Method for rapid high-throughput extraction of fungal genome DNA by using glass bead grinding

Abstract

The invention discloses a method for rapid high-flux extraction of fungal genome DNA by glass bead grinding, and belongs to the technical field of molecular biology. According to the method, a CTAB method (cetyltrimethylammonium bromide method) is adopted, the required reagent is few, and expensive enzymes and other biochemical reagents do not need to be used. The method for extracting the DNA is low in cost, the whole method is easy to operate, the DNA extraction flux is high, and the needed time is short. The obtained genome DNA can be widely applied to experiments such as gene cloning, genotype detection and the like which require large-scale genome extraction.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for rapidly and high-throughput extraction of fungal genomic DNA suitable for PCR amplification by grinding with glass beads. Background Art

[0002] Fungi are widely distributed in nature, with many species. Fungi vary greatly in morphology. Some are so small that they can only be seen in single-cell form under a microscope, such as yeast colonies; some are so large that they can be seen with the naked eye, such as the fruiting bodies of mushrooms such as Ganoderma lucidum. Some metabolites of fungi are widely used in industry and are produced on a large scale, such as ethanol, citric acid, glycerol, enzyme preparations, sterols, fats, growth hormones, vitamins, etc. Fungi also play an important role in the transformation of substances in nature. It can degrade various organic matter in the environment into simple complexes and inorganic small molecules, allowing many important chemical elements to be recycled. In addition, fungi are also important research tools for basic biological research. However, fungi also have a harmful side to humans. They are pathogenic fungi that parasitize on the surface of animals and plants and cause animal and plant diseases. Animal pathogenic fungi can often cause diseases in humans, livestock and insects. Many fungi produce toxins in food and grains. For example, acute aflatoxin poisoning can cause liver necrosis; chronic aflatoxin poisoning can cause liver cancer. These have brought great harm and losses to humans. In recent years, both at home and abroad, more and more attention has been paid to the genetic resources of fungi, and all countries have increased their research investment. In the agricultural field, fungal pathogenic genes that interact with hosts are excavated and cloned from important food crops such as rice, wheat, corn, and sorghum, and research on anti-fungal diseases is conducted to ultimately obtain new disease-resistant varieties; in the medical field, pathogenic genes are found in pathogenic fungi to better serve human health.

[0003] Functional studies of fungal genomic DNA have already begun, but the premise of these studies is the extraction of high-quality fungal genomic DNA. Because fungi have special cell structures, some fungi are multinucleated cells rich in secondary metabolites such as polysaccharides and proteins. Conventional traditional methods use liquid nitrogen grinding to destroy cell walls, and there are also methods such as liquid nitrogen combined with proteinase K and snail enzyme enzymatic destruction of cell walls. These methods reported at home and abroad currently have cumbersome experimental steps and long extraction time, which cannot meet the needs of current high-throughput fungal genome research. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to provide a simple, safe, high-throughput and rapid method for extracting fungal genomic DNA.

[0005] The technical solution of the present invention is: a method for rapid high-throughput extraction of fungal genomic DNA using glass beads, comprising the following steps:

[0006] (1) Place the fungal sample to be extracted in a container, add glass beads and preheated CTAB extraction solution, vortex for 20 seconds, and then place the container in a grinder and grind at 60 Hz for 35 seconds;

[0007] (2) adding an equal amount of preheated CTAB extract as in step (1) to the product after treatment in step (1), mixing the mixture, and keeping the mixture at 65° C. for 10 min;

[0008] (3) adding a pre-cooled mixture of chloroform and isoamyl alcohol to the product after treatment in step (2), mixing, centrifuging at 10,000 rpm for 10 min, and collecting the supernatant;

[0009] (4) adding RNase A solution to the supernatant obtained in step (3) and mixing, vortexing for 10 seconds and then standing at room temperature for 10 minutes, then adding a mixture of chloroform and isoamyl alcohol, centrifuging at 10000 rpm for 10 minutes, and collecting the supernatant;

[0010] (5) adding an equal volume of isopropanol to the supernatant obtained in step (4) and mixing, centrifuging at 10,000 rpm for 5 min, and discarding the supernatant;

[0011] (6) Add 75% ethanol to the precipitate of step (5) to wash the precipitate, centrifuge at 10,000 rpm for 5 min, and discard the supernatant; repeat this step once;

[0012] (7) Add ddH2O to the precipitate treated in step (6), vortex for 10 seconds, and store at low temperature.

[0013] Furthermore, the amount of glass beads added is 0.3 g, and the diameter of the glass beads is 0.2-0.4 mm.

[0014] Furthermore, the CTAB extract comprises, by volume, 50 parts of 1xCTAB, 50 parts of ddH2O, and 1 part of 2% β-mercaptoethanol.

[0015] Furthermore, the composition of the 1xCTAB is: 10 g CTAB, 40.9 g NaCl, 50 mL 1 mol / L Tris-HCl with a pH of 8, 20 mL 0.5 mol / L EDTA, and the volume is adjusted to 500 ml with water.

