Nucleic acid extraction method for endangered medicinal mangrove plant acanthus ilicifolius

Abstract

The invention relates to the field of molecular biology, and discloses a method for extracting nucleic acid of endangered medicinal mangrove plant acanthus ilicifolius, which comprises the following steps: splitting cells by using an improved splitting solution, extracting by using a chloroform-isoamyl alcohol mixed solution in a certain proportion, precipitating DNA by using 1 / 10 volume of sodium acetate and the same volume of isopropanol, purifying the DNA, and purifying the purified DNA to obtain the nucleic acid of the endangered medicinal mangrove plant acanthus ilicifolius. Therefore, the high-quality genome DNA is obtained. The DNA extracted by adopting the method for extracting the nucleic acid of the endangered medicinal mangrove plant acanthus ilicifolius is high in concentration and purity and not easy to degrade, meanwhile, the damage of superoxide anion free radicals or active oxygen to the DNA can be prevented, and a foundation is laid for subsequent molecular biology experiments such as PCR (Polymerase Chain Reaction) amplification, molecular marker technology development, genome library construction and the like.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and more particularly, to a method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl. Background Art

[0002] Acanthus ebracteatus Vahl is an upright shrub of the genus Acanthus in the family Acanthaceae. The extract of Acanthus ebracteatus Vahl leaves is extremely viscous and easily browns, so nucleic acid extraction is difficult.

[0003] In the prior art, the SDS method and the kit method 1, 2 are usually used for nucleic acid extraction, but both have certain defects: (1) During the extraction of genomic DNA from Acanthus ebracteatus Vahl plants using the traditional SDS method, problems such as viscous supernatant, easy browning, and difficult nucleic acid extraction occur; when using ordinary plant genomic DNA extraction kits 1, 2, a viscous lysis solution phenomenon is likely to occur during the extraction process, clogging the column pores, making it difficult for the liquid to pass through the column, and resulting in extremely low DNA extraction concentration and serious contamination.

[0004] Therefore, the present invention proposes a method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl to solve the above technical problems. Summary of the Invention

[0005] In order to overcome the above-mentioned defects existing in the prior art, the present invention provides a method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl. The DNA extracted by the method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl provided by the present invention has a high concentration, high purity, is not easily degraded, and can prevent damage to DNA by superoxide anion radicals or reactive oxygen species, laying a foundation for subsequent molecular biology experiments such as PCR amplification, molecular marker technology development, and genomic library construction.

[0006] The above technical object of the present invention is achieved through the following technical solutions: A method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl, comprising the following steps:

[0007] S1. Take about 0.2 g of young and tender leaves of Acanthus ebracteatus Vahl, put them into a mortar for grinding, add 1 mL of lysis solution preheated to 65 °C during the grinding process, grind the leaves into a paste, transfer them to a 2 mL centrifuge tube, and add 2 μL of RNase A solution with a concentration of 100 mg / mL;

[0008] S2. Incubate in a water bath at 70 °C for 30 min, and gently invert and mix once every 10 min;

[0009] S3. After cooling, add an equal volume of chloroform-isoamyl alcohol solution, shake vigorously until milky white, and centrifuge at 10000 rpm for 10 min;

[0010] S4. Take 600 μL of the supernatant into a 1.5 mL centrifuge tube, add an equal volume of isopropanol solution pre-cooled at -20°C and 1 / 10 volume of 3 mol / L NaAC solution, mix well, and place at -20°C for 30 min.

[0011] S5. Centrifuge at 10000 rpm for 10 min, discard the supernatant to obtain a white precipitate, add 1 mL of 75% ethanol solution to wash the precipitate, air-dry and dissolve it in 0.1 mL of TE buffer, and store at -20°C.

[0012] Further, in step S1, the composition of the lysis solution is: cetyltrimethylammonium bromide with a ratio of unit mass to solution volume of 2%, 75 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, pH value of 8.0, ethylenediaminetetraacetic acid with a concentration of 15 mmol / L and pH value of 8.0, sodium chloride solution with a concentration of 1 mol / L and pH value of 8.0, β-mercaptoethanol with a ratio of unit volume to solute volume of 2%, and polyvinylpyrrolidone with a ratio of unit mass to volume of 1%.

