CN111217896A

CN111217896A - Application of ginseng PgWRKY4X transcription factor in regulating and controlling ginsenoside compound content in ginseng

Info

Publication number: CN111217896A
Application number: CN202010129213.XA
Authority: CN
Inventors: 高文远; 王娟; 姚陆; 贺军平; 刘静静; 许馨丹
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-02-28
Filing date: 2020-02-28
Publication date: 2020-06-02
Anticipated expiration: 2040-02-28
Also published as: CN111217896B

Abstract

The invention discloses application of a ginseng PgWRKY4X transcription factor in regulating and controlling the content of ginsenoside compounds in ginseng; the amino acid sequence of the ginseng PgWRKY4X transcription factor is shown in SEQ ID No. 1. Experiments prove that the transcription factor PgWRKY4X of the ginseng can be combined with the promoter of the PgSE gene of the ginseng, so that the expression of key enzyme PgSE on a ginsenoside synthesis path is improved, and the content of ginsenoside compounds in the ginseng is further improved. The ginseng PgWRKY4X transcription factor, the coding gene thereof and the overexpression recombinant vector containing the coding gene can be used for regulating and improving the content of ginsenoside compounds in ginseng.

Description

Application of ginseng PgWRKY4X transcription factor in regulating and controlling ginsenoside compound content in ginseng

Technical Field

The invention belongs to the field of biochemistry, relates to a plant gene regulation secondary metabolite, and particularly relates to an application of a ginseng PgWRKY4X transcription factor and a coding gene in regulating and controlling the content of ginsenoside compounds in ginseng.

Background

Ginseng (panaxginngcc.a.mey) belongs to a perennial herbaceous plant of the family araliaceae, and in china, ginseng has historically been regarded as the king of centella asiatica. As a traditional precious Chinese medicinal material, ginseng has rich pharmacological actions including antidiabetic activity, anti-stress activity, antioxidant capacity, immune system regulating action, anticancer activity and the like. Wherein, the triterpenoid ginsenoside plays an important role. Ginsenosides in ginseng are classified into three groups according to the difference in structure: one type is protopanaxadiol type saponin, including ginsenoside Rb₁，Rb₂，Rc，Rd，Rg₃，Rh₂(ii) a One type is protopanaxatriol saponin, including ginsenoside Re, Rf, and ginsenoside Rg₁，Rg₂，Rh₁(ii) a Yet another class is oleanolic type saponins, including ginsenoside Ro. The pharmacopeia contains ginsenoside Rb₁，Rg₁The Re content is used as the index of ginsenoside substances to judge the quality of the ginseng medicinal material.

The synthesis of ginsenoside in ginseng is regulated by transcription factors, and the analysis of the regulation mechanism has some problems, so that the research on the regulation mechanism of ginsenoside compound accumulation in ginseng has important significance for production and academic research, but no report on the application of the transcription factor PgWRKY4X and coding gene in regulating the content of ginsenoside compound in ginseng exists at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the transcription factor PgWRKY4X of ginseng in regulating and controlling the content of ginsenoside compounds in ginseng.

The second purpose of the invention is to provide the application of the coding gene of the transcription factor PgWRKY4X of ginseng in regulating and controlling the content of ginsenoside compounds in ginseng.

The third purpose of the invention is to provide the application of the over-expression recombinant vector containing the coding gene of the transcription factor PgWRKY4X of ginseng in regulating and controlling the content of ginsenoside compounds in ginseng.

The technical scheme of the invention is summarized as follows:

application of transcription factor PgWRKY4X in regulating ginsenoside compound content in Ginseng radix; the amino acid sequence of the ginseng PgWRKY4X transcription factor is shown in SEQ ID No. 1.

The ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re.

The application of the coding gene of the ginseng PgWRKY4X transcription factor in regulating and controlling the content of ginsenoside compounds in the ginseng is disclosed, wherein the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

The ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re.

An application of an overexpression recombinant vector containing a ginseng PgWRKY4X transcription factor coding gene in regulating and controlling the content of ginsenoside compounds in ginseng is disclosed, wherein a nucleotide sequence of the overexpression recombinant vector is shown as SEQ ID No. 3.

The ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re.

Has the advantages that:

experiments prove that the transcription factor PgWRKY4X of the ginseng can be combined with the promoter of the PgSE gene of the ginseng, so that the expression of key enzyme PgSE on a ginsenoside synthesis path is improved, and the content of ginsenoside compounds in the ginseng is further improved. The ginseng PgWRKY4X transcription factor, the coding gene thereof and the overexpression recombinant vector containing the coding gene can be used for regulating and improving the content of ginsenoside compounds in ginseng.

Drawings

FIG. 1 is an overexpression recombinant vector containing the coding gene of the transcription factor PgWRKY4X of ginseng;

FIG. 2 is a pCAMBIA1300-PgWRKY4X-RNAi interference recombinant vector;

FIG. 3 is a fluorescent detection map of PgWRKY4X overexpression suspension cells (PgWRKY4X-OE) and PgWRKY4X-RNAi interference suspension cells (PgWRKY4X-R), wherein DAPI is a nuclear localization micrograph, GFP fluorescence, Brightfield and Merge are respectively fluorescence, optical lens and combined micrographs;

FIG. 4 shows the transcription levels of PgWRKY4X overexpression suspension cells (PgWRKY4X-OE), PgWRKY4X-RNAi interference suspension cells (PgWRKY4X-R) and PgWRKY4X (a) and PgSE (b) genes in control group;

FIG. 5 is a graph showing the expression levels of PgWRKY4X overexpression suspension cells (PgWRKY4X-OE), PgWRKY4X-RNAi interference suspension cells (PgWRKY4X-R) and PgSE proteins in a control group;

FIG. 6 shows PgWRKY4X overexpression suspension cells (PgWRKY4X-OE), PgWRKY4X-RNAi interference suspension cells (PgWRKY4X-R) and ginsenoside Rb in control group₁(a)，Rg₁(b) The content of Re (c);

FIG. 7 is a verification diagram of binding site of transcription factor PgWRKY4X, in which probe is a biotin labeled probe, GST-PgWRKY4X is a protein expressed by PgWRKY4X, and completter is a Competitor.

Detailed Description

The invention is further described with reference to the following figures and examples, which do not limit the scope of the invention.

First, experimental material and instrument

Plant total RNA extraction kit (B511311, Shanghai Biotechnology, Ltd., China), first-strand cDNA Synthesis kit (B639252, Shanghai Biotechnology, Ltd., China), ginsenoside Rb₁，Rg₁Re standard (Dalian Meiren Biotechnology Co., Ltd.). Fluorescence microscopy (LEICADM5000B, Buffalo Grove, IL, France).

Second, Experimental methods

1. Construction of recombinant vector containing ginseng PgWRKY4X transcription factor coding gene

Construction of an overexpression recombinant vector containing a ginseng PgWRKY4X transcription factor coding gene:

through PCR technology, specific primers PgWRKY4X-F-R (SEQ ID No.4 and SEQ ID No.5 in Table 1) are used, cDNA obtained by reverse transcription of total RNA extracted from adventitious roots of ginseng is used as a template, a PgWRKY4X encoding gene (shown as SEQ ID No.2, and a corresponding amino acid sequence is SEQ ID No.1) is amplified and inserted into a pCAMBIA1300 vector (Beijing Huayue), and a pCAMBIA1300-PgWRKY4X recombinant vector, namely an overexpression recombinant vector (shown as SEQ ID No. 3) containing the ginseng PgWRKY4X transcription factor encoding gene is constructed (shown as FIG. 1).

Construction of pCAMBIA1300-PgWRKY4X-RNAi interference recombinant vector:

firstly, a gene sequence of a PgWRKY4X forward fragment is amplified by using a PCR technology and a specific primer PgWRKY4X-P (SEQ ID No.6 and SEQ ID No.7 in Table 1) as a template and an encoding gene sequence of PgWRKY4X as a template, and is inserted into a pCAMBIA1300 vector to obtain a pCAMBIA1300-PgWRKY4X-P recombinant vector, then a gene sequence of a PgWRKY4X-N (SEQ ID No.8 and SEQ ID No.9 in Table 1) as a specific primer is amplified by using a PCR technology and an encoding gene sequence of PgWRKY4X as a template to obtain a gene sequence of a PgWRKY4 5631 reverse fragment, and finally the reverse fragment is constructed on the pCAMBIA1300-PgWRKY4X-P recombinant vector containing the forward fragment to obtain the pCAMBIA1300-PgWRKY 4-X-N recombinant vector (pCAMBIA 1300-X-PgWRKY 4-2-P recombinant vector).

