Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer

A kind of artemisinin and transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of difficulty in artificial synthesis, limitations of large-scale commercial production, and no detection of artemisinin , to achieve the effect of stabilizing new drug sources

Active Publication Date: 2012-09-19
SHANGHAI JIAO TONG UNIV SUBEI RES INST
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Problems solved by technology

[0003] At present, the main source of artemisinin is extracted from the aerial parts of Artemisia annua plants. However, the content of artemisinin in Artemisia annua is very low. The average content of artemisinin in different planting environments and planting varieties is 0.01-1% of the dry weight of leaves of Artemisia annua. , making the large-scale commercial production of this drug limited
Due to the complex structure of artemisinin, the artificial synthesis is difficult, the yield is low, and the cost is high, so it is not feasible
Some people try to produce artemisinin by tissue culture and cell engineering. However, the content of artemisinin in callus is less than 0.1% of dry weight, and the highest in shoot is only 0.16% of dry weight. Artemisinin was not detected in roots
Therefore, the feasibility of using tissue culture and cell engineering to produce artemisinin is not high.

Method used

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  • Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer
  • Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer
  • Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer

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Experimental program
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Embodiment

[0027] Step 1, cloning of Artemisia annua DBR2 gene

[0028] (1) Extraction of total RNA from Artemisia annua genome

[0029] Take 100-200 mg young leaves of Artemisia annua, quick-freeze them with liquid nitrogen, grind them quickly with a mortar, add them to a 1.5 mL Eppendorf tube filled with 1 mL TRlzol (TRlzol Reagents, GIBCOBRL, USA), shake them fully, and place them at room temperature Stand for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 sec, place at room temperature for 2-3 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C; pipette the supernatant (about 600 μL) into a clean 1.5 mL Eppendorf tube, add an equal volume of isopropyl Alcohol, mix by inverting, place at room temperature for 10min, then centrifuge at 12,000rmp for 10min at 4°C; discard the supernatant, add 1mL of 75% ethanol to wash, shake, and centrifuge at 7,500rmp for 5min at 4°C; dry at room temperature for 10-15min Dissolve in an appropriate amount (30-40μL) of RNAase-free ...

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Abstract

The invention provides a method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer in the field of biotechnology. The method includes the steps: cloning genes of artemisinic aldehyde delta 11(13) DBR2 from the sweet wormwood, constructing a plant expression vector containing the DBR2 genes, using agrobacterium tumefaciens for mediation to transfer the DBR2 genes into the sweet wormwood so that plants regenerate, using PCR (polymerase chain reaction) to detect integration conditions of the exogenous targeted DBR2 genes, determining the artemisinin content in the transgenic sweet wormwood by means of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and screening the transgenic sweet wormwood plants with the improved artemisinin content. The artemisinin content in the obtained transgenic sweet wormwood can be remarkably increased and is 2.83 times maximally of that of a non-transgenic control plant, and accordingly the method for increasing the artemisinin content in the sweet wormwood lays a foundation for large-scale artemisinin production by the aid of the transgenic sweet wormwood.

Description

technical field [0001] The invention relates to a method for increasing the content of artemisinin in the field of biotechnology, in particular to a method for increasing the content of artemisinin in Artemisia annua by transfecting DBR2 gene. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin (artemisinin) is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from its aerial part. It is currently the most effective drug for treating malaria in the world, especially for cerebral malaria and anti-malaria Chloroquine malaria has the characteristics of quick action and low toxicity. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapy (ACTs). In addition, with the deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammator...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 唐克轩沈乾陈韵斐王涛吴韶龑陆续
Owner SHANGHAI JIAO TONG UNIV SUBEI RES INST
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