CN112538475A

CN112538475A - Extraction method, sequencing method and kit of plant genome DNA

Info

Publication number: CN112538475A
Application number: CN201910898449.7A
Authority: CN
Inventors: 杨彦; 刘清正; 王然; 陈翠; 高普霞
Original assignee: Wuhan Bgi Medical Laboratory Co ltd
Current assignee: Wuhan Bgi Medical Laboratory Co ltd
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2021-03-23

Abstract

The invention relates to the technical field of biology, in particular to an extraction method, a sequencing method and a kit of plant genome DNA. The extraction method comprises the following steps: grinding the plant sample, mixing the obtained ground powder with the cell nucleus extracting solution, filtering, and centrifuging the obtained filtrate to obtain a precipitate containing cell nuclei; performing density gradient centrifugation on the sediment containing the cell nucleus by using the cell nucleus separating medium to obtain a separating medium containing the cell nucleus; removing protein and RNA in the separation solution containing the cell nucleus to obtain a solution containing DNA; the solution containing the DNA is dialyzed to obtain plant genomic DNA. The provided kit comprises a NIBMT solution; percoll separating medium; a proteinase K solution; an RNase; and a semi-permeable membrane. The plant genome DNA fragments extracted by the method or the kit are longer, so that the requirement of sequencing can be met, and the sequencing quality is improved.

Description

Extraction method, sequencing method and kit of plant genome DNA

Technical Field

The invention relates to the technical field of biology, in particular to an extraction method, a sequencing method and a kit of plant genome DNA.

Background

In obtaining plant genomic DNA, the CTAB method is a commonly used mainstream technique. CTAB (cetyltrimethylammonium bromide) is a detergent that dissolves cell membranes and forms complexes with nucleic acids, which complexes are soluble in solutions of high ionic strength and can be separated from proteins and polysaccharides by centrifugation. Under the condition of phenol/chloroform/isoamyl alcohol denaturation, removing residual CTAB, protein and other impurities, then utilizing isopropanol or absolute ethyl alcohol to precipitate DNA molecules from a supernatant solution, finally using an resolution Buffer to dissolve the DNA, and adding RNase to remove RNA in a genome, thereby obtaining the plant genome DNA.

However, the integrity of the plant genome DNA obtained by the CTAB method is poor, and the fragment is short, so that the requirement of sequencing is not met.

Therefore, how to obtain plant genome DNA with longer fragments and good integrity to meet the requirement of sequencing needs further improvement.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method for extracting plant genomic DNA, a method for sequencing plant genomic DNA, and a plant genomic DNA extraction kit.

The inventor of the invention finds out in the research process that:

in the process of extracting plant genome DNA, the CTAB method is widely applied as a common extraction method. The CTAB method is used for extracting plant genome DNA, and usually a certain proportion of PVP (polyvinylpyrrolidone) is added into a CTAB solution to dissolve cell membranes so as to separate DNA and macromolecular substances combined with the DNA, such as polysaccharide, polyphenol, protein and the like. Then removing macromolecular substances by repeated extraction. However, such repeated extractions can result in significant DNA loss and DNA degradation. However, if the macromolecular substances are not extracted and removed, the yield of the extracted DNA is low or even the extracted DNA cannot be extracted due to incomplete removal of the macromolecular substances, so that a subsequent library can not be built, and the sequencing quality is seriously influenced. In addition, highly toxic reagents such as phenol/chloroform/isoamyl alcohol are also used in the extraction process, which is harmful to the environment. The CTAB method is not mild enough, and it is difficult to extract a long-fragment DNA from a plant, and it is difficult to satisfy the requirement of sequencing.

In order to obtain plant genome DNA with longer fragments and better integrity, the invention provides an extraction method of the plant genome DNA, which comprises the steps of grinding a plant sample, mixing with a cell nucleus extracting solution, obtaining a solution containing a cell nucleus by a density gradient centrifugation mode, removing other macromolecular substances except DNA molecules, such as protein, by enzyme digestion, and finally removing small molecular substances by dialysis to obtain the plant genome DNA. Compared with a CTAB method, the method provided by the invention is milder, can obtain a longer DNA fragment, has the integrity of more than 50Kb, is used for sequencing, and can improve the sequencing quality by using a 10X Genomics platform for sequencing.

