A method for biosynthesis of ginsenosides by using tobacco BY-2 cells as a chassis

Abstract

The application discloses a method for biosynthesizing ginsenoside by using tobacco BY-2 cells as a chassis, and belongs to the technical field of bioengineering. The method comprises the following steps: firstly, a recombinant expression vector containing a key gene set of a ginsenoside synthesis pathway is constructed, wherein the gene set comprises tHMGR, SE, SS and other genes derived from Panax ginseng and Pn1-31 and Pn3-31 genes derived from Panax notoginseng; secondly, the recombinant vector is introduced into tobacco BY-2 cells, and a pure positive cell line is obtained through solid culture medium screening; finally, the positive cell line is cultured in liquid or solid culture medium to obtain ginsenoside Rh2 and / or Rg3. The application successfully reconfigures a rare ginsenoside synthesis pathway by taking advantage of the fast growth and stable culture of tobacco BY-2 cells, realizes efficient heterologous synthesis of the rare ginsenoside, and provides a repeatable technical scheme for industrial production of the rare ginsenoside.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to genetic engineering, plant cell culture and natural product biosynthesis technology, and more particularly to a method for biosynthesis of ginsenosides by using tobacco BY-2 cells as a chassis. BACKGROUND

[0002] Ginsenosides are the main active secondary metabolites of Panax plants (such as Panax ginseng, Panax notoginseng and Panax quinquefolium), and have significant pharmacological activities, including anti-tumor, immune regulation, cardiovascular and cerebrovascular protection, and neuroprotection, and have wide application value in the fields of medicine, health care products and cosmetics. Among them, rare ginsenosides (such as Rh2, Rg3 and CK) have higher bioavailability and stronger pharmacological activities, and have become the focus of natural product research in recent years.

[0003] However, the content of rare ginsenosides in natural Panax plants is extremely low, usually only one ten-thousandth to one hundred-thousandth, and is limited by factors such as long plant growth cycle (3-5 years), large influence of cultivation environment (soil, climate), and resource scarcity. The traditional method of obtaining rare ginsenosides by directly extracting natural plants has problems such as low efficiency, high cost, and serious resource dependence, and cannot meet the needs of industrial production and market demand.

[0004] In order to break through the limitations of natural sources, researchers have tried to produce rare ginsenosides through chemical synthesis, microbial heterologous synthesis or plant cell engineering. Although chemical synthesis can achieve the artificial preparation of some ginsenosides, it has problems such as complicated reaction steps, poor stereoselectivity, serious pollution by organic solvents, and toxic residues of products, which makes it difficult to be applied to the production of medicinal grade products. Although microbial heterologous synthesis (such as using Escherichia coli and yeast as chassis) can improve the synthesis efficiency to some extent by introducing key synthesis genes to construct metabolic pathways, as prokaryotes or lower eukaryotes, microorganisms lack the complex post-translational modification system unique to plants, resulting in poor compatibility of metabolic pathways, low accumulation of products, and difficulty in synthesizing complex rare ginsenosides.

[0005] Plant cell culture technology has become an ideal platform for the biosynthesis of rare ginsenosides because plant cells have a complete eukaryotic metabolic system and can accurately achieve modification reactions such as hydroxylation and glycosylation required for ginsenoside synthesis. Although the plant cell chassis (such as Panax ginseng suspension cells and Panax notoginseng hairy roots) reported so far can synthesize some ginsenosides, it has problems such as slow growth, low genetic transformation efficiency, poor product stability, and difficulty in large-scale culture, which limits its industrial application.

[0006] Therefore, developing a method for constructing an efficient and stable rare ginsenoside biosynthesis system through genetic engineering is of great significance for breaking through the limitation of natural resources, reducing production costs, and realizing the industrialized production of rare ginsenosides. SUMMARY

[0007] The present application aims to solve the problems of the natural scarcity of rare ginsenosides, the low efficiency and poor stability of existing synthesis methods in the background art, and provides a method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis by reconstructing the complete ginsenoside synthesis pathway in tobacco BY-2 cells through genetic engineering means.