TABLE 1 primer List

2. Genetic transformation mediated by agrobacterium tumefaciens GV3101

2.1 transformation and activation of Agrobacterium strains

Respectively transferring an overexpression recombinant vector containing a ginseng PgWRKY4X transcription factor coding gene and a pCAMBIA1300-PgWRKY4X-RNAi interference recombinant vector into GV3101 agrobacterium (Beijing Huayu biology), and culturing on YEB culture medium (1g/L yeast extract, 5g/L peptone, 5g/L beef extract, 5g/L sucrose, 0.5g/L magnesium sulfate, pH7.0) added with 50mg/L kanamycin and 50mg/L rifampicin to respectively obtain the transformation agrobacterium of the overexpression recombinant vector containing the ginseng PgWRKY4X transcription factor coding gene and the transformation agrobacterium containing the pCAMBIA1300-PgWRKY4X-RNAi interference recombinant vector. Single colonies of the transformed Agrobacterium containing the recombinant vectors were picked up and inoculated into 1mL of YEB medium containing the corresponding antibiotic, and cultured overnight at 28 ℃ with shaking at 200 rpm. Adding 200 μ L of the above bacterial liquid into 10mL YEB culture medium containing corresponding antibiotic, shaking at 200rpm and 28 deg.CCulturing overnight to make the concentration of bacterial liquid OD₆₀₀The value was 0.6. Centrifuging at room temperature at 4000rpm for 10min, discarding supernatant, and diluting with 1/2MS (adding acetosyringone AS, final concentration 100 μ M) liquid culture medium to OD₆₀₀And continuing suspension culture for 2h with the value of 0.4 to obtain the agrobacterium culture solution.

2.2 Pre-culture of suspension cells of Ginseng radix

Inoculating the new subculture ginseng suspension cells to a pre-culture solid medium 1/2MS (added with 100 mu M acetosyringone AS), and performing dark culture at 25 ℃ for 2 d.

2.3 Co-culture of Agrobacterium and Ginseng suspension cells

Before infection, the pre-cultured ginseng suspension cells are taken out and placed into the culture solution obtained in the step 2.1 for dip dyeing for 10min, and the ginseng suspension cells are gently shaken to be in full contact with the bacterial solution. Taking out infected ginseng suspension cells, sucking residual bacteria on the surface of the ginseng suspension cells by using sterile absorbent paper, transferring the ginseng suspension cells to a co-culture medium (solid) 1/2MS (adding 100 mu M acetosyringone AS), and co-culturing for 2d in the dark.

2.4 screening of transformed Ginseng suspension cells

And (3) transferring the cells obtained in the step (2.3) into a sterile culture medium 1/2MS + Cef mycin Cef250mg/L (solid), carrying out dark culture at 25 ℃ for 3 days, washing the ginseng suspension cells with sterile water, carrying out suction drying with sterile absorbent paper, transferring into the sterile culture medium, continuing the culture, and repeating for 3 times until no obvious thalli grow out. Then selecting the ginseng suspension cells with good growth state, transferring the ginseng suspension cells to a 1/2MS culture medium without antibiotics for continuous recovery culture for 20 days, and then performing normal subculture on a solid culture medium and a liquid culture medium respectively to obtain transformed ginseng suspension cells: PgWRKY4X overexpression suspension cells (PgWRKY4X-OE) and PgWRKY4X-RNAi interference suspension cells (PgWRKY 4X-R).

