CN114634939B

CN114634939B - PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof

Info

Publication number: CN114634939B
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: 2023-06-16
Anticipated expiration: 2042-05-07
Also published as: ZA202206777B; CN114634939A

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 the 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 a protein encoded by the PgJMT1 gene is shown as SEQ ID No. 2. The protein coded by the PgJMT1 gene has an obvious functional gene sequence of jasmonic acid carboxymethyl transferase and a typical S-adenosyl-L-methionine binding domain, belongs to jasmonic acid methyl transferase (JMT), is a key enzyme for catalyzing JA to synthesize MeJA in a MeJA synthesis pathway, and controls the conversion of jasmonic acid substances in plants. The constructed PgJMT1 gene overexpression vector is used for transforming ginseng leaves through agrobacterium mediation, and the content of MeJA and ginsenoside in the ginseng leaves with proper expression of the PgJMT1 gene is obviously improved. The invention has potential application value in improving the yield of ginsenoside and improving the quality of ginseng by using PgJMT1 gene in 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 gene 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 rare Chinese medicinal material in China, has a history of thousands of years in many Asian countries, and the secondary metabolite ginsenoside in ginseng is the most main medicinal effect component. Jasmonic acid and its derivatives such as methyl jasmonate (MeJA) and jasmonic acid isoleucine complex (jasmoyl-L-isoleucine, JA-lle) belong to the class of Jasmonates (JAs), with MeJA and JA-Ile being the most active signaling molecules. Research shows that MeJA has very remarkable influence on controlling plant secondary metabolism as an environmental signal molecule, can be used as an exogenous inducer for controlling plant specific metabolic pathways, and can also respond to defense reaction by controlling plant secondary metabolites. Research shows that MeJA is often exogenously added for research of increasing ginsenoside content because of its ability to induce expression of a series of synthase genes in triterpene ginsenoside biosynthesis. However, due to the complexity of plant growth metabolism, the timing and amount of application of exogenous MeJA are difficult to precisely control, and inhibition of growth of plant cells is very easy to occur, thereby affecting the yield of ginsenoside. Meanwhile, 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 molecular biology level, thereby realizing durable and efficient synthesis and accumulation of ginsenoside, improving the content of ginsenoside and the medicinal value of ginseng, and having important application value for accurately regulating and controlling the accumulation of ginsenoside and producing ginsenoside in large scale and high efficiency.

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, and regulate mass synthesis and accumulation of ginsenoside by endogenous MeJA so as to achieve the purposes of improving yield of ginsenoside and improving quality of ginseng.

In order to achieve the above purpose, the invention firstly provides a PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng, wherein the PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng is shown as SEQ ID No.1, and the PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng is from ginseng.

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

Preferably, the amplification primer sequences of the PgJMT1 genes for regulating the synthesis of methyl jasmonate in ginseng are shown as SEQ ID No.3 and SEQ ID No. 4.

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

Preferably, the expression vector is pCAMBIA1302.

Preferably, the recombinant vector is constructed as follows: the open reading frame of PgJMT1 gene synthesized by methyl jasmonate in the regulated ginseng is used as an over-expression sequence, and the cDNA fragment is inserted into a plant expression vector pCAMBIA1302 to prepare the plant expression vector pCAMBIA1302.

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

Preferably, the application mode is to transform ginseng tissue by using agrobacterium to mediate PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng.

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 ginseng JMT family genes, and then screens out genes PgJMT1 with the catalysis JA of MeJA from a plurality of ginseng JMT genes through an in vitro expression experiment and an agrobacterium rhizogenes A4-mediated ginseng leaf transformation experiment, wherein the protein coded by the PgJMT1 genes has obvious functional motif of jasmonate carboxymethyltransferase and typical S-adenosyl-L-methionine binding domain, and belongs to plant jasmonate methyltransferase JMT. The protein coded by the ginseng PgJMT1 gene can catalyze JA to synthesize MeJA, the content of endogenous MeJA in ginseng is regulated and controlled by the screened PgJMT1 gene and the protein coded by the same, and the biosynthesis and accumulation of ginsenoside are regulated by the endogenous MeJA, so that the purposes of improving the yield of the ginsenoside and improving the quality of the ginseng are achieved. The gene and the protein coded by the gene are efficient, specific and feasible methods for improving the ginsenoside.

