CN115725620A - Method for synthesizing panax japonicus saponin in panax notoginseng cells - Google Patents

Method for synthesizing panax japonicus saponin in panax notoginseng cells Download PDF

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CN115725620A
CN115725620A CN202211106516.5A CN202211106516A CN115725620A CN 115725620 A CN115725620 A CN 115725620A CN 202211106516 A CN202211106516 A CN 202211106516A CN 115725620 A CN115725620 A CN 115725620A
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panax
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notoginseng
saponin
pnds
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CN115725620B (en
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葛锋
胡泽群
陈勤
王志龙
刘迪秋
崔秀明
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Kunming University of Science and Technology
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Abstract

The invention discloses a method for synthesizing panax japonicus saponin in panax notoginseng cells, which is constructed byPnDSThe gene RNAi expression vector is introduced into the pseudo-ginseng cells to obtain pseudo-ginseng cells for synthesizing panax japonicus saponin, wherein the nucleotide sequence of RNAi fragments is shown as SEQ ID NO. 1; the method is based on reverse regulation, and can inhibit the first key enzyme gene related to dammarane type triterpenoid saponin synthesis branch in notoginsenoside biosynthesis pathwayPnDSExpression of (2), decreaseThe synthesis of the little dammarane type triterpenoid saponin promotes the panax notoginseng cells to synthesize oleanane type saponin-panax japonicus saponin which does not contain originally, and the invention provides a new way for the industrial large-scale production of panax notoginseng cells.

Description

Method for synthesizing panax japonicus saponin in panax notoginseng cells
Technical Field
The invention belongs to the technical field of saponin synthesis, and particularly relates to a method for synthesizing panax japonicus saponin in panax notoginseng cells.
Background
Araliaceae (Araliaceae) Panax (Panax) Comprises multiple medicinal plants, wherein ginseng (A), (B), (C)Panax ginseng) American ginseng (American ginseng)Panax quinquefolius) Notoginseng (A), notoginseng (A)Panax notoginseng) And bead ginseng (A)Panax japonicus) The application is the most extensive. The main active component of the compounds is triterpenoid saponin, and the triterpenoid saponin has various pharmacological effects of preventing and treating cardiovascular and cerebrovascular diseases, resisting fatigue and oxidation, enhancing immunity, protecting liver and the like. The biosynthesis routes of the triterpenoid saponins of the panax medicinal plant are basically the same in the initial stage and the skeleton construction stage, and the main difference of saponin synthesis among various species is that after 2,3-oxidosqualene is formed, 2,3-oxidosqualene is taken as a common precursor, and two independent triterpenoid saponin synthesis branches are respectively generated. 2,3-oxidosqualene is catalyzed by Dammarenediol Synthetase (DS) and enters dammarane type saponin synthesis branch; catalyzed by beta-amyrin synthase (beta-AS), the oleanane type saponin enters a synthesis branch of oleanane type saponin.
To date, triterpene saponins separated from ginseng genus herbs are mainly dammarane type saponins, and ginseng, american ginseng and rhizoma panacis majoris all contain two types of triterpene saponins of dammarane type and oleanane type, but panax notoginseng only contains dammarane type saponins, and can not synthesize oleanane type saponins, which is the characteristic that panax notoginseng is different from other ginseng genus species. At present, it is known that panax japonicus saponins IV and iv a belong to oleanane type saponins, and panax notoginseng in a natural state does not have the capacity of synthesizing panax japonicus saponins IV and iv a.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synthesizing panax japonicus saponin in panax notoginseng cellsBy constructingPnDSGene RNAi expression vector, and transferring it into notoginseng cell to inhibit the first key enzyme of dammarane type triterpenoid saponin synthesis branch in notoginsengPnDSThe gene expression weakens the metabolic flow of a dammarane type saponin synthesis branch, promotes pseudo-ginseng cells to synthesize panax japonicus saponin, wherein the nucleotide sequence of an RNAi fragment is shown as SEQ ID NO 1;
the purpose of the invention is realized by the following technical scheme:
1. extracting total RNA from radix Notoginseng, reverse transcribing to synthesize radix Notoginseng cDNA, and PCR amplifying with the synthesized first strand cDNA as templatePnDSGene interference fragment, gene thereofPnDSThe nucleotide sequence of (A) is shown as SEQ ID NO. 1;
2. construction ofPnDSGene RNAi interference vector pHellsamgte-PnDSTransforming agrobacterium, and screening out positive monoclonal through PCR;
3. pHellsamgte-PnDSIntroducing pseudo-ginseng cell expression, and screening a positive transgenic cell line through antibiotic screening and qRT-PCR (quantitative reverse transcription-polymerase chain reaction);
4. extracting saponin from transgenic cell and non-transgenic cell line of Notoginseng radix, and analyzing the difference of saponin types and contents between transgenic cell and non-transgenic cell line.
