Method for biosynthesizing ginsenoside by using tobacco BY-2 cells as chassis

Abstract

The invention discloses a method for biosynthesizing ginsenoside by taking tobacco BY-2 cells as a chassis, and belongs to the technical field of bioengineering. The method comprises the following steps that firstly, a recombinant expression vector containing a ginsenoside synthesis pathway key gene set is constructed, and the gene set comprises genes such as tHMGR, SE and SS from ginseng and Pn1-31 and Pn3-31 genes from panax notoginseng; secondly, introducing the recombinant vector into tobacco BY-2 cells, and screening through a solid culture medium to obtain a pure positive cell line; and finally, culturing the positive cell line in a liquid or solid culture medium to obtain ginsenoside Rh2 and / or Rg3. By utilizing the advantages of fast growth and stable culture of the tobacco BY-2 cells, a rare ginsenoside synthesis pathway is successfully reconstructed, efficient heterologous synthesis of the rare ginsenoside is realized, and a repeatable technical scheme is provided for industrial production of the rare ginsenoside.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically involving genetic engineering, plant cell culture and natural product biosynthesis technology, and particularly a method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis. Background Technology

[0002] Ginsenosides are the main active secondary metabolites of plants in the genus Panax (such as ginseng, Panax notoginseng, and American ginseng) belonging to the Araliaceae family. They possess significant pharmacological activities, including anti-tumor, immunomodulatory, cardiovascular and cerebrovascular protection, and neuroprotective effects, and have wide applications in the pharmaceutical, health product, and cosmetic fields. Among them, rare ginsenosides (such as Rh2, Rg3, and CK) have become a hot topic in natural product research in recent years due to their higher bioavailability and stronger pharmacological activity.

[0003] However, the content of rare ginsenosides in natural ginseng plants is extremely low, usually only one ten-thousandth to one hundred-thousandth. Furthermore, due to factors such as the long growth cycle of plants (3-5 years), the great influence of cultivation environment (soil, climate), and the scarcity of resources, the traditional method of obtaining rare ginsenosides by directly extracting natural plants has problems such as low efficiency, high cost, and serious resource dependence, making it difficult to meet the needs of industrial production and the market.

[0004] To overcome the limitations of natural sources, researchers have attempted to produce rare ginsenosides through techniques such as chemical synthesis, heterologous microbial synthesis, or plant cell engineering. While chemical synthesis can achieve the artificial preparation of some saponins, it suffers from problems such as cumbersome reaction steps, poor stereoselectivity, severe organic solvent pollution, and residual toxicity of the products, making it difficult to apply to the production of pharmaceutical-grade products. Heterologous microbial synthesis (such as using E. coli and yeast as chassis) improves synthesis efficiency to some extent by introducing key synthetic genes to construct metabolic pathways. However, as prokaryotes or lower eukaryotes, microorganisms lack the complex post-translational modification systems unique to plants, resulting in poor metabolic pathway compatibility, low product accumulation, and difficulty in synthesizing structurally complex rare ginsenosides.

[0005] Plant cell culture technology, due to the complete eukaryotic metabolic system of plant cells, can precisely realize the hydroxylation, glycosylation and other modification reactions required for saponin synthesis, making it an ideal platform for the biosynthesis of rare ginsenosides. Although currently reported plant cell chassis (such as ginseng suspension cells and Panax notoginseng hairy roots) can synthesize some ginsenosides, they suffer from problems such as slow growth, low genetic transformation efficiency, poor product stability, and difficulty in large-scale culture, which limit their industrial application.