According to the invention, the plant super-expression vector of the PgJMT1 gene is constructed, the Agrobacterium rhizogenes A4 is used for mediating the PgJMT1 gene to transform the ginseng leaf, and the PgJMT1 gene is expressed in the ginseng leaf at a high level, so that compared with the control ginseng leaf, the content of the total ginsenoside in the obtained PgJMT1 gene transient super-expression ginseng leaf is obviously improved. Therefore, the ginseng tissue or plant with obviously improved total ginsenoside content can be obtained by utilizing gene editing or over-expression PgJMT1 gene, and an efficient technical means is provided for improving the quality of ginseng or improving the yield of total ginsenoside.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of PCR products of PgJMT1 gene in example 1 of the present invention, wherein lane 1 shows PCR amplification products, and M shows DNA standard molecular weight;

FIG. 2 shows the detection of the expression level of PgJMT1 gene in ginseng hairy roots after 100. Mu. Mol/L exogenous MeJA treatment by fluorescence quantitative PCR (qRT-PCR) in experimental example 1 according to the present invention, taking beta-actin as an internal reference;

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

FIG. 4 shows the SDS-PAGE result of the PgJMT1 gene expressed in Escherichia coli according to experimental example 2 of the present invention;

FIG. 5 is a measurement result of PgJMT1 recombinant protein and catalytic activity thereof in experimental example 3 of the present invention, wherein FIGS. 5A and 5B are results of UPLC-MS/MS analysis of JA and MeJA standards; FIGS. 5C and 5D are results of UPLC-MS/MS analysis of a crude extract containing PgJMT1 recombinant protein reacted with JA; FIGS. 5E and 5F show UPLC-MS/MS analysis results of the reaction product of the crude protein extract extracted from the bacterial liquid containing the empty carrier and JA;

FIG. 6 is a schematic diagram of the expression cassette of the PgJMT1 gene plant supervector in experimental example 4 of the invention;

FIG. 7 shows qRT-PCR detection of PgJMT1 gene expression levels in Agrobacterium rhizogenes A4-mediated transformation of ginseng leaves in Experimental example 4 of the present invention;

FIG. 8 shows the MeJA content of leaves of ginseng transformed with the PgJMT1 gene mediated by Agrobacterium rhizogenes A4 in experimental example 4 of the present invention;

FIG. 9 shows the ginsenoside content of the leaves of the Ginseng radix of the Agrobacterium rhizogenes A4-mediated transformation PgJMT1 gene in experimental example 4 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

Example 1

Cloning of PgJMT1 Gene

1. Ginseng RNA extraction and reverse transcription synthesis cDNA

(1) Ginseng RNA extraction

Taking ginseng hairy roots cultured in a 1/2MS solid culture medium, inoculating the ginseng hairy roots into a 1/2MS liquid culture medium, carrying out dark culture at 120rpm and 25 ℃ for 3 weeks, adding MeJA with the final concentration of 100 mu mol/L into the culture medium, and then carrying out culture under the same conditions for 24 hours to induce gene expression related to the MeJA.

Taking ginseng hairy roots induced by MeJA for 24 hours, quickly placing the ginseng hairy roots in a mortar precooled by liquid nitrogen, immediately adding the liquid nitrogen, and quickly grinding the ginseng hairy roots into fine powder. Taking 40mg in a 1.5mL centrifuge tube without RNase, adding 1mL TRIzol and 40 mu L beta-mercaptoethanol, quickly mixing, and standing at room temperature for 5-10min; adding 0.2mL of chloroform, shaking for 5s, and standing at room temperature for 10min; centrifuging at 12000 Xg for 15min at 4deg.C, collecting the upper layer, placing in a 1.5mL centrifuge tube, and discarding the precipitate; then adding 0.4mL of 3mol/L ammonium acetate (pH 5.2) and 0.6mL of isopropanol, mixing, standing at room temperature for 10min, centrifuging at 4deg.C and 12000 Xg for 10min, and discarding the supernatant; adding 1mL of 75% ethanol, and uniformly mixing; centrifuging at 4deg.C and 10000 Xg for 5min, discarding supernatant, and adding 20 μl DEPC water to dissolve RNA.

