CN115725620B - Method for synthesizing panax japonicus saponins in pseudo-ginseng cells - Google Patents

Abstract

The invention discloses a method for synthesizing panax japonicus saponins in pseudo-ginseng cells, which comprises the following steps of constructingPnDSGene RNAi expression vector and method for introducing the same into pseudo-ginseng cells to obtain synthetic panax japonicus soapThe nucleotide sequence of the RNAi fragment of the notoginseng cell of the glycoside is shown as SEQ ID NO. 1; the method can inhibit the first key enzyme gene related to dammarane type triterpene saponin synthesis branch in Notoginseng radix saponin biosynthesis pathway from reverse regulation point of viewPnDSThe expression of the dammarane type triterpenoid saponin is reduced, so that the pseudo-ginseng cells are promoted to synthesize the oleanane type saponin which is not originally contained, namely the panax japonicus saponin.

Description

A method for synthesizing bamboo ginseng saponins in Panax notoginseng cells

Technical field

The invention belongs to the technical field of saponin synthesis, and specifically relates to a method for synthesizing bamboo saponins in Panax notoginseng cells.

Background technique

The Panax genus of the Araliaceae family includes a variety of medicinal plants, among which Panaxginseng, Panax quinquefolius, Panax notoginseng and Panaxjaponicus are the most widely used. Their main active ingredient is triterpene saponins, which have various pharmacological effects such as preventing and treating cardiovascular and cerebrovascular diseases, anti-fatigue, antioxidant, enhancing immunity, and protecting the liver. The biosynthetic pathways of triterpenoid saponins in medicinal plants of the genus Ginseng are basically the same in the initial stage and skeleton construction stage. The main difference in saponin synthesis between species is that after the formation of 2,3-oxysqualene, 2,3-oxysqualene is used as the From the common precursor, two independent triterpene saponin synthesis branches emerged. 2,3-Oxisqualene is catalyzed by dammarenediol synthase (DS) and enters the dammarane-type saponin synthesis branch; catalyzed by β-amyrin alcohol synthase (β-AS), it enters oleanane Type saponin synthesis branch.

So far, the triterpene saponins isolated from Panax genus medicinal materials are mainly dammarane saponins. Ginseng, American ginseng, and ginseng all contain two types of triterpene saponins: dammarane type and oleanane type, but notoginseng It only contains dammarane-type saponins and cannot synthesize oleanane-type saponins. This is the characteristic that distinguishes Panax notoginseng from other ginseng species. It is currently known that bamboo saponin IV and bamboo saponin IVa are oleanane-type saponins, and Panax notoginseng in the natural state does not have the ability to synthesize bamboo saponin IV and bamboo saponin IVa.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a method for synthesizing ginseng saponins in Panax notoginseng cells. This method constructs a PnDS gene RNAi expression vector and transfers it into Panax notoginseng cells to inhibit Panax notoginseng. The expression of the PnDS gene, the first key enzyme of the dammarane-type triterpene saponin synthesis branch, weakens the metabolic flow of the dammarane-type triterpene saponin synthesis branch and promotes Panax notoginseng cells to synthesize bamboo saponins, in which the nucleotides of the RNAi fragment The sequence is shown in SEQ ID NO:1;

The object of the present invention is achieved through the following technical solutions:

1. Extract total RNA from the roots of Panax notoginseng, synthesize Panax notoginseng cDNA by reverse transcription, and use the synthesized first-strand cDNA as a template to amplify the PnDS gene interference fragment by PCR. The nucleotide sequence of the gene PnDS is as SEQ ID NO: 1 shown;

2. Construct the PnDS gene RNAi interference vector pHellsagte-PnDS, transform Agrobacterium, and screen out positive single clones through PCR;

3. Use Agrobacterium-mediated genetic transformation method to introduce pHellsagte-PnDS into Panax notoginseng cells for expression, and screen positive transgenic cell lines through antibiotic screening and qRT-PCR;

4. Extract saponins from Panax notoginseng transgenic cells and non-transgenic cell lines, and analyze the differences in types and contents of saponins between transgenic cells and non-transgenic cell lines.

