CN114634939A

CN114634939A - PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof

Info

Publication number: CN114634939A
Application number: CN202210491395.4A
Authority: CN
Inventors: 张儒; 李昭影; 张变玲; 谭时泉
Original assignee: Hunan Institute of Engineering
Current assignee: Hunan Institute of Engineering
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-06-17
Anticipated expiration: 2042-05-07
Also published as: ZA202206777B; CN114634939B

Abstract

The invention provides a PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof, wherein the PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng is derived from ginseng, the sequence of the PgJMT1 gene is shown as SEQ ID No.1, and the amino acid sequence of the protein coded by the PgJMT1 gene is shown as SEQ ID No. 2. The protein coded by the PgJMT1 gene has an obvious jasmonic acid carboxymethyltransferase specific functional gene sequence and a typical S-adenosine-L-methionine binding domain, belongs to jasmonic acid methyltransferase (JMT), is a key enzyme for catalyzing JA to synthesize MeJA in a MeJA synthesis path, and controls the conversion of jasmonic acid substances in plants. The constructed overexpression vector of the PgJMT1 gene is used for transforming the ginseng leaf blade through agrobacterium mediation, and the MeJA and ginsenoside content in the ginseng leaf blade for properly expressing the PgJMT1 gene are both obviously improved. The invention has potential application value in the aspects of improving the yield of the ginsenoside and improving the quality of the ginseng by utilizing the PgJMT1 gene in the ginseng.

Description

PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof

Technical Field

The invention relates to the technical field of biological genetic engineering, in particular to a PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof.

Background

Ginseng is a plant of the genus Panax of the family Araliaceae, is a famous and precious Chinese medicinal material in China, has been used for thousands of years in many Asian countries, and a secondary metabolite ginsenoside in the ginseng is the main active ingredient of the ginseng. Jasmonic acid and derivatives thereof such as methyl jasmonate (MeJA) and jasmonic acid isoleucine complex (JA-lle) belong to jasmonic acids (jasmonates, JAs), wherein MeJA and JA-Ile are the most active signal molecules. Research shows that MeJA as an environmental signal molecule has obvious influence on the regulation of plant secondary metabolism, can be used as an exogenous inducer to regulate a specific metabolic pathway of a plant, and can also respond to a defense reaction in a mode of regulating a plant secondary metabolite. Research shows that MeJA is often added externally to improve ginsenoside content because it can induce the expression of a series of synthetase genes in triterpene ginsenoside biosynthesis. However, due to the complexity of plant growth metabolism, the timing and amount of exogenous MeJA application are difficult to control precisely, and inhibition of plant cell growth is very easy to occur, which further affects the yield of ginsenoside. At the same time, exogenous application of MeJA et al JAs is not suitable for large-scale field planting. How to regulate the synthesis of endogenous MeJA of ginseng through the molecular biology level and further realize the synthesis and accumulation of the ginsenoside with lasting and high efficiency so as to improve the content of the ginsenoside and the medicinal value of ginseng, and the method has important application value for accurately regulating and controlling the accumulation of the ginsenoside and efficiently producing the ginsenoside in a large scale.

Disclosure of Invention

In order to solve the technical problems, the invention provides a PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof, and aims to promote increase of endogenous MeJA content of ginseng cells by transforming and expressing ginseng tissues through the screened PgJMT1 gene, regulate large-scale synthesis and accumulation of ginsenoside through endogenous MeJA and achieve the purposes of improving yield of ginsenoside and improving quality of ginseng.

In order to achieve the above object, the present invention firstly provides a PgJMT1 gene regulating methyl jasmonate synthesis in ginseng, the PgJMT1 gene regulating methyl jasmonate synthesis in ginseng is represented by SEQ ID No.1, and the PgJMT1 gene regulating methyl jasmonate synthesis in ginseng is derived from ginseng.

Preferably, the amino acid sequence of the protein coded by the PgJMT1 gene for regulating the synthesis of methyl jasmonate in ginseng is shown as SEQ ID No. 2.

Preferably, the sequence of the amplification primer of the PgJMT1 gene for regulating the synthesis of methyl jasmonate in the ginseng is shown as SEQ ID No.3 and SEQ ID No. 4.

Preferably, the PgJMT1 gene for regulating the synthesis of methyl jasmonate in the ginseng and the expression vector form a recombinant vector.

Preferably, the expression vector is pCAMBIA 1302.

