Method for enhancing arteannuin content in southernwood using gene hmgr and fps co-transformation

A technology of co-transformation of artemisinin, applied in botany equipment and methods, biochemical equipment and methods, measuring devices, etc., can solve undiscovered problems and achieve the effect of stabilizing new drug sources

Inactive Publication Date: 2008-05-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No relevant reports have been found that the co-transformation of hmgr and fps mentioned in the subject of the present invention improves the content of artemisinin in Artemisia annua

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Cloning of hmgr and fps 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 (30-50μL) ...

Embodiment 2

[0027] Construction of Plant Binary Expression Vector Containing hmgr and fps 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 expression cassette of the gfp*gus fusion gene was amplified by PCR, connected to the pMD18-T vector, transformed and screened, and single clones were picked and sequenced to confirm that they were correct.

[0030] 2. Construction of intermediate vectors pMD18-T::p35S-hmgr-nos and pMD18-T::p35S-fps-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 with hmgr and fps genes to obtain transgenic Artemisia annua plants

[0041] 1. Obtaining the dual plant expression vector Agrobacterium tumefaciens containing hmgr and fps genes

[0042] The plant binary expression vector containing hmgr and fps gene in embodiment 2 is transformed into Agrobacterium tumefaciens (such as EHA105, there is the biological material that sells publicly for the market, can buy from Australia CAMBIA company, bacterial strain number is Gambar 1), And carry out PCR verification. The results showed that the plant binary expression vector containing hmgr and fps genes has been successfully constructed into the strain of Agrobacterium tumefaciens.

[0043] 2. Agrobacterium tumefaciens mediates hmgr and fps genes to transform Artemisia annua

[0044] 2.1. Preculture of explants

[0045] Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% NaC...

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Abstract

The invention relates to a method for co-transforming hmgr and fps in the field of biotechnology to increase the content of artemisinin in artemisia annua. The present invention clones hmgr and fps genes from Artemisia annua, constructs a plant expression vector containing said DNA molecules, uses Agrobacterium tumefaciens to mediate, simultaneously introduces hmgr and fps genes into Artemisia annua and regenerates plants, and detects exogenous objects by PCR The integration of genes hmgr and fps was determined, and the content of artemisinin in Artemisia annua was determined by high performance liquid chromatography-evaporative light scattering detector, and the transgenic plants of Artemisia annua with increased artemisinin content were screened. The content of artemisinin in the transgenic Artemisia annua obtained by the present invention is significantly increased, up to 2.32 times that of the non-transformed control plants.

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 co-transforming double key enzyme genes hmgr and fps to increase the content of artemisinin in Artemisia annua. 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-based combination therapies (ACTs). In addition, with the deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29C12Q1/68G01N30/02
Inventor 唐克轩景福远张凌王国丰
Owner SHANGHAI JIAO TONG UNIV
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