阅读说明：本技术 快速简易提取厌氧真菌dna的方法及应用 (Method for quickly and simply extracting DNA of anaerobic fungi and application ) 是由 魏亚琴 王霄霄 苟琦敏 于 2020-12-30 设计创作，主要内容包括：本发明涉及菌株DNA的提取技术领域,具体涉及快速简易提取厌氧真菌DNA的方法及应用。所述的方法包括如下步骤：(1)菌株培养；(2)收集菌体；(3)破壁；(4)抽提DNA；(5)沉淀DNA；所述的厌氧真菌DNA提取方法,提取DNA的纯度和浓度高,且采用液氮研磨冷冻和钢珠振荡破碎厌氧真菌细胞壁,破碎过程中所需样本量较少,不易损失样本,破壁效果良好,简单易行。同时,本发明所述的抽提缓冲液配方简单,成本低廉,提取DNA的效果良好；采用本方法提取厌氧真菌的DNA操作简易,实验重复性好,能够获得高质量的DNA,对于促进厌氧真菌的研究具有重大意义。(The invention relates to the technical field of extraction of DNA of strains, in particular to a method for quickly and simply extracting DNA of anaerobic fungi and application thereof. The method comprises the following steps: (1) culturing the strain; (2) collecting thalli; (3) breaking cell wall; (4) extracting DNA; (5) precipitating DNA; the method for extracting the DNA of the anaerobic fungi has high purity and concentration of the extracted DNA, and adopts liquid nitrogen grinding and freezing and steel ball oscillation to break the cell wall of the anaerobic fungi, so that the required sample amount is less in the breaking process, the sample is not easy to lose, the wall breaking effect is good, and the method is simple and easy to implement. Meanwhile, the extraction buffer solution has the advantages of simple formula, low cost and good DNA extraction effect; the method for extracting the DNA of the anaerobic fungi has the advantages of simple operation, good experimental repeatability, capability of obtaining high-quality DNA and great significance for promoting the research of the anaerobic fungi.)

1. The method for quickly and simply extracting the DNA of the anaerobic fungi is characterized by comprising the following steps:

(1) strain culture: inoculating anaerobic fungi into an anaerobic culture medium, adding compound antibiotics, and standing for culture;

(2) and (3) collecting thalli: centrifuging the bacterial liquid obtained in the step (1) to obtain mycelia;

(3) wall breaking: grinding the mycelium obtained in the step (2) by using quick liquid nitrogen, adding 0.1-1g of the mycelium into a centrifugal tube, adding steel balls, cooling, and oscillating the centrifugal tube, wherein the centrifugal tube is kept in a frozen state during oscillation so as to break the cell wall of anaerobic fungi;

(4) extracting DNA: adding an extraction buffer solution into the solution obtained in the step (3), oscillating, adding ammonium acetate after constant-temperature water bath, uniformly mixing, cooling, centrifuging, taking supernate, and removing precipitate and steel balls; the supernatant was purified with equal volumes of Tris saturated phenol: chloroform: extracting with isoamyl alcohol solution for 2 times, mixing, centrifuging, and removing foreign protein;

(5) and (3) precipitating DNA: putting the upper-layer water phase obtained in the step (4) into another centrifugal tube, adding isopropanol, uniformly mixing, and standing at low temperature to precipitate DNA; centrifuging, removing supernatant, washing precipitate with ethanol, and dissolving precipitate with sterile double distilled water to obtain anaerobic fungus DNA.

2. The method of claim 1, wherein the anaerobic culture medium of step (1) is prepared by the following steps: yeast extract 1.0g, peptone 1.0g, glucose 1.0g, NaHCO₃7.0g, 1.0 g/L1 mL of resazurin, 1.7g of L-cysteine hydrochloride, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation at 4 ℃ for 20min, 165mL of salt solution I, 165mL of salt solution II and constant volume of distilled water to 1000 mL; preparation of salt solution IThe method comprises the following steps: NaCl 6g, (NH)₄)₂SO₄ 3g，KH₂PO₄3g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: k₂HPO₄4g, distilled water to 1000 mL.