Advantages and technical effects of the present invention:

The invention provides a new method for producing bamboo ginseng saponin IVa and bamboo ginseng saponin IV. Bamboo ginseng saponin IVa and bamboo ginseng saponin IV are the main active ingredients of ginseng and are typical oleanane-type saponins. , not contained in Panax notoginseng medicinal materials. The present invention synthesizes Bamboo ginseng saponin IVa and Bamboo ginseng saponin IV in Panax notoginseng cells through gene regulation methods, thereby realizing heterologous expression of saponins among species. The method of the present invention is simple, easy to operate, and has the potential for industrial production and marketing. application potential.

Description of the drawings

Figure 1 shows the RNAi fragment sequence amplification results;

Figure 2 shows the amplification product of RNAi fragment with attB recombination site, where 1: RNAi fragment, M: MarkerDL2501;

Figure 3 is the identification electrophoresis diagram of pHellsagte-PnDS vector transferred into E. coli DH5α, in which 1-8: bacterial liquid sample, M: MarkerDL2501, +: pHellsagte-PnDS plasmid, -: negative control;

Figure 4 is the identification electrophoresis diagram of pHellsagte-PnDS vector transferred into Agrobacterium LBA4404, in which 1-5: bacterial liquid sample; M: MarkerDL2502; -: negative control;

Figure 5 shows the electrophoresis pattern of PCR detection of transgenic Panax notoginseng cells, in which 1-6: transgenic Panax notoginseng cell line, M: MarkerDL2502;

Figure 6 shows the detection results of real-time fluorescence quantitative PCR detection of the PnDS gene and five genes related to the synthesis of bamboo saponin IVa and bamboo saponin IV in transgenic Panax notoginseng cells, in which WT is a wild-type cell line. , T1 to T4 are transgenic Panax notoginseng cell lines with 4 PnDS genes;

Figure 7 shows the saponin content in the transgenic Panax notoginseng cell line, in which WT is the wild-type Panax notoginseng cell line, and T1-T4 is the transgenic Panax notoginseng cell line).

Detailed ways

The present invention will be further described in detail below through examples, but the scope of protection of the present invention is not limited to the content described. The methods in the examples are all conventional methods unless otherwise stated, and the reagents used are all conventional commercially available reagents or Reagents prepared according to conventional methods.

Example 1: Cloning of RNAi fragment of PnDS gene

(1) Extraction of total RNA from Panax notoginseng

The present invention adopts an improved guanidinium isothiocyanate method to extract total RNA from Panax notoginseng callus. The specific operation is as follows: the mortar and mortar rod are subjected to RNase removal and high-temperature dry heat sterilization, and then cooled to room temperature. Weigh an appropriate amount of Panax notoginseng callus cells and place them in a treated pestle. Use liquid nitrogen to grind the Panax notoginseng callus into powder. Add 10% (w/v) pre-cooled RNA extraction buffer and 1.0% ( w/v) of β-mercaptoethanol and grind thoroughly. Transfer 1.0 mL of the grinding solution to a 2 mL centrifuge tube, add 500 μL RNA extraction phenol, 100 μL chloroform and 1/10 volume of 2M sodium acetate solution (pH 4.0), shake vigorously to mix, and let stand on ice for 5 minutes before adding 4 Centrifuge at 12500g for 15 minutes at ℃, slowly transfer the supernatant to a new 2mL centrifuge tube, add 1:1 equal volume of RNA extraction phenol/chloroform, shake vigorously to mix, let stand on ice for 5 minutes, and centrifuge at 4℃ and 12500g 15min (repeat this step until the middle layer almost disappears). Slowly absorb the supernatant and transfer it to a new 2mL centrifuge tube, add an equal volume of chloroform, shake vigorously and mix, let stand on ice for 5 minutes, centrifuge at 4°C and 12500g for 15 minutes, slowly absorb the supernatant and transfer it to a new 1.5mL centrifuge tube. In the centrifuge tube, add 1/10 volume of 3M sodium acetate solution (pH 5.2), then add an equal volume of isopropanol, mix slowly by inverting, let stand at -20°C for 1.5h, and after sufficient precipitation, centrifuge at 4°C and 12500g 25 minutes; discard the supernatant and pipet and wash the precipitate with 75% ethanol solution. After two pipetting and washing, use a pipette to suck off the liquid and place it on a clean workbench for air drying. After the ethanol has completely evaporated, add 15-30 μL RNaes dissolve RNA pellet in water. The integrity of the total RNA extracted from Panax notoginseng callus cells was detected by agarose gel electrophoresis, and then the concentration and purity of the extracted RNA were detected using a UV spectrophotometer.

