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Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana

A technology of caffeoylquinic acid and stevia, which is applied in the field of genetic engineering, can solve the problems of low content and not yet effectively utilized, and achieve cost reduction, growth improvement and accumulation of secondary metabolites, without occupying arable land area Effect

Active Publication Date: 2014-04-02
JIANGXI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Stevia also contains a class of valuable components, chlorogenic acid and dicaffeoylquinic acid, but the content is low and has not been effectively utilized

Method used

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  • Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana
  • Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana
  • Production method for chlorogenic acid and dicaffeoylquinic acid through induction and culture of hairy roots of stevia rebaudiana

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 Obtaining of stevia rebaudiana sterile explant

[0038] Manually remove the pappus on stevia seeds, incubate in a water bath at 20-40°C for 1-28 hours, blot dry with filter paper, sterilize stevia seeds with 70% alcohol for 20-60S, rinse with sterile water 2-3 times , and then disinfected with 0.1% mercury solution for 2-20 minutes, and rinsed with sterile water for 5-6 times. Inoculate on solid medium, the medium formula is: MS basic medium, 10-60g / L sucrose, 0.01-1mg / L NAA and 0-1mg / L BA, and then add 7-8g / L agar powder. Cultivate at a temperature of 25±2°C, cultivate in the dark for 10-20 days, and then move to a light room with a light intensity of 2000lx and a light time of 12h / d to obtain sterile seedlings.

Embodiment 2

[0039] Example 2 Obtaining hairy roots by genetically transforming Stevia rebaudiana with Agrobacterium rhizogenes

[0040] 1. Activation of Agrobacterium rhizogenes

[0041] Inoculate Agrobacterium rhizogenes on YEB solid medium containing rifampin antibiotics, pick a single colony, and inoculate it in YEB liquid medium containing rifampin antibiotics, at a temperature of 28-30°C, at a speed of 50-400r / min, and shake Cultivate overnight, then take 10-500 μL of the activated bacterial solution and add it to 10-50 mL of YEB (LB) liquid medium containing Rif and other antibiotics, culture at 28°C, shake at 50-400 rpm in the dark until the OD value is 0.5-1.0.

[0042] 2. Resuspension of Agrobacterium rhizogenes

[0043] Centrifuge at 10000-5000rpm at room temperature for 2-20min, discard the supernatant, resuspend the bacteria with an equal volume of 1 / 2MS, culture at 25-35℃, 50-200rpm in the dark for half an hour, so that the concentration of the bacteria solution reaches OD60...

Embodiment 3

[0052]Example 3 Molecular detection of hairy roots of Stevia rebaudiana

[0053] 1, the extraction of Stevia rebaudiana hairy root genomic DNA, the method is as follows:

[0054] 1) Take 100-200 mg of young stevia hairy roots, add liquid nitrogen and grind them into powder;

[0055] 2) Put the powdered stevia hairy root into a 1.5ml Eppendorf centrifuge tube, add 600μl of 2×CTAB preheated at 60°C and 10μl of mercaptoethanol, mix well and place in a 60°C water bath for 40- 50min, invert and mix from time to time;

[0056] 3) Add an equal volume of phenol (pH 8.0) to the lysate after cooling to room temperature, gently invert and mix, and let stand for 10 minutes;

[0057] 4) Centrifuge at 12000rpm for 20min, then pipette the milky white supernatant into a new tube, record the volume when pipetting;

[0058] 5) Add an equal volume of phenol:chloroform (1:1), gently invert and mix, and let stand for 10 minutes;

[0059] 6) Centrifuge at 12000rpm for 20min and transfer the mil...

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Abstract

The invention belongs to the field of genetic engineering, specifically to a production method for chlorogenic acid and dicaffeoylquinic acid by establishing a hairy root culture system through genetic transformation of stevia rebaudiana with agrobacterium rhizogenes. The method comprises the following steps: preparation of an explant of stevia rebaudiana, activation and culture of agrobacterium rhizogenes, suspension culture and production of chlorogenic acid and dicaffeoylquinic acid. PCR detection results show that hairy roots of stevia rebaudiana are generated through transformation; HPLC detection results show that the hairy roots of stevia rebaudiana can produce chlorogenic acid and dicaffeoylquinic acid. Since the hairy roots used in the invention have the characteristic of rapid growth on a hormone-free medium, the method provided by the invention has the advantages of simple operation, low cost, no restriction by natural conditions like climate and land, etc. A stable and sustainable novel drug source and food function factors are provided for production of chlorogenic acid and dicaffeoylquinic acid through culture of the hairy roots, and a reliable source and a reliable substance base are provided for large scale production of chlorogenic acid and dicaffeoylquinic acid by using a bioreactor in the future.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for infecting Stevia rebaudiana with Agrobacterium rhizogenes to induce hairy roots, and using the hairy roots to produce secondary metabolites chlorogenic acid and dicaffeoylquinic acid. Background technique [0002] Plants can produce natural medicinal ingredients, but it also brings about a situation of massive consumption of plant resources. The use of plant bioengineering to produce active ingredients of medicinal plants can effectively protect natural resources and save land, which is of great significance to the sustainable development of modern society. The plant genetic transformation Agrobacterium rhizogenes vector system has the unique advantages of genetic and biosynthetic stability, fast growth of hairy roots, easy cultivation, and high active ingredients, which provides broad prospects for the industrial production of plant secondary ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C07C69/732
Inventor 陈继光上官新晨尹忠平吴少福付晓张清峰洪艳平
Owner JIANGXI AGRICULTURAL UNIVERSITY
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