Construction method and application of tobacco plant for expressing rare ginsenoside

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a tobacco plant for expressing rare ginsenoside. According to the method, dammarendiol synthase DDS, cytochrome P450 CYP716A47 and glycosyl transferase UGTPg1 / UGTPg45 are added into tobacco, heterologous synthesis of rare ginsenosides CK and Rh2 is achieved, dependence on traditional ginseng cultivation extraction is avoided, and cost is remarkably reduced and efficiency is remarkably improved. The characteristics of short growth cycle and easy culture of tobacco are utilized to overcome the problems of low efficiency and low yield of traditional synthesis; positive transgenic tobacco leaves are dedifferentiated to form callus cells, so that materials are provided for large-scale fermentation, and an industrial production foundation is laid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing a tobacco plant for expressing rare ginsenosides and application thereof. BACKGROUND

[0002] As the core active ingredient of ginseng, ginsenosides can be clearly divided into dammarane-type and oleanane-type according to the aglycone skeleton. The dammarane-type is further divided into protopanaxadiol (PPD-type) and protopanaxatriol (PPT-type). Among the ginsenosides contained in ginseng, Rb1, Rb2, Rc, Rd, Rg1 and Re are the main components, belonging to PPD-type or PPT-type, and usually account for more than 90% of the total ginsenoside content in ginseng roots, constituting the material basis for the efficacy of ginseng.

[0003] Rare ginsenosides, on the other hand, have completely different characteristics: their content in ginseng is only one ten-millionth, but their activity is stronger and their molecular weight is smaller, and their absorption and utilization rate in the human body is significantly improved. Studies have shown that after oral administration of ginseng roots (tablets) and their processed products, the intestinal flora of the human body will convert a large amount of ordinary ginsenosides into rare ginsenosides through glycosyl hydrolysis, among which PPD-type ginsenosides are hydrolyzed into rare ginsenosides CK and Rh2 for human body absorption and utilization. These two rare ginsenosides exhibit extremely strong biological activity, including anti-cancer effect of inhibiting tumor cell proliferation and metastasis, anti-inflammatory effect of reducing inflammatory response, as well as multiple effects of anti-allergy, blood glucose regulation, abnormal angiogenesis inhibition, aging delay, nerve and liver protection, etc., which have immeasurable application value in the fields of medicine and health care. However, the low abundance of rare ginsenosides in natural ginseng, combined with the complex chemical structure, makes the chemical synthesis of rare ginsenosides face great challenges, which seriously restricts the large-scale application and industrial development of this kind of high-activity compounds.

[0004] At present, the industrialized production of ginsenosides still takes traditional ginseng cultivation and extraction process as the core, but this mode has many bottlenecks that are difficult to break through: first, ginseng has a growth cycle of 4-6 years, and the long cultivation period not only increases the time cost, but also greatly limits the expansion of production scale; second, ginseng has strict requirements for growing environment, is easily affected by pests and diseases and climate change, resulting in severe fluctuations in yield; third, the ginsenoside extraction process is complex, which needs multiple extraction and purification processes, not only with high energy consumption, but also with low yield, directly causing high production cost.

[0005] More importantly, the content of rare ginsenosides in natural ginseng is only about one ten-thousandth, and such a low proportion makes it impossible for traditional separation and extraction methods to meet the needs of industrial production. Taking CK and Rh2 with high activity as examples, although their pharmacological values are significant, the scarcity of their natural content makes it difficult for them to be clinically applied and commercially developed. Even if the most advanced separation and purification technology is used, it is still difficult to obtain such rare ginsenosides from natural ginseng due to the problems of extremely low yield, high cost, and unfeasible large-scale production.

[0006] In recent years, with the rapid development of biotechnology, the use of modern biological means to synthesize ginsenosides and improve the yield has become a research focus. Researchers mainly promote research through three technical paths: one is tissue culture technology, which constructs ginseng cell or hairy root culture system to realize ginsenoside production in a controllable environment; the second is biological transformation method, which uses the catalytic action of microorganisms or enzymes to convert common ginsenosides into rare ginsenosides; the third is synthetic biology method, which uses artificial biosynthesis pathway to produce target ginsenosides. These explorations not only deepen the understanding of the biosynthesis mechanism of ginsenosides, but also accumulate theoretical basis and technical reserves for industrial production. However, the current synthesis methods still have problems such as low efficiency, low yield, and complex process, and there is still a long way to go to large-scale industrial application.

