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Method for directly transforming foreign DNA (Deoxyribonucleic Acid) to enter trichoderma reesei resting spores without depending on medium

A technology of Trichoderma reesei and dormant spores, which is applied in the biological field to achieve 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 foreign DNA (Deoxyribonucleic Acid) to enter trichoderma reesei resting spores without depending on medium

Examples

Experimental program
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Effect test

Embodiment 1

[0058] A method for directly transforming exogenous DNA into dormant spores of Trichoderma reesei without relying on a medium, comprising the following steps:

[0059] 1) Trichoderma reesei culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Trichoderma reesei CICC 13052 on the surface of the solid agar medium at a temperature of 24°C and a humidity of 50-60%, and cultivate for 7 days to allow the surface of the medium to grow. full of Trichoderma reesei spores.

[0061] Pour sterile water onto the surface of the culture medium, wash down (shake, or gently scrape with a smooth sterile glass applicator) the Trichoderma reesei 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 directly transforming exogenous DNA into dormant spores of Trichoderma reesei without relying on a medium, comprising the following steps:

[0086] 1) Trichoderma reesei culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Trichoderma reesei CICC 13052 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 Trichoderma reesei spores.

[0088] Pour sterile water onto the surface of the culture medium, wash down (shake, or gently scrape with a smooth sterile glass applicator) the Trichoderma reesei 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 precipita...

Embodiment 3

[0102] A method for directly transforming exogenous DNA into dormant spores of Trichoderma reesei without relying on a medium, comprising the following steps:

[0103] 1) Trichoderma reesei culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Trichoderma reesei CICC 13052 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 Trichoderma reesei spores.

[0105] Pour sterile water onto the surface of the culture medium, wash down (shake, or gently scrape with a smooth sterile glass applicator) the Trichoderma reesei 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 precipitat...

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Abstract

The invention discloses a method for directly transforming a foreign DNA (Deoxyribonucleic Acid) to enter trichoderma reesei resting spores without depending on a medium. The method comprises three steps of culturing trichoderma reesei and colleting spores; pretreating trichoderma reesei spores; electrically shocking the trichoderma reesei spores by using an HDEN (High-Density Distributed Electrode Network) method, thus obtaining the trichoderma reesei spores into which plasmids to be transferred are introduced. According to the method disclosed by the invention, spores which are not germinated are used as starting materials for introducing foreign molecules; the foreign DNA is introduced into the trichoderma reesei resting spores by applying an HDEN electrotransformation technology, so that a complex step of germinating the spores can be omitted, and the step of preparing protoplast or transforming agrobacterium in a traditional method is omitted; in addition, the transformation rate is high; each conversion reaction system at least can achieve an 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 directly transforming exogenous DNA into dormant spores of Trichoderma reesei without relying on a medium. Background technique [0002] Trichoderma reesei is a eukaryotic microorganism belonging to the filamentous fungi. Trichoderma reesei has an excellent ability to synthesize and secrete proteins, and its industrial scale fermentation conditions are relatively mature, which promotes the research on genetic modification of Trichoderma reesei, which can be used as a host for expressing foreign proteins. However, it is very difficult to transform exogenous DNA to Trichoderma reesei, which restricts its development. [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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/885
CPCC12N15/80
Inventor 林峻
Owner FUZHOU UNIV