CN113549649B - Preparation method of ginsenoside F1 - Google Patents

Abstract

The invention discloses ginsenoside F₁The method of preparing (1) is to use the genePnDDS、P12H、D6HAndUGTPn20simultaneously transferring into tobacco to obtain synthetic ginsenoside F₁The transgenic tobacco of (1), wherein the genePnDDSThe nucleotide sequence of (A) is shown as SEQ ID NO. 1, geneP12HThe nucleotide sequence of (A) is shown as SEQ ID NO. 2, geneD6HThe nucleotide sequence of (A) is shown as SEQ ID NO. 3, geneUGTPn20The nucleotide sequence of (A) is shown as SEQ ID NO. 4; the experimental results show thatThe obtained transgenic tobacco plant can synthesize ginsenoside F₁To make ginsenoside F₁The method is more efficient and convenient, simple, easy to operate and suitable for large-scale production and market popularization and application, and the method is used for obtaining the ginsenoside F₁Provides a new approach.

Description

Ginsenoside F1Preparation method of (1)

Technical Field

The invention belongs to the technical field of biological medicine preparation, and particularly relates to heterologous synthesis of ginsenoside F in tobacco₁The method of (1).

Background

Panax plants have been used for the treatment of various diseases and improvement of physical functions, wherein triterpenoid saponins are main active ingredients of Panax plants. The triterpene saponin has various pharmacological effects of promoting cell proliferation, inhibiting apoptosis, inhibiting oxidative stress, resisting inflammation, increasing cell activity, etc., and can be used for protecting nerve cell, myocardial cell, liver cell, lung epithelial cell, etc.

Ginsenoside F₁( ginsenoside F₁ ) Is rare dammarane type triterpenoid saponin component in medicinal plants such as ginseng, pseudo-ginseng, American ginseng and the like, is protopanaxatriol type saponin generated by catalyzing PPT and glycosyl donor UDP-glucose (UDP-glucose) through UDP-glucosyltransferase, and has the effects of resisting tumors, aging, oxidation and the like. Ginsenoside F₁Has strong inhibiting effect on proliferation and migration of skin cancer cell B16 and melanin production, and can protect human stratum corneum HaCaT from damage caused by ultraviolet rays. Despite ginsenoside F₁Has important pharmacological activity, but has very low content in Panax medicinal materials, and the Panax species is perennial plant with long growth period, so that the ginsenoside F is obtained₁Is not easy to obtain and expensive, and the development and the acquisition of the ginsenoside F are urgently needed₁Novel methods and novel approaches.

Tobacco is a wide-range economic crop with mature cultivation technology, has abundant natural product synthesis systems in vivo, and has a mature transgenic technology system. Although tobacco may have the potential to synthesize natural compounds, the synthesis of ginsenoside F in tobacco has not been seen₁And no synthesis including ginsenoside F is found in the tobacco genome₁The complete pathway of dammarane type tetracyclic triterpene saponins in the tobacco plant indicates that the tobacco plant does not have the capability of synthesizing the saponins.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides ginsenoside F₁The method comprises constructing and synthesizing ginsenoside F in tobacco₁Thereby producing ginsenoside F in tobacco₁The invention provides a method for obtaining ginsenoside F₁The new approach of (1);

the method of the invention is to use the genePnDDS、P12H、D6HAndUGTPn20simultaneously transferring into tobacco to obtain synthetic ginsenoside F₁The transgenic tobacco of (1), wherein said genePnDDSThe nucleotide sequence of (A) is shown as SEQ ID NO. 1, geneP12HThe nucleotide sequence of (A) is shown as SEQ ID NO. 2, geneD6HThe nucleotide sequence of (A) is shown as SEQ ID NO. 3, geneUGTPn20The nucleotide sequence of (A) is shown in SEQ ID NO. 4.

The purpose of the invention is realized by the following technical scheme:

(1) from notoginseng (A)Panaxnotoginseng) Extracting total RNA from root, reverse transcription synthesizing notoginseng cDNA, using the synthesized first strand cDNA as template, PCR amplifying notoginsenoside skeleton synthetase genePnDDSSkeleton-modified enzyme geneP12HSkeleton-modified enzyme geneD6HAnd a backbone-modifying enzyme geneUGTPn20The amplification primers were as follows:

PnDDS-Kpn I -F：5'-GGGTACCCCTGCAGATGTGGAAGCTGAA-3'；

PnDDS-Pst I-R：5'-TGCACTGCAGTGCACCCGGGTTAAATTTTGAGCT-3'；

P12H-Sac I-F：5'-CGAGCTCGATGGTGTTGTTTTTCTCCCTATCT-3'；

P12H-Pst I -R：5'-TGCACTGCAGTGCATTAATTGTGGGGATGTAGATGAAT-3'；

D6H-Sac I-F：5'-CGAGCTCATGGATCTCTTTATCTCATCTC-3'；

D6H-BamH I-R：5'-CGCGGATCCCAAGGTGATAGACGAATAGG-3'；

UGTPn20-EcoR I-F：5'-GGAATTCCATAAATCTGGGAAAGGG-3'；

UGTPn20-Sac I–R：5'-CGAGCTCGCCAACAATAAAATCGTCACT-3'；

(restriction sites corresponding to the respective primers are underlined)

(2) Respectively connecting the target fragments to pCAMBIA2300s plant expression vectors, and transforming agrobacterium; screening out positive monoclonal by PCR;

(3) infecting tobacco leaf with positive agrobacterium containing target gene, and regenerating plant to obtain tobacco leaf containing target genePnDDS、P12H、D6HAndUGTPn20transgenic tobacco plants of four genes;

(4) extracting total RNA from positive transgenic tobacco, performing reverse transcription to obtain cDNA, and performing RT-PCR to determine whether four genes are expressed;

(5) drying and grinding the leaves of the transgenic tobacco, soaking the leaves in methanol for 24 hours, then ultrasonically extracting saponin, and detecting the types and the content of the ginsenoside in the transgenic tobacco by using HPLC.

The invention has the advantages and technical effects that:

the invention is based on the characteristics of easy planting and rapid growth of tobacco, and separates the gene from notoginsengPnDDS、P12H、D6HAndUGTPn20simultaneously transferring into tobacco and obtaining the expression of the samePnDDS、P12H、D6HAndUGTPn20the experimental results of the four-gene transgenic tobacco show that the transgenic plant obtained by the invention canCan synthesize ginsenoside F₁To make ginsenoside F₁The method is simple, easy to operate and suitable for large-scale production and market popularization and application, and the method is used for obtaining the ginsenoside F₁Provides a new approach.

Drawings

FIG. 1 shows genesPnDDS、P12H、D6HAndUGTPn20 detecting the electrophoresis detection result of the PCR product, wherein M is DNA Marker;

FIG. 2 is the identification electrophoretogram of four genes transferred into Agrobacterium LBA 4404;

wherein the A diagram is pCAMBIA2300s-DS(ii) a The B diagram is pCAMBIA2300s-P12H(ii) a The C diagram is pCAMBIA2300s-D6H(ii) a The diagram D is pCAMBIA2300s-UGTPn20In the figure, M is a DNA Marker, and 1-5 are positive agrobacterium strains; "-" is water as template; "+" is cDNA as template;

FIG. 3 is a PCR-verified electrophoresis of DNA in transgenic tobacco, wherein M is DNA Marker, WT is wild type tobacco, T1-T6 is transgenic tobacco, and "+" is cDNA as template;

FIG. 4 is a PCR-verified electrophoresis of cDNA in transgenic tobacco, wherein M is DNA Marker, WT is wild type tobacco, T1-T6 is transgenic tobacco, and "+" is cDNA as template;

FIG. 5 is a graph showing the saponin content in transgenic tobacco, wherein WT is wild type tobacco, T4-T5 is transgenic tobacco, DD is dammarenediol, PPD is protopanaxadiol, and PPT is protopanaxatriol.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the present invention, and the methods used in the examples are all conventional methods unless otherwise specified, and reagents used therein are all conventional commercially available reagents or reagents prepared by conventional methods.

Example 1:PnDDS、P12H、D6HandUGTPn20cloning of genes

Grinding Notoginseng radix with liquid nitrogen into powder, transferring into centrifuge tube, extracting total RNA from Notoginseng radix root by guanidinium isothiocyanate method,the first strand cDNA is synthesized by reverse transcription, and the reaction system and the operation process are as follows: taking 5 μ g of Total RNA, adding 50ng oligo (dT), 2 μ L dNTP (2.5 mM each) and DEPC water in sequence to the reaction volume of 14.5 μ L; after mixing uniformly, heating and denaturing at 70 ℃ for 5min, then quickly cooling on ice for 5min, then sequentially adding 4 mu L of 5 XFirst-stand buffer, 0.5 mu LRNasin (200U) and 1 mu L M-MLV (200U), mixing uniformly and centrifuging for a short time, carrying out warm bath at 42 ℃ for 1.5h, taking out, heating at 70 ℃ for 10min, and terminating the reaction; amplifying ginsenoside F by PCR using the synthesized first strand cDNA as template₁Four genes in the synthetic pathwayPnDDS、P12H、D6HAndUGTPn20using CE Design software to Design primers with homology arms, and the primers used for amplification are respectively as follows:

PnDDS-Kpn I -F：5'-GGGTACCCCTGCAGATGTGGAAGCTGAA-3'；

PnDDS-Pst I-R：5'-TGCACTGCAGTGCACCCGGGTTAAATTTTGAGCT-3'；

P12H-Sac I-F：5'-CGAGCTCGATGGTGTTGTTTTTCTCCCTATCT-3'；

P12H-Pst I -R：5'-TGCACTGCAGTGCATTAATTGTGGGGATGTAGATGAAT-3'；

D6H-Sac I-F：5'-CGAGCTCATGGATCTCTTTATCTCATCTC-3'；

D6H-BamH I-R：5'-CGCGGATCCCAAGGTGATAGACGAATAGG-3'；

UGTPn20-EcoR I-F：5'-GGAATTCCATAAATCTGGGAAAGGG-3'；

UGTPn20-Sac I–R：5'-CGAGCTCGCCAACAATAAAATCGTCACT-3'；

(restriction sites corresponding to the respective primers are underlined)

The PCR reaction system is 50 μ L and comprises: 25 μ L of LPrime STAR MAX Premix, 1 μ L of forward primer F, 1 μ L of reverse primer R1 μ L, 1 μ L of cDNA, 22 μ L of ddH₂O; and (3) PCR reaction conditions: 3min at 98 ℃; 10 s at 98 ℃, 15 s at 60 ℃, 15 s at 72 ℃ and 35 cycles; 5min at 72 ℃; after PCR is finished, agarose gel electrophoresis detectionThe detection result is shown in figure 1, and the sizes of the four gene amplification bands are respectively as follows: 2310bp, 1503bp, 1407bp and 1423 bp. After successful amplification, the target band was recovered using a SanPrep column DNA gel recovery kit (Shanghai Biotech): taking 1 microliter of the recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for later use.

Example 2: plant expression vector construction

The plant expression vector pCAMBIA2300s was linearized with restriction enzymes, which were 15. mu.L pCAMBIA2300s plasmid, 5. mu.L 10 XM buffer, 2.5. mu.L each of enzymes at the front and rear cleavage sites, 25. mu.L ddH₂O, mixing uniformly, centrifuging for a short time, and placing at 37 ℃ for enzyme digestion for 3.5 h; dropping the enzyme digestion product in agarose gel for electrophoresis, then carrying out gel recovery on the pCAMBIA2300s carrier large fragment, and using a SanPrep column type DNA gel recovery kit (Shanghai production) in the whole process; taking 1 microliter of the recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for later use. In the case of homologous recombination, the collected product is assembled using the Clonexpressing Multi S One Step Cloning KitPnDDS、P12H、D6HAndUGTPn20respectively connected to pCAMBIA2300s plant expression vector, reaction system (10 μ L) and operation process: 4 mu.L pCAMBIA2300S vector DNA, 2 mu.L target fragment, 2 mu.L 5 xcell buffer, and 1 mu. L, ddH of Exnase II₂O1 mu L, mixing uniformly, centrifuging for a short time, and then carrying out water bath reaction at 37 ℃ for 30 min; then transferring the ligation product into escherichia coli DH5 alpha by adopting a heat shock transformation method, coating the escherichia coli DH5 alpha on an LB solid culture medium containing 50mg/L kanamycin, and culturing for 24h at 37 ℃; selecting single bacterial colony in LB liquid culture medium containing 50mg/L kanamycin, shaking bacteria at 37 ℃ and 200rpm, respectively carrying out PCR by using bacterial liquid as a template and using specific primers for amplifying four genes, respectively selecting clones of the four genes successfully connected with pCAMBIA2300s, if the detected bacterial strain is positive, sending the positive to a sequencing company for further final confirmation, and finally adding glycerol into the escherichia coli bacterial liquid containing the target gene and storing the escherichia coli bacterial liquid at-80 ℃ for later use.

Extraction of pCAMBIA2300 s-containing protein from Escherichia coli by using SanPrep column type plasmid DNA small extraction kitDS、pCAMBIA2300s-P12H、pCAMBIA2300s-D6HAnd pCAMBIA2300s-UGTPn20Plasmid is extracted according to the instruction of the kit; taking 1 μ L of the extract, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 deg.C for use.

