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Method for directly delivering exogenous DNA into monascus resting spores

A technology of deoxyribose nucleic acid and dormant spores, which is applied in the biological field to achieve the effect of high transformation rate

Inactive 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 directly delivering exogenous DNA into monascus resting spores

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Effect test

Embodiment 1

[0058] A method for directly delivering exogenous deoxyribonucleic acid to the inside of Monascus dormant spores, comprising the following steps:

[0059] 1) Monascus culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Monascus CICC 41234 on the surface of the solid agar medium, at a temperature of 30°C and a humidity of 50-60%, and cultivate for 5 days, so that the surface of the medium is covered with red Aspergillus 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 Monascus spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use sterilized Filter through 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 precipitated dormant spores and re...

Embodiment 2

[0085] A method for directly delivering exogenous deoxyribonucleic acid to the inside of Monascus dormant spores, comprising the following steps:

[0086] 1) Monascus culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Monascus CICC 41234 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%, so that the surface of the medium grows Full of Monascus spores.

[0088] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass spreader) the Monascus spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterilized Filter through 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 precipitated dormant spores and remove the supernatant. ...

Embodiment 3

[0102] A method for directly delivering exogenous deoxyribonucleic acid to the inside of Monascus dormant spores, comprising the following steps:

[0103] 1) Monascus culture and spore collection

[0104] Prepare a solid agar medium (PDA medium) in a 15cm petri dish, inoculate Monascus CICC 41234 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% to allow the surface of the medium to grow Full of Monascus 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 Monascus spores on the surface of the culture medium, suck out the spore suspension with a pipette, and use sterilized Filter through 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 precipitated dormant spores and remove t...

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Abstract

The invention discloses a method for directly delivering exogenous DNA into monascus resting spores. The method includes three steps: performing monascus culture and spore collection; pre-treating monascus spores; using a HDEN method to apply electric shock to the monascus sports to obtain monascus spores with to-be-converted plasmids guided in. Non-germinal spores are used as a starting material for guiding in exogenous molecules, and HDEN electrotransformation technology is applied to guide the exogenous DNA into the monascus resting spores, so that a complex step of spore germination can be omitted, and steps of protoplast preparation or argrobacterium-mediated transformation in conventional methods are omitted; transformation rate is high, and each transformation reaction system can realize effect of at least 6000 positive transformants at least.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for directly delivering exogenous deoxyribonucleic acid to the inside of resting spores of Monascus. Background technique [0002] Monascus can be used in wine making, vinegar making, coloring and seasoning of fermented bean curd, and traditional Chinese medicine, which has important industrial and economic value. To genetically modify it, it is necessary to transform exogenous DNA into cells, but it is very difficult to transform exogenous DNA into Monascus at present. [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, int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12R1/645
Inventor 林峻
Owner FUZHOU UNIV
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