[0008] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0009] A method for biosynthesizing ginsenosides using tobacco BY-2 cells as a chassis, comprising the following steps:

[0010] (1) Constructing a recombinant expression vector: introducing a key gene set of ginsenoside synthesis pathway into a basic vector pYLTAC380GW to obtain a recombinant expression vector

[0011] pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI; the key gene set includes truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene tHMGR, squalene epoxidase gene SE, squalene synthase gene SS, dammarenediol synthase gene PgDDS, cytochrome P450 gene PgPPDs, farnesyl pyrophosphate synthase gene FPS, isopentenyl pyrophosphate isomerase gene IDI, and genes Pn1-31 and Pn3-31 derived from Panax notoginseng;

[0012] (2) Transforming tobacco BY-2 cells: introducing the recombinant expression vector of step (1) into tobacco BY-2 cells, adding infection solution, and dark incubating for 2 days; collecting cells and washing cells with liquid medium;

[0013] (3) Positive cell line screening: spreading the washed cells on solid culture medium, dark incubating for 2 weeks, picking callus and transferring to solid culture medium for screening to obtain pure transgenic positive cell line;

[0014] (4) Ginsenoside synthesis culture: inoculating the positive cell line into tobacco BY-2 cell liquid or solid culture medium for culture, collecting the culture and detecting to obtain ginsenosides.

[0015] Further, the recombinant expression vector of step (2) is introduced into tobacco BY-2 cells by Agrobacterium-mediated method.

[0016] Further, the ginsenoside is ginsenoside Rh2 and / or Rg3.

[0017] Further, the construction of the recombinant expression vector in step (1) comprises:

[0018] (1) Gene acquisition: obtain the original gene sequences of FPS, IDI, SS, SE, PgDDS, PgPPDs, Pn1-31, Pn3-31 and HMGR from the NCBI database, obtain tHMGR through signal peptide prediction, amplify and recover the above 9 gene fragments;

[0019] (2) Intermediate vector construction:

[0020] ① Use restriction endonuclease KpnI and SalI to double enzyme cut the donor plasmid pYL322d1, and perform homologous recombination of tHMGR, SS, Pn3-31-Pn1-31 gene fragments and the enzyme cut pYL322d1 to obtain pYL322d1-A donor vector;

[0021] ② Use restriction endonuclease KpnI and PstI to double enzyme cut the donor plasmid pYL322d2, and perform homologous recombination of SE, PgDDS-PgPPDs gene fragments and the enzyme cut pYL322d2 to obtain pYL322d2-B donor vector;

[0022] ③ Use restriction endonuclease NcoI and HindIII to double enzyme cut the donor plasmid pYLMF-H, and perform homologous recombination of FPS-IDI gene fragments and the enzyme cut pYLMF to obtain pYLMF-FPS-IDI donor vector;

[0023] (3) Recombinant expression vector assembly:

[0024] ① Take pYLTAC380GW skeleton vector and pYL322d1-A donor vector to co-transform E. coli NS3529, and obtain pYLTAC380GW-tHMGR vector through kanamycin and chloramphenicol double resistance screening, I-SceI enzyme cutting and NotI enzyme cutting verification;

[0025] ② Take pYLTAC380GW-tHMGR vector and pYL322d2-B donor vector to co-transform E. coli NS3529, and obtain pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 vector through kanamycin and ampicillin double resistance screening, PI-SceI enzyme cutting and NotI enzyme cutting verification;

[0026] ③ Take the vector obtained in step ② and the pYLMF-FPS-IDI donor vector, add Gateway BP Clonase II Enzyme MIX for recombination reaction, screen by 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.

[0027] Further, the infection solution is 10 mM MES, 10 mM MgCl2, 115℃ high-temperature high-pressure sterilization for 20 minutes, and 150 μM AS is added after cooling.

[0028] Further, the liquid medium of tobacco BY-2 cells is MS Salt 4.4 g / L, sucrose 30 g / L, potassium dihydrogen phosphate 0.255 g / L, pH adjusted to 5.0, 115℃ high-pressure sterilization for 20 minutes, and 1000x sterile hormone stock solution is added after cooling to 65℃, and the hormone stock solution contains 0.2 mg / mL 2,4-D, 1 mg / mL thiamine, and 100 mg / mL myo-inositol.

[0029] Further, the solid medium of tobacco BY-2 cells is 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 is added, 115℃ high-pressure sterilization for 20 minutes, and 1000x sterile hormone stock solution is added after cooling to 65℃, and the hormone stock solution contains 0.2 mg / mL 2,4-D, 1 mg / mL thiamine, and 100 mg / mL myo-inositol.