Preferably, the recombinant vector is constructed as follows: the cDNA fragment is inserted into a plant expression vector pCAMBIA1302 by taking the open reading frame of the PgJMT1 gene synthesized by regulating methyl jasmonate in the ginseng as an overexpression sequence.

Based on a general inventive concept, the invention also provides an application of the PgJMT1 gene for regulating the synthesis of methyl jasmonate in ginseng in regulating the synthesis and accumulation of ginsenoside.

Preferably, the application mode is that the PgJMT1 gene for regulating the synthesis of methyl jasmonate in ginseng is mediated by agrobacterium to transform ginseng tissues.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts a MeJA-induced ginseng root transcriptome sequencing method to preliminarily screen out a ginseng JMT family gene, and then screens out a gene PgJMT1 which catalyzes JA to be MeJA from the ginseng JMT gene through an in vitro expression experiment and an agrobacterium rhizogenes A4-mediated ginseng leaf transformation experiment, wherein the protein coded by the gene PgJMT1 has an obvious specific functional motif of jasmonic acid carboxymethyltransferase and a typical S-adenosine-L-methionine binding domain, and belongs to plant jasmonic acid methyltransferase JMT. The protein coded by the gene PgJMT1 of the ginseng can catalyze JA to synthesize MeJA, the content of endogenous MeJA of the ginseng is regulated through the screened gene PgJMT1 and the coded protein thereof, and the biosynthesis and accumulation of ginsenoside are regulated through the endogenous MeJA, so that the aims of increasing the yield of the ginsenoside and improving the quality of the ginseng are fulfilled. The gene and the protein coded by the gene are a high-efficiency, specific and feasible method for improving the ginsenoside.

According to the invention, through constructing a plant overexpression vector of the PgJMT1 gene, the agrobacterium rhizogenes A4 mediates the PgJMT1 gene to transform the ginseng leaf, the PgJMT1 gene is expressed in the ginseng leaf at a high level, and compared with a control ginseng leaf, the content of total ginsenosides in the obtained PgJMT1 gene transient overexpression ginseng leaf is obviously improved. Therefore, the ginseng tissue or plant with the significantly improved content of the total ginsenoside can be obtained by utilizing the gene editing or over-expression PgJMT1 gene, and an efficient technical means is provided for improving the quality of the ginseng or improving the yield of the total ginsenoside.

Drawings

FIG. 1 shows the result of electrophoresis of the PCR product of the PgJMT1 gene in example 1 of the present invention, in which lane 1 shows the PCR amplification product and M shows the DNA standard molecular weight;

FIG. 2 shows the expression level of PgJMT1 gene in ginseng hairy roots after 100. mu. mol/L exogenous MeJA treatment for different time periods in fluorescent quantitative PCR (qRT-PCR) assay of Experimental example 1 of the present invention, with beta-actin as the internal reference;

FIG. 3 is a schematic diagram of an expression cassette of a prokaryotic expression vector of the PgJMT1 gene in Experimental example 2 of the present invention;

FIG. 4 is a SDS-PAGE result of a protein in which PgJMT1 gene was expressed in E.coli in Experimental example 2 of the present invention;

FIG. 5 shows the results of measuring PgJMT1 recombinant protein and its catalytic activity in Experimental example 3 of the present invention, wherein FIGS. 5A and 5B show the results of UPLC-MS/MS analysis of JA and MeJA standards; FIGS. 5C and 5D are the results of UPLC-MS/MS analysis of the product of the reaction of crude extract containing PgJMT1 recombinant protein with JA; FIGS. 5E and 5F are UPLC-MS/MS analysis results of reaction products of crude protein extract extracted from empty vector-containing bacterial suspension and JA;

FIG. 6 is a schematic diagram showing a plant hyper-vector expression cassette of PgJMT1 gene in Experimental example 4 of the present invention;

FIG. 7 shows the expression level of PgJMT1 gene in leaves of Agrobacterium rhizogenes A4-mediated transformed ginseng detected by qRT-PCR in Experimental example 4 of the present invention;

FIG. 8 shows MeJA content in leaves of Panax ginseng C.A. Pat. No.4 of the present invention, which was transformed with PgJMT1 gene mediated by Agrobacterium rhizogenes A4;

FIG. 9 shows the ginsenoside content in the ginseng leaves transformed with PgJMT1 gene mediated by Agrobacterium rhizogenes A4 in Experimental example 4.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Example 1

Cloning of PgJMT1 Gene

1. Ginseng RNA extraction and its reverse transcription synthesis cDNA

(1) Ginseng RNA extraction

Ginseng radix hairy roots cultured in 1/2MS solid medium were inoculated into 1/2MS liquid medium, cultured in dark at 120rpm and 25 ℃ for 3 weeks, and MeJA was added to the medium at a final concentration of 100. mu. mol/L, and cultured under the same conditions for 24 hours to induce gene expression related to MeJA.

