Method for increasing content of astragalus methglycoside through exogenous gene transfer technology

A technology of astragaloside IV and exogenous genes, which is applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, plant cells, etc., can solve problems such as unseen applications, and achieve improved survival rate, increased yield, and improved astragaloside IV. The effect of content

Inactive Publication Date: 2005-11-23
SHANGHAI UNIV OF T C M
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Problems solved by technology

But so far, there is no application of this technology in Chinese herbal medicine

Method used

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  • Method for increasing content of astragalus methglycoside through exogenous gene transfer technology
  • Method for increasing content of astragalus methglycoside through exogenous gene transfer technology
  • Method for increasing content of astragalus methglycoside through exogenous gene transfer technology

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Embodiment 1

[0018] 1) Extraction of plasmid pBI121

[0019] Preparation of reaction reagents: LB liquid medium; solution 1 (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH 8.0); solution 2 (0.2M NaOH, 1% SDS); solution 3 (5M KAc 60ml, HAc 11.5ml, H 2 O 28.5ml); phenol:chloroform:isoamyl alcohol (25:24:1) mixture; RNAse (20μg / ml); absolute ethanol; 70% ethanol.

[0020] Operating procedure: Take 2ml of antibiotic-containing LB liquid medium, add it to a 15ml well-ventilated test tube, then pick a single colony from the transformation plate, 37℃, 300rpm, culture overnight; take 1.5ml culture, 4℃, 12000rpm Centrifuge for 0.5min, pour out the supernatant, and make the precipitate as dry as possible; add 100μl of solution 1 to resuspend the bacteria and shake vigorously; add 200μl of solution 2, close the lid, quickly invert to mix, and place on ice; add 150μl of ice to pre-cool Tighten the lid, mix upside down, place on ice for 3~5min; 12000rpm, 4℃, centrifuge for 5min, transfer the supernatant; add a...

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Abstract

The invention discloses a method for increasing content of astragalus methglycoside through exogenous gene transfer technique, which comprises transforming vitreoscilla hemoglobin gene (vgb), inserting the vgb gene into plasmid pBI121 by employing enzyme cutting technology in combination with PCR, obtaining intermediate expression vector pBI121-vgb using 35s-CaMV strong promotor as the starting element, and transferring into bacillus coli DH5 alpha, employing three-parent hybridizing method (pRK2013, DH5 alpha and LBA9402), obtaining trans-vgb gene Agrobacterium rhizogenes LBA9402-vgb, disseminating astragalus root sprout, thus obtaining transgenic Radix Astragali hairy-root, whose Radix Astragali Astragaloside content is five times higher then the non-transgenic hairy-root.

Description

Technical field: [0001] The invention belongs to the technical field of traditional Chinese medicine. Specifically, it relates to a technique of transferring foreign genes to increase the content of astragaloside IV. Background technique: [0002] Astragalus membranaceus (Astragalus membranaceus or A. mongholicus) is one of the most important Chinese medicinal materials. It has the functions of replenishing and solidifying the surface, diuresis and detoxification, inducing muscles and ulcers, hydrating and reducing swelling. It is the main ingredient of many traditional Chinese medicine compounds, patent medicines and health products. With the strengthening of people’s growing health concepts, the demand for astragalus will increase year by year; but at present, the wild resources of astragalus are decreasing year by year, the quality of cultivated varieties is declining, and the problems of pesticide pollution and heavy metal residues are facing problems. , Export brings certain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
Inventor 杜旻胡之璧王子艳刘涤周吉燕倪跃元
Owner SHANGHAI UNIV OF T C M
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