[0013] Further, in step S3, the ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol solution is 24:1.

[0014] Further, in step S4, the pH value of the NaAC solution is 5.2.

[0015] In summary, the present invention has the following beneficial effects: (1) Using an improved lysis solution to lyse cells. Compared with ordinary kits, CTAB is used and PVP is increased; compared with the traditional SDS method, CTAB is used, Tris-Hcl and EDTA are reduced, and NaCl is increased. CTAB can form complexes with proteins and polysaccharides but cannot precipitate nucleic acids; PVP, chemically named polyvinylpyrrolidone, is an antioxidant with strong solubility, which can combine with polyphenols and polysaccharides to form complexes, separating polyphenol polysaccharides from DNA, thereby removing impurities; Tris-Hcl can provide a buffer environment to prevent nucleic acids from being damaged; EDTA can chelate Mg 2+ or Mn 2+ to inhibit DNase activity; NaCl can provide a high-salt environment;

[0016] (2) Extracting with a 24:1 chloroform-isoamyl alcohol mixture can effectively eliminate foam during the extraction process and improve the extraction efficiency;

[0017] (3) Using sodium acetate and isopropanol to precipitate DNA, changing absolute ethanol to isopropanol. Isopropanol has the characteristics of less dosage and fast speed, and is suitable for precipitating DNA with low concentration and large volume. Alcohol substances can precipitate DNA and remove polysaccharides.

[0018] (4) The DNA extracted by using the nucleic acid extraction method of the endangered medicinal mangrove plant Acanthus ebracteatus provided by the present invention has a high concentration, high purity, is not easily degraded, and can prevent the damage of superoxide anion radicals or reactive oxygen species to DNA, laying a foundation for subsequent molecular biology experiments such as PCR amplification, molecular marker technology development, and genomic library construction. Description of the Drawings

[0019] Figure 1 It is a comparison diagram of the samples in Example 1 and Example 2 of the present invention. Note: The two tubes on the left are the samples in Example 1 added to the lysis solution without heating, and the two tubes on the right are the samples in Example 2 added to the lysis solution without heating;

[0020] Figure 2 It is a comparison diagram of the samples in Example 1 and Example 2 of the present invention. Note: The two tubes on the left are the samples in Example 1 added to the lysis solution after heating, and the two tubes on the right are the samples in Example 2 added to the lysis solution after heating;

[0021] Figure 3 It is the actual effect diagram of adding chloroform-isoamyl alcohol to the sample in Example 2 of the present invention;

[0022] Figure 4 It is a comparison diagram of the samples in Example 1 and Example 2 of the present invention. Note: The two tubes on the left are the samples in Example 1 centrifuged after extraction, and the two tubes on the right are the samples in Example 2 centrifuged after extraction;

[0023] Figure 5 It is a comparison diagram of the samples in Example 1 and Example 2 of the present invention. Note: The two tubes on the left are the samples in Example 1 adding absolute ethanol to precipitate DNA, and the two tubes on the right are the samples in Example 2 adding isopropanol and sodium acetate to precipitate DNA;

[0024] Figure 6 It is the agarose gel electrophoresis diagram of Acanthus ebracteatus DNA in Example 4 of the present invention. Note: Ordinary kit 1 is the agarose gel electrophoresis diagram of the extract of the Solarbio plant genomic DNA extraction kit, ordinary kit 2 is the agarose gel electrophoresis diagram of the extract of the Kediyuan plant DNA small sample extraction kit, SDS is the agarose gel electrophoresis diagram of the extract by the method of Example 1 of the present invention, and CTAB is the agarose gel electrophoresis diagram of the extract by the method of Example 2 of the present invention. Detailed Description of the Embodiments

[0025] The following further elaborates on the present invention with reference to the Figure 1-6 drawings.

