Extraction method of polysaccharide polyphenol plant super-long DNA

Abstract

The invention relates to the technical field of molecular biology, in particular to an extraction method of polysaccharide and polyphenol plant super-long DNA. The invention provides an extraction method of polysaccharide and polyphenol plant super-long DNA (deoxyribonucleic acid), which comprises the following steps: after extracting plant cell nucleuses, splitting by using SDS (sodium dodecyl sulfate), adding chloroform for extraction, and precipitating to obtain the super-long DNA. According to the extraction method provided by the invention, after the components of the lysate are optimized and extracted, impurities such as protein are removed in cooperation with chloroform extraction, so that the problem of low purity of DNA extracted from polysaccharide and polyphenol plants at present is solved. According to the method for extracting the super-long DNA of the polysaccharide and polyphenol plant, provided by the invention, the DNA with high purity and relatively long fragments can be obtained, the requirement of a third-generation sequencing platform on the DNA quality can be met, and good sequencing output can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a method for extracting super-long DNA from polysaccharide and polyphenol plants. Background Art

[0002] T2T genome (Telomere-to-Telomere) refers to a high-quality genome from telomere to telomere with high genome accuracy, high continuity, and high integrity. Ultra-long DNA (ultra-long DNA), with a fragment length of hundreds of Kb or even several Mb, combined with Oxford Nanopore PromethION platform sequencing, can effectively span large fragment duplications of hundreds of Kb in the genome, significantly improving the genome assembly problems such as high heterozygosity, polyploidy, and ultra-large genomes. In order to obtain high-quality ultra-long DNA, Wuhan Future Group Biotechnology Co., Ltd. invented a method for extracting ultra-long DNA from plants (patent number: CN 112143776A), but due to the complexity and diversity of plant materials, especially the high content of polysaccharides and polyphenols in forest trees, the purity of the extracted DNA is poor, and the sequencing output of N50=100Kb does not reach an average of 20G.

[0003] The main problems encountered in the extraction of super-long DNA from plants are the extraction of the cell nucleus and the purification of the DNA in the nucleus. During the extraction of the cell nucleus, some plants are difficult to break and have many impurities, especially those with high polysaccharide and polyphenol content, where the nucleus adheres to the endoplasmic reticulum and forms a network. At the same time, the nuclear membrane of some plant cells is connected to some reticular cell components, especially polysaccharides, which will adhere to the nucleus and be recovered. These impurities are lysed by SDS together with the cell nucleus.

[0004] The third-generation sequencing platform has the following requirements for DNA quality: OD260 / 280 ratio ranges from 1.8 to 2.0, OD260 / 230 ratio ranges from 2.0 to 2.5; Nanodrop / Qubit ratio is close to 1.0. Although the ultra-long DNA of polysaccharide and polyphenol plants was extracted according to the existing method, the quality inspection results showed that the DNA length and purity could meet the requirements of the sequencing platform, but the sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process. The output after the machine was completely unable to reach the average sequencing output of 50 to 60G, indicating that the DNA quality inspection data can only be used as a reference and does not necessarily obtain good sequencing results.

[0005] Therefore, there is an urgent need for a method to extract ultra-long plant DNA suitable for third-generation sequencing. Summary of the invention

[0006] The invention provides a method for extracting super-long DNA of polysaccharide and polyphenol plants.

[0007] In the first aspect, the ultra-long DNA extraction method provided by the present invention is suitable for the extraction of ultra-long DNA from polysaccharide and polyphenol plants, and the extraction method includes: crude extraction of cell nuclei, purification of cell nuclei, cell nuclei lysis and DNA purification; the polyvinyl pyrrolidone concentration in the lysis buffer used for cell nucleus lysis is 1.5-2.5wt%, and the SDS concentration is 1.5-3.0wt%.

