Method for extracting DNA (deoxyribonucleic acid) from pericarpium zanthoxyli

Abstract

The invention belongs to the technical field of DNA extraction, and discloses a method for extracting DNA from pericarpium zanthoxyli. The method comprises the following steps: adding CTAB (Cetyltrimethyl Ammonium Bromide) lysate into zanthoxylum bungeanum peel powder, and Centrifuging a mixed solution obtained after cracking, taking supernate, adding chloroform isoamyl alcohol into the supernate, and carrying out protein precipitation and centrifugation; adding isopropanol into the centrifuged mixed solution to carry out DNA precipitation, and incubating; and centrifuging the incubated mixed solution, removing supernate, and cleaning. Compared with a traditional CTAB method, the method has the advantages that the defect that pepper is rich in polysaccharide, polyphenol and grease can be overcome, high-quality genome DNA is stably and efficiently extracted from pepper peel, and the method is used for analysis and detection such as PCR and high-throughput sequencing database building.

Description

Technical Field

[0001] The invention relates to the technical field of DNA extraction, and in particular to a method for extracting DNA from Zanthoxylum bungeanum peel. Background Art

[0002] Zanthoxylum bungeanum Maxim. is the pericarp of the Zanthoxylum bungeanum plant of the Rutaceae family.

[0003] With the continuous development of the Sichuan pepper industry, there are many and complex varieties of Sichuan pepper on the market, and the phenomenon of homonymous and heteronymous products is common. It is crucial to accurately identify Sichuan pepper varieties and have a deep understanding of their genetic characteristics. At present, the existing Sichuan pepper identification and analysis methods have their own advantages and disadvantages. Although the traditional morphological identification method is simple and intuitive, it is greatly affected by environmental factors and has limited ability to distinguish varieties with similar morphology. Based on the high stability of DNA and the continuous development of molecular biology, DNA molecular marker technology has brought new opportunities for the identification of Sichuan pepper varieties.

[0004] Developing molecular biological methods for identifying Zanthoxylum bungeanum varieties and efficiently extracting DNA from Zanthoxylum bungeanum are the most critical and basic steps. The most commonly used method for extracting DNA from plant tissues is the CTAB method. CTAB (cetyltrimethylammonium bromide) is a cationic detergent used to lyse cells and release genomic DNA. The CTAB method is currently the most commonly used plant tissue DNA extraction scheme and has been used for more than 30 years. However, the secondary metabolite composition of different plant species varies. The traditional CTAB extraction scheme cannot be applied to all materials and needs to be improved according to different materials. Zanthoxylum bungeanum peel contains rich polysaccharides, phenolic compounds, proteins, oils and pigments. During DNA extraction, phenolic compounds will cause oxidation and discoloration. Polysaccharides and phenolic compounds will covalently bind to DNA, causing coprecipitation and affecting the efficiency of DNA extraction. After the Zanthoxylum bungeanum peel is ground and crushed, oil will precipitate and float on the upper layer, which is not conducive to the absorption of the lysis supernatant. At present, there is no report on the DNA extraction method for Zanthoxylum bungeanum peel. Exploring the extraction method suitable for the characteristics of Zanthoxylum bungeanum peel has certain scientific value and practical application value. Summary of the invention

[0005] In order to solve the problems in the background technology, the present invention provides a method for extracting DNA from Zanthoxylum bungeanum peel.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A method for extracting DNA from Zanthoxylum bungeanum peel comprises the following steps: Add CTAB lysis solution to the Zanthoxylum bungeanum peel powder for full lysis; The mixed solution obtained after lysis was centrifuged, the supernatant was taken, chloroform isoamyl alcohol was added to the supernatant to precipitate protein, and then centrifuged; Add isopropanol to the centrifuged mixture to precipitate DNA and incubate; The mixed solution after incubation was centrifuged, and the supernatant was removed and washed.

[0007] Preferably, the CTAB lysis solution comprises: 2% (v / v) CTAB, 100 mM pH8.0 Tris-HCl, 2 M NaCl, 20 mM pH8.0 EDTA, 2% (w / v) PVP-K40, 2% (w / w) diatomaceous earth, 0.2% (v / v) β-mercaptoethanol, and 100 μg / mL proteinase K.

[0008] Preferably, the volume ratio of Zanthoxylum bungeanum peel powder to CTAB lysis solution is (80-120): 1 mg / mL.

[0009] Preferably, the volume of chloroform-isoamyl alcohol is the same as the volume of the supernatant.

[0010] Preferably, chloroform-isoamyl alcohol is a solution prepared by mixing chloroform and isoamyl alcohol in a volume ratio of 24:1.

