Method for increasing content of artemisinin in artemisia annua through ICS1 gene transferring

A kind of artemisinin and transgenic technology, applied in horticultural methods, genetic engineering, plant genetic improvement and other directions, can solve the problems of difficult artificial synthesis, low artemisinin content, unable to fully meet market demand, etc., and achieve a stable new drug source. Effect

Active Publication Date: 2016-06-22
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 through ICS1 gene transferring
  • Method for increasing content of artemisinin in artemisia annua through ICS1 gene transferring
  • Method for increasing content of artemisinin in artemisia annua through ICS1 gene transferring

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Embodiment 1

[0031] Step 1, cloning of Artemisia annua ICS1 gene

[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 liquid,...

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Abstract

The invention relates to a method for increasing the content of artemisinin in artemisia annua through ICS1 gene transferring, and belongs to the biotechnology field.The method comprises the steps that isochorismate synthase ICS1 genes are cloned from the artemisia annua, plant expression vectors containing the ICS1 genes are constructed, the ICS1 genes are transferred into the artemisia annua through agrobacterium tumefaciens mediation to regenerate plants, the integrating condition of the exogenous target genes ICS1 is detected through PCR, the content of the artemisinin in the transgenic artemisia annua is determined through high-performance liquid chromatography and an evaporative light scattering detector (HPLC-ELSD), and the transgenic artemisia annua plants of which the artemisinin content is increased are obtained through screening.According to the method, the content of the artemisinin in the obtained transgenic artemisia annua is significantly increased, the highest content reaches 1.90 times of that of non-transgenic control plants, therefore, the method for increasing the content of the artemisinin in the artemisia annua is supplied, and a foundation is laid for massively producing the artemisinin through the transgenic artemisia annua.

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 ICS1 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 artemisini...

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
CPCA01H4/00C12N9/90C12N15/8205C12N15/8243C12Y504/04002
Inventor 赵静雅张婷婷张颖王路尧张利达钱虹妹唐克轩
Owner SHANGHAI JIAO TONG UNIV
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