Method of enabling exogenous DNA to penetrate cell barriers to enter penicillium notatum resting spores

Abstract

The invention discloses a method of enabling exogenous DNA to penetrate cell barriers to enter penicillium notatum resting spores. The method includes three steps: penicillium notatum culturing and spore collection, penicillium notatum spore pretreatment and using of a HDEN method to electrically shock penicillium notatum spores to obtain the penicillium notatum spores with to-be transformed plasmids guided in. Ungerminated spores are used as a starting material for guiding in exogenous molecules, and HDEN electric transformation technology is applied to guide exogenous DNA into the penicillium notatum resting spores, so that a complex step of spore germination can be omitted, steps of preparing protoplast or agrobacterium-mediated transformation in conventional methods are omitted, transformation rate is high, and each transformation reaction system at least can realize 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 allowing exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis. Background technique

[0002] Penicillium pointis is a eukaryotic microorganism belonging to the filamentous fungi. Penicillium pointis is an important fermentation industrial strain, which can produce antibiotics. But it is very difficult to transform foreign DNA into Penicillium pointis.

[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, introducing an enzyme gene with important economic value into Penicillium pointis c...

Examples

Embodiment 1

[0058] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0059] 1) Point Penicillium culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Penicillium SIIM M80 on the surface of the solid agar medium, at a temperature of 26°C and a humidity of 50-60%, and cultivate for 4 days to allow the surface of the medium to be covered with Point Penicillium spores.

[0061] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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...

Embodiment 2

[0085] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0086] 1) Point Penicillium culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Penicillium SIIM M80 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 Penicillium spores.

[0088] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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 supernatant. The obtained spores wi...

Embodiment 3

[0102] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0103] 1) Point Penicillium culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Penicillium SIIM M80 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 Penicillium spores.

[0105] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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 supernatant. The obtained spores wil...

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for allowing exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis. Background technique

[0002] Penicillium pointis is a eukaryotic microorganism belonging to the filamentous fungi. Penicillium pointis is an important fermentation industrial strain, which can produce antibiotics. But it is very difficult to transform foreign DNA into Penicillium pointis.

[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, introducing an enzyme gene with important economic value into Penicillium pointis c...

Examples

Embodiment 1

[0058] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0059] 1) Point Penicillium culture and spore collection

[0060] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Penicillium SIIM M80 on the surface of the solid agar medium, at a temperature of 26°C and a humidity of 50-60%, and cultivate for 4 days to allow the surface of the medium to be covered with Point Penicillium spores.

[0061] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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...

Embodiment 2

[0085] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0086] 1) Point Penicillium culture and spore collection

[0087] In a 15cm petri dish, prepare a solid agar medium (YPD medium), inoculate Penicillium SIIM M80 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 Penicillium spores.

[0088] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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 supernatant. The obtained spores wi...

Embodiment 3

[0102] A method for exogenous DNA to penetrate cell barriers and enter dormant spores of Penicillium pointis, comprising the following steps:

[0103] 1) Point Penicillium culture and spore collection

[0104] In a 15cm petri dish, prepare a solid agar medium (PDA medium), inoculate Penicillium SIIM M80 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 Penicillium spores.

[0105] Pour sterile water onto the surface of the medium, wash off (shake, or gently scrape with a smooth sterile glass applicator) the Penicillium spores on the surface of the medium, suck out the spore suspension with a pipette, and use a sterile 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 supernatant. The obtained spores wil...

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