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Preparation method of ginsenoside F1

A technology of ginsenoside and P12H, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problem that there is no synthesis, no synthesis has been seen, and no complete dammarane-type tetracyclic triterpene saponins have been found. Access and other issues, to achieve the effect of easy planting, efficient and convenient acquisition, and easy operation

Active Publication Date: 2021-10-26
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although tobacco may have the potential to synthesize natural compounds, the synthesis of ginsenoside F in tobacco has not been seen 1 reported, and no synthesis including ginsenoside F was found in the tobacco genome 1 The complete pathway of dammarane-type tetracyclic triterpene saponins, indicating that tobacco itself does not have the ability to synthesize such saponins

Method used

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  • Preparation method of ginsenoside F1
  • Preparation method of ginsenoside F1
  • Preparation method of ginsenoside F1

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: PnDDS , P12H , D6H and UGTPn20 gene cloning

[0032] Using Panax notoginseng root as material, grind it into powder with liquid nitrogen, then transfer it to a centrifuge tube, extract total RNA from the root of Panax notoginseng by using guanidine isothiocyanate method, reverse transcribe to synthesize the first strand of cDNA, and the reaction system The operation process is as follows: take 5 μg TotalRNA, add 50ng oligo (dT), 2 μL dNTP (2.5mM each), and DEPC water in sequence to a reaction volume of 14.5 μL; after mixing, heat and denature at 70°C for 5 minutes, then quickly cool on ice 5min, then sequentially add 4μL 5×First-standbuffer, 0.5μL RNasin (200U), 1μL M-MLV (200U), mix well and centrifuge for a short time, incubate at 42℃ for 1.5h, take it out and heat at 70℃ for 10min to terminate the reaction; The synthesized first-strand cDNA was used as a template to amplify ginsenoside F by PCR 1 Four genes in the synthetic pathway PnDDS , P12H ...

Embodiment 2

[0043] Embodiment 2: plant expression vector construction

[0044] The plant expression vector pCAMBIA2300s was linearized with restriction endonucleases, and the enzyme digestion system was 15 μL of pCAMBIA2300s plasmid, 5 μL of 10×M buffer, 2.5 μL of enzymes at the front and rear restriction sites, and 25 μL of ddH 2 O, after mixing, centrifuge for a short time, place at 37°C for enzyme digestion for 3.5 hours; spot the enzyme digestion product on agarose gel for electrophoresis, and then perform gel recovery on the large fragment of pCAMBIA2300s vector, and use SanPrep column DNA gel for the whole process Recovery kit (Shanghai Sangong); take 1 μL of the recovered product to detect the size and concentration of the recovered fragment by agarose gel electrophoresis, and store it at -20°C for future use. For homologous recombination, the ClonExpressMultiS One Step Cloning Kit was used for assembly, and the recycled PnDDS , P12H , D6H and UGTPn20 Connect to pCAMBIA2300s...

Embodiment 3

[0046] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0047] The plant expression vector pCAMBIA2300s- DS , pCAMBIA2300s- P12H , pCAMBIA2300s- D6H and pCAMBIA2300s- UGTPn20 Transfer into competent cells of Agrobacterium tumefaciens LBA4404, the operation steps are: take 2 μg of plasmid and add it to a centrifuge tube containing 200 μL of competent cells, mix gently, and ice-bath for 5 minutes, then transfer to liquid nitrogen to freeze for 1 minute, and then quickly Put it in a water bath at 37°C for 5 minutes, then immediately ice-bath for 2 minutes, add 800 μL LB liquid medium, and shake and culture at 28°C, 200 rpm for 4 hours; apply the activated Agrobacterium to a medium containing 50 mg / L kanamycin and 25 mg / L lysine On the LB solid medium of Fuping, culture at 28°C for about 48 hours; select a single colony and shake it, shake it in the LB liquid medium containing 50mg / L kanamycin and 25mg / L rifampicin at 28...

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Abstract

The invention discloses a preparation method of ginsenoside F1. The method comprises the steps that genes PnDDS, P12H, D6H and UGTPn20 are simultaneously transferred into a tobacco to obtain a transgenic tobacco capable of synthesizing the ginsenoside F1, wherein the nucleotide sequence of the gene PnDDS is as shown in SEQ ID NO: 1, the nucleotide sequence of the gene P12H is as shown in SEQ ID NO: 2, the nucleotide sequence of the gene D6H is as shown in SEQ ID NO: 3, and the nucleotide sequence of the gene UGTPn20 is as shown in SEQ ID NO: 4. Experimental results show that an obtained transgenic tobacco plant can synthesize the ginsenoside F1, so that the ginsenoside F1 can be obtained more efficiently and conveniently, the method is simple, easy to operate and suitable for large-scale production and market popularization and application, and a new way is provided for obtaining the ginsenoside F1.

Description

technical field [0001] The invention belongs to the technical field of biomedicine preparation, in particular to a heterologously synthesized ginsenoside F in tobacco 1 Methods. Background technique [0002] Panax ginseng has long been used to treat various diseases and improve body functions, and triterpene saponins are the main active ingredients of Panax ginseng. Triterpene saponins have various pharmacological effects such as promoting cell proliferation, inhibiting cell apoptosis, inhibiting oxidative stress, anti-inflammation, increasing cell viability, etc., and can protect nerve cells, cardiomyocytes, liver cells, lung epithelial cells and other cells effect. [0003] Ginsenoside F 1 (ginsenoside F 1 ) is a rare dammarane-type triterpene saponin component in medicinal plants such as ginseng, Panax notoginseng, American ginseng, etc. A protopanaxatriol-type saponin, which has anti-tumor, anti-aging, anti-oxidation and other effects. Ginsenoside F 1 It has a str...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/52C12N15/54A01H5/00A01H6/82
CPCC12N15/8243C12N15/8205C12N9/00C12N9/1288
Inventor 葛锋雷君陈勤王志龙胡泽群刘迪秋崔秀明
Owner KUNMING UNIV OF SCI & TECH
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