[0026] Example 1: Using the SDS method

[0027] (1) Weigh 0.2 g of the sample and put it into a mortar. Add 1 mL of the lysis solution preheated to 65 °C, grind the sample into a paste, transfer it to a 2 mL centrifuge tube. The composition of the lysis solution is: 100 mmol / L Tris-HCl, pH 8.0; 50 mmol / L EDTA, pH 8.0; 500 mmol / L NaCl; 2% β-mercaptoethanol (v / v); 1% PVP (m / v); 2% sodium dodecyl sulfate (SDS) (m / v). Place it in a water bath at 65 °C and incubate for 20 min.

[0028] (2) Centrifuge at 10000 rpm for 10 min, transfer 700 μL of the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of 5 mol / L KAC (pH 4.8), gently rotate and mix well, let it stand for a while, and then centrifuge at 10000 rpm for 10 min.

[0029] (3) Transfer 500 μL of the supernatant to a new 1.5 mL centrifuge tube, add 2 volumes of anhydrous ethanol pre-cooled to -20 °C, gently mix, let it stand at -20 °C for 30 min, then centrifuge at 10000 rpm for 10 min, and discard the supernatant.

[0030] (4) Wash the precipitate with 500 μL of 75% ethanol, repeat 2 - 3 times, and then air-dry the precipitate.

[0031] (5) Add 100 μL of TE buffer to dissolve the dried DNA, add 2 μL (100 mg·ml-1) RNase A, incubate at 37 °C for 30 min, and then store at -20 °C for later use.

[0032] Example 2: Using a nucleic acid extraction method for the endangered medicinal mangrove plant Acanthus ebracteatus proposed in this application

[0033] (1) Take about 0.2 g of the young leaves of Acanthus ebracteatus, put them into a mortar, add 1 mL of the lysis solution preheated to 65 °C, grind the leaves into a paste, transfer it to a 2 mL centrifuge tube, and add 2 μL of RNase A (100 mg / mL); the composition of the lysis solution is: 75 mmol / L Tris-HCl, pH 8.0; 15 mmol / L EDTA, pH 8.0; 1 mol / L NaCl; 2% (v / v) β-mercaptoethanol, 2% (m / v) CTAB, 1% (m / v) PVP.

[0034] (2) Incubate in a water bath at 70 °C for 30 min, gently invert and mix well every 10 min.

[0035] (3) After cooling, add an equal volume of chloroform-isoamyl alcohol (24:1), shake vigorously until it turns milky white, and centrifuge at 10000 rpm at room temperature for 10 min.

[0036] (4) Transfer 600 μL of the supernatant to a 1.5 mL centrifuge tube, add an equal volume of isopropanol pre-cooled at -20°C and 1 / 10 volume of 3 mol / L NaAC (pH 5.2), mix well, and place at -20°C for 30 min.

[0037] (5) Centrifuge at 10000 rpm for 10 min at room temperature, discard the supernatant, obtain a white precipitate, add 1 mL of 75% ethanol to wash the precipitate, air-dry and dissolve in 0.1 mL of TE buffer, and store at -20°C for later use.

[0038] Example 3: Using a kit method

[0039] (1) Weigh 0.2 g of the sample, grind it thoroughly in liquid nitrogen until it becomes a fine powder, and let the liquid nitrogen evaporate naturally.

[0040] (2) Quickly transfer the ground plant tissue powder to a centrifuge tube pre-filled with 400 μL of solution PA, 20 μL of RNase A (10 mg / mL), and 5 μL of a thiol reducing agent, invert and mix well, and let it stand at room temperature for 10 min.

[0041] (3) Add 140 μL of solution PB, invert and mix well, centrifuge at 10000 rpm for 10 min, and transfer 300 μL of the supernatant to a new centrifuge tube.

[0042] (4) Add the same volume of solution PC as the supernatant, invert and mix well, then add the same volume of absolute ethanol as solution PC, centrifuge at 10000 rpm for 5 min, and discard the waste liquid.