Taking the ginseng hairy roots induced by MeJA for 24h, quickly placing the ginseng hairy roots in a mortar precooled by liquid nitrogen, immediately adding the liquid nitrogen, and quickly grinding the mixture into fine powder. Taking 40mg, putting into a 1.5mL centrifuge tube without RNase, adding 1mL TRIzol and 40 mu L beta-mercaptoethanol, quickly mixing uniformly, and placing at room temperature for 5-10 min; adding 0.2mL of chloroform, shaking for l5s, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 12000 Xg for 15min, collecting the upper layer, placing in 1.5mL centrifuge tube, and discarding the precipitate; adding 0.4mL of 3mol/L ammonium acetate (pH5.2) and 0.6mL of isopropanol, mixing, standing at room temperature for 10min, centrifuging at 4 deg.C and 12000 Xg for 10min, and discarding the supernatant; adding 1mL of 75% ethanol, and mixing uniformly; centrifuging at 4 deg.C and 10000 Xg for 5min, discarding supernatant, adding 20 μ L DEPC water to dissolve RNA, and keeping.

(2) Reverse transcription to synthesize cDNA

Synthesizing a first cDNA chain by using reverse transcriptase with oligo d (T)18 as a primer, wherein a reverse transcription reaction system is shown in table 1, and adding the components according to the system in table 1, and then stirring and mixing the components evenly; standing at room temperature for 10min, transferring to a constant temperature water bath box, and reacting at 42 ℃ for 1 h; after the reaction is finished, quickly placing on ice to cool for 2min, and finally placing at-20 ℃ for standby.

TABLE 1 reverse transcription reaction System

Components	Sample addition amount
		Template mRNA(200ng/μL)	10μL
5×1st strand synthesis buffer	4μL
		dNTP mixture(10mmol/L)	1μL
RNase inhibitor	1μL
		oligo(dT)(50μmol/L)	2μL
M-MLV(200U/μL)	1μL
		RNase-free H₂O	1μL
Total volume	20μL

2. PCR amplification of PgJMT1 Gene

Based on the candidate PgJMT1 gene sequence information obtained by MeJA-induced ginseng transcriptome sequencing, PCR amplification primers are designed by Primer Premier 5 software, and PgJMT1 gene PCR amplification primers are as follows.

PgJMT1-F：5′-ATGGATTCTGAGAAAGTTTCTGC-3′；(SEQ ID No.3)

PgJMT1-R：5′-TCATAATTTCCTAAGAAC-3′；(SEQ ID No.4)

The reaction conditions for PCR amplification of the PgJMT1 gene are shown in Table 2. The PCR products were analyzed by agarose gel electrophoresis.

TABLE 2 PCR reaction conditions

FIG. 1 shows the result of 1% agarose gel electrophoresis of the PCR product of the PgJMT1 gene, wherein lane 1 in FIG. 1 shows the PCR amplification product, M shows the DNA standard molecular weight, and the result shows that the size of the PCR product is about 1119bp, which is consistent with the expected theoretical size.

3. Subcloning of PgJMT1 gene and sequencing analysis thereof

Recovering an electrophoresis strip gel with the size of 1119bp in the PCR product, connecting the gel recovered product to a pGEM-T Easy subcloning vector, converting competent escherichia coli DH5 alpha, screening, extracting a plasmid of a positive bacterial colony, sequencing the plasmid, comparing and analyzing a sequencing result in Blast in NCBI, and displaying that the protein coded by the gene has an obvious specific functional motif of jasmonic acid carboxymethyltransferase and a typical S-adenosyl-L-methionine binding domain, belongs to plant jasmonic acid methyltransferase JMT, and the molecular docking analysis also proves that the PgJMT1 gene is obtained.

