CN108624588B - Method for extracting agilawood high-quality DNA - Google Patents

Method for extracting agilawood high-quality DNA Download PDF

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CN108624588B
CN108624588B CN201810466650.3A CN201810466650A CN108624588B CN 108624588 B CN108624588 B CN 108624588B CN 201810466650 A CN201810466650 A CN 201810466650A CN 108624588 B CN108624588 B CN 108624588B
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魏建和
冯剑
刘洋洋
杨云
康勇
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Abstract

The invention discloses a method for extracting high-quality DNA of agilawood, which comprises the steps of agilawood sample disinfection and impurity removal, liquid nitrogen grinding, warm bath, centrifugation, alcohol precipitation, washing dissolution, directional PCR amplification and the like, and can be used for extracting high-quality DNA from agilawood containing secondary metabolites such as resin and the like, particularly agilawood with high resin content, long forming (storing) time and deep-processed products (or residues). The invention not only improves the quality and quantity of the agilawood DNA, but also can extract the DNA from deep processing residues, can be directly applied to the research of molecular biology and Chinese medicine identification, and provides a new method for identifying agilawood species.

Description

Method for extracting agilawood high-quality DNA
Technical Field
The invention belongs to the field of molecular biology and Chinese medicine identification, relates to an agilawood DNA extraction method, and particularly relates to a method for extracting DNA from agilawood which is high in resin content, long in forming (storing) time and deep-processed products (or residues).
Background
The lignum Aquilariae Resinatum is resin-containing wood of Aquilaria of Thymelaeaceae, and is a rare Chinese medicinal material. The Chinese eaglewood serving as a medicine is recorded in famous medical records of Tandong pottery hong Jing, has the effects of promoting qi circulation to relieve pain, warming middle energizer to arrest vomiting and receiving qi to relieve asthma, and is mainly used for treating diseases such as fullness and pain in chest and abdomen, vomiting due to stomach cold, hiccup, asthma caused by qi deficiency due to kidney deficiency and the like. In the circulation process, information such as species and formation of agilawood is lost, and the agilawood is processed, so that the quality of the agilawood can be judged only by identification. The traditional 'seeing, hearing and burning' method has subjective consciousness, and agilawood detection method based on 'Chinese pharmacopoeia' is difficult to objectively judge the basic species, so that the phenomenon of adulteration or substitution is frequently rare, and great troubles are caused to clinical medication, customs law enforcement and market specifications.
Agilawood is formed for a long time, generally, the agilawood needs several years or even decades, the wood is aged seriously, most of the wood is composed of dead cells, a large amount of secondary metabolites and pigments are contained, and DNA degradation is serious. The extraction difficulty of the high-purity DNA is larger, the conventional method has poor extraction effect, and the requirements in the fields of germplasm resources, agilawood formation mechanism, species identification, genetic evolution, phylogeny, variety breeding and the like are difficult to meet, and the development of the research work often needs to obtain the high-quality DNA of agilawood, so the extraction of the high-quality DNA is an essential step. In addition, the cost of the agilawood is too high, and the cost of buying the experimental agilawood sample again is high. Therefore, a method suitable for extracting high-quality DNA of lignum Aquilariae Resinatum is urgently needed.
Disclosure of Invention
The invention aims to: the method for extracting the high-quality DNA of the agilawood is provided, the inhibitor of DNA extraction is removed or reduced as far as possible, the release and enrichment of the high-quality DNA of the agilawood in the high resin content, the long forming (storing) time and the deep processing product (or residue) are realized, and an effective technical means is provided for the research of the molecular marker technology and other related fields of the agilawood.
In order to realize the purpose, the invention discloses a method for extracting agilawood high-quality DNA, which comprises the following steps:
(1) sterilizing lignum Aquilariae Resinatum sample, removing impurities, charging nitrogen, and grinding into lignum Aquilariae Resinatum powder;
(2) adding a DNA extracting solution into the sample obtained in the step (1), uniformly mixing, carrying out warm bath in a water bath, cooling at room temperature, adding a DNA separating solution, uniformly mixing, centrifuging, and collecting a supernatant;
(3) adding precooled DNA precipitation liquid into the supernatant, uniformly pumping and standing for precipitation;
(4) cooling at room temperature, adding a DNA washing solution, transferring to a DNA adsorption column after ice bath, centrifuging, and removing a waste liquid;
(5) adding an ethanol solution into the DNA adsorption column in the step (4), centrifuging, and discarding waste liquid;
(6) volatilizing the solvent in the DNA adsorption column in the step (5), adding a preheated DNA dissolving solution, centrifuging, and taking the supernatant to obtain total agilawood DNA;
(7) and adding the DNA extracting solution serving as template DNA into a PCR reaction system, and performing directional amplification to obtain a DNA target fragment.