[0030] Further, the solid medium contains 50 mg / L hygr and 200 mg / L timentin.

[0031] Further, the liquid culture condition in step (4) is 25℃, 130 rpm shaker culture for 7-14 days, and the solid culture condition is 25℃ dark culture for 14 days.

[0032] Further, the volume ratio of the infection solution to BY-2 cells in step (2) is 3:50, and the centrifugal collection condition is 180g centrifugation for 2 minutes.

[0033] Further, the positive cell line in step (3) is identified by PCR and needs to contain the complete tHMGR, SE, SS, PgPPDs, PgDDS, Pn1-31, Pn3-31, FPS, and IDI gene set.

[0034] A ginsenoside obtained by a method of biosynthesizing ginsenoside by taking tobacco BY-2 cells as a chassis.

[0035] The present application obtains the following core technical effects by taking tobacco BY-2 cells as a chassis to biosynthesize rare ginsenosides:

[0036] 1. Breaking through the limitation of natural resources, realizing the heterologous synthesis of rare ginsenosides

[0037] The complete synthesis pathway from the basic metabolic precursor to the rare ginsenosides Rh2 and Rg3 is successfully reconstructed in tobacco BY-2 cells, which breaks the dependence on natural Panax plants and solves the problem of low content and difficulty in obtaining natural saponins.

[0038] 2. Utilizing the advantages of the chassis to improve synthesis efficiency and stability

[0039] Relying on the characteristics of fast growth, stable suspension culture state and high genetic transformation efficiency of tobacco BY-2 cells, the positive cell line obtained by screening can stably synthesize target saponins.

[0040] 3. Optimizing the culture and screening system to ensure controllable production of products

[0041] By determining the culture medium formula and culture conditions, efficient screening and stable subculture of transgenic cell lines are realized, which solves the problem of unstable product accumulation in heterologous synthesis and provides a repeatable technical solution for industrial scale production. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 : PCR amplification electrophoretogram of key genes in ginsenoside synthesis pathway;

[0043] Figure 2 : PCR positive identification results of pYL322d1-tHMGR, -SS, -Pn3-31-Pn1-31 bacterial liquid;

[0044] Figure 3 : PCR positive identification results of pYL322d2-SE, PgDDS-PgPPDs bacterial liquid;

[0045] Figure 4 : PCR positive identification results of pYLMF-FPS-IDI bacterial liquid;

[0046] Figure 5 : PCR positive identification results of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31 bacterial liquid;

[0047] Figure 6: pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31-FPS-IDI PCR positive identification electrophoretogram of bacterial liquid;

[0048] Figure 7 : pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31-FPS-IDI PCR positive identification electrophoretogram of bacterial liquid;

[0049] Figure 8 : pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31-FPS-IDI PCR positive identification electrophoretogram of bacterial liquid;

[0050] Figure 9 : BY-2 cell transformation positive clone PCR identification results (lane mark: positive CK is recombinant plasmid control, negative CK is wild type BY-2 cell, 1-6 is candidate positive cell line);

[0051] Figure 10: Rh2 (upper) and Rg3 (lower) content in liquid culture of positive cell line;

[0052] Figure 11: PCR identification results of target genes after subculture of positive cell line (lanes T1-T3 and T7 are positive cell lines of different subculture times). DETAILED DESCRIPTION

[0053] The present application will be further described below through specific examples.

[0054] Example 1: Ginsenoside synthesis vector

[0055] Construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI:

[0056] I. Experimental materials

[0057] Donor plasmid: pYL322d1, pYL322d2, pYLMF-H, pYLTAC380GW;

[0058] Restriction endonuclease: KpnI, SalI, PstI, NcoI, HindIII, I-SceI, PI-SceI, NotI (purchased from NEB company);

[0059] Tool enzymes: Gateway BP ClonaseII Enzyme MIX (purchased from Thermo Fisher), high-fidelity DNA polymerase (purchased from Takara);

[0060] E. coli strains: NS3529 competent cells, NEB-10β competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.);

[0061] Culture medium: LB solid medium (containing 25 mg / L kanamycin Kan, 15 mg / L chloramphenicol Chi, or 15 mg / L ampicillin Amp), LB liquid medium.

