for 30min;

② Wash the sample wells 3-5 times with ddH ₂ O and electrophoresis buffer before loading;

③Electrophoresis: After the reaction is completed, add Loading buffer and mix well, and then electrophoresis. When the bromophenol blue electrophoresis reaches the 2/3-3/4 position of the gel, stop the electrophoresis (100V, 60min). The specific time can be adjusted according to the gel concentration and probe size. , the unbound probe followed bromophenol blue.

(4) Transfer film

①Before transferring, the membrane needs to be soaked in 0.5×TBE for at least 10min;

②When transferring the membrane, a small corner of the membrane should be clamped with very clean tweezers and powder-free gloves;

③The transfer film must use a clean sponge, not the sponge used in WB;

④Transfer membrane voltage 100V, 30min.

(5) Cross-linking

Gel imager UV cross-linking for 15min, face down.

(6) Wash the membrane

①The volume of the reagents used below is suitable for a 10×10cm membrane. If the membrane used is too large or too small, the volume of the reagent can be adjusted appropriately; put the membrane in a clean glass dish;

②Put the blocking solution and 4×Wash Buffer in a 37-50°C water bath to preheat in advance until all components are fully dissolved; add 16 mL of blocking solution to the glass dish, and slowly shake to seal for 15 minutes (the time can be extended);

③ Prepare binding solution/blocking solution: Take 50 μL of Stabilizde Streptavidin-Horseradish Peroxidase Conjugate and add it to 16 mL of blocking solution (1:300 dilution);

④ Pour off the blocking solution in ②, add the binding/blocking solution in ③, and incubate with slow shaking for 15 min (the time can be extended to 45 min);

⑤Prepare 1×Wash solution: add 40mL of 4×Wash Buffer to 120mL of ultrapure water, transfer the membrane to a new container, add 20mL of 1×Wash solution, shake and rinse slowly for 5min;

⑥ Repeat step ⑤ 3 times (a total of 4 rinses);

⑦ Transfer the membrane to another new container, add 30 mL of Substrate Equilibration Buffer, and incubate with slow shaking for 5 minutes;

(7) Development

①Preparation of chemiluminescence reaction solution: Add 6mL Luminol/Enhancer solution to 6mL StablePeroxide solution (sunlight or any strong light will damage the luminescence working solution, it is best to keep the luminescence working solution in a brown bottle, and avoid long-term light. Short Time exposure will not affect the working fluid under standard laboratory fluorescent lamps);

②Take out the membrane, carefully blot it dry, spread the membrane flat on the surface of a clean plastic film, add the chemiluminescence solution to the membrane surface (so that the solution completely covers the membrane surface), and incubate for 5 minutes;

③Take out the film and absorb the residual buffer, but do not let the film dry, wrap the wet film with plastic wrap to avoid bubbles and wrinkles. Using a gel imager, exposure to X-rays for 2-5min.

Example 5

Analysis of the regulatory effect of PgMYB2 on PgDDS in Arabidopsis protoplasts

1PCR amplification

A. DDS promoter sequence amplification

Use Primer Premier 5.0 software to design primers to amplify the full length of DDS promoter, forward primer with Spe restriction site, reverse primer with NcoI restriction site, synthesized by Invitrogen company, the sequence is as follows:

Primer Sequence (5'→3')

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

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

reaction system:

PCR reaction conditions:

B. PgMYB2 sequence amplification

Use Primer Premier 5.0 software to design primers to amplify the full length of PgMYB2, forward primer with EcoRI restriction site, reverse primer with BamHI restriction site, synthesized by Invitrogen company, the sequence is as follows:

Primer Sequence (5'→3')

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

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

reaction system:

PCR reaction conditions:

2 PCR product gel recovery

The kit used was Wizard SV Gel and PCR Clean-up system kit (Promega), and the gel was cut and recovered according to the instructions. 3 vector plasmid digestion

The pGreenII 0800-LUC plasmid was digested with Spe and NcoI, and the pEGAD MYC plasmid was digested with EcoRI and BamHI. The enzyme reaction solution was placed in a 37°C water bath for 2 hours. The system is as follows:

4 The PCR purified product is ligated with the double-enzyme digestion product of the vector

5Transformation, screening and plasmid extraction of ligation products

Steps 4.5-4.7 as in Example 1 were implemented.