The invention has the advantages and technical effects that:
the invention provides a new method for producing panax japonicus saponin IVa and panax japonicus saponin IV, wherein the panax japonicus saponin IVa and the panax japonicus saponin IV are main active ingredients of panax japonicus, belong to typical oleanane type saponin and do not contain panax notoginseng medicinal materials. The invention synthesizes panax japonicus saponin IVa and panax japonicus saponin IV in panax notoginseng cells by a gene regulation method, realizes the heterologous expression of the saponins in interspecies, has simple method and easy operation, and has the potential of industrial production and market popularization and application.
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FIG. 1 shows the result of RNAi fragment sequence amplification;
FIG. 2 is an amplification product of an RNAi fragment with attB recombination sites, wherein 1: RNAi fragment, M: markerDL2501;
FIG. 3 shows pHellsamgte-PnDSThe carrier is transformed into an identification electrophoretogram of Escherichia coli DH5 alpha, wherein the ratio of 1-8: bacterial liquid sample, M: markerDL2501, +: pHellsamgte-PnDSPlasmid-negative control;
FIG. 4 shows pHellsamgte-PnDSIdentification electrophoretogram of vector transferred agrobacterium LBA4404, wherein 1-5: a bacterial liquid sample; m: markerDL2502; negative control;
FIG. 5 is an electrophoresis diagram of PCR detection of transgenic Panax notoginseng cells, wherein 1-6: transgenic pseudo-ginseng cell line, M: markerDL2502;
FIG. 6 is real-time fluorescent quantitative PCR detection in transgenic notoginseng cellsPnDSGenes and detection results of relative expression quantity of 5 genes related to synthesis of panax japonicus saponin IVa and panax japonicus saponin IV, wherein WT is a wild cell line, and T1-T4 are 4 genesPnDSA transgenic notoginseng cell line of the gene;
FIG. 7 is the saponin content in transgenic Panax notoginseng cell lines, wherein WT is a wild type Panax notoginseng cell line and T1-T4 are transgenic Panax notoginseng cell lines).
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the present invention, and the methods used in the examples are all conventional methods unless otherwise specified, and reagents used therein are all conventional commercially available reagents or reagents prepared by conventional methods.
Example 1:PnDScloning of RNAi fragments of genes
(1) Extraction of panax notoginseng total RNA
The invention adopts an improved guanidinium isothiocyanate method to extract panax notoginseng callus total RNA, and the specific operation is as follows: and (4) removing RNA enzyme from a mortar and a mortar rod, performing high-temperature dry heat sterilization treatment, and cooling to room temperature. Weighing appropriate amount of Notoginseng radix callus cells, placing in the treated bowl, grinding Notoginseng radix callus into powder with liquid nitrogen, adding 10% (w/v) precooled RNA extraction buffer and 1.0% (w/v) beta-mercaptoethanol, and fully grinding. Transferring 1.0mL of grinding fluid into a 2mL centrifuge tube, adding 500 μ L of RNA extraction phenol, 100 μ L of chloroform and 1/10 volume of 2M sodium acetate solution (pH is 4.0), shaking vigorously and mixing uniformly, standing on ice for 5min, centrifuging at 4 ℃ and 12500g for 15min, slowly sucking supernatant and transferring into a new 2mL centrifuge tube, adding 1:1 isovolume of RNA extraction phenol/chloroform, shaking vigorously and mixing uniformly, standing on ice for 5min, centrifuging at 4 ℃ and 12500g for 15min (repeating the steps until the middle layer almost disappears). Slowly sucking the supernatant, transferring the supernatant into a new 2mL centrifuge tube, adding chloroform with the same volume, violently shaking and mixing uniformly, standing on ice for 5min, centrifuging at 4 ℃ for 15min at 12500g, slowly sucking the supernatant, transferring the supernatant into a new 1.5mL centrifuge tube, adding 3M sodium acetate solution (pH is 5.2) with the volume of 1/10, adding isopropanol with the same volume, slowly reversing and mixing uniformly, standing at-20 ℃ for 1.5h for full precipitation, and centrifuging at 4 ℃ for 25min at 12500 g; discarding the supernatant, blowing and washing the precipitate with 75% ethanol solution, blowing and washing twice, sucking off the liquid with a pipette gun, placing on a super clean bench for air drying, and adding 15-30 μ L of RNase water to dissolve the RNA precipitate after ethanol is completely volatilized. Detecting the integrity of the total RNA of the panax notoginseng callus cells by agarose gel electrophoresis, and then detecting the concentration and purity of the extracted RNA by using an ultraviolet spectrophotometer.
