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Method for allowing exogenous DNA to penetrate cell walls and cell membranes of ungerminated spores of mucor circinelloides

A technology of Mucor sclerotiorum and cell wall, which is applied in the biological field and achieves the effects of simple steps and low transformation rate

Pending Publication Date: 2016-12-07
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] At present, there is no method or report that can directly introduce exogenous DNA molecules into dormant (non-germinated) fungal spores without mediation

Method used

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  • Method for allowing exogenous DNA to penetrate cell walls and cell membranes of ungerminated spores of mucor circinelloides

Examples

Experimental program
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Effect test

Embodiment 1

[0058] A method for allowing exogenous DNA to pass through the ungerminated spore cell wall and cell membrane of Mucor circiniferii, comprising the following steps:

[0059] 1) Culture and spore collection of Mucor circiniferatum

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate the surface of the solid agar medium with Mucor circinatus CICC 40242 at a temperature of 28°C and a humidity of 50-60%, and cultivate for 5 days to allow the surface of the medium to grow. Full of Mucor volvulus spores.

[0061] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass spreader) the Mucor circinii spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use Filter with sterilized lens cleaning paper (or sand core funnel, filter paper, etc.) to remove mycelium and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, co...

Embodiment 2

[0085] A method for allowing exogenous DNA to pass through the ungerminated spore cell wall and cell membrane of Mucor circiniferii, comprising the following steps:

[0086] 1) Culture and spore collection of Mucor circiniferatum

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Mucor circinarius CICC 40242 on the surface of the solid agar medium, and cultivate it for 15 days at a temperature of 16°C and a humidity of 15-50%. The surface is covered with spores of Mucor volvulus.

[0088] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass spreader) the Mucor circinii spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use Filter with sterilized lens cleaning paper (or sand core funnel, filter paper, etc.) to remove mycelium and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, collect the precipi...

Embodiment 3

[0102] A method for allowing exogenous DNA to pass through the ungerminated spore cell wall and cell membrane of Mucor circiniferii, comprising the following steps:

[0103] 1) Culture and spore collection of Mucor circiniferatum

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Mucor circinarius CICC 40242 on the surface of the solid agar medium, and cultivate it for 3 days at a temperature of 40°C and a humidity of 60-85%. The surface is covered with spores of Mucor volvulus.

[0105] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass spreader) the Mucor circinii spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use Filter with sterilized lens cleaning paper (or sand core funnel, filter paper, etc.) to remove mycelium and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, collect the precipit...

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Abstract

The invention discloses a method for allowing exogenous DNA to penetrate the cell walls and cell membranes of the ungerminated spores of mucor circinelloides. The method includes: culturing the mucor circinelloides and collecting the spores, pretreating the mucor circinelloides spores and using an HDEN method to perform electric shock on the mucor circinelloides spores to obtain the mucor circinelloides spores imported with to-be-transformed plasmids. The method has the advantages that the ungerminated spores are used as the initial materials for importing exogenous molecules, the HDEN electric transformation technology is used to import the exogenous DNA into the resting mucor circinelloides spores, the complex step of spore germination can be omitted, steps such as protoplast preparation or agrobacterium-mediated transformation in a traditional method are omitted, the method is high in transformation rate, and each transformation reaction system can at least reach the effect of not less than 6000 positive transformants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for allowing exogenous DNA to pass through the cell wall and cell membrane of ungerminated spores of Mucor circiniferii. Background technique [0002] Mucor circiniferii is an industrial fermentation strain that can produce various important industrial products such as viscolytic enzymes. But it is very difficult to transform foreign DNA, which restricts its genetic engineering. [0003] Genetic engineering is based on the theory of molecular genetics, using modern methods of molecular biology as a means, to construct DNA molecules in vitro according to the pre-designed blueprint of genes from different sources, and then introduce them into cells to change the original genetic characteristics of organisms , obtain new varieties, and produce new products (for example, introducing an enzyme gene with important economic value into the cells of Mucor circiniferii fo...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/645
CPCC12N15/80
Inventor 林峻
Owner FUZHOU UNIV
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