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Method for co-transforming sps, hmgr and dxs genes to cultivate Artemisia annua with high artemisinin content in flower buds

A technology of co-transformation and artichoke, applied in the field of genetic engineering to cultivate plants, can solve the problems of limited drug effect and low toxicity

Inactive Publication Date: 2017-08-08
CHONGQING MEDICAL & PHARMA COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The drug resistance of Plasmodium limits the effect of the antimalarial drug chloroquine, while artemisinin has the characteristics of low toxicity and quick action against malaria

Method used

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  • Method for co-transforming sps, hmgr and dxs genes to cultivate Artemisia annua with high artemisinin content in flower buds
  • Method for co-transforming sps, hmgr and dxs genes to cultivate Artemisia annua with high artemisinin content in flower buds
  • Method for co-transforming sps, hmgr and dxs genes to cultivate Artemisia annua with high artemisinin content in flower buds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Extraction and purification of Artemisia annua total RNA. Follow the instructions of the TRIZOL kit provided by Invitrogen. Grind the upper young leaves of Artemisia annua with liquid nitrogen, add 3mL Trizol to the EP tube, centrifuge at 15,000r / min for 15min at 5°C to discard the precipitate; add 3ml of water-saturated phenol, centrifuge at 5°C, 15,000r / min for 10min, take Add 250 μL of 5M NaCl solution to the supernatant, add 300 μL of chloroform, shake, mix well, and centrifuge at 15,000 r / min for 10 min at 4°C, and take the supernatant. Add 0.3mL isopropanol, mix well to get crude RNA extract; add 75% ethanol to wash and dry, and dissolve with DEPC-H2O. Extract according to the operating instructions of the kit RNAiso for polysaccharide-rich plant tissue RNA. Extracted total RNA was tested for integrity and purity by agarose gel electrophoresis and UV spectrophotometer. The mortar, pestle and spoon used should be baked at 160°C for more than 10 hours....

Embodiment 2

[0055] Example 2 Construction of a plant binary expression vector containing Sps, Hmgr and Dxs genes.

[0056]2.1 Construction of intermediate vector pMD19-T-p35s-gfp*gus-nos

[0057] Using pMD19-T and pCAMBIA2301 as basic construction elements, construct pMD19-T-p35s-gfp*gus-nos intermediate vector. The specific implementation steps are as follows. A pair of primers were designed based on the base sequence of p35s-gfp*gus-nos on pCAMBIA2301, and restriction endonuclease sites were introduced on the upstream and downstream primers to facilitate the construction of expression vectors. Using the pCAMBIA2301 plasmid as a template, the expression cassette of the gfp*gus fusion gene was amplified by PCR, and then connected to the pMD19-T vector. After transformation and screening, single clones were picked and sequenced for comparison and confirmation.

[0058] 2.2 Construction of intermediate vectors

[0059] Construction of pMD19-T-p35s-Dxs-nos, pMD19-T-p35s-Sps-nos and pMD19-T...

Embodiment 3

[0067] Example 3, Agrobacterium-mediated transformation of young embryos of Artemisia annua and acquisition of resistant plants

[0068] Using YEB solid medium, pick a single colony of Agrobacterium carrying the recombinant plasmid the night before infection and inoculate it on YEB medium containing 45 mg / L spectinomycin, shake the bacteria at 220 rpm, and keep the temperature at 25°C overnight. Streak culture of Agrobacterium on YEB solid medium until the diameter of the circle becomes a single colony of about 1.5mm, after PCR verification, then re-streak culture on YEB solid medium, culture at 20°C for 4 days, collect the bacteria, and suspend In IM, acetosyringone (AS) was used to adjust the final concentration to 180 μmol / L, which was used to prepare bacterial solutions with different concentrations for future use. The OD of the bacterium solution used by the immature embryo 600nm They are 0.1, 0.3, 0.5, 0.7, 0.9, 1.1 respectively, and the infection time is set to 1, 4, 7...

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Abstract

The invention discloses a method for cultivating sweet wormwood herb with high artemisinin content in flower buds by co-transforming Sps, Hmgr and Dxs genes, relating to the technical field of biology. The method comprises the following steps: cloning three genes namely Anti-Sps, Anti-Hmgr and Anti-Dxs genes from sweet wormwood herb, constructing a plant expression vector containing three genes namely the Sps, Hmgr and Dxs genes which are combined in pairs, performing mediation by using agrobacterium, transferring the three genes namely the Sps, Hmgr and Dxs genes into sweet wormwood herb, and cultivating and screening regenerated plants to obtain a transgenic sweet wormwood herb plant with increased artemisinin content in the flower buds. When the content of non-transformed comparison sweet wormwood herb is 6.20mg / g of dry weight, the maximum artemisinin content in the flower buds of an obtained gene engineering sweet wormwood herb plant disclosed by the invention is 12.10mg / g of dry weight, and the artemisinin content in the flower buds is 1.95 times of the content of non-transformed comparison sweet wormwood herb. The method disclosed by the invention has great significance in saving costs for pharmaceutical factories which take sweet wormwood herb as a raw material.

Description

technical field [0001] The invention relates to the field of cultivating plants by genetic engineering, in particular to a method for cultivating Artemisia annua with high artemisinin content by using transgenic technology. Background technique [0002] Artemisiae annie L., Asteraceae, Artemisia. The scientific name is Artemisia annua, also known as Artemisia stinky, Artemisia annua, and Artemisia annua. It grows on hillsides, forest margins and wastelands, and is an annual herb. Ordinary people use it to relieve summer heat, headache, fever and cold. The extraction of artemisinin is to use the aerial parts of Artemisia annua leaves and unopened flower buds to extract and produce effective physiologically active components such as artemisinin and other products. It is a new antimalarial drug with low toxicity, high efficiency and quick effect (Chinese Pharmacopoeia, 2005; Zhong Guoyue , et al., 2007). Factors that affect the artemisinin content of Artemisia annua include...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/84C12N15/53C12N15/60A01H5/00
Inventor 陈俊意朱照静杨治国杨延音谭丽田数高管琴张宝勇彭坤曾祥琼
Owner CHONGQING MEDICAL & PHARMA COLLEGE
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