(2)Synthesis of the first strand of cDNA

Select RNA with better quality and use Promega's GoScript reverse transcription system to synthesize the first strand of cDNA. First, add 1.0 μL of Oligo(dT) ₁₅ , 5.0 μg of total RNA, and Nuclease-free Water to 10 μL and mix thoroughly and gently. Place it in a 70°C water bath for 5 minutes for pre-denaturation, then immediately place it on ice for 5 minutes, then add the following reagents: Nuclease-free Water 1.6μL, GoScript ^TM 5×Reaction Buffer 4.0μL, PCR NucleotideMix 1.0μL, MgCl ₂ (25mM) 2.0μL, Recombinant Ribonuclease Inhibitor 0.4 μL, GoScript ^TM Reverse Transcriptase 1.0 μL, mix well, centrifuge briefly, anneal at 25°C for 5 minutes, extend in a water bath at 42°C for 90 minutes, and finally, place in a 70°C water bath for 15 minutes to terminate the reverse transcriptase activity to obtain Panax notoginseng. cDNA first strand.

(3)Synthesis of RNAi fragments

According to the full-length sequence of Panax notoginseng PnDS gene (KJ804174.1), use PrimerPremier5.0 to design its specific primers PnDS _F : 5'-TCCCCTTATCATTGCCCT-3' and PnDS _R : 5'-GCTTTTTCCCCATTTCCT-3', using the three obtained above The first strand of seven cDNA is used as a template, and high-fidelity PCR amplification is performed under the action of DNA polymerase Ex Taq. PCR reaction conditions: 98°C, 3min; 98°C, 10s, 60°C, 15s, 72°C, 15s, 35cycles; 72°C, 5 min; separate the amplified product obtained by 1% agarose gel electrophoresis, and the recovered fragment is the 503-1246th nucleotide sequence of the gene PnDS;

After the target fragment is recovered, it is connected to the pGEM T-easy vector and transferred into E. coli DH5α. Single colonies on the plate are randomly selected and amplified by bacterial liquid PCR to detect positive clones. The single clones that were positive in the bacterial liquid PCR test were sent for sequencing, and the sequenced sequence was compared with the Panax notoginseng PnDS gene sequence using the Blast software in NCBI. The results are shown in Figure 1. The nucleotide sequence is shown in SEQ ID NO: 1. , the comparison result is correct, inoculate the E. coli single clone with the correct result into the liquid LB medium containing spectinomycin and expand it, culture it with shaking at 37°C and 200 rpm for 15 hours, and use the SanPrep column plasmid extraction kit to extract the plasmid (T -PnDS).

Example 2: Construction of PnDS gene RNAi expression vector

(1) Cloning of RNAi fragments connected to attB recombination site

According to the technical principle of GatewayTM, the primers BP-C _F (5'-GGGG ACAAGTTTGTACAAAAAAGCAGG CT TCCCCTTATCATTGCCCT-3') and BP-C _R (5'-GGGG ACCACTTTGTACAAGAAAGCTGGGT GCTTTTTCCCCATTTCCT-3') were designed (the underlined part is the attB recombination site), Use PCR to add adapters to both ends of the RNAi fragment, allowing a homologous recombination reaction between the target fragment and the vector. Use the above extracted plasmid as a template, BP-C _F /BP- _CR as primers, and use Ex Taq to perform attB- PCR amplification, the PCR reaction conditions were the same as in step (3) of Example 1. After this amplification, attB recombination sites were connected to both sides of the RNAi fragment; the PCR product was recovered by gel, and the results are shown in Figure 2;