[0007] Therefore, developing new and efficient synthesis methods to improve the yield of rare ginsenosides has become a key breakthrough for realizing the industrial production of ginsenosides. At the same time, it is urgent to explore new ways to synthesize ginsenosides using abundant natural resources, to reduce production costs and improve production efficiency, and to promote the leap-forward development of rare ginsenosides from laboratory research to industrial application. SUMMARY In view of the above problems, the present application provides a precursor substance 2,3-oxidosqualene for synthesizing ginsenosides, and a tobacco as a chassis host, which can realize the synthesis of rare ginsenosides CK and Rh2 in tobacco by adding cyclase DDS, hydroxylase CYP716A47 and glycosyltransferase UGT. At the same time, the positive transgenic tobacco leaf is de-differentiated to form callus to realize the synthesis of rare ginsenosides CK and Rh2 in tobacco cells, which provides material basis for large-scale fermentation and lays a foundation for realizing the industrial production of rare ginsenosides.

[0008] The technical scheme of the present application is as follows: On the one hand, the present application provides a construction method of a plant for expressing rare ginsenosides, which comprises introducing three expression units into the plant: (1) the first expression unit comprises 35S promoter, dammaradienol synthase DDS and NOS terminator connected in sequence; (2) a second expression unit comprising 35S promoter, cytochrome P450 CYP716A47 and NOS terminator connected in sequence; (3) a third expression unit comprising 35S promoter, glycosyltransferase UGTPg1 or UGTPg45 and NOS terminator connected in sequence.

[0009] Specifically, the 35S promoter is a cauliflower mosaic virus 35S promoter.

[0010] Specifically, the NOS terminator is a nopaline synthase gene terminator.

[0011] Specifically, the three expression units are arranged in the tandem mode of 35S-DDS-NOS—35S-CYP716A47-NOS—35S-UGTPg1-NOS and / or 35S-DDS-NOS—35S-CYP716A47-NOS—35S-UGTPg45-NOS.

[0012] Specifically, the sequence of the dammarenediol synthase DDS is shown in SEQ ID NO. 1; the sequence of the cytochrome P450 CYP716A47 is shown in SEQ ID NO. 3; the sequence of the glycosyltransferase UGTPg1 is shown in SEQ ID NO. 5; and the sequence of the glycosyltransferase UGTPg45 is shown in SEQ ID NO. 7.

[0013] Specifically, the plant is tobacco.

[0014] Preferably, the tobacco is selected from Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica or Nicotiana attenuata.

[0015] Specifically, the rare ginsenoside is ginsenoside CK and ginsenoside Rh2.

[0016] In another aspect, the present application provides an expression vector comprising the three expression units in the aforementioned construction method.

[0017] In another aspect, the present application provides a host cell comprising the three expression units in the aforementioned construction method or the expression vector of claim 5.

[0018] Specifically, the host cell is a microorganism or a plant cell.

[0019] Preferably, the host cell is Agrobacterium or a tobacco cell.

[0020] Preferably, the Agrobacterium is selected from Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105 or Agrobacterium C58. The tobacco cells are selected from N. benthamiana, N. tabacum, N. rustica or N. sylvestris.

[0021] Preferably, the Agrobacterium can be Agrobacterium GV3101. The tobacco cells can be N. benthamiana.

[0022] In another aspect, the present application provides a method for synthesizing rare ginsenosides, comprising the following steps: S1, transferring the expression vector of claim 8 into tobacco cells or explants to obtain transgenic tobacco cells or explants; S2, culturing the transgenic tobacco cells or explants of step S1 to obtain callus; S3, differentiating the callus of step S2 to obtain regenerated plants; S4, synthesizing rare ginsenosides through the regenerated plants of step S3.

[0023] Specifically, it further comprises dedifferentiating the leaf tissue of the regenerated plants of step S3 to form callus, and synthesizing rare ginsenosides by culturing the callus.

[0024] Specifically, the transferring of step S1 is achieved by Agrobacterium-mediated transformation, gene gun method or CRISPR-Cas9 gene editing.

[0025] Preferably, the transferring of step S1 is achieved by Agrobacterium-mediated transformation.

[0026] Specifically, the tobacco cells or explants of step S1 are N. benthamiana cells or explants.