[0011] Preferably, the incubation is at -20°C for 30 to 60 minutes.

[0012] Preferably, sufficient lysis is achieved by incubating at 65°C for 10-20 minutes and homogenizing.

[0013] Compared with the prior art, the present invention has the following effects: The method of the present invention is applicable to the formulation of lysis extract and extraction operation flow of genomic DNA from polysaccharide and polyphenol plant materials. The uniquely formulated lysis extract can precipitate and remove impurities such as proteins, polysaccharides and other secondary metabolites in plant samples. The extracted genomic DNA has high purity and stable and reliable quality, and can be directly used in various downstream analysis experiments such as PCR, enzyme digestion, and library construction. Compared with the traditional CTAB method, this method can overcome the characteristics of Zanthoxylum bungeanum rich in polysaccharides, polyphenols and oils, and stably and efficiently extract high-quality genomic DNA from Zanthoxylum bungeanum peel for analysis and detection such as PCR and high-throughput sequencing library construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 a is the actual state of the sample after lysis and centrifugation in Example 1, and b is the actual state of the emulsion after the upper aqueous phase and CIA are fully vortexed; Figure 2a is an enlarged view of the state of sample DNA extracted from experimental groups 1, 2, and 4 in Example 1, and b is a state diagram of sample DNA extracted from experimental groups 1, 2, and 4 (left) and a state diagram of sample DNA extracted from experimental groups 3 and 5 (right); Figure 3 The PCR amplification results of DNA 1-5 extracted from each experimental group in Example 1; Figure 4 The PCR amplification results of different specific genes in the extracted DNA in Example 2; Figure 5 The PCR amplification results of genomic DNA of different varieties of Zanthoxylum bungeanum in Example 3 are shown. DETAILED DESCRIPTION

[0015] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention should be included in the protection scope of the present invention.

[0016] The present invention provides a method for extracting DNA from Zanthoxylum bungeanum peel, comprising the following steps: Add CTAB lysis solution to the Zanthoxylum bungeanum peel powder for full lysis; The mixed solution obtained after lysis was centrifuged, the supernatant was taken, chloroform isoamyl alcohol was added to the supernatant to precipitate protein, and then centrifuged; Add isopropanol to the centrifuged mixture to precipitate DNA and incubate; The mixed solution after incubation was centrifuged, and the supernatant was removed and washed.

[0017] In the embodiment of the present invention, diatomaceous earth is innovatively added for more complete grinding. Diatomaceous earth is a porous biological siliceous sedimentary rock with a hard siliceous shell and a rich pore structure. During the grinding process, the rough surface and sharp edges of diatomaceous earth can assist in destroying the cell structure under the action of mechanical force, making it easier to release the cell contents. The porous structure of diatomaceous earth also gives it a certain adsorption capacity, which can adsorb some polysaccharides, reduce the interference of polysaccharides with subsequent DNA purification steps, and improve the purity of DNA.

[0018] In an embodiment of the present invention, the CTAB lysis solution comprises: 2% (v / v) CTAB, 100 mM pH8.0 Tris-HCl, 2 M NaCl, 20 mM pH8.0 EDTA, 2% (w / v) PVP-K40, 2% (w / w) diatomaceous earth, 0.2% (v / v) β-mercaptoethanol, and 100 μg / mL proteinase K.

[0019] In order to better protect DNA from degradation, β-mercaptoethanol is added to the lysis solution. β-mercaptoethanol is a strong reducing agent that can destroy the disulfide bonds of intracellular proteins, inhibit the activity of nucleases in cells and in the extraction environment, effectively reduce the degradation of DNA during the extraction process, and improve the quality of extracted DNA; in order to further protect DNA from degradation, proteinase K is added to the lysis solution. Proteinase K can degrade proteins bound to nucleic acids when extracting DNA, release them from the DNA sample, and free them in the extraction solution. At the same time, proteinase K can also quickly inactivate nucleases to prevent them from degrading the target product DNA and improve the extraction efficiency.

[0020] In some preferred embodiments, the volume ratio of Zanthoxylum bungeanum peel powder to CTAB lysis solution is (80-120): 1 mg / mL.

[0021] In some preferred embodiments, the volume of chloroform-isoamyl alcohol is the same as the volume of the supernatant.

[0022] In some preferred embodiments, chloroform-isoamyl alcohol is a solution of chloroform and isoamyl alcohol in a volume ratio of 24:1.

[0023] In some preferred embodiments, the incubation is at -20°C for 30 to 60 minutes.

