Application of panax japonicus beta-amyrin synthase gene Pjbeta-AS

A technology of balsamol and synthesizing enzymes, applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve the problems of high prices of medicinal materials, high time costs, low land utilization, etc., and overcome the long period of artificial cultivation Effect

Active Publication Date: 2019-02-01
KUNMING UNIV OF SCI & TECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] Panax notoginseng and pearl ginseng are perennial medicinal materials. Their roots are used as medicine and require crop rotation. The time cost of cultivation is high, and the land utilization rate is low. In addition, the demand for medicinal materials has increased year by year in recent years, resulting in varying degrees of supply and demand for both panax notoginseng and pearl ginseng. Contradictions, prices of medicinal materials remain high
[0006] Our previous work has studied the biosynthetic pathway of notoginseng saponins and the regulation technology of this pathway in detail, but we have never found oleanane-type saponins in Panax notoginseng cells

Method used

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  • Application of panax japonicus beta-amyrin synthase gene Pjbeta-AS
  • Application of panax japonicus beta-amyrin synthase gene Pjbeta-AS
  • Application of panax japonicus beta-amyrin synthase gene Pjbeta-AS

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1: Cloning of the full-length ORF of the Panax ginseng β-amyresinol synthase gene

[0018] (1) Extraction of total RNA from Panax ginseng

[0019] Select Ginseng cells that have grown for 35 days, and use the guanidine isothiocyanate method to extract their total RNA. The mortar, pipette tip, and centrifuge tube used are all washed, and soaked in 0.1% volume fraction after overnight shaking. Soak in diethyl pyrocarbonate (DEPC) aqueous solution for more than 24 hours, take it out at 121°C, 1.034×10 5 Pa steam sterilize for 40 minutes, and dry at 50°C for later use. Add an appropriate amount of alcohol to the cleaned mortar and burn the mortar and medicine spoon. After the mortar cools down, take 1.0 g of beaded ginseng cells, grind them into powder in liquid nitrogen, and add 5.0 mL of guanidine isothiocyanate extraction buffer and 50.0 µL of β-mercaptoethanol. Transfer the grinding solution to a 2.0 mL EP tube, add 1 / 10 volume of 2 M sodium acetate (pH 4.0)...

Embodiment 2

[0027] Example 2: Construction of a plant expression vector containing the ginseng β-arsinol synthase gene

[0028] Using pCAMBIA1300S as the plant expression vector, use Sac I and Xba Ⅰ Double enzyme digestion of the pGEM T-easy vector and pCAMBIA1300S plant expression vector containing the β-amyresinol synthase gene of G. , extract the plasmid for polymerase chain reaction and enzyme digestion verification, and obtain the plant expression vector containing the gene.

[0029] The above-mentioned linking of the ginseng β-amyresinol synthase gene to the regulatory sequence of the plant expression vector forms a plant expression vector containing the gene, and the expression vector can be used to produce oleanane-type saponins ( bamboo ginsenoside IV and bamboo ginseng saponin IVa).

Embodiment 3

[0030] Example 3: Obtaining of Agrobacterium tumefaciens strains containing the plant expression vector of Ginseng ginseng β-arsinol synthase gene

[0031] The plant expression vector containing the β-arsinol synthase gene of Panax ginseng was transformed into Agrobacterium tumefaciens EHA105, and polymerase chain reaction was performed to verify that the plant containing the β-arsinol synthase gene of Panax chinensis The expression vector has been successfully constructed into the strain of Agrobacterium tumefaciens. The specific construction method is as follows:

[0032] Prepare competent Agrobacterium EHA105 by calcium chloride method, add 5mL LB liquid medium (containing rifamycin 25mg / L) into a 50mL sterile centrifuge tube, and pick well-grown cells from the plate with a sterilized toothpick A single colony of EHA105 was inoculated in the centrifuge tube, cultured in a constant temperature shaker at 28°C at 180 rpm for 16-18 hours, and the next day, 2 mL of the culture ...

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Abstract

The invention discloses an application of panax japonicus beta-amyrin synthase gene Pjbeta-AS, which is the application of the panax japonicus beta-amyrin synthase gene Pjbeta-AS for promoting the Panax notoginseng cells to synthesize oleanolic saponin, and a nucleotide sequence of the panax japonicus beta-amyrin synthase gene Pjbeta-AS is shown in SEQ ID NO: 1. Gene cloning is used to obtain thepanax japonicus beta-amyrin synthase gene Pjbeta-AS; a plant expression vector containing the Pjbeta-AS gene is constructed; the above vector is used for transforming Agrobacterium tumefaciens EHA105to obtain a strain of Agrobacterium tumefaciens containing the gene; the Panax notoginseng cell strain is transformed with the Agrobacterium tumefaciens strain, and the Panax notoginseng cell which positively transfects the Pjbeta-AS gene is obtained, the type and content of the main oleanolic saponin in the transgenic Panax notoginseng cells can be detected; in the transgenic Panax notoginseng cell obtained by the present invention, and the contents of the panax japonicas IV and the panax japonicus IVa belonging to the oleanolic saponin are 0.20 mg/g and 0.42 mg/g respectively.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of medicinal plants, and in particular relates to the gene of β-amyresinol synthase of Panax ginseng Pjβ-AS The application of the invention, and the method for transferring the β-amyresinol synthase gene of Panax notoginseng into Panax notoginseng cells, so as to synthesize oleanane-type saponins in the Panax notoginseng cells. Background technique [0002] Panax notoginseng ( Panax notoginseng ) is a plant of the genus Ginseng of Araliaceae. Its roots and rhizomes are used as medicine. It is an authentic medicinal material in Yunnan Province and an important component of "Yunnan Baiyao". Panax notoginseng is a traditional and precious Chinese herbal medicine, which has dual effects of promoting blood circulation and stopping bleeding. It is recorded in the Qing Dynasty pharmacy book "Compendium of Materia Medica Supplements" that "ginseng is the first in nourishing qi, and Sanqi is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12P33/00
CPCC12N9/90C12P33/00C12Y504/99054
Inventor 葛锋张翔于怡琳姜森向盈盈刘迪秋
Owner KUNMING UNIV OF SCI & TECH
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