Method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators

Abstract

The present invention discloses a method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators. The method includes three steps of culture of Aspergillus niger and collection of spores, pretreatment of Aspergillus niger spores, and electroporation of Aspergillus niger spores by using HDEN method, to obtain Aspergillus niger 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 Aspergillus niger 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.

Claims

1. A method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators, comprising steps of: 1) culture of Aspergillus niger and collection of spores Aspergillus niger is inoculated onto a surface of a solid agar medium, and cultured until Aspergillus niger spores are overgrown on the surface of the solid agar medium, the Aspergillus niger spores are washed off from the surface of the solid agar 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 Aspergillus niger spores 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 Aspergillus niger spore suspension with a spore concentration of 10.sup.4-10.sup.11 spores/ml, in which the electroporation buffer consists of 4-hydroxyethyl piperazine ethanesulfonic 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 Aspergillus niger spores by using high-density distributed electrode network (HDEN) method the Aspergillus niger 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 the electroporation, and subsequently the mixture of the spores and the plasmid is aspirated off, to obtain resting spores of Aspergillus niger with introduction of exogenous DNA, in which ratio of the Aspergillus niger spore suspension to the plasmid to be transformed is 6-600000 l of Aspergillus niger spore suspension to 0.1-10000 g of plasmid to be transformed; and 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 Aspergillus niger independent of mediators according to claim 1, wherein the solid agar 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 Aspergillus niger independent of mediators according to claim 1, wherein in the step 1), Aspergillus niger 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 Aspergillus niger independent of mediators according to claim 3, wherein in the step 1), Aspergillus niger is cultured at a temperature of 30 C. with 50-60% humidity for 5 days.

5. The method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators according to claim 1, wherein in the step 2), the electroporation buffer consists of 4-hydroxyethyl piperazine ethanesulfonic 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 Aspergillus niger independent of mediators according to claim 1, wherein the Aspergillus niger 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 Aspergillus niger independent of mediators according to claim 1, wherein 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 Aspergillus niger independent of mediators according to claim 7, wherein in the step 3), the ratio of the Aspergillus niger spore suspension to the recombinant plasmid AnEp8-hygro is 60 l of Aspergillus niger spore suspension to 1 g of recombinant plasmid AnEp8-hygro.

9. The method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators according to claim 1, wherein in the step 3), the parameters for the electroporation comprise: a voltage of 400 V, pulse duration of 2000 ms, and repeat for 3 times at an interval of 500 ms.

10. The method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators according to claim 1, wherein 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 200 g/ml, culturing the mixture at a temperature of 16-40 C. with 15-85% humidity until single colonies are formed, and counting the single colonies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an agarose gel electrophoresis to confirm whether a sample contains the hygromycin resistance gene after PCR amplification in Example 1.

DESCRIPTION OF THE EMBODIMENTS

(2) The following examples are provided for a better understanding of the present invention; however, the present invention is not limited thereto.

(3) All the experimental process should follow the principle of aseptic condition while conducting the microbial experiments, and the instruments, consumable materials, and reagents should be sterilized.

Example 1

(4) A method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators includes the following steps:

(5) 1) Culture of Aspergillus niger and Collection of Spores

(6) In a 15-cm Petri dish, a solid agar medium (PDA medium) was prepared. Aspergillus niger CBS 513.88 was inoculated onto the surface of the solid agar medium and cultured at a temperature of 30 C. with 50-60% humidity for 5 days, to allow Aspergillus niger spores to overgrow on the surface of the medium (where the spores appeared dark brown and was easy to visually identified).

(7) Sterilized water was poured onto the surface of the medium, to wash down the Aspergillus niger spores off from the surface of the medium (by vibrating, or gently scratching with a sterilized smooth glass spreading rod). The spore suspension was aspirated with a pipette and filtered using sterilized lens paper (or fitted glass filter, filter paper, etc.) to remove the mycelia and retain the spores. The filtered liquid was placed in a centrifuge tube and centrifuged to collect the pelleted resting spores, and the supernatant was discarded. The collected spores were subjected to chromosome staining, to observe and confirm that the chromosomes in the spores are haploid.

(8) 2) Pretreatment of Aspergillus niger Spores

(9) The spore pellets were re-suspended in an electroporation buffer (where the volume of the electroporation buffer added should fill up the centrifuge tube), centrifuged again to collect the spore pellets, and the supernatant was discarded. After repeating the above steps twice, the spores were re-suspended in the electroporation buffer again, and observed under a microscope, to confirm that the spore suspension was free of contamination with mycelia and the spores were non-germinated. When the spores were finally re-suspended in the electroporation buffer, the volume of the electroporation buffer was controlled to maintain a spore concentration in the Aspergillus niger spore suspension of 10.sup.8 spores/ml.

