CN102978233B - Rhizopus nigricans hypha liposome direct transformation method - Google Patents

Abstract

The invention relates to a direct transformation method of Rhizopus niger mycelium liposome, which comprises the following steps: 1) Transformation of Rhizopus niger mycelium liposome: 1) Mixing young Rhizopus niger wet mycelium with mannitol aqueous solution and then grinding , to obtain the bacterial suspension, and set aside; 2) Mix liposome 2000 and plasmid pEGFP-C1, place it at -5-5°C for 15-60min, then transfer it to the bacterial suspension, mix it, and store it at -5-5°C Place at 5°C for 15-60 minutes; 3) Spread the mixture obtained in step 2) on a PDA plate, and incubate at 25-30°C for 1-5 days; 2) Subculture: 4) Pick the fluorescent cells in step 3) and detect them with a fluorescence microscope Transformants were inoculated on PDA plates and cultured at 25-30°C for 1-5 days, and subcultured for 5-8 times. The method is simple and quick, has high conversion rate, and the transformants are genetically stable, and does not need to prepare protoplasts or complex devices.

Description

A kind of rhizopus niger mycelia liposome direct transformation method

technical field

The invention belongs to the technical field of liposome transformation, and in particular relates to a direct transformation method of rhizopus niger hyphae liposome.

Background technique

At present, the genetic transformation methods of filamentous fungi mainly include protoplast electroporation, biolistic transformation, lithium acetate-mediated transformation, Agrobacterium-mediated transformation, PEG-mediated protoplast transformation and protoplast liposome transformation. law etc. These methods generally have disadvantages such as cumbersome operation, complicated equipment, low conversion rate, and unstable transformants. If some need to prepare protoplasts, the preparation process is complicated, including protoplast electroporation, PEG-mediated protoplast transformation and protoplast liposome transformation. Some methods do not require the preparation of protoplasts, but the equipment is complicated and the transformation cost is high, such as the gene gun transformation method. Some are limited to the transformation of a few types of filamentous fungi, such as lithium acetate-mediated transformation. Some transformation rates are unstable, and the transformation efficiency is affected by many factors. For example, the transformation efficiency of Agrobacterium transformation method is affected by many factors such as the type of Agrobacterium strain, the type of plasmid vector and the matching between the two, the composition of the medium, and the induction conditions. Impact.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a direct transformation method of Rhizopus niger mycelium liposome, which is simple, fast, high transformation rate, genetically stable transformants, and does not require the preparation of protoplasts and complex devices.

To achieve the above object, the present invention adopts the following technical solutions:

A kind of rhizopus niger mycelia liposome direct conversion method, it comprises the following steps:

a) Liposome transformation of Rhizopus niger hyphae:

1) Mix the wet hyphae of young Rhizopus nigricans with mannitol aqueous solution and grind to obtain a bacterial suspension for later use;

2) Mix liposome 2000 and plasmid pEGFP-C1, place it at -5-5°C for 15-60min, then transfer it into the bacterial suspension, mix well, and place it at -5-5°C for 15-60min;

3) Spread the mixture obtained in step 2) on a PDA plate, and incubate at 25-30°C for 1-5 days;

2) Subculture:

4) Picking step 3) The transformant with fluorescence detected by fluorescence microscope was inoculated on a PDA plate and cultured at 25-30°C for 1-5 days, and subcultured for 5-8 times in total.

Preferably, the step 1) specifically includes: mixing 0.05-0.2 g of young Rhizopus niger wet mycelium with 0.5-2 ml of mannitol aqueous solution with a concentration of 0.5-1.0 mol/l.

The step 2) is specifically: take 30-100 μl liposome 2000 and mix with 30-100 μl plasmid pEGFP-C1.

Compared with the prior art, the rhizopus niger hyphae liposome direct transformation method of the present invention has the advantages of simplicity, quickness, high transformation rate, genetically stable transformants, no need to prepare protoplasts, no need for complex devices and the like. Using the green fluorescent protein gene as a selection marker can directly observe the living body without adding other selective drugs such as antibiotics, and can be easily used to monitor the elimination of the selection marker gene in the transformant.