[0024] In some preferred embodiments, sufficient lysis is performed by incubating at 65° C. for 10 to 20 minutes and homogenizing.

[0025] In order to make the scheme of the present invention more efficient, the extraction method and effect of Zanthoxylum bungeanum peel DNA are described in detail through multiple specific examples below.

[0026] The specific method used for the quality detection of the extracted DNA in the following examples is as follows: (1) Primer sequences: Universal primers for two nuclear gene fragments ITS2 and ETS and one chloroplast gene fragment psbA-trnH were selected from the Zanthoxylum bungeanum genome sequence. Qualitative PCR detection was performed on the extracted Zanthoxylum bungeanum genomic DNA. The primer sequences are shown in Table 1.

[0027] Table 1 .

[0028] (2) PCR reaction system: 2×PCR Master Mix (catalog number: KMM-101) produced by TOYOBO was used for detection. The reaction system is shown in Table 2.

[0029] Table 2 .

[0030] (3) Amplification conditions: Set the amplification program in the ProFlex™ three-chamber gradient PCR instrument as shown in Table 3.

[0031] Table 3 .

[0032] (4) Electrophoresis conditions: Take 5 μL of PCR amplification product and mix it thoroughly with 1 μL of 6× nucleic acid loading buffer as the gel loading solution. The components of agarose gel include 1.5% (w / v) agarose, 1× TAE buffer and 1× GelRed dye. Then add the above gel loading solution to the starting microwell of the gel to ensure that the entire gel is placed in 1× TAE buffer, and analyze the mixture at a constant voltage of 180 V for 30 min. Visualization of the gel is achieved by taking pictures with the Champ Gel-7000 gel imager.

[0033] Example 1 Weigh 100 mg of dried Zanthoxylum bungeanum peel and grind it into powder using a tissue grinder; Add 1 mL of lysis buffer to allow the pepper powder to fully contact the lysis buffer, and incubate at 65°C for 10 min. During the whole process, stir to keep the suspension in the tube homogeneous and prevent it from sinking to the bottom, so that the tissue cells are fully lysed. After sufficient lysis, centrifuge at 13000 rpm for 10 min. Figure 1 Take the upper aqueous phase, add an equal volume of CIA (chloroform, isoamyl alcohol volume ratio 24:1) in a fume hood, and vortex thoroughly to obtain an emulsion (as shown in Figure 1 b) This step can precipitate the protein, and then centrifuge at 13000 rpm for 10 min; Remove the upper aqueous phase, add an equal volume of pre-cooled isopropanol to the tube in a fume hood, mix thoroughly to precipitate DNA, and then incubate at -20°C for 30 min; After incubation, centrifuge at 12,000 rpm for 10 min and discard the supernatant; Add 1 mL of rinse buffer, centrifuge at 12,000 rpm for 1 min, and discard the supernatant.

[0034] Repeat the rinsing operation twice, discard the supernatant, open the lid and invert at room temperature to evaporate the residual ethanol in the tube; Add 50 μL of deionized water, pipette to dissolve the DNA, and store at -20°C.

[0035] The following different experiments were set up for the added lysate, as follows: Experimental group 1: 1× CTAB solution; Experimental group 2: 1× CTAB solution, with 0.2% (v / v) β-mercaptoethanol added when Zanthoxylum bungeanum powder was added for lysis; Experimental group 3: 1× CTAB solution, with 2% (w / v) diatomaceous earth added when adding Zanthoxylum bungeanum powder for lysis; Experimental group 4: 1× CTAB solution, with 100 μg / mL proteinase K added when adding Zanthoxylum bungeanum powder for lysis; Experimental group 5: 1× CTAB solution, with additional 0.2% (v / v) β-mercaptoethanol, 2% (w / w) diatomaceous earth, and 100 μg / mL proteinase K added when Zanthoxylum bungeanum powder was added for lysis; The above CTAB solution components are: 2% (v / v) CTAB, 100 mM Tris-HCl, pH 8.0, 2 M NaCl, 20 mM EDTA, pH 8.0, added to deionized water to dissolve and then sterilized by high pressure, and after cooling, 2% (w / v) PVP-K40 was added and mixed.