Specifically, the invention provides the following technical scheme:

according to one aspect of the present invention, the present invention provides a method for extracting plant genomic DNA, comprising: grinding the plant sample, mixing the obtained ground powder with the cell nucleus extracting solution, filtering, and centrifuging the obtained filtrate to obtain a precipitate containing cell nuclei; performing density gradient centrifugation on the sediment containing the cell nucleus by using the cell nucleus separating medium so as to obtain a separating medium containing the cell nucleus; removing protein and RNA in the separation solution containing the cell nucleus to obtain a solution containing DNA; subjecting the DNA-containing solution to a dialysis treatment to obtain the plant genomic DNA.

A plant sample is ground (for example, liquid nitrogen grinding is carried out), the obtained ground powder is mixed with a cell nucleus extracting solution, large-fragment tissue is removed through filtration, a precipitate containing cell nuclei is obtained, then the cell nucleus separating solution is used for carrying out density gradient centrifugation on the precipitate containing the cell nuclei, after the cell nuclei are obtained, macromolecular substances except DNA molecules, such as protein and RNA, are removed, and then small-molecular substances are dialyzed and removed through a dialysis method, so that the DNA molecules with high purity and long fragments are obtained.

The plant genome DNA extraction method is different from a phenol/chloroform/isoamylol extraction method of a CTAB method, and the degradation caused by physical strand breakage damage brought by mechanical shearing force to large-segment DNA in the extraction process is avoided; the extraction method of the plant genome DNA provided by the invention is mild, and the nucleic acid is protected from the influence of the shearing force of a solution system.

The extraction method of the plant genome DNA provided by the invention can obtain DNA molecules with longer segments, can meet the requirements of a 10X Genomics platform on a sequencing library, and improves the sequencing data quality of the 10X Genomics platform.

According to an embodiment of the present invention, the above-mentioned method for extracting plant genomic DNA may further include the following technical features:

in some embodiments of the invention, the cell nucleus extract is a NIBMT solution. Therefore, the cell can be quickly cracked, and the cell nucleus in the cell can be extracted.

In some embodiments of the invention, the filtration comprises at least two filtration treatments with a filter membrane, wherein the filter membrane pore size of the second filtration treatment is smaller than the filter membrane pore size of the first filtration treatment. Through at least two times of filtration treatment, the aperture of the filter membrane used in the next filtration treatment is smaller than that of the filter membrane used in the previous filtration treatment, so that impurities such as debris tissues and the like can be effectively removed, and the purity of the target cell nucleus solution is further improved.

In some embodiments of the invention, the filtering comprises at least: performing a first filtration treatment with a 70 micron pore size filter membrane to obtain a first filtrate; a second filtration treatment was performed using a 40 micron pore size filter membrane to obtain a second filtrate. Thereby remarkably improving the purity of the target cell nucleus solution.

In some embodiments of the invention, the nuclear separation fluid is a Percoll separation fluid. Percoll is a silica gel particle coated with vinylpyrrolidone, and the resulting solution has a low diffusion constant and a very stable gradient. And Percoll does not penetrate biological membranes and is harmless to cells, so that the Percoll separating medium can be used for separating cells, subcellular components, bacteria and viruses, and can also be used for separating damaged cells and fragments thereof from intact living cells.

In some embodiments of the invention, the density gradient centrifugation comprises at least: the Percoll separating medium with volume fraction of 75% and the Percoll separating medium with volume fraction of 70% are used for density gradient centrifugation. Generally, for most plants, separation is achieved by two-step gradient centrifugation using 70% by volume of Percoll and 75% by volume of the Percoll medium, with nuclei floating between 70% by volume and 75% by volume. Some plants may require more gradients to separate the nuclei from debris, so there may be at least multiple gradients of Percoll.

In some embodiments of the invention, the nuclear-containing separation is digested with proteases and rnases to remove proteins and RNA from the nuclear-containing separation. Therefore, proteins and RNA in the separation liquid containing cell nucleus can be removed gently without affecting the size of DNA molecule.

