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Artemisia japonica mesophyll protoplast separation and conversion method

A technology for protoplasts and Artemisia annua is applied in the field of separation and instantaneous transformation of Artemisia mesophyll protoplasts, which can solve the problems of accelerating the research and development process of biosynthesis of natural active ingredients, and achieve the effect of good state and high yield.

Active Publication Date: 2021-02-23
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 Artemisia annua protoplasts to verify the function of the target gene. The use of Artemisia annua protoplasts to verify the function of the target gene is conducive to the rapid identification of active ingredients in Compositae medicinal plants such as Artemisia genus (such as artemisinin, etc.) biosynthetic key enzyme gene, so it is very important to establish a protoplast isolation and transformation system suitable for Artemisia annua, which can speed up the biosynthesis research and development process of natural active ingredients

Method used

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  • Artemisia japonica mesophyll protoplast separation and conversion method
  • Artemisia japonica mesophyll protoplast separation and conversion method
  • Artemisia japonica mesophyll protoplast separation and conversion method

Examples

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Embodiment 1

[0037] This embodiment relates to a method for isolating protoplasts of Artemisia annua, comprising the following steps:

[0038] Preparation of protoplasts from leaves of Artemisia annua;

[0039] (1) Select Artemisia annua cuttage seedlings that have grown robustly in 3-4 weeks. At this time, each plant seedling has 8-10 young leaves, and the lower epidermis of the leaves is torn off with adhesive tape, such as figure 1 shown;

[0040] (2) Preplasma separation: soak the leaves with the lower epidermis removed in 0.4mol / L mannitol solution for 1 hour;

[0041] (3) Enzymolysis: Put the leaves soaked in the mannitol solution into the enzymolysis solution, about 5-10 leaves. Vacuum for 30 minutes in the dark, and put into a horizontal shaker for dark enzymatic hydrolysis for 4 hours. The enzymatic hydrolysis solution is 1.75% cellulase R10, 0.5% dissociated enzyme R10, 0.4mol / L mannitol, 0.02mol / L potassium chloride, 0.02mol / L MES (pH=5.7) and placed in a 55°C water bath Aft...

Embodiment 2

[0051] For the treatment of the young leaves of Artemisia annua in the method of Example 1, the present invention provides two processing methods: cutting the leaves of Artemisia annua into thin strips and putting them into the enzymatic hydrolysis solution, tearing off the lower epidermis of the leaves with adhesive tape and putting them into the enzymatic hydrolysis solution, The treated young leaves were subjected to enzymolysis and purification according to the method in Example 1. The results showed that the lower epidermis of the young leaves should be torn off with adhesive tape (Table 1).

[0052] Table 1 Cutting into 5mm thin strips and tearing off the lower epidermis of leaves with adhesive tape, protoplast yield and vitality

[0053]

[0054]

[0055] Such as figure 2 , the young leaves were cut into 5mm thin strips and the lower epidermis of the leaves were torn off with adhesive tape, and the yield and vitality of the protoplasts obtained by cutting the le...

Embodiment 3

[0057] For the preplasma separation in the method of Example 1, the present invention sets a control group without preplasma separation in the process of preplasma separation, and the specific results are shown in Table 2.

[0058] Table 2 Effect of preplasma separation on protoplast yield and vigor

[0059]

[0060] As shown in Table 2, pretreatment with 0.4 mol / L mannitol for 1 h on the leaves without the lower epidermis significantly increased the yield of protoplasts, and the activity of protoplasts increased to 80.0%.

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Abstract

The invention discloses an artemisia japonica mesophyll protoplast separation and instantaneous conversion method. The method comprises the following steps that 1) tender artemisia japonica leaves areselected to remove lower epidermis, and the leaves without the lower epidermis are soaked in a mannitol solution; 2) the leaves soaked in the mannitol solution are placed into artemisia japonica enzymatic hydrolysate and vacuumized in darkness, and enzymolysis is carried out in a dark place to obtain enzymolysis mixed liquid containing mesophyll protoplast; 3) the enzymolysis mixed liquid is washed, filtered and centrifuged to obtain protoplast precipitate, and resuspending is carried out to obtain a protoplast suspension; and 4) plasmids carrying green fluorescent protein GFP labels are transferred into the protoplast by using the instantaneous conversion method, and culturing is carried out to express the plasmids in the artemisia japonica protoplast. The yield of the obtained protoplast is 1.93*10<6> / g FW, the activity is 87.5%, and the obtained protoplast is high in yield and good in state. The technology lays an experimental foundation for research on protein-protein interaction,subcellular localization and the like.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a method for isolating and instantaneously transforming protoplasts of Artemisia annua mesophyll. Background technique [0002] Artemisia japonica Tumb., also known as Artemisia japonica Tumb., is a perennial herb belonging to the genus Artemisia in the family Asteraceae. The plant contains volatile oil, the main components of which are piperin, picretene, α-thujone, 1,8-acylolein, artemisinin, etc. The whole herb of Artemisia annua is used as medicine, which has the effects of clearing heat, detoxifying, relieving summer heat, dehumidification, hemostasis, anti-inflammatory, and dispelling blood stasis; it is also used as an alternative to "Qinghao". Research reports in recent years have shown that Artemisia annua plants do not produce artemisinin themselves, but can promote the synthesis of artemisinic acid, dihydroartemisinic acid and artemisinin in Artemisia annua through ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206C12N2509/00
Inventor 潘琪芳邓伯富彭柏文唐克轩
Owner SHANGHAI JIAO TONG UNIV
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