Preferably, the agilawood sample is any sample containing agilawood DNA.
More preferably, the agilawood sample is one or more of agilawood containing resin, an agilawood medicinal material, agarwood, agilawood plant tissue, an agilawood deep processing product or agilawood deep processing residue.
Preferably, the centrifugal working temperature is 4-20 ℃, the centrifugal speed is 4500-; the temperature of the warm bath is 50-65 ℃, and the reaction time is 3-8 h; the precipitation temperature is-20 ℃, and the reaction time is 12-18 h; ice bath time is 5-10 min; the ethanol solution in the step (5) is 70-80% ethanol solution; repeating the steps (4), (5) and (6) for 1-2 times.
Preferably, the DNA extracting solution is 3% CTAB, 100mM Tris-HCl, 2mM EDTA, 1.4mM NaCl, 1% PVP-40, 0.3M PTB, 10mg/mL proteinase K and beta-mercaptoethanol; the DNA separation solution is chloroform-isoamylol with the ratio of 24: 1; the DNA precipitation solution is isopropanol or absolute ethyl alcohol; the DNA washing solution comprises 5-8M ammonium acetate; the DNA dissolving solution is water or TE solution;
more preferably, 5-10. mu.L/mg of DNA extract and an equal volume of DNA isolate are added in step (2); adding 2 times of DNA precipitation solution; and (4) adding an equal volume of washing solution.
Preferably, the PCR amplification reaction conditions in step (7) are as follows: pre-denaturation at 94-95 deg.C for 0.4-5 min; denaturation at 94 deg.C for 0.4-1min, annealing at 50-56 deg.C for 0.5-1min, extension at 72 deg.C for 0.5-2min, and circulating for 35-55 times; extending for 5-20min at 72 ℃; keeping the temperature at 4 ℃;
the PCR reaction system is as follows: the total volume is 15-50 muL, 2 × Easy Taq PCR Supermix 10-15 muL, 10-100mM primer 1-2 muL, 5-1200ng DNA template, and the rest is dd H2O。
Preferably, step (2) is: adding a 3% tannic acid solution into the sample obtained in the step (1), shaking up, standing, adding chitosan, shaking up, standing, adding a DNA extracting solution, uniformly mixing, carrying out water bath warm bath, cooling at room temperature, adding a DNA separating solution, uniformly mixing, centrifuging, and collecting a supernatant.
More preferably, the operation steps of the warm bath in the step (2) are as follows: the temperature of the warm bath is 60-65 ℃, and the reaction time is 1-2 h; then cooling to 50-60 ℃, and reacting for 1-2 h; then the temperature is increased to 65 ℃ for reaction for 1-5 h.
The invention has the beneficial effects that:
(1) the method improves the quantity and quality of the agilawood DNA on the basis of the traditional CTAB method, and avoids the defects that the extraction effect of the CTAB method is unstable, the imported kit (such as QIAGEN and OMEGA) is expensive and the extraction rate is low, the extraction time of the traditional PTB method is long (5-7 days) and the interference of phenol, polypeptide and carbohydrate is introduced in the extraction process;
(2) a large amount of protein, polysaccharide, phenols, chromone and other substances in the agilawood sample can interfere the extraction of the agilawood DNA, tannic acid and chitosan are sequentially added for pretreatment before the DNA extracting solution is added, and the influence of inhibitors on the extraction of the agilawood DNA is reduced through the pretreatment step;
(3) the temperature and the reaction time are adjusted in the step of water bath and warm bath, and a multi-stage cracking mode is adopted, so that the DNA extraction efficiency is improved;
(4) the invention uses less sample, 0.1-0.2g sample can meet the requirement of extracting genome DNA. The minimally invasive detection method is more suitable for extracting DNA of precious agilawood and precious agilawood products, and avoids damaging the real objects in a large area;
(5) the method is also suitable for effectively extracting DNA from residues in the deep processing process of the agilawood;
(6) the method has simple operation steps, the extracted DNA meets the requirements of downstream application, the practicability is strong, and the large-scale operation can be carried out.