3. The method as claimed in claim 1, wherein the amount of the anaerobic fungus inoculated in step (1) is 10% v/v and the amount of the antibiotic complex is 1% v/v, wherein the antibiotic complex is penicillin sodium and streptomycin sulfate, and the final concentrations are 1600IU/mL and 2000IU/m L, respectively.

4. The method of claim 1, wherein the rotating speed of the bacterial liquid centrifugation in the step (2) is 5000g/min, and the centrifugation time is 5 min.

5. The method of claim 1, wherein the cooling in step (3) is performed by rapidly grinding the mycelia with liquid nitrogen, placing 0.1g of the ground mycelia in a centrifuge tube, adding 2 steel balls, cooling the centrifuge tube in liquid nitrogen, and shaking the centrifuge tube for 3-5min while keeping the centrifuge tube in a frozen state.

6. The method of claim 1, wherein the extraction buffer of step (4) is prepared by the following steps: tris2.4228g, NaCl 1.461g, ethylene diamine tetraacetic acid 0.9306g, sodium dodecyl sulfate 2g, distilled water to a constant volume of 100mL, and HCl to adjust the pH value to 8.5.

7. The method of claim 1, wherein the ammonium acetate in step (4) is added at a concentration of 10M 200. mu.L, Tris saturated phenol: chloroform: the amount of isoamyl alcohol solution added is equal to the volume of the supernatant.

8. The process of claim 1 wherein the isopropanol in step (5) is added in an amount of 0.5 to 0.6 times the volume of the upper aqueous phase and the isopropanol is pre-cooled at-20 ℃.

9. The method of claim 1, wherein the sterile double distilled water of step (5) is added in an amount of 50 μ L.

10. The method of claim 1, wherein the ethanol of step (5) is added in an amount of 1mL of 75%.

Technical Field

The invention relates to the technical field of extraction of DNA of strains, in particular to a method for quickly and simply extracting DNA of anaerobic fungi and application thereof.

Background

DNA extraction of anaerobic fungi is an important task in the molecular biology research of anaerobic fungi. At present, the extraction method of fungal genome mainly comprises CTAB method, SDS extraction method, urea extraction method and enzymolysis method. The extraction steps of the above extraction method are substantially similar, and the key point is how to effectively break cell walls. At present, the commonly used method for breaking the cell wall of the anaerobic fungi comprises (1) a classical liquid nitrogen grinding method. (2) Liquid nitrogen freeze-thaw (and glass bead shaking) methods. The method uses liquid nitrogen, which increases the cost and equipment requirement; (3) and (4) performing an enzymatic hydrolysis method. Enzymes used for breaking the walls of filamentous fungi are expensive and are not economical in processing large amounts of samples; (4) chemical reagent (benzyl chloride) method. The chemical reagent has hidden trouble in environmental protection.

Most of the methods for extracting the DNA of the anaerobic fungi are liquid nitrogen grinding and breaking the cell walls of the anaerobic fungi, but the wall breaking effect is poor, or a special cell breaker is used for breaking the cell walls of the anaerobic fungi, but the cell breaker has high cost, a complex wall breaking process and low popularization rate. The most commonly used method for extracting the fungal genome by a person skilled in the art is a CTAB method, but the extraction process of the CTAB method is complicated, the variety of required reagents is various, and the extraction effect is general. In view of the above technical problems, those skilled in the art have developed a kit for extracting DNA from anaerobic fungi, which has a good extraction effect, but the kit is expensive and has limited extraction times, and cannot satisfy the requirement of researchers performing a large number of experiments in the research process or repeatedly verifying the experiment effect, and some kits need to be prepared and added with a specific reagent to improve the extraction effect during the extraction process.

During the research process, the inventor finds that anaerobic fungi contain chitin, namely chitin, and are structurally homologous polysaccharides formed by polymerizing N-acetylglucosamine through beta connection. It is insoluble in general weak inorganic acid or organic solvent, and insoluble in alkaline solution, and only soluble in strong inorganic acid. The wall breaking of the cell wall of the fungus is difficult, and the extraction effect is poor because the wall breaking can not be carried out when the skilled person extracts the DNA of the anaerobic fungus.