(2) First Strand cDNA Synthesis
Selecting RNA with better quality, synthesizing a first strand cDNA by using GoScript reverse transcription system of Promega company, and firstly, mixing Oligo (dT) 15 1.0 muL, 5.0 mug of total RNA, and 10 muL of nucleic-free Water, fully and gently mixing, placing the mixture in a Water bath kettle at 70 ℃ for 5min in a Water bath for pre-denaturation, immediately placing the mixture in an ice bath for 5min, and then adding the following reagents: nuclear-free Water 1.6 mu L, goScript TM 5×Reaction Buffer 4.0µL、PCR Nucleotide Mix 1.0µL、MgCl 2 (25 mM) 2.0µL、Recombinant RNasin ® Ribonuclease Inhibitor 0.4µL、GoScript TM 1.0 mu L of Reverse Transcriptase, mixing uniformly, carrying out instantaneous centrifugation, annealing at 25 ℃ for 5min, extending in water bath at 42 ℃ for 90min, and finally placing in water bath in a water bath kettle at 70 ℃ for 15min to terminate the activity of the Reverse Transcriptase to obtain the first chain of the pseudo-ginseng cDNA.
(3) Synthesis of RNAi fragments
According to the pseudo-ginsengPnDSGene full-length sequence (KJ 804174.1), and its specific primer was designed using PrimerPremier5.0PnDS F :5'-TCCCCTTATCATTGCCCT-3' andPnDS R :5'-GCTTTTTCCCCATTTCCT-3', using the first strand of cDNA of notoginseng obtained as template, and making high fidelity PCR amplification under the action of DNA polymerase Ex Taq, the PCR reaction condition is: at 98 ℃ for 3min; at 98 ℃ for 10s, at 60 ℃ for 15s, at 72 ℃ for 15s, at 35 cycles;72 ℃ for 5min; separating the obtained amplification product by 1% agarose gel electrophoresis, and recovering the fragment as genePnDSThe nucleotide sequence of positions 503-1246 of (a);
and recovering the target fragment, connecting the recovered target fragment with a pGEM T-easy vector, transferring the target fragment into Escherichia coli DH5 alpha, randomly selecting a single colony on a plate, carrying out PCR amplification on a bacterial liquid, and detecting positive clone. Sequencing the monoclonal antibody with positive PCR detection of the bacterial liquid, and sequencing the sequence and the pseudo-ginseng by using Blast software in NCBIPnDSComparing the gene sequences, the result is shown in figure 1, the nucleotide sequence is shown in SEQ ID NO:1, the comparison result is correct, the Escherichia coli with the correct result is inoculated in a liquid LB culture medium containing spectinomycin for propagation, the culture is carried out at 37 ℃ and 200rpm for 15h in a shaking way, and the plasmid (T-PnDS）。
Example 2:PnDSconstruction of Gene RNAi expression vector
(1) Cloning of RNAi fragments with attB recombination sites
Designing a primer BP-C according to the technical principle of gateway F (5’-GGGGACAAGTTTGTACAAAAAAGCAGG CTTCCCCTTATCATTGCCCT-3') and BP-C R (5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGCTTTTTCCCCATTTCCT-3') (the underlined part is attB recombination site), adapters are added to both ends of RNAi fragments by PCR means to allow homologous recombination between the target fragments and the vector, the extracted plasmid is used as template, BP-C is used as template F /BP-C R Using Ex Taq to carry out attB-PCR amplification as a primer, wherein the PCR reaction condition is the same as that in the step (3) of the embodiment 1, and after the amplification, attB recombination sites are connected to both sides of an RNAi fragment; PCR products were recovered from the gel and the results are shown in FIG. 2;
（2）PnDSconstruction of Gene RNAi expression vector
According to Gateway ® BP Clonase TM II, constructing an RNAi vector by the Enzyme Mix kit instruction, wherein the reaction system is set as follows: 0.15 mug of attB-PCR glue recovery product, 0.15 mug of pHellsamge 2 carrier plasmid and BP clone TM II Enzyme Mix 2.00 μ L, TE buffer (pH = 8.0) to 10 μ L; sequentially adding the solutions, blowing and uniformly mixing by using a liquid transfer gun, placing the mixture in a water bath kettle at 25 ℃, reacting for 4 hours, then adding 1 mu L of proteinase K into the reaction solution, carrying out a water bath for 10 minutes in a water bath kettle at 37 ℃, terminating the reaction, converting the reaction product into escherichia coli competent cells, picking out monoclonal cells, carrying out amplification culture on the escherichia coli competent cells, and then carrying out plasmid (pHellsgate-PnDS) Extracting; the obtained plasmids were treated with restriction enzymes respectivelyXbaI andXhoi, performing single enzyme digestion, wherein the enzyme digestion system is as follows: pHellsamgte-PnDSPlasmid 5.0 mug, 10 Xbuffer 2.0 mug L, restriction endonucleaseXbaⅠ/XhoⅠ1.5 µL、ddH 2 Supplementing water to 20 mu L;