[0027] In another aspect, the present application provides the use of the aforementioned construction method or expression vector or host cell or method in the preparation of rare ginsenosides.

[0028] The present application has the following beneficial effects: (1) The present application adds dammarenediol synthase DDS, cytochrome P450 CYP716A47 and glycosyltransferase UGTPg1 / UGTPg45 Synthesizing rare ginsenosides CK and Rh2 in tobacco realizes the heterologous synthesis of ginsenosides, does not need to rely on traditional cultivation of ginseng extraction, can significantly reduce the production cost, and improves the production efficiency.

[0029] (2) The present application uses tobacco as a chassis host, utilizes its natural short growth cycle, easy to culture and other characteristics, realizes the efficient synthesis of rare ginsenosides CK and Rh2, and overcomes the shortcomings of low synthesis efficiency and low yield of traditional ginsenosides.

[0030] (3) The present application forms the callus cells after the positive transgenic tobacco leaves are dedifferentiated, realizes the synthesis of the rare ginsenoside CK and Rh2 in the tobacco cells, provides the material basis for the large-scale fermentation, and lays a solid foundation for realizing the industrialized production of the rare ginsenoside.

[0031] (4) The method of the present application is simple in operation, short in process flow, easy to scale up, overcomes the defects of complex process and tedious operation in the prior art, is beneficial to reduce the production cost and improve the production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a schematic diagram of the overexpression vector.

[0033] Figure 2 It is the positive screening result of the rare ginsenoside CK, wherein the molecular sizes of the makers of the upper two figures are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in order; the molecular sizes of the makers of the lower six figures are 2000bp, 1000bp, 750bp, 500bp, 200bp and 100bp from top to bottom in order.

[0034] Figure 3 It is the positive screening result of the rare ginsenoside Rh2, wherein the molecular sizes of the makers of the upper two figures are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in order; the molecular sizes of the makers of the lower six figures are 2000bp, 1000bp, 750bp, 500bp, 200bp and 100bp from top to bottom in order.

[0035] Figure 4 It is the content detection result of the rare ginsenoside CK and Rh2.

[0036] Figure 5 It is the obtaining process of the tobacco cells. DETAILED DESCRIPTION

[0037] The present application will be further clarified by the following examples, which are only a part of the present application, and are not used to limit the present application, but are used to illustrate the present application. The experimental methods used in the following examples are conventional experiments, and the materials, reagents and the like used in the following examples are commercially available, unless otherwise specified.

[0038] Example 1 1.1 Construction of multi-gene overexpression vector Three rare ginsenoside synthesis key enzyme genes dammarenediol synthase DDS (GenBank ID: GU183405.1), cytochrome P450 CYP716A47 (GenBank ID: JN604536.1) and glycosyltransferase UGTPg1 (GenBank ID: KF377585.1) / UGTPg45 (GenBank ID: KM401918.1), by sequence synthesis, introduce the cauliflower mosaic virus 35S promoter sequence at the 5'-end of each gene, add the nopaline synthase gene terminator sequence (NOS) at the 3'-end, form the expression frame of 35S-DDS-NOS-35S-CYP716A47-NOS-35S-UGT-NOS, and the schematic diagram of the vector is shown as Figure 1 .

[0039] The nucleotide sequence of dammarenediol synthase DDS is shown as SEQ ID NO. 1:

[0040] Dammarenediol synthase DDS The amino acid sequence of the Dammarenediol synthase is shown as SEQ ID NO. 2: MWKLKVAQGNDPYLYSTNNFVGRQYWEFQPDAGTPEEREEVEKARKDYVNNKKLHGIHPCSDMLMRRQLIKESGIDLLSIPPLRLDENEQVNYDAVTTAVKKALRLNRAIQAHDGHWPAENAGSLLYTPPLIIALYISGTIDTILTKQHKKELIRFVYNHQNEDGGWGSYIEGHSTMIGSVLSYVMLRLLGEGLAESDDGNGAVERGRKWILDHGGAAGIPSWGKTYLAVLGVYEWEGCNPLPPEFWLFPSSFPFHPAKMWIYCRCTYMPMSYLYGKRYHGPITDLVLSLRQEIYNIPYEQIKWNQQRHNCCKEDLYYPHTLVQDLVWDGLHYFSEPFLKRWPFNKLRKRGLKRVVELMRYGATETRFITTGNGEKALQIMSWWAEDPNGDEFKHHLARIPDFLWIAEDGMTVQSFGSQLWDCILATQAIIATNMVEEYGDSLKKAHFFIKESQIKENPRGDFLKMCRQFTKGAWTFSDQDHGCVVSDCTAEALKCLLLLSQMPQDIVGEKPEVERLYEAVNVLLYLQSRVSGGFAVWEPPVPKPYLEMLNPSEIFADIVVEREHIECTASVIKGLMAFKCLHPGHRQKEIEDSVAKAIRYLERNQMPDGSWYGFWGICFLYGTFFTLSGFASAGRTYDNSEAVRKGVKFFLSTQNEEGGWGESLESCPSEKFTPLKGNRTNLVQTSWAMLGLMFGGQAERDPTPLHRAAKLLINAQMDNGDFPQQEITGVYCKNSMLHYAEYRNIFPLWALGEYRKRVWLPKHQQLKI.