Example 3: agrobacterium-mediated genetic transformation of plants and transgenic plant screens

The plant expression vector pCAMBIA2300s constructed above is frozen and thawed by liquid nitrogenDS、pCAMBIA2300s-P12H、pCAMBIA2300s-D6HAnd pCAMBIA2300s-UGTPn20Transferring the agrobacterium tumefaciens LBA4404 competent cells into an agrobacterium tumefaciens LBA4404 competent cell, and comprising the following operation steps: adding 2 mu g of plasmid into a centrifuge tube containing 200 mu L of competent cells, gently mixing uniformly, performing ice bath for 5min, then transferring into liquid nitrogen for freezing for 1min, then rapidly placing in a 37 ℃ water bath for 5min, then immediately performing ice bath for 2min, adding 800 mu L of LB liquid culture medium, and performing shake culture at 28 ℃ and 200rpm for 4 h; coating the activated agrobacterium on LB solid culture medium containing 50mg/L kanamycin and 25mg/L rifampicin, and standing and culturing at 28 ℃ for about 48 h; selecting single colony shake bacteria, shake culturing in LB liquid culture medium containing 50mg/L kanamycin and 25mg/L rifampicin at 28 deg.C and 200rpm for 24h, finally performing PCR with specific primer for amplifying four genes to determine pCAMBIA2300s-DS、pCAMBIA2300s-P12H、pCAMBIA2300s-D6HAnd pCAMBIA2300s-UGTPn20The plasmid has been transformed into Agrobacterium; the results of PCR detection are shown in FIG. 2, and 5 strains of transgenic pCAMBIA2300s-DS、pCAMBIA2300s-P12H、pCAMBIA2300s-D6HAnd pCAMBIA2300s-UGTPn20Positive agrobacterium strain of plasmid, and the sequence is identical to the original sequence through sequencing verification, and the strain glycerol is preserved at-80 ℃ for standby.

Example 4: agrobacterium-mediated genetic transformation of tobacco

The transgenic recipient in this experiment was tobacco, tobacco seeds were soaked in 75% ethanol for 30s, washed with sterile water and then washed with 0.1% HgCl₂Soaking for 8min, washing with sterile water for several times, sowing on 1/2MS culture medium, dark culturing at 28 deg.C for 6d, germinating, transferring to light incubator (25 deg.C, 16 h/d light), and subculturing with 1/2MS culture medium every monthNext, the process is carried out.

The preserved liquid containing pCAMBIA2300s was taken out from the-80 ℃ refrigeratorDS、pCAMBIA2300s-P12H、pCAMBIA2300s-D6HAnd pCAMBIA2300s-UGTPn20Agrobacterium LBA4404 strain of plasmid, inoculating Agrobacterium containing target gene in LB liquid culture medium containing 50mg/L kanamycin and 20mg/L rifampicin, culturing at 28 deg.C and 200rpm until the culture medium is turbid; sucking 1mL of turbid mixed bacteria liquid to an LB solid culture medium containing 50mg/L kanamycin and 20mg/L rifampicin, and culturing for 48h at 28 ℃; then scraping off the agrobacterium on an LB solid culture medium, inoculating the agrobacterium on an MGL liquid culture medium added with 20mg/L acetosyringone, and carrying out shake culture at 28 ℃ for 2-3h to activate the agrobacterium.

Mixing 1cm²Placing tobacco leaf with size into MGL liquid culture medium containing four target genes Agrobacterium tumefaciens, and shake-culturing at 120rpm and 28 deg.C for 20 min; after infection, sucking the taken bacterial liquid of the tobacco leaves by using sterile filter paper, transferring the bacterial liquid into an MS solid culture medium containing 40mg/L acetosyringone, and performing dark culture for 48 hours at about 25 ℃;

transferring the tobacco leaves after the co-culture to a screening culture medium (MS +0.5 mg/L6-BA +0.1mg/L NAA +50mg/L kanamycin +300 mg/L cephamycin) for selective culture, transferring a culture bottle to a light incubator for culture (25 ℃, 16 h/d light and 8 h/d dark) during the screening culture, carrying out subculture on the tobacco leaves after the tobacco leaves are differentiated and germinated by using an MS culture medium containing 50mg/L kanamycin and 300mg/L cephamycin, wherein the regeneration plants need to be further screened because the callus differentiation rate of the tobacco is higher, transferring the regeneration seedlings of the tobacco to the MS culture medium containing 50mg/L kanamycin to root the regeneration plants, and finally selecting the regeneration seedlings with better rooting for further detection.

