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Preparation method of ginsenoside Rh2

A technology of ginsenoside and nucleotide sequence, which is applied in the field of biomedicine preparation, can solve the problems that there is no complete pathway for dammarane-type tetracyclic triterpene saponins and the ability to synthesize saponins, and achieve efficient, convenient, easy-to-operate, planting easy effect

Active Publication Date: 2021-10-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although tobacco may have the potential to synthesize natural compounds, there is no report on the synthesis of ginsenoside Rh2 in tobacco, and there is no complete pathway for the synthesis of dammarane-type tetracyclic triterpene saponins including ginsenoside Rh2 in the tobacco genome. Tobacco itself does not have the ability to synthesize such saponins

Method used

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  • Preparation method of ginsenoside Rh2
  • Preparation method of ginsenoside Rh2
  • Preparation method of ginsenoside Rh2

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Gene DS and transcription factor genes PnWRKY the acquisition of

[0025] Using notoginseng root as material, grind notoginseng root into powder with liquid nitrogen, then transfer it into a centrifuge tube, use guanidine isothiocyanate method to extract total RNA, use reverse transcriptase M-MLV (promega) to extract total RNA as The template is used to synthesize the first strand of cDNA. The reaction system and operation process are as follows: take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM 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 for 5 minutes, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), and 1 μL M-MLV (200 U) in sequence, mix well and centrifuge for a short time, and incubate at 42 °C After 1.5 h, take it out and heat at 70°C for 10 minutes to terminate the reaction; after the first strand of cDNA is synthesized, store ...

Embodiment 2

[0032]Example 2: Genes DS with PnWRKY Construction of overexpression vector

[0033] Based on Panax notoginseng gene DS , PnWRKY The nucleic acid sequence of the two genes is combined with the insertion position of the two genes in the plant expression vector pCAMBIA2300s, and the primers with homology arms are designed using CE Design software;

[0034] DS The upstream primer is tacgaattcgagctcggtaccATGTGGAAGCTGAAGGTTGCTC, and the downstream primer is gccaagcttgcatgcctgcagTCGGATCTCTCTCGGCCTG;

[0035] PnWRKY The upstream primer is tacgaattcgagctcggtaccTCAGTTGACCTTGTTAGGTTTCTGA, and the downstream primer is caggtcgactctagaggatccTTAGTCTCAATGAATGAATGACATCTTT;

[0036] with high-fidelity enzymes DS , PnWRKY For gene cloning, the total PCR reaction system is 50 μL, including: PrimeSTAR MAX Premix 25 μL, upstream primer 1 μL, downstream primer 1 μL, cDNA 1 μL, ddH 2 O 22 μL; PCR reaction conditions: 98°C for 3 min; 98°C for 10 s, 60°C for 15 s, 72°C for 15 s, 35 cycles...

Embodiment 3

[0038] Example 3: Transformation of Agrobacterium cells

[0039] Extract and purify pCAMBIA2300S- PnWRKY and pCAMBIA2300S- PnDS Plasmid: Competent cells of Agrobacterium LBA4404 strain were prepared and divided into 1.5mL centrifuge tubes, 150μL per tube, and stored at -80°C after quick freezing in liquid nitrogen. The plant expression vector pCAMBIA2300S- PnWRKY and pCAMBIA2300S- PnDS The plasmid was transferred into the prepared Agrobacterium LBA4404 competent cells; the operation steps were: take 3 μg pCAMBIA2300S- PnWRKY and pCAMBIA2300S- PnDS Add the plasmid into a centrifuge tube containing 150 μL of competent cells, mix gently, and then put it in an ice bath for 30 minutes, then transfer it to liquid nitrogen for quick freezing for 5 minutes, then place it in a 37°C water bath for 5 minutes, and then immediately put it in an ice bath for 2 minutes; The state cells were transferred to LB liquid medium (without any antibiotics), cultured with shaking at 28°C an...

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Abstract

The invention discloses a preparation method of ginsenoside Rh2, the method is as follows: a gene DS and a transcription factor gene PnWRKY are simultaneously transferred into tobacco to obtain transgenic tobacco for synthesizing ginsenoside Rh2, the nucleotide sequence of the gene DS is as shown in SEQ ID NO: 1, and the nucleotide sequence of the transcription factor gene PnWRKY is as shown in SEQ ID NO: 2; the transgenic plant obtained by the method disclosed by the invention can be used for synthesizing the ginsenoside Rh2; and 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 Rh2.

Description

technical field [0001] The invention relates to a preparation method of ginsenoside Rh2, which belongs to the technical field of biomedicine preparation. Background technique [0002] Ginsenoside Rh2 is a rare ginsenoside. Ginsenoside Rh2 has regulatory effects on the immune system, central nervous system, endocrine system, and cardiovascular system. It has anti-tumor, anti-allergy, anti-depression, anti-aging and improves myocardial ischemia. and other effects. In terms of anti-tumor, Rh2 can not only inhibit the development of various tumors by promoting tumor cell apoptosis and blocking the tumor cell cycle, but also enhance the body's anti-tumor immune response and improve the immunosuppressive effect of chemotherapy drugs. Rh2 also has a significant anti-inflammatory effect, can inhibit the inflammatory response induced by lipopolysaccharide (LPS), and has potential therapeutic effects on Alzheimer's disease, allergic asthma, allergic dermatitis, etc. [0003] Rh2 was...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/60C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N9/88C12N15/8243C12Y402/01125
Inventor 葛锋陈勤雷君胡泽群王志龙刘迪秋崔秀明
Owner KUNMING UNIV OF SCI & TECH
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