Drawings
FIG. 1 is the absorption spectrum of total DNA extracted from lignum Aquilariae Resinatum in example 1;
FIG. 2 is the total DNA extracted from Aquilaria agallocha in example 1 of the present invention and PCR amplification electrophoretogram;
in FIGS. 1 and 2, the left side is DNA marker, and 1, 2, 3 and 4 are respectively made of dried lignum Aquilariae Resinatum with 10% extract content, lignum Aquilariae Resinatum with 30% extract content, and lignum Aquilariae Resinatum sample with 6h residue after water bath heating.
FIG. 3 is an electrophoretogram of total DNA of lignum Aquilariae Resinatum obtained by different extraction methods;
FIG. 4 is the ITS2 sequence PCR amplification electrophoresis chart of total DNA of lignum Aquilariae Resinatum obtained by different extraction methods;
in FIGS. 3 and 4, the leftmost DNA marker is shown, and 1, 2, 3, 4 and 5 are respectively agilawood samples with extract content of 15% -20%.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Reagents for experiments:
CTAB (cetyltrimethylammonium), Tris-Hcl (pH 8.0), EDTA (pH 8.0), NaCl, PVP-40 (polyvinylpyrrolidone), PTB, ammonium acetate, proteinase K, β -mercaptoethanol, chloroform isoamyl alcohol (24:1), absolute ethanol, 80% ethanol, TE buffer, agarose, 1 × TBE solution, DNA maker, DNA Ladding buffer, nucleic acid dye, chitosan, and tannic acid.
TE buffer components used: 10mM Tris-HCl and 1mM EDTA mixture (pH 8.0).
In the embodiment of the invention, the DNA extracting solution is 3% CTAB, 100mM Tris-HCl, 2mM EDTA, 1.4mM NaCl, 1% PVP-40, 0.3M PTB, 10mg/mL proteinase K and beta-mercaptoethanol; the DNA separation solution is chloroform-isoamylol with the ratio of 24: 1; the DNA precipitation solution is isopropanol or absolute ethyl alcohol; the DNA washing solution comprises 5-8M ammonium acetate; the DNA dissolving solution is water or TE solution.
Example 1:
a method for extracting high-quality DNA of agilawood comprises the following steps:
(1) wiping 200mg of dried aquilaria sinensis wood with 75% alcohol to remove surface dirt, drying, cutting, placing in a 5mL centrifuge tube, adding liquid nitrogen, sealing, and grinding for 5 min.
(2) Adding 1000 μ L preheated DNA extract rapidly, shaking; carrying out warm bath for 8h in a water bath at 60 ℃, and shaking the centrifuge tube for several times during the warm bath; an equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 9000rpm for 20min at room temperature.
(3) Transferring the supernatant obtained in the step (2) into a new 5mL centrifuge tube to ensure that no residue is sucked; adding 2 times of DNA precipitation solution, pumping and mixing uniformly, and standing for 18h at-20 ℃.
(4) And (4) taking out the centrifugal tube in the step (3) for cooling at room temperature, adding an isovolumetric DNA detergent, carrying out ice bath for 5min, absorbing 650 mu L of solution to a DNA adsorption column, placing the DNA adsorption column in a 2mL collecting tube, centrifuging for 1min at 10000rmp, discarding the liquid in the collecting tube, and repeating the process for the rest samples.
(5) Placing the adsorption column of step (4) in a new collection tube, adding 650 μ L of 80% ethanol, centrifuging at 10000rmp for 1min, discarding the centrifugation liquid of the collection tube, repeating for 2 times, and centrifuging at 12000rpm for 1 min.
(6) And (5) placing the DNA adsorption column in the step (5) on a new 1.5mL centrifuge tube, opening the cover, and placing for 2 min. Add 100. mu.L of preheated sterile water or TE solution to the DNA adsorption column, warm-bath for 2min, centrifuge, and repeat this step. The concentration and purity were determined on a NanoDrop 2000DNA concentration meter.
(7) And (4) sucking the total DNA of the agilawood obtained in the step (6) as a DNA template, and performing directional PCR amplification. After the PCR was completed, the detection was carried out by 1.5% agarose gel electrophoresis.