[0006] Therefore, developing a method for constructing an efficient and stable biosynthetic system for rare ginsenosides through genetic engineering is of great significance for overcoming the limitations of natural resources, reducing production costs, and realizing the industrial production of rare ginsenosides. Summary of the Invention

[0007] The purpose of this invention is to address the problems of scarce natural sources of rare ginsenosides, low efficiency and poor stability of existing synthesis methods in the background art. By using genetic engineering to reconstruct a complete ginsenoside synthesis pathway in tobacco BY-2 cells, this invention provides a highly efficient and stable method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis includes the following steps: (1) Construction of recombinant expression vector: The key gene set of ginsenoside synthesis pathway was introduced into the basic vector pYLTAC380GW to obtain the recombinant expression vector. pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI; the key gene set includes the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMGR, squalene epoxidase gene SE, squalene synthase gene SS, dammarene diol synthase gene PgDDS, cytochrome P450 gene PgPPDs, farnesyl pyrophosphate synthase gene FPS, isopentenyl pyrophosphate isomerase gene IDI, and the genes Pn1-31 and Pn3-31 from Panax notoginseng; (2) Transformation of tobacco BY-2 cells: The recombinant expression vector described in step (1) was introduced into tobacco BY-2 cells, an invasion staining solution was added, and the cells were cultured in the dark for 2 days; the cells were collected and washed with liquid culture medium. (3) Screening of positive cell lines: After washing, the cells were spread on a solid culture medium and cultured in the dark for 2 weeks. Callus tissue was then transferred to a solid culture medium for screening to obtain pure transgenic positive cell lines. (4) Ginsenoside synthesis culture: Positive cell lines were inoculated into tobacco BY-2 cell liquid or solid culture medium, and the culture was collected and tested to obtain ginsenosides.

[0009] Further, in step (2), the recombinant expression vector was introduced into tobacco BY-2 cells via Agrobacterium-mediated transformation.

[0010] Furthermore, the ginsenosides mentioned are ginsenosides Rh2 and / or Rg3.

[0011] Furthermore, the construction of the recombinant expression vector in step (1) includes: (1) Gene acquisition: The original gene sequences of FPS, IDI, SS, SE, PgDDS, PgPPDs, Pn1-31, Pn3-31 and HMGR were obtained from the NCBI database. tHMGR was obtained by signal peptide prediction. The above 9 gene fragments were amplified and recovered. (2) Construction of intermediate carrier: ① The donor plasmid pYL322d1 was digested with restriction endonucleases KpnI and SalI. The tHMGR, SS, and Pn3-31-Pn1-31 gene fragments were then homologously recombinated with the digested pYL322d1 to obtain the pYL322d1-A donor vector. ② The donor plasmid pYL322d2 was digested with restriction endonucleases KpnI and PstI. The SE, PgDDS-PgPPDs gene fragments were then homologously recombinated with the digested pYL322d2 to obtain the pYL322d2-B donor vector. ③ The donor plasmid pYLMF-H was digested with restriction endonucleases NcoI and HindIII, and the FPS-IDI gene fragment was homologously recombined with the digested pYLMF to obtain the pYLMF-FPS-IDI donor vector. (3) Assembly of recombinant expression vector: ① The pYLTAC380GW backbone vector and the pYL322d1-A donor vector were co-transformed into Escherichia coli NS3529. After screening with double antibiotics of kanamycin and chloramphenicol, and verification by I-SceI and NotI enzyme digestion, the pYLTAC380GW-tHMGR vector was obtained. ② The pYLTAC380GW-tHMGR vector and the pYL322d2-B donor vector were co-transformed into Escherichia coli NS3529. After screening with double antibiotics of kanamycin and ampicillin, and verification by PI-SceI and NotI restriction enzyme digestion, the pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 vector was obtained. ③ Take the vector obtained in step ② and the pYLMF-FPS-IDI donor vector, add GatewayBPClonaseIIEnzymeMIX for recombination reaction, screen for kanamycin resistance and 5% sucrose, and verify by enzyme digestion to obtain the final recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI.

[0012] Furthermore, the infection solution is: 10mM MES, 10mM MgCl2, sterilized at 115℃ for 20 minutes under high temperature and pressure, and then cooled before adding 150μM AS.

[0013] Furthermore, the tobacco BY-2 cell liquid culture medium consisted of MS Salt 4.4 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 0.255 g / L, pH adjusted to 5.0, autoclaved at 115°C for 20 minutes, cooled to 65°C, and then 1000× sterile hormone stock solution was added. The hormone stock solution contained 0.2 mg / mL 2,4-D, 1 mg / mL thiamine, and 100 mg / mL myo-inositol.

