Method for preparation and transient gene expression of jatropha curcas tissue culture seedling protoplast

A protoplast and transient expression technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of lack of technical information on Jatropha protoplasts, save steps and time, and improve transformation efficiency

Inactive Publication Date: 2016-02-17
SOUTH CHINA AGRI UNIV
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Problems solved by technology

Currently, technical information on obtaining Jatropha protoplasts is scarce

Method used

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  • Method for preparation and transient gene expression of jatropha curcas tissue culture seedling protoplast

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Embodiment 1

[0034] Example 1 A method for the preparation of protoplasts of Jatropha japonicus tissue culture seedlings and transient gene expression

[0035] (1) Weigh 1.82g of mannitol and dissolve it in 20ml of double-distilled water, prepare the mannitol solution and place it in a sterile small beaker;

[0036] (2) Take the leaves of Jatropha vulgaris tissue cultured seedlings on the ultra-clean table, cut into 0.5-1mm wide leaf strips with a blade, and put the cut leaf strips into the mannitol solution;

[0037] (3) Remove the leaf strips and put them in a pre-prepared 50ml conical flask containing 15ml of enzymatic hydrolysis solution, avoid light, and leave it at 22~25℃ for 6-7h; when the enzymatic hydrolysis solution turns green, shake Erlenmeyer flask to promote the release of protoplasts;

[0038] (4) Add 15ml equal volume of pre-cooled W5 solution, filter with 100 mesh filter, and place the filtrate on ice in a 50ml round bottom centrifuge tube;

[0039] (5) Centrifuge with a centrifuga...

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Abstract

The invention belongs to the technical field of molecular biology and gene engineering, and discloses a method for preparation and transient gene expression of a jatropha curcas tissue culture seedling protoplast. The method comprises the following operation steps that jatropha curcas tissue culture seedling leaves are cut into leaf strips with the width of 0.5-1 mm, the leaf strips are placed into an enzymatic hydrolysate for light shading, standing is performed at the temperature of 22-25 DEG C for enzymolysis for 6-7 h, filtration washing is performed on the protoplast, a certain amount of plasmid DNA, the proroplast and the PEG4000 / Ca2+ solution are added for processing, culture is performed for 18-40 h and then observation is performed under a confocal laser scanning microscope.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a method for preparing the protoplasts of Jatropha paulownia tissue culture seedlings and transient gene expression. Background technique [0002] Jatrophacarcas L., also known as Jatropha, is a deciduous shrub or small tree in the Euphorbiaceae Jatropha genus. Jatropha seeds have a high oil content and can be processed into biodiesel. It has strong market development potential and application prospects. Transient expression technology is a technology that transfers a target gene into target cells in a relatively short time and obtains a short-term high-level expression of the gene. It is a fast and convenient method for studying gene-protein. Currently, technical information about obtaining Jatropha protoplasts is very scarce. Therefore, we adjusted and improved the existing methods of Arabidopsis protoplast preparation to obtain highly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
Inventor 彭昌操杨紫薇刘思雯韩鸿均刘应波任陈希白雪
Owner SOUTH CHINA AGRI UNIV
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