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Method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum

A protoplast and high-efficiency technology, applied in the field of cell biology and biology, can solve the problems of mechanism research limitations and achieve high plasmid transformation efficiency

Pending Publication Date: 2020-01-10
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, like most coniferous tree species, due to the immature genetic transformation system, further research on its mechanism is limited. At present, most studies on protoplasts focus on the mesophyll cells of the model plant Arabidopsis thaliana and some broad-leaved plants. Leaf plants are only reported in a small amount in Pinaceae plants, and the related research on the protoplasts of Taxus chinensis is still blank at home and abroad. Therefore, using the protoplasts of Taxus cypresses will provide us with a convenient and powerful tool for analyzing the molecular mechanism of trees of the genus Taxus cypress. Provide technical basis for transient expression detection of bald cypress gene, protoplast fusion, etc.

Method used

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  • Method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum
  • Method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum
  • Method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 prepares the selection of the bald cypress material of protoplast

[0030] 1) collect the bald cypress seeds in the classification garden of Nanjing Zhongshan Botanical Garden in autumn and winter, and place the seeds in a dry and ventilated place to dry naturally;

[0031] 2) Wash the seeds with tap water, soak the seeds in deionized water for 3 weeks, and change the water every 3 days;

[0032] 3) Put the washed seeds in a specific substrate (V (peat soil): V (perlite) = 1: 1), then cover the surface of the seeds with fine soil with a thickness of 1-2 cm, pour water once, and cover with straw , to promote germination by keeping warm and moisturizing;

[0033] 4) After the bald cypress emerges, remove the covering grass, and place the seedlings in a light constant temperature incubator at a temperature of 25±2°C and a strong light of 50 μEm -2 the s -1 , the light time is 16h / d. Water once every 2 days until the seedlings grow a round of complete leave...

Embodiment 2

[0035]Example 2 Separation and purification of different tissue protoplasts

[0036] 1) Optimal enzyme solution system for the separation of protoplasts from different tissues

[0037] The present invention optimizes the protoplast enzymatic hydrolysis system of Taxus thaliana based on the study on the optimization of the separation conditions of the mesophyll protoplasts published by Liao Jiaming et al. in 2010. A gradient is set for every 1% of the cellulase from 1% to 8% vol. Set up a gradient for each 1% of pectinase from 1% to 5% vol, three different tissues of root, stem, and leaf, a total of 120 sets of enzyme solution ratios, observe the enzymolysis situation and count after 5 hours of enzymolysis, and count each time Repeated three times, the results are shown in Table 1-3, showing: when the cellulase is 2% vol, pectinase is 1% vol, the root enzymolysis effect is the best, and the protoplast output is 4.32 × 10 6 / gFW; when cellulase is 6% vol, pectinase is 4% vol, t...

Embodiment 3

[0054] Embodiment 3 Utilizes PEG-mediated method to transform different tissue protoplasts

[0055] Expression of plasmids transformed into protoplasts

[0056] The vector used for transformation in this example is p2FGW7.0, which is a commercial vector, and the vector diagram is as follows figure 2 As shown, the vector contains a constitutive strong expression promoter P35S, which can be highly expressed in protoplasts, contains the green fluorescent protein GFP tag, and can observe the green fluorescent signal under the irradiation of ultraviolet light. In the early stage, the Gateway and TA cloning techniques have been used to fuse the Baldacea tree-Cedar chinensis ThRAP2.1 (GenBank: KY463469) gene and p2FGW7.0 to obtain the target plasmid vector.

[0057] 1) Pipette 10 μL of the target plasmid vector (>10 μg) containing the exogenous gene into a 2 mL round-bottom EP tube;

[0058] 2) Take 100 μL of the protoplasts of the isolated and purified bald cypress roots, stems a...

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Abstract

The invention discloses a method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum. The method comprises the following steps: 1) a taxodium distichum seedling with 1-3 rounds of grown leaves is selected and cultured for 24 hours in a dark place, cleaning is conducted once with 75% alcohol firstly, then cleaning is conducted with deionized water for three times, and then the leaves, stem segments and roots of the taxodium distichum seedling are separated by a blade; (2) the roots, stems and the leaves of the taxodium distichum are subjected to enzymolysis through cellulase and pectinase to obtain the protoplasts, and the obtained protoplasts are separated, purified and pretreated; and (3) the protoplasts of different tissue are taken as receptors, a transient expression vector containing an exogenous gene is transferred into the protoplasts through a PEG-mediated transformation method, and through culturing, the exogenous gene is expressed in the protoplasts. The method is easy to operate and easy to implement, a large number of the complete and pure root, stem and leaf protoplasts of the taxodium distichumcan be obtained, the conversion efficiency reaches up to 50% or above, and the technical basis is provided for transient expression detection of a taxodium distichum gene, protoplast fusion and the like.

Description

technical field [0001] The invention belongs to the field of cell biology and biotechnology, in particular to a method for separating, purifying and instantaneously transforming protoplasts of bald cypress roots, stems and leaves. Background technique [0002] Plant cells form protoplasts after removal of the cell wall, which includes the cell membrane, cytoplasm, and nucleus. In short, plant protoplasts are "naked cells" surrounded by plasma membranes. With the continuous development of science and technology, protoplasts have gradually become an effective tool for researchers to study the biological functions of genes and proteins and analyze plant signal transduction pathways. Protoplast isolation is the first step in protoplast cultivation, which directly affects the success of protoplast cultivation. At present, the separation of protoplasts by enzymatic hydrolysis is recognized as the most convenient and effective method. Cellulase is used to digest the cell wall, p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8214C12N2509/00
Inventor 华建峰宣磊杨颖郭金博裴笑笑王芝权於朝广殷云龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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