[0043] (5) Add 600 μL of the wash solution to the adsorption column (first check if absolute ethanol has been added), centrifuge at 10000 rpm for 1 min, discard the waste liquid, and place the adsorption column back into the collection tube.

[0044] (6) Repeat step (5).

[0045] (7) Centrifuge at 10000 rpm for 2 min without adding sample.

[0046] (8) Place the adsorption column into a clean centrifuge tube, suspend and add 50 μL of the elution buffer pre-heated in a 65°C water bath to the center of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 10000 rpm for 2 min, and then the plant genomic DNA can be obtained and stored at -20°C for later use.

[0047] Example 4: Generate a gel electrophoresis diagram of the plant genomic DNA extracted in the above Examples 1 - 3, as Figure 6As shown, where the ordinary kit 1 is the agarose gel electrophoresis pattern of the extract of the Solarbio plant genomic DNA extraction kit; the ordinary kit 2 is the agarose gel electrophoresis pattern of the extract of the Kedi source plant DNA small sample extraction kit B; SDS refers to the agarose gel electrophoresis pattern of the extract by the method of Example 1; CTAB is the agarose gel electrophoresis pattern of the extract by the method of Example 2. It can be seen from the figure that there are almost no DNA bands extracted by the ordinary kit 1, the ordinary kit 2 and the traditional SDS method, while the DNA bands extracted by the improved CTAB method are bright and the sample loading wells are also clear.

[0048] In summary, the conclusion is drawn that the DNA extracted by the nucleic acid extraction method for the endangered medicinal mangrove plant Acanthus ebracteatus proposed in this application has the characteristics of high concentration and high purity.

[0049] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A method for extracting nucleic acids from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl, characterized in that, It includes the following steps: S1. Take about 0.2 g of young leaves of Acanthus ebracteatus Vahl, put them into a mortar for grinding, add 1 mL of lysate preheated at 65 °C during the grinding process, grind the leaves into a paste, transfer them to a 2 mL centrifuge tube, and add 2 μL of RNase A solution with a concentration of 100 mg / mL; S2. Incubate in a water bath at 70 °C for 30 min, gently invert and mix once every 10 min; S3. After cooling, add an equal volume of chloroform-isoamyl alcohol solution, shake vigorously until it turns milky white, and centrifuge at 10000 rpm for 10 min; S4. Take 600 μL of the supernatant into a 1.5 mL centrifuge tube, add an equal volume of isopropanol solution pre-cooled at -20 °C and 1 / 10 volume of 3 mol / L NaAC solution, mix well, and place at -20 °C for 30 min; S5. Centrifuge at 10000 rpm for 10 min, discard the supernatant to obtain a white precipitate, add 1 mL of 75% ethanol solution to wash the precipitate, air-dry and dissolve it in 0.1 mL of TE buffer, and store at -20 °C.

2. The nucleic acid extraction method of the endangered medicinal mangrove plant Acanthus ebracteatus Vahl according to claim 1, wherein, In step S1, the composition of the lysate is: cetyltrimethylammonium bromide with a ratio of unit mass to solution volume of 2%, tris(hydroxymethyl)aminomethane hydrochloride at 75 mmol / L, pH 8.0, ethylenediaminetetraacetic acid at a concentration of 15 mmol / L and pH 8.0, sodium chloride solution at a concentration of 1 mol / L and pH 8.0, β-mercaptoethanol with a ratio of unit volume to solute volume of 2%, and polyvinylpyrrolidone with a ratio of unit mass to volume of 1%.

3. The nucleic acid extraction method of the endangered medicinal mangrove plant Acanthus ebracteatus Vahl according to claim 1, characterized in that, In step S3, the ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol solution is 24:

1.

4. A method for extracting nucleic acid from the endangered medicinal mangrove plant Acanthus ebracteatus Vahl, according to claim 1, characterized in that, In step S4, the pH value of the NaAC solution is 5.2.

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