Description of drawings

Figure 1 is the fluorescent detection of Rhizopus niger liposome-transformed GFP gene (magnification 10×20);

Fig. 2 is the total DNA electrophoresis figure of Rhizopus niger transformant; G1, G2, G3, G4, G5, G6 are the mycelia DNA of six generations of Rhizopus niger spore subculture after transformation, M is hind-Ⅲ, and total genome is about 20kbp;

Fig. 3 is the electrophoresis figure of PCR detection GFP gene in the total DNA of Rhizopus niger transformants; G1, G2, G3, G4, G5, G6 are the total genome PCR amplification GFP gene electrophoresis figures of six generations of Rhizopus niger spore subculture mycelia after transformation , M is 1kb plus;

Fig. 4 is the result of southern hybridization of the Rhizopus niger transformant; CK is the untransformed Rhizopus niger mycelium genome, and G1, G2, G3, G4, G5, G6 are the six generations of Rhizopus niger spore subculture mycelium genomes after transformation through Sac I single Southern hybridization diagram after enzyme digestion.

Detailed ways

The present invention will be further described by the following examples, but the protection scope of the present invention is not limited thereto.

Example 1

1. Experimental materials

1. Strains and plasmids Rhizopus stolonifer (Rhizopus niger) Rhizopus stolonifer

Rhizopus stolonifer (Rhizopus stolonifer) was purchased from China General Microorganisms Collection and Management Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3.4098. The plasmid pEGFP-C1 was donated by the Cell Biology Laboratory of Zhengzhou University.

2. culture medium

PDA medium (1L): 200g of potatoes (peeled and cut into pieces, boiled in water for 15 minutes, and filtered with four layers of gauze to obtain the filtrate), 20g of agar (Agar), 20g of glucose, and 1000ml of water, aliquoted and autoclaved.

3. Liposomes

Lipofectamine 2000, Invitrogen, Carlsbad, California, USA.

2. Experimental method

A kind of rhizopus niger mycelia liposome direct conversion method, it comprises the following steps:

a) Liposome transformation of Rhizopus niger hyphae:

1) Take 0.1g of young Rhizopus nigricans wet mycelia in a sterilized mortar, add 1ml of mannitol aqueous solution with a concentration of 0.6mol/l, mix and grind to obtain a bacterial suspension, and transfer it to a 1.5ml centrifuge tube in, spare;

2) Mix 50 μl liposome 2000 with 50 μl plasmid pEGFP-C1, place at 0°C for 30 minutes, then transfer to the bacterial suspension, mix well, and place at 0°C for 30 minutes to obtain a mixture;

3) Smear 100 μl of the mixture obtained in step 2) on a PDA plate (10 plates in total), and incubate at 28°C for 2 days;

2) Subculture:

4) Picking step 3) Transformants with fluorescence detected by fluorescence microscope were inoculated on PDA plates and cultured at 28°C for 3 days, and subcultured for 6 times in total.

PCR detection of transformants

(1) Primer design

 Specific primers designed according to the EGFP sequence of the target gene in pEGFP-C1:

GFP-S atggtgagcaagggcgaggagc

GFP-X ttatctagatccggtggatccc

(2) Extract the total DNA of Rhizopus niger transformants by CTAB method

(3) PCR amplification of the target gene (ie fluorescent protein gene - GFP gene)

 Using the transformed Rhizopus niger genome as a template, the PCR reaction system is as follows, with a total volume of 25 microliters:

DNA 1 μl

dNTP mixture 4 μl

Upstream primer GFP-S (25mM) 0.5μl

Downstream primer GFP-X (25mM) 0.5μl

10×LA PCR Buffer (Mg2+Plus) 2.5μl

TaLaRa LA Taq(5u/ul) 0.5μl

ddH ₂ O 16μl

The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min.

After the PCR reaction, take 5 μl of the PCR reaction solution and use 1.0% agarose gel for electrophoresis detection (results shown in Figures 2 and 3).

hybridization detection

(1) Recovery and purification of PCR products

1) Electrophoresis the PCR product in 1% agarose gel, and use the AxyPrep DNA gel extraction kit to recover the target gene. The specific operation steps are as follows:

2) Cut off the agarose gel containing the target DNA under ultraviolet light, absorb the liquid on the surface of the gel with a paper towel and chop it up. Calculate the weight of the gel (record the weight of the 1.5 ml centrifuge tube in advance), and use this weight as a gel volume (eg 100 mg = 100 μl volume)

3) Add 3 gel volumes of Buffer DE-A, mix well and heat at 75°C with intermittent mixing (every 2-3 min) until the gel block is completely melted (about 6-8 min).