Detecting DNA level, extracting DNA of transgenic tobacco leaf by CTAB method, taking genome DNA of transgenic plant as template, and amplifying genePnDDS、P12H、D6HAndUGTPn20carrying out PCR on the specific primer; the PCR reaction system is 2 μ L DNA, 0.4 μ L upstream primer (10 μ M), 0.4 μ L downstream primer (10 μ M), 10 μ L PCR Mix, 7.2 μ L ddH 2O; and (3) PCR reaction conditions: 5min at 94 ℃; 94 ℃ for 30s, 60 ℃ for 90s, 72 ℃ for 1min, 32 cycles; 7min at 72 ℃; after the PCR is finished, 10 μ L of the product is used for agarose gel electrophoresis to detect positive transgenic plants, the amplification result of part of tobacco transgenic plants is shown in FIG. 3, and 6 positive plants are obtained by detection on the DNA level.

RNA level detection, total RNA is extracted from positive transgenic single plants and young leaves of non-transgenic tobacco (wild type) on DNA level, the method for extracting total RNA is the same as that in example 1, reverse transcription is carried out to generate a first cDNA chain, and the first cDNA chain is used as a template for amplificationPnDDS、P12H、D6HAndUGTPn20carrying out PCR on specific primers of the genes; the PCR reaction system was 1.5. mu.L LcDNA, 0.4. mu.L of the forward primer (10. mu.M), 0.4. mu.L of the reverse primer (10. mu.M), 10. mu.L of PCR Mix, 7.7. mu.L of ddH₂O; and (3) PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 90s at 72 ℃ and 32 cycles; 7min at 72 ℃; after the PCR was completed, 10. mu.L of the resulting mixture was subjected to agarose gel electrophoresis, and each transgenic individual was analyzed based on the PCR resultsPnDDS、P12H、D6HAndUGTPn20expression of four gene transcription levels, after PCR was completed, 10. mu.L of the DNA was subjected to agarose gel electrophoresis, and the results of detection of some individuals were shown in FIG. 4, and co-detected in T4 and T5 transgenic individualsPnDDS、P12H、D6HAndUGTPn20four genes are expressed in large amounts at the transcriptional level.

Example 5:PnDDS、P12H、D6HandUGTPn20gene expression for synthesizing ginsenoside F from tobacco₁Influence of (2)

Drying and grinding the leaves of the transgenic tobacco, weighing 1.0g of tobacco leaf powder, soaking the tobacco leaf powder in 50mL of methanol for 24 hours, then performing ultrasonic treatment for 60min, extracting saponin in the transgenic tobacco, and detecting the type and content of ginsenoside in the transgenic tobacco by using HPLC; the specific chromatographic conditions are as follows: high performance liquid chromatography (Agilent 1260), chromatographic column GRACE Vision HT C18 HL (250 mm x 4.6 mm, 5 μm), mobile phase acetonitrile (A)/water (B), flow rate of 1.0mL/min, column temperature of 30 ℃, detection wavelength of 203nm, gradient elution (v/v) by acetonitrile (A)/water (B), gradient elution program: 0-20min, 20% acetonitrile; 20-30min, 20% -35% acetonitrile; 30-40min, 35% acetonitrile; 40-50min, 35-40%Acetonitrile; 50-60min, 40-100% acetonitrile; the results are shown in FIG. 5; wherein in T5 plant, ginsenoside F₁The content of (a) is the highest and reaches 88.7 mug/g DW (dry weight); small amounts of the precursors dammarenediol (DD), protopanaxadiol (PPD) and protopanaxatriol (PPT) were also detected.