Experimental example 1

Quantitative fluorescent PCR (qRT-PCR) analysis of expression level of PgJMT1 gene

1. RNA extraction and reverse transcription

The method is similar to example 1, and comprises the steps of taking the hairy roots of 1/2MS solid culture medium after dark culture for 3 weeks at 25 ℃, inoculating the hairy roots into 1/2MS liquid culture medium, dark culture for 21 days at 25 ℃ and 110rpm, adding 100 mu mol/L MeJA for induction treatment, and respectively taking out the hairy roots after different treatment times for extracting RNA. The RNA was used as a primer for synthesizing cDNA using an oligo (D) (T)18 as a primer by reverse transcriptase. qRT-PCR analysis primers for beta-actin and PgJMT1 genes were as follows:

beta-actin fluorescent quantitative primer F: 5'-TGCCCCAGAAGAGCACCCTGT-3'; (SEQ ID No.5)

Beta-actin fluorescent quantitative primer R: 5'-AGCATACAGGGAAAGATCGGCTTGA-3', respectively; (SEQ ID No.6)

PgJMT1 fluorescent quantitative primer F: 5'-TGGCTCAGGACACAACACTT-3', respectively; (SEQ ID No.7)

PgJMT1 fluorescent quantitative primer R: 5'-TCGTTTAGGTACACGCGGAA-3', respectively; (SEQ ID No.8)

2. qRT-PCR analysis of PgJMT1 Gene expression level

The PgJMT1 gene expression level is analyzed and detected by a CFX Connect fluorescent quantitative PCR instrument, amplification is carried out according to a SYBR Premix Ex Taq fluorescent quantitative PCR kit, a qRT-PCR reaction system is shown in a table 3, and reaction conditions are shown in a table 4.

TABLE 3 qRT-PCR reaction System

TABLE 4 qRT-PCR reaction conditions

Three replicates per sample. After completion of the reaction, the amplification curve and the dissolution curve were confirmed, using 2^-ΔΔCtThe method calculates the difference of the expression level of the PgJMT1 gene. FIG. 2 shows that qRT-PCR detects 100. mu. mol/L exogenous MeJA treatment at different timesThe result of the expression level of the PgJMT1 gene in the hairy root of the later ginseng is that beta-actin is used as an internal reference. The result shows that the expression level of the PgJMT1 gene in the ginseng hairy roots is obviously improved after being induced by MeJA, when MeJA is treated for 48 hours, the expression level of the PgJMT1 gene is the highest and is 2.48 times of the expression level in the control ginseng hairy roots, and then the expression level of the PgJMT1 gene is reduced, which indicates that the PgJMT1 gene is related to a MeJA-mediated signal pathway.

Experimental example 2

Construction of prokaryotic expression vector of PgJMT1 gene and induced expression thereof

1. Construction of prokaryotic expression vector of PgJMT1 gene (the schematic diagram of the expression frame of the vector is shown in FIG. 3)

And (3) carrying out PCR amplification on PgJMT1, recovering a PCR product, and connecting the PgJMT1 gene to a pET32a vector in a homologous recombination mode. The ligation product was transformed into E.coli DH 5. alpha. and plated on LB solid medium containing ampicillin and cultured overnight at 37 ℃. And (4) selecting positive clones to perform colony PCR detection, and sequencing and verifying.

2. PgJMT1 recombinant protein induction expression

(1) Selecting a single colony of a positive recombinant bacterium containing a PgJMT1 gene, inoculating the single colony into 2mL of LB liquid culture medium containing ampicillin, activating at 180rpm and 37 ℃ overnight, inoculating the single colony into 100mL of LB culture medium according to a ratio of 1: 100, culturing at 180rpm and 37 ℃ until OD600 is between 0.6 and 0.8, adding IPTG until the final concentration is 0.4mmol/L, inducing and expressing at 20 ℃, and sampling once every 2 hours of induction, thereby determining the optimal induction time for obtaining recombinant protein expression.

(2) After induction expression is finished, 1mL of bacterial liquid is taken, centrifuged for 5min at 4 ℃ and 12000rpm, and thalli are collected; washing twice with deionized water; adding 200 μ L cell lysate, reacting at 4 deg.C for 10min, placing in refrigerator at-20 deg.C for 30min, heat-shocking at 37 deg.C for 10min, centrifuging at 12000rpm for 30min when the solution is clear, and collecting supernatant.

(3) Adding the supernatant into 2 xSDS loading buffer solution, and boiling for 10 min; centrifuging at 12000rpm for 1min at 4 deg.C; and taking 10 mu L of supernatant for loading. 10% of separation gel and 5% of concentrated gel are prepared and analyzed by SDS-PAGE gel electrophoresis detection.

