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Method for increasing content of artemisinin in Artemisia annua

A technology of artemisinin and artemisinin, applied in the field of genetic engineering, can solve the problems of difficult maintenance, limited output, high equipment cost, etc., and achieve the effect of enhancing metabolic pathways

Inactive Publication Date: 2017-03-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although a major breakthrough has been made through the chemical semi-synthesis of artemisinin through synthetic biology, the multi-step key enzyme genes in the artemisinin biosynthetic pathway have been introduced into the yeast genome, and high-yield artemisinic acid and artemisinic acid have been successfully obtained in yeast. Artemisinin is then synthesized in vitro by chemical methods. This method is successful in the laboratory stage, but the semi-synthetic equipment through yeast fermentation is costly in the early stage, difficult to maintain, and the output is limited, which cannot fully meet the market demand.

Method used

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  • Method for increasing content of artemisinin in Artemisia annua
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  • Method for increasing content of artemisinin in Artemisia annua

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1, Cloning of Artemisia annua ADS, CYP71AV1, CPR and ALDH1 Genes

[0028] Amorphadiene synthase (ADS) is the first key enzyme in the unique pathway of artemisinin biosynthesis. It is the catalysis of ADS enzyme, which transfers the terpenoid metabolism in Artemisia annua to the direction of artemisinin synthesis. The monooxygenase CYP71AV1 (Amorpha-4,11-diene oxidase) of the P450 family is a multifunctional enzyme that can catalyze three consecutive steps in the artemisinin synthesis pathway, and finally catalyze the formation of amorphadiene Artemisinic acid. Its molecular chaperone CPR enzyme (cytochrome P450 reductase) was found in in vitro experiments to help CYP71AV1 enzyme catalyze the above reaction process. Aldehyde dehydrogenase ALDH1 can convert dihydroartemisinic aldehyde into dihydroartemisinic acid.

[0029] This example adopts the method of gene cloning to obtain the key enzyme genes ADS, CYP71AV1, CPR and ALDH1 of artemisinin biosynthesis with...

Embodiment 2

[0037] Embodiment 2, the construction of the plant expression vector containing ADS, CYP71AV1, CPR and ALDH1 gene

[0038] 1. Construction of intermediate carrier

[0039] The pMD18T vector (Takara, Dalian) was selected as the cloning vector to construct intermediate vectors pMD18T-ADS, pMD18T-CYP71AV1, pMD18T-CPR and pMD18T-ALDH1. On both sides of ADS, CYP71AV1, CPR and ALDH1 genes, primers ADS-SpeI-F and ADS-BstEII-R, CYP71AV1-SpeI-F and CYP71AV1-BstEII-R, CPR-SpeI-F and CPR-BstEII-R and ALDH1-SpeI-F and ALDH1-BstEII-R introduced SpeI and BstEII restriction sites, and the primer sequences are shown in Table 2. Those skilled in the art know that the restriction endonuclease sites can also be changed according to different subsequent applicable vectors.

[0040] The full-length gene with restriction sites was ligated to the pMD18T vector by T4 ligase, and the sequencing was completed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0041] Table 2: PCR Primers

[0042]...

Embodiment 3

[0052] Example 3, Agrobacterium tumefaciens mediated ADS, CYP71AV1, CPR and ALDH1 gene genetic transformation Artemisia annua obtained transgene due to plant

[0053] 1. Acquisition of plant expression vector Agrobacterium tumefaciens containing ADS, CYP71AV1, CPR and ALDH1 genes

[0054] The plant expression vectors containing ADS, CYP71AV1, CPR and ALDH1 genes obtained in Example 2 are transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA, Australia. , and the strain number is Gambar 1), and was verified by PCR. The results showed that the plant expression vectors containing ADS, CYP71AV1, CPR and ALDH1 genes had been successfully transformed into Agrobacterium tumefaciens strains.

[0055] When using the freeze-thaw method to transfer the plant expression vector into Agrobacterium tumefaciens, add the plant expression vector to the competent cells of Agrobacterium ...

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Abstract

The invention discloses a method for increasing the content of artemisinin in Artemisia annua. The method includes jointly transforming the Artemisia annua by the aid of amorpha fruticosa diene synthetase ADS genes, amorpha fruticosa-4, 11-diene oxidase CYP71AV1 genes, cytochrome P450 reductase CPR genes and acetaldehyde dehydrogenase ALDH1 genes; measuring the content of the artemisinin in the Artemisia annua by the aid of high-performance liquid chromatography-evaporative light-scattering detectors and acquiring transgenic Artemisia annua plants with the increased artemisinin content by means of screening. The method has the advantages that the content of the artemisinin in the transgenic Artemisia annua can be obviously increased by the aid of the method, and the maximum content of the artemisinin can reach 3.4 times the content of artemisinin in non-transformation comparison plants.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for co-transforming four key enzyme genes ADS, CYP71AV1, CPR and ALDH1 to increase the content of artemisinin in Artemisia annua and the prepared transgenic Artemisia annua. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin, a sesquiterpene lactone compound 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. However, the content of artemisinin in the plant Artemisia annua is very low, which cannot fully meet the global market demand. Artemisia annua has secretory glandular trichomes and non-glandular trichomes. There a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
CPCC12N9/0073C12N9/0008C12N9/0081C12N9/88C12N15/8205C12N15/8243C12Y102/0101C12Y114/13158C12Y114/15006C12Y402/03024
Inventor 唐克轩石璞付雪晴沈乾江伟民马亚男郝小龙孙小芬
Owner SHANGHAI JIAO TONG UNIV
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