Sequence listing

<110> university of Kunming science

<120> a method for preparing ginsenoside F1

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2310

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 1

atgtggaagc tgaaggttgc tcaaggaaat gatccatatt tgtatagcac taacaacttt 60

gttggcagac aatattggga gtttcagccc gatgctggta ctccagaaga gagggaagag 120

gttgaaaatg cacgcaagga ttatgtaaac aataaaaagc tacatggagt tcatccatgc 180

agtgatatgc tgatgcgcag gcagcttatt aaagaaagtg gaatcgatct cctaagcata 240

ccgccggtga gattagatga aaacgaacaa gtgaactacg atgcagttac aaccgctgtg 300

aagaaagctc ttcgattgaa ccgggcaatt caagcacacg atggtcactg gccagctgaa 360

aatgcaggct ctttacttta tacacctccc cttatcattg ccctatatat cagcggaacg 420

attgacacta ttctgacaaa acaacacaag aaggaactga ttcgcttcgt ttacaaccat 480

caaaatgagg atggtggatg gggatcctat attgaggggc acagcacgat gattgggtca 540

gtacttagct tcgtgatgtt acgtttgcta ggagaaggat tagctgaatc tgatgatgga 600

aatggtgcag ttgagagagg ccggaagtgg atacttgatc atggaggtgc agccagcata 660

ccctcttggg gaaagactta tctagcggtg cttggagtat atgagtggga agggtgcaac 720

ccgctgcccc cagaattctg gcttttccct tcaagttttc cttttcatcc agcaaaaatg 780

tggatctact gccggtgtac ctacatgcca atgtcgtatt tgtatgggaa gagatatcat 840

ggaccaataa ccgatcttgt tttatctttg agacaagaaa tttacaacat tccttatgag 900

cagataaagt ggaatcaaca gcgccataac tgttgcaagg aggatctcta ctaccctcat 960

tcccttgtac aagacctggt ttgggatggt cttcactact ttagtgaacc attcctcaaa 1020

cgttggccct tcaacaaact gcgaaaaaga ggtctaaaaa gagtggttga actaatgcgc 1080

tatggtgcca ccgagaccag attcataacc acaggaaatg gggaaaaagc tttacaaata 1140

atgagttggt gggcagaaga tcccaatggt gatgagttta aacatcacct tgctagaatt 1200

cctgatttct tatggattgc tgaggatgga atgacagtac agagttttgg tagtcaacta 1260

tgggactgta ttcttgctac tcaagcaatt atcgccacca atatggttga agaatacgga 1320

gattctctta agaaggcgca tttcttcatc aaagaatcgc agataaaaga aaatccaaga 1380

ggagacttct taaaaatgtg tcgacagttt actaaaggtg cgtggacttt ctctgatcaa 1440

gatcatggtt gcgttgtctc ggactgcaca gctgaagcac taaagtgcct tctgttactt 1500

tcacaaatgc cacaggacat tgtcggagaa aaacctaagg ttgagcgatt atatgaggct 1560

gtgaatgttc ttctctattt gcagagtcgt gtaagtggtg gcttcgcagt ttgggagcct 1620

ccagttccaa aaccatattt ggagatgttg aatccttcag aaatttttgc agacattgtt 1680

gttgagagag agcacattga atgcactgca tctgtaatca aaggtctgat ggcatttaaa 1740

tgcttgcatc ctgggcatcg tcagaaagag atagaggatt ctgtggcgaa agccatccgt 1800

tatcttgaaa gaaaccaaat gcctgatggt tcatggtatg gcttttgggg aatttgtttc 1860

ctctatggga cattttttac cctatcaggg tttgcttctg ctgggaggac ttatgacaac 1920

agtgaagcag ttcgtaaggg tgttaaattt ttcctttcaa cacaaaatga agaaggtggt 1980

tggggggaga gtcttgaatc atgcccaagc gagaaattta caccactcaa gggaaacagg 2040

acaaatctag tacaaacatc atgggctatg ctaggtctta tgtttggtgg acaggccgag 2100

agagatccga cacctctgca tagagcagcg aagttgttga tcaatgcgca aatggataat 2160

ggagatttcc ctcaacagga aattactgga gtatactgta aaaatagtat gttacattat 2220

gcggagtaca gaaatatatt tcctctttgg gcactcggag aatatcggaa acgtgtttgg 2280

ttgccaaagc accagcagct caaaatttaa 2310

<210> 2

<211> 1503

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 2

atgcacatta cattcaatcc aaccaaaacc ttaggtgata tggcagcagc aatggtgttg 60

tttttctccc tatctcttct tctccttccc cttcccctat tattgtttgc ctatttttct 120

tatacaaaac gcatccccca gaaagaaaat gattcaaaag ctcccctccc ccccggtcaa 180