FIG. 4 is an electrophoresis chart of PgJMT1 protein expression after IPTG induction, lanes 1-9 show total protein extracted from bacterial liquid after 0, 2, 4, 6, 8, 10, 12, 14 and 16h induction with 0.4mmol/L IPTG, lane 10 shows total protein extracted from empty carrier as control bacteria, M shows protein standard molecular weight; the result shows that the induction time has a relatively obvious effect on the expression of the PgJMT1 protein, the protein is already obviously expressed at 4h, the expression level of the protein is gradually increased along with the increase of the time, and the expression level of the protein reaches a higher level at 12 h.

Experimental example 3

1. PgJMT1 recombinant protein activity assay

(1) Extracting protein from the recombinant strain after IPTG induction for 14h, taking the escherichia coli transformed with the empty vector as an experimental control group (pET32a-BL21), and respectively taking JA as a candidate substrate for reaction. Separately adding MgCl₂S-adenosyl-L-methionine (SAM), JA, ATP, DDT and appropriate amount of crude protein extract, the reaction system is detailed in table 5.

TABLE 5 PgJMT1 recombinant protease activity assay reaction System

(2) Mixing the above components, reacting at 25 deg.C for 12h, adding 100 μ L anhydrous ethanol to terminate reaction, lyophilizing the reaction solution, extracting MeJA as possible product by the method in example 5, redissolving with 100 μ L mass spectrum methanol, treating with ultrasound for 10min, filtering with 0.22 μm filter membrane, and detecting MeJA by UPLC-MS/MS.

FIG. 5 shows the result of activity assay of PgJMT1 recombinant protein, wherein FIG. 5A and FIG. 5B show the results of UPLC-MS/MS analysis of JA and MeJA standards; FIGS. 5C and 5D show that the reaction product of crude extract containing PgJMT1 recombinant protein and JA is analyzed by UPLC-MS/MS, and the result shows that the content of JA in the substrate is obviously reduced and the content of MeJA is obviously increased; FIGS. 5E and 5F show the reaction product of crude protein extract extracted from empty vector-containing bacterial suspension and JA, after UPLC-MS/MS analysis, showing that the content of JA is unchanged and no MeJA is produced in the target product; the results indicate that the recombinant PgJMT1 protein can catalyze JA to MeJA.

Experimental example 4

Construction of plant overexpression vector of PgJMT1 gene and transient overexpression of plant overexpression vector in ginseng leaf

1. Construction of plant overexpression vector of PgJMT1 gene, and expression cassette of the overexpression vector is shown in FIG. 6.

(1) PCR amplification of PgJMT1 gene fragment for homologous recombination

Homologous recombination primers such as PgJMT1-F1(SEQ ID No.9) and PgJMT1-R1(SEQ ID No.10) are designed according to the sequence of the PgJMT1 gene to expand the full length of cDNA. And Bgl II restriction endonuclease is used for enzyme digestion of pCAMBIA1302 to prepare a linearized vector, and a PCR amplification product and linearized vector gel are recovered.

PgJMT1-F1：

5′-GGACTCTTGACCATGGATTCTGAGAAAGTTTCTGC-3′；(SEQ ID No.9)

PgJMT1-R1：

5′-TCGCCTTTGGAAGTTGAATGCCTCATAATTTCCTAAGAAC-3′；(SEQ ID No.10)

The PCR conditions are shown in Table 6.

TABLE 6 PCR reaction conditions

(2) Construction of pCAMBIA1302-PgJMT1 expression vector and transformation of Agrobacterium rhizogenes A4

The PgJMT1 gene is recombined to a pCAMBIA1302 vector by using In-Fusion HD Cloning Kit, and the constructed recombinant expression vector is named as pCAMBIA1302-PgJMT1 after the correct connection of the recombinant vector is identified by PCR and sequencing. The constructed pCAMBIA1302-PgJMT1 vector is transformed into Agrobacterium rhizogenes A4 by a freeze-thaw method, and positive clones are identified by PCR after transformation. And then the agrobacterium containing the PgJMT1 gene over-expression vector is obtained after sequencing and identification are successful.