[0041] Cytochrome P450 CYP716A47 The nucleotide sequence of the Cytochrome P450 is shown as SEQ ID NO. 3:

[0042] Cytochrome P450 CYP716A47 The amino acid sequence of the cytochrome P450 is shown as SEQ ID NO. 4: MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMFPRSHGESNADNFSKVRGKMMFLLKVDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPINIPGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYTTLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPGVGTFREAITDFTYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVVTNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN.

[0043] Glycosyltransferase UGTPg1 The nucleotide sequence of the glycosyltransferase is shown as SEQ ID NO. 5:

[0044] Glycosyltransferase UGTPg1 The amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 6: MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNLPETDPQNYMLKPRHAIFPSVIETQKTHVRDIISGMTQSESTQVVGLLADLLFINIMDIANEFNVPTYVYSPAGAGHLGLAFHLQTLNDKKQDVTEFRNSDTELLVPSFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEELEPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLERSGHRFLWSLRPSIPKGETKLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESVWCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKEMKEKSRAAMSENGSSYNSLAKLFEEIM.

[0045] Glycosyltransferase UGTPg45 The nucleotide sequence of the glycosyltransferase is shown as SEQ ID NO. 7:

[0046] Glycosyltransferase UGTPg45 The amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 8: MEREMLSKTHIMFIPFPAQGHMSPMMQFAKRLAWKGLRITIVLPAQIRDFMQITNPLINTECISFDFDKDDGMPYSMQAYMGVVKLKVTNKLSDLLEKQRTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNARLGKLKIPPEEGLTSVSLPSIPLLGRDDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSQWPITSIGPLIPSMYLDKQLPNDKDNGINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKYHFIWVVIESERTKLSSDFLAEAEAEEKGLIVSWCPQLQVLSHKSIGSFMTHCGWNSTVEALSLGVPMVALPQQFDQPANAKYIVDVWQIGVRVPIGEEGVVLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKLVSS.

[0047] 1.2 Obtaining of transgenic tobacco plants and identification of positive lines Transgenic lines were obtained by genetic transformation of tobacco. The constructed vector was transformed into Agrobacterium GV3101 (Shanghai Weidi, CAT#: AC1001) by freeze-thaw method. The transformed Agrobacterium was inoculated into 5 mL YEB liquid medium (containing 25 mg / L hygromycin) and cultured at 28°C, 200 rpm until OD 600 ≈0.8, centrifuged at 4°C, 5000 rpm for 10 min to collect the bacteria, resuspended with equal volume of MS liquid medium (containing 100 μM acetosyringone), and adjusted OD 600To 0.3-0.5. Take the leaf of 4-6 weeks old aseptic N. benthamiana seedlings, cut into 0.5-1 cm2small pieces, immediately inoculate the explants into pre-culture medium (MS + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA), 25℃ dark culture for 2 days. Immerse the pre-cultured explants into the infection solution, gently shake for 10 min. Absorb the excess bacterial solution on the surface of the explants with sterile filter paper, inoculate into co-culture medium (MS + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA + 100 μM acetosyringone), 20-22℃ dark culture for 2-3 days. Transfer the co-cultured explants into debacterialization medium (MS + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA + 500 mg / L cefotaxime), 25℃ dark culture for 7 days. Subculture to selection medium (MS + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA + 500 mg / L cefotaxime + 25 mg / L hygromycin) every 2 weeks. After 3-4 generations, transfer the resistant calli into differentiation medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 25 mg / L hygromycin) to induce bud differentiation. Culture the resistant calli on the differentiation medium for 4-6 weeks, and cut off the 3-5 cm high bud seedlings after the differentiation of the adventitious buds. Transfer to rooting medium (1 / 2MS + 0.5 mg / L IBA + 25 mg / L hygromycin), 25℃, 16 h light culture for 4-6 weeks to induce rooting. When the roots of the regenerated plants are well developed, open the culture bottle cap, and acclimate in the greenhouse for 3-5 days. Take out the plants, wash the root medium, and transplant into the mixed medium of sterilized peat soil: perlite = 3:1. Maintain the relative humidity of 80-90%, and culture at 25℃. Finally obtain the T2 generation of N. benthamiana transgenic seedlings by subculture.