The PCR reaction system is as follows:
(1) the total volume of the reaction is 25 mu L;
(2)2×Easy Taq PCR SuperMix(-dye)(+dye)12.5μL；
(3) 1. mu.L each of 10mM primers (I2F, I2R);
(4)dd H2O 8.5μL；
(5) DNA template 2. mu.L.
A pair of universal primer sequences for amplifying the rRNA encoding gene fragment ITS 2:
(1) upstream primer I2F: 5'-ATGCGATACTTGGTGTGAAT-3', respectively;
(2) downstream primer I2R: 5'-GACGCTTCTCCAGACTACAAT-3', respectively;
(3) the expected length of the amplified product fragment is 500 bp.
The PCR amplification procedure includes: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s for 40 cycles; extension at 72 ℃ for 10 min.
Detecting the concentration and purity of total DNA of agilawood:
taking aquilaria sinensis dried wood, agilawood with 10% of extract content, agilawood with 30% of extract content and residual agilawood as samples after heating in water bath for 6h, determining concentration and purity according to the method of example 1, and finding out the results in table 1 and figures 1-2.
TABLE 1 concentration and purity of total DNA extracted from Aquilaria sinensis according to the invention
Figure BDA0001662242100000051
Figure BDA0001662242100000061
According to the experimental results, the total DNA concentration of different agilawood samples extracted by the method in the embodiment 1 of the invention reaches 96.6-499.8 ng/. mu.L, A260/A280The value is 1.87-2.11. After PCR amplification, a clear band was obtained at 500 nm.
Example 2:
a method for extracting high-quality DNA of agilawood comprises the following steps:
(1) taking 200mg of dried aquilaria sinensis wood, wiping the surface and removing surface dirt by a bleaching agent, airing, cutting into pieces, placing in a 5mL centrifuge tube, adding liquid nitrogen, fully sealing and grinding for 5 min.
(2) Adding 2000 μ L preheated DNA extract rapidly, shaking; carrying out warm bath for 3h in a water bath at 50 ℃, and shaking the centrifuge tube for several times during the warm bath; an equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 4500rpm for 1min at room temperature.
(3) Transferring the supernatant obtained in the step (2) into a new 5mL centrifuge tube to ensure that no residue is sucked; adding 2 times of DNA precipitation solution, pumping and mixing uniformly, and standing for 12h at-20 ℃.
(4) And (4) taking out the centrifugal tube in the step (3) for cooling at room temperature, adding an isovolumetric DNA detergent, carrying out ice bath for 10min, sucking 650 mu L of solution to a DNA adsorption column, placing the DNA adsorption column in a 2mL collecting tube, carrying out 4500rmp, centrifuging for 20min, discarding the liquid in the collecting tube, and repeating the process for the rest samples.
(5) Placing the adsorption column of step (4) in a new collection tube, adding 650 μ L70% ethanol, 4500rmp, centrifuging for 20min, discarding the centrifugation solution of the collection tube, repeating for 1 time, and centrifuging at 4500rpm for 20 min.
(6) And (5) placing the DNA adsorption column in the step (5) on a new 1.5mL centrifuge tube, opening the cover, and standing for 1 min. Add 100. mu.L of preheated sterile water or TE solution to the DNA adsorption column, warm-bath for 1min, centrifuge, and repeat this step. The concentration and purity were determined on a NanoDrop 2000DNA concentration meter.
(7) And (4) sucking the total DNA of the agilawood obtained in the step (6) as a DNA template, and performing directional PCR amplification. After the PCR was completed, the detection was carried out by 1.5% agarose gel electrophoresis.
The PCR reaction system is as follows:
(1)2×Easy Taq PCR SuperMix(-dye)(+dye)15μL；
(2) 2. mu.L each of 10mM primers (I2F, I2R);
(3) 1200ng of DNA template.
By dd H2O make up volume to 50 μ L;
a pair of universal primer sequences for amplifying the rRNA encoding gene fragment ITS 2:
(1) upstream primer I2F: 5'-ATGCGATACTTGGTGTGAAT-3', respectively;
(2) downstream primer I2R: 5'-GACGCTTCTCCAGACTACAAT-3', respectively;
(3) the expected length of the amplified product fragment is 500 bp.