6 Recombinant plasmids were co-transfected into Arabidopsis protoplasts and detected dual-luciferase activity

Carry out co-rotation according to the following combination. All operations on protoplasts must reduce the tip of the pipette tip and keep it smooth, so as to reduce the damage to the protoplast caused by the shear force generated during pipetting and mixing; the steps are as follows:

(1) Take the plasmid of the corresponding quality in the following table groupings to carry out the experiment:

Table 3 Design components of dual-luciferase reporter gene experimental group and control group

(2) Weigh 0.2g celluLase R-10 (solarbio) and 0.08g macerozyme R-10 (solarbio) into 20mL enzyme stock solution to dissolve, in 55℃ water bath for 10min, add 200μL 1M CaCl ₂ and 0.02g BSA after cooling (0.1%);

(3) Picking the leaves of Arabidopsis thaliana, selecting healthy, tender green hypertrophic leaves (about 10 pieces per transformation) after 4 weeks and before bolting, cutting off the leading edge and petiole of the leaves, and cutting the leaves with a blade Cut into 1mm wide strips, put them in the enzymolysis solution, protect from light, 23°C, 40-50rpm for 2.5-3h enzymolysis;

(4) Filter the enzymatic hydrolysate with a 100-200 mesh sieve into a 50mL centrifuge tube, centrifuge for 5min at 4°C, 70×g, brake=4;

(5) Discard the supernatant, wash the precipitate gently with 4 mL of W5 solution in ice bath, centrifuge for 5 min at 4°C, 70×g, brake=4;

(6) Discard the supernatant, gently blow the precipitate with 4 mL of W5 solution in ice bath again, and place on ice for 30 min in the dark;

(7) 23°C/room temperature, 70×g, brake=4, centrifuge for 5 min, discard the supernatant, add 100 μL of MMG solution for each transformation, and gently resuspend the pellet with MMG solution;

(8) Take the corresponding mass of plasmid according to the components in Table 3 into a 1.5 mL centrifuge tube, add 100 μL of the protoplasts in step 6, and mix gently;

(9) Add 110 μL of PEG-Ca solution, mix quickly and gently to prevent protoplasts from clumping, and leave at room temperature for 20-30min;

(10) Add 440 μL of W5 solution to the centrifuge tube, mix gently, 23°C/room temperature, 70×g, brake=4, centrifuge for 5 min;

(11) Discard the supernatant, add 500 μL of W5 solution, mix and wash gently, 23°C/room temperature, 70×g, brake=4, centrifuge for 5 min;

(12) Discard the supernatant, add 500 μL of W5 solution, mix gently, transfer to a 12-well plate that has been cleaned and rinsed with W5 solution, make up to 1 mL with W5 solution, and mix slightly;

(13) 23°C (for Arabidopsis incubator), culture in dark for 6-18h;

Reporter Assay System(Promega)检测双荧光素酶活性。(14) with

Reporter Assay System (Promega) detects dual luciferase activity.

Example 6

Analysis of the effect of transient expression of PgMYB2 in ginseng leaves

1 Pick the EHA105 colony containing the pCAMBIA1302-PgMYB2 overexpression plasmid in Example 2, put it in 1 mL of liquid LB medium containing the corresponding antibiotics, place it in a constant temperature shaker at 28 °C, and cultivate it at 250 rpm for 24 h. Pick the EHA105 containing the pCAMBIA1302 plasmid as The control group was treated in the same way;

2. Add 100 μL of 0.5M MES (pH=5.7) and 2 μL of 100 mM acetosyringone solution to 5 mL of LB with the corresponding antibiotic, and then inoculate 50 μL of the bacterial solution cultured in step 1, at 28°C and 250rpm to expand about 16h to about OD ₆₀₀ =1.0;

3 Centrifuge at 4,000 × g for 10 min at 23°C, discard the supernatant, collect the bacterial pellet, and resuspend the bacterial pellet with 10 mM MgCl ₂ solution to OD ₆₀₀ = 1.0. According to the volume of the resuspension solution, press 2 μL per milliliter of resuspension solution. Proportionally add an appropriate amount of 100mM acetosyringone, mix gently, and let stand at room temperature for more than 3 hours;

4 Take ginseng leaves that are about two months old and are in the vigorous growth stage, suck the Agrobacterium suspension with a 1mL sterile syringe, gently touch the front of the leaves with your fingers, and inject the bacterial liquid from the back of the leaves. For convenience of injection, a sterile blade can be used. A small wound is drawn on the leaf, and the bacterial liquid is injected along the wound. At least three leaves of each bacterial liquid are injected;

5. The injected ginseng plants were placed in a 25°C incubator for 2 days, and 16 hours of light and 8 hours of dark treatment were given every day; ginseng leaves were collected from each group, RNA was extracted, and the expression levels of related genes were detected by RT-PCR and Real time-PCR. , the steps are the same as in Example 2.

SEQUENCE LISTING

<110> Central South University

<120> Ginseng PgMYB2 transcription factor and its application in regulating ginsenoside synthesis

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Claims (2)

1. The application of ginseng PgMYB2 gene in promoting the expression of ginseng damalediol synthase gene, characterized in that the sequence of the PgMYB2 gene is shown in SEQ ID NO.2. 2 . The application according to claim 1 , wherein the application is by transfecting an overexpression plasmid having the PgMYB2 gene into ginseng suspension cells. 3 .

Images