[0048] Identify the T2 generation of N. benthamiana transgenic seedlings. Use the rapid plant genomic DNA extraction kit (Beijing Code Imagene, CAT#: DE140) and the GenePure polysaccharide and polyphenol plant RNA rapid extraction kit (Beijing Code Imagene, CAT#: RE124-01) to extract the DNA and RNA of the tobacco seedlings, respectively. The primers in Table 1 are subjected to the following procedures: ① pre-denaturation 94℃ 2 min; ② denaturation 94℃ 30 s; ③ annealing 58℃; ④ extension 72℃ 2 min 40 s; cycle 34 times for steps ②-④; ⑤ final extension 72℃ 5 min. Perform PCR and RT-PCR, respectively, and finally perform 0.8% agarose gel electrophoresis for detection. The results are shown in Figure 2-3Among them, the tobacco positive lines for synthesizing CK are 6, respectively N8-7, N14-1, N14-7, N14-8, N14-9, N14-10; the tobacco positive lines for synthesizing Rh2 are 8, respectively N18-1, N18-8, 19-5, N26-2, N26-3, N26-6, N26-9, N26-10.

[0049] Table 1 Primers required for transgenic positive seedling detection

[0050] 1.3 Determination of the content of rare ginsenosides CK and Rh2 About 20 g of fresh tobacco leaves of the above positive strains were rapidly frozen in liquid nitrogen for 10 min, then placed in a freeze dryer for freeze-drying, and pulverized into powder by a high-speed pulverizer. 0.3 g of the sample was accurately weighed into a 15 mL centrifuge tube, 80% methanol was added according to the ratio of 1:29 (g:mL), ultrasonic extraction was performed for 2 h, and the ultrasonic process was shaken. The extraction solution was centrifuged at high speed (15000 rpm, 10 min), and the supernatant was taken for standby. The content of rare ginsenosides CK and Rh2 was detected by liquid chromatography. Among them, the chromatographic column was BEHC18 (50x2.1mm, 1.7 μm); the mobile phase was A phase: 0.1% (v / v) formic acid; B phase: 0.1% (v / v) formic acid-methanol; chromatographic gradient: 0 min, 50% B, 3.5 min, 100% B, 5.2 min 100% B, 5.3 min, 50% B, 6.7 min 50% B; column temperature: 40℃; injection volume: 5 μL; flow rate: 0.4 mL / min; mass spectrometry conditions: positive ion mode: electrospray ionization source (ESI), ion source temperature 550℃, ion source voltage 5500V, gas curtain gas 40psi, nebulizing gas 45psi, auxiliary gas 45 psi. Scanning was performed by multiple reaction monitoring (MRM). The detection results are as follows Figure 4 , wherein the highest content of CK is 24.36 μg / g DW, and the highest content of Rh2 is 1.07 μg / g DW, wherein N18-8, N26-6, N26-10 do not detect the content of Rh2 Figure 4 ).

