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Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene

A kind of artemisinin and transgenic technology, applied in the direction of genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of artificial synthesis difficulty, infeasibility, no detection of artemisinin, etc., to achieve Stabilize the effect of new drug sources

Inactive Publication Date: 2011-11-16
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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, which limits the large-scale commercial production of this drug.
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% by weight, and the highest in shoot is only 0.16% by 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 improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene
  • Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Cloning of AOC Gene of Artemisia annua

[0023] 1. Extraction of Total RNA from Artemisia annua Genome

[0024] Take a small amount of young leaves of Artemisia annua (the species of Artemisia annua with high artemisinin content produced in Youyang, Chongqing), freeze them quickly with liquid nitrogen, grind them quickly with a mortar, and add 1 mL of TRIzol (TRIzol Reagents, GIBCO BRL , USA) in a 1.5mL Eppendorf tube, shake fully, place at room temperature for 5min, add 200μL chloroform, shake vigorously for 15sec, place at room temperature for 2-3min, then centrifuge at 12,000g for 15min at 4°C; the supernatant (approx. 600μL) into a clean 1.5mL Eppendorf tube, add an equal volume of isopropanol, mix by inversion, place at room temperature for 10min, then centrifuge at 4°C, 12,000g for 10min; discard the supernatant, add 1mL of 75% ethanol to wash, shake Finally, centrifuge at 4°C and 7,500g for 5 minutes; dry at room temperature for 15-20 minutes and dissolve in an ...

Embodiment 2

[0035] Construction of Plant Binary Expression Vector Containing AOC Gene

[0036] 1. Construction of the intermediate vector pCAMBIA2300::p35S-gus-nos

[0037]The binary plant expression vector pCAMBIA2300::p35S-gus-nos was constructed by selecting pBI121 and pCAMBIA2300 as basic elements. Specifically, pBI121 and pCAMBIA2300 plasmids were digested with HindIII and EcoRI; the gus expression cassette of pBI121 and the large fragment of pCAMBIA2300 were recovered; the recovered products were ligated, transformed and screened, and verified by plasmid digestion.

[0038] 2. Construction of plant expression vector pCAMBIA2300::p35S-AOC-nos

[0039] The pCAMBIA2300::p35S-gus-nos was used as the expression vector, and the AOC gene in Example 1 was used to replace the gus gene on it. Specifically, BamHI / SacI double digestion of pGEM T-easy+AOC and pCAMBIA2300::p35S-gus-nos, recovery of AOC and pCAMBIA2300::p35S-gus-nos large fragments, ligation transformation, picking single clones...

Embodiment 3

[0042] Agrobacterium tumefaciens mediated genetic transformation of Artemisia annua to obtain transgenic Artemisia annua plants

[0043] 1. Obtaining AOC gene-containing binary plant expression vector Agrobacterium tumefaciens engineering bacteria

[0044] The plant binary expression vector containing the AOC gene in Example 2 was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1), and carried out PCR validation. The results showed that the plant binary expression vector containing AOC gene had been successfully constructed into the strain of Agrobacterium tumefaciens.

[0045] 2. Agrobacterium tumefaciens mediated AOC gene transformation of Artemisia annua

[0046] 2.1. Preculture of explants

[0047] Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% NaClO for 20 min, rinsed with s...

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Abstract

The invention belongs to the technical field of biology, and relates to a method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene. The method is characterized by: cloning the AOC gene from the artemisia annua L; constructing a plant expression vector containing the AOC gene; transferring the AOC gene into the artemisia annua L through adopting agrobacterium tumefaciens mediation, and regenerating plants; detecting integration situations of the exogenous target gene AOC through PCR, detecting the artemisinin content in the transgenic artemisia annua L through a high performance liquid chromatography and a evaporative light scattering detector; carrying out screening to obtain the transgenic artemisia annua L plants with improved artemisinin content. The artemisinin content in the transgenic artemisia annua L provided by the present invention is substantially improved, the artemisinin content in the non-transformed normal artemisia annua L is 1.53 mg / gDW while the artemisinin content in the AOC-transferred artemisia annua L is up to 10.17 mg / gDW, such that the artemisinin content in the AOC-transferred artemisia annua L is 6.65 times as much as the artemisinin content in the non-transformed artemisia annua. Therefore, with the present invention, a method for improving the artemisinin content in the artemisia annua L t is provided, base for producing the artemisinin on a large scale through the transgene is established.

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 an AOC 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-inflammat...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 唐克轩陆续江伟民张凌张芳源钱虹妹陈韵斐沈乾王涛吴韶龑
Owner SHANGHAI JIAO TONG UNIV
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