(2) Reverse transcription synthesis of cDNA

Synthesizing a cDNA first strand by using oligo (T) 18 as a primer and using reverse transcriptase, wherein a reverse transcription reaction system is shown in a table 1, and after adding each component according to the table 1 system, gently stirring and uniformly mixing; after being placed at room temperature for 10min, the mixture is moved to a constant temperature water bath box for reaction for 1h at 42 ℃; after the reaction is finished, the mixture is rapidly placed on ice for cooling for 2min, and finally placed at the temperature of minus 20 ℃ for standby.

TABLE 1 reverse transcription reaction system

Component (A)	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 H ₂ O	1μL
Total volume of	20μL

2.PCR amplification of PgJMT1 Gene

According to the sequence information of candidate PgJMT1 genes obtained by MeJA-induced ginseng transcriptome sequencing, a Primer Premier 5 software is used for designing PCR amplification primers, and the PCR amplification primers of the PgJMT1 genes 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 PCR products of PgJMT1 gene by electrophoresis on 1% agarose gel, and FIG. 1 shows that lane 1 shows PCR amplification products, M shows DNA standard molecular weight, and the result shows that the size of the PCR products is about 1119bp, which is consistent with the expected theoretical size.

3. Subcloning of PgJMT1 gene and sequencing analysis thereof

The electrophoresis strip with the size of about 1119bp in the PCR product is recovered, then the gel recovery product is connected to pGEM-T Easy subclone vector, competent escherichia coli DH5 alpha is transformed, plasmids of positive colonies are extracted after screening, sequencing is carried out on the plasmids, blast is compared and analyzed in NCBI as a sequencing result, the result shows that the protein coded by the gene has obvious functional motif of jasmonic acid carboxymethyl transferase and typical S-adenosyl-L-methionine binding domain, belongs to plant jasmonic acid methyl transferase JMT, and the PgJMT1 gene is also proved to be obtained through molecular docking analysis.

Experimental example 1

Fluorescent quantitative PCR (qRT-PCR) analysis of PgJMT1 Gene expression level

1. RNA extraction and reverse transcription

Taking hairy roots after dark culture for 3 weeks at 25 ℃ in a 1/2MS solid culture medium, inoculating the hairy roots into a 1/2MS liquid culture medium, dark culturing for 21d at 25 ℃ and 110rpm, adding 100 mu mol/L MeJA for induction treatment, and respectively taking the hairy roots after different treatment time for extracting RNA, wherein the extraction method is the same as in example 1. The RNA was used as a primer for oligo (T) 18, and cDNA was synthesized using reverse transcriptase. qRT-PCR analysis primers of beta-actin and PgJMT1 genes are as follows:

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

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

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

PgJMT1 fluorescent quantitative primer R:5'-TCGTTTAGGTACACGCGGAA-3'; (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 Table 3, and reaction conditions are shown in Table 4.

TABLE 3 qRT-PCR reaction System

TABLE 4 qRT-PCR reaction conditions

Each sample was replicated in triplicate. After completion of the reaction, the amplification curve and the dissolution curve were confirmed by using 2 ^-ΔΔCt The method calculates the difference of the expression level of PgJMT1 genes. FIG. 2 is a qRT-PCR assayThe result of measuring the expression level of PgJMT1 gene in ginseng hairy roots after 100 mu mol/L exogenous MeJA treatment for different time is taken as an internal reference. The result shows that the expression level of the PgJMT1 gene in ginseng hairy roots is obviously improved after being induced by MeJA, and when the MeJA is treated for 48 hours, the expression level of the PgJMT1 gene is highest and is 2.48 times that of the expression level of control ginseng hairy roots, and then the expression level of the PgJMT1 gene is reduced, so that the PgJMT1 gene is related to a MeJA-mediated signal path.

Experimental example 2

Construction of PgJMT1 gene prokaryotic expression vector and induction expression thereof

1. Construction of PgJMT1 Gene prokaryotic expression vector (schematic representation of vector expression frame is shown in FIG. 3)

PCR amplification of PgJMT1, recovery of PCR product, and homologous recombination to connect PgJMT1 gene to pET32a vector. The ligation product was transformed into E.coli DH 5. Alpha. And spread on ampicillin-containing LB solid medium and incubated overnight at 37 ℃. Positive clones were picked for colony PCR detection and sequenced for verification.