[0016] Furthermore, in steps (1) and (2), the preheating temperature is 65°C.

[0017] Furthermore, in step (3), the precooling temperature is 4°C.

[0018] Furthermore, in step (3), in the mixed solution of chloroform and isoamyl alcohol, the volume ratio of chloroform to isoamyl alcohol is 24:1.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) It eliminates the complicated procedure of grinding with special reagent liquid nitrogen in the existing method.

[0021] (2) It avoids the use of toxic carcinogenic reagents such as phenol in existing methods.

[0022] (3) This method only takes 1 hour, shortening the extraction time by 2 hours, greatly speeding up the experiment and achieving high-throughput rapid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis diagrams are of different genomic DNAs extracted respectively by the method of the present invention; M represents Marker (DNA sizes from top to bottom are 15000 bp, 10000 bp, 7500 bp, 5000 bp, 2500 bp, 2000 bp, and 1000 bp, respectively), 1 is genomic DNA of Rhizoctonia solani, 2 is genomic DNA of Pichia pastoris, 3 is genomic DNA of Soil mold, 4 is genomic DNA of Colletotrichum kaoliang, 5 is genomic DNA of Puccinia strepsirrhiza, and 6 is genomic DNA of Blumeria graminis.

[0024] Figure 2 The electrophoresis diagram is of PCR amplification of fungal ribosomal 5.8SrDNA using different genomic DNAs extracted respectively by the method of the present invention; M represents DL 2000 Marker (DNA sizes from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 200 bp, and 100 bp, respectively), 1 is DNA amplified using genomic DNA of Rhizoctonia solani as a template, 2 is DNA amplified using genomic DNA of Pichia pastoris as a template, 3 is DNA amplified using genomic DNA of Soil mold as a template, 4 is DNA amplified using genomic DNA of Colletotrichum sorghum as a template, 5 is DNA amplified using genomic DNA of Puccinia strepsirrhizae as a template, and 6 is DNA amplified using genomic DNA of Blumeria graminis as a template. DETAILED DESCRIPTION

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0026] Example 1 Extraction of fungal genome

[0027] 1. Experimental materials, reagents (preparation) and instruments and equipment:

[0028] Materials: Collect the fungi to be extracted in 2 mL centrifuge tubes (Ustilaginoidea virens, Pichia pastoris, Stachybotrys chartarum, Colletotrichum graminicola, Puccinia striiformis West. f. sp. Tritici, Blumeria graminis f. sp. tritici)

[0029] Experimental medicine:

[0030] 1xCTAB, 2% β-mercaptoethanol, isopropanol, chloroform·isoamyl alcohol, 10 mg / mL RNase A solution, 75% alcohol, ddH2O.

[0031] (1) CTAB extraction solution (prepared and used immediately): 1xCTAB + ddH2O + 2% β-mercaptoethanol (volume ratio 50:50:1)

[0032] (2) Chloroform and isoamyl alcohol: chloroform + isoamyl alcohol (volume ratio 24:1)

[0033] (3) 1xCTAB: CTAB 10 g, NaCl 40.9 g, 1 mol / L Tris-HCl 50 mL (PH = 8.0), 0.5 mol / L ETA 20 mL, dilute to 500 ml with water

[0034] Instruments and equipment: sterile blades, 2mL sterile EP tubes, 0.2-0.4mm transparent glass beads, pipette gun, grinder, vortex mixer, water bath, centrifuge, ice box, 4℃ refrigerator, paper towels

[0035] 2. Experimental steps:

[0036] (1) Preheat the CTAB extraction solution to 65°C, precool the isopropanol to 4°C, weigh 0.3 g of 0.2-0.4 mm transparent glass beads and pour them into the EP tube containing the fungus.

[0037] (2) Add 400 μL of preheated CTAB extraction solution to the EP tube containing the fungal colonies to be extracted, vortex for 20 seconds, and then place it in a grinder and grind at 60 Hz for 35 seconds.

[0038] (3) After taking out, add 400 μL of preheated CTAB extract, invert to mix, and place in a 65°C water bath for 10 min.

[0039] (4) Add 560 μL of chloroform / isoamyl alcohol (precooled at 4°C) and mix thoroughly by inversion. Centrifuge at 10,000 rpm for 10 min and transfer about 600 μL of the supernatant to a new EP tube.

[0040] (5) Add 9 μL of RNase A solution, mix thoroughly by inversion and vortex for 10 seconds, and leave at room temperature for 10 minutes. Add 300 μL of chloroform / isoamyl alcohol, and centrifuge at 10,000 rpm for 10 minutes.

[0041] (6) Pipette 400 μL of supernatant into the corresponding EP tube, add 400 μL of isopropanol (isopropanol: supernatant = 1:1), invert and mix until white flocs appear in the tube. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.

[0042] (7) Add 1 mL of 75% alcohol to wash, invert several times, centrifuge at 10,000 rpm for 5 min, discard the supernatant, and repeat twice.

