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Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes

A kind of artemisinin and transgenic technology, applied in horticultural methods, genetic engineering, plant genetic improvement, etc., can solve the problems of difficulty in artificial synthesis, limitations of large-scale commercial production, and inability to meet demand, and achieve the goal of stabilizing new drug sources Effect

Inactive Publication Date: 2016-06-29
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0003] However, the content of artemisinin in the plant Artemisia annua is very low, large-scale commercial production is limited, and it cannot fully meet the global market demand
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
Production of artemisinin through yeast engineering, the initial investment cost is high, and the limited output cannot meet the demand

Method used

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  • Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes
  • Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes
  • Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes

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Embodiment 1

[0031] Step 1, cloning of the CHI gene of Artemisia annua

[0032] (1) Extraction of total RNA from Artemisia annua genome

[0033]The total RNA of Artemisia annua genome was extracted using RNA kit from TIANGEN Company. Take 50-100 mg young leaves of Artemisia annua and quickly grind them into powder in liquid nitrogen, add 550 μL of lysis solution RL (mercaptoethanol has been added), and vortex vigorously to mix. Centrifuge at 9800rpm for 5min, transfer 450μL to filter column CS, place CS in a collection tube, centrifuge at 12000rpm for 5 minutes, carefully draw 400μL of the supernatant in the collection tube into an RNase-free centrifuge tube, slowly add 0.5 times the volume of the supernatant mixed with absolute ethanol, transferred to the adsorption column CR3, centrifuged at 12,000 rpm for 1 min, discarded the waste liquid, and put CR3 back into the collection tube. Add 350 μL protein-removing solution RW1 to CR3, centrifuge at 12,000 rpm for 1 min, discard the waste l...

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Abstract

The invention relates to the field of biological technologies and provides a method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes. According to the method, the CHI genes are cloned from Artemisia annua L., a plant expression carrier containing the CHI genes is established, the CHI genes are mediated by agrobacterium tumefaciens to be transferred into Artemisia annua L. for regeneration of a plant, the integration condition of an exogenous target gene CHI is detected through the PCR (polymerase chain reaction), the content of artemisinin in transgenic Artemisia annua L. is detected through an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and the transgenic Artemisia annua L. plant with the content of artemisinin increased is screened out. The content of artemisinin in transgenic Artemisia annua L. is increased and is 1.5 times that of a non-transgenic contrast plant in the highest level, and the method for increasing the content of artemisinin in Artemisia annua L. is provided and lays a foundation for large-scale production of artemisinin from transgenic Artemisia annua L..

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 a CHI gene. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin, a sesquiterpene lactone oxide containing a peroxy bridge extracted from its aerial part, is currently the most widely used and effective antimalarial drug, especially for cerebral malaria and anti-chloroquine malaria. . Currently, artemisinin combination therapies (ACTs) are the most effective treatment for malaria recommended by the World Health Organization. With the gradual deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammatory, anti-tumor, anti-cancer and immunomodulatory functions. [0003] However, the content of artemisin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N9/90C12N15/61A01H5/00A01H4/00
CPCA01H4/00C12N9/90C12N15/8205C12N15/8243C12Y505/01006
Inventor 赵静雅王路尧张颖付雪晴张婷婷张利达钱虹妹唐克轩
Owner SHANGHAI JIAO TONG UNIV
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