[0008] When the present invention extracts DNA from polysaccharide and polyphenol plants, it is found that the DNA precipitate is attached with a transparent colloid. The present invention increases the concentration of SDS in the lysis buffer to enhance the lysis effect, and draws on the idea of ​​adding PVP40 to the CTAB lysis buffer to add PVP40 to the SDS lysis buffer. It is found that the addition of PVP40 can effectively separate the polysaccharide substance from the DNA.

[0009] Specifically, adding PVP40 at a concentration of 2 wt % to the lysis buffer can form an insoluble complex with plant polyphenols, effectively remove polyphenols, and reduce phenol contamination in DNA; at the same time, PVP40 can also remove certain polysaccharides.

[0010] In the method for extracting ultra-long DNA provided by the present invention, when purifying the cell nucleus, Triton X-100 with a final concentration of 0.1-0.5 vol% is added alone for lysis, and then the plant cell nucleus is obtained by filtration and centrifugation. Triton X-100, as a surfactant, can dissolve most organelles while maintaining the integrity of the cell nucleus. For different plant varieties and tissue parts, especially polysaccharides and polyphenols, the final concentration of Triton X-100 can be appropriately increased to improve the purity of the cell nucleus.

[0011] In the ultra-long DNA extraction method provided by the present invention, the lysis buffer comprises: Tris-HCl with a concentration of 45-55 mM, NaCl with a concentration of 480-520 mM, EDTA with a concentration of 45-55 mM, SDS with a concentration of 1.5-3.0 wt% and PVP40 with a concentration of 1.5-2.5 wt%.

[0012] In the ultra-long DNA extraction method provided by the present invention, the homogenization buffer comprises: Tris-HCl with a concentration of 15-25 mM, KCl with a concentration of 75-85 mM, sucrose with a concentration of 480-520 mM, EDTA with a concentration of 8-12 mM, PVP10 with a concentration of 0.8-1.2 wt%, β-mercaptoethanol with a concentration of 0.2-0.5 vol%, spermine with a concentration of 0.8-1.2 mM and spermidine with a concentration of 0.8-1.2 mM.

[0013] The ion concentration of the Buffer A solution (homogenization buffer) used in the present invention is close to the level in the plant cell nucleus, and the cell nucleus can remain intact in this solution. In addition, the extract contains 500mM sucrose, which is close to the level in plant cells with high polysaccharide and polyphenol content. The cell nucleus is not easy to break in this environment, which is convenient for homogenization treatment.

[0014] In the ultra-long DNA extraction method provided by the present invention, the extracting solution used for DNA purification is chloroform:isoamyl alcohol, chloroform and isopropanol in a volume ratio of 24:1 respectively.

[0015] As a specific embodiment of the present invention, a method for extracting ultra-long DNA comprises: (1) Crude extraction of cell nuclei: The ground sample is evenly mixed with a homogenization buffer to obtain a homogenate solution I; (2) Purification of cell nuclei: After adding an equal volume of homogenization buffer to the homogenate I of step (1), add TritonX-100 with a final concentration of 0.1-0.5 vol% to obtain a homogenate lysate II, filter the homogenate lysate II through a filter membrane and centrifuge it at a low speed for the first time, collect the supernatant after the first centrifugation and centrifuge it at a high speed for the second time, resuspend and wash the precipitate after the second centrifugation with a homogenization buffer, and centrifuge it again to obtain the plant cell nucleus; The use of membrane filtration can remove some impurities, but the cell nuclei of different plants vary in size, which will affect the recovery rate of the cell nuclei. Preferably, the filter membrane used in the present invention is 70 μm or 40 μm.

[0016] (3) Cell nucleus lysis: adding lysis buffer to the plant cell nucleus obtained in step (2) to obtain a DNA lysis solution; (4) Purification of ultra-long DNA: Add an equal volume of chloroform / isopropanol (24:1) to the DNA lysis solution for extraction. After centrifugation to collect the supernatant, continue to add an equal volume of chloroform to the supernatant for extraction. Centrifuge again to collect the supernatant, add 80% volume of isopropanol to the supernatant, slowly invert and mix to obtain flocculent DNA, which is the ultra-long DNA.

