阅读说明：本技术 一种植物基因组dna的提取方法及其应用 (Extraction method and application of plant genome DNA ) 是由 郑洪坤 毕经德 骆晨 朱艳荣 王瑞 于 2019-09-11 设计创作，主要内容包括：本发明涉及分子生物学技术领域,具体涉及一种植物基因组DNA的提取方法及其应用。本发明提供一种植物基因组DNA的提取方法,其为在利用CTAB裂解植物细胞后,加入SDS和EDTA溶液进行抽提,经沉淀获得基因组DNA。该方法采用CTAB裂解试剂进行植物组织细胞裂解、配合SDS和EDTA作为抽提试剂沉淀去除蛋白质等杂质。利用本发明提供的植物基因组DNA提取方法能够获得高浓度、高纯度和高完整性的植物基因组DNA,可满足基因组测序的要求,同时避免了酚/氯仿有毒化学试剂的使用；该方法还具有成本低廉、操作步骤简单、提取耗时短等优势,适用于大量植物样本基因组测序的基因组DNA的提取。(The invention relates to the technical field of molecular biology, in particular to an extraction method of plant genome DNA and application thereof. The invention provides a method for extracting plant genome DNA, which is characterized in that after a CTAB is utilized to crack plant cells, SDS and EDTA solution are added for extraction, and the genome DNA is obtained through precipitation. The method adopts a CTAB lysis reagent to perform plant histiocyte lysis, and adopts SDS and EDTA as extraction reagents to precipitate and remove impurities such as protein and the like. By utilizing the plant genome DNA extraction method provided by the invention, the plant genome DNA with high concentration, high purity and high integrity can be obtained, the requirement of genome sequencing can be met, and the use of phenol/chloroform toxic chemical reagents is avoided; the method also has the advantages of low cost, simple operation steps, short extraction time and the like, and is suitable for extracting the genome DNA of the genome sequencing of a large number of plant samples.)

1. A method for extracting plant genome DNA is characterized in that after a CTAB lysate is used for cracking plant cells, an extracting agent is added for extraction, and the genome DNA is obtained through precipitation;

the extractant comprises SDS and EDTA.

2. The extraction method according to claim 1, wherein the extraction agent is added in an amount such that the mass-to-volume ratio g of SDS in the extraction system: ml is 0.9-1.1%, and the concentration of EDTA is 4-6 mM.

3. The extraction method according to claim 1 or 2, wherein the extraction is performed by standing at-15 to-20 ℃ for 5 to 10min, and then centrifuging at low temperature to collect the lower solution.

4. The extraction method according to any one of claims 1 to 3, wherein the CTAB lysate comprises CTAB, NaCl, PVP40, Tris-HCl, EDTA, D-Sorbitol, anhydrous sodium sulfite and beta-mercaptoethanol.

5. The extraction method according to claim 4, wherein the CTAB lysate is added in an amount such that the concentration of NaCl in the lysis system is 1.8-2.2M;

preferably, the CTAB lysate is added in an amount such that the mass-to-volume ratio g of CTAB in the lysis system: ml is 1.8-2.2%, and the mass-volume ratio g of PVP40 is as follows: 1.8-2.2% of ml, 90-110 mM of Tris-HCl, 22.5-27.5 mM of EDTA, 315-385 mM of D-Sorbitol, 1.8-2.2M of NaCl, 90-110 mM of anhydrous sodium sulfite and 1.5-3% of beta-mercaptoethanol by volume.

6. The extraction method according to any one of claims 1 to 5, wherein the cracking is carried out at 60 to 65 ℃ for 30 to 50 min.

7. The extraction method according to any one of claims 1 to 6, wherein the precipitation is isopropanol precipitation;

preferably, the isopropanol precipitation is to add sodium acetate and isopropanol and place for 20-30 min at-20 ℃;

more preferably, the addition amount of the sodium acetate is that the concentration of the sodium acetate in a precipitation system is 150-200 mM; the addition amount of the isopropanol is 35-40% of the total volume of the precipitation system.

8. The extraction method according to any one of claims 1 to 7, comprising the steps of:

(1) fully grinding plant tissues in liquid nitrogen to obtain plant tissue powder;

(2) adding the CTAB lysate into the plant tissue powder in the step (1) until the mass-volume ratio g of CTAB in a lysis system is as follows: ml is 1.8-2%, and the mass-volume ratio g of PVP40 is as follows: 1.8-2% of ml, 90-100 mM of Tris-HCl, 22.5-25 mM of EDTA, 315-350 mM of D-Sorbitol, 1.8-2M of NaCl, 90-100 mM of anhydrous sodium sulfite and 1.5-2% of beta-mercaptoethanol by volume, and treating the mixture at 65 ℃ for 30-50 min;

(3) standing at room temperature for 3-5 min, and centrifuging at low temperature to collect supernatant; adding the extracting agent into the collected supernatant, and standing at-20 ℃ for 5-10 min for extraction; the addition amount of the extraction agent is that the mass volume ratio g of SDS in an extraction system is: ml is 0.9-1%, and the concentration of EDTA is 4-5 mM;

(4) centrifuging at low temperature, and collecting the lower layer solution; adding sodium acetate and isopropanol into the collected lower-layer solution, and precipitating at-20 ℃ for 20-30 min; the addition amount of the sodium acetate is such that the concentration of the sodium acetate in a precipitation system is 150-200 mM; the addition amount of the isopropanol is 35-40% of the total volume of the precipitation system;

(5) centrifuging at low temperature, removing supernatant, washing precipitate with ethanol for 2 times, drying, dissolving genome DNA, and adding RNase to remove RNA.