[0051] 1.4 Obtaining of tobacco callus cells The T2 generation tobacco seeds of N14-7 and N19-5 were respectively sterilized in 10% sodium hypochlorite solution for 10 min, washed with sterile water for 4-5 times, and then inoculated in MS medium. When 2-3 leaves were grown, the leaves were transferred to culture bottles containing MS + 25 mg / L hygromycin medium, and grown for 60 days. Then, the DNA and RNA of the leaves were extracted for identification to obtain positive plants, and the method was the same as above. The leaves of the positive plants were taken in the clean bench, and the leaves were cut into 0.5 cm x 0.5 cm using sterilized scissors. The cut tobacco leaves were transferred to the induction callus medium, and the leaf callus induction medium was MS + 1.0 mg / L NAA + 1.0 mg / L 6-BA. The medium was replaced every two weeks, and after 15-20 days, callus cells grew on the edge of the leaves. After about 45 days, the callus cells were peeled off and transferred to the subculture medium (MS + 1.0 mg / L NAA + 1.0 mg / L 6-BA) for subsequent subculture of tobacco callus cells. Figure 5 After 1 month of culture, the cells were collected, and liquid nitrogen freezing and freeze-drying were performed for rare ginsenoside content detection. The method and detection conditions were the same as 1.3. HPLC-MS was used to detect the contents of CK and Rh2 in tobacco cells, which were 20.52 μg / g DW and 0.98 μg / g DW, respectively.

[0052] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for constructing a plant expressing rare ginsenosides, characterized in that: This involves introducing three expression units into the plant: (1) The first expression unit comprises a 35S promoter, a dammarene diol synthase (DDS), and a NOS terminator linked in sequence; (2) The second expression unit comprises a 35S promoter, cytochrome P450 CYP716A47, and NOS terminator connected in sequence; (3) The third expression unit comprises a 35S promoter, a glycosyltransferase UGTPg1 or UGTPg45, and a NOS terminator connected in sequence.

2. The construction method according to claim 1, characterized in that The 35S promoter is the cauliflower mosaic virus 35S promoter.

3. The construction method according to claim 1, characterized in that The NOS terminator is a nopaline synthase gene terminator.

4. The construction method according to claim 1, characterized in that The three expression units are arranged in series in the form of 35S-DDS-NOS—35S-CYP716A47-NOS—35S-UGTPg1-NOS and / or 35S-DDS-NOS—35S-CYP716A47-NOS—35S-UGTPg45-NOS.

5. The construction method according to claim 4, characterized in that The sequence of the dammarene diol synthase DDS is shown in SEQ ID NO.1; the sequence of the cytochrome P450 CYP716A47 is shown in SEQ ID NO.3; the sequence of the glycosyltransferase UGTPg1 is shown in SEQ ID NO.5; and the sequence of the glycosyltransferase UGTPg45 is shown in SEQ ID NO.

7.

6. The construction method according to claim 1, characterized in that The plant is tobacco.

7. The construction method according to claim 6, characterized in that: The tobacco is selected from Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica or Nicotiana scabra.

8. The construction method according to any one of claims 1 to 7, characterized in that: The rare ginsenosides are ginsenoside CK and ginsenoside Rh2.

9. An expression vector comprising the three expression units according to the construction method of any one of claims 1 to 5.

10. A host cell comprising the three expression units according to the construction method of any one of claims 1 to 5 or the expression vector according to claim 5.

11. The host cell according to claim 10, characterized in that The host cell is a microorganism or a plant cell.

12. The host cell according to claim 11, characterized in that The host cell is Agrobacterium or tobacco cell.

13. The host cell according to claim 12, characterized in that The Agrobacterium is selected from Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105 or Agrobacterium C58; The tobacco cells are selected from Nicotiana benthamiana, Nicotiana tabacum, Nicotiana rustica or Nicotiana lanuginosa.

14. A method for synthesizing rare ginsenosides, characterized in that: The method comprises the following steps: S1. Transforming the expression vector according to claim 9 into tobacco cells or explants to obtain transgenic tobacco cells or explants; S2. culturing the transgenic tobacco cells or explants described in step S1 to obtain callus tissue; S3, differentiating the callus tissue from step S2 to obtain regenerated plants; S4. Synthesizing rare ginsenosides through the regenerated plants of step S3.

15. The method according to claim 14, characterized in that The method further includes dedifferentiating the leaf tissue of the regenerated plant in step S3 to form callus tissue, and culturing the callus tissue to synthesize rare ginsenosides.

16. The method according to claim 14, characterized in that The transfer described in step S1 is achieved by Agrobacterium-mediated transformation, gene bombardment or CRISPR-Cas9 gene editing.

17. The method according to claim 14, characterized in that The tobacco cells or explants in step S1 are Nicotiana benthamiana cells or explants.

18. Use of the construction method according to any one of claims 1 to 8, the expression vector according to claim 9, the host cell according to any one of claims 10 to 13, or the method according to any one of claims 14 to 17 in the preparation of rare ginsenosides.

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