[0017] The plant material used in the present invention is a fresh plant, which can be a whole plant or a part of a plant tissue.

[0018] In the ultra-long DNA extraction method provided by the present invention, during the cell nucleus purification in step (2), the homogenate lysate II is filtered using 70 μm and 40 μm filter membranes, and the intact cells and / or cell fragments are removed by centrifugation to obtain plant cell nuclei.

[0019] In the ultra-long DNA extraction method provided by the present invention, RNase A and proteinase K are omitted in the cell nucleus lysis stage, and SDS is used to destroy non-covalent bonds such as ionic bonds and hydrogen bonds in proteins. 2.5% SDS can effectively remove proteins while enhancing the lysis effect. The extracted nucleic acid is resuspended and dissolved in TE Buffer. Adding an appropriate amount of RNase A to TE Buffer can remove residual RNA and greatly shorten the lysis time. The existing ultra-long DNA extraction process often uses 1.25% SDS lysis solution to add RNase A digestion and proteinase K enzymolysis.

[0020] In the second aspect, the present invention provides a plant ultra-long DNA extraction kit, which includes: a homogenization buffer, a lysis buffer, TritonX-100 and an extracting solution; the homogenization buffer contains 20mM Tris-HCl, 80mM KCl, 500mM sucrose, 10mM EDTA, 1wt% PVP10, 0.5vol% β-mercaptoethanol, 1mM spermine and 1mM spermidine; the lysis buffer contains 50mM Tris-HCl, 500mM NaCl, 50mM EDTA, 2.5wt% SDS and 2wt% PVP40; the extracting solution contains chloroform:isoamyl alcohol, chloroform and isopropanol in a volume ratio of 24:1.

[0021] By using the extraction kit provided by the present invention to replace the conventional DNA extraction kit, high-quality plant genomic DNA can be obtained, which fully meets the requirements of the third-generation sequencing machine and obtains more sequencing data output.

[0022] According to the understanding of those skilled in the art, the present invention also provides the use of the above-mentioned ultra-long DNA extraction method or the above-mentioned plant ultra-long DNA extraction kit in shortening the plant ultra-long DNA extraction time.

[0023] And the use of the above-mentioned ultra-long DNA extraction method or the above-mentioned plant ultra-long DNA extraction kit in improving the quality of plant DNA extraction.

[0024] The beneficial effects of the present invention are: By using the method for extracting ultra-long DNA from polysaccharide and polyphenol plants provided by the present invention, high-purity and long-fragment DNA can be obtained, which can meet the requirements of the third-generation sequencing platform for DNA quality and obtain good sequencing output. At the same time, the method for extracting ultra-long DNA from polysaccharide and polyphenol plants provided by the present invention also has the advantages of simple operation steps and short extraction time, and 6 to 8 samples can be extracted within one day. The sequencing results show that the data volume of N50 100Kb of grape DNA extracted according to the method provided by the present invention can reach 28.2G, while the data volume of N50 100Kb of extracted oak leaf DNA can reach 29.7G. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is the result of pulsed electrophoresis, wherein M1 is maker Lamda PFG Ladder, M2 is Quick-Load 1kbExtend DNA Ladder, and the lanes indicated by numbers are test samples, specifically: Lane 1 is the grape leaf genomic DNA sample extracted by the method of Example 1; Lane 2 is the oak leaf genomic DNA sample extracted by the method of Example 2; Lane 3 is the nanmu leaf genomic DNA sample extracted by the method of Example 3; Lane 4 is the selfheal bud genomic DNA sample extracted by the method of Example 4.

[0027] Figure 2 It is the result of pulsed electrophoresis, where M1 is maker Lamda PFG Ladder, M2 is Quick-Load 1kbExtend DNA Ladder, and the lanes marked with numbers are test samples. Specifically: Lane 5 is the DNA sample extracted by the method of Example 1; Lane 6 is the DNA sample extracted by the method of Example 2; Lane 7 is the DNA sample extracted by the method of Example 3.