acaggttggc ctttgatagg cgaaactctt aattatttat cttgtgtcaa aagtgggttt 240

tctgaaaatt ttgtgaaata taggaaggaa aagtattccc ccaaagtttt caggacatct 300

cttttaggag aaccgatggc aatcttgtgt gggccggagg ggaacaaatt cctctactca 360

acggaaaaaa agctagtcca aacttggttc ccgagcagtg ttgaaaagat gttccccaga 420

tctcatggcg aatccaacgc agacaacttc tccaaagtac gcggcaaaat gatgtttcta 480

ctcaaggtgg acgggctgaa aaaatatgtt ggcctaatgg acagggtgat gaaacagttt 540

ttagagacgg attggaatcg ccaacaacaa atcaacgttc acaacacggt taagaaatac 600

acggtcacga tgtcgtgtcg ggtgtttatg agtatcgatg atgaagagca agtcagaaga 660

cttggcagct caattcagaa catagaggcc ggactcctcg ccgtgcctat aaatataccg 720

gggactgcta tgaatcgtgc cattaagacc gtaaagttgc tatctagaga ggttgaggcg 780

gtgattaagc aaagaaaagt ggatcttttg gagaataagc aagcgtccca accgcaagat 840

ttattgtcac acttgctact tacggccaat caggatggcc agtttttgag cgaatcggat 900

attgctagcc acttgatagg cttgatgcaa ggtggttata ccaccttaaa tggtacaatc 960

accttcgtta tcaactatct tgcagagttt cctgatgtct acaatcaagt ccttaaagag 1020

caagtggaaa tagcaaactc aaaacaccca aaagagttgc ttaattggga ggatttgagg 1080

aagatgaagt attcgtggaa tgttgctcaa gaggtattga gaataatacc accaggagtt 1140

ggaacattca gagaggctat taccgacttc acctatgctg gatatttaat tccaaaggga 1200

tggaagatgc atctgattcc acatgacacg cacaagaacc caacatattt tccaaatcca 1260

gaaaaattcg atccaaccag gtttgaagga aatggtccgg ctccatatac atttactcct 1320

ttcggaggag gacctcgaat gtgtccggga attgaatatg cacgtctagt aatactcatt 1380

tttattcaca atgtggttac aaacttcaga tgggagaagc tcatccctag tgaaaaaatt 1440

ctcaccgatc cgattccaag atttgcgcat ggacttccaa ttcatctaca tccccacaat 1500

taa 1503

<210> 3

<211> 1407

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 3

atggatctct ttatctcatc tcaattactt cttctactag tcttttgctt attcctcttt 60

tggaatttca aaccaagtag ccaaaacaaa cttccccccg gcaaaacagg atggcccata 120

attggagaaa cactagaatt catctcctgt ggccaaaaag gtaaccctga aaagttcgta 180

acacaaagaa tgaaaaaata ctcccctgat gtcttcacaa catccttagc aggcgagaaa 240

atggtagttt tctgcggtgc ctyggggaac aaattcwttt tctccaasga aaacaagctt 300

gttgtgtcct ggkggccccc tgccawwtcm aaaatcctaa ctgcaacaat accttcggta 360

gagaaaagca aagccttgcg gagtctaatt gttgaattct taaaacccga agcaytccac 420

aagtttattt cggtcatgga tcggacaacg aggcagcact ttgaagccaa atggaacggg 480

agtacagaag tgaaagcttt cgctatgtca gagacgctga cttttgagtt ggcctgttgg 540

ctgctcttta gcataagtga tccggtgcag gtgcagaagc tttctcatct ttttgagaag 600

gttaaagcgg gattattgtc tttaccttta aactttccgg gcacggcttt taaccgtggg 660

atcaaggccg ccaatcttat tagaaaagag ctttcggtgg tgataaaaca gaggagaagt 720

gataaatcag agactcgaaa ggatcttttg tcccacgtta tgatttccaa tggcgagggc 780

gagaaatttt tcagcgaaat ggatattgcg gacgttgttc ttaatatact gattgctagc 840

catgatacca ctagcagtgc catgggctct gtggtctact ttcttgcaga tcatcctcac 900

atctatgcta aagtcctcac agaacaaatg gagatcgcaa agtcgaaagg ggcaggagaa 960

cttttgagct gggacgacat caagaggatg aagtattccc gcaatgttat aaatgaagct 1020

atgagattag tacctccttc tcaaggaggt tttaaagtag ttacaagtaa attcmgttac 1080

gcmaacttca tcwttcccaa aggatggaar atcttttgga gcgkwwwctc gacacataaa 1140

gatcccaaat actttaaaaa tccagaggag tttgatcctt caagatttga aggagatgga 1200

cctatgccat tcacatttat accatttgga ggaggaccaa ggatgtgccc tgggagtgag 1260

tttgctcgtc tggaggttct aatattcatg caccatttgg ttaccaattt taggtgggag 1320

aaggtgtttc ccaatgaaaa gattatttat actccatccc tcccggagaa tggtcttcct 1380