2. Agrobacterium-mediated PgJMT1 gene transformation of ginseng leaf

(1) Agrobacterium rhizogenes culture containing pCAMBIA1302-PgJMT1

Single colonies of Agrobacterium containing the pCAMBIA1302-PgJMT1 vector and control Agrobacterium (containing the empty vector pCAMBIA1302) were inoculated into 10mL of YEB liquid medium containing the corresponding antibiotic and cultured for 16-24 h. 1mL of the bacterial solution was transferred to 100mL of YEB liquid medium containing the corresponding antibiotic, and 10. mu.L of 100mmol/L acetosyringone (AS; mother liquor prepared with DMSO so that the final concentration was 20. mu. mol/L) was added. Incubated overnight at 28 ℃. The culture solution was centrifuged at 5000rpm and 4 ℃ for 10min, and the cells were collected, washed 3 times with 1/2MS medium, and then diluted with 1/2MS + AS (final concentration of 20. mu. mol/L) medium to an OD600 of about 0.8 to give an invaded solution for transformation of ginseng leaf.

(2) Agrobacterium rhizogenes A4 mediated PgJMT1 gene transformed ginseng leaf

Sucking the agrobacterium tumefaciens staining solution by using a 1mL needle-free sterile syringe, and injecting the agrobacterium tumefaciens staining solution into the ginseng leaves from the lower epidermis of the ginseng leaves; after 3 days of injection, ginseng leaves were cut, and the expression level of PgJMT1 gene, MeJA and ginsenoside content were analyzed.

3. qRT-PCR analysis of transient expression level of PgJMT1 gene in ginseng leaf

The expression level of the PgJMT1 gene in the ginseng leaf was analyzed by qRT-PCR in the method of example 2, using the ginseng leaf infected with Agrobacterium rhizogenes A4 for 3 days, and the results are shown in FIG. 7.

FIG. 7 shows the expression level of PgJMT1 gene in ginseng leaf after 3 days of Agrobacterium rhizogenes A4 mediated transformation of PgJMT1 gene by fluorescent quantitative PCR (qRT-PCR); beta-actin is used as an internal reference; in the figure, the control shows the ginseng leaf blade transformed with the empty vector, and T5, T9, T12 and T16 respectively show the ginseng leaf blade transformed with PgJMT1 gene; the results showed that the expression level of PgJMT1 gene in the leaf of ginseng transformed with PgJMT1 gene was up-regulated, and that the expression levels of PgJMT1 gene in the leaf of T5, T9, T12 and T16 were 1.26, 1.81, 2.45 and 1.83 times of the control, respectively. The PgJMT1 gene is shown to be expressed in the transient over-expression ginseng leaves.

4. MeJA and ginsenoside content determination in ginseng leaf of transient over-expression PgJMT1 gene

(1) MeJA extraction and assay

Taking ginseng leaves infected by agrobacterium rhizogenes A4 for 3 days, grinding the ginseng leaves into fine powder in liquid nitrogen, weighing a proper amount of fine powder of the ginseng leaves, adding an isopropanol-water-hydrochloric acid mixed extracting solution, adding 8 mu L of an internal standard solution with the concentration of 1 mu g/mL, and oscillating for 30min at 4 ℃; adding dichloromethane, and oscillating at low temperature for 30 min; centrifuging at 13000 Xg for 5min at 4 ℃, and taking the lower organic phase; the organic phase was dried under nitrogen in the dark, redissolved in methanol (0.1% formic acid), centrifuged at 13000 Xg for 10min at 4 ℃ and the supernatant was filtered through a 0.22 μm filter and examined by UPLC-MS/MS for MeJA, the results of which are shown in FIG. 8.

FIG. 8 shows MeJA content in leaves of Panax ginseng 3 days after Agrobacterium rhizogenes A4 mediated transformation of PgJMT1 gene, wherein the control shows the leaves of Panax ginseng transformed with empty vector, and T5, T9, T12 and T16 respectively show the leaves of Panax ginseng transformed with PgJMT1 gene; the results show that the MeJA content in the ginseng leaf blade transformed with the PgJMT1 gene is obviously up-regulated, and the MeJA content in the leaf blades of T5, T9, T12 and T16 is respectively 2.91, 2.21, 3.32 and 1.98 times of that in the control