[0028] Figure 3 It is the result of pulsed electrophoresis, wherein M1 is maker Lamda PFG Ladder, M2 is Quick-Load 1kbExtend DNA Ladder, lane 8 is the DNA sample extracted by the method of Example 4; lane 9 is the DNA sample extracted by the method of Example 5. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The buffer used in the embodiment of the present invention is: Buffer A solution (homogenization buffer): Tris-HCl concentration is 20mM, KCl concentration is 80mM, sucrose concentration is 500mM, EDTA concentration is 10mM, PVP10 concentration is 1wt%, β-mercaptoethanol concentration is 0.5vol%, spermine concentration is 1mM, and spermidine concentration is 1mM.

[0031] Buffer B solution (lysis buffer): Tris-HCl concentration is 50 mM, NaCl concentration is 500 mM, EDTA concentration is 50 m M, SDS concentration is 2.5wt%, and PVP40 concentration is 2wt%.

[0032] Example 1 This embodiment provides a method for extracting ultra-long DNA from grape leaves, which is as follows: (1) Crude extraction of nuclei Take 1g of sample and put it into a mortar precooled with liquid nitrogen. Keep half the volume of liquid nitrogen in the mortar. Use a pestle to grind evenly for about 2 minutes until the sample is a particle of about 5 mm. Transfer the ground sample to a 50mL centrifuge tube, add 15mL of buffer A solution to the centrifuge tube, mix the sample and Buffer A solution (homogenization buffer) evenly, and use Qiagen Tissue Ruptor to homogenize at 10000rpm for 3 times, each time for 18s.

[0033] (2) Cell nucleus purification 1) Add 15 mL of buffer A solution again, homogenize once at 8000 rpm, then add TritonX-100 with a final concentration of 0.5 vol%, invert to mix, and lyse on ice for 30 min.

[0034] 2) After the lysis is completed, the plant homogenate is filtered using 70μm and 40μm filter membranes respectively; centrifuged at 4°C and 60rcf for 2 minutes to remove intact cells, cell fragments and other impurities.

[0035] 3) Collect the supernatant after centrifugation and centrifuge at 4°C and 1200rcf for 15 minutes. Wash the precipitate twice with buffer A solution and collect the precipitate by centrifugation to obtain the plant cell nucleus.

[0036] (3) Nuclear lysis The collected precipitate was first resuspended in 100 μL of 1x PBS solution, and then lysed by adding 5 mL of buffer B at 50 °C for 30 min; (4) Purification of ultra-long DNA 1) Add an equal volume of chloroform / isoamyl alcohol (24:1) to extract once, ensure that the lysate and 24:1 are mixed by inversion, centrifuge at 8000rcf for 15 minutes at room temperature, and take the supernatant; 2) Add an equal volume of chloroform to extract once, ensure that the lysate is mixed evenly with 24:1 by inversion, centrifuge at 8000rcf for 15 minutes at room temperature, and take the supernatant; 3) Add 80% volume of isopropanol and slowly invert to mix until flocculent DNA precipitates. 4) Wash the DNA precipitate twice with 75% ethanol. After the ethanol evaporates completely, add 100ul of TE Buffer containing RNase A (1ul / mL) to dissolve the DNA.

[0037] (5) The DNA quality test results are as follows:

[0038] The quality inspection results show that the DNA extracted in this example meets the requirements for the third-generation sequencing machine. The sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process, and the N50 100K data output was 28.2G. The pulsed electrophoresis results of the grape leaf genomic DNA sample are shown in Figure 1 .

[0039] Example 2 This example provides a method for extracting super-long DNA from oak leaves. This example uses the same extraction method as in Example 1 to extract DNA from oak leaves. The DNA quality test results are as follows:

[0040] The quality inspection results show that the DNA extracted in this example meets the requirements for the third-generation sequencing machine. The sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process, and the N50 100K data output was 29.7G. The pulsed electrophoresis results of the DNA samples are shown in Figure 1 .