The PCR amplification procedure includes: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, extension at 72 ℃ for 2min, 55 cycles; extension at 72 ℃ for 20 min.
Example 3:
a method for extracting high-quality DNA of agilawood comprises the following steps:
(1) taking 100mg of dried aquilaria sinensis wood, wiping the surface and removing dirt on the surface by 75% alcohol, drying, cutting, putting into a 5mL centrifuge tube, adding liquid nitrogen, sealing and grinding for 5 min.
(2) Add 500. mu.L of preheated DNA extract quickly and shake well. The water bath at 65 ℃ is used for warm bath for 8h, and the centrifuge tube is shaken for a plurality of times during the warm bath. An equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 12000rpm for 1min at room temperature.
(3) The supernatant from step (2) was transferred to a new 5mL centrifuge tube to ensure that no debris was aspirated. Adding 2 times of DNA precipitation solution, pumping and mixing uniformly, and standing for 12h at-20 ℃.
(4) And (4) taking out the centrifugal tube obtained in the step (3) for cooling at room temperature, adding an isovolumetric DNA detergent, carrying out ice bath for 10min, sucking 650 mu L of solution to a DNA adsorption column, placing the DNA adsorption column in a 2mL collecting tube, carrying out 12000rmp, centrifuging for 10min, discarding the liquid in the collecting tube, and repeating the process on the residual sample.
(5) Placing the adsorption column of step (4) in a new collection tube, adding 650 μ L of 80% ethanol, centrifuging at 12000rmp for 5min, discarding the centrifugation liquid of the collection tube, repeating for 2 times, and centrifuging at 10000rpm for 5 min.
(6) And (5) placing the DNA adsorption column in the step (5) on a new 1.5mL centrifuge tube, opening the cover, and placing for 2 min. Add 100. mu.L of preheated sterile water or TE solution to the DNA adsorption column, warm-bath for 2min, centrifuge, and repeat this step. The concentration and purity were determined on a NanoDrop 2000DNA concentration meter.
(7) And (4) sucking the total DNA of the agilawood obtained in the step (6) as a DNA template, and performing directional PCR amplification. After the PCR was completed, the detection was carried out by 1.5% agarose gel electrophoresis.
The PCR reaction system is as follows:
(1)2×Easy Taq PCR SuperMix(-dye)(+dye)10μL；
(2) 1. mu.L each of 10mM primers (I2F, I2R);
(3) 5ng of DNA template.
By dd H2O supplements the total reaction volume to 15 mu L;
a pair of universal primer sequences for amplifying the rRNA encoding gene fragment ITS 2:
(1) upstream primer I2F: 5'-ATGCGATACTTGGTGTGAAT-3', respectively;
(2) downstream primer I2R: 5'-GACGCTTCTCCAGACTACAAT-3', respectively;
(3) the expected length of the amplified product fragment is 500 bp.
The PCR amplification procedure includes: pre-denaturation at 95 ℃ for 0.4 min; denaturation at 94 deg.C for 0.4min, annealing at 50 deg.C for 0.5min, extension at 72 deg.C for 0.5min, and 35 cycles; extension at 72 ℃ for 5 min.
Example 4:
a method for extracting high-quality DNA of agilawood comprises the following steps:
step (2): adding 3% tannic acid solution 3mL into the sample obtained in the step (1), shaking up, standing for 5min, adding 2mg of chitosan, shaking up, standing, adding 1000 μ L of preheated DNA extracting solution, uniformly mixing, carrying out water bath at 60 ℃ for 6h, cooling at room temperature, and shaking the centrifuge tube for several times; an equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 4500rpm for 1min at room temperature.
The rest of the procedure was the same as in example 1.
Example 5:
a method for extracting high-quality DNA of agilawood comprises the following steps:
the step (2) is as follows: adding 3% tannic acid solution 3mL into the sample obtained in the step (1), shaking up, standing for 5min, adding 2mg of chitosan, shaking up, standing, rapidly adding 1000 mu L of preheated DNA extracting solution, shaking up, carrying out warm bath at 60 ℃ for 2h, then cooling to 50 ℃ for 1h, then heating to 65 ℃ for 5h, and shaking a centrifuge tube for several times; an equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 9000rpm for 20min at room temperature.
The rest of the procedure was the same as in example 1.