4) Add 0.5 Buffer DE-A volume of Buffer DE-B and mix well; when the separated DNA fragment is less than 400bp, add 1 gel volume of isopropanol.

5) Pipette the mixture in 3, transfer it to a DNA preparation tube (placed in a 2 ml centrifuge tube), and centrifuge at 12000g for 1 min. Discard the filtrate.

6) Put the preparation tube back into the centrifuge tube, add 0.5 ml Buffer W1, centrifuge at 12000g for 30 s, and discard the filtrate.

7) Put the preparation tube back into the centrifuge tube, add 0.7 ml Buffer W2, centrifuge at 12000g for 30 s, discard the filtrate, wash once with 0.7 ml Buffer W2 and centrifuge at 12000g for 1 min in the same way.

8) Place the preparation tube in a 2 ml centrifuge tube and centrifuge at 12000g for 1 min.

9) Place the preparation tube in a clean 1.5 ml centrifuge tube, add 25-30 μl of water or Eluent to the center of the DNA preparation membrane, and let stand at room temperature for 1 min. Centrifuge at 12000g for 1 min to elute DNA.

10) Take an appropriate amount of electrophoresis to detect the recovery efficiency.

(2) Southern hybridization detection

Use high-efficiency DNA digoxin labeling and detection kit I (DIG-high prime DNA labeling and detection Starter kit I, purchased from Roche, USA), and the operation steps are as follows (refer to the kit manual for details):

1) Extract Rhizopus genomic DNA by CTAB method, digest 5-10 μg Rhizopus genomic DNA with Sac I, divide 50 μl system into 5 tubes, digest overnight at 37°C for 12 hours;

2) Precipitate with ethanol, concentrate the DNA, and after complete digestion, combine 5 tubes of digestion solution and constant volume, add 1/5 volume of 5M NaCl (50μl), 2 times the volume of absolute ethanol (500μl); after settling at -20℃ , centrifuged to remove the supernatant, washed once with 70% ethanol, and left at room temperature for several minutes. After the alcohol evaporated, add 25 μl of sterile water to dissolve. The concentration was detected by NanoDrop ND1000 UV spectrophotometer, and the DNA concentration was controlled to be about 0.5 μg/μl;

3) Electrophoresis: add 10 μl of each sample, 1.0% agarose gel, voltage 60 volts, electrophoresis for 4 hours, and take pictures;

4) DNA denaturation: Cut off excess gel and mark a corner, soak the gel in alkaline buffer for 15 minutes, and shake gently; change the medium once, and continue soaking for 20 minutes;

5) Nylon membrane preparation: cut a positively charged nylon membrane, each side is 1mm larger than the gel, mark the same corner as the gel, and soak in the transfer buffer for 5 minutes;

6) Upward capillary transfer membrane blotting: put a support in a glass dish, place the filter paper on the surface of the support, the ends of the filter paper droop, pour alkaline transfer buffer, and the liquid level is flush with the support; The gel is turned over and placed on the filter paper with the bottom face up to ensure that there are no air bubbles between the gel and the filter paper; place the positively charged nylon membrane on the gel so that the two marks overlap, then put in two wet filter papers and a stack of absorbent paper , thickness 5-8cm, then put in about 400g weight, transfer for 16h, remove the nylon membrane;

7) DNA fixation: immerse the nylon membrane in the neutralization buffer, place it at room temperature for 15 minutes, take it out and put it on a dry filter paper, fix the DNA with ultraviolet crosslinking method, and irradiate with ultraviolet light for 5 minutes;

8) Probe preparation: The probe is the PCR product obtained by using the plasmid pEGFP-C1 as a template and using the probe primers GFP-S atggtgagcaagggcgaggagc and GFP-X ttatctagatccggtggatccc. After recovery, it is boiled in boiling water for 10 minutes, cooled rapidly, and centrifuged. 4 μl, add 1 μl DIG labeling enzyme mixture according to the instructions of the DIG High Prime DNA Labeling and Detection Starter Kit, mix and store at 37°C overnight (16h), and put it at -20°C for later use after labeling.