(10) The electroporation buffer consisted of HEPES 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 was 7.0.

(11) 3) Electroporation of the Aspergillus niger Spores by Using HDEN Method

(12) 60 l of the Aspergillus niger spore suspension and 1 g of recombinant plasmid AnEp8-hygro were added to wells of a 96-well cell culture plate and mixed evenly, to obtain a mixture of the spores and the plasmid. The cell culture plate was placed on an ice bath for 10 min, electroporation was carried out subsequently 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 was placed on the ice bath again for 10 min after electroporation, and subsequently the mixture of the spores and the plasmid was aspirated off, to obtain resting spores of Aspergillus niger with introduction of exogenous DNA.

(13) In this example, the parameters for electroporation include: a voltage of 400 V, pulse duration of 2000 ms, and repeat for 3 times at an interval of 500 ms.

(14) 4) Confirmatory Experiment

(15) The aspirated mixture of the spores and the plasmid was coated onto a plate containing YPD solid agar medium with a final concentration of hygromycin B of 200 g/ml, and cultured at a temperature of 28 C. with 50-60% humidity until single colonies were formed. The colonies were counted and the transformation efficiency was calculated.

(16) At the same time when the above experimental steps were performed, a control group was prepared. The same mixture of the spores and the plasmid that was not electroporated was coated onto another plate containing YPD solid agar medium with a final concentration of hygromycin B of 200 g/ml, and cultured under the same conditions. No single colony was formed in the control group, and the single colonies in the experimental group were determined to be positive clones.

(17) When single colonies were formed in the experimental group, DNA was extracted from the single colonies in the experimental group, and the exogenous hygromycin B resistance gene which may contained in the DNA was amplified by PCR. The band size of the amplified product was determined by agarose gel electrophoresis. The result of electrophoresis is shown in FIG. 1. The direction of electrophoresis is from bottom to top. The Marker is Takara 250 bp DNA ladder marker, lane 1 is negative control, and a clear band is observed at about 1 kb in lane 2, which is consistent with the band of hygromycin B resistance gene, indicating successful transformation. That is to say the lane 2 is the band of the amplified hygromycin B resistance gene. Sanger DNA sequencing result shows that the DNA sequence is consistent with the exogenous hygromycin B resistance gene, hence it can be accurately determined that the positive clone in this example is a successful transformant.

(18) The volume of the AnEp8-hygro plasmid in this example was 12.4 kb, and 1 plasmid (about 124.26 fmol plasmid molecules) was used, with which 610.sup.6 non-germinated resting spores of Aspergillus niger were electroporated, to produce no less than 8000 positive transformants.

(19) The recombinant plasmid AnEp8-hygro in this example was constructed with a hygromycin B resistance gene and an AnEp8 plasmid. The construction of the recombinant plasmid AnEp8-hygro was as follows:

(20) The hygromycin B resistance gene is as shown in SEQ ID NO. 1.

(21) The protein sequence encoded is as shown in SEQ ID NO. 2.

(22) The hygromycin B resistance gene was amplified by PCR using primers:

(23) TABLE-US-00001 F: (SEQIDNO.4) CATTAGCTAGCATGAAAAAGCCTGAACTCACCG R: (SEQIDNO.5) TCTGGCGCGCCCTATTCCTTTGCCCTCGG

(24) PCR system (50 L): template 3 L, primer F (10 M) 2 L, primer R (10 M) 2 L, 2 Taq PCR mix 25 L, and ddH.sub.2O top up to 50 L.

(25) PCR program: 94 C. for 10 min, 35 cycles of (94 C. for 30 s, 61.8 C. for 30 s, and 72 C. for 90 s), and 72 C. for 10 min.

(26) The PCR product was confirmed by agarose gel electrophoresis, and after correct detection, the PCR product was recovered by using Thermo GeneJET Gel Extraction and DNA Cleanup Micro Kit.

(27) The AnEp8 plasmid was a gift from U.S. Fungal Genetics Stock Center (FGSC), and was described in literature 2: STORMS R, ZHENG Y, LI H, SILLAOTS S, MARTINEZ-PEREZ A, TSANG A. Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger [J]. Plasmid, 2005, 53(3): 191-204.