Example 6:
a method for extracting high-quality DNA of agilawood comprises the following steps:
the step (2) is as follows: adding 3% tannic acid solution 3mL into the sample obtained in the step (1), shaking up, standing for 5min, adding 2mg of chitosan, shaking up, standing, rapidly adding 1000 mu L of preheated DNA extracting solution, shaking up, carrying out warm bath at 65 ℃ for 1h, then cooling to 60 ℃ for 2h, then heating to 65 ℃ for 1h, and shaking a centrifuge tube for several times; an equal volume of DNA isolate was added, vortexed for 30s and centrifuged at 9000rpm for 20min at room temperature.
The rest of the procedure was the same as in example 1.
Detecting the concentration and purity of total DNA of agilawood:
the total DNA of lignum Aquilariae Resinatum obtained in step (6) of examples 1-6 was collected, and the concentration and purity were determined, and the results are shown in Table 2.
TABLE 2 concentration and purity of total DNA extracted from lignum Aquilariae Resinatum in examples 1-6 of the present invention
Concentration ng/. mu.L A260/A280Ratio of
Example 1 499.8 1.92
Example 2 450.6 2.05
Example 3 474.8 2.01
Example 4 504.6 1.87
Example 5 517.6 1.85
Example 6 516.4 1.87
Comparative example: extraction of total DNA of lignum Aquilariae Resinatum by different methods
1. Improved CTAB method
(1) Respectively sucking 0.2g lignum Aquilariae Resinatum sample, placing in 5mL centrifuge tube, grinding into powder with liquid nitrogen, adding 1000 μ L preheated 3% CTAB extraction buffer solution, mixing well, water bathing at 65 deg.C for 8 hr, shaking well occasionally, and standing at room temperature.
(2) The mixture was extracted 2 times with an equal volume of chloroform isoamyl alcohol (24:1) at 9000rpm and centrifuged for 10 min.
(3) Transferring the supernatant obtained in the step (2) twice to a 5mL centrifuge tube, adding equal volume of precooled isopropanol, mixing uniformly, and placing at-20 ℃ overnight.
(3) Taking out the sample in the step (3), returning to the room temperature, centrifuging at 9000rpm for 10min, discarding the supernatant, washing with CTAB washing buffer solution 500 μ L for 2 times, centrifuging, sucking the liquid, and drying at the room temperature.
(4) The reaction mixture was washed 2 times with 500. mu.L of 70% ethanol, and the supernatant was discarded and dried at room temperature.
(5) Add 50 u L sterile water dissolved dried DNA, repeat 1 times.
2. OMEGA HP Plant DNAkit DNA extraction kit
0.2g of agilawood sample is put into a 5mL centrifuge tube and ground into powder by liquid nitrogen, and the other steps are carried out according to the manufacturer's instructions except that 1000 mul of preheated CPL and 10 mul of beta-mercaptoethanol are added in the step (1).
3. PTB method
(1) Placing 0.2g lignum Aquilariae Resinatum in 5mL centrifuge tube, grinding with liquid nitrogen to obtain lignum Aquilariae Resinatum powder, adding 1mL 0.5M EDTA, soaking the powder completely, and standing at room temperature for 48 hr for demineralization.
(2) Add 500. mu.L proteinase K and 1mL PTB solution, vortex and mix, water bath at 65 ℃ for 12 h.
(3) The mixture was extracted with an equal volume of phenol/chloroform (1:1), centrifuged at 12000rpm for 10min, and the supernatant was transferred to a new 2mL centrifuge tube.
(4) An equal volume of chloroform isoamyl alcohol (24:1) was added to step (3), centrifuged at 12000rpm for 10min, the supernatant was pipetted into a new 2mL centrifuge tube, and the procedure was repeated.
(5) 2 volumes of pre-cooled absolute ethanol and 550. mu.L of 7.5M ammonium acetate were added and mixed well. The DNA was precipitated by storing at-20 ℃ for 12 h.
(6) After returning to room temperature, centrifuge at 12000rmp for 20min, discard the liquid, wash 2 times with 300. mu.L of 80% ethanol and precipitate overnight with 80% ethanol.
(7) Centrifuging at 12000rpm for 20min, discarding the liquid, drying at room temperature, and adding 100. mu.L of sterile water to dissolve the DNA.