[0014] Furthermore, the solid culture medium for tobacco BY-2 cells was prepared as follows: MS Salt 4.4 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 0.255 g / L, pH adjusted to 5.8, 7 g / L plant gel added, autoclaved at 115°C for 20 minutes, cooled to 65°C, and then 1000× sterile hormone stock solution was added. The hormone stock solution contained 0.2 mg / mL 2,4-D, 1 mg / mL thiamine, and 100 mg / mL myo-inositol.

[0015] Furthermore, the solid culture medium contains 50 mg / L hygromycin (Hygr) and 200 mg / L termethin.

[0016] Furthermore, the liquid culture conditions in step (4) are 25°C and 130 rpm shaker culture for 7-14 days, and the solid culture conditions are 25°C and dark culture for 14 days.

[0017] Furthermore, in step (2), the volume ratio of the infection solution to BY-2 cells is 3:50, and the centrifugation collection conditions are 180g centrifugation for 2 minutes.

[0018] Furthermore, the positive cell lines described in step (3) must be identified by PCR and contain the complete set of tHMGR, SE, SS, PgPPDs, PgDDS, Pn1-31, Pn3-31, FPS, and IDI genes.

[0019] A method for obtaining ginsenosides by biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis.

[0020] This application achieves the following core technical effects through a technical solution for the biosynthesis of rare ginsenosides using tobacco BY-2 cells as a chassis: 1. Overcoming the limitations of natural resources to achieve heterologous synthesis of rare ginsenosides We successfully reconstructed the complete synthetic pathway from basal metabolic precursors to rare ginsenosides Rh2 and Rg3 in tobacco BY-2 cells, freeing us from dependence on natural ginseng plants and solving the problems of extremely low content and difficulty in obtaining natural saponins.

[0021] 2. Utilize chassis advantages to improve synthesis efficiency and stability Based on the characteristics of tobacco BY-2 cells, such as rapid growth rate, stable suspension culture, and high genetic transformation efficiency, the positive cell lines obtained through screening can stably synthesize the target saponins.

[0022] 3. Optimize the cultivation and screening system to ensure controllable product production. By clarifying the culture medium formula and culture conditions, efficient screening and stable subculturing of transgenic cell lines were achieved, solving the problem of unstable product accumulation in heterologous synthesis and providing a reproducible technical solution for industrial-scale production. Attached Figure Description

[0023] Figure 1 Electrophoresis diagram of PCR amplification of key genes in the ginsenoside synthesis pathway; Figure 2 : PCR identification results of pYL322d1-tHMGR, -SS, -Pn3-31-Pn1-31 bacterial culture; Figure 3 : Positive PCR identification results for pYL322d2-SE and PgDDS-PgPPDs bacterial cultures; Figure 4 : pYLMF-FPS-IDI bacterial culture PCR positive identification result; Figure 5 : The bacterial culture PCR of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31 was positive. Figure 6 Electrophoresis image of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31NotI restriction enzyme digestion; Figure 7 Electrophoresis image of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31-FPS-IDI bacterial culture for positive PCR identification; Figure 8 Electrophoresis image of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31-FPS-IDINotI enzyme digestion; Figure 9PCR identification results of positive clones after BY-2 cell transformation (lane labels: positive CK is the recombinant plasmid control, negative CK is wild-type BY-2 cells, 1-6 are candidate positive cell lines). Figure 10: Rh2 (top) and Rg3 (bottom) content in liquid culture of positive cell lines; Figure 11: PCR identification results of the target gene after subculture of positive cell lines (lanes T1-T3 and T7 are positive cell lines after different subcultures). Detailed Implementation

[0024] The present application will be further described below through specific embodiments.

[0025] Example 1: Ginsenoside Synthesis Carrier Construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI: I. Experimental Materials Donor plasmids: pYL322d1, pYL322d2, pYLMF-H, pYLTAC380GW; Restriction endonucleases: KpnI, SalI, PstI, NcoI, HindIII, I-SceI, PI-SceI, NotI (purchased from NEB); Enzymes: Gateway BP Clonase II Enzyme MIX (purchased from Thermo Fisher Scientific), high-fidelity DNA polymerase (purchased from Takara Scientific); Escherichia coli strains: NS3529 competent cells and NEB-10β competent cells (purchased from Tiangen Biotech Co., Ltd.). Culture media: LB solid medium (containing 25 mg / L kanamycin Kan, 15 mg / L chloramphenicol ChI or 15 mg / L ampicillin Amp), LB liquid medium.