(28) The AnEp8 plasmid in the literature 2 was used. The AnEp8 plasmid has a sequence as shown in SEQ ID NO. 3. The AnEp8 plasmid was extracted using plasmid extraction kit available from Shanghai Sangon Biotech. The AnEp8 plasmid and purified PCR product of hygromycin B resistance gene were double-digested by Fastdigest restriction endonucleases NheI and AscI from Fermentas, subjected to agarose gel electrophoresis, and extracted (by using Thermo GeneJET Gel Extraction and DNA Cleanup Micro Kit). The hygromycin B gene was ligated to the AnEp8 plasmid by using T4 DNA ligase (product of Fermentas). The enzymatic cleavage and ligation operations were carried out strictly according to manufacturer's instruction. Subsequently, Escherichia coli transformation was performed, to construct a recombinant plasmid Anep8-hygro. The recombinant plasmid Anep8-hygro was subjected to Sanger sequencing and after confirmed by double-digestion, the Escherichia coli cells were mass cultured, and the recombinant plasmid was extracted by using a plasmid extraction kit (EndoFree Plasmid Maxi Kit) from Qiagen (according to manufacturer's instruction).

Example 2

(29) A method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators includes the following steps:

(30) 1) Culture of Aspergillus niger and Collection of Spores

(31) In a 15-cm Petri dish, a solid agar medium (YPD medium) was prepared. Aspergillus niger CBS 513.88 was inoculated onto the surface of the solid agar medium and cultured at a temperature of 16 C. with 15-50% humidity for 15 days, to allow Aspergillus niger spores to overgrow on the surface of the medium (where the spores appeared dark brown and was easy to visually identified).

(32) Sterilized water was poured onto the surface of the medium, to wash down the Aspergillus niger spores off from the surface of the medium (by vibrating, or gently scratching with a sterilized smooth glass spreading rod). The spore suspension was aspirated with a pipette and filtered using sterilized lens paper (or fritted glass filter, filter paper, etc.) to remove the mycelia and retain the spores. The filtered liquid was placed in a centrifuge tube and centrifuged to collect the pelleted resting spores, and the supernatant was discarded. The collected spores were subjected to chromosome staining, to observe and confirm that the chromosomes in the spores are haploid.

(33) 2) Pretreatment of Aspergillus niger Spores

(34) The spore pellets were re-suspended in an electroporation buffer (where the volume of the electroporation buffer added should fill up the centrifuge tube), centrifuged again to collect the spore pellets, and the supernatant was discarded. After repeating the above steps twice, the spores were re-suspended in the electroporation buffer again, and observed under a microscope, to confirm that the spore suspension was free of contamination with mycelia and the spores were non-germinated. When the spores were finally re-suspended in the electroporation buffer, the volume of the electroporation buffer was controlled to maintain a spore concentration in the Aspergillus niger spore suspension of 10 spores/ml.

(35) The electroporation buffer consisted of HEPES having a final concentration of 0.01 mmol/L and mannitol having a final concentration of 0.5 mmol/L, and the pH of the electroporation buffer was 3.0.

(36) 3) Electroporation of the Aspergillus niger Spores by Using HDEN Method

(37) 6 l of the Aspergillus niger spore suspension and 0.1 g of recombinant plasmid AnEp8-hygro were added to the wells of a 96-well cell culture plate and mixed evenly, to obtain a mixture of the spores and the plasmid. The cell culture plate was placed on an ice bath for 15 min, electroporation was carried out subsequently 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 was placed on the ice bath again for 15 min after electroporation, and subsequently the mixture of the spores and the plasmid was aspirated off, to obtain resting spores of Aspergillus niger with introduction of exogenous DNA.

(38) In this example, the parameters for electroporation include: a voltage of 1 V, pulse duration of 2000000 ms, and repeat for 100 times at an interval of 5 ms.

(39) 4) Confirmatory Experiment

(40) The aspirated mixture of the spores and the plasmid was coated onto a plate containing YPD solid agar medium with a final concentration of hygromycin B of 200 g/ml, and cultured at a temperature of 16 C. with 15-50% humidity until single colonies were formed. The colonies were counted and the transformation efficiency was calculated.

(41) At the same time when the above experimental steps were performed, a control group was prepared (as described in Example 1).

(42) When single colonies were formed in the experimental group, DNA was extracted from the single colonies in the experimental group, and the positive clone in this example was confirmed as a successful transformant by the method as described in Example 1.

(43) The volume of the AnEp8-hygro plasmid in this example was 12.4 kb, and 0.1 g plasmid (about 12.426 fmol plasmid molecules) was used, with which 610.sup.8 non-germinated resting spores of Aspergillus niger were electroporated, to produce no less than 6000 positive transformants.