2. PgJMT1 recombinant protein induced expression

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

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

(3) Adding the supernatant into a 2 XSDS loading buffer solution, and boiling for 10min; centrifuging at 12000rpm at 4deg.C for 1min; 10. Mu.L of the supernatant was sampled. 10% of separation gel and 5% of concentrated gel are prepared and subjected to SDS-PAGE gel electrophoresis detection analysis.

FIG. 4 is an electrophoresis chart of PgJMT1 protein expression after IPTG induction, lanes 1-9 show total proteins extracted from bacterial solutions after 0.4mmol/L IPTG induction at final concentrations of 0.4mmol/L, 2, 4, 6, 8, 10, 12, 14 and 16h, lane 10 shows total proteins extracted from a control bacterium of a transfer vector, and M shows the standard molecular weight of the proteins; the results show that the induction time has obvious influence on the expression of PgJMT1 protein, the protein has obvious expression at 4 hours, the expression quantity of the protein gradually increases along with the time increase, and the expression quantity of the protein reaches higher level at 12 hours.

Experimental example 3

1. PgJMT1 recombinant protein activity determination

(1) Taking recombinant bacteria after IPTG induction for 14h to extract protein, taking escherichia coli transformed with an empty vector as an experimental control group (pET 32a-BL 21), and respectively taking JA as a candidate substrate to react. Respectively adding MgCl ₂ S-adenosylmethionine (SAM), JA, ATP, DDT and appropriate amount of crude protein extract, the reaction system is shown in Table 5.

TABLE 5 PgJMT1 recombinant protease activity determination reaction system

(2) After mixing the above components, reacting for 12 hours at 25 ℃, adding 100 mu L of absolute ethyl alcohol to terminate the reaction, freeze-drying the reaction solution, extracting a possible product MeJA by the method in example 5, re-dissolving by using 100 mu L of mass spectrum grade methanol, performing ultrasonic treatment for 10 minutes, filtering by using a 0.22 mu m filter membrane, and detecting the MeJA by UPLC-MS/MS.

FIG. 5 is a measurement result of PgJMT1 recombinant protein activity, wherein FIG. 5A and FIG. 5B are results of UPLC-MS/MS analysis of JA and MeJA standards; FIGS. 5C and 5D are results of reaction of a crude extract containing PgJMT1 recombinant protein with JA, and the results of UPLC-MS/MS analysis show that the content of substrate JA is obviously reduced and the content of MeJA is obviously increased; FIGS. 5E and 5F show the results of the reaction between the crude protein extract extracted from the bacterial liquid containing empty carrier and JA, and the results of UPLC-MS/MS analysis show that the JA content of the substrate is not changed and the MeJA is not generated in the target product; the results indicate that recombinant PgJMT1 protein can catalyze JA as MeJA.

Experimental example 4

Construction of PgJMT1 gene plant super-expression vector and transient super-expression of plant super-expression vector in ginseng leaf

1. The PgJMT1 gene plant super-expression vector is constructed, and the super-expression vector expression frame is shown in figure 6.

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

Homologous recombination primers were designed based on the PgJMT1 gene sequence to extend its cDNA full length, such as PgJMT1-F1 (SEQ ID No. 9) and PgJMT1-R1 (SEQ ID No. 10). And preparing a linearization vector by using Bgl II restriction endonuclease to cleave pCAMBIA1302, and recovering PCR amplification products and linearized vector gel.

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 into a pCAMBIA1302 vector by In-Fusion HD Cloning Kit, and the constructed recombinant expression vector is named pCAMBIA1302-PgJMT1 after the recombinant vector is connected correctly by PCR and sequencing identification. The constructed pCAMBIA1302-PgJMT1 vector is transformed into agrobacterium rhizogenes A4 by a freeze thawing method, and positive clones after transformation are identified by PCR. And then obtaining the agrobacterium containing the PgJMT1 gene super-expression vector after successful sequencing identification.

