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Method for preparation and transformation of wheat endosperm cell protoplast

A protoplast and wheat germ technology, applied in the field of protoplast preparation and transformation of wheat endosperm cells, can solve problems such as inapplicability

Pending Publication Date: 2020-08-18
CAPITAL NORMAL UNIVERSITY
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Problems solved by technology

Obviously, the preparation and transformation methods of mesophyll cell protoplasts are not applicable to wheat endosperm cell protoplasts

Method used

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  • Method for preparation and transformation of wheat endosperm cell protoplast
  • Method for preparation and transformation of wheat endosperm cell protoplast
  • Method for preparation and transformation of wheat endosperm cell protoplast

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Embodiment 1

[0082] Embodiment 1, preparation and transformation of wheat endosperm cell protoplast

[0083] 1. Experimental method

[0084] The flow chart of the preparation and transient transformation of the wheat endosperm cell protoplast of the present embodiment is as follows figure 1 . It mainly includes the following steps:

[0085] S1: Harvest fresh wheat grains 8-11 days after flowering, cut them open along the ventral seam, and obtain endosperm tissue;

[0086] S2: Cut the endosperm tissue into thin strips, soak and wash in buffer 1;

[0087] S3: placing the washed endosperm tissue in buffer 2 to obtain crude wheat endosperm cell protoplasts;

[0088] S4: The crude endosperm cell protoplasts were treated by the ice bath standing time gradient sedimentation method to obtain the purified wheat endosperm cell protoplasts;

[0089] S5: suspending the purified wheat endosperm cell protoplasts with MMg buffer;

[0090] S6: Transformation of wheat protoplasts obtained above was i...

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Abstract

The invention discloses a method for preparation and transformation of a wheat endosperm cell protoplast. The invention provides a method for preparing the wheat endosperm cell protoplast, comprisingthe following steps of: taking fresh wheat grains 8-11 days after flowering, splitting along abdominal seams to obtain endosperm tissues, soaking and washing in a buffer solution 1, then putting in abuffer solution 2 for enzymolysis, and carrying out ice bath standing time gradient sedimentation to obtain a purified wheat endosperm cell protoplast. The invention provides an efficient, economicaland rapid protoplast preparation and transformation method for wheat endosperm cells containing a large number of stored starch particles. According to the method, the endosperm cell protoplast can berapidly and effectively separated; the method can be used for subcellular localization, transcription factor activity analysis, protein interaction identification and enzyme activity analysis of wheat endosperm expression factors, creates conditions for in-vivo molecular biological research of the factors, and effectively avoids experimental deviation caused by error expression of endosperm specific expression genes in other cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing and transforming protoplasts of wheat endosperm cells. Background technique [0002] Wheat endosperm is a nutrient storage organ, containing about 75% storage starch, 15% storage protein and 1-2% fat. During the development of endosperm, a large number of genes are involved in the synthesis, transportation and storage of nutrients. Understanding the regulation and function of these endosperm-expressed genes is a prerequisite for improving wheat yield and quality through genetic means. Due to the long growth cycle of wheat and the difficulty of stable genetic transformation, it is still difficult to study the biological functions of endosperm-expressed genes in wheat on a large scale. Therefore, there is an urgent need to develop alternative technologies. [0003] Protoplast transient expression is a routine technique for studying protein subcellular localiza...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206
Inventor 李小辉韩市陈晏月明
Owner CAPITAL NORMAL UNIVERSITY
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