[0062] Key target gene recombination primers and PCR program:

[0063] Table 1 Key target gene homologous recombination primers

[0064]

[0065] PCR reaction was performed using Takara high-fidelity enzyme PrimeSTAR:

[0066] Prepare 50 μL PCR reaction system (template 2 μL, upstream and downstream primers 1 μL each, 2 × Taq Mix 25 μL, ddH2O to 50 μL);

[0067] 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, a total of 35 cycles; 72℃ final extension for 3 min.

[0068] Table 2 PCR program for target gene amplification

[0069]

[0070] Verification primers and PCR program in multi-fragment construction process:

[0071] 1: The verification primer sequences involved in the multi-fragment construction process are shown in Table 3:

[0072] Table 3 pYLTAC380GW verification primers at each round

[0073]

[0074] 2: PCR program for each round of verification in the multi-fragment construction process:

[0075] 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55-65℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; 72℃ final extension for 2 min.

[0076] II. Acquisition and amplification of key genes

[0077] 1. Retrieve and acquire the key gene sequences of ginsenoside synthesis pathway from NCBI database: Artemisia annua farnesyl pyrophosphate synthase gene (AaFPS), Arabidopsis thaliana isopentenyl pyrophosphate isomerase gene (AtIDI), Panax ginseng squalene synthase gene (PgPSS, SS), Panax ginseng squalene epoxidase gene (PgSE, SE), Panax ginseng dammarenediol synthase gene (PgDDS), Panax ginseng cytochrome P450 gene (PgPPDS), Panax notoginseng genes Pn1-31 and Pn3-31, and Panax ginseng 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMGR).

[0078] 2. Use online signal peptide prediction software SignalP5.0 to predict the N-terminal signal peptide of HMGR gene, and obtain truncated gene tHMGR without cell localization after truncating the signal peptide coding sequence.

[0079] 3. Design specific primers using Panax ginseng and Panax notoginseng cDNA as templates, and amplify the above 8 key genes (tHMGR, SE, SS, PgDDS, PgPPDs, Pn1-31, Pn3-31, and FPS-IDI) by PCR. The PCR products were verified by agarose gel electrophoresis, and the results showed that the sizes of the amplified fragments were consistent with the expected sizes, as shown in Figure 1 Ginsenoside synthesis pathway key gene PCR amplification electrophoretogram, the target fragments were recovered for future use.

[0080] III. Construction of intermediate donor vector

[0081] 1. Construction of donor vector pYL322d1-A

[0082] (1) Take the donor plasmid pYL322d1, and perform double enzyme digestion with restriction endonuclease KpnI and SalI (37℃ reaction for 2h). The enzyme digestion products were recovered by agarose gel electrophoresis to obtain the linearized vector backbone;

[0083] (2) Linearized pYL322d1 was ligated with the recovered tHMGR, SS, Pn3-31-Pn1-31 gene fragments by homologous recombination enzyme. The reaction system was as follows: 50 ng of linearized vector, 100 ng of each gene fragment, 2 μL of recombination enzyme, and ddH2O to make up to 10 μL. The reaction was carried out at 37℃ for 30 min;

[0084] (3) The recombination product was transformed into E. coli NEB-10 β competent cells, and plated on LB solid medium containing Kan (25 mg / L) and incubated at 37℃ overnight. Single colonies were picked for bacterial liquid PCR identification. The results showed that positive clones amplified bands with sizes consistent with the target genes, as shown in Figure 2pYL322d1-tHMGR, -SS, -Pn3-31-Pn1-31 PCR positive identification results of bacterial liquid, the extraction of plasmid is pYL322d1-A donor vector.

[0085] 2. Construction of donor vector pYL322d2-B

[0086] (1) Take the donor plasmid pYL322d2, double enzyme cut with restriction endonuclease Kpnl and Pstl (37℃ reaction for 2h), recover the linearized vector skeleton;

[0087] (2) Linearized pYL322d2 and recovered SE, PgDDS-PgPPDs gene fragments are connected by homologous recombination, and the reaction conditions are the same as those of the construction of donor vector pYL322d1-A;

[0088] (3) Transform E. coli NEB-10β competent cells, and coat on LB solid medium containing Kan (25mg / L), and incubate at 37℃ overnight. PCR identification of bacterial liquid shows that the positive clones amplify the target band, see Figure 3 pYL322d2-SE, PgDDS-PgPPDs PCR positive identification results of bacterial liquid, the extraction of plasmid is pYL322d2-B donor vector.