The detection method comprises the following steps: taking 2 mu L of total DNA of Chinese eaglewood, determining the concentration and purity in a NanoDrop 2000DNA concentration determinator, detecting by using 1.5% agarose gel electrophoresis, respectively taking a total DNA sample of the Chinese eaglewood or a PCR amplification product, uniformly mixing the total DNA sample or the PCR amplification product with 2 mu L of DNAlading buffer, carrying out sample application, carrying out electrophoresis for 15min, and observing under a gel imager. The results are shown in Table 3 and FIGS. 3-4.
TABLE 3 Total DNA concentration and purity of lignum Aquilariae Resinatum obtained by different extraction methods
Figure BDA0001662242100000101
The results show that compared with different extraction methods, the method provided by the invention has the advantages that the concentration and the purity of the total DNA of the agilawood are higher, and clear bands can be obtained for 5 samples after PCR amplification.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.

Claims (5)

1. A method for extracting agilawood high-quality DNA is characterized by comprising the following steps:
(1) sterilizing lignum Aquilariae Resinatum sample, removing impurities, charging nitrogen, and grinding into lignum Aquilariae Resinatum powder;
(2) adding a 3% tannic acid solution into the sample obtained in the step (1), shaking up, standing, adding chitosan, shaking up, standing, adding a 5-10 mu L/mg DNA extracting solution, uniformly mixing, carrying out warm bath on the mixture in a water bath at 50-65 ℃ for 3-8h, cooling at room temperature, adding an isovolumetric DNA separating solution, uniformly mixing, centrifuging, and collecting a supernatant;
(3) adding 2 times volume of precooled DNA precipitation solution into the supernatant, uniformly pumping, standing and precipitating for 12-18h at-20 ℃;
(4) cooling at room temperature, adding an isovolumetric DNA washing solution, carrying out ice bath for 5-10min, transferring to a DNA adsorption column, centrifuging, and removing waste liquid;
(5) adding 70-80% ethanol solution into the DNA adsorption column in the step (4), centrifuging, and discarding waste liquid;
(6) volatilizing the solvent in the DNA adsorption column in the step (5), adding a preheated DNA dissolving solution, centrifuging, and taking the supernatant to obtain total agilawood DNA;
(7) adding the DNA obtained in the step (6) as template DNA into a PCR reaction system, and performing directional amplification to obtain a DNA target fragment;
the centrifugation working temperature is 4-20 ℃, the centrifugation speed is 4500-12000rpm, and the time is 1-20 min;
repeating the steps (4), (5) and (6) for 1-2 times;
the DNA extracting solution consists of 3% CTAB, 100mM Tris-HCl, 2mM EDTA, 1.4mM NaCl, 1% PVP-40, 0.3M PTB, 10mg/mL proteinase K and beta-mercaptoethanol;
the DNA separation solution is chloroform-isoamylol with the ratio of 24: 1;
the DNA precipitation solution is isopropanol or absolute ethyl alcohol;
the DNA washing solution comprises 5-8M ammonium acetate;
the DNA dissolving solution is water or TE solution.
2. The method for extracting agilawood high-quality DNA as claimed in claim 1, wherein the agilawood sample is any sample containing agilawood DNA.
3. The method for extracting the high-quality DNA of the agilawood according to claim 2, wherein the agilawood sample is one or more of aquilaria sinensis wood, agilawood plant tissue, agilawood deep-processing products or agilawood deep-processing residues.
4. The method for extracting agilawood high-quality DNA according to claim 1,
the PCR amplification reaction conditions in the step (7) are as follows: pre-denaturation at 94-95 deg.C for 0.4-5 min; denaturation at 94 deg.C for 0.4-1min, annealing at 50-56 deg.C for 0.5-1min, extension at 72 deg.C for 0.5-2min, and circulating for 35-55 times; extending for 5-20min at 72 ℃; keeping the temperature at 4 ℃;
the PCR reaction system is as follows: the total volume is 15-50. mu.L, 2 × Easy Taq PCR Supermix 10-15. mu.L, 10-100mM primer 1-2. mu.L, 5-1200ng DNA template, and the balance dd H2O.
5. The method for extracting agilawood high-quality DNA (deoxyribonucleic acid) according to claim 1, wherein the temperature of the warm bath is 60-65 ℃, and the reaction time is 1-2 h; then cooling to 50-60 ℃, and reacting for 1-2 h; then the temperature is increased to 65 ℃ for reaction for 1-5 h.
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