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A kind of separation and instantaneous transformation method of tripterygium wilfordii protoplast

A technology of protoplasts and separation methods, applied in the field of plant tissue culture, can solve the problems of restricting the in-depth study of tripterygium wilfordii functional genes and synthetic biology, research blanks of tripterygium wilfordii protoplasts and transient transformation, etc.

Active Publication Date: 2019-09-24
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Claims
  • Application Information

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Problems solved by technology

In recent years, the study of Tripterygium wilfordii has attracted widespread attention from experts in the fields of biomedicine and agronomy at home and abroad, but the research on the protoplasts and transient transformation of Tripterygium wilfordii is still blank, which limits the functional genes and synthesis of tripterygium wilfordii to a certain extent. in-depth study of biology

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  • A kind of separation and instantaneous transformation method of tripterygium wilfordii protoplast
  • A kind of separation and instantaneous transformation method of tripterygium wilfordii protoplast
  • A kind of separation and instantaneous transformation method of tripterygium wilfordii protoplast

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Embodiment 1

[0056] Embodiment 1, a kind of separation and purification method of Tripterygium wilfordii suspension cell protoplast and its activity detection

[0057] 1. Preparation of Tripterygium wilfordii suspension cells

[0058] Tripterygium wilfordii suspension cells are derived from callus tissue, have a large number of intercellular spaces, cell walls are easily hydrolyzed by enzymes, have the advantages of fast growth and rapid accumulation of secondary metabolites, and are ideal materials for preparing protoplasts. The specific preparation method is as follows:

[0059] 1. Obtaining callus tissue

[0060] Sterilize the young shoots of Tripterygium wilfordii, cultivate aseptic seedlings from cuttings, then take the new leaves of the aseptic seedlings, cut them into small pieces of about 0.5cm×0.5cm, and inoculate them with 0.5mg / L 2,4-D ( Beijing Dingguo Changsheng Biotechnology Co., Ltd., DH101-1) on MS agar medium (Caisson Labs, MSP22-50LT) at 25°C in the dark to induce callu...

Embodiment 2

[0124] Example 2, Application of Tripterygium wilfordii Suspension Cell Protoplasts in Transient Transformation and Expression of Exogenous Genes

[0125] The transient transformation of tripterygium wilfordii suspension cell protoplasts was studied by using PEG-mediated transformation method, the specific steps are as follows:

[0126] 1. Adjust the concentration of the purified protoplasts obtained in step 3 of Example 1 to about 5×10 5 individual / mL to obtain the protoplast solution;

[0127] 2. Tap and mix 200 μl of protoplast solution, 20 μg of E3025 plasmid (the E3025 plasmid has its own GFP coding sequence, and the nucleotide sequence of the E3025 plasmid is shown in sequence 1) and 220 μL of PEG solution to obtain the transformation mixture. The transformation mixture was induced for 20 minutes under the conditions to obtain the transformation mixture;

[0128] 3. Dilute the transformation mixture with 880 μL W5 solution at room temperature, then gently shake the cen...

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Abstract

The invention discloses a Tripterygium wilfordii protoplast separation and instantaneous conversion method. Experiments prove that the yield of protoplasts obtained by the separation method is 4.24*10<5> / g FW, and the activity is 94.5%; and exogenous plasmids are successfully converted into the protoplasts extracted by the invention through a PEG 4000 mediated conversion method and are smoothly expressed, thereby successfully establishing a Tripterygium wilfordii suspension cell protoplast instantaneous conversion system. The invention lays a foundation for carrying out functional genomics research of Tripterygium wilfordii in a deep-going way, and simultaneously provides a material basis for research of Tripterygium wilfordii such as gene editing, subcellular localization, gene overexpression and interference and the like.

Description

technical field [0001] The invention belongs to the field of plant tissue culture, and specifically relates to a method for separating and transiently transforming protoplasts of Tripterygium wilfordii, in particular to a method for separating and purifying protoplasts of Tripterygium wilfordii and a method for transiently transforming exogenous genes. Background technique [0002] The traditional Chinese medicine Tripterygium wilfordii Hook.f. is a root medicinal material, which is derived from the root or xylem of the root of Tripterygium wilfordii Hook.f.. and other effects. The main active substances of Tripterygium wilfordii are alkaloids, diterpenes, triterpenes, sesquiterpenes and sugars. It has anti-inflammatory, anti-tumor, immunosuppressive and other effects, and is mainly used in rheumatoid, anti-tumor, glomerular diseases, systemic lupus erythematosus and other diseases in modern clinical treatment. In recent years, the study of Tripterygium wilfordii has attra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04C12N15/87
Inventor 高伟黄璐琦胡添源王睿周家伟
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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