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Tea tree mesophyll protoplast separating and purifying method

A technology of protoplast and purification method, applied in plant cells and other directions, can solve the problems of poor integrity and difficult separation of tea leaf protoplasts, and achieve the effects of good integrity, easy acquisition, and simple separation and purification operations.

Inactive Publication Date: 2018-05-29
INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

[0004] The present invention aims at the existing tea tree leaf protoplast separation difficulties, poor integrity and time-sensitive extraction of petal protoplasts, etc., and provides a separation and purification method for tea tree mesophyll protoplasts

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  • Tea tree mesophyll protoplast separating and purifying method
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  • Tea tree mesophyll protoplast separating and purifying method

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Embodiment Construction

[0017] The present invention will be further described in detail below in conjunction with specific embodiments.

[0018] see Figure 1 to Figure 4 , a method for separating and purifying tea tree mesophyll protoplasts, specifically comprising the following steps:

[0019] Step 1. Collect the fully expanded first leaves of tea tree seedlings, wash the surface with distilled water, then disinfect with 70% ethanol for 3 minutes, then rinse with sterile water for 5 times, and then dry them with absorbent paper; then immerse the leaves in the protoplast rinse solution , protoplast pre-separation 30min. Disinfect the surface of leaves to prevent contamination in subsequent experiments; pre-separation of protoplasts can improve the efficiency of enzymatic hydrolysis.

[0020] Step 2, remove the main vein and leaf margin with a sharp blade sterilized by 70% ethanol through the blade of step 1, and cut into 5mm 2 Small pieces, and then quickly placed in a sterilized vial; and enzym...

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Abstract

The invention discloses a tea tree mesophyll protoplast separating and purifying method. Young leaves of tea tree seedlings are used as materials, and a protoplast preparing system is established through enzymolysis separation and density gradient purification. The young leaves are easy to obtain; protoplast separation and purification are simple in operation and can be carried out all year round;the separated and purified protoplasts are relatively high in yield and good in integrity; and a foundation is laid for a transient protoplast expression system to carry out researches such as protein subcellular localization, protein interaction, gene expression regulation, subcellular organelle preparation and the like.

Description

technical field [0001] The invention relates to a separation and purification method, more specifically to a method for separation and purification of tea tree mesophyll protoplasts, and belongs to the technical field of plant protoplasts. Background technique [0002] Tea tree (Camellia sinensis L.) is an important foliage woody economic crop in my country and a resource plant for one of the world's three major non-alcoholic beverages. Because tea is rich in various valuable characteristic secondary metabolites such as catechin, caffeine and theanine, the molecular biology research of tea tree is deepening day by day, and a large number of functional genes have been identified and cloned one after another. However, since the establishment of the tea tree genetic transformation system has been difficult to truly break through, and the metabolic pathways of some tea tree-specific substances are missing in the model plants Arabidopsis thaliana and tobacco, the research on func...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 刘艳丽金孝芳马林龙曹丹
Owner INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI
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