[0026] Key target gene recombination primers and PCR procedure: Table 1. Primers for homologous recombination of key target genes

[0027] PCR reaction was performed using the Takara high-fidelity enzyme PrimeSTAR: Prepare a 50 μL PCR reaction mixture (2 μL template, 1 μL each of forward and reverse primers, 25 μL 2×Taq Mix, and ddH2O to bring the total volume to 50 μL). Reaction conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing temperature and extension time are shown in Table 2, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 3 min.

[0028] Table 2 PCR Procedure for Target Gene Amplification

[0029] Validation of primers and PCR procedures during multi-fragment construction: 1. The validation primer sequences involved in the multi-fragment construction process are shown in Table 3. Table 3. Validation primers for pYLTAC380GW in each round.

[0030] 2: PCR procedures for each round of validation during multi-fragment construction: Pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55-65℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; final extension at 72℃ for 2 min.

[0031] II. Acquisition and Amplification of Key Genes 1. Retrieved key gene sequences of the ginsenoside synthesis pathway from the NCBI database: Artemisia annua farnesyl pyrophosphate synthase gene (AaFPS), Arabidopsis thaliana isopentenyl pyrophosphate isomerase gene (AtIDI), ginseng squalene synthase gene (PgPSS, i.e. SS), ginseng squalene epoxidase gene (PgSE, i.e. SE), ginseng damarene diol synthase gene (PgDDS), ginseng cytochrome P450 gene (PgPPDS), Panax notoginseng genes Pn1-31 and Pn3-31, and ginseng 3-hydroxy-3-methylglutaryl-CoA reductase gene (HMGR).

[0032] 2. The N-terminal signal peptide of the HMGR gene was predicted using the online signal peptide prediction software SignalP5.0. After truncating the signal peptide coding sequence, the truncated gene tHMGR was obtained without cell localization.

[0033] 3. Using ginseng and Panax notoginseng cDNA as templates, specific primers were designed to amplify the above eight key genes (tHMGR, SE, SS, PgDDS, PgPPDs, Pn1-31, Pn3-31, FPS-IDI) by PCR. The PCR products were verified by agarose gel electrophoresis, and the results showed that the amplified fragment sizes were consistent with expectations. (See attached image) Figure 1 Electrophoresis images of PCR amplification of key genes in the ginsenoside synthesis pathway were obtained, and the target fragments were recovered for later use.

[0034] III. Construction of the intermediate donor carrier 1: Construction of donor vector pYL322d1-A (1) Take the donor plasmid pYL322d1 and double digest it with restriction endonucleases KpnI and SalI (react at 37℃ for 2h). The digestion product is recovered by agarose gel electrophoresis to restore the linearized vector backbone.

[0035] (2) Linearized pYL322d1 was ligated with the recovered tHMGR, SS, and Pn3-31-Pn1-31 gene fragments using homologous recombinase. The reaction system was: 50 ng of linearized vector, 100 ng of each gene fragment, 2 μL of recombinase, and ddH2O to 10 μL. The reaction was carried out at 37℃ for 30 min.

[0036] (3) The recombinant product was transformed into *E. coli* NEB-10 β competent cells, plated on LB agar containing Kan (25 mg / L), and incubated overnight at 37°C. Single colonies were picked for colony PCR identification. The results showed that positive clones amplified bands of the same size as the target gene. Figure 2 The positive PCR results of pYL322d1-tHMGR, -SS, and -Pn3-31-Pn1-31 bacterial cultures indicate that the extracted plasmids are the pYL322d1-A donor vector.