9. A plant genome DNA extraction kit is characterized by comprising a reagent A, a reagent B and a reagent C; the reagent A comprises CTAB in a mass-to-volume ratio g: 1.8-2.2% of ml, and the mass-volume ratio g of PVP 40: 1.8-2.2% ml, 90-110 mM Tris-HCl, 22.5-27.5 mM EDTA, 315-385 mM D-Sorbitol, 1.8-2.2M NaCl, 90-110 mM anhydrous sodium sulfite; the reagent B comprises beta-mercaptoethanol; the reagent C comprises the following components in mass-volume ratio g: ml 9-11% SDS and 45-55 mM EDTA.

10. Use of the method for extracting plant genomic DNA according to any one of claims 1 to 8 or the kit for extracting plant genomic DNA according to claim 9 for constructing a plant genomic sequencing library.

Technical Field

The invention relates to the technical field of molecular biology, in particular to an extraction method of plant genome DNA and application thereof.

Background

With the development of genome sequencing technology, the research on the sequence, structure and function of plant genome is rapidly developed, and the acquisition of plant genome DNA with high purity, high content and high integrity is the first prerequisite for the application of genome sequencing technology. The extraction of plant genome DNA is more difficult than the extraction of microorganism, animal tissue or blood sample genome because of the large amount of polysaccharide, polyphenol substances and other secondary metabolites with complex structures in the plant tissue. In addition, the requirements of genome sequencing library construction on the integrity, purity and concentration of genome DNA are higher than those of common experiments such as a template for detection amplification experiments, so that the plant genome DNA capable of meeting the genome sequencing requirements is more difficult to obtain.

The CTAB method is currently the most economical and effective method and the most common method for extracting plant genome DNA. However, in the extraction process, phenol, chloroform or chloroform, isoamyl alcohol extraction steps are used to achieve the purpose of removing impurities such as protein, so that the purity of the extracted genomic DNA meets the application requirements of subsequent NGS sequencing and the like. In 27.10.2017, in the precautionary reference of carcinogen list published by international cancer research institution of world health organization, chloroform is in the category 2B carcinogen list. As phenol and chloroform extraction reagents used in the traditional CTAB method are toxic, the safety problem of experimenters is greatly threatened. However, the alternative method of the CTAB method reported in the prior art has problems of high cost and unsuitability for processing a large amount of samples (such as magnetic bead purification and column purification), or has a problem of low DNA purity (such as SDS lysis method) caused by incomplete removal of impurities such as polysaccharides and polyphenols. How to obtain plant genome DNA with high purity, high concentration and high integrity and capable of meeting the requirement of genome sequencing by utilizing safer reagents, a simple extraction process and lower reagent cost is a technical problem which needs to be solved at present.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a plant genome DNA extraction method which can obtain genome DNA with high purity, high concentration and high integrity and meeting the requirement of genome sequencing without using toxic reagents of phenol and chloroform.

In order to achieve the purpose, the technical scheme of the invention is as follows:

through a large amount of research and practice, the invention discovers that SDS and EDTA as extraction reagents can well replace phenol/chloroform/isoamylol extraction reagents in the traditional CTAB method to obtain plant genome DNA with quality (purity, concentration and integrity) equivalent to or better than that of the traditional CTAB method, and provides the plant genome DNA extraction method which uses CTAB lysis reagent for plant tissue cell lysis and uses SDS and EDTA as extraction reagents for removing impurities such as protein and the like through precipitation based on the discovery.

The invention provides a method for extracting plant genome DNA, which comprises the steps of cracking plant cells by CTAB lysate, adding an extracting agent for extraction, and obtaining genome DNA by precipitation; the extractant comprises SDS and EDTA.

Preferably, the addition amount of the extraction agent is such that the mass-to-volume ratio g of SDS in the extraction system: ml is 0.9-1.1%, and the concentration of EDTA is 4-6 mM.

The invention discovers that the extraction by SDS and EDTA with the concentration can better play the role of removing protein and other impurities by precipitation and ensure the integrity of genome DNA.

Further preferably, the extractant is an aqueous solution of SDS and EDTA.

Further preferably, the extraction is carried out at a temperature of between 15 ℃ below zero and 20 ℃ below zero for 5 to 10min, and then the lower layer solution is collected by low-temperature centrifugation. Extraction under the low temperature condition can ensure the integrity of the genome DNA while SDS and EDTA can better play a role in removing protein and other impurities by precipitation.

In the invention, the CTAB lysate contains CTAB, NaCl, PVP40, Tris-HCl, EDTA, D-Sorbitol, anhydrous sodium sulfite and beta-mercaptoethanol.

Preferably, the CTAB lysate is added in an amount such that the concentration of NaCl in the lysis system is 1.8-2.2M. The concentration of NaCl