2.5 identification of Positive transformed Ginseng suspension cells

2.5.1qRT-PCR method for identifying positively transformed ginseng suspension cells

Extracting total RNA in ginseng suspension cells by using a method of a plant total RNA extraction kit, performing reverse transcription to generate cDNA (refer to the kit use instruction), and analyzing the transcription level of PgWRKY4X (SEQ ID No.12 and SEQ ID No.13 in Table 2) by using a qRT-PCR (Table 2) method to identify, wherein the PCR operation conditions are as follows: at 95 ℃ for 10 min; the actin (DQ243702.1) gene was used as an internal reference gene (SEQ ID No.10 and SEQ ID No.11 in Table 2) after cycling 40 times at 95 ℃, 10 seconds, 60 ℃, 30 seconds.

TABLE 2 primer List

2.5.2 Green fluorescence identification of Positive transformed Ginseng suspension cells

Under a fluorescence microscope, the successfully transformed ginseng suspension cells PgWRKY4X emit green fluorescence, which indicates that the group of cells are positive transformed ginseng suspension cells.

qRT-PCR analysis of PgSE Gene expression

The enzyme expressed by PgSE is a key enzyme in the synthesis pathway of ginsenoside substances in ginseng, so that the transcription level of PgSE is measured to confirm whether the PgWRKY4X transcription factor has a regulating effect on the synthesis of ginsenoside substances. The procedure is as in step 2.5.1, primers for PgSE are shown in Table 2 as SEQ ID No.14 and SEQ ID No. 15.

SDS-PAGE analysis of PgSE protein expression

The enzyme expressed by the PgSE is a key enzyme in the synthesis pathway of ginsenoside substances in ginseng, so that the protein expression level of the PgSE is determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and whether the synthesis of the ginsenoside substances is regulated after the overexpression or interference of the PgWRKY4X transcription factor can be proved.

5. Through the method of EMSA, the binding site of the transcription factor PgWRKY4X is explored

And (3) according to the PgWRKY4X transcription factor family, predicting the binding site of the PgSE promoter of the target gene, designing a corresponding double-stranded oligonucleotide probe, and carrying out biotin labeling on the oligonucleotide probe to obtain a biotin labeled probe. According to the use instruction of a chemiluminescence method EMSA kit (Shanghai Biyuntian biotechnology, Co., Ltd.), whether the PgWRKY4X transcription factor can activate the promoter of the key gene PgSE is verified through an EMSA experiment.

6. Determination of ginsenoside substance content in positive transformed ginseng suspension cells

Precisely weighing 0.2g of dry and crushed ginseng suspension cells, placing the dry and crushed ginseng suspension cells in a 100mL triangular flask with a plug, adding 40mL of methanol, carrying out water bath at 60 ℃ for 2h, carrying out suction filtration while the solution is hot, collecting filtrate, adding 20mL of methanol into the triangular flask, and carrying out water bath on filter paper and filter residues again at 60 ℃ for 2 h. After the filtrate was evaporated to dryness, the filtrate was dissolved in 10mL of distilled water, extracted with 20mL of n-butanol, and the n-butanol layer was collected and evaporated by rotation (temperature 70 ℃ C., rotation speed 50rpm) to a constant volume of 1 mL. Filtering with 0.22 μm microporous membrane to obtain sample. The liquid phase system used was Shimadzu LC-2030C development system (Shimadzu, Kyoto, Japan). Chromatographic conditions are as follows: column Kromasil C18(4.6 mm. times.250 mm,5 μm); mobile phase: a-acetonitrile, B-water; flow rate: 1.0 mL/min; detection wavelength: 203nm, column temperature: 35 ℃; sample introduction amount: 20 μ L. Gradient: A-ACN: 35min, 20%; 40min, 30%; 50min, 31%; 60min, 32%; 45% for 70 min; 100min, 60%; 110min, 60%; 111min, 20%; 120min, 20 percent.

