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Method for obtaining Tibetan Sinopodophyllum hexandrum hairy root which produce podophyllotoxin through agrobacterium rhizogenes genetic transformation, and its product

A technology of Agrobacterium rhizogenes and podophyllotoxin, which is applied in the fields of molecular biology, physiology, breeding and genetic engineering, and can solve the problems of scarcity of peach seven in Tibet and low yield of podophyllotoxin

Inactive Publication Date: 2006-05-10
TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(Three issues need to be further elaborated here: 1. The scarcity of naturally grown Tibetan peach seven, 2. The contradiction between using naturally grown Tibetan peach seven to produce podophyllotoxin and protecting natural resources, 3, and existing methods Podophyllotoxin is obtained in low yield

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Obtaining the aseptic explants of Tibet Taoerqi

[0023] Method 1: Using explants to establish sterile explants of Tibet Taoerqi

[0024] Take the young shoots germinated from the rhizomes of Tibet Taoerqi (provided by the Tibetan medicinal material plantation of the Forestry Department of the College of Agriculture and Animal Husbandry, Tibet University) and wash them with running water for 1 hour; Rinse with bacterial water 3 times; then use 0.1% (M / V) mercuric chloride (HgCl 2 ) solution soaked for 15 minutes, rinsed 6 times with sterile water; then inoculated in the induction medium (the medium was contained in a 150ml Erlenmeyer flask and sterilized at 121°C for 20 minutes) with the addition of sterile cluster bud induction medium. For: MS basic medium, add plant growth regulator 2.0mg / L BA (benzyl adenine), 0.3mg / L NAA (naphthalene acetic acid), 30g / L sucrose and 0.6g / L PVP (polyvinylpyrrolidone) to adjust The pH value of the medium was 5.8, and 5% agar powder...

Embodiment 2

[0028] Genetic Transformation of Agrobacterium rhizogenes to Obtain Hairy Roots

[0029] 1. Activation of Agrobacterium rhizogenes:

[0030] A. Agrobacterium rhizogenes A4, R1000, R1601 (cultivated in Plant Physiology Teaching and Research Section, Department of Forestry, College of Agriculture and Animal Husbandry, Tibet University). Take it out from the refrigerator before use, inoculate it into 50ml YEB liquid culture (add kanamycin to reach a final concentration of 100mg / L), and culture twice at 28°C with shaking at 200rpm;

[0031] B second activation OD 600 When it reaches 0.3, add 100 μmol / mL acetosyringone, continue to culture at 28°C with shaking at 200 rpm, OD 600 When reaching 0.6, centrifuge at 4000 rpm for 10 minutes at room temperature.

[0032] Discard the supernatant at 3C, suspend the bacteria with MS liquid medium (100 μmol / mL acetosyringone), dilute to 5 times the original volume, and culture at 28°C with shaking at 200 rpm, so that the concentration of...

Embodiment 3

[0037] Molecular detection of the hairy roots of Peach chinensis in Tibet

[0038] 1. Extraction of genomic DNA from the seven hairy roots of Tibet peach, the method is as follows:

[0039] 1) Take a small amount of Tibetan peach seven-haired root, put it into a 1.5ml Eppendorf tube, add 500 microliters of extraction buffer.

[0040] 2) After fully grinding with a small glass rod, put it in a water bath at 60°C for 50 minutes, during which it is often inverted and mixed;

[0041] 3) Centrifuge at room temperature for 10 minutes at 12000 rpm;

[0042] 4) Take the supernatant, add 500ul saturated phenol [Tris-HCl (pH8.0) saturated, absorb the lower layer], mix gently, and stand at 4°C for 5 minutes until the layers are separated;

[0043] 5) 12000rpm, centrifuge at room temperature for 10min;

[0044] 6) Aspirate the supernatant (about 250 microliters), add 2 times the volume of absolute ethanol (stored at -20°C), mix well, and let stand at room temperature until the DNA prec...

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PUM

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Abstract

The invention provides a method of producing hair roots of Tibet peaches for podophyllinic acid lactone by gene engineering to convert Tibet peache genes. The invention relates to a method of converting Tibet peache genes with hair root agricillins to gain hair roots of Tibet peaches for podophyllinic acid lactone. Its process is: convert Tibet peache genes with hair root agricillins to gain hair roots of Tibet peaches for podophyllinic acid lactone, ascertain said hair roots by molecule detection, and the results of HPLC detection show that hair roots of Tibet peaches produced by gene convertion could be used to produce podophyllinic acid lactone. The invention offers a lasting new pattern drug-source to the production of podophyllinic acid lactone.

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 podophyllotoxin-producing Tibetan peach seven hairy roots by using transgenic technology, in particular to the genetic transformation of Tibet peach seven by Agrobacterium rhizogenes And obtain the specific process of producing podophyllotoxin-like seven-haired root of Tibetan peach. The present invention also provides podophyllotoxin-producing Tibetan chinensis root obtained by genetic engineering and its cultured progeny, regenerated plant, plant tissue or seed. Background technique [0002] Plant secondary metabolites have extremely complex chemical structures, and no effective 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 abilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01H5/00C12N5/04C12N15/82C12Q1/68
Inventor 兰小中鲍隆友廖志华李春燕
Owner TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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