(44) The construction method of the recombinant plasmid AnEp8-hygro in this example was the same as that in Example 1.

Example 3

(45) A method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators includes the following steps:

(46) 1) Culture of Aspergillus niger and Collection of Spores

(47) In a 15-cm Petri dish, a solid agar medium (PDA medium) was prepared. Aspergillus niger CBS 513.88 was inoculated onto the surface of the solid agar medium and cultured at a temperature of 40 C. with 60-85% humidity for 3 days, to allow Aspergillus niger spores to overgrow on the surface of the medium (where the spores appeared dark brown and was easily to visually identified).

(48) Sterilized water was poured onto the surface of the medium, to wash down the Aspergillus niger spores off from the surface of the medium (by vibrating, or gently scratching with a sterilized smooth glass spreading rod). The spore suspension was aspirated with a pipette and filtered using sterilized lens paper (or flitted glass filter, filter paper, etc.) to remove the mycelia and retain the spores. The filtered liquid was placed in a centrifuge tube and centrifuged to collect the pelleted resting spores, and the supernatant was discarded. The collected spores were subjected to chromosome staining, to observe and confirm that the chromosomes in the spores are haploid.

(49) 2) Pre-Treatment of Aspergillus niger Spores

(50) The spore pellets were re-suspended in an electroporation buffer (where the volume of the electroporation buffer added should fill up the centrifuge tube), centrifuged again to collect the spore pellets, and the supernatant was discarded. After repeating the above steps twice, the spores were re-suspended in the electroporation buffer again, and observed under a microscope, to confirm that the spore suspension was free of contamination with mycelia and the spores were non-germinated. When the spores were finally re-suspended in the electroporation buffer, the volume of the electroporation buffer was controlled to maintain a spore concentration in the Aspergillus niger spore suspension of 10.sup.4 spores/ml.

(51) The electroporation buffer consisted of HEPES having a final concentration of 100 mmol/L and mannitol having a final concentration of 5000 mmol/L, and the pH of the electroporation buffer was 7.0.

(52) 3) Electroporation of the Aspergillus niger Spores by Using HDEN Method

(53) 600000 l of the Aspergillus niger spore suspension and 10000 g of recombinant plasmid AnEp8-hygro were added to wells of a 96-well cell culture plate and mixed evenly, to obtain a mixture of the spores and the plasmid. The cell culture plate was placed on an ice bath for 10 min, electroporation was carried out subsequently 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 was placed on the ice bath again for 10 min after electroporation, and subsequently the mixture of the spores and the plasmid was aspirated off, to obtain resting spores of Aspergillus niger with introduction of exogenous DNA.

(54) In this example, the parameters for electroporation include: a voltage of 6000 V, pulse duration of 2 ms, and repeat for 1 time at an interval of 50000 ms.

(55) 4) Confirmatory Experiment

(56) The aspirated mixture of the spores and the plasmid was coated onto a plate containing YPD solid agar medium with a final concentration of hygromycin B of 200 g/ml, and cultured at a temperature of 40 C. with 60-85% humidity until single colonies were formed. The colonies were counted and the transformation efficiency was calculated.

(57) At the same time when the above experimental steps were performed, a control group was prepared (as described in Example 1).

(58) When single colonies were formed in the experimental group, DNA was extracted from the single colonies in the experimental group, and the positive clone in this example was confirmed as a successful transformant by the method as described in Example 1.

(59) The volume of the AnEp8-hygro plasmid in this example was 12.4 kb, and 10000 g plasmid (about 1242600 fmol plasmid molecules) was used, with which 610.sup.6 non-germinated resting spores of Aspergillus niger were electroporated, to produce no less than 7000 positive transformants.

(60) The construction method of the recombinant plasmid AnEp8-hygro in this example was the same as that in Example 1.

Example 4

(61) In this example, the parameters for electroporation were the same as those in the Example 1 except that the voltage was 30 V, the pulse duration was 1000000 ms, electroporated for 50 times at an interval of 5000 ms, and no less than 6500 positive transformants were produced.

Example 5

(62) In this example, the parameters for electroporation were the same as those in Example 1 except that the voltage was 3000 V, the pulse duration was 100 ms, electroporated for 5 times at an interval of 25000 ms, and no less than 7200 positive transformants were produced.

(63) The method for introducing exogenous DNA into resting spores of Aspergillus niger provided in the present invention can be used to introduce not only the recombinant plasmid AnEp8-hygro, but also any other plasmids into resting spores of Aspergillus niger.