(2) Construction of PnDS gene RNAi expression vector

in accordance with BP Clonase ^TM II Enzyme Mix kit instructions are used to construct RNAi vectors. The reaction system settings are as follows: attB-PCR gel recovery product 0.15 μg, pHellsagte 2 vector plasmid 0.15 μg, BPClonase ^TM II Enzyme Mix 2.00 μL, TE buffer (pH= 8.0) Make up to 10 μL; add the above solutions in sequence, pipet and mix with a pipette, place in a 25°C water bath, react for 4 hours, then add 1 μL of proteinase K to the reaction solution, and wait in a 37°C water bath for 10 minutes to terminate. reaction, the reaction product was transformed into Escherichia coli competent cells, single clones were selected, the single clone PCR verification results are shown in Figure 3, they were expanded and cultured, and then the plasmid (pHellsgate-PnDS) was extracted; the obtained plasmids were used with restriction Endonucleases XbaI and XhoI were used for single enzyme digestion. The enzyme digestion system was as follows: pHellsagte-PnDS plasmid 5.0 μg, 10×Buffer 2.0 μL, restriction endonuclease XbaⅠ/XhoⅠ 1.5 μL, and ddH ₂ O water was added to 20 μL;

The enzyme digestion system was placed in a 37°C water bath for 5 hours of enzyme digestion reaction. After the reaction, the enzyme digestion products were detected by 1% agarose gel electrophoresis. The PnDS gene was judged based on the size of the gel bands of the enzyme digestion products of the two enzymes. The RNAi expression vector pHellsagte-PnDS was successfully constructed; the vector was then transformed into Agrobacterium LBA4404 competent cells by liquid nitrogen freezing and thawing. At the same time, the empty pHellsagte 2 was also transformed into LBA4404 competent cells as an empty control. The results are shown in Figure 4. It can be seen from the figure that the amplified band is about 750 bp, which is in line with expectations, indicating that the RNAi fragment of the PnDS gene has been successfully transformed into Agrobacterium.

Example 3: Genetic transformation of Panax notoginseng mediated by Agrobacterium tumefaciens

1. Preculture of Panax notoginseng cells

(1) Collect Panax notoginseng stems and leaves, use Panax notoginseng callus culture medium (MS medium + 2,4-D 2mg/L + KT 1mg/L, medium pH 5.6), the culture conditions are solid culture, temperature 25±1°C, cultured in the dark, with a culture period of 28 days, to obtain the callus of Panax notoginseng; use MS solid medium containing 2,4-D 2mg/L and KT 2mg/L to obtain the callus of Panax notoginseng. Subculture for 15 days to obtain Panax notoginseng subcultured cells;

(2) Select Panax notoginseng callus cells in good growth status and transfer them to the Panax notoginseng callus cell pre-culture medium (add acetosyringone to the Panax notoginseng callus culture medium to 40 mg/L), and spread them flat. Cover the entire surface of the culture medium and culture it in the dark at 25°C for 3 days;

(2) Panax notoginseng cell infection

Pipette 100-200 μL of the activated LBA4404 Agrobacterium bacterial liquid carrying the PnDS gene RNAi expression vector plasmid pHellsagte-PnDS and spread it on the LB solid medium plate containing 50 mg/L kanamycin and 25 mg/L rifampicin (the This step can be performed at the same time as the pre-culture of Panax notoginseng callus cells). Incubate upside down in a 28°C incubator for 2-3 days until the plate is covered with a thick layer of bacteria. Use an inoculation needle to scrape a certain size of the grown bacterial mass into MGL liquid culture medium containing 40 mg/L acetosyringone, and culture it with shaking on a shaking table at 28°C and 200 rpm until the OD ₆₀₀ of the bacterial liquid is 0.6-0.8, and then connect to the above In the Panax notoginseng callus cells pre-cultured for 3 days, the Panax notoginseng callus cells were completely immersed in the bacterial solution, and cultured on a shaker at 25°C and 105 rpm for 20 minutes;

(3) Cell collection and co-culture

After the infection, the Panax notoginseng callus cells were suction-filtered using a Buchner funnel to remove the bacterial liquid, and then the remaining bacterial liquid on the surface of the Panax notoginseng callus cells was sucked up with sterile filter paper, and then the Panax notoginseng callus cells were removed. Transfer to the Panax notoginseng co-culture medium with sterile filter paper on the surface (to prevent direct contact between Agrobacterium and the culture medium, resulting in overgrowth of Agrobacterium), and place it in a dark condition at 25°C for 3 days, in which Panax notoginseng co-culture The base is the same as the pre-culture medium of Panax notoginseng callus cells.