In order to solve the above technical problems, the inventors extracted the genomic DNA of anaerobic fungi in order to better study the genetic information of anaerobic fungi. The method is suitable for extracting the anaerobic fungus genome DNA, has the characteristics of low cost, small sample amount, quick extraction and simple and convenient operation, and solves the problem of long extraction time of the DNA sample obtained by using the conventional method.

Disclosure of Invention

The invention aims to provide a novel method for extracting anaerobic fungus DNA with low cost, small required sample amount, rapidness, simplicity, feasibility and high efficiency aiming at the defects of the existing technology for extracting the anaerobic fungus DNA.

The method for quickly and simply extracting the DNA of the anaerobic fungi comprises the following steps:

(1) strain culture: inoculating anaerobic fungi into an anaerobic culture medium, adding compound antibiotics, and standing for culture;

(2) and (3) collecting thalli: centrifuging the bacterial liquid obtained in the step (1) to obtain mycelia;

(3) wall breaking: quickly grinding the mycelium obtained in the step (2) by liquid nitrogen, adding a small amount of the mycelium into a centrifuge tube, adding steel balls, quickly cooling, and oscillating the centrifuge tube to break the cell wall of anaerobic fungi;

(4) extracting a DNA solution: adding an extraction buffer solution into the solution obtained in the step (3), oscillating, carrying out constant-temperature water bath, adding ammonium acetate, uniformly mixing, cooling, centrifuging, taking supernate, and removing precipitate and steel balls; the supernatant was purified with equal volumes of Tris saturated phenol: chloroform: extracting with isoamyl alcohol solution for 2 times, mixing, centrifuging, and removing foreign protein;

(5) and (3) precipitating DNA: putting the upper-layer water phase obtained in the step (4) into another centrifugal tube, adding isopropanol, uniformly mixing, and standing at low temperature to precipitate DNA; centrifuging, removing supernatant, washing precipitate with ethanol, and dissolving precipitate with sterile double distilled water to obtain anaerobic fungus DNA.

Preferably, the anaerobic culture medium of step (1) is prepared by the following steps: yeast extract 1.0g, peptone 1.0g, glucose 1.0g, NaHCO₃7.0g of resazurin (1.0g/L)1mL, L-cysteine hydrochloride 1.7g, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation at 4 ℃ for 20min, 165mL of salt solution I, 165mL of salt solution II and distilled water to reach the constant volume of 1000 mL; the preparation of the salt solution I is as follows: NaCl 6g, (NH)₄)₂SO₄ 3g，KH₂PO₄ 3g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: k₂HPO₄4g, distilled water to constant volume of 1000mL。

Preferably, the inoculation amount of the anaerobic fungus in the step (1) is 10% v/v, and the inoculation amount of the compound antibiotic is 1% v/v, wherein the compound antibiotic is penicillin sodium and streptomycin sulfate, and the final concentration is 1600IU/mL and 2000IU/mL respectively.

Preferably, the rotating speed of the bacterium liquid centrifugation in the step (2) is 5000g/min, and the centrifugation time is 5 min.

Preferably, the cooling in the step (3) is that liquid nitrogen quickly grinds mycelium, then a small amount of ground mycelium (about 0.1g) is placed in a centrifuge tube, 2 steel balls are added, the centrifuge tube is placed in liquid nitrogen for cooling, the centrifuge tube is vibrated, the centrifuge tube is kept in a frozen state during the vibration, and the vibration time is 3-5 min.

Preferably, the extraction buffer of step (4) is prepared by the following steps: 2.4228g of Tris (pH 8.5), 1.461g of NaCl, 0.9306g of ethylene diamine tetraacetic acid disodium salt, 2g of sodium dodecyl sulfate and 100mL of distilled water with constant volume. And the pH was adjusted to 8.5 with HCl.

Preferably, the ammonium acetate added in step (4) is 200 μ L of ammonium acetate with a concentration of 10M, Tris saturated phenol: chloroform: the amount of isoamyl alcohol solution added is equal to the volume of the supernatant. (the preparation method and volume ratio of Tris saturated phenol: chloroform: isoamyl alcohol solution are 25mL Tris saturated phenol: 24mL chloroform: 1mL isopropanol).

