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Method for building Chinese cabbage genetic transformation system by cell-penetrating peptide and microspore culture

A genetic transformation system and microspore culture technology, applied in the field of plant cell engineering technology and molecular biology, can solve the problems of long experimental period and low transformation efficiency.

Inactive Publication Date: 2015-08-05
SHENYANG AGRI UNIV
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Problems solved by technology

However, this conversion method has the disadvantages of low conversion efficiency and long experimental cycle.

Method used

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  • Method for building Chinese cabbage genetic transformation system by cell-penetrating peptide and microspore culture
  • Method for building Chinese cabbage genetic transformation system by cell-penetrating peptide and microspore culture
  • Method for building Chinese cabbage genetic transformation system by cell-penetrating peptide and microspore culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0048] Example 2 1. Obtaining the target gene expression components of transfected microspores

[0049] Transfer the vector pBI221-GFP plasmid DNA into Escherichia coli DH5α competent cells. The specific steps are: ①Take out the competent cell suspension (stored in -80℃ refrigerator) and thaw it on ice; ②Add 2 μL of plasmid DNA to 100 μL DH5α competent cells , mix gently, and place on ice for 30 minutes; ③42°C water bath for 90s, and immediately place on ice for 3-5 minutes after heat shock; ④Add 500 μL LB liquid medium (without antibiotics), mix well, 37°C, 150rpm Shake culture for 1 hour, the purpose is to restore the normal growth state of the bacteria, and express the corresponding antibiotic resistance gene encoded by the plasmid; ⑤4000rpm, centrifuge for 5min, discard 400μL supernatant, and the remaining resuspended strain; Amp 50μgmL-1 LB plate, placed on the front side for 20min, and then cultured upside down at 37°C for 16-20 hours.

[0050] Single-clonal coloni...

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Abstract

The invention belongs to the field of a plant cell engineering technology and molecular biology and relates to a method for building a Chinese cabbage genetic transformation system by cell-penetrating peptide and microspore culture. The method comprises carrying out in-vitro synthesis of a transfection complex by film-penetrating functions of cell-penetrating peptide, adding the transfection complex into a separated microspore suspension liquid, preparing a regeneration plant by a free microspore culture technology and carrying out identification on the regeneration plant. The method builds the Chinese cabbage genetic transformation system based on free microspore culture, provides a study platform for Chinese cabbage genetic breeding and lays the method foundation of Chinese cabbage functional genomics.

Description

technical field [0001] The invention belongs to the fields of plant cell engineering technology and molecular biology, and specifically relates to a method for establishing a Chinese cabbage genetic transformation system in combination with the membrane-penetrating function of cell-penetrating peptides during microspore culture. Background technique [0002] Chinese cabbage is one of the most important vegetable crops in my country. With the release of the genome sequence of Chinese cabbage, more and more attention has been paid to the study of its functional genomics. The construction of an efficient genetic transformation system of Chinese cabbage plays an important role in the verification of Chinese cabbage gene function. [0003] In the research of transgenic Chinese cabbage, many scholars at home and abroad have established the genetic transformation system of Chinese cabbage by means of tissue culture and other means. However, such conversion methods have disadvantag...

Claims

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Application Information

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IPC IPC(8): C12N15/87A01H4/00A01H5/00
Inventor 冯辉孟茜刘志勇章云李承彧冀瑞琴王玉刚
Owner SHENYANG AGRI UNIV
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