(2) Ginsenoside extraction and content determination

And (3) infecting the ginseng leaves for 3 days with agrobacterium rhizogenes A4, washing the ginseng leaves for 2min with tap water, washing the ginseng leaves twice with double distilled water, and drying the ginseng leaves to constant weight at 60 ℃. Grinding into fine powder, extracting with 80% methanol at 60 deg.C (1 g: 40mL), and treating with ultrasonic wave for 3 times (each time for 15 min); evaporating methanol in water bath at 60 deg.C, washing with water, ultrasonic dissolving, extracting with diethyl ether twice, collecting water phase, extracting with water saturated n-butanol, and collecting n-butanol layer. Evaporating n-butanol in water bath at 60 deg.C to obtain total ginsenoside, dissolving with appropriate amount of methanol under ultrasonic wave, fixing volume to scale, and filtering with 0.45 μm microporous membrane to obtain sample solution. Detecting the content of the ginsenoside by adopting liquid chromatography: the content of each saponin monomer in the sample is respectively determined by taking the total saponins Rb1, Rb2, Rc, Rd, Re, Rg1 and Rg3 of ginseng as standard substances, and the sum of the content of each saponin monomer represents the content of the total saponins in the ginseng cells, and the result is shown in fig. 9.

FIG. 9 shows the ginsenoside content in the leaves of ginseng after 3 days of Agrobacterium rhizogenes A4 mediated transformation of PgJMT1 gene, wherein the control shows the leaves of ginseng transformed with empty vector, and T5, T9, T12 and T16 respectively show the leaves of ginseng transformed with PgJMT1 gene; the result shows that the content of the total ginsenoside in the ginseng leaf blade transformed with the PgJMT1 gene is obviously up-regulated, and the ginsenoside content in the T5, T9, T12 and T16 leaf blades is 3.11 times, 2.03 times, 2.58 times and 1.72 times of that in the control blade respectively. Although the MeJA content in the T12 leaf blade is the highest, the ginsenoside content in the T5 leaf blade with relatively low MeJA content is the highest, and the content of the ginsenoside is 3.11 times that of the control. The results show that the PgJMT1 gene can effectively promote the synthesis of MeJA in the ginseng cells, and endogenous MeJA with proper concentration can promote the synthesis and accumulation of ginsenoside.

Sequence listing

<110> Hunan institute of engineering

<120> PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1119

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggattctg agaaagtttc tgctaacacc atggattctg agaaagtttt ccacatggct 60

ggaggagttg gagagactag ctatgccaaa aattccttga ttcagaaaaa ggcatctgat 120

atggccaaga acataaccct ccaaaccatt caacaagttt ataatctcca tacaacaact 180

tcaaagagtt ttggcatagc tgacttgggg tgtggctcag gacacaacac tttatcaatc 240

atcaaacaaa tgcttgaagc atgcagttat gatgaatatg atgacaacat taacaatgag 300

ttccgcgtgt acctaaacga ccttccaaac aacgatttca acgcaatctt caaggtgttg 360

ccggatttct acacagagtt gaggagggat gggaagggaa agtttgatca tatatatata 420

ggtgcttatc ctggctcttt ctatggaaga ctttttccag aaaagtgctt gcacttcatt 480

tattccaaca acagcttgca ttggctttct aaggtaccac catccattta tgacaagcaa 540

aacaattcta cgaacaaggg caacatttac ataacagaat caagccccct agaggtgtct 600

caggcatact ttaggcagtt ccaagaggac ctctggctgt ttctacggtc ccgatctgaa 660

gaacttgttg ccggaggacg tatggtgttg atcgtgtcgg gcagaagtgg ccggaatcat 720

gacgacagag gcattacatt tttatgggca cttctttcta aatcactcgc aattttagtt 780

tctcagggac tagttgaaga ggaaaagctt gatgggtaca atgttcagtt ttatgcacca 840

tcagaagatg aaataaaaga tgaagtaata agagagggat ctttccaaat ggaccgtttt 900

gaaatgtttg aaatagacaa ggttgttgac ggtggtgcaa gctacggaac ggcggtggca 960

aagacggcta aggcggtgca aggaccgatg atatgccaac attttggcga tggagttcta 1020

gacagtgtgt ttgagaacta tggaagatta gttgatgaaa acatggctgt agaggagata 1080

aggcctatat attttgtttt tgttcttagg aaattatga 1119

<210> 2

<211> 372

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Ser Glu Lys Val Ser Ala Asn Thr Met Asp Ser Glu Lys Val