attcgtctat caccttgtac gctttaa 1407

<210> 4

<211> 1423

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 4

atgaagtcag aattgatatt cttccttacg acggctcgga cacctcgtgg gatggtggag 60

atggctaaac tcttcatcag tcgacatgaa aacctctcgg tcaccgtcct catcgcgaaa 120

ttctacatgg atacgggggt agacaactac aataaatcac tcttaacaaa gcctaccccg 180

cgtctcacaa ttgtaaatct cccggaaagc gacccccaaa actatatgct caaaccacgc 240

cacgccatct ttcctagcgt catcgagact cagaagacac acgtgcgaga cataatatca 300

ggcatgactc agtccgagtc gactcgggtc gttggtttgc tggctgacct tttgttcatc 360

ammmywwtkg rcattgccaa tgagttcaat gttccaactt atgtatactc ccctgccgga 420

gcaggtcaty ttggcctygs gttccatytc cagacactca acgacaaaaa gcaagatgtg 480

accgagttca ggcactcgga cactgagtta ttggtaccga gttttgcaaa cccggttccc 540

gccgaggtct tgccgtcgat gtatgtggat aaagaaggtg ggtatgatta tttgttttca 600

ttgttccgga ggtgcagaga gtcaaaggca attattatta acacgtttga ggagctggaa 660

ccctatgcga tcaattccct ccggatggat agtatgatcc ctccgatcta cccggtggga 720

cccatactaa atctcaacgg tgatggccaa aactccgatg aggctgctgt gatccttggt 780

tggttagacg atcaaccacc ttcatctgtg gtgtttttgt gctttggtag ctatggaacc 840

tttcaagaaa accaggtgaa ggagattgca atgggtctag agcgcagtgg gcatcgcttc 900

ttgtggtcct tgcgtccgtc tatccctaaa ggcgagacaa agcttcagct taaatactca 960

aatttggaag aaattctccc agtcggattc ttggacagga catcatgcgt cggaaaagtt 1020

attggatggg ccccgcaagt ggcggtgcyc gracacgagg cagtcggagg gttcctgtct 1080

cattgtggtt ggaattcgac attagagagt gtgtggtgtg gcgtgcccgt cgcaacatgg 1140

ccaatgtacg gcgagcaaca actcaatgct tttgagatgg ttaaggagtt gggtattgcg 1200

gtggaaattg aggtggacta taagaatgaa tattttaaca tgacgaatga ttttattgtt 1260

agggcagaag aaatcgagac gaaaataaag aagctgatga tggatgaaaa gaatagtgaa 1320

ataaggaaga aggtaaagga aatgaaagaa aagagtaggc ttgcaatgtc tgagaatgga 1380

tcatcttata atcatgacga aggtatttga ggaaattatg taa 1423

<210> 5

<211> 28

<212> DNA

<213> Artificial sequence (Artificial)

<400> 5

gggtacccct gcagatgtgg aagctgaa 28

<210> 6

<211> 34

<212> DNA

<213> Artificial sequence (Artificial)

<400> 6

tgcactgcag tgcacccggg ttaaattttg agct 34

<210> 7

<211> 32

<212> DNA

<213> Artificial sequence (Artificial)

<400> 7

cgagctcgat ggtgttgttt ttctccctat ct 32

<210> 8

<211> 38

<212> DNA

<213> Artificial sequence (Artificial)

<400> 8

tgcactgcag tgcattaatt gtggggatgt agatgaat 38

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence (Artificial)

<400> 9

cgagctcatg gatctcttta tctcatctc 29

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence (Artificial)

<400> 10

cgcggatccc aaggtgatag acgaatagg 29

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 11

ggaattccat aaatctggga aaggg 25

<210> 12

<211> 28

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

cgagctcgcc aacaataaaa tcgtcact 28

Claims (1)

1. Ginsenoside F₁The preparation method is characterized by comprising the following steps: the genePnDDS、P12H、D6HAndUGTPn20simultaneously transferring into tobacco to obtain synthetic ginsenoside F₁The transgenic tobacco of (1), wherein the genePnDDSThe nucleotide sequence of (A) is shown as SEQ ID NO. 1, geneP12HThe nucleotide sequence of (A) is shown as SEQ ID NO. 2, geneD6HThe nucleotide sequence of (A) is shown as SEQ ID NO. 3, the geneUGTPn20The nucleotide sequence of (A) is shown in SEQ ID NO. 4.

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