CN113549649B - Preparation method of ginsenoside F1 - Google Patents

Abstract

The invention discloses a preparation method of ginsenoside F _1. The method is to simultaneously transfer genes PnDDS , P12H , D6H and UGTPn20 into tobacco to obtain transgenic tobacco capable of synthesizing ginsenoside F ₁ , wherein the nucleotide sequence of the gene PnDDS is As shown in SEQ ID NO:1, the nucleotide sequence of gene P12H is shown in SEQ ID NO:2, the nucleotide sequence of gene D6H is shown in SEQ ID NO:3, and the nucleotide sequence of gene UGTPn20 is shown in SEQ ID NO:3 ID NO: 4; the experimental results show that the transgenic tobacco plants obtained by the present invention can synthesize ginsenoside F ₁ , which makes the acquisition of ginsenoside F ₁ more efficient and convenient, and the method of the invention is simple, easy to operate, and suitable for large-scale production and Market promotion and application, the invention provides a new way for obtaining ginsenoside F ₁ .

Description

A kind of preparation method of ginsenoside F1

technical field

The invention belongs to the technical field of biological medicine preparation, in particular to _a method for heterologous synthesis of ginsenoside F1 in tobacco.

Background technique

Ginseng plants have been used to treat various diseases and improve bodily functions all the time, and triterpenoid saponins are the main active components of ginseng plants. Triterpenoid saponins have various pharmacological effects such as promoting cell proliferation, inhibiting cell apoptosis, inhibiting oxidative stress, anti-inflammatory, and increasing cell viability, and can protect nerve cells, cardiomyocytes, liver cells, lung epithelial cells and other cells. effect.

Ginsenoside F ₁ ( ginsenoside F ₁ ) is a rare dammarane-type triterpenoid saponin component in medicinal plants such as ginseng, Panax notoginseng and American ginseng. It is composed of PPT and glycosyl donor UDP-glucose (UDP-glucose) through UDP- A protopanaxatriol saponin produced by the catalysis of glucosyltransferase, has anti-tumor, anti-aging, anti-oxidation and other effects. Ginsenoside F ₁ has a strong inhibitory effect on the proliferation and migration of skin cancer cells B16 and the production of melanin, and can also protect the damage to human stratum corneum HaCaT caused by ultraviolet rays. Although ginsenoside F ₁ has important pharmacological activities, its content in the medicinal materials of the genus Ginseng is very low, and the species of ginseng is a perennial plant with a long growth cycle, which makes ginsenoside F ₁ difficult to obtain and expensive. There is an urgent need to develop and obtain ginseng. Novel methods and routes for saponin F ₁ .

Tobacco, as an economic crop with extensive planting and mature cultivation technology, has a rich natural product synthesis system in the body, and tobacco has a mature transgenic technology system. Although tobacco may have the potential to synthesize natural compounds, no reports of synthesizing ginsenoside F ₁ in tobacco have been reported, and no dammarane-type tetracyclic triterpenoid saponins, including ginsenoside F ₁ , have been found in the tobacco genome. The complete pathway indicates that tobacco itself does not have the ability to synthesize such saponins.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a preparation method of ginsenoside F ₁ , which produces ginsenoside F ₁ in tobacco by constructing a method for synthesizing ginsenoside F ₁ in tobacco. A new way to obtain ginsenoside F ₁ ;

The method of the invention is to simultaneously transfer genes PnDDS , P12H , D6H and UGTPn20 into tobacco to obtain transgenic tobacco capable of synthesizing ginsenoside F ₁ , wherein the nucleotide sequence of the gene PnDDS is shown in SEQ ID NO: 1, the gene P12H The nucleotide sequence of the gene is shown in SEQ ID NO: 2, the nucleotide sequence of the gene D6H is shown in SEQ ID NO: 3, and the nucleotide sequence of the gene UGTPn20 is shown in SEQ ID NO: 4.

