A method for efficient isolation and transient transformation of Artemisia annua protoplasts

A protoplast, transient transformation technology, applied in the field of plant biology, can solve problems affecting the progress of scientific research of Artemisia annua

Active Publication Date: 2021-11-09
SHANGHAI JIAOTONG 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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  • A method for efficient isolation and transient transformation of Artemisia annua protoplasts
  • A method for efficient isolation and transient transformation of Artemisia annua protoplasts
  • A method for efficient isolation and transient transformation of Artemisia annua protoplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The present embodiment relates to a method for the separation of protoplast Artemisia annua, comprising the steps of:

[0071] Protoplast preparation:

[0072] (1) Select the vigorous growth of A. annua 2-3 weeks seedlings, when seedlings have 4-6 leaves per plant, leaves tear tape under the skin into the enzyme solution, about 30-40 leaves into . Dark under vacuum 30-40min, placed in a horizontal shaker in the dark enzymatic 4-6h. Enzymatic hydrolyzate of: 1.65% cellulase R10,0.44% macerozyme R10,0.4mol / L mannitol, 0.02mol / L potassium chloride, 0.02mol / L MES pH 5.7, and placed in water bath for 10min 55 ℃ (and so increase the solubility of inactivating the protease enzyme solution), cooled to room temperature and then added 0.01mol / L calcium chloride, 0.1% BSA, the volume to 20mL, filtered stirring to a Petri dish with a 0.45μm filter uniformly middle. The pH is 5.6-5.8 hydrolysates.

[0073] (2) W5 solution was added to 20mL of enzymatic hydrolysis was terminated,...

Embodiment 2

[0082] The present embodiment relates to a method of transiently transformed protoplasts annua PEG-mediated kind, comprising the steps of:

[0083] (1) preparing a plasmid expression vector: The vector used to pCambia1300-GFP, available CAMBIA company from Australia, the plasmid was extracted with endotoxin-free plasmid TianGen large mentioned kit, the extracted plasmid was adjusted to a concentration of about 1μg / μL standby;

[0084] (2) A 10μL plasmid DNA (1μg / μL) was added to 200μL -MMG protoplast suspension, mixed flicking the tube bottom;

[0085] (3) solution was added 220μL of 40% PEG4000, flicking bottom of the tube mixed and placed in the dark at 23 ℃ 20-25min;

[0086] (4) Add 880μL of W5 solution termination transformation mix flicking the tube bottom;

[0087] (5) centrifugation 1000rpm 3min, the supernatant was discarded, the solution was added 1ml W5 of resuspended protoplasts, 23 ℃ light for 12-16 h culture;

[0088] (6) detecting the fluorescent protein: confoc...

Embodiment 3

[0091] The present embodiment relates to the transient expression of the reporter gene in LUC and REN artesunate protoplasts.

[0092] Protoplast Isolation:

[0093] Protoplast Isolation same manner as in Example 2.

[0094] In transient transformation methods PEG-mediated protoplasts annua:

[0095] Expression vector plasmid preparation: The carrier is pGreenII 0800-ALDH1 pro -LUC, pGreenII 0800 promega carrier obtained from the company, ALDH1 pro Artemisinin synthesis promoter last step ALDH1 key enzyme gene, which is NCBI Accession No. proALDH1 (KC118525.1), the plasmid was extracted with the root of the days without toxin kits provide large plasmids and adjust the concentration of the extracted about 1μg / μL standby.

[0096] (1) in 6μL the pGreenII 0800-ALDH1 pro -LUC plasmid (1μg / μL) was added to 200μL -MMG protoplast suspension, mixed flicking the tube bottom;

[0097] (2) was added 220μL 40% PEG4000, flicking bottom of the tube mixed and placed in the dark at 23 ℃ 20-25...

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Abstract

The invention discloses a method for efficiently separating and instantaneously transforming protoplasts of Artemisia annua, comprising: 1) selecting young leaves of Artemisia annua and removing the lower epidermis to obtain the leaves of Artemisia annua without the epidermis; 2) removing the epidermis from the leaves Putting the leaves of Artemisia annua into the enzymolysis solution for enzymolysis to obtain a mixture of enzymolysis; 3) filtering and centrifuging the mixture of enzymolysis to obtain protoplast precipitation, and obtaining protoplasts after resuspension; 4) decomposing the protoplasts Instant transformation. The present invention establishes the method for protoplast separation and transformation in Artemisia annua for the first time, and the output of protoplast is 2.749×10 5 1 / g FW, the activity is 96%, the transformation efficiency of green fluorescent protein is 80%, and the obtained protoplasts have large yield and high activity. Moreover, the protoplasts of Artemisia annua were used as receptors for transformation, and the green fluorescent protein GFP, luciferase LUC and sea cucumber luciferase REN could be successfully expressed.

Description

Technical field [0001] The present invention relates to plant biotechnology, and in particular relates to separation of a foreign gene and a method for transient transformation of Artemisia annua leaf protoplasts. Background technique [0002] Artemisia annua (Artemisia annua L.) Artemisia Asteraceae annual herb plants have strong volatile aroma, which contains a variety of plant secondary metabolites, such as: artemisinin, artemisinic acid, artesunate ketone, caryophyllene [beta], [beta] farnesene, germacrene, alpha] pinene, camphor, volatile oil. Artemisinin (the Artemisinin) is isolated from A. annua leaves sesquiterpene lactone compound containing peroxide bridges in the structure, the World Health Organization (WHO) recommended antimalarial drug combination therapy (Artemisinin-based combination therapies , the main active ingredient ACTs) are. In recent years, artemisinin and its derivatives have been reported to treat lupus, Alzheimer's disease, tuberculosis, diabetes, ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 JIAOTONG UNIV
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