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Rhizopus nigricans hypha liposome direct transformation method

A technology of Rhizopus niger and liposome, which is applied in the field of liposome transformation, can solve problems such as unstable transformation rate, and achieve the effect of simple elimination, high transformation rate and stable genetics

Inactive Publication Date: 2013-03-20
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

Some transformation rates are unstable, and the transformation efficiency is affected by many factors. For example, the transformation efficiency of Agrobacterium transformation method is affected by many factors such as the type of Agrobacterium strain, the type of plasmid vector and the matching between the two, the composition of the medium, and the induction conditions. Impact

Method used

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  • Rhizopus nigricans hypha liposome direct transformation method
  • Rhizopus nigricans hypha liposome direct transformation method
  • Rhizopus nigricans hypha liposome direct transformation method

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Embodiment 1

[0021] 1. Experimental materials

[0022] 1. Strains and plasmids Rhizopus stolonifer (Rhizopus niger) Rhizopus stolonifer

[0023] Rhizopus stolonifer (Rhizopus stolonifer) was purchased from China General Microbiology Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3.4098. The plasmid pEGFP-C1 was donated by the Cell Biology Laboratory of Zhengzhou University.

[0024] 2. culture medium

[0025] PDA medium (1L): 200g of potatoes (peeled and cut into pieces, boiled in water for 15 minutes, and filtered with four layers of gauze to obtain the filtrate), 20g of agar (Agar), 20g of glucose, and 1000ml of water, aliquoted and autoclaved.

[0026] 3. Liposomes

[0027] Lipofectamine 2000, Invitrogen, Carlsbad, California, USA.

[0028] 2. Experimental method

[0029] A kind of rhizopus niger mycelia liposome direct conversion method, it comprises the following steps:

[0030] a) Liposome transformation of Rhizopus niger hyphae:

[0031] ...

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Abstract

The invention relates to a rhizopus nigricans hypha liposome direct transformation method. The method includes the steps of transformation of rhizopus nigricans hypha liposome and subculture. The steps of the transformation of the rhizopus nigricans hypha liposome comprise firstly mixing the tender rhizopus nigricans hypha and mannitol water solutions, grinding and obtaining bacterium suspension for use; secondly uniformly mixing liposome 2000 and plasmid pEGFP-C1, standing 15-60 minutes at the temperature of -5-5 DEG C, then transferring the mixture to the bacterium suspension, uniformly mixing, and standing 15-60 minutes at the temperature of -5-5 DEG C; and thirdly coating the mixing liquid obtained from the second step on a potato dextrose agar (PDA) plate, and cultivating the mixing liquid for 1-5 days at the temperature of 25-30 DEG C. The steps of the subculture comprise picking transformant which is provided with fluorescent though detection of a fluorescent microscope in the third step, inoculating the transformant onto the PDA plate to cultivate the transformant for 1-5 days at the temperature of 25-30 DEG C, and carrying out the subculture for 5-8 times. The method is easy and convenient to carry out, and high in conversion rate, transformant genetic stability is achived, protoplast is needless to be prepared, and complicated devices are needless.

Description

technical field [0001] The invention belongs to the technical field of liposome transformation, and in particular relates to a direct transformation method of rhizopus niger hyphae liposome. Background technique [0002] At present, the genetic transformation methods of filamentous fungi mainly include protoplast electroporation, biolistic transformation, lithium acetate-mediated transformation, Agrobacterium-mediated transformation, PEG-mediated protoplast transformation and protoplast liposome transformation. law etc. These methods generally have disadvantages such as cumbersome operation, complicated equipment, low conversion rate, and unstable transformants. If some need to prepare protoplasts, the preparation process is complicated, including protoplast electroporation, PEG-mediated protoplast transformation and protoplast liposome transformation. Some methods do not require the preparation of protoplasts, but the equipment is complicated and the transformation cost i...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/845
Inventor 邱立友孙强崔继红柴冉高玉千戚元成申进文
Owner HENAN AGRICULTURAL UNIVERSITY
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