[0037] 2. Construction of donor vector pYL322d2-B (1) Take the donor plasmid pYL322d2, digest it with restriction endonucleases KpnI and PstI (react at 37℃ for 2h), and recover the linearized vector backbone;

[0038] (2) Linearized pYL322d2 was ligated with the recovered SE, PgDDS-PgPPDs gene fragments via homologous recombination, under the same reaction conditions as the construction of the donor vector pYL322d1-A;

[0039] (3) Transformed *E. coli* NEB-10β competent cells, plated on LB solid medium containing Kan (25 mg / L), and incubated overnight at 37°C. Colony PCR identification showed that positive clones amplified the target band, see [see details]. Figure 3 The positive PCR results of pYL322d2-SE and PgDDS-PgPPDs bacterial cultures indicate that the extracted plasmid is the pYL322d2-B donor vector.

[0040] 3. Construction of donor vector pYLMF-FPS-IDI (1) Take the donor plasmid pYLMF-H, digest it with restriction endonucleases NcoI and HindIII (react at 37℃ for 2h), and recover the linearized vector backbone.

[0041] (2) Linearized pYLMF-H and the recovered FPS-IDI gene fragment were ligated by homologous recombination under the same conditions as the construction of the donor vector pYL322d1-A.

[0042] (3) Transformed *E. coli* NEB-10β competent cells, plated on LB solid medium containing Kan (25 mg / L), and incubated overnight at 37°C. Colony PCR identification showed that positive clones amplified the target band, see [see details]. Figure 4 The pYLMF-FPS-IDI bacterial culture PCR positive identification result indicates that the extracted plasmid is the pYLMF-FPS-IDI donor vector.

[0043] IV. Multiple rounds of assembly of recombinant expression vectors 1. First round of assembly: Construction of pYLTAC380GW-tHMGR (1) Take 300 ng pYLTAC380GW plasmid and 500 ng pYL322d1-A plasmid and mix them together to transform into Escherichia coli NS3529 competent cells. Spread the mixture on LB solid medium containing Kan (25 mg / L) and ChI (15 mg / L) and incubate at 37℃ for 72 h.

[0044] (2) Collect colonies and extract plasmids. Take 50 ng of plasmid, add 1 μL of L-SceI enzyme and 1 μL of buffer, and add ddH2O to make up to 10 μL. React at 37℃ for 5 h. Transform the reaction product into Escherichia coli NEB-10β competent cells, spread on LB solid medium containing Kan (25 mg / L), and culture overnight at 37℃.

[0045] (3) Single colonies were selected for PCR screening. Positive clones were amplified and plasmids were extracted. NotI restriction enzyme digestion was used for verification (37℃ for 30 min). Agarose gel electrophoresis (150V, 2-3 h, on ice) showed the expected fragments. See [link to relevant documentation]. Figure 5 The bacterial culture PCR of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31 showed a positive result, and the plasmid pYLTAC380GW-tHMGR was obtained.

[0046] 2. Second round assembly: Construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 (1) Take 300ngpYLTAC380GW-tHMGR plasmid and 500ngpYL322d2-B plasmid, mix them, transform them into Escherichia coli NS3529 competent cells, spread them on LB solid medium containing Kan (25mg / L) and Amp (15mg / L), and incubate at 37℃ for 48h.

[0047] (2) Collect colonies and extract plasmids. Take 50 ng of plasmid, add 1 μL of PI-SceI enzyme and 1 μL of buffer, and add ddH2O to make up to 10 μL. React at 37℃ for 5 h. Transform the reaction product into Escherichia coli NEB-10β competent cells, spread on LB solid medium containing Kan (25 mg / L), and culture overnight at 37℃.

[0048] (3) Positive clones were screened by PCR and verified by NotI restriction enzyme digestion, see Figure 6 The plasmid pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 was obtained.

[0049] 3. Final assembly: Construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI (1) Take 150 ng pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 plasmid, 100 ng pYLMF-FPS-IDI plasmid and 1 μL Gateway BP ClonaseII Enzyme MIX, add ddH2O to make up to 5 μL, react at 25℃ for 5 h; add 2 μL Proteinase K, react at 37℃ for 15 min to terminate the reaction.

[0050] (2) The reaction product was transformed into Escherichia coli NEB-10β competent cells, spread on LB solid medium containing Kan (25 mg / L) and 5% sucrose, and cultured overnight at 37°C.

