Method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense

Patent metadata

Field	Value
Publication number	US-11236362-B2
Application number	US-201916296253-A
Country	US
Kind code	B2
Filing date	Mar 8, 2019
Priority date	Sep 9, 2016
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

Abstract

The present invention discloses a method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense. The method includes three steps of culture of Penicillium amagasakiense and collection of spores, pretreatment of Penicillium amagasakiense spores, and electroporation of Penicillium amagasakiense spores by using HDEN method, to obtain Penicillium amagasakiense spores with introduction of plasmids to be transformed. In the present invention, non-germinated spores are used as a starting material for introduction of an exogenous molecule, and exogenous DNA is introduced into the resting spores of Penicillium amagasakiense by employing the HDEN electrotransformation technique, whereby the complex step of spore germination is omitted, and steps of protoplast preparation or Agrobacterium-mediated transformation in conventional methods etc. are omitted. Moreover, the transformation efficiency is high, and at least an effect of no less than 6000 positive transformants per transformation reaction system can be achieved.

First claim

I claim:

1. A method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense , characterized in that, the method comprising steps of:
1) culture of Penicillium amagasakiense and collection of spores, wherein
Penicillium amagasakiense is inoculated onto a surface of a solid agar medium, and cultured until Penicillium amagasakiense spores are overgrown on the surface of the medium, the Penicillium amagasakiense spores are washed off from the surface of the medium, suspension of the spores is aspirated off and filtered to remove mycelia, and filtrate containing the spores is collected, and centrifuged to collect the pelleted resting spores;
2) pretreatment of Penicillium amagasakiense spores, wherein
the spores are re-suspended in an electroporation buffer, and centrifuged to collect the spore pellets, the re-suspension and centrifugation steps are repeated 3-4 times, and the last collected spore pellets are re-suspended in the electroporation buffer, to obtain an Penicillium amagasakiense spore suspension with a spore concentration of 10 4 -10 11 spores/ml,
in which the electroporation buffer consists of 4-hydroxyethyl piperazineethanesulfonic acid having a final concentration of 0.01-100 mmol/L and mannitol having a final concentration of 0.5-5000 mmol/L, and the pH of the electroporation buffer is 3.0-9.5; and
3) electroporation of Penicillium amagasakiense spores by using HDEN method, wherein
the Penicillium amagasakiense spore suspension prepared in the above steps and a plasmid to be transformed are added to wells of a cell culture plate and mixed uniformly, to obtain a mixture of the spores and the plasmid, the cell culture plate is placed on an ice bath for 10-15 min, subsequently electroporation is carried out by using the HDEN method using an Etta Biotech X-Porator H1 electroporator, by inserting an electroporator head fitted with a matrix electrode into the mixture of the spores and the plasmid, and energizing, to generate an electric field inside the mixture of the spores and the plasmid, the cell culture plate is placed on the ice bath again for 10-15 min after electroporation, and subsequently the mixture of the spores and the plasmid is aspirated off, to obtain resting spores of Penicillium amagasakiense with introduction of exogenous DNA,
in which ratio of the Penicillium amagasakiense suspension to the plasmid to be transformed is 6-600000 μl of Penicillium amagasakiense spore suspension to 0.1-10000 μg of plasmid to be transformed; and
wherein the parameters for the electroporation comprise: a voltage of 1-6000 V, pulse duration of 2-2000000 ms, and repeat for 1-100 times at an interval of 5-50000 ms.

2. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, the medium in the step 1) is PDA medium, YPD medium, or Czapek-Dox medium.

3. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, in the step 1) Penicillium amagasakiense is cultured at a temperature of 16-40° C. with 15-85% humidity for 3-15 days.

4. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 3 , characterized in that, in the step 1) of claim 1 , Penicillium amagasakiense is cultured at a temperature of 25° C. with 50-60% humidity for 8 days.

5. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, in the step 2), the electroporation buffer consists of 4-hydroxyethyl piperazineethanesulfonic acid having a final concentration of 1 mmol/L and mannitol having a final concentration of 50 mmol/L, and the pH of the electroporation buffer is 7.0.

6. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, the Penicillium amagasakiense spore suspension in the step 2) is observed under a microscope before electroporation, to confirm that the spore suspension is free of contamination with mycelia and the spores are non-germinated, and subsequently electroporation is carried out.

7. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, in the step 3), the plasmid to be transformed is recombinant plasmid AnEp8-hygro, and the recombinant plasmid AnEp8-hygro is constructed with a hygromycin B resistance gene and an AnEp8 plasmid.

8. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 7 , characterized in that, in the step 3) of claim 1 , the ratio of the Penicillium amagasakiense suspension to the recombinant plasmid AnEp8-hygro is 60 μl of Penicillium amagasakiense spore suspension to 1 μg of recombinant plasmid AnEp8-hygro.

9. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, in the step 3), the parameters for the electroporation comprise: a voltage of 450 V, pulse duration of 2500 ms, and repeat for 3 times at an interval of 400 ms.

10. The method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense according to claim 1 , characterized in that, the step 3) further comprises aspirating the mixture of the spores and the plasmid off, coating the mixture onto a plate containing YPD solid agar medium with a final concentration of hygromycin B of 600 μg/ml, culturing the mixture at a temperature of 16-40° C. with 15-85% humidity until single colonies are formed, and counting the colonies.

Assignees

• Univ Fuzhou

Inventors

• Lin Jun

Classifications

• C12N1/14 Primary

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C12R2001/80

  Penicillium · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/87 Primary

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title