[0089] 3. Construction of donor vector pYLMF-FPS-IDI

[0090] (1) Take the donor plasmid pYLMF-H, double enzyme cut with restriction endonuclease Ncol and HindIII (37℃ reaction for 2h), recover the linearized vector skeleton.

[0091] (2) Linearized pYLMF-H and recovered FPS-IDI gene fragments are connected by homologous recombination, and the reaction conditions are the same as those of the construction of donor vector pYL322d1-A.

[0092] (3) Transform E. coli NEB-10β competent cells, and coat on LB solid medium containing Kan (25mg / L), and incubate at 37℃ overnight. PCR identification of bacterial liquid shows that the positive clones amplify the target band, see Figure 4 pYLMF-FPS-IDI PCR positive identification results of bacterial liquid, the extraction of plasmid is pYLMF-FPS-IDI donor vector.

[0093] Four, multiple rounds of assembly of recombinant expression vector

[0094] 1. First round of assembly: construction of pYLTAC380GW-tHMGR

[0095] (1) Take 300 ng of pYLTAC380GW plasmid and 500 ng of pYL322d1-A plasmid, and transform them into E. coli NS3529 competent cells, and spread them on LB solid medium containing Kan (25 mg / L) and Chi (15 mg / L), and incubate them at 37°C for 72 h;

[0096] (2) Collect the colonies and extract the plasmid, take 50 ng of the plasmid, add 1 μL I-SceI enzyme and 1 μL Buffer, and ddH2O to make up to 10 μL, and react at 37°C for 5 h; the reaction product is transformed into E. coli NEB-10β competent cells, and spread on LB solid medium containing Kan (25 mg / L), and incubate overnight at 37°C;

[0097] (3) Pick single colonies for PCR screening, and after expansion of positive clones, extract the plasmid, and cut it with NotI enzyme (37°C for 30 min), and perform agarose gel electrophoresis (150 V, 2-3 h, electrophoresis on ice) to show the expected fragment, see Figure 5 PCR positive identification result of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn3-31-Pn1-31 bacterial liquid, and obtain plasmid pYLTAC380GW-tHMGR.

[0098] 2. Second round of assembly: construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31

[0099] (1) Take 300 ng of pYLTAC380GW-tHMGR plasmid and 500 ng of pYL322d2-B plasmid, and transform them into E. coli NS3529 competent cells, and spread them on LB solid medium containing Kan (25 mg / L) and Amp (15 mg / L), and incubate them at 37°C for 48 h.

[0100] (2) Collect the colonies and extract the plasmid, take 50 ng of the plasmid, add 1 μL I-SceI enzyme and 1 μL Buffer, and ddH2O to make up to 10 μL, and react at 37°C for 5 h; the reaction product is transformed into E. coli NEB-10β competent cells, and spread on LB solid medium containing Kan (25 mg / L), and incubate overnight at 37°C.

[0101] (3) Positive clones are screened by PCR and verified by NotI enzyme cutting, see Figure 6 , and obtain plasmid pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31.

[0102] 3. Final assembly:

[0103] Construction of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI

[0104] (1) Mix 150 ng of pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 plasmid, 100 ng of pYLMF-FPS-IDI plasmid and 1 μL of Gateway BP Clonase II Enzyme MIX, add ddH2O to 5 μL, and react at 25°C for 5 h; add 2 μL of Proteinase K, and react at 37°C for 15 min to terminate the reaction.

[0105] (2) Transform the reaction product into E. coli NEB-10β competent cells, and coat on LB solid medium containing Kan (25 mg / L) and 5% sucrose, and culture at 37°C overnight.

[0106] (3) Pick single colonies for bacterial liquid PCR identification, and electrophoresis shows positive bands, see Figure 7 ; extract plasmids for Not I enzyme digestion verification, and the results show that each gene fragment is correctly inserted, see Figure 8 , and finally obtain the recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI.