In some embodiments of the invention, the proteinase is a proteinase K solution comprising proteinase K, EDTA and SDS. In the process of removing protein by using the proteinase K solution, EDTA in the proteinase K solution can inhibit the activity of DNase, can avoid DNA degradation, and SDS contained in the EDTA can help to lyse cells. Therefore, protein can be removed at a high speed without affecting DNA.

In some embodiments of the invention, the dialysis treatment is performed using a semi-permeable membrane having a pore size of 0.1 microns. Thus, small molecular substances can be removed by dialysis, and DNA with a long fragment and high purity can be obtained.

In some embodiments of the present invention, the dialysis treatment time is 16-48 h, and may be 17h, 18h, 19h, 20h, 22h, 24h, or 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48 h. Thus, small molecular substances can be removed by dialysis, and DNA with a long fragment and high purity can be obtained.

In some embodiments of the invention, the plant genomic DNA is greater than 50 Kb. By the method, DNA molecules with longer segments can be obtained, for example, the obtained plant genome DNA is larger than 50Kb, the requirement of 10X Genomics platform library establishment can be met, the extracted genome DNA segment is long, the segment length of a 10X Genomics ex-library can be increased, and the sequencing data quality of the 10X Genomics platform is improved.

According to a second aspect of the present invention, there is provided a plant genomic DNA extraction kit comprising: a NIBMT solution; percoll separating medium; a proteinase K solution; an RNase; and a semi-permeable membrane. Through the reagent kit containing the reagent and the semipermeable membrane, the plant sample is extracted, and DNA with longer fragments and better purity can be mildly obtained, so that the reagent kit is more suitable for the requirement of sequencing.

In some embodiments of the invention, the kit extracts plant genomic DNA according to the extraction method of any embodiment of the first aspect of the invention.

According to a third aspect of the invention, there is provided a method of sequencing plant genomic DNA comprising: extracting genomic DNA from a plant sample by the extraction method according to any of the embodiments of the first aspect of the present invention; and (3) carrying out library construction on the plant genome DNA, and carrying out high-throughput sequencing on the obtained library so as to obtain a sequencing result.

In some embodiments of the invention, the high throughput sequencing is performed using a 10X Genomics platform.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of genomic DNA of Lotus japonicus/forsythia suspense/tuber mustard extracted by CTAB method according to the embodiment of the present invention.

FIG. 2 is a diagram showing the result of electrophoresis of genomic DNA of Lotus japonicus/forsythia suspense/tuber mustard provided by the method of the present invention according to an embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings, and it should be noted that the described embodiments are exemplary and are intended to be illustrative of the present invention and should not be construed as limiting the present invention.

The invention provides a plant genome DNA extraction kit, which comprises an NIBMT solution; percoll separating medium; a proteinase K solution; an RNase; and a semi-permeable membrane. When the kit is used for extracting plant genome DNA, NIBMT solution can help to extract cell nuclei from a plant sample, and Percoll separation solution is used for density gradient centrifugation to obtain separation solution containing the cell nuclei; macromolecular substances such as protein, RNA and the like in the separation solution containing the cell nucleus can be removed by using the proteinase K solution and the RNase. Finally, the semipermeable membrane is used for dialysis to remove small molecular substances, so that DNA molecules with longer fragments are extracted from the plant sample.

When the Percoll separating medium is used, the Percoll separating medium with volume fraction of 75% and the Percoll separating medium with volume fraction of 70% can be prepared, and the separation of most plant cell nucleuses and other substances can be basically realized through density gradient centrifugation. In at least some embodiments, different volume fractions of Percoll isolates can be prepared using a nuclear extract (e.g., NIBMT solution) mixed with Percoll. The volume fraction here means the volume of Percoll as a percentage of the total volume of the separation liquid.

