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Method for efficiently separating and instantaneously converting protoplast of artemisia annua L.

A protoplast and instantaneous transformation technology, applied in the field of plant biology, can solve problems affecting the progress of scientific research of Artemisia annua, improve the efficiency of scientific research and shorten the experimental period

Active Publication Date: 2019-01-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, before the application of the present invention, there was no report on the use of transient transformation of Artemisia annua protoplasts to carry out the functional verification of the target gene, which greatly affected the progress of scientific research on this important medicinal plant of Artemisia annua. Transformation system is very important

Method used

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  • Method for efficiently separating and instantaneously converting protoplast of artemisia annua L.
  • Method for efficiently separating and instantaneously converting protoplast of artemisia annua L.
  • Method for efficiently separating and instantaneously converting protoplast of artemisia annua L.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] This embodiment relates to a method for isolating protoplasts of Artemisia annua, comprising the following steps:

[0071] Preparation of protoplasts:

[0072] (1) Select young Artemisia annua seedlings that have grown vigorously for 2-3 weeks. At this time, each seedling has 4-6 leaves. Use tape to tear off the lower epidermis of the leaves and put them into the enzymatic hydrolysis solution, and put about 30-40 leaves. . Vacuumize in the dark for 30-40min, and put it in a horizontal shaker for 4-6h in the dark for enzymatic hydrolysis. The enzymolysis solution is: 1.65% cellulase R10, 0.44% resolase R10, 0.4mol / L mannitol, 0.02mol / L potassium chloride, 0.02mol / L MES with pH 5.7, placed in a water bath at 55°C for 10min (to inactivate the protease and increase the solubility of the enzyme solution), after cooling down to room temperature, add 0.01mol / L calcium chloride, 0.1% BSA, set the volume to 20mL, stir well and filter it into a petri dish with a 0.45μm filter ...

Embodiment 2

[0082] This embodiment relates to a method for transient transformation of Artemisia annua protoplasts mediated by PEG, comprising the following steps:

[0083] (1) Preparation of the expression vector plasmid: the vector used is pCambia1300-GFP, which was purchased from CAMBIA Company in Australia. The Tiangen Endotoxin-Free Plasmid Extraction Kit was used for plasmid extraction, and the extracted plasmid concentration was adjusted to about 1 μg / μL for later use;

[0084] (2) Add 10 μL of plasmid DNA (1 μg / μL) to 200 μL of protoplast-MMG suspension, flick the bottom of the tube to mix;

[0085] (3) Add 220 μL of 40% PEG4000 solution, flick the bottom of the tube to mix, and place in the dark at 23°C for 20-25 minutes;

[0086] (4) Add 880 μL of W5 solution to stop the transformation, flick the bottom of the tube to mix;

[0087] (5) Centrifuge at 1000rpm for 3min, discard the supernatant, add 1ml of W5 solution to resuspend the protoplasts, and culture at 23°C for 12-16h und...

Embodiment 3

[0091] This example relates to the transient expression of reporter genes LUC and REN in protoplasts of Artemisia annua.

[0092] Isolation of protoplasts:

[0093] Same as the method for protoplast isolation in Example 2.

[0094] Transient transformation method of Artemisia annua protoplasts mediated by PEG:

[0095] Expression vector plasmid preparation: the vector used is pGreenII 0800-ALDH1 pro -LUC, pGreenII 0800 vector obtained from Promega, ALDH1 pro It is the promoter of the key enzyme gene ALDH1 in the last step in the synthesis of artemisinin. Its NCBI accession number is proALDH1 (KC118525.1). The plasmid is extracted with the Tiangen Endotoxin-free Plasmid Extraction Kit, and the concentration of the extracted plasmid is adjusted to It is about 1 μg / μL for use.

[0096] (1) Add 6 μL of pGreenII 0800-ALDH1 pro - Add LUC plasmid (1 μg / μL) to 200 μL protoplast-MMG suspension, flick the bottom of the tube to mix;

[0097] (2) Add 220 μL of 40% PEG4000, flick the...

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Abstract

The invention discloses a method for efficiently separating and instantaneously converting a protoplast of artemisia annua L., comprising the following steps: 1) selecting leaves of young and tender artemisia annua and removing lower epidermis, to obtain the leaves of the artemisia annua L. with the lower epidermis removed; 2) placing the leaves of the artemisia annua L. with the lower epidermis removed in an enzymatic hydrolysate for enzymatic hydrolysis to obtain a mixed solution of enzymatic hydrolysis; 3) filtering and centrifuging the mixed solution of enzymatic hydrolysis to obtain a protoplast precipitate, and resuspending to obtain a protoplast; and 4) instantaneously converting the protoplast. The method for separating and converting the protoplast is constructed in the artemisia annua L. for the first time, to obtain the protoplast with yield of 2.749 is multiplied by 10<5> / g FW, viability of 96%, and conversion efficiency of green fluorescent protein of 80%, and the obtained protoplast has large yield and high viability. The protoplast of the artemisia annua L. is utilized as a receptor for conversion, and green fluorescent protein GFP, luciferase LUC and sea cucumberluciferase REN can be successfully expressed.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a method for isolating protoplasts of leaves of Artemisia annua and transiently transforming exogenous genes. Background technique [0002] Artemisia annua L. is an annual herb of the genus Artemisia in the Asteraceae family. The plant has a strong volatile aroma and contains a variety of secondary metabolites, such as: artemisinin, artemisinic acid, artemisinone, β-Caryophyllene, β-farnesene, myrcene, α-pinene, camphor, volatile oil, etc. Artemisinin is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from the leaves of Artemisia annua. It is an antimalarial drug combination therapy (Artemisinin-based combination therapies) recommended by the World Health Organization (WHO). , the main active ingredient of ACTs). In recent years, artemisinin and its derivatives have been reported to have the effects of treating lupus erythematosus, Alzheimer's...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8201Y02A40/146
Inventor 唐克轩马亚男徐东北刘航吴张宽玉付雪晴钟旖珺谢利辉秦维陈甜甜王宇婷孙小芬
Owner SHANGHAI JIAO TONG UNIV
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