[0041] Example 3 This embodiment provides a method for extracting super-long DNA from Phoebe nanmu leaves. This embodiment uses the same extraction method as in Example 1 to extract DNA from Phoebe nanmu leaves. The DNA quality test results are as follows:

[0042] The quality inspection results show that the DNA extracted in this example meets the requirements for the third-generation sequencing machine. The sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process, and the N50 100K data output was 40.3G. The pulsed electrophoresis results of the DNA samples are shown in Figure 1 .

[0043] Example 4 This example provides a method for extracting super-long DNA from Prunella vulgaris flower buds. This example uses the same extraction method as in Example 1 to extract DNA from Prunella vulgaris flower buds. The DNA quality test results are as follows:

[0044] The quality inspection results showed that the DNA extracted in this example met the requirements for the third-generation sequencing machine. The sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process, and the N50 100K data The mass production is 20.5G. The pulsed electrophoresis results of DNA samples are shown in Figure 1 .

[0045] Comparative Example 1 This comparative example is consistent with the existing published patent method. The experimental materials of this comparative example are the same as those of Example 1. This comparative example uses grape leaves as samples, and the specific scheme for extracting ultra-long DNA is as follows.

[0046] The buffer used in this comparative example is: Homogenization buffer: Tris-HCl 10 mM; EDTA 10 mM; potassium chloride 80 mM; sucrose 0.5 M; Triton X-100 0.50%; polyvinyl pyrrolidone 1%; spermine 1 mM; spermidine 1 mM; β-mercaptoethanol 0.2%; pH value is 8.0.

[0047] Nuclear lysis buffer: Tris-HCl 10mM; EDTA 20mM; NaCl 100mM; SDS 1.25%; Nuclear Wash Buffer: Tris-HCl 50 mM, EDTA 10 mM.

[0048] 1. Isolation of Nuclei 1.1 Crude Isolation of Cell Nuclei (1) Take 1 g of frozen leaves and cut into pieces; (2) Add 15 mL of homogenization buffer; then homogenize using a Qiagen Tissue Ruptor at 35,000 rpm for 4 times, each time for 20 seconds.

[0049] 1.2 Purification of cell nuclei separation fluid (3) Add an equal volume of homogenization buffer and filter the plant tissue homogenate using 100 μm and 40 μm filter membranes in turn; (4) Centrifuge at 80 rcf for 3 min at 4°C to remove intact cells, cell debris, and other residues; (5) Discard the supernatant and add 3 ml of homogenization buffer to the crude cell nucleus pellet. After the cell nuclei are completely dispersed with a brush, add homogenization buffer to 30 ml. (6) Place on ice for 30 min to fully lyse the cell membrane; (7) Centrifuge at 2500 rcf for 20 min to obtain a crude nuclear pellet, and wash the crude nuclei three times with homogenization buffer; (8) Centrifuge at 2500 rcf for 20 min and wash the pellet three times with nuclear wash buffer; 2. Nuclear Lysis 2.1 Digestion of cells (9) Lyse the product of step (8) with 15 mL of nuclear lysis buffer; (10) Add RNase A (3 μL / mL) and react at 37°C for 1 h. (11) Add Proteinase K (20 μL / mL) and react at 50°C for 3 h; 2.2 Ultralong DNA purification (12) Extract once with an equal volume of phenol, centrifuge at 4500 rcf for 10 min at room temperature, and collect the supernatant; (13) Extract once with an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), centrifuge at 4500 rcf for 10 min at room temperature, and collect the supernatant; (14) Extract once with an equal volume of chloroform / isoamyl alcohol (24:1), centrifuge at 4500 rcf for 10 min at room temperature, and collect the supernatant; (15) Precipitate with an equal volume of anhydrous ethanol; (16) Wash the DNA pellet twice with 70% ethanol; (17) After the ethanol has evaporated completely, add 100 μL of EB to dissolve the DNA.