2. Agrobacterium-mediated PgJMT1 gene transformed 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 pCAMBIA 1302) were picked and inoculated into 10mL of YEB liquid medium with the corresponding antibiotics for 16-24h, respectively. 1mL of the bacterial liquid is transferred to 100mL of YEB liquid culture medium added with corresponding antibiotics, and 10 mu L of 100mmol/L acetosyringone (AS; mother liquor is prepared by DMSO so that the final concentration is 20 mu mol/L) is added. Incubated overnight at 28 ℃. The culture solution was centrifuged at 5000rpm at 4℃for 10min, and the cells were collected, washed 3 times with 1/2MS medium, and then diluted to an OD600 of about 0.8 with 1/2MS+AS (final concentration of 20. Mu. Mol/L) medium to obtain an infected liquid for transforming ginseng leaves.

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

Sucking the agrobacteria invasion solution by a 1mL sterile injector without a needle, and injecting the agrobacteria invasion solution into the ginseng leaf from the lower epidermis of the ginseng leaf; after 3 days of injection, the leaves of ginseng were cut off and analyzed for PgJMT1 gene expression level, meJA and ginsenoside content.

3. Analysis of transient expression level of PgJMT1 Gene in Ginseng radix leaf by qRT-PCR

The expression level of PgJMT1 gene in the ginseng leaf after 3 days of infection of Agrobacterium rhizogenes A4 was analyzed by qRT-PCR in the method of example 2, and the result is shown in FIG. 7.

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

4. MeJA and ginsenoside content determination in ginseng leaf with transient overexpression PgJMT1 gene

(1) MeJA extraction and content determination

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 ginseng leaf fine powder, adding isopropanol-water-hydrochloric acid mixed extract, adding 8 mu L of 1 mu g/mL of internal standard solution, and oscillating for 30min at 4 ℃; adding dichloromethane, and oscillating for 30min at low temperature; centrifuging at 13000 Xg at 4deg.C for 5min, and collecting the lower organic phase; the organic phase was dried in nitrogen in the dark, redissolved in methanol (0.1% formic acid), centrifuged at 13000 Xg at 4℃for 10min, the supernatant was filtered through a 0.22 μm filter and the MeJA was detected by UPLC-MS/MS, the results are shown in FIG. 8.

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

(2) Ginsenoside extraction and content determination

Taking ginseng leaves infected by agrobacterium rhizogenes A4 for 3 days, washing with tap water for 2min, washing with double distilled water twice, and drying at 60 ℃ to constant weight. Grinding it into fine powder, leaching with 80% methanol at 60deg.C (1 g:40 mL), and ultrasonic treating for 3 times each for 15min; evaporating methanol in water bath at 60deg.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 60deg.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 ginsenoside by liquid chromatography: the total saponins Rb1, rb2, rc, rd, re, rg1 and Rg3 were used as standard substances to measure the content of each saponin monomer in the sample, and the sum of the content of each saponin monomer was used to represent the total saponins in the ginseng cells, and the result is shown in FIG. 9.

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

Sequence listing

<110> Hunan engineering institute

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

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1119

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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

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<213> Artificial sequence (Artificial Sequence)

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

3. A primer for amplifying the PgJMT1 gene regulating methyl jasmonate synthesis in ginseng according to claim 1, wherein the primer sequences are shown in SEQ ID No.3 and SEQ ID No. 4.

4. A recombinant vector for regulating the synthesis of methyl jasmonate in ginseng according to claim 1, wherein the recombinant vector consists of the PgJMT1 gene and an expression vector.

5. The recombinant vector of PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng according to claim 4, wherein the expression vector is pCAMBIA1302.

6. The recombinant vector for regulating the synthesis of methyl jasmonate in ginseng, pgJMT1 gene according to claim 5, wherein the recombinant vector is constructed in the following manner: the open reading frame of PgJMT1 gene synthesized by methyl jasmonate in the regulated ginseng is used as an over-expression sequence, and the cDNA fragment is inserted into a plant expression vector pCAMBIA1302 to prepare the plant expression vector pCAMBIA1302.

7. Use of a PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng according to claim 1 for regulating synthesis and accumulation of ginsenoside.

8. The use according to claim 7, wherein the mode of use is the transformation of ginseng tissue with agrobacterium mediating the PgJMT1 gene regulating methyl jasmonate synthesis in ginseng.