9) Hybridization

Pre-hybridization: Soak the DNA-bound nylon membrane in 6×SSC solution for 2 minutes; put the membrane into the hybridization bag, add the kit’s pre-hybridization solution DIG EasyHyb, squeeze out the air, seal it, and bathe in water at 42°C for 1 hour;

Probe preparation: Take 2 μl of probe, add it to a PCR tube containing about 100 μl of hybridization solution DIG EasyHyb, close the lid, prick the hole with a needle on the top of the lid, heat in boiling water for 5 minutes, quickly put it on ice to cool, and then add an appropriate amount Preheated hybridization solution;

Hybridization: Take out the hybridization bag from the water bath, pour out the pre-hybridization solution; add the denatured DNA probes to an appropriate amount of pre-hybridization solution DIG EasyHyb, and transfer the solution into the hybridization bag, squeeze out the air, seal it, and set it at 42 ℃ water bath shaker (hybridization oven) overnight;

Membrane washing: Take out the hybridization membrane from the hybridization bag, wash twice with a solution containing 2×SSC and 0.1% SDS at room temperature, each time for 5 minutes, then wash twice with a solution containing 0.5×SSC and 0.1% SDS at 65°C , 15 minutes each time. Rinse the membrane with washing solution for 3-5 minutes;

Blocking: Incubate with 10mL Blocking Solution of the kit at room temperature for 30min, then incubate with enzyme-linked antibody solution anti-DIG-AP solution for 30min at room temperature, then wash twice with washing solution for 15min each time, and then in the detection solution Balance 2-5min;

10) Color development: add NBT/BCIP color development solution to the front of the membrane, and develop color in the dark. After the hybridization signal is clear, transfer the membrane to TE (or deionized water) and soak for 5 minutes to terminate the reaction;

11) Picture scanning and storage: scan the picture with a scanner, put the membrane on filter paper to dry naturally after scanning, and store it in a sealed bag.

3. Experimental results

1. Fluorescent detection of transformants

 The hyphae were picked and observed under a fluorescent microscope. The control mycelia had no fluorescence under ultraviolet excitation, while the transformant transfected with the GFP gene through liposomes produced strong green fluorescence after ultraviolet excitation (see Figure 1). The average number of transformants was 122, and the conversion rate was 8.19/μg.

2 PCR detection of transformants

A transformant was randomly selected, subcultured by 6 generations of spores, fluorescence could be detected under a fluorescent microscope, the total DNA of mycelia was extracted (see Figure 2), and PCR detection was performed with fluorescent protein gene primers GFP-S and GFP-X. The fluorescent protein gene can be detected in 6 passages (see Figure 3).

3. Southern hybridization results

The total DNA of the extracted 6th generation spore subcultured mycelium was purified and then subjected to Southern hybridization. The results showed that after liposome transformation, the GFP gene was integrated into the Rhizopus niger genome, with only one copy number, and it was stably passaged (see Figure 4) .

Claims (2)

1. a direct transformation method of Rhizopus niger mycelium liposome, is characterized in that, comprises the following steps: a) Liposome transformation of Rhizopus niger hyphae: 1) Mix the wet hyphae of young Rhizopus nigricans with mannitol aqueous solution and grind to obtain a bacterial suspension for later use; 2) Mix liposome 2000 and plasmid pEGFP-C1, place it at -5-5°C for 15-60min, then transfer it into the bacterial suspension, mix well, and place it at -5-5°C for 15-60min; 3) Spread the mixture obtained in step 2) on a PDA plate, and incubate at 25-30°C for 1-5 days; 2) Subculture: 4) Picking step 3) Inoculate the fluorescent transformants detected on the PDA plate at 25-30°C for 1-5 days, and subculture for 5-8 times in total; The step 1) specifically comprises: mixing 0.05-0.2 g of young Rhizopus niger wet mycelium with 0.5-2 ml of mannitol aqueous solution with a concentration of 0.5-1.0 mol/l. 2. The method for direct transformation of Rhizopus niger hyphae liposomes according to claim 1, characterized in that the step 2) is specifically: take 30-100 μl of liposome 2000 and mix with 30-100 μl of plasmid pEGFP-C1.

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