Preferably, the isopropanol is added in the step (5) in an amount of 0.5 to 0.6 times the volume of the upper aqueous phase, and the isopropanol is pre-cooled at-20 ℃.

Preferably, the addition amount of the sterile double distilled water in the step (5) is 50. mu.L.

Preferably, the ethanol added in the step (5) is 1mL of 75%.

The invention has the beneficial effects that: the method for extracting the DNA of the anaerobic fungi has high purity and concentration of the extracted DNA, and adopts liquid nitrogen grinding and freezing and steel ball oscillation to break the cell wall of the anaerobic fungi, so that the required sample amount in the breaking process is less, the sample is not easy to lose, the wall breaking effect is good, and the method is simple and easy to implement. Meanwhile, the extraction buffer solution has the advantages of simple formula, low cost and good DNA extraction effect. The method for extracting the DNA of the anaerobic fungi has the advantages of simple operation, good experimental repeatability, capability of obtaining high-quality DNA and great significance for promoting the research of the anaerobic fungi.

Drawings

FIG. 1 electrophoresis chart of total DNA of anaerobic fungi extracted by different methods

1. The CTAB method is commonly used; 2. extracting with plant genome kit; 3. the method of the invention; fast DNA soil kit extraction method

Detailed Description

In order to facilitate an understanding of the present invention, the present invention will be described more fully and in detail with reference to the preferred embodiments, but the scope of the present invention is not limited to the specific embodiments described below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The media used in the following examples are as follows:

the preparation steps of the anaerobic culture medium are as follows: yeast extract 1.0g, peptone 1.0g, glucose 1.0g, NaHCO₃7.0g, 1mL of resazurin (1.0g/L), 1.7g of L-cysteine hydrochloride, 8000 Xg of rumen fluid collected before morning feeding, 170mL of supernatant after centrifugation for 20min at 4 ℃, 165mL of salt solution I, 165mL of salt solution II and distilled water to reach the constant volume of 1000 mL.

The preparation steps of the salt solution I are as follows: NaCl 6g, (NH)₄)₂SO₄ 3g，KH₂PO₄ 3g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂O0.6 g and distilled water to 1000 mL.

The preparation steps of the salt solution II are as follows: k₂HPO₄4g, distilled water to 1000 mL.

And removing oxygen. Sterilizing at high temperature and high pressure.

EXAMPLE I extraction of DNA from anaerobic fungi

(1) Strain culture: inoculating 5mL of anaerobic fungus liquid for extracting DNA into an anaerobic bottle containing 45mL of anaerobic culture medium in an inoculation amount of 10% v/v, simultaneously adding 1% v/v compound antibiotics (penicillin sodium 1600IU/mL and streptomycin sulfate 2000IU/mL), standing, and carrying out anaerobic culture at 39 ℃ for 72 hours to obtain the anaerobic fungus liquid for extracting genes.

(2) And (3) collecting thalli: centrifuging 5000g/min of the anaerobic fungus liquid obtained by culturing in the step (1) for 5min, and taking mycelium; (3) wall breaking: and (3) grinding the mycelium obtained in the step (2) by using liquid nitrogen quickly, putting a small amount (about 0.1g) of the mycelium into a 1.5mL centrifuge tube, adding 2 steel balls (d is 5mm), quickly freezing the centrifuge tube by using the liquid nitrogen, and oscillating the centrifuge tube on an oscillator for 3-5min, wherein the centrifuge tube keeps a frozen state during oscillation so as to break the cell wall of the anaerobic fungus.

(4) Extracting a DNA solution: adding 0.5mL extraction buffer solution into the broken sample, shaking for 15min on a shaker, and soaking in water bath at 65 deg.C for 10 min. Then 200 μ L of 10M ammonium acetate was added, the mixture was inverted and mixed, the mixture was left on ice for 8min, centrifuged at 13000rpm for 10min, and the supernatant was taken, and the precipitate and steel balls were discarded.

