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Method for directly transforming exogenous DNA to enter aspergillus nidulans resting spores through non-medium dependency

A medium-dependent technology of Aspergillus nidulans, applied in the biological field, achieves the effect of low conversion rate and simple steps

Inactive Publication Date: 2016-11-23
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 directly transforming exogenous DNA to enter aspergillus nidulans resting spores through non-medium dependency

Examples

Experimental program
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Effect test

Embodiment 1

[0058] A method for directly transforming exogenous DNA into dormant spores of Aspergillus nidulans without medium dependence, comprising the following steps:

[0059] 1) Aspergillus nidulans culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Aspergillus nidulans CICC 2288 on the surface of the solid agar medium, at a temperature of 28°C and a humidity of 50-60%, and cultivate for 6 days to allow the surface of the medium to be covered with Aspergillus nidulans spores.

[0061] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Aspergillus nidulans spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use sterile water to remove the spores. Filter with sterilized lens tissue (or sand core funnel, filter paper, etc.) to remove mycelia and retain spores. The filtered liquid is put into a centr...

Embodiment 2

[0085] A method for directly transforming exogenous DNA into dormant spores of Aspergillus nidulans without medium dependence, comprising the following steps:

[0086] 1) Aspergillus nidulans culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Aspergillus nidulans CICC 2288 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%. Overgrown with Aspergillus nidulans spores.

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

Embodiment 3

[0101] A method for directly transforming exogenous DNA into dormant spores of Aspergillus nidulans without medium dependence, comprising the following steps:

[0102] 1) Aspergillus nidulans culture and spore collection

[0103] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Aspergillus nidulans CICC 2288 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%. Overgrown with Aspergillus nidulans spores.

[0104] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Aspergillus nidulans spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use sterile water to remove the spores. Filter with sterilized lens tissue (or sand core funnel, filter paper, etc.) to remove mycelia and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, co...

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Abstract

The invention discloses a method for directly transforming exogenous DNA to enter aspergillus nidulans resting spores through non-medium dependency. The method comprises three steps: aspergillus nidulans culture and spore collection, aspergillus nidulans spore pretreatment and electric shock of aspergillus nidulans spores using a HDEN process to obtain aspergillus nidulans spores with introduced to-be-transformed plasmid. In the invention, the non-terminated spores are used as a start material for introducing exogenous molecules, the exogenous DNA is introduced into the aspergillus nidulans resting spores by the HDEN electrotransformation technology, and a complicated step of spore germination can be saved; the steps such as preparation of protoplast and agrobacterium-mediated transformation in traditional method are saved, and the transformation rate is high; and in each transformation reaction system, an effect of at least 6000 positive transformants can be realized.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for directly transforming exogenous DNA into dormant spores of Aspergillus nidulans without medium dependence. Background technique [0002] Aspergillus nidulans is a fungus belonging to the genus Aspergillus. Distributed in food, soil and air. It has been used as a material for fungal genetics research, and can be used as an expression vector for foreign genes to express recombinant proteins. But the current technology is very difficult for A. nidulans to transform exogenous DNA. [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 produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12R1/66
Inventor 林峻
Owner FUZHOU UNIV
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