Third, experimental results

1. Identification of positively transformed Ginseng suspension cells

1.1 identification of PgWRKY4X overexpressing suspension cells

Identification of PgWRKY4X overexpressing suspension cells (PgWRKY4X-OE) there are three methods. The first method is to observe whether the PgWRKY4X overexpression suspension cells emit green fluorescence or not under a fluorescence microscope, and the PgWRKY4X overexpression suspension cells can be seen from FIG. 3 to emit green fluorescence under the fluorescence microscope. The second method is to detect the transcription level of PgWRKY4X in the over-expression transformed ginseng suspension cells, and the relative expression quantity of the PgWRKY4X gene in the over-expression group is 4-5 times that of the control group as shown in figure 4 (a). The third method is to measure the protein level of PgWRKY4X, and as shown in FIG. 5, the expression level of PgWRKY4X protein in the overexpression group is obviously higher than that in the control group. The result shows that the PgWRKY4X overexpression suspension cell is the PgWRKY4X overexpression positive transformation ginseng suspension cell.

1.2 PgWRKY4X-RNAi interference suspension cell identification

PgWRKY4X-RNAi interference suspension cell (PgWRKY4X-R) identification has the following two methods. The first method is to measure the transcription level of PgWRKY4X gene interfering suspension cells, and the relative expression level of the PgWRKY4X gene in the interfering group is 2-3 times lower than that in the control group as shown in figure 4 (b). The second method is to observe whether the interfering suspension cells emit green fluorescence under a fluorescence microscope, and as shown in fig. 3, the interfering suspension cells emit green fluorescence under the fluorescence microscope. Therefore, the PgWRKY4X-RNAi interference suspension cell is the PgWRKY4X-RNAi interference positive transformation ginseng suspension cell.

2. Determination of ginsenoside substance content

Determination of ginsenoside Rb as representative substance in ginsenoside substances₁，Rg₁Re content (FIG. 6), ginsenoside Rb found in positively overexpressed suspension cells₁The content of ginsenoside Rb is about 6.8 times of that of the control group and the content of ginsenoside Rb in the interference group is about 6.8 times of that of ginsenoside Rb in the control group₁Is about 2.1 times lower than the control group (fig. 6 a); ginsenoside Rg₁The content of the ginsenoside is about 3.2 times of that of the control group, and the ginsenoside Rg of the interference group₁Is about 2.6 times lower than the control group (fig. 6 b); the content of ginsenoside Re was about 3.1 times that of the control group, and the content of ginsenoside Re in the interfering group was about 1.8 times lower than that of the control group (fig. 6 c). The obtained PgWRKY4X transcription factor can increase ginsenoside content in Ginseng radix.

Regulating expression of relevant enzymes for synthesizing ginsenoside substances by PgWRKY4X transcription factor

In order to investigate whether the PgWRKY4X transcription factor has a regulating effect on the synthesis of ginsenosides in ginseng, a key enzyme PgSE on the synthesis pathway of ginsenosides was studied. First, we measured the expression of PgSE protein, and as shown in fig. 5, the expression of PgSE protein in over-expressed suspension cells was significantly increased and the expression of PgSE protein in RNAi-interfered group was significantly decreased, compared to the control group. We detected the transcription level of PgSE gene (FIG. 4b), consistent with the protein expression results, over-expressed suspension cells have obviously increased PgSE gene transcription, and RNAi interference groups have obviously decreased PgSE gene transcription. It is speculated that the PgWRKY4X transcription factor may bind to the PgSE gene promoter to regulate PgSE expression. To confirm this hypothesis, we performed EMSA verification (fig. 7), which revealed that the PgWRKY4X transcription factor did bind to the promoter of the PgSE gene and regulate the synthesis of ginsenosides by regulating the expression of the PgSE gene.

Experiments prove that the ginseng transcription factor PgWRKY4X influences the synthesis and yield of ginsenoside substances by regulating and controlling the gene expression and protein expression of the ginseng squalene epoxidase (PgSE).

