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Method for genetic transforming scutellaria viscidula to obtain hairy roots producing baicalin by agrobacteriitm rhizogenes

A technology of Agrobacterium rhizogenes and Scutellaria glutinosa is applied to other methods of inserting foreign genetic materials, using vectors to introduce foreign genetic materials, and cells modified by introducing foreign genetic materials, etc. It can solve the problem of uncertain commercial prospects and effective ingredients. Low, low survival rate, etc.

Inactive Publication Date: 2008-05-21
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the main technology for obtaining Scutellaria baicalensis is artificial cultivation, but there are disadvantages such as long production cycle, low survival rate, excessive pesticide residues and low active ingredients, making the commercial prospect of this method unclear.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Obtainment of Sterile Explants of Scutellaria baicalensis

[0031] Method 1: Using explants to establish aseptic explants of Scutellaria baicalensis

[0032] The newly germinated seedling leaves of Scutellaria lanceolata were collected and washed with running water for 1 hour; then soaked in 75% (V / V) ethanol for 45 seconds, and washed with sterile water for 3 times; 2 ) solution was soaked for 9 minutes, rinsed with sterile water for 5 times; then inoculated in the added sterile cluster bud induction medium (the medium was filled in a 150mL conical flask, sterilized at 121 ° C for 20 minutes), the medium formula is: MS basic medium, 30g / L sucrose-adjusted medium, pH 5.8, then add 8.5g·L -1 of agar powder. The leaves of Scutellaria lanceolata were cultured in a light incubator, and the culture conditions were: 25 °C, 12 hours of light, and the light intensity was 55 μmol.m -2 .s -1 . After 17 days, sterile seedlings of Scutellaria pilosa can be obtained, which ca...

Embodiment 2

[0036] Genetic transformation of Agrobacterium rhizogenes to obtain hairy roots

[0037] 1. Agrobacterium rhizogenes C58C1. Take it out from the ultra-low temperature freezer before use, and inoculate it in 50ml YEB liquid medium (the final concentration of rifampicin is 40mg·L). -1 ), 28 °C, 200 rpm shaking culture twice, the recovery of bacteria;

[0038] 2. Add acetosyringone two hours before the end of the second activation culture to make the final concentration reach 100 μmol·L -1 OD of bacterial solution 600 When it reaches 0.4, add acetosyringone, continue to 28 ° C, 200 rpm shaking culture, bacterial liquid OD 600 When it reaches 0.6, it can be used for transformation;

[0039] 3. Centrifuge at 4000 rpm for 10 minutes at room temperature, discard the supernatant, and use an equal volume of MS liquid medium (containing 100 μmol L -1 acetosyringone) suspension, at 28 ° C, 200rp shaking culture, so that the concentration of bacteria reaches the OD of 600 = about ...

Embodiment 3

[0044] Molecular detection of hairy roots of Scutellaria baicalensis

[0045] 1. Extraction of genomic DNA from the hairy root of Scutellaria lanceolata, the method is as follows:

[0046] 1) Take 200 mg of hairy roots that have been cultured in liquid for two weeks, filter and wash with 10 mL of distilled water, fully suck dry, quick-freeze in liquid nitrogen, and grind into powder.

[0047] 2) In a 1.5 mL Eppendorf tube, add 500 μL of extraction buffer (3% mercaptoethanol), fully shake in a water bath at 65° C. for 50 minutes, and mix by inversion every 5 minutes.

[0048] 3) 4°C, 12,000 rpm, centrifugation for 10 minutes.

[0049] 4) Aspirate the supernatant, add 500 μL of phenol:chloroform:isoamyl alcohol (25:24:1), mix gently, and let stand for 5 minutes until layers are separated.

[0050] 5) Centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0051] 6) Aspirate about 350 μL of the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix...

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Abstract

The invention provides a method for utilizing the gene engineering technology to genetically transform the Scutellaria viscidula Bge and obtaining the hairy root of the Scutellaria viscidula Bge which is used for producing baicalin. The method relates to a method which comprises rooting agrobacterium is used for genetically transforming the Scutellaria viscidula Bge; the hairy root of the Scutellaria viscidula Bge which can be used for producing baicalin is obtained. The process is that rooting agrobacterium is used for genetically transforming the Scutellaria viscidula Bge to obtain the hairy root of the Scutellaria viscidula Bge and then the hairy root is confirmed to be the genetically transformed hairy one through the molecular detection and the genetically transformed hairy root of the Scutellaria viscidula Bge is proven to be able to be used for producing baicalin through HPLC detection. The method can provide a novel continuously medicine source for producing baicalin.

Description

technical field [0001] The invention belongs to the fields of molecular biology, physiology, breeding, genetic engineering and the like, and relates to a method for obtaining baicalin-producing hairy roots of Scutellaria baicalensis by utilizing transgenic technology, in particular to the genetic transformation of Agrobacterium rhizogenes and obtaining Specific procedures for baicalin-producing hairy roots of Scutellaria glutinosa. The invention also provides the baicalin-producing hairy root of Scutellaria glutinosa obtained by genetic engineering technology and its cultured progeny. Background technique [0002] Plant secondary metabolites have extremely complex chemical structures, and no efficient or economical synthesis methods have been found so far. Compared with conventional cell culture, the hairy root culture system has the advantages of rapid growth, no need for exogenous plant hormones, strong and stable ability to synthesize secondary metabolites, and the relea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/87C12N5/10C12Q1/68
Inventor 孙敏雷桅王淑芳
Owner SOUTHWEST UNIV
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