Process for raising gene transferring rate of cotton pollen tube way leading-in

A technology of pollen tube passage and conversion rate, which is applied in the biological field and can solve the problems of low conversion rate and instability

Inactive Publication Date: 2005-06-08
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, due to many factors affecting the cotton pollen tube passage technology, the conversion rate of this technology is low and unstable (in cotton, its conversion rate is about 1%) (Wang Changhai et al., using the pollen tube passage method to convert the exogenous plasmid Improved method of DNA injection into cotton, Journal of Cotton Science, 1999, 11(4): 220-221)

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  • Process for raising gene transferring rate of cotton pollen tube way leading-in

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Embodiment Construction

[0016] 1. Extraction of DNA

[0017] When carrying out the genetic transformation of the target gene, extract the plasmid DNA, then electrophoresis on 0.8% agarose gel, check the purity and concentration of the plasmid DNA, use TE buffer (10mmol / L Tris-HCl, 0.5mmol / L EDTA, 10mmol / L NaCl) to dilute plasmid DNA to 1 μg·μl -1 According to the needs of the experiment, dilute to 0.01-0.02μg·μl when used -1 . When carrying out the genetic transformation of total DNA, extract the total DNA of the plant, after the DNA is purified, dilute the total DNA to 1 μg·μl with TE buffer (10mmol / L Tris-HCl, 0.5mmol / L EDTA, 10mmol / L NaCl) -1 According to the needs of the experiment, dilute to 0.1-0.2μg·μl when used -1 .

[0018] 2. Preparation of EMS solution

[0019] Ethyl methane sulfonate (EMS for short) is a product of Sigma Company, and it is made into 1 mg·ml with phosphate buffer solution. -1 The stock solution was filter sterilized and stored at 4°C.

[0020] 3. Preparation of tra...

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Abstract

A method for increasing the transform rate of the genes introduced to the pollen tube channel of cotton features use of an improved buffering liquid prepared from the buffer liquid TE and ethyl methane sulfonate. Its advantage is high effect to increase said transform rate by 2 times.

Description

(1) Technical field [0001] The invention relates to a method for improving the conversion rate of cotton pollen tube channel introduction gene, which belongs to the field of biotechnology. (2) Background technology [0002] The technology of using the pollen tube channel to introduce genes is a direct transformation technology using the germplasm cells or fertilized eggs of plants as the transformation object. This technology was created and first applied by Mr. Zhou Guangyu in my country (Zhou Guangzi et al., Inheritance of exogenous DNA into cotton embryos, Methods in Enzymology, 1983, 101: 433-488). In 1978, Zhou Guangyu fully investigated the mutant offspring of distant hybridization of plants at home and abroad, and found that the offspring of distant hybridization of plants can produce phenotypic variation, but no morphological changes at the chromosome level can be observed, so it is speculated that the phenotypic variation is the hybridization of DNA fragments the re...

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Application Information

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IPC IPC(8): C12N15/09C12N15/67
Inventor 沈法富韩秀兰李静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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