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

(1) Extract total RNA from the root of Panaxnotoginseng , reverse transcription to synthesize Panax notoginseng cDNA, and use the synthesized first-strand cDNA as a template to amplify the Panax notoginseng backbone synthase gene PnDDS , backbone modification enzyme gene P12H , The backbone modification enzyme gene D6H and the backbone modification enzyme gene UGTPn20 , the amplification primers are as follows:

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

PnDDS-Pst I- R: 5'- TGCACTGCAGTGCA CCCGGGTTAAATTTTGAGCT-3';

P12H-Sac I- F: 5'- CGAGCTCG ATGGTGTTGTTTTTCTCCCTATCT-3';

P12H-Pst I- R: 5'- TGCACTGCAGTGCA TTAATTGTGGGGATGTAGATGAAT-3';

D6H - Sac I -F:5'- CGAGCTC ATGGATCTCTTTATCTCATCTC-3';

D6H - BamH I -R: 5'- CGCGGATCCCAA GGTGATAGACGAATAGG-3';

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

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

(underlined are the corresponding restriction sites of the respective primers)

(2) The target fragments were connected to the pCAMBIA2300s plant expression vector respectively, and transformed into Agrobacterium; positive single clones were screened by PCR;

(3) Infect tobacco leaves with positive Agrobacterium containing the target gene, and obtain transgenic tobacco plants containing four genes of PnDDS , P12H , D6H and UGTPn20 through plant regeneration;

(4) Extract the total RNA from the positive transgenic tobacco, reverse-transcribe it into cDNA, and perform RT-PCR to determine whether the four genes are expressed;

(5) The leaves of transgenic tobacco were dried and ground, soaked in methanol for 24 hours, and then ultrasonically extracted saponins. The types and contents of ginsenosides in transgenic tobacco were detected by HPLC.

Advantages and technical effects of the present invention:

Based on the characteristics of easy planting and rapid growth of tobacco, the present invention simultaneously transfers genes PnDDS , P12H , D6H and UGTPn20 isolated from Panax notoginseng into tobacco, and obtains transgenic tobacco capable of simultaneously expressing four genes of PnDDS , P12H , D6H and UGTPn20 . , the experimental results show that the transgenic plant obtained by the present invention can synthesize ginsenoside F ₁ , which makes the acquisition of ginsenoside F ₁ more efficient and convenient. The method of the invention is simple, easy to operate, and suitable for large-scale production and market promotion and application. Ginsenoside F ₁ provides a new approach.

Description of drawings

Fig. 1 is the electrophoresis detection result of gene PnDDS , P12H , D6H and UGTPn20 PCR products, and M in the figure is DNAMarker;

Fig. 2 is the identification electropherogram of four genes transferred into Agrobacterium LBA4404;

A picture is pCAMBIA2300s- DS ; B picture is pCAMBIA2300s -P12H ; C picture is pCAMBIA2300s- D6H ; D picture is pCAMBIA2300s -UGTPn20 , M in the figure is DNA Marker, 1-5 are positive Agrobacterium strains; "-" is water is the template; "+" is the cDNA template;

Fig. 3 is a PCR verification electropherogram 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 verification electropherogram 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;

Figure 5 is a graph of saponin content in transgenic tobacco, wherein WT is wild-type tobacco, T4-T5 is transgenic tobacco, DD is dammarediol, PPD is protopanaxadiol, and PPT is protopanaxatriol.