(4) Sterilization culture

After the co-culture is completed, transfer the Panax notoginseng callus cells to a sterilized beaker and wash them 5-6 times with sterile water containing 400 mg/L cephalosporin to fully remove Agrobacterium. After cleaning, use a cloth The Panax notoginseng callus cells were suction-filtered using a funnel to remove the liquid, and then the remaining liquid on the surface of the Panax notoginseng callus cells was sucked up with sterile filter paper. Transfer the Panax notoginseng callus cells to sterilized medium (add cephalosporin and kanamycin to the Panax notoginseng callus medium to a final concentration of 400 mg/L and 50 mg/L) and place at 25 15 days of sterilization culture was carried out under dark conditions.

(5) Screening, culture and subculture

After 15 days of sterilization and culture, the Panax notoginseng callus cells were transferred to the Panax notoginseng callus selection medium (add kanamycin to the Panax notoginseng callus medium to a final concentration of 50 mg/L), approximately Subculture once every 35 days (the subgeneration cycle can be adjusted according to growth conditions). After 4-5 subculture screenings, a PnDS gene RNAi transgenic Panax notoginseng cell line with kanamycin resistance can be obtained.

Example 4: Detection of expression levels of PnDS genes and genes related to saponin synthesis in transgenic Panax notoginseng cells

(1) Detection of genomic DNA level of transgenic Panax notoginseng cell line

The modified CTAB method was used to extract the genomic DNA of the transgenic Panax notoginseng cell line, and the upstream and downstream primers npt-F (5'-CTCTGATGCCGCCGTGTT-3') were designed based on the kanamycin npt II resistance gene sequence on the T-DNA in the pHellsagte 2 vector. and npt-R(5'-CCCTGATGCTCTTCGTCCA-3'), using the proposed Panax notoginseng genomic DNA as a template for PCR detection, and screening the positive transgenic Panax notoginseng cells. The results are shown in Figure 5. In the figure, a strip of approximately The specific band of 430 bp is consistent with the expected size, indicating that the six Panax notoginseng transgenic cell lines have introduced exogenous DNA and integrated it into the genomic DNA for stable inheritance. It is preliminarily confirmed that the PnDS gene RNAi transgenic cell lines have been obtained.

(2) Fluorescence quantitative PCR detection of transgenic Panax notoginseng cells

Extract the total RNA of the positive transgenic Panax notoginseng cell line and the non-transgenic Panax notoginseng cell line, and reverse-transcribe it into cDNA. The specific operation steps are the same as in Example 1;

