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Method for medium-independent direct transforming of exogenous DNA into Rhizopus oryzae resting spore

A technology of dormant spores and Rhizopus oryzae, applied in the direction of recombinant DNA technology, microbial-based methods, biochemical equipment and methods, etc., to achieve a high 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 medium-independent direct transforming of exogenous DNA into Rhizopus oryzae resting spore

Examples

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Effect test

Embodiment 1

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

[0059] 1) Rhizopus oryzae culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Rhizopus oryzae CICC 3084 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 to allow the surface of the medium to be covered with Rhizopus oryzae spores.

[0061] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Rhizopus oryzae spores on the surface of the 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, collect ...

Embodiment 2

[0085] A method for directly transforming exogenous DNA into dormant spores of Rhizopus oryzae without relying on medium, comprising the following steps:

[0086] 1) Rhizopus oryzae culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Rhizopus oryzae CICC 3084 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%. Covered with Rhizopus oryzae 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 Rhizopus oryzae 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 mycelium and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, collect the precipitated do...

Embodiment 3

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

[0103] 1) Rhizopus oryzae culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Rhizopus oryzae CICC 3084 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%. Covered with Rhizopus oryzae spores.

[0105] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Rhizopus oryzae spores on the surface of the 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, collect the precipitated dormant spores and r...

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Abstract

The invention discloses a method for medium-independent direct transforming of exogenous DNA into Rhizopus oryzae resting spore, wherein the method comprises three steps such as Rhizopus oryzae culturing and spore collecting, Rhizopus oryzae spore pre-treating, and electric shocking of the Rhizopus oryzae spore with a HDEN method so as to obtain the Rhizopus oryzae spore introduced into plasmid to be transformed. According to the present invention, the non-germinating spore is adopted as the starting material for introducing exogenous molecules and the exogenous DNA is introduced into the Rhizopus oryzae resting spore by using the HDEN electrotransformation technology, such that the complex step spore germination can be eliminated, the steps such as protoplast preparation or Agrobacterium transformation, and the like in the traditional method can be eliminated, and the transformation rate is high, wherein at least not less than 6000 positive transformants can be achieved 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 transforming exogenous DNA into dormant spores of Rhizopus oryzae without relying on a medium. Background technique [0002] Rhizopus oryzae is one of the important molds in Chinese medicine and koji. It is also common in soil, air and other substances. It can saccharify starch, transform sucrose, produce lactic acid, fumaric acid and trace alcohol. The ability to produce L(+) lactic acid is strong, reaching about 70%. But it is very difficult to transform exogenous DNA to Rhizopus oryzae, which restricts its genetic engineering transformation and industrial application. [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 chang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12R1/845
CPCC12N15/80C12N2800/10
Inventor 林峻
Owner FUZHOU UNIV
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