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Method for directly importing foreign DNA into fusarium moniliforme resting spores

A technology of Fusarium moniliformes, dormant spores, applied in the biological field

Inactive Publication Date: 2017-02-08
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 importing foreign DNA into fusarium moniliforme resting spores

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Effect test

Embodiment 1

[0058] A method for directly introducing exogenous DNA into dormant spores of Fusarium moniliforme, comprising the following steps:

[0059] 1) Fusarium moniliforme culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Fusarium moniliforme ATCC 46493 on the surface of the solid agar medium, and cultivate it for 5 days at a temperature of 24°C and a humidity of 50-60%. Overgrown with Fusarium moniliform 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 Fusarium moniliforme spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use a sterile 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, collect the precipitated dormant spores and remove the ...

Embodiment 2

[0085] A method for directly introducing exogenous DNA into dormant spores of Fusarium moniliforme, comprising the following steps:

[0086] 1) Fusarium moniliforme culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Fusarium moniliforme ATCC 46493 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 Fusarium moniliform 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 Fusarium moniliforme spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use a sterile 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, collect the precipitated dormant spores and remove the...

Embodiment 3

[0102] A method for directly introducing exogenous DNA into dormant spores of Fusarium moniliforme, comprising the following steps:

[0103] 1) Fusarium moniliforme culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Fusarium moniliforme ATCC 46493 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 Fusarium moniliform spores.

[0105] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Fusarium moniliforme spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use a sterile 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, collect the precipitated dormant spores and remove the ...

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Abstract

The invention discloses a method for directly importing foreign DNA into fusarium moniliforme resting spores. The method comprises the following three steps: performing fusarium moniliforme culture and spore collection, pretreating fusarium moniliforme spores and performing electric shock on the fusarium moniliforme spores by using a high-density distributed electrode network (HDEN) method, so that the fusarium moniliforme spores imported with to-be-transformed plasmids are obtained. Non-germinated spores serve as starting materials of importing foreign molecules, and the foreign DNA is imported into the fusarium moniliforme resting spores by employing the HDEN electroporation technology, so that a complicated step of germinating the spores can be saved, and the steps of preparing protoplast or performing agrobacterium-mediated transformation and the like in the traditional method are saved. Moreover, the transformation rate is high, and an effect of obtaining not less than 6000 positive transformants can be achieved at least in each transformation reaction system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for directly introducing exogenous DNA into resting spores of Fusarium moniliforme. Background technique [0002] Fusarium moniliforme ( Fusarium moniliforme ) belongs to the subphylum Deuteromycota, Hyphospora, Tumorosporum, Tumorosporidae, and Fusarium genus. The fungus can cause diseases in agriculture, and often parasitizes crops such as corn, oats, wheat, and rice. In addition to causing plant root rot and stem base rot, it can also cause symptoms such as top rot and ear rot. The metabolites of the fungus are moniliformin and zearalenone, which are toxic and are closely related to human gastric cancer and esophageal cancer. Fusarium moniliforme and its toxins can easily contaminate human food. Therefore, the study of the molecular mechanism of toxin production by Fusarium moniliforme, as well as the physiology and biochemistry of cells, is of great signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12R1/77
CPCC12N15/80C12N2800/10
Inventor 林峻
Owner FUZHOU UNIV
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