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

An astragaloside IV and exogenous gene technology, which is applied in the directions of recombinant DNA technology, introduction of foreign genetic material, plant cells, etc. using vectors, can solve problems such as unseen applications, and achieve the improvement of survival rate, yield, and improvement of astragaloside IV. The effect of content

Inactive Publication Date: 2007-11-21
SHANGHAI UNIV OF T C M
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  • Description
  • Claims
  • Application Information

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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 pBI 121

[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 (20 μg / ml); absolute ethanol; 70% ethanol.

[0020] Operating procedures: Take 2ml of LB liquid medium containing antibiotics, add it to a 15ml well-ventilated test tube, then pick a single colony from the transformation plate, culture at 37°C, 300rpm overnight; take 1.5ml of culture, 4°C, 12000rpm , centrifuge for 0.5min, discard the supernatant, and make the pellet as dry as possible; add 100μl solution 1 to resuspend the bacteria, and shake vigorously; add 200μl solution 2, cover tightly, quickly invert and mix, and place on ice; add 150μl ice to pre-cool 3, cover tightly, mix by inverting, place on ice for 3-5min; centrifuge at 12000rpm, 4°C for 5min, transfer the super...

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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 technology of exogenous gene transfer to increase the content of astragaloside IV. Background technique: [0002] Astragalus membranaceus or A.mongholicus is one of the important Chinese herbal medicines in my country. It has the functions of tonifying and solidifying the skin, diuresis and detoxification, promoting muscle and reducing sores, diuresis and detumescence, and is the main component of many traditional Chinese medicine compounds, patent medicines and health care products. With the strengthening of people's growing health care concept, the demand for Astragalus membranaceus will increase year by year; but at present, the wild resources of Astragalus membranaceus are decreasing year by year, the quality of cultivated varieties is declining, and they are facing problems such as pesticide pollution and heavy metal residues. , Export bri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N15/74C12N15/82C12N5/04A01H4/00
Inventor 杜旻胡之璧王子艳刘涤周吉燕倪跃元
Owner SHANGHAI UNIV OF T C M