The NIBMT solution is used herein as an explanation of the general meaning in the art. Proteinase K solution contains proteinase K, EDTA and SDS. In the process of removing protein by using the proteinase K solution, EDTA in the proteinase K solution can inhibit the activity of DNase, can avoid DNA degradation, and SDS contained in the EDTA can help to lyse cells. Therefore, protein can be removed at a high speed without affecting DNA. In at least some embodiments, the proteinase K solution contains proteinase K at a concentration of 1-3 mg/mL, EDTA at a concentration of 0.1-0.5M, and SDS at a mass concentration of 1-5%. The mass concentration here means the percentage of the mass of SDS to the total volume of the proteinase K solution.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Embodiment 1 provides a plant genome DNA extraction method, the method through containing plant tissue cell nucleus lysis supernatant using density gradient centrifugation principle separation release to get the cell nucleus, utilize protease and RNase digestion except DNA molecular macromolecular substance, this step is completely different from CTAB method phenol/chloroform/isoamyl alcohol extraction method, has avoided the mechanical shear force in the extraction process to large fragment DNA brought physical twist damage, and then cause the large fragment DNA degradation; finally, the small molecular substances are dialyzed and removed by using a semipermeable membrane dialysis method, thereby obtaining the genome DNA. Wherein in the process of extracting the plant genome DNA, the used reagents are as follows:

1 xnib, 1 xnibt and 1 xnibtm, the formulations of which are shown in table 1, table 2 and table 3, respectively:

TABLE 11 XNIB (HB homogenization buffer, HB homogene buffer)

After the NIB was prepared according to the above table, the pH was adjusted to 9.0 using 2M NaOH, and filtration was performed using a 0.2 μ M filter instead of autoclaving.

TABLE 21 XNIBT buffer (Triton Nuclear extraction buffer with Triton)

TABLE 31 XNIBTM buffer (nuclear extraction buffer with Triton and. beta. -mercaptoethanol added Nuclear isolation buffer with Triton and. beta. -ME)

The following matters need to be noted in the process of preparing the reagent:

1. PVP10 is less soluble and should be added in small amounts until completely dissolved.

2. Before use, 2-Mercaptoethanol was added to the fume hood and placed on ice. The experiment after milling was conducted in a fume hood, as the 1X NIBTM contained 2-BEM.

3. Centrifugation speed varies with genome size. Generally, smaller genomes are centrifuged at greater speeds, such as 4000g for algal 63M and 1800g for cotton 880M.

The method for extracting plant genome DNA provided in this example is as follows:

1. homogenizing the tissue and isolating and purifying the cell nuclei

(1) Placing the mortar and pestle into a refrigerator at minus 80 ℃ for precooling before starting the experiment, pouring liquid nitrogen for further cooling before grinding, placing about 5-10 g of tender plant leaves, and pouring sufficient liquid nitrogen for proper grinding until fine powder is formed;

(2) taking a certain amount of freshly prepared NIBTM in a 200mL beaker, and putting the beaker on ice, wherein 10mL of NIBTM is required to be used for every 1g of sample;

(3) transferring the plant sample ground in the step (1) into a 200mL beaker containing NIBTM, and gently stirring the plant sample on ice for 10min by using a magnetic stirrer to obtain homogenate;

(4) filtering the homogenate obtained in the step (3) into a new 50ml centrifuge tube by using a filter membrane with the aperture of 70 mu m, and then filtering the filtrate into another new 50ml centrifuge tube by using a filter membrane with the aperture of 40 mu m;

(5) and (3) centrifuging the filtrate obtained after filtration in the step (4) for 20min at 1800g and 4 ℃. The supernatant was removed and 1mL of precooled NIBTM was added.

(6) The pellet was gently resuspended with a small brush and 20mL of ice-cold NIBTM were added to obtain a resuspension.

(7) The resuspension from the previous step was gravity filtered through a 40 μm pore filter into a 50mL centrifuge tube. Centrifugation was carried out at 57g at 4 ℃ for 2min to remove intact cells and residual tissue.

(8) The supernatant was transferred to a new centrifuge tube and centrifuged at 1800g for 15min at 4 ℃ to pellet the nuclei.

(9) Two Percoll isolates of different concentrations were prepared in 2 50mL centrifuge tubes according to Table 4.

TABLE 4 Percoll separating medium preparation table

And (3) uniformly mixing the Percoll separating medium with different concentrations, namely slowly pouring the 70% Percoll separating medium into the 75% Percoll separating medium along the pipe wall, and standing.

(10) And (8) pouring out the supernatant after the centrifugation, and adding 4mL of NIBTM to resuspend the cell nucleus to obtain a cell nucleus resuspension solution.