[0107] Example 2: Transformation of tobacco BY-2 cells and screening of positive cell lines

[0108] I. Test materials

[0109] Tobacco BY-2 cells: wild type BY-2 cell line preserved in the laboratory;

[0110] Recombinant expression vector: pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI constructed in Example 1;

[0111] pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI;

[0112] Agrobacterium strain: GV3101 (containing vir helper plasmid, preserved in the laboratory);

[0113] Culture medium: BY-2 liquid standard medium, BY-2 solid screening medium (containing 50 mg / L hygromycin (Hygr) and 200 mg / L timentin (TM)), formula referring to Example 1;

[0114] Reagents: sterile water, ddH2O, DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.), PCR reagent (purchased from Takara Company); infection solution formula: 10 mM MES, 10 mM MgCl2, 115°C high pressure sterilization for 20 minutes, after cooling, add 150 μM AS (acetyl-syringone).

[0115] II. Suspension culture and pretreatment of tobacco BY-2 cells

[0116] 1. Take 1-2 g of wild type BY-2 cells normally growing on the plate, gently crush with tweezers, and disperse into a 250 mL wide-mouth conical flask containing 50 mL of BY-2 liquid standard medium, seal with a sterile breathable film, and place in a 25°C shaker at a speed of 130 rpm for 7 days of culture;

[0117] 2. Subculture according to the subculture scheme: take 5 mL of BY-2 suspension cells cultured for 7 days, add 45 mL of fresh BY-2 liquid standard medium to a 250 mL wide-mouth conical flask, continue to culture at 25°C, 130 rpm for 3-5 days, and obtain a well-conditioned BY-2 cell line.

[0118] III. Agrobacterium-mediated transformation of tobacco BY-2 cells with recombinant vectors

[0119] 1. Agrobacterium pretreatment: inoculate Agrobacterium GV3101 containing a recombinant expression vector into LB liquid medium containing 50 mg / L Kan, culture at 30°C, 220 rpm until OD600=0.5-0.8, centrifuge at 5000 rpm for 5 minutes to collect the bacterial cells, resuspend in the above-mentioned infection solution to OD600=0.6, and reserve for use;

[0120] 2. Infection treatment: take the BY-2 cell line cultured for 3-5 days, mix according to the ratio of cells to Agrobacterium infection solution of 3:50, transfer to a 250 mL wide-mouth conical flask, seal with a sterile breathable film, and place in a 25°C, 130 rpm shaker for dark culture for 2 days;

[0121] 3. Cell washing: after 48 h, transfer the mixed solution to a 50 mL centrifuge tube, centrifuge at 180 g for 2 minutes to collect the cells, and discard the supernatant; wash the cells with BY-2 liquid standard medium for 3 times (180 g centrifugation for 2 minutes after each washing), and completely remove the residual Agrobacterium.

[0122] IV. Screening and purification of positive cell lines

[0123] 1. Uniformly spread the washed cells on the BY-2 solid selection medium (containing 50 mg / L Hygr and 200 mg / L TM), seal with a sterile breathable film, and place in a 25°C dark culture;

[0124] 2. After 2 weeks of culture, the bright yellow calli in good condition were selected and transferred to new BY-2 solid screening medium for the second round of screening, and cultured in dark at 25°C for 2 weeks;

[0125] 3. The screening steps 1-2 were repeated for 1-2 times to obtain a pure transgenic positive cell line with uniform morphology and stable growth.

[0126] V. PCR identification of the positive cell line

[0127] 1. The wild-type BY-2 cells (negative control) and the positive cell line obtained by screening were taken respectively, and the genomic DNA was extracted using a DNA extraction kit, and the concentration was adjusted to 200 ng / μL;

[0128] 2. The extracted DNA was used as a template, and specific primers for tHMGR, SE, SS, PgPPDs, PgDDS, Pn1-31, Pn3-31, FPS, and IDI genes were designed, and a 50 μL PCR reaction system was prepared (2 μL of template, 1 μL of each upstream and downstream primer, 2×Taq Mix 25 μL, and ddH2O to make up to 50 μL), and the reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles; and 72°C final extension for 10 min;

[0129] 3. The PCR products were detected by 1% agarose gel electrophoresis, and the results showed that the positive cell line amplified bands consistent with the size of the target gene, while the wild-type cells had no corresponding bands, as shown in Figure 9 , indicating that the target gene set has been integrated into the tobacco BY-2 cell genome.