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Sequence listing

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Pro Cys Ser Thr Ser Leu Gly Ser Ser Gly Pro Thr Ile Thr Leu Asp

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cttccttggt tgttaaggat ggttatcaat ggcgtaagta cggtcaaaag gttactagag 1140

ataatccatc tccaagagct tacttcaagt gttcttttgc tccaacttgt ccagttaaga 1200

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ataatcatcc acatccatct aaggttgaag ccaactcttc atctaataga tgtgctgctc 1320

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tggctgctgc tatttctggt aaaatcttgc aacacaatca aaccgatggt gagatggtta 1560

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aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1680

acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 1740

agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1800

cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1860

agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 1920

actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 1980

tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2040

acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2100

tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2160

acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2220

ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2280

ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaactgcag gttcgagtat 2340

tatggcattg ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt 2400

ttttattcgg ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa 2460

tgatatggtc cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg 2520

tgtgttgaat ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc 2580

aaatcgtggc ctctaatgac cgaagttaat atgaggagta aaacactaag cttggcactg 2640

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2700

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2760

tcccaacagt tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt 2820

gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac 2880

ctaagagaaa agagcgttta ttagaataac ggatatttaa aagggcgtga aaaggtttat 2940

ccgttcgtcc atttgtatgt gcatgccaac cacagggttc ccctcgggat caaagtactt 3000

tgatccaacc cctccgctgc tatagtgcag tcggcttctg acgttcagtg cagccgtctt 3060

ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc ctgccctttt 3120

cctggcgttt tcttgtcgcg tgttttagtc gcataaagta gaatacttgc gactagaacc 3180

ggagacatta cgccatgaac aagagcgccg ccgctggcct gctgggctat gcccgcgtca 3240

gcaccgacga ccaggacttg accaaccaac gggccgaact gcacgcggcc ggctgcacca 3300

agctgttttc cgagaagatc accggcacca ggcgcgaccg cccggagctg gccaggatgc 3360

ttgaccacct acgccctggc gacgttgtga cagtgaccag gctagaccgc ctggcccgca 3420

gcacccgcga cctactggac attgccgagc gcatccagga ggccggcgcg ggcctgcgta 3480

gcctggcaga gccgtgggcc gacaccacca cgccggccgg ccgcatggtg ttgaccgtgt 3540

tcgccggcat tgccgagttc gagcgttccc taatcatcga ccgcacccgg agcgggcgcg 3600

aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc taccctcacc ccggcacaga 3660

tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag gcggctgcac 3720

tgcttggcgt gcatcgctcg accctgtacc gcgcacttga gcgcagcgag gaagtgacgc 3780

ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc attgaccgag gccgacgccc 3840

tggcggccgc cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc aggacggcca 3900

ggacgaaccg tttttcatta ccgaagagat cgaggcggag atgatcgcgg ccgggtacgt 3960

gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat gaaatcctgg ccggtttgtc 4020

tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg 4080

tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat 4140

gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt 4200

aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa 4260

ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg 4320

gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac 4380

cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg 4440

gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca 4500

agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag 4560

gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc 4620

atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt 4680

atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca 4740

gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa 4800

ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg 4860

gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag 4920

ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg 4980

cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag 5040

agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg 5100

gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc 5160

ggttggccag gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc 5220

aagcccgagg aatcggcgtg acggtcgcaa accatccggc ccggtacaaa tcggcgcggc 5280

gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc ggcaacgcat 5340

cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa tccgcaaaga 5400

atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg aagccgccca agggcgacga 5460

gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata gtcgcagcat 5520

catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg aggtgatccg 5580

ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca tggccagtgt 5640

gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca tgaaccgata 5700

ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg cggacgtact 5760

caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag aaacctgcat 5820

tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga acggccgcct 5880

ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa agagcgaaac 5940

cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg agatcacaga 6000

aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg atcccggcat 6060

cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag ccagatggtt 6120

gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt tctgtttcac 6180

cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg aggaggcggg 6240

gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg aagcatccgc 6300

cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg aaaaaggtcg 6360

aaaaggtctc tttcctgtgg atagcacgta cattgggaac ccaaagccgt acattgggaa 6420

ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac acatgtaagt 6480

gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa aacttattaa 6540

aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag ccgaagagct 6600

gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc cccgccgctt cgcgtcggcc 6660

tatcgcggcc gctggccgct caaaaatggc tggcctacgg ccaggcaatc taccagggcg 6720

cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca catcaaggca ccctgcctcg 6780

cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 6840

cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 6900

gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct 6960

taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 7020

gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 7080

ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 7140

acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 7200

aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 7260

tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 7320

aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 7380

gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 7440

acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 7500

accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 7560

ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 7620

gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 7680

gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 7740

ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 7800

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 7860

cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgcattcta ggtactaaaa 7920

caattcatcc agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa 7980

gtcaaaaaat agctcgacat actgttcttc cccgatatcc tccctgatcg accggacgca 8040

gaaggcaatg tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact 8100

tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag 8160

ttcctcttcg ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg 8220

agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc 8280

caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga 8340

gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc 8400

ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc 8460

atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat 8520

catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt 8580

taaatatagg ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc 8640

cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat 8700

tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct 8760

aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag 8820

aaaacagctt tttcaaagtt gttttcaaag ttggcgtata acatagtatc gacggagccg 8880

attttgaaac cgcggtgatc acaggcagca acgctctgtc atcgttacaa tcaacatgct 8940

accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata 9000

gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc 9060

ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct 9120

gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta 9180

ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattaatt cgggggatct 9240

ggattttagt actggatttt ggttttagga attagaaatt ttattgatag aagtatttta 9300

caaatacaaa tacatactaa gggtttctta tatgctcaac acatgagcga aaccctatag 9360

gaaccctaat tcccttatct gggaactact cacacattat tatggagaaa ctcgagcttg 9420

tcgatcgaca gatccggtcg gcatctactc tatttctttg ccctcggacg agtgctgggg 9480

cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt 9540

ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat 9600

cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg 9660

tcaagaccaa tgcggagcat atacgcccgg agtcgtggcg atcctgcaag ctccggatgc 9720

ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag 9780

atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct 9840

gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc 9900

cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac 9960

gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg 10020

catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac 10080

ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc gaccggttgt 10140

agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg 10200

gagatgcaat aggtcaggct ctcgctaaac tccccaatgt caagcacttc cggaatcggg 10260

agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag 10320

ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct 10380

tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc 10440

tcgacagacg tcgcggtgag ttcaggcttt ttcatatctc attgcccccc cggatctgcg 10500

aaagctcgag agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa 10560

atgaaatgaa cttccttata tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca 10620

tcccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg 10680

tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca 10740

gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct 10800

tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg 10860

tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta 10920

tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttatc acatcaatcc 10980

acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg 11040

ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg 11100

caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag 11160

atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca 11220

atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg 11280

tgctccacca tgttggcaag ctgctctagc caatacgcaa accgcctctc cccgcgcgtt 11340

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 11400

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 11460

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 11520

atgaccatga ttacg 11535

<210>4

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

caccatttct ccgtctgtct gat 23

<210>5

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gccgcggcca tctca 15

<210>6

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gacttccaag ttccaacaac tac 23

<210>7

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

cgttaaaaac aagaagaaga ag 22

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

caaagttaat agtgatcagg cg 22

<210>9

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

cgaatttgcc tcgacttttg aag 23

<210>10

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

tcggaagttg gctcggttt 19

<210>11

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

acttttgagc acgcgagaga a 21

<210>12

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

caccatttct ccgtctgtct gat 23

<210>13

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gccgcggcca tctca 15

<210>14

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

atgggaagtt tgggggcaat tct 23

<210>15

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

gttctcactg tttgttcagt agtaggtt 28

Claims

1. Application of transcription factor PgWRKY4X in regulating ginsenoside compound content in Ginseng radix; the amino acid sequence of the ginseng PgWRKY4X transcription factor is shown in SEQ ID No. 1.

2. The use of claim 1, wherein the ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re.

3. The application of the coding gene of the ginseng PgWRKY4X transcription factor in regulating and controlling the content of ginsenoside compounds in the ginseng is disclosed, wherein the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

4. The use of claim 3, wherein the ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re。

5. An application of an overexpression recombinant vector containing a ginseng PgWRKY4X transcription factor coding gene in regulating and controlling the content of ginsenoside compounds in ginseng is disclosed, wherein a nucleotide sequence of the overexpression recombinant vector is shown as SEQ ID No. 3.

6. The use of claim 5, wherein the ginsenoside compound is ginsenoside Rb₁Or Rg₁Or Re.