(11) 4mL of the nuclear resuspension obtained in step (10) was carefully applied to the top of the separation obtained in step (9) using a 1mL pipette, and centrifuged at 650g for 30min at 4 ℃.

According to experimental results, the two-step gradient is carried out by using the Percoll separating medium with the volume fraction of 70% and the Percoll separating medium with the volume fraction of 75%, the separation effect is good, cell nuclei float in the middle of the Percoll separating medium with the volume fraction of 70% and the Percoll separating medium with the volume fraction of 75%, a white layer is formed, and fragments are usually precipitated at the bottom of a centrifugal tube.

(12) The upper yellow liquid was first carefully removed with a 5mL tip and the white layer collected with a large bore tip and placed in a new tube. The pellet that is sucked above or below the white layer (nuclei) will not cause damage to the nuclei because the subsequent washing step will remove the Percoll pellet. The nuclei containing Percoll were placed in a new centrifuge tube and diluted to 20mL with NIBTM, centrifuged at 1800g for 15min at 4 ℃.

(13) The precipitate was washed three times with 20mL of NIBTM in the same manner.

(14) After the last wash, 500. mu.L of cell suspension buffer (or added as appropriate depending on the size of the pellet) was added to the pelleted nuclei, and the pellet was gently suspended with a small brush.

2. Protease digestion

(1) According to the formulations of tables 5 and 6, Proteinase K Solution (protease K Solution) and RNA digest (RNA digest Solution) were prepared.

TABLE 5 Protease K Solution preparation Table

TABLE 6 RNA digest Solution formulation Table

Wherein the digest buffer is formulated as per table 7:

TABLE 7 digest buffer formulation Table

Wherein the protease K solution is prepared and then placed in a water bath at 50 ℃ for preheating for 2-5min to activate the enzyme activity, and the diagnostic solution is prepared and then placed on ice.

(2) Add 70. mu.L of the prepared RNA digest solution to the nuclear pellet in step 1(14)1.5mL tube and gently shake the tube to allow the pellet to exit the bottom of the tube. Note that the nuclear pellet should float freely in the RNA diagnostic solution, and if the pellet is not floating, the tip is used to gently pull up one side of the nuclear pellet until the pellet is free from the bottom of the tube.

(3) The tube was placed in a 50 ℃ water bath, then 70 μ L of incubated protease K solution was added and the tube wall was flicked and mixed well.

(4) The tube caps were capped and incubated at 25 ℃ for approximately 2hours, during which time the tube walls were flicked every 20min or mixed with a wide-mouthed tip. The incubation time may be suitably extended if necessary to allow the precipitate to be sufficiently dispersed and transparent.

3. Purification by dialysis

(1) Pouring at least 500mL of TE buffer into the beaker, putting the beaker into a rotor, and placing the beaker on a magnetic stirring instrument;

(2) transferring the DNA digested in the step 2(4) to a dialysis cup by using a wide-mouth 1000-microliter tip, wherein the pore size of a dialysis membrane in the dialysis cup is 1 micrometer;

(3) the dialysis cup was placed on a float and placed in a beaker containing TE buffer (TE buffer) whose composition is shown in Table 8 below;

(4) and adjusting the speed of the magnetic stirrer, mixing uniformly at a medium speed, and dialyzing by 16-48 hours. If the dialysis exceeds 24 hours, the TE buffer needs to be replaced with fresh one. The dialysis is carried out under medium speed condition, and a better dialysis effect can be obtained. The rotating speed is slow, the molecular diffusion is slow, the concentration of dialysate under the membrane is too high, and the dialysis effect is influenced; the rotation speed is too fast and violent, so that the liquid can splash, the sample loss is caused, and the yield is reduced.