According to the 18S rRNA gene (accession number: D85171.1), β-amysyrinsynthase (β-AS) gene (accession number: KP658156), oleanolic acid synthase (Oleanic acidsynthase, CYP716A52v2) gene (Accession number: JX036032.1), oleanolic acid glucuronosyltra nsferase (OAGT) gene (Accession number: MH819287.1), PjmUGT1 gene and PjmUGT2 gene to design primers for fluorescence quantitative PCR : 18S-F: 5'-GGGGAGTATGGTCGCAAGG-3', 18S-R: 5'-CAGAACAT CTAAGGGCATCACAG-3'; β-AS-F: 5'-GTATTCCCTGTAGAGCATCGCAT-3', β-AS-R: 5'-GGCACAGGCGTTGTTTTCAC -3'; CYP716A52v2-F: 5'-AGGAGCAAATGGAGATAGTGA-3', CYP716A52v2-R: 5'-GATTGAGAAACCGTTGTAGG-3'; OAGT-F: 5'-GCATAATCTCGGACA AGTAC-3', OAGT-R: 5'-AAAGGTTGGGAGTCTGAAGT- 3'; PjmUGT1-F: 5'-TCACATAAA TCCGATGGTCC-3', PjmUGT1-R: 5'-AGAAATCCCTGAAATCCTCC-3'; PjmUGT2-F: 5'-GC ATTCTCCCTTTGTTTCAG-3', PjmUGT2-R: 5'-CGACTTGCCTCACTCTTCCT- 3'. The cDNA synthesized by reverse transcription was diluted 5 times, that is, 20 μL cDNA was diluted to 100 μL. Use the diluted cDNA as a template and use the above primers for fluorescence quantitative PCR. According to Follow the instructions of qPCRMaster Mix and repeat the detection three times for each gene corresponding to each sample. The 2 ^-ΔΔCt method was used to process the fluorescence quantitative data, and the relative expression of the PnDS gene in the transgenic Panax notoginseng cell line compared to the non-transgenic Panax notoginseng cell line was analyzed and detected. The results are shown in Figure 6. The results show that in the selected T1- The expression level of the key enzyme PnDS gene in the four transgenic Panax notoginseng cell lines of T4 is significantly lower than that of the ordinary Panax notoginseng cell line, while the genes related to the synthesis of bamboo ginseng saponin IVa and bamboo ginseng saponin IV are β-AS, CYP716A52v2, and OAGT. , the relative expression levels of PjmUGT1 and PjmUGT2 were significantly higher than those of ordinary Panax notoginseng cell lines.

Example 5: Detection of saponin content in transgenic Panax notoginseng cells

(1) Preparation of sample solution

Collect the callus cells of the transgenic Panax notoginseng cell line and the ordinary Panax notoginseng cell line in step (1) of Example 4, mark them with numbers respectively and place them in a 55°C oven for drying (about 12 hours). After drying, Fully grind into powder, weigh 0.5g of transgenic and ordinary Panax notoginseng cell line powder respectively, place them in 50mL centrifuge tubes, add 50mL of 70% methanol solution to each and soak overnight, and then use ultrasonic waves for crushing treatment (60W, ultrasonic 4s, 2s interval) for 1.5~2.0h until the Panax notoginseng cell powder is fully broken. After ultrasonic, transfer to a centrifuge and centrifuge at 4000rpm for 30min. Collect the supernatant and place it in an oven at 50~55°C. Dry it. After drying, dissolve it with 10 mL of distilled water, and extract it three times with an equal volume of water-saturated n-butanol. Collect the extract and place it in an oven at 50-55°C for drying. After drying, dissolve it with 70% methanol solution and adjust the volume to 25 mL. After filtering through a 0.45 μM microporous membrane, the saponin solution is obtained. .

(2) High performance liquid chromatography analysis

HPLC was used to detect the types and contents of saponins in transgenic Panax notoginseng cells. The HPLC detection conditions were as follows: the chromatographic column was Waters-XTerra-MS-C18 (5 μm, 250 mm × 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 1.0mL/min, the column temperature is 30°C, and the detection wavelength is set to 203nm; the results are shown in Figure 7. The results show that the Panax notoginseng cells after transgenic operation contain oleanane-type saponins (Bamboo ginseng saponin IVa, Bamboo ginseng saponin IVa, Ginsenoside IV), while unmodified wild-type Panax notoginseng cells do not produce oleanane-type saponins (Bamboo ginseng saponin IVa, Bamboo ginseng saponin IV).

Claims (1)

1. A method for synthesizing bamboo ginseng saponins in Panax notoginseng cells, characterized by: transferring the RNAi fragment of the interfering gene PnDS into Panax notoginseng cells to obtain Panax notoginseng cells that synthesize bamboo ginseng saponins, wherein the RNAi fragment of the interfering gene PnDS The nucleotide sequence of the RNAi fragment is shown in SEQ ID NO: 1; Transgenic Panax notoginseng cells can synthesize bamboo ginseng saponin IV and bamboo ginseng saponin IVa.