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Method for allowing transformed-external-source desoxyribonucleic acid to enter rhizopus stolonifer resting spores

A technology of deoxyribonucleic acid and Rhizopus staphylococcus, which is applied in the biological field and achieves the effects of simple steps and low conversion rate

Inactive Publication Date: 2016-12-21
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 transformed-external-source desoxyribonucleic acid to enter rhizopus stolonifer resting spores

Examples

Experimental program
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Effect test

Embodiment 1

[0058] A method for transforming exogenous deoxyribonucleic acid into the dormant spores of Rhizopus staphylococcus, comprising the following steps:

[0059] 1) Rhizopus staphylococcus culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate the surface of the solid agar medium with Rhizopus staphylococcus CICC 40325, the temperature is 28°C, the humidity is 50-60%, and cultured for 6 days, let the surface of the medium grow. Full of Rhizopus staphylococcus spores.

[0061] Pour sterile water on the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Rhizopus staphylococcus on the surface of the medium, suck out the spore suspension with a pipette, and use Filter the sterilized lens paper (or sand core funnel, filter paper, etc.) to remove the mycelium and retain the spores. The filtered liquid is placed in a centrifuge tube. After centrifugation, the precipitate...

Embodiment 2

[0085] A method for transforming exogenous deoxyribonucleic acid into the dormant spores of Rhizopus staphylococcus, comprising the following steps:

[0086] 1) Rhizopus staphylococcus culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate the surface of the solid agar medium with Rhizopus staphylococcus CICC 40325, and cultivate for 15 days at a temperature of 16°C and a humidity of 15-50%, and let the medium Surface covered with Rhizopus staphylococcus spores.

[0088] Pour sterile water on the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Rhizopus staphylococcus on the surface of the medium, suck out the spore suspension with a pipette, and use Filter the sterilized lens paper (or sand core funnel, filter paper, etc.) to remove the mycelium and retain the spores. The filtered liquid is placed in a centrifuge tube. After centrifugation, the precipitated...

Embodiment 3

[0102] A method for transforming exogenous deoxyribonucleic acid into the dormant spores of Rhizopus staphylococcus, comprising the following steps:

[0103] 1) Rhizopus staphylococcus culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate the surface of the solid agar medium with Rhizopus staphylococcus CICC 40325, and cultivate it for 3 days at a temperature of 40°C and a humidity of 60-85%, and let the medium Surface covered with Rhizopus staphylococcus spores.

[0105] Pour sterile water on the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Rhizopus staphylococcus on the surface of the medium, suck out the spore suspension with a pipette, and use Filter the sterilized lens paper (or sand core funnel, filter paper, etc.) to remove the mycelium and retain the spores. The filtered liquid is placed in a centrifuge tube. After centrifugation, the precipitat...

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Abstract

The invention discloses a method for allowing transformed-external-source desoxyribonucleic acid to enter rhizopus stolonifer resting spores. The method includes the steps that rhizopus stolonifer is cultivated, and spores are collected; the rhizopus stolonifer spores are pretreated; the rhizopus stolonifer spores are subjected to electric shock with the HDEN method, and the rhizopus stolonifer spores with led-in to-be-transformed plasmids are obtained. According to the method, the non-germinated spores serve as initiative materials for leading in external source molecules, external source DNA is led into the rhizopus stolonifer resting spores with the HDEN electrotransformation technology, the complex spore germination step can be omitted, the protoplast preparing step or the agrobacterium-mediated transformation step and the like in the traditional method is omitted, the transformation rate is high, and the effect that 6,000 positive transformants or more exist in each transformation reaction system is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for transforming exogenous deoxyribonucleic acid into dormant spores of Rhizopus staphylococcus. Background technique [0002] Rhizopus staphylococcus is a common mold that can be easily isolated from soil or air. It can be used to produce a series of important economical compounds such as pectinase, endoglucanase, lipase, and testosterone. The product is an important industrial fermentation strain. However, it is very difficult to transform exogenous DNA into Rhizopus staphylococcus, which restricts its genetic engineering. [0003] Genetic engineering is based on molecular genetics and uses modern methods of molecular biology as a means to construct DNA molecules in vitro from genes from different sources according to a pre-designed blueprint, and then introduce them into cells to change the original genetic characteristics of organisms. , to obtain new varie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12R1/845
Inventor 林峻
Owner FUZHOU UNIV
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