[0050] 3. Quality inspection results

[0051] The quality inspection results show that the DNA extracted in this comparative example meets the requirements for the third-generation sequencing machine, but the purity is slightly lower than that of Example 1. According to the standard process, the Oxford Nanopore PromethION platform is used for sequencing, and the N50 100K data volume only produces 11.2G. The pulsed electrophoresis results of the DNA samples are shown in Figure 2 .

[0052] Comparative Example 2 Different concentrations of SDS in lysis buffer The experimental materials used in this comparative example are the same as those in Example 1. In this comparative example, grape leaves are used as samples. The difference between the DNA extraction method used in this comparative example and that in Example 1 is that the SDS concentration in the lysis buffer used for cell nucleus lysis is 1.25 wt%, and the lysis is performed for 30 min. The DNA quality test results are as follows:

[0053] The quality inspection results show that the nucleic acid extracted in this comparative example does not meet the requirements for the third-generation sequencing machine, with low purity and small total amount. The pulsed electrophoresis results of the DNA samples are shown in Figure 2 .

[0054] Comparative Example 3 Different concentrations of SDS in lysis buffer The experimental materials used in this comparative example are the same as those in Example 1. In this comparative example, grape leaves are used as samples. The difference between the DNA extraction method used in this comparative example and that in Example 1 is that the SDS concentration in the lysis buffer used for cell nucleus lysis is 2 wt%, and the lysis is performed for 30 min. The DNA quality inspection results are as follows:

[0055] The quality inspection results show that the nucleic acid extracted in this comparative example meets the requirements for the third-generation sequencing machine, but the total amount of nucleic acid extracted is less than that of 2.5% SDS lysis. The pulsed electrophoresis results of the DNA samples are shown in Figure 2 Comparative Example 4 Different concentrations of SDS in lysis buffer The experimental materials used in this comparative example are the same as those in Example 1. In this comparative example, grape leaves are used as samples. The difference between the DNA extraction method used in this comparative example and that in Example 1 is that the SDS concentration in the lysis buffer used for cell nucleus lysis is 3 wt%, and the lysis is performed for 30 min. The DNA quality inspection results are as follows:

[0056] The quality inspection results show that the nucleic acid extracted in this comparative example meets the requirements for the third-generation sequencing machine, but from the electrophoresis gel image, it can be seen that the DNA fragments extracted using 3% SDS are shorter than those extracted using 2.5% SDS. The pulsed electrophoresis results of the DNA samples are shown in Figure 3 Comparative Example 5 PVP40 was not added to the lysis buffer The experimental materials used in this comparative example are the same as those in Example 1. Grape leaves are used as samples in this comparative example. The difference between the DNA extraction method used in this comparative example and that in Example 1 is that PVP40 is not added to the lysis solution, and the lysis is performed for 30 minutes. The DNA quality test results are as follows:

[0057] The quality inspection results show that the nucleic acid extracted in this comparative example meets the requirements for the third-generation sequencing machine. However, when sequencing was performed using the Oxford Nanopore PromethION platform according to the standard process, the sequencing chip was severely blocked, and the N50 100K data volume only produced 9.8G. The pulsed electrophoresis results of the DNA samples are shown in Figure 3 .

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting ultra-long DNA, characterized in that: The extraction method is suitable for extracting ultra-long DNA from polysaccharide and polyphenol plants, and comprises: crude extraction of cell nuclei, purification of cell nuclei, cell nuclei lysis and DNA purification; the polyvinyl pyrrolidone concentration in the lysis buffer used for cell nuclei lysis is 1.5-2.5wt%, and the SDS concentration is 1.5-3.0wt%.

2. The method for extracting ultra-long DNA according to claim 1, characterized in that: When purifying the cell nucleus, add a homogenization buffer, then add TritonX-100 with a final concentration of 0.1~0.5 vol% for lysis, and then obtain the plant cell nucleus by filtration and centrifugation.