placing the enzyme digestion system in a 37 ℃ water bath kettle for enzyme digestion reaction for 5h, detecting the enzyme digestion product by 1% agarose gel electrophoresis after the reaction is finished, and judging the size of the enzyme digestion product gel running belt according to the size of the enzyme digestion products of the two enzymesPnDSGene RNAi expression vector pHellsamgte-PnDSThe construction is successful; then the vector is transferred into agrobacterium LBA4404 competent cells by a liquid nitrogen freeze thawing method, and simultaneously the unloaded pHellsamgte 2 is also transferred into the LBA4404 competent cells as an unloaded control, the result is shown in figure 4, the amplified band is about 750bp, which is in line with the expectation, and the result shows that the amplified band is about 750bpPnDSThe RNAi fragments have been successfully transformed into Agrobacterium.
Example 3: agrobacterium tumefaciens mediated panax notoginseng genetic transformation
1. Pre-culture of notoginseng cell
(1) Collecting stem and leaf of Notoginseng radix, and culturing with Notoginseng radix callus culture medium (MS culture medium + 2,4-D2 mg/L + KT 1mg/L, culture medium pH5.6) at 25 + -1 deg.C in dark for 28 days to obtain Notoginseng radix callus; subculturing the obtained Notoginseng radix callus with MS solid culture medium containing 2,4-D2 mg/L, KT mg/L for 15 days to obtain Notoginseng radix subculture cell;
(2) Selecting panax notoginseng callus cells with good growth state, transferring the panax notoginseng callus cells to a panax notoginseng callus cell pre-culture medium (acetosyringone is added to a panax notoginseng callus culture medium to 40 mg/L), laying the panax notoginseng callus cells to cover the whole surface of the culture medium, and placing the panax notoginseng callus cells in the dark culture at 25 ℃ for 3 days;
(2) Cell infection of notoginseng
Sucking 100-200 mu L for carryingPnDSGene RNAi expression vector plasmid pHellsamgtte-PnDSThe activated LBA4404 Agrobacterium liquid is coated on an LB solid medium plate containing 50mg/L kanamycin and 25mg/L rifampicin (the step can be carried out simultaneously with the preculture of the panax notoginseng callus cells), and is inversely cultured in an incubator at 28 ℃ for 2-3 days until the plate is full of thick bacteria. Scraping the well-grown fungus mass with an inoculating needle to a MGL liquid culture medium containing 40mg/L acetosyringone, and performing shaking culture at 28 deg.C and 200rpm on a shaking table until the OD of the bacterial liquid 600 0.6-0.8, inoculating into the above Notoginseng radix callus cells pre-cultured for 3 days, completely immersing Notoginseng radix callus cells in bacterial liquid, and shake culturing on shaking table at 25 deg.C and 105rpm for 20min;
(3) Cell collection and Co-culture
After infection, carrying out suction filtration on the pseudo-ginseng callus cells by using a Buchner funnel to remove bacterial liquid, then carrying out suction drying on residual bacterial liquid on the surfaces of the pseudo-ginseng callus cells by using sterile filter paper, then transferring the pseudo-ginseng callus cells to a pseudo-ginseng co-culture medium with the surfaces paved with the sterile filter paper (preventing agrobacterium from directly contacting with the culture medium to cause overgrowth of the agrobacterium), and placing the pseudo-ginseng co-culture medium under the dark condition of 25 ℃ for co-culture for 3 days, wherein the pseudo-ginseng co-culture medium is the same as a pseudo-ginseng callus cell pre-culture medium.