[0043] (8) Add 80 μL ddH2O and vortex for 10 seconds to ensure that the precipitate is fully dissolved.

[0044] The genome quality was checked by 1% agarose gel electrophoresis. Figure 1 shown.

[0045] The extracted fungal genomic DNA was detected by ultraviolet spectrophotometry. The extracted DNA samples were measured using a NanoDrop 2000 UV-visible spectrophotometer (Nano-Drop Technologies, USA), and the OD values ​​of the samples were measured at 230 nm, 260 nm, and 280 nm, respectively. The results are shown in Table 1

[0046] Table 1 Purity and concentration of DNA extracted by the method of the present invention

[0047]

[0048] In Table 1: 1 and 2 are genomic DNAs of rice green sclerotium fungus, rice Aspergillus oryzae; 3 and 4 are genomic DNAs of Pichia pastoris; 5 and 6 are genomic DNAs of black mold; 7 and 8 are genomic DNAs of sorghum anthracnose; 9 and 10 are genomic DNAs of wheat stripe rust; 11 and 12 are genomic DNAs of wheat powdery mildew.

[0049] Example 2 Amplification of fungal genomes

[0050] 1. Experimental materials, reagents (preparation) and instruments

[0051] Materials: Genomic DNA extracted from Example 1

[0052] Experimental reagents: H2O (DI), 0.01mol / L ITS1, 0.01mol / L ITS4, PCR mix (2X M5 HiPerplus Taq HiFI)

[0053] Instruments and equipment: sterilized PCR tubes, 7200r / min centrifuge (PCR tubes), vortex mixer, pipette gun, PCR instrument 2. Experimental steps:

[0054] (1) Take a sterilized PCR tube and add 6ul H2O (DI), 1ul ITS1 primer (TCC GTA GGT GAA CCTGCG G), 1ul ITS4 primer (TCC TCC GCT TAT TGA TAT GC), and 10ul PCR mix in sequence. Vortex for 10 seconds.

[0055] (2) Add 1ul genomic DNA and vortex for 10 seconds

[0056] (3) Place in a centrifuge and centrifuge for 10 seconds, then place in a PCR instrument

[0057] (4) Set the PCR instrument program: pre-denaturation at 95°C for 3 min, 30 cycles of (denaturation at 94°C for 25 s, annealing at 61°C for 25 s, and extension at 72°C for 30 s), and then extension at 72°C for 5 min. Set the buffer temperature to 4°C and run the PCR instrument.

[0058] (5) Use 1% agarose gel electrophoresis to detect the quality of the DNA fragment after PCR amplification. The results are as follows Figure 2 shown.

Claims

1. A method for rapid high-throughput extraction of fungal genomic DNA using glass bead grinding, characterized in that: The following steps are involved: (1) Place the fungal sample to be extracted in a container, add glass beads and preheated CTAB extraction solution, vortex for 20 seconds, and then place the container in a grinder and grind at 60 Hz for 35 seconds; (2) adding an equal amount of preheated CTAB extract as in step (1) to the product after treatment in step (1), mixing the mixture, and keeping the mixture at 65° C. for 10 min; (3) adding a pre-cooled mixture of chloroform and isoamyl alcohol to the product after treatment in step (2), mixing, centrifuging at 10,000 rpm for 10 min, and collecting the supernatant; (4) adding RNase A solution to the supernatant obtained in step (3) and mixing, vortexing for 10 seconds and leaving at room temperature for 10 minutes, then adding a mixture of chloroform and isoamyl alcohol, centrifuging at 10,000 rpm for 10 minutes, and collecting the supernatant; (5) adding an equal volume of isopropanol to the supernatant obtained in step (4) and mixing, centrifuging at 10,000 rpm for 5 min, and discarding the supernatant; (6) Add 75% ethanol to the precipitate of step (5) to wash the precipitate, centrifuge at 10,000 rpm for 5 min, and discard the supernatant; repeat this step once; (7) Add ddH2O to the precipitate treated in step (6), vortex for 10 seconds, and store at low temperature.

2. The method according to claim 1, characterized in that The amount of glass beads added is 0.3 g, and the diameter of the glass beads is 0.2-0.4 mm.

3. The method according to claim 1, characterized in that The CTAB extraction solution is composed of: 50 parts of 1xCTAB, 50 parts of ddH2O, and 1 part of 2% β-mercaptoethanol by volume.

4. The method according to claim 3, characterized in that The composition of the 1xCTAB is: 10 g CTAB, 40.9 g NaCl, 50 mL 1 mol / L Tris-HCl with a pH of 8, 20 mL 0.5 mol / L EDTA, and the volume is adjusted to 500 ml with water.

5. The method according to claim 1, characterized in that In steps (1) and (2), the preheating temperature is 65°C.

6. The method according to claim 1, characterized in that In step (3), the precooling temperature is 4°C.

7. The method according to claim 1, characterized in that In step (3), in the mixed solution of chloroform and isoamyl alcohol, the volume ratio of chloroform to isoamyl alcohol is 24:1.

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