[0036] The DNA samples extracted from the above experimental groups are as follows Figure 2 As shown. The DNA3 and DNA5 samples extracted from pepper powder in experimental groups 3 and 5 were Figure 2 b The right side of the state, DNA precipitation can be completely dissolved, DNA extraction samples are clear and transparent; while the samples DNA1, DNA2, and DNA4 extracted from pepper powder in experimental groups 1, 2, and 4 are Figure 2 b The left state, the extracted precipitate is light brown, it is difficult to dissolve with water, and the dissolved DNA sample is colloid and difficult to absorb. This is because the peel cells of Zanthoxylum bungeanum contain a large number of secondary metabolites such as polysaccharides and polyphenols. These substances co-precipitate with DNA during the DNA extraction process to form a viscous colloid, which is difficult to dissolve or oxidize to produce browning, seriously affecting the yield and quality of DNA extraction, as well as subsequent PCR amplification experiments. DNA is brown, difficult to precipitate and viscous, which will affect subsequent enzyme reaction experiments such as PCR.

[0037] The DNA samples were used as templates and the primers ITS2 were used for PCR amplification. The agarose gel electrophoresis results of the above experimental groups were as follows: Figure 3, DNA1, DNA2, DNA3, and DNA4 groups did not successfully amplify the ITS2 band. The lane of DNA5 group had the target band.

[0038] Example 2 Maoxian red prickly ash was used as material, genomic DNA was extracted using the method of Experimental Group 5 of Example 1, and universal primers of two nuclear gene fragments ITS2 and ETS and one chloroplast gene fragment psbA-trnH were selected from the prickly ash genome sequence for PCR amplification detection.

[0039] The genomic DNA sample of Maoxian red prickly ash was used as a template and the universal primers of ITS2, ETS and psbA-trnH of prickly ash gene were used for PCR amplification. The results of agarose gel electrophoresis were as follows: Figure 4 , the target bands were successfully amplified, indicating that the extracted DNA samples can meet the analysis requirements.

[0040] Example 3 Maoxian red pepper (MX), Hanyuan red pepper (HY), Sichuan Guangyuan red pepper (GY), Gansu Longnan red pepper (LN), Shandong Zaozhuang red pepper (ZZ) were used as materials, and genomic DNA was extracted using the method of experimental group 5 in Example 1, and primer ITS2 was used for PCR amplification detection.

[0041] The agarose gel electrophoresis results of PCR amplification products are shown in Figure 5 The amplification success rate can reach 100%, indicating that the DNA extraction method of the present application can extract genomic DNA of different varieties of Zanthoxylum bungeanum, and the quality of the extracted DNA samples can meet the detection requirements.

[0042] The above example successfully achieved DNA extraction from 5 groups of Zanthoxylum bungeanum samples, and used two nuclear gene fragments ITS2 ETS and a pair of chloroplast gene fragments psbA-trnH universal primers to extract Zanthoxylum bungeanum genomic DNA for PCR product amplification, verifying the effectiveness and reliability of the DNA extraction method of the present application.

[0043] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A method for extracting DNA from Zanthoxylum bungeanum peel, characterized in that: The following steps are involved: Add CTAB lysis solution to the Zanthoxylum bungeanum peel powder for full lysis; The mixed solution obtained after lysis was centrifuged, the supernatant was taken, chloroform isoamyl alcohol was added to the supernatant to precipitate protein, and then centrifuged; Add isopropanol to the centrifuged mixture to precipitate DNA and incubate; The mixed solution after incubation was centrifuged, and the supernatant was removed and washed.

2. The method for extracting DNA from Zanthoxylum bungeanum peel as claimed in claim 1, wherein The CTAB lysis solution comprises: 2% (v / v) CTAB, 100 mM pH8.0 Tris-HCl, 2 M NaCl, 20 mM pH8.0 EDTA, 2% (w / v) PVP-K40, 2% (w / w) diatomaceous earth, 0.2% (v / v) β-mercaptoethanol, and 100 μg / mL proteinase K.

3. The method for extracting DNA from Zanthoxylum bungeanum peel as claimed in claim 1, characterized in that, The volume ratio of Zanthoxylum bungeanum peel powder to CTAB lysis solution is (80-120): 1 mg / mL.

4. The method for extracting DNA from Zanthoxylum bungeanum peel as claimed in claim 1, characterized in that The volume of chloroform-isoamyl alcohol was the same as the volume of the supernatant.

5. The method for extracting DNA from Zanthoxylum bungeanum peel according to claim 1 or 4, characterized in that: Chloroform-isoamyl alcohol is a solution prepared by mixing chloroform and isoamyl alcohol in a volume ratio of 24:

1.

6. The method for extracting DNA from Zanthoxylum bungeanum peel as claimed in claim 1, characterized in that The incubation is carried out at -20°C for 30 to 60 minutes.

7. The method for extracting DNA from Zanthoxylum bungeanum peel as claimed in claim 1, characterized in that: Complete lysis is achieved by incubating at 65°C for 10-20 minutes and homogenizing.

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