TABLE 8 TE buffer composition

4. Disruption of genomic DNA

(1) After the dialysis, all the liquid on the dialysis membrane was transferred to a 1.5mL centrifuge tube with a wide-mouthed tip, and DNA was observed as a pellet. (Note that the DNA after dialysis is relatively aggregated and in a viscous state);

(2) using a 1mL wide-mouthed tip, the pipettor was adjusted to 150. mu.L, and the most viscous portion of the DNA was aspirated and transferred to a new 1.5mL centrifuge tube. (note that, at this time, the DNA needs to be slowly sucked up, and at the same time, since the DNA is sticky and relatively aggregated, more DNA adhesion is still present after sucking up, and at this time, the tube cover can be closed to cut off the DNA);

(3) slowly blowing and beating the DNA for 8-10 times by using an uncut 1mL gun head to preliminarily break the DNA, and paying attention to the fact that the DNA is difficult to absorb at the moment, so that the blowing and beating process is slow at first, but the DNA is more and more easy to blow and beat along with the increase of the blowing and beating times;

(4) using a 200 mu L pipette, adjusting the range to 150 mu L, further blowing and beating the DNA for about 10 times by using a 200 mu L uncut gun head to realize further breaking, and judging the breaking condition of the DNA: the DNA is aspirated with a 200. mu.L pipette tip and gently ejected, and normally interrupted DNA should be in a state of one droplet at a time. If a series of droplets are continuously ejected, the droplets need to be blown again several times, and the droplets are not broken and are small.

FIG. 1 shows that the main band of the genomic DNA extracted by the method is above 50Kb, the sample integrity is good, the degradation is avoided, and the requirement of 10X Genomics platform library establishment can be met. Wherein M1 and M2 in FIG. 1 represent different DNA markers, 1 represents the extracted genomic DNA by the method of the invention, and BR represents the positive reference DNA with good integrity.

Comparative example 1

At present, the CTAB method is a mainstream technology for extracting long-fragment DNA of plants. CTAB (cetyltrimethylammonium bromide) is a detergent that dissolves cell membranes and forms complexes with nucleic acids, which complexes are soluble in solutions of high ionic strength (0.7mol/L) and can be separated from proteins and polysaccharides by centrifugation. Under the condition of phenol chloroform isoamyl alcohol denaturation, removing residual CTAB, protein and other impurities, then utilizing isoamyl alcohol or absolute ethyl alcohol to precipitate DNA molecules from a supernatant solution, finally using an resolution Buffer to dissolve DNA, and adding RNase to remove RNA in a genome.

The prior art, namely the CTAB method extraction, has the following specific scheme:

a) starting a constant-temperature water bath kettle, setting the temperature to be 65 ℃ for later use (when the room temperature is lower than 15 ℃, carrying out temperature bath on CTAB to extract lysate);

b) taking about 2g of plant sample (sample same as example 1), grinding into flour under liquid nitrogen, transferring into a 50mL centrifuge tube, adding 15mL of 2% CTAB lysate, and shaking and mixing uniformly;

c) incubating at 65 deg.C for 60-90min, and mixing by reversing for 5-10 times;

d) after incubation, centrifuging for 10min at the room temperature of 4000 g;

e) transferring the supernatant to a new corresponding centrifuge tube, adding phenol/chloroform/isoamylol (25:24:1) with equal volume of the supernatant, reversing and uniformly mixing for 20 times, and centrifuging for 10min at the room temperature of 4000 g;

[ Note ] when the supernatant was aspirated, the supernatant was aspirated as much as possible without aspirating the intermediate protein layer, but without aspirating impurities such as the intermediate protein layer.

f) Transferring the supernatant to a new corresponding centrifuge tube, adding phenol/chloroform/isoamylol (25:24:1) with equal volume of the supernatant, reversing and uniformly mixing for 20 times, and centrifuging for 12min at the room temperature of 4000 g;

[ notes ] use centrifuge correctly, balance well before centrifugation. Phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol are volatile, pungent odor organic solvents that require access in a biosafety cabinet or fume hood. When the mask is taken, gloves, masks and the like need to be worn, and protective measures are taken.