1 5 10 15

Phe His Met Ala Gly Gly Val Gly Glu Thr Ser Tyr Ala Lys Asn Ser

20 25 30

Leu Ile Gln Lys Lys Ala Ser Asp Met Ala Lys Asn Ile Thr Leu Gln

35 40 45

Thr Ile Gln Gln Val Tyr Asn Leu His Thr Thr Thr Ser Lys Ser Phe

50 55 60

Gly Ile Ala Asp Leu Gly Cys Gly Ser Gly His Asn Thr Leu Ser Ile

65 70 75 80

Ile Lys Gln Met Leu Glu Ala Cys Ser Tyr Asp Glu Tyr Asp Asp Asn

85 90 95

Ile Asn Asn Glu Phe Arg Val Tyr Leu Asn Asp Leu Pro Asn Asn Asp

100 105 110

Phe Asn Ala Ile Phe Lys Val Leu Pro Asp Phe Tyr Thr Glu Leu Arg

115 120 125

Arg Asp Gly Lys Gly Lys Phe Asp His Ile Tyr Ile Gly Ala Tyr Pro

130 135 140

Gly Ser Phe Tyr Gly Arg Leu Phe Pro Glu Lys Cys Leu His Phe Ile

145 150 155 160

Tyr Ser Asn Asn Ser Leu His Trp Leu Ser Lys Val Pro Pro Ser Ile

165 170 175

Tyr Asp Lys Gln Asn Asn Ser Thr Asn Lys Gly Asn Ile Tyr Ile Thr

180 185 190

Glu Ser Ser Pro Leu Glu Val Ser Gln Ala Tyr Phe Arg Gln Phe Gln

195 200 205

Glu Asp Leu Trp Leu Phe Leu Arg Ser Arg Ser Glu Glu Leu Val Ala

210 215 220

Gly Gly Arg Met Val Leu Ile Val Ser Gly Arg Ser Gly Arg Asn His

225 230 235 240

Asp Asp Arg Gly Ile Thr Phe Leu Trp Ala Leu Leu Ser Lys Ser Leu

245 250 255

Ala Ile Leu Val Ser Gln Gly Leu Val Glu Glu Glu Lys Leu Asp Gly

260 265 270

Tyr Asn Val Gln Phe Tyr Ala Pro Ser Glu Asp Glu Ile Lys Asp Glu

275 280 285

Val Ile Arg Glu Gly Ser Phe Gln Met Asp Arg Phe Glu Met Phe Glu

290 295 300

Ile Asp Lys Val Val Asp Gly Gly Ala Ser Tyr Gly Thr Ala Val Ala

305 310 315 320

Lys Thr Ala Lys Ala Val Gln Gly Pro Met Ile Cys Gln His Phe Gly

325 330 335

Asp Gly Val Leu Asp Ser Val Phe Glu Asn Tyr Gly Arg Leu Val Asp

340 345 350

Glu Asn Met Ala Val Glu Glu Ile Arg Pro Ile Tyr Phe Val Phe Val

355 360 365

Leu Arg Lys Leu

370

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggattctg agaaagtttc tgc 23

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcataatttc ctaagaac 18

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgccccagaa gagcaccctg t 21

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcatacagg gaaagatcgg cttga 25

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tggctcagga cacaacactt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcgtttaggt acacgcggaa 20

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggactcttga ccatggattc tgagaaagtt tctgc 35

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcgcctttgg aagttgaatg cctcataatt tcctaagaac 40

Claims

1. A PgJMT1 gene for regulating the synthesis of methyl jasmonate in ginseng is characterized in that the PgJMT1 gene for regulating the synthesis of methyl jasmonate in ginseng is shown as SEQ ID No. 1.

2. The PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to claim 1, wherein the amino acid sequence of the protein encoded by the PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng is shown as SEQ ID No. 2.

3. The PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to claim 1, wherein the amplification primer sequence of the PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng is shown as SEQ ID No.3 and SEQ ID No. 4.

4. The PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to claim 1, wherein the PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng and an expression vector constitute a recombinant vector.

5. The PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to claim 4, wherein the expression vector is pCAMBIA 1302.

6. The PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to claim 4, wherein the recombinant vector is constructed as follows: the cDNA fragment is inserted into a plant expression vector pCAMBIA1302 by taking the open reading frame of the PgJMT1 gene synthesized by regulating methyl jasmonate in the ginseng as an overexpression sequence.

7. The use of the PgJMT1 gene for regulating methyl jasmonate synthesis in ginseng according to any one of claims 1 to 6 for regulating ginsenoside synthesis and accumulation.

8. The use of claim 7, wherein said means of using is to transform ginseng tissue using an agrobacterium-mediated transformation of the PgJMT1 gene that regulates methyl jasmonate synthesis in ginseng.