[0051] (3) Single colonies were picked for bacterial culture PCR identification. Electrophoresis showed a positive band. (See...) Figure 7 The extracted plasmid was digested with NotI enzyme for verification, and the results showed that all gene fragments were correctly inserted. (See attached image.) Figure 8 Finally, the recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI was obtained.

[0052] Example 2: Transformation of Tobacco BY-2 Cells and Screening of Positive Cell Lines I. Experimental Materials Tobacco BY-2 cells: Wild-type BY-2 cell line preserved in the laboratory; Recombinant expression vector: constructed in Example 1 pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI; Agrobacterium strain: GV3101 (containing vir helper plasmid, preserved in the laboratory). Culture media: BY-2 liquid standard medium and BY-2 solid screening medium (containing 50 mg / L hygromycin (Hygr) and 200 mg / L termethin (TM)), the formulation is as described in Example 1; Reagents: sterile water, ddH2O, DNA extraction kit (purchased from Tiangen Biotech Co., Ltd.), PCR reagent (purchased from Takara Corporation); Infection solution formula: 10mM MES, 10mM MgCl2, autoclaved at 115℃ for 20 minutes, and 150μM AS (acetylsyleugenol) added after cooling.

[0053] II. Suspension Culture and Pretreatment of Tobacco BY-2 Cells 1. Take 1-2g of wild-type BY-2 cells that are growing normally on the plate, gently crush them with tweezers, disperse them into a 250mL wide-mouth conical flask containing 50mL of BY-2 liquid standard culture medium, seal the flask with a sterile breathable membrane, and place it on a shaker at 25℃ for 7 days at 130rpm. 2. Passage according to the subculture protocol: Take 5 mL of BY-2 suspension cells cultured to day 7 and add them to a 250 mL wide-mouth conical flask containing 45 mL of fresh BY-2 liquid standard culture medium. Continue culturing at 25℃ and 130 rpm for 3-5 days to obtain a healthy BY-2 cell line.

[0054] III. Agrobacterium-mediated transformation of tobacco BY-2 cells using recombinant vectors 1. Agrobacterium pretreatment: Agrobacterium GV3101 containing the recombinant expression vector was inoculated into LB liquid medium containing 50 mg / L Kan, cultured at 30℃ and 220 rpm until OD600 = 0.5-0.8, centrifuged at 5000 rpm for 5 minutes to collect the cells, and resuspended with the above infection solution until OD600 = 0.6 for later use; 2. Infection treatment: Take the BY-2 cell line cultured for 3-5 days, mix it with Agrobacterium infection solution at a volume ratio of 3:50, transfer it to a 250mL wide-mouth conical flask, seal it with a sterile breathable membrane, and incubate it in the dark at 25℃ and 130rpm on a shaker for 2 days. 3. Cell washing: After 48 hours, transfer the mixture to a 50 mL centrifuge tube, centrifuge at 180 g for 2 minutes to collect the cells, and discard the supernatant; wash the cells 3 times with BY-2 liquid standard medium (centrifuge at 180 g for 2 minutes after each wash) to completely remove residual Agrobacterium.

[0055] IV. Screening and purification of positive cell lines 1. Spread the washed cells evenly on BY-2 solid selection medium (containing 50 mg / L Hygr and 200 mg / L TM), seal with a sterile breathable membrane, and incubate in the dark at 25°C. 2. After 2 weeks of culture, select bright yellow callus tissues in good condition on the culture medium and transfer them to a new BY-2 solid screening medium for a second round of screening. Incubate in the dark at 25°C for 2 weeks. 3. Repeat the screening step 1-2 times to obtain pure transgenic positive cell lines with uniform morphology and stable growth.

[0056] V. PCR Identification of Positive Cell Lines 1. Take wild-type BY-2 cells (negative control) and the selected positive cell lines respectively, and extract genomic DNA using a DNA extraction kit, adjusting the concentration to 200 ng / μL; 2. Using the extracted DNA as a template, design specific primers for the tHMGR, SE, SS, PgPPDs, PgDDS, Pn1-31, Pn3-31, FPS, and IDI genes. Prepare a 50 μL PCR reaction system (2 μL template, 1 μL each of forward and reverse primers, 25 μL 2×Taq Mix, and ddH2O to 50 μL). Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min. 3. PCR products were detected by 1% agarose gel electrophoresis. The results showed that positive cell lines all amplified bands of the same size as the target gene, while wild-type cells did not show corresponding bands. (See attached image) Figure 9 This indicates that the target gene set has been integrated into the genome of the tobacco BY-2 cells.