The supernatant was purified with equal volumes of Tris saturated phenol: chloroform: extracting with isoamyl alcohol solution (25mL Tris saturated phenol: 24mL chloroform: 1mL isopropanol, mixing) for 2 times, mixing, centrifuging for 5min, and removing impurity protein to obtain upper water phase.

Preparation of extraction buffer: 2.4228g of Tris (Tris-HCl pH 8.5), 1.461g of NaCl, 0.9306g of disodium ethylene diamine tetraacetate (EDTA-2Na), 2g of Sodium Dodecyl Sulfate (SDS) and 100mL of distilled water. And the pH was adjusted to 8.5 with HCl.

TABLE 1 concentration of the various components of the extraction buffer

	Relative molecular mass	Concentration of	Unit of
				Tris	121.14	200	mM
NaCl	58.44	250	mM
				EDTA-2Na (disodium edetate)	372.24	25	mM
SDS (sodium dodecyl sulfate)	288.38	2％	g/mL

(5) And (3) precipitating DNA: putting the upper-layer water phase obtained in the step (4) into another centrifugal tube, adding 0.5-0.6 volume of isopropanol precooled at-20 ℃, reversing and uniformly mixing, and standing at-20 ℃ for 30min to precipitate DNA; centrifuging at 13000rpm for 15min, discarding the supernatant, washing the precipitate with 1mL 75% ethanol for 2 times, and dissolving the precipitate with 50. mu.L sterile double distilled water. The extracted DNA of the anaerobic fungi is detected by gel electrophoresis and stored at the temperature of minus 20 ℃ for standby.

Example II study of extraction Effect of the extraction method of the present invention

The method further proves the advantages of the method for extracting the total DNA of the anaerobic fungi by adopting the purity and the concentration of the total DNA of the anaerobic fungi extracted by different 3 common methods and the effect of amplifying the ITS1 complete sequence of the anaerobic fungi to further detect and comprehensively evaluate the quality of the total DNA of the anaerobic fungi extracted by different methods.

mu.L of each DNA sample was taken, and the concentration of DNA (ng. mu.L) was determined by a nucleic acid protein analyzer using the corresponding buffer as a blank^－1) And purity A₂₆₀/A₂₈₀And the ITS1 complete sequence of the anaerobic fungi is amplified by adopting primers Neo18S For (5'-AAT CCT TCG GAT TGG CT-3') and Neo5.8S Rev (5'-CGA GAA CCA AGA GAT CCA-3') special For ITS1 of the anaerobic fungi. The assay was repeated 3 times for each sample, 3 replicates each time. The results are shown in table 2 and fig. 1.

TABLE 2 comparison of advantages and disadvantages of total DNA of anaerobic fungi extracted by different methods

Note:^a，^b，^c，^dindicates the statistical difference (p)<0.05)。

As can be seen from Table 2 and FIG. 1, by comparing the purity and concentration of the total DNA of the extracted anaerobic fungi and the effect of amplifying the ITS1 complete sequence of the anaerobic fungi, and considering the time consumption, cost, etc., compared with the 3 conventional methods for extracting total DNA of anaerobic fungi, it can be demonstrated that: the method for extracting the total DNA of the anaerobic fungi has the characteristics of high purity, high concentration, small required sample amount, good effect of amplifying the ITS1 of the fungi, low cost and the like, and can be used for extracting the DNA of the anaerobic fungi.

In conclusion, the method for extracting the DNA of the anaerobic fungi has high purity and concentration of the extracted DNA, and the cell walls of the anaerobic fungi are crushed by adopting liquid nitrogen grinding and freezing and steel ball oscillation, so that the required sample amount is less in the crushing process, the sample is not easy to lose, the wall breaking effect is good, and the method is simple and easy to implement. Meanwhile, the extraction buffer solution has the advantages of simple formula, low cost and good DNA extraction effect. The method for extracting the DNA of the anaerobic fungi is simple to operate, has good experimental repeatability, can obtain the high-quality DNA, and has important significance for the research of promoting the anaerobic fungi to degrade lignocellulose.