Detailed ways

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

Example 1: PnDDS , P12H , D6H and UGTPn20 gene cloning

Using Panax notoginseng root as material, grind Panax notoginseng root into powder with liquid nitrogen, then transfer it into a centrifuge tube, extract total RNA from Panax notoginseng root by guanidine isothiocyanate method, reverse transcription to synthesize the first strand of cDNA, the reaction system And the operation process is as follows: take 5μg TotalRNA, add 50ng oligo (dT), 2μL dNTP (2.5mM each), DEPC water in turn to the reaction volume of 14.5μL; after mixing, heat denaturation at 70°C for 5min, then quickly cool on ice 5 min, then add 4 μL 5×First-standbuffer, 0.5 μL LRNasin (200 U), 1 μL M-MLV (200 U) in sequence, mix well, centrifuge for a short time, incubate at 42 °C for 1.5 h, take out and heat at 70 °C for 10 min to terminate the reaction; The synthesized first-strand cDNA was used as the template, and the open reading frame sequences of the four genes PnDDS , P12H , D6H and UGTPn20 in the synthetic pathway of ginsenoside F ₁ were amplified by PCR, and primers with homology arms were designed using CE Design software. The primers used are as follows:

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

PnDDS-Pst I- R: 5'- TGCACTGCAGTGCA CCCGGGTTAAATTTTGAGCT-3';

P12H-Sac I- F: 5'- CGAGCTCG ATGGTGTTGTTTTTCTCCCTATCT-3';

P12H-Pst I- R: 5'- TGCACTGCAGTGCA TTAATTGTGGGGATGTAGATGAAT-3';

D6H - Sac I -F:5'- CGAGCTC ATGGATCTCTTTATCTCATCTC-3';

D6H - BamH I -R: 5'- CGCGGATCCCAA GGTGATAGACGAATAGG-3';

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

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

(underlined are the corresponding restriction sites of the respective primers)

The PCR reaction system was 50 μL, including: 25 μL Prime STAR MAX Premix, 1 μL upstream primer F, 1 μL downstream primer R 1 μL , 1 μL cDNA, 22 μL ddH ₂ O; PCR reaction conditions: 98°C for 3 min; 98°C for 10 s, 60°C for 15 s, 72°C 15 s, 35 cycles; 72°C for 5 min; after PCR, agarose gel electrophoresis was used to detect the detection results. After successful amplification, use the SanPrep column DNA gel recovery kit (Shanghai Shenggong) to recover the target band: Take 1 μL of the recovered product to detect the size and concentration of the recovered fragments by agarose gel electrophoresis, and store at -20°C for later use.

Example 2: Plant expression vector construction

The plant expression vector pCAMBIA2300s was linearized with restriction endonucleases. The restriction enzyme digestion system was 15 μL pCAMBIA2300s plasmid, 5 μL 10×M buffer, 2.5 μL and 25 μL ddH ₂ O for the enzymes at the front and rear restriction sites, and centrifuged for a short time after mixing. Digestion at 37°C for 3.5h; spot the digested product on agarose gel for electrophoresis, and then gel recovery of the large fragment of the pCAMBIA2300s vector. The whole process uses the SanPrep column DNA gel recovery kit (Shanghai Shenggong); Take 1 μL of the recovered product to detect the size and concentration of the recovered fragments by agarose gel electrophoresis, and store at -20°C for later use. During homologous recombination, the ClonExpressMultiS One Step Cloning Kit was used for assembly, and the recovered PnDDS , P12H , D6H and UGTPn20 were respectively connected to the pCAMBIA2300s plant expression vector. , 5×cell buffer 2μL, Exnase II 1μL, ddH ₂ O 1μL, mix well, centrifuge for a short time, and then react in a water bath at 37°C for 30min; then use the heat shock transformation method to transfer the ligation product into Escherichia coli DH5α, spread on the The LB solid medium containing 50 mg/L kanamycin was cultured at 37°C for 24 hours; a single colony was selected in the LB liquid medium containing 50 mg/L kanamycin, and the bacteria were shaken at 37°C and 200 rpm. Amplify the specific primers of the four genes and perform PCR, and select the clones that successfully connect the four genes to pCAMBIA2300s. If the detected strains are positive, send them to a sequencing company for further final confirmation. Add glycerol to the solution and store at -80°C for later use.

The plasmids pCAMBIA2300s- DS , pCAMBIA2300s -P12H , pCAMBIA2300s- D6H and pCAMBIA2300s -UGTPn20 in Escherichia coli were extracted by SanPrep column plasmid DNA mini-extraction kit. The size and concentration of the recovered fragments were detected by electrophoresis, and stored at -20°C for later use.

Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants

The above-constructed plant expression vectors pCAMBIA2300s- DS , pCAMBIA2300s -P12H , pCAMBIA2300s- D6H and pCAMBIA2300s -UGTPn20 were transferred into Agrobacterium tumefaciens LBA4404 competent cells by liquid nitrogen freeze-thaw method. In the centrifuge tube of competent cells, mix gently and then ice bath for 5 minutes, then transfer to liquid nitrogen to freeze for 1 minute, then quickly place in 37°C water bath for 5 minutes, then immediately ice bath for 2 minutes, add 800 μL of LB liquid medium, and freeze at 28 Cultivate with shaking at 200rpm for 4h; spread the activated Agrobacterium on LB solid medium containing 50mg/L kanamycin and 25mg/L rifampicin, and incubate at 28°C for about 48h; select a single colony and shake the bacteria , in LB liquid medium containing 50mg/L kanamycin and 25mg/L rifampicin for 24h with shaking at 28°C and 200rpm. Finally, PCR was performed with specific primers for amplifying the four genes, and it was clear that pCAMBIA2300s- DS , The pCAMBIA2300s -P12H , pCAMBIA2300s- D6H and pCAMBIA2300s -UGTPn20 plasmids have been transformed into Agrobacterium; the PCR detection results are shown in Figure 2, and 5 strains were positive for the pCAMBIA2300s- DS , pCAMBIA2300s -P12H , pCAMBIA2300s- D6H and pCAMBIA2300s -UGTPn20 plasmids Agrobacterium strain, and verified by sequencing, the sequence is exactly the same as the original sequence, and the strain glycerol is stored at -80 ℃ for future use.

Example 4: Agrobacterium-mediated genetic transformation of tobacco

The transgenic receptor in this experiment is tobacco. Tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water, soaked in 0.1% HgCl for _8min , then washed with sterile water for several times, and sown in 1/2 The cells were cultured in the dark at 28 °C for 6 d on MS medium, transferred to a light incubator (25 °C, 16 h/d light) after germination, and then subcultured with 1/2 MS medium once a month.

Take out the preserved Agrobacterium LBA4404 strain containing pCAMBIA2300s- DS , pCAMBIA2300s -P12H , pCAMBIA2300s- D6H and pCAMBIA2300s -UGTPn20 plasmids from the -80℃ refrigerator, and inoculate the Agrobacterium containing the target gene in a mixture containing 50 mg/L kanamycin In the LB liquid medium containing 50 mg/L rifampicin and 20 mg/L rifampicin, cultivate at 28°C and 200 rpm until the medium is turbid; pipette 1 mL of the turbid mixed bacterial solution into LB containing 50 mg/L kanamycin and 20 mg/L rifampicin. Then, the Agrobacterium on the LB solid medium was scraped and inoculated into the MGL liquid medium supplemented with 20 mg/L acetosyringone, and the Agrobacterium was activated by shaking at 28 °C for 2-3 hours. Bacillus.

Put 1cm ² size tobacco leaves into the MGL liquid medium containing four kinds of target gene Agrobacterium, 120rpm, 28 ℃ of shaking culture for 20min; after the infection is finished, the bacterial liquid of the tobacco leaves taken out is sucked dry with sterile filter paper , and then transferred to MS solid medium containing 40 mg/L acetosyringone, about 25 °C, and cultured in the dark for 48 h;

The tobacco leaves after co-cultivation were transferred to the selection medium (MS+0.5mg/L 6-BA+0.1mg/L NAA+50mg/L kanamycin+300 mg/L cephalosporin) for selection culture , transfer the culture flask to a light incubator (25°C, 16 h/d light, 8 h/d dark) during screening and culture. Subculture on the MS medium containing 50 mg/L of kanamycin, because the callus differentiation rate of tobacco is high, it is necessary to further screen the regenerated plants. Finally, the regenerated seedlings with better rooting were selected for further testing.