3. The method for extracting ultra-long DNA according to claim 1, characterized in that: The lysis buffer comprises: Tris-HCl with a concentration of 45-55 mM, NaCl with a concentration of 480-520 mM, EDTA with a concentration of 45-55 mM, SDS with a concentration of 1.5-3.0 wt % and PVP40 with a concentration of 1.5-2.5 wt %.

4. The method for extracting ultra-long DNA according to claim 2, characterized in that: The homogenization buffer comprises: Tris-HCl with a concentration of 15-25 mM, KCl with a concentration of 75-85 mM, sucrose with a concentration of 480-520 mM, EDTA with a concentration of 8-12 mM, PVP10 with a concentration of 0.8-1.2 wt%, β-mercaptoethanol with a concentration of 0.2-0.5 vol%, spermine with a concentration of 0.8-1.2 mM and spermidine with a concentration of 0.8-1.2 mM.

5. The method for extracting ultra-long DNA according to claim 1, characterized in that: The extraction solvents used for DNA purification were chloroform:isoamyl alcohol, chloroform and isopropanol in a volume ratio of 24:1, respectively.

6. The method for extracting ultra-long DNA according to any one of claims 1 to 5, characterized in that: include: (1) Crude extraction of cell nuclei: The ground sample is uniformly mixed with a homogenization buffer for homogenization to obtain a homogenate solution I; (2) Purification of cell nuclei: After adding an equal volume of homogenization buffer to the homogenate I of step (1), add TritonX-100 with a final concentration of 0.1-0.5 vol% to obtain a homogenate lysate II, filter the homogenate lysate II through a filter membrane and centrifuge it at a low speed for the first time, collect the supernatant after the first centrifugation and centrifuge it at a high speed for a second time, resuspend and wash the precipitate after the second centrifugation with a homogenization buffer, and centrifuge it at a high speed again to obtain the plant cell nucleus; (3) Cell nucleus lysis: adding lysis buffer to the plant cell nucleus obtained in step (2) to obtain a DNA lysis solution; (4) Purification of ultra-long DNA: Add an equal volume of chloroform / isoamyl alcohol to the DNA lysate for extraction. After centrifugation to collect the supernatant, continue to add an equal volume of chloroform to the supernatant for extraction. After centrifugation to collect the supernatant, add 80% volume of isopropanol to the supernatant, slowly invert and mix to obtain flocculent DNA, which is the ultra-long DNA.

7. The method for extracting ultra-long DNA according to claim 6, characterized in that: In step (2) of cell nucleus purification, the homogenate lysate II is filtered using 70 μm and 40 μm filter membranes, and the intact cells and / or cell fragments are removed by low-speed centrifugation, and the plant cell nuclei are obtained by high-speed centrifugation.

8. A kit for extracting ultra-long DNA from plants, characterized in that: The plant ultra-long DNA extraction kit comprises: a homogenization buffer, a lysis buffer, TritonX-100 and an extracting solution; the homogenization buffer contains 20mM Tris-HCl, 80mM KCl, 500mM sucrose, 10mM EDTA, 1wt% PVP10, 0.5vol% β-mercaptoethanol, 1mM spermine and 1mM spermidine; the lysis buffer contains 50mM Tris-HCl, 500mM NaCl, 50mM EDTA, 2.5wt% SDS and 2wt% PVP40; the extracting solution contains chloroform:isoamyl alcohol, chloroform and isopropanol in a volume ratio of 24:

1.

9. Use of the ultra-long DNA extraction method according to any one of claims 1 to 7 or the ultra-long DNA extraction kit for plants according to claim 8 in shortening the ultra-long DNA extraction time for plants.

10. Use of the ultra-long DNA extraction method according to any one of claims 1 to 7 or the ultra-long DNA extraction kit for plants according to claim 8 in improving the quality of plant DNA extraction.

Images