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Nucleic acid sequence, carrier and method for increasing artemisinin content in Artemisia annua

A technology of nucleic acid sequence and Artemisia annua, applied in the field of genetic engineering, can solve problems such as limited output, high cost, and high input cost, and achieve the effect of increasing artemisinin content, increasing content, and promoting synthesis

Active Publication Date: 2020-01-07
SHANGHAI JIAOTONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Nucleic acid sequence, carrier and method for increasing artemisinin content in Artemisia annua

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Experimental program
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Embodiment 1

[0082] Step 1, cloning of Artemisia annua pCYP, CRY1 and GUS genes

[0083] (1) Extraction of total RNA from Artemisia annua genome

[0084] 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 t...

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Abstract

The invention relates to the field of genetic engineering and specifically relates to a nucleotide sequence, a carrier and a method for increasing artemisinin content in sweet wormwood. The nucleotide sequence is formed in the manner of connecting pCYP and GUS genes in sweet wormwood and CCT1 gene at C end of CRY1 gene in sweet wormwood in turn. The sequence is capable of effectively promoting the synthesis of artemisinin in sweet wormwood and increasing the artemisinin content in sweet wormwood. The invention also provides the carrier containing the nucleotide sequence and establishes an excellent basis for nucleotide sequence transfer. The invention also provides the method for increasing artemisinin content in sweet wormwood. The method comprises the following steps: transferring the nucleotide sequence or the carrier into sweet wormwood; detecting the integration condition of exogenous target gene by PCR; determining the artemisinin content in transgenic sweet wormwood through a high performance liquid chromatography and an evaporative light-scattering detector; screening, thereby acquiring the transgenic sweet wormwood with increased content of artemisinin. A basis is established for producing artemisinin in a large scale by utilizing the transgenic sweet wormwood.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a nucleic acid sequence, a carrier and a method for increasing the content of artemisinin in Artemisia annua. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin in its secondary metabolites is an important antimalarial drug, especially effective against cerebral malaria and chloroquine-resistant malaria. At the same time, Artemisia annua essential oil is also rich in a large number of monoterpenes and sesquiterpenes with different functions, such as 1,8-cineole, caryophyllene, artemisinone and so on. 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,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/82C12N15/66A01H5/12A01H6/14
CPCC07K14/415C07K2319/00C12N9/2402C12N15/8205C12N15/8243C12Y302/01031
Inventor 赵静雅李杉张婷婷马嘉伟付雪晴黎凌孙小芬唐克轩张雷
Owner SHANGHAI JIAOTONG UNIV
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