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Method for enhancing arteannuin content in southernwood using gene cyp71av1 and cpr co-transformation

A technology of artemisinin and co-transformation, applied in the field of genetic engineering, can solve the problems of difficulty in artificial synthesis, low feasibility of artemisinin, low content of artemisinin, etc., and achieve the effect of stabilizing the new drug source

Inactive Publication Date: 2008-05-21
SHANGHAI JIAO TONG UNIV SUBEI RES INST
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  • Summary
  • 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 (0.01%-1%), 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% of dry weight, and the highest in shoot is only 0.16% of dry 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Cloning of cyp71av1 and cpr genes of Artemisia annua

[0021] 1. Extraction of Total RNA from Artemisia annua Genome

[0022] A small amount of young leaves of Artemisia annua (the species with high artemisinin content produced in Youyang, Chongqing) were taken, quick-frozen with liquid nitrogen, and quickly ground with a mortar, and added with 1 mL of TRIzol (TRIzol Reagents, GIBCOBRL, 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 4°C, 12,000g for 15min; supernatant (about 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 12,000g for 10min at 4°C; discard the supernatant, add 1mL of 75% ethanol to wash, shake , centrifuged at 4°C, 7,500g for 5min; dried at room temperature for 15-20min, dissolved in an appropriate amount (30-50...

Embodiment 2

[0027] Construction of plant binary expression vector containing cyp71av1 and cpr genes

[0028] 1. Construction of the intermediate vector pMD18-T::p35S-gfp*gus-nos

[0029] Select pMD18-T and pCAMBIA1304 as the basic elements to construct the intermediate vector pMD18-T::p35S-gfp*gus-nos. Specifically, a pair of primers were designed according to the sequence of p35S-gfp*gus-nos on pCAMBIA1304, and restriction endonuclease sites were respectively introduced into the upstream and downstream primers, so as to construct the expression vector. Using the pCAMBIA1304 plasmid as a template, the gfp*gus fusion gene expression cassette was amplified by PCR, connected to the pMD18-T vector, transformed and screened, and the single clone was picked and sequenced to confirm that it was correct.

[0030] 2. Construction of intermediate vectors pMD18-T::p35S-cyp71av1-nos and pMD18-T::p35S-cpr-nos

[0031]Based on the pMD18-T::p35S-gfp*gus-nos, the gfp*gus fusion gene on it was replaced ...

Embodiment 3

[0040] Genetic transformation of Artemisia annua mediated by Agrobacterium tumefaciens cyp71av1 and cpr genes to obtain transgenic Artemisia annua plants

[0041] 1. Obtaining the engineering bacteria of Agrobacterium tumefaciens containing the binary plant expression vector of cyp71av1 and cpr genes

[0042] The plant binary expression vector containing cyp71av1 and cpr genes 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 Australia CAMBIA company, and the strain number is Gambar1), and Perform PCR verification. The results show that the plant binary expression vector containing cyp71av1 and cpr genes has been successfully constructed into the strain of Agrobacterium tumefaciens.

[0043] 2. Agrobacterium tumefaciens mediates cyp71av1 and cpr genes to transform Artemisia annua

[0044] 2.1. Preculture of explants

[0045] Artemisia annua seeds were soak...

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Abstract

The invention is a method for adopting the cotransformation of genes cyp71av1 and cpr to increase the arteannuin content in southernwood in the gene engineering technology field. The invention clones the gene cyp71av1 of the CYP71AV1 and the gene cpr of the CPR from the southernwood to construct the plant expression vector which contains DNA molecules and agrobacterium tumefaciems is used for mediation; the genes of cyp71av1 and cpr are admitted into the southernwood for regenerating plant; a PCR is used for detecting the integration situation of the foreign object genes of the cyp71av1 and the cpr and a high-efficiency liquid phase chromatography-evaporative light scattering detector is used for detecting the arteannuin content in southern wood and the transgenic southernwood plant with increased arteannuin content is selected. The invention obtains the transgenic southernwood with obviously increased arteannuin content and the highest content is 1.82 times the content of the non-transgenic plant.

Description

technical field [0001] The invention relates to a method in the technical field of genetic engineering, in particular to a method for increasing the content of artemisinin in Artemisia annua by co-transformation of double key enzyme gene cyp71av1 and cpr. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from its aerial part. It is currently recognized as the most effective drug for treating malaria in the world, especially for cerebral malaria and chloroquine-resistant malaria. Quick-acting and low toxicity characteristics. 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-inflammatory...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/53C12Q1/68
Inventor 唐克轩景福远张凌王国丰
Owner SHANGHAI JIAO TONG UNIV SUBEI RES INST
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