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Method for directly introducing exogenous deoxyribonucleic acid (DNA) into resting spores of Pyricularia oryzae

A technology of deoxyribonucleic acid and dormant spores, which is applied to nucleic acid carriers, biochemical equipment and methods, and the use of carriers to introduce foreign genetic materials, etc., to achieve high conversion rates

Pending 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 introducing exogenous deoxyribonucleic acid (DNA) into resting spores of Pyricularia oryzae

Examples

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Embodiment 1

[0058] A method for directly introducing exogenous deoxyribonucleic acid in the dormant spores of Pyrus spp. comprising the following steps:

[0059] 1) Oryza spore culture and spore collection

[0060] Prepare a solid agar medium (PDA medium) in a 15cm petri dish, inoculate Pyrium oryzae ATCC 208987 on the surface of the solid agar medium, and cultivate it for 6 days at a temperature of 24°C and a humidity of 40-60%. Covered with oryzae spores.

[0061] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Pyrium oryzae 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 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 deoxyribonucleic acid in the dormant spores of Pyrus spp. comprising the following steps:

[0086] 1) Oryza spore culture and spore collection

[0087] Prepare a solid agar medium (YPD medium) in a 15cm petri dish, inoculate Pyrium oryzae ATCC 208987 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 pear spores.

[0088] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Pyrium oryzae 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 mycelia and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, collect the precipitated dormant spores and remove th...

Embodiment 3

[0102] A method for directly introducing exogenous deoxyribonucleic acid in the dormant spores of Pyrus spp. comprising the following steps:

[0103] 1) Rice pear spore culture and spore collection

[0104] Prepare a solid agar medium (PDA medium) in a 15cm petri dish, inoculate Pyrium oryzae ATCC 208987 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 pear spores.

[0105] Pour sterile water onto the surface of the culture medium, wash off (shake, or gently scrape with a smooth sterile glass coating rod) the spores of Pyrium oryzae 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 mycelia and retain spores. The filtered liquid is put into a centrifuge tube. After centrifugation, collect the precipitated dormant spores and remove...

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Abstract

The invention discloses a method for directly introducing exogenous deoxyribonucleic acid (DNA) into resting spores of Pyricularia oryzae. The method comprises the three steps, i.e., carrying out Pyricularia oryzae culture and spore collection, pretreating Pyricularia oryzae spores and carrying out electroshock on the Pyricularia oryzae spores by using an HDEN (High-density Distributed Electrode Network) method, thereby obtaining the Pyricularia oryzae spores which are used for being introduced into plasmid to be transformed. According to the method, un-germinated spores serve as a starting material for introducing exogenous molecules, and the exogenous DNA is introduced into the resting spores of the Pyricularia oryzae by using an HDEN electrotransformation technology, so that a complicated step, i.e., carrying out spore germination can be avoided, and a step of preparing protoplast or transforming Agrobacterium tumefaciens and the like in the traditional methods can be avoided; the transformation ratio is high, and each transformation reaction system at least can achieve the effect of no less than 6,000 positive transformants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for directly introducing exogenous deoxyribonucleic acid into dormant spores of Pyrus spp. Background technique [0002] Rice blast is one of the important diseases of rice, which is mainly caused by the fungus Pyrosporium oryzae parasitizing on rice. It is necessary to transform exogenous DNA into its cells for molecular biology research and genetic modification, but it is very difficult to transform exogenous DNA 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 , to obtain new varieties and produce new products (such as introducing an enzyme gene with important economic...

Claims

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Application Information

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IPC IPC(8): C12N15/80
CPCC12N15/80C12N2800/10
Inventor 林峻
Owner FUZHOU UNIV
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