[0130] Example 3: Culture and detection of human ginsenoside by tobacco BY-2 positive cell line

[0131] I. Test materials

[0132] Positive cell line: positive cell lines 1, 2, 3, and 7 obtained by screening in Example 2;

[0133] Culture medium: BY-2 liquid standard medium, BY-2 solid standard medium (formula same as in Example 2);

[0134] Reagents: ginsenoside Rh2, Rg3, Rd standard (purchased from Sigma Company), methanol (chromatographically pure), acetonitrile (chromatographically pure), ultrapure water.

[0135] II. Expansion culture of the positive cell line

[0136] 1. Liquid culture: 1-2 g of callus of positive cell lines 1, 2, 3, and 7 were inoculated into 250 mL wide-mouthed conical flasks containing 50 mL of BY-2 liquid standard medium, respectively, and cultured at 25°C with 130 rpm shaking, with subculture every 7 days. Cell samples cultured for 5 d, 7 d, and 14 d were collected, respectively.

[0137] 2. Solid culture: 0.5 g of callus of positive cell lines 1, 2, 3, and 7 were inoculated onto BY-2 solid standard medium, respectively, and cultured in the dark at 25°C for 14 days. Callus samples were collected.

[0138] III. Extraction and detection of ginsenosides

[0139] ① Sample pretreatment: The collected cell or callus samples were freeze-dried, ground into powder, and accurately weighed 100 mg of the powder. Then, 1 mL of 70% methanol solution was added, and ultrasonic extraction was performed for 30 min (power 300 W, temperature 40°C). Centrifugation was performed at 4°C and 12000 rpm for 10 min. The supernatant was passed through a 0.22 μm organic filter membrane and was ready for detection.

[0140] ② HPLC detection conditions: The chromatographic column was a C18 column (250 x 4.6 mm, 5 μm). The mobile phase was acetonitrile: water = 30:70 (v / v). The flow rate was 1 mL / min. The detection wavelength was 203 nm. The column temperature was 30°C. The injection volume was 10 μL.

[0141] ③ Standard curve preparation: 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL of Rh2, Rg3, and Rd standard solutions were prepared, respectively. Detection was performed according to the above chromatographic conditions. The standard concentration was taken as the abscissa, and the peak area was taken as the ordinate to draw the standard curve (R²>0.999).

[0142] IV. Analysis of detection results

[0143] ① Liquid culture samples: The Rh2 content of positive cell lines 1, 2, and 3 was the highest (about 10 μg / g) at 14 d of culture, and the Rg3 content was about 5 μg / g. With the extension of culture time, the Rd content gradually decreased, and the Rh2 content reached a peak at 14 d. Figure 10 ;

[0144] ② Solid culture samples: The Rg3 content in the callus of positive cell lines was significantly higher than that in liquid culture cells. The Rg3 content in the callus of cell line 2 reached 150 μg / g, and Rh2 and Rd were not detected or had very low content.

[0145] Table 4: Detection results of ginsenosides Rh2, Rg3, and Rd in tobacco BY-2 callus

[0146]

[0147] ③ Stability verification: after 5 passages of positive cell lines, PCR identification still showed complete gene set, see Figure 11 , HPLC detection showed that the content of Rg3 and Rh2 fluctuated <10%, indicating that the synthesis system had good stability.

[0148] The above are preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Equivalent changes or modifications made according to the features and principles of the patent application range described herein should be included in the patent application range.