(4) Sterilization culture
After the co-culture is finished, transferring the panax notoginseng callus cells into a sterilized beaker, washing the panax notoginseng callus cells for 5-6 times by using sterile water containing 400mg/L of cefomycin to fully remove agrobacterium, performing suction filtration on the panax notoginseng callus cells by using a Buchner funnel after the washing is finished, removing liquid, and then sucking residual liquid on the surfaces of the panax notoginseng callus cells by using sterile filter paper. The panax notoginseng callus cells are transferred to a sterilization culture medium (the panax notoginseng callus culture medium is added with cefamycin and kanamycin to the final concentration of 400mg/L and 50 mg/L), and the sterilization culture is carried out for 15 days at the temperature of 25 ℃ in the dark.
(5) Selection culture and subculture
Sterilizing for 15 days, transferring Notoginseng radix callus cells to Notoginseng radix callus screening culture medium (adding kanamycin to obtain final concentration of 50 mg/L), subculturing for about 35 days (subculture period can be adjusted according to growth condition), and screening for 4-5 times to obtain kanamycin resistant tissuePnDSGene RNAi transgenic notoginseng cell line.
Example 4: in transgenic cells of Notoginseng radixPnDSGene and detection of gene expression level related to synthesis of panax japonicus saponin
(1) Transgenic panax notoginseng cell line genome DNA level detection
Extracting transgenic notoginseng cell line genome DNA by improved CTAB method according to kanamycin on T-DNA in pHellsamge 2 carriernptII resistance gene sequence design upstream and downstream primersnpt-F (5'-CTCTGATGCCGCCGTGTT-3') andnpt-R (5'-CCCTGATGCTCTTCGTCCA-3'), PCR detection is carried out by taking the genomic DNA of the panax notoginseng as a template, and the panax notoginseng cells with positive transgenes are screened, the result is shown in figure 5, a specific band with the size of about 430bp is amplified in the figure and is consistent with the expected size, the results show that the six panax notoginseng transgenic cell lines are introduced with exogenous DNA and integrated on the genomic DNA for stable inheritance, and the initial determination obtainsPnDSGene RNAi transgenic cell lines.
(2) Fluorescent quantitative PCR detection of transgenic notoginseng cell
Extracting total RNA of the positive transgenic panax notoginseng cell line and the non-transgenic panax notoginseng cell line, and performing reverse transcription to obtain cDNA, wherein the specific operation steps are the same as those in the embodiment 1;
according to 18S rRNA gene (accession number: D85171.1), beta-amyrin synthase (beta-AS) gene (accession number: KP 658156), oleanolic acid synthase (CYP 716A52v 2) gene (accession number: JX 036032.1)Oleanolic acid glucuronic acid transferase (OAGT) gene (accession No. MH 819287.1), pjmUGT1 gene and PjmUGT2 gene were designed as primers for fluorescent quantitative PCR: 18S-F:5'-GGGGAGTATGGTCGCAAGG-3',18S-R:5'-CAGAACATCTAAGGGCATCACAG-3'; beta-AS-F: 5'-GTATTCCCTGTAGAGCATCGCAT-3', β -AS-R:5'-GGCACAGGCGTTGTTTTCAC-3'; CYP716A52v2-F:5'-AGGAGCAAATGGAGATAGTGA-3', CYP716A52v2-R:5'-GATTGAGAAACCGTTGTAGG-3'; OAGT-F:5'-GCATAATCTCGGACAAGTAC-3', OAGT-R: 5'-AAAGGTTGGGAGTCTGAAGT-3'; pjmUGT1-F:5'-TCACATAAATCCGATGGTCC-3', pjmUGT1-R:5'-AGAAATCCCTGAAATCCTCC-3'; pjmUGT2-F:5'-GCATTCTCCCTTTGTTTCAG-3', pjmUGT2-R:5'-CGACTTGCCTCACTCTTCCT-3'. And diluting the cDNA synthesized by reverse transcription by 5 times, namely diluting the 20 mu L cDNA to 100 mu L. Using the diluted cDNA as template, performing fluorescent quantitative PCR with the above primer, and performing quantitative PCR according to GoTaq ® The qPCRMaster Mix instructions were run and 3 replicates of each gene per sample were tested. By use of 2 -∆∆Ct The method processes the fluorescence quantitative data, analyzes and detectsPnDSThe results of the relative expression amount of the gene in the transgenic panax notoginseng cell line relative to that in the non-transgenic panax notoginseng cell line are shown in FIG. 6, and the results show that key enzymes in the selected T1-T4 four transgenic panax notoginseng cell linesPnDSThe expression level of the gene is obviously lower than that of the common panax notoginseng cell line, and the genes related to the synthesis of panax japonicus saponin IVa and panax japonicus saponin IVβ-AS、CYP716A52v2、OAGT、PjmUGT1AndPjmUGT2the relative expression quantity is obviously higher than that of the common panax notoginseng cell line.
Example 5: detection of saponin content in transgenic notoginseng cell
(1) Preparation of sample solution
Collecting callus cells of the transgenic panax notoginseng cell line and the common panax notoginseng cell line in the step (1) in the example 4, marking the callus cells respectively, placing the cells in an oven at 55 ℃, drying the cells (about 12 h), grinding the cells into powder fully after drying, weighing 0.5g of transgenic panax notoginseng cell line powder and 0.5g of common panax notoginseng cell line powder, placing the powder in a 50mL centrifuge tube respectively, adding 50mL of 70% methanol solution respectively, soaking the solution overnight, then carrying out crushing treatment (60W, ultrasonic for 4s, and intermittent for 2 s) by using ultrasonic waves for 1.5 to 2.0h until the panax notoginseng cell powder is fully crushed, transferring the obtained powder to a centrifuge for 30min at 4000rpm after the ultrasonic treatment, collecting supernatant, placing the supernatant in an oven at 50 to 55 ℃, drying the obtained product, dissolving the obtained product by using 10mL of distilled water, and carrying out n-butyl alcohol saturation extraction by using water with the same volume for 3 times. Collecting the extract, placing the extract in an oven at 50-55 ℃ for drying, dissolving the extract by using 70% methanol solution after drying, fixing the volume to 25mL, and filtering the solution by using a 0.45 mu M microporous filter membrane to obtain the saponin solution.
(2) High performance liquid chromatography
And (3) detecting the types and the contents of the saponins in the transgenic panax notoginseng cells by using HPLC (high performance liquid chromatography), wherein the HPLC detection conditions are as follows: the column was Waters-XTerra-MS-C18 (5 μm,250 mm. Times.4.6 mm, USA). The mobile phase was water and acetonitrile. Gradient elution: 0-20min,20% acetonitrile; 20-30min, 20-35% acetonitrile; 30-40min,35% acetonitrile; 40-50min,35-40% acetonitrile; 50-60min,40-100% acetonitrile. The flow rate is 1.0 mL/min, the column temperature is 30 ℃, and the detection wavelength is set to 203nm; the results are shown in fig. 7, and show that the transgenic panax notoginseng cells contain oleanane-type saponins (panax japonicus saponin iva and panax japonicus saponin iv), while the wild type unmodified panax notoginseng cells do not produce oleanane-type saponins (panax japonicus saponin iva and panax japonicus saponin iv).

Claims (2)

1. A method for synthesizing panax japonicus saponin in panax notoginseng cells is characterized in that: the genePnDSTransferring the fragment into Notoginseng radix cell to obtain Notoginseng radix cell for synthesizing rhizoma Panacis Japonici saponin, wherein the genePnDSThe nucleotide sequence of the fragment is shown as SEQ ID NO. 1.
2. The method for synthesizing panax japonicus saponin in panax notoginseng cells according to claim 1, wherein: the transgenic panax notoginseng cells can synthesize panax japonicus saponin IV and panax japonicus saponin IVa.
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