DNA level detection, the DNA of transgenic tobacco leaves was extracted by CTAB method, and the genomic DNA of transgenic plants was used as the template to conduct PCR with specific primers for amplifying genes PnDDS , P12H , D6H and UGTPn20 ; the PCR reaction system was 2 μL DNA, 0.4 μL upstream primers (10μM), 0.4μL downstream primer (10μM), 10μL PCR Mix, 7.2 μL ddH2O; PCR reaction conditions: 94°C 5min; 94°C 30s, 60°C 90s, 72°C 1min, 32 cycles; 72°C 7min; PCR end Afterwards, 10 μL of the product was taken for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some tobacco transgenic plants are shown in Figure 3, and 6 positive plants were obtained at the DNA level.

RNA level detection, take the positive transgenic individual plant and non-transgenic tobacco (wild type) young leaves at the DNA level to extract total RNA, the method of total RNA extraction is the same as in Example 1, reverse transcription to generate the first strand of cDNA, and use this method PCR was performed with specific primers for the amplification of PnDDS , P12H , D6H and UGTPn20 genes as templates; the PCR reaction system was 1.5 μL cDNA, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 10 μL PCR Mix, 7.7 μL ddH ₂ O; PCR reaction conditions: 94°C for 5 min; 94°C for 30s, 60°C for 30s, 72°C for 90s, 32 cycles; 72°C for 7min; after PCR, take 10 μL for agarose gel electrophoresis, and analyze each transgene according to PCR results The expression levels of the four genes PnDDS , P12H , D6H and UGTPn20 in the individual plant, after the PCR, 10 μL was taken for agarose gel electrophoresis. The detection results of some individual plants are shown in Figure 4. A total of T4 and T5 were detected. Four genes, PnDDS , P12H , D6H and UGTPn20 , were abundantly expressed at the transcriptional level in transgenic plants.

Example 5: Effects of PnDDS , P12H , D6H and UGTPn20 gene expression on tobacco synthesis of ginsenoside F ₁

The leaves of the transgenic tobacco were dried and ground into powder, and 1.0 g of tobacco leaf powder was weighed and soaked in 50 mL of methanol for 24 h, and then ultrasonicated for 60 min to extract the saponins in the transgenic tobacco. The types and contents of ginsenosides in the transgenic tobacco were detected by HPLC; Conditions are: high performance liquid chromatograph (Agilent 1260), column GRACE VisionHT C18 HL (250mm×4.6mm, 5µm), mobile phase is acetonitrile (A)/water (B), flow rate is 1.0mL/min, column temperature 30°C, detection wavelength 203nm, gradient elution (v/v) with 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 Figure 5; among them, ginsenoside F ₁ has the highest content in T5 plants , reaching 88.7 µg/g DW (dry weight); a small amount of its precursors dammarediol (DD), protopanaxadiol (PPD) and protopanaxatriol (PPT) were also detected.

sequence listing

<110> Kunming University of Science and Technology

<120> A kind of preparation method of ginsenoside F1

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2310

<212> DNA

<213> Panax notoginseng

<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

<400> 2

atgcacatta cattcaatcc aaccaaaacc ttaggtgata tggcagcagc aatggtgttg 60

ttttttctccc 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

ctttttaggag 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 taagaaatc 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

<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 ttaccttta 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

<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. a preparation method of ginsenoside F ₁ , is characterized in that: gene PnDDS , P12H , D6H and UGTPn20 are transferred into tobacco simultaneously to obtain the transgenic tobacco that can synthesize ginsenoside F ₁ , wherein the nucleotide sequence of gene PnDDS such as As shown in SEQ ID NO:1, the nucleotide sequence of gene P12H is as shown in SEQ ID NO:2, the nucleotide sequence of gene D6H is as shown in SEQ ID NO:3, and the nucleotide sequence of gene UGTPn20 is as shown in SEQ ID NO: 4 shown.

Images