Claims

1. A method for biosynthesis of ginsenosides using tobacco BY-2 cells as a chassis, characterized in that, Comprising the following steps: (1) Constructing a recombinant expression vector: introducing a set of human ginsenoside synthesis pathway key genes into a vector pYLTAC380GW to obtain a recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI; the set of human ginsenoside synthesis pathway key genes comprises a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene tHMGR, a Panax notoginseng squalene epoxidase gene SE, a Panax notoginseng squalene synthase gene SS, a Panax notoginseng dammarenediol synthase gene PgDDS, a Panax notoginseng cytochrome P450 gene PgPPDs, a Artemisia annua farnesyl pyrophosphate synthase gene FPS, an Arabidopsis thaliana isopentenyl pyrophosphate isomerase gene IDI, and genes Pn1-31 and Pn3-31 derived from Panax notoginseng; (2) Transforming tobacco BY-2 cells: introducing the recombinant expression vector of step (1) into tobacco BY-2 cells, adding an infection solution, dark incubating, and collecting cells; the volume ratio of the infection solution to BY-2 cells is 3:50, and the centrifugal collection condition is 180g centrifugation for 2 minutes; the infection solution is 10mM MES, 10mM MgCl2, high-temperature high-pressure sterilization at 115℃ for 20 minutes, and after cooling, 150μM AS is added; (3) Screening a positive cell line; (4) Ginsenoside synthesis culture: inoculating the positive cell line into tobacco BY-2 cell liquid or solid culture medium for culture, and collecting the culture to obtain ginsenosides; the liquid culture condition is 25℃, 130rpm shaker culture for 7-14 days, and the solid culture condition is 25℃ dark culture for 14 days; The tobacco BY-2 cell liquid culture medium is MS Salt 4.4g / L, sucrose 30g / L, potassium dihydrogen phosphate 0.255g / L, pH adjusted to 5.0, high-pressure sterilization at 115℃ for 20 minutes, and after cooling to 65℃, 1000× sterile hormone stock solution is added, and the hormone stock solution contains 0.2mg / mL 2,4-D, 1mg / mL thiamine, and 100mg / mL inositol; The tobacco BY-2 cell solid culture medium is MS Salt 4.4g / L, sucrose 30g / L, potassium dihydrogen phosphate 0.255g / L, pH adjusted to 5.8, 7g / L plant gel added, high-pressure sterilization at 115℃ for 20 minutes, and after cooling to 65℃, 1000× sterile hormone stock solution is added, and the hormone stock solution contains 0.2mg / mL 2,4-D, 1mg / mL thiamine, and 100mg / mL inositol; the solid culture medium also contains 50mg / L hygromycin and 200mg / L timentin.

2. The method of claim 1, wherein, The construction of the recombinant expression vector in step (1) comprises: (1) Gene acquisition: obtaining the original gene sequences of FPS, IDI, SS, SE, PgDDS, PgPPDs, Pn1-31, Pn3-31, and HMGR from the NCBI database, obtaining tHMGR through signal peptide prediction, amplifying and recovering the above 9 gene fragments; (2) Intermediate vector construction: ① The donor plasmid pYL322d1 was digested with restriction endonuclease Kpnl and Sail, and the tHMGR, SS, Pn3-31-Pn1-31 gene fragments were subjected to homologous recombination with the digested pYL322d1 to obtain the pYL322d1-A donor vector; ② The donor plasmid pYL322d2 was digested with restriction endonuclease Kpnl and Pstl, and the SE, PgDDS-PgPPDs gene fragments were subjected to homologous recombination with the digested pYL322d2 to obtain the pYL322d2-B donor vector; ③ The donor plasmid pYLMF-H was digested with restriction endonuclease Ncol and Hindlll, and the FPS-IDI gene fragments were subjected to homologous recombination with the digested pYLMF to obtain the pYLMF-FPS-IDI donor vector; (3) Recombinant expression vector assembly: ① The pYLTAC380GW backbone vector and the pYL322d1-A donor vector were co-transformed into E. coli NS3529, and the kanamycin and chloramphenicol double-antibiotic-resistant colonies were screened, and the I-SceI enzyme digestion and NotI enzyme digestion were verified to obtain the pYLTAC380GW-tHMGR vector; ② The pYLTAC380GW-tHMGR vector and the pYL322d2-B donor vector were co-transformed into E. coli NS3529, and the kanamycin and ampicillin double-antibiotic-resistant colonies were screened, and the PI-SceI enzyme digestion and NotI enzyme digestion were verified to obtain the pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31 vector; ③ The vector obtained in step ② and the pYLMF-FPS-IDI donor vector were added with Gateway BP Clonase II Enzyme MIX for recombination reaction, and the kanamycin-resistant and 5% sucrose-resistant colonies were screened, and the enzyme digestion was verified to obtain the final recombinant expression vector pYLTAC380GW-tHMGR-SE-SS-PgPPDs-PgDDS-Pn1-31-Pn3-31-FPS-IDI.

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