Method for obtaining hairy roots of Tibetan Dysosma versipellis for producing podophyllotoxin, hairy roots and descendant

A podophyllotoxin and hairy root technology, applied in the fields of breeding, genetic engineering, physiology, and molecular biology, can solve the problems of waste of raw materials, low acquisition rate, and aggravated damage to wild Tibetan star anise

Inactive Publication Date: 2006-03-22
TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is precisely because the Tibetan star anise lotus has these important medicinal values ​​and good market development prospects, the market demand for the Tibetan star anise lotus is increasing, and researchers are collecting wild resources more and more frequently, and the number of collections is increasing. More and more, coupled with the carpet-like over-collection of farmers and herdsmen, the wild population of the originally rare Tibetan star anise is shrinking day by day. According to the data obtained from our field resource survey, the population of Tibetan star anise has already reached the basic level of 3 years ago. has shrunk by more than two-thirds, and its endangered status is really worrying
In addition, the traditional extraction process mainly relies on the use of crude raw material powder for ethanol percolation, concentration, precipitation, filtration, evaporation to dryness, and ether treatment to obtain podophyllotoxin. Great waste, aggravating the damage to the wild Tibetan star anise
Generally speaking, the traditional method of extracting podophyllotoxin is at the expense of wild resources, because, with the continuous expansion of the application of traditional methods, the degree of destruction of wild resources will continue to increase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Obtaining sterile explants of Tibetan star anise

[0024] Method 1: Using explants to establish sterile explants of Tibetan star anise

[0025] Take the young shoots germinated from the rhizomes of the Tibetan star anise lotus (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 water 3 times; then use 0.1% (M / V) mercury 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% a...

Embodiment 2

[0029] Genetic Transformation of Tibetan Star Anise Lily with Agrobacterium rhizogenes to Obtain Hairy Roots

[0030] 1. Agrobacterium rhizogenes A4, R1000, R1601 (cultivated by Plant Physiology Department, Department of Forestry, College of Agriculture and Animal Husbandry, Tibet University).

[0031] 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;

[0032] 2. The second activation of 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;

[0033] 3. Discard the supernatant, suspend the bacteria with MS liquid medium (100 μmol / mL acetosyringone), dilute to 5 times the original volume, and culture at 28°C and 200 rpm to make the concentration of the bacteria reach the OD 600 =0.3 or so; ...

Embodiment 3

[0038] Molecular detection of hairy roots of the Tibetan star anise

[0039] 1. Extraction of genomic DNA from hairy root of Tibetan star anise lotus, the method is as follows:

[0040] 1) Take a small amount of hairy root of Tibetan star anise, put it into a 1.5ml Eppendorf tube, and add 500 microliters of extraction buffer.

[0041] 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;

[0042] 3) Centrifuge at room temperature for 10 minutes at 12000 rpm;

[0043] 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;

[0044] 5) 12000rpm, centrifuge at room temperature for 10min;

[0045] 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 p...

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PUM

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Abstract

The present invention provides genetic engineering technological process of transforming Tibetan Dysosma versipellis to obtain hairy roots of Tibetan Dysosma versipellis for producing podophyllotoxin. The technological process includes genetic transformation of rooting agrobacterium to Tibetan Dysosma versipellis to obtain hairy roots of Tibetan Dysosma versipellis. The hairy roots of Tibetan Dysosma versipellis is, molecular detection shows, genetically transformed hairy roots, and may be, HPLC detection shows, used in producing podophyllotoxin. The present invention provides one new way of producing podophyllotoxin.

Description

technical field [0001] The present invention relates to the fields of molecular biology, physiology, breeding, genetic engineering and the like, and relates to a method for obtaining podophyllotoxin-producing Tibetan star anise root by transgenic technology, in particular to Agrobacterium rhizogenes genetic transformation of Tibetan star anise and obtaining Specific process of podophyllotoxin-producing hairy roots of the Tibetan star anise. The present invention also provides podophyllotoxin-producing Tibetan star anise hairy 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 ability to syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A01H1/00
Inventor 李春燕兰小中
Owner TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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