g) After the two extractions, if the supernatant is viscous or turbid, repeating the step f;

h) transferring the supernatant to a new corresponding centrifuge tube, adding isopropanol with the volume of 1 time that of the supernatant and 100 mu L of sodium acetate solution, slightly reversing and uniformly mixing;

i) placing the mixture into a refrigerator with the temperature of 20 ℃ below zero for standing overnight;

j) centrifuging at 4000g for 10-20min at room temperature, and after centrifuging, obviously precipitating at the bottom of a centrifugal tube;

k) discarding the supernatant, adding 500-750 mu L75% ethanol, slightly shaking or blowing and washing the precipitate to float the precipitate, and transferring the precipitate into a 1.5mL centrifuge tube;

l) centrifuging at room temperature of 12000g for 3min, removing the supernatant, adding 750 mu L of 75% ethanol, slightly shaking or blowing and washing the precipitate, and allowing the precipitate to float;

m) centrifuging at the room temperature of 12000g for 3min, removing supernatant, and quickly throwing at the room temperature of 12000g for 30 s; removing residual liquid, and air drying for 3-15 min;

n) adding appropriate amount of TE solution (RNAse added, TE solution: RNAse ═ 100:1) in sample tubes, incubate at 37 ℃ for 10-90min until the samples are completely solubilized and briefly centrifuge.

The result is shown in fig. 2, the main band of the genomic DNA is about 50Kb, and the main band has a relatively obvious dispersion phenomenon, which indicates that the DNA is degraded to different degrees, and the integrity of the genomic DNA is relatively poor, which directly results in a relatively short ex-warehouse fragment of the whole library, thereby affecting the assembly effect of subsequent sequencing. In the figure 2, M1 and M2 represent different DNA markers respectively, 1 represents genomic DNA extracted by a CTAB method, and BR represents positive reference DNA with good integrity.

It can be seen from the results obtained by combining the above example 1 and comparative example 1 that the integrity of the obtained genome is significantly improved, the main band is significant and the band is substantially free from dispersion phenomenon by using the method of the present invention compared with the CTAB method for extracting plant genome DNA.

The terms "first", "second" and "first" are used herein for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A method for extracting plant genome DNA, which is characterized by comprising the following steps:

grinding the plant sample, mixing the obtained ground powder with the cell nucleus extracting solution, filtering, and centrifuging the obtained filtrate to obtain a precipitate containing cell nuclei;

subjecting the pellet containing the cell nuclei to density gradient centrifugation using a cell nucleus separating medium to obtain a cell nucleus-containing separating medium;

removing protein and RNA in the separation solution containing the cell nucleus to obtain a solution containing DNA;

subjecting the DNA-containing solution to a dialysis treatment to obtain the plant genomic DNA.

2. The method of claim 1, wherein the cell nucleus extract is a NIBMT solution.

3. The extraction method according to claim 1, wherein the filtration comprises at least two filtration treatments with a filter membrane, wherein the filter membrane pore size of the second filtration treatment is smaller than the filter membrane pore size of the first filtration treatment;

optionally, the filtering comprises: performing a first filtration treatment with a 70 micron pore size filter membrane to obtain a first filtrate; a second filtration treatment was performed using a 40 micron pore size filter membrane to obtain a second filtrate.

4. The extraction method according to claim 1, wherein the cell nucleus separation liquid is Percoll separation liquid.

5. The extraction method according to claim 1, characterized in that said density gradient centrifugation comprises at least:

the Percoll separation solution with volume fraction of 75% and the Percoll separation solution with volume fraction of 70% are used for density gradient centrifugation.

6. The extraction method according to claim 1, wherein the separation liquid containing cell nuclei is subjected to a digestion treatment using protease and rnase to remove proteins and RNA from the separation liquid containing cell nuclei;

optionally, the protease is a proteinase K solution comprising proteinase K, EDTA and SDS.

7. The extraction method according to claim 1, wherein the dialysis treatment is performed using a semipermeable membrane having a pore size of 0.1 μm;

optionally, the dialysis treatment time is 16-48 hours.

8. The extraction method according to claim 1, wherein the plant genomic DNA is greater than 50 Kb.

9. A plant genome DNA extraction kit is characterized by comprising:

a NIBMT solution;

percoll separating medium;

a proteinase K solution;

an RNase;

a semi-permeable membrane;

optionally, the kit is used for extracting plant genomic DNA according to the extraction method of any one of claims 1-8.

10. A method for sequencing genomic DNA of a plant, comprising:

extracting genomic DNA of a plant sample by using the extraction method according to any one of claims 1 to 8;

performing library construction on the plant genome DNA, and performing high-throughput sequencing on the obtained library so as to obtain a sequencing result;

optionally, the high throughput sequencing is performed using a 10X Genomics platform.