[0057] Example 3: Culture and detection of ginsenoside synthesis by the tobacco BY-2 positive cell line I. Experimental Materials Positive cell lines: Positive cell lines 1, 2, 3, and 7 obtained from screening in Example 2; Culture media: BY-2 liquid standard culture medium, BY-2 solid standard culture medium (formula same as in Example 2); Reagents: Ginsenoside Rh2, Rg3, Rd standards (purchased from Sigma), methanol (chromatographic grade), acetonitrile (chromatographic grade), and ultrapure water.

[0058] II. Expanded Culture of Positive Cell Lines 1. Liquid culture: Take 1-2g of callus tissue from positive cell lines 1, 2, 3, and 7, and inoculate them into 250mL wide-mouth conical flasks containing 50mL BY-2 liquid standard medium. Culture at 25℃ and 130rpm in a shaker. Subculture every 7 days and collect cell samples after 5d, 7d, and 14d of culture. 2. Solid culture: Take 0.5g of callus tissue from each of the positive cell lines 1, 2, 3 and 7, and inoculate them onto BY-2 solid standard medium. Incubate in the dark at 25℃ for 14 days, and collect callus tissue samples.

[0059] III. Extraction and Detection of Ginsenosides ① Sample pretreatment: Collected cell or callus samples, freeze-dry and grind into powder. Accurately weigh 100 mg of powder, add 1 mL of 70% methanol solution, and extract by sonication for 30 min (power 300 W, temperature 40 ℃). Centrifuge at 4 ℃ and 12000 rpm for 10 min, and take the supernatant and filter it through a 0.22 μm organic filter membrane for analysis. ②HPLC detection conditions: chromatographic column: C18 column (250×4.6mm, 5μm); mobile phase: acetonitrile:water = 30:70 (v / v); flow rate: 1mL / min; detection wavelength: 203nm; column temperature: 30℃; injection volume: 10μL; ③ Standard curve preparation: Prepare Rh2, Rg3, and Rd standard solutions of 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL respectively, and detect them under the above chromatographic conditions. Plot the standard curve with the standard concentration as the abscissa and the peak area as the ordinate (R² > 0.999 for all).

[0060] IV. Analysis of Test Results ① Liquid culture samples: Positive cell lines 1, 2, and 3 showed the highest Rh2 content (approximately 10 μg / g) and Rg3 content (approximately 5 μg / g) after 14 days of culture. With prolonged culture time, Rd content gradually decreased, while Rh2 content peaked at 14 days. (See attached image) Figure 10 ; ② Solid culture samples: The Rg3 content in the callus of positive cell lines was significantly higher than that in liquid culture cells. Among them, the Rg3 content in the callus of cell line 2 reached 150 μg / g, while Rh2 and Rd were not detected or had extremely low content. Table 4: Detection results of ginsenosides Rh2, Rg3, and Rd in tobacco BY-2 callus

[0061] ③ Stability verification: After five subcultures, the positive cell lines still showed a complete gene set upon PCR identification. (See...) Figure 11HPLC analysis showed that the content of Rg3 and Rh2 fluctuated by less than 10%, indicating that the synthesis system has good stability.

[0062] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Equivalent changes or modifications made to the features and principles described in the claims of this invention should be included within the scope of this invention.

Claims

1. A method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis, characterized in that, Includes the following steps: (1) Construction of recombinant expression vector: The key gene set of ginsenoside synthesis pathway was introduced into the vector pYLTAC380GW to obtain the recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI; (2) Transformation of tobacco BY-2 cells: The recombinant expression vector described in step (1) was introduced into tobacco BY-2 cells, an invasion staining solution was added, the cells were cultured in the dark, and the cells were collected. (3) Screening for positive cell lines; (4) Ginsenoside synthesis culture: Positive cell lines were inoculated into tobacco BY-2 cell liquid or solid culture medium and cultured. The culture was collected to obtain ginsenosides.

2. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, The set of key genes for the ginsenoside synthesis pathway includes the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMGR, squalene epoxidase gene SE, squalene synthase gene SS, dammarene diol synthase gene PgDDS, cytochrome P450 gene PgPPDs, farnesyl pyrophosphate synthase gene FPS, isopentenyl pyrophosphate isomerase gene IDI, and genes Pn1-31 and Pn3-31 from Panax notoginseng.

3. The method according to claim 2, characterized in that, The construction of the recombinant expression vector in step (1) includes: (1) Gene acquisition: The original gene sequences of FPS, IDI, SS, SE, PgDDS, PgPPDs, Pn1-31, Pn3-31 and HMGR were obtained from the NCBI database. tHMGR was obtained by signal peptide prediction. The above 9 gene fragments were amplified and recovered. (2) Construction of intermediate carrier: ① The donor plasmid pYL322d1 was digested with restriction endonucleases KpnI and SalI. The tHMGR, SS, and Pn3-31-Pn1-31 gene fragments were then homologously recombinated with the digested pYL322d1 to obtain the pYL322d1-A donor vector. ② The donor plasmid pYL322d2 was digested with restriction endonucleases KpnI and PstI. The SE, PgDDS-PgPPDs gene fragments were then homologously recombinated with the digested pYL322d2 to obtain the pYL322d2-B donor vector. ③ The donor plasmid pYLMF-H was digested with restriction endonucleases NcoI and HindIII, and the FPS-IDI gene fragment was homologously recombined with the digested pYLMF to obtain the pYLMF-FPS-IDI donor vector. (3) Assembly of recombinant expression vector: ① The pYLTAC380GW backbone vector and the pYL322d1-A donor vector were co-transformed into Escherichia coli NS3529. After screening with double antibiotics of kanamycin and chloramphenicol, and verification by I-SceI and NotI enzyme digestion, the pYLTAC380GW-tHMGR vector was obtained. ② The pYLTAC380GW-tHMGR vector and the pYL322d2-B donor vector were co-transformed into Escherichia coli NS3529. After screening with double antibiotics of kanamycin and ampicillin, and verification by PI-SceI and NotI restriction enzyme digestion, the pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 vector was obtained. ③ Take the vector obtained in step ② and the pYLMF-FPS-IDI donor vector, add GatewayBPClonaseIIEnzymeMIX for recombination reaction, screen for kanamycin resistance and 5% sucrose, and verify by enzyme digestion to obtain the final recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI.

4. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, The infection solution consisted of 10 mM MES and 10 mM MgCl2, sterilized at 115°C for 20 minutes under high temperature and pressure, and then 150 μM MES was added after cooling.

5. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, The tobacco BY-2 cell liquid culture medium consisted of MS Salt 4.4 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 0.255 g / L, pH adjusted to 5.0, autoclaved at 115°C for 20 minutes, cooled to 65°C, and then 1000× sterile hormone stock solution was added. The hormone stock solution contained 0.2 mg / mL 2,4-D, 1 mg / mL thiamine, and 100 mg / mL inositol.

6. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, Tobacco BY-2 cell solid culture medium: MS Salt 4.4 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 0.255 g / L, adjust pH to 5.8, add 7 g / L plant gel, autoclave at 115℃ for 20 minutes, cool to 65℃ and add 1000× sterile hormone stock solution, the hormone stock solution containing 0.2 mg / mL 2,4-D, 1 mg / mL thiamine and 100 mg / mL inositol.

7. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 6, characterized in that, The solid culture medium contains 50 mg / L hygromycin and 200 mg / L termethin.

8. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, Step (4) Liquid culture conditions are 25℃ and 130rpm shaker culture for 7-14 days, and solid culture conditions are 25℃ dark culture for 14 days.

9. The method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis according to claim 1, characterized in that, In step (2), the volume ratio of the infection solution to BY-2 cells is 3:50, and the centrifugation collection conditions are 180g centrifugation for 2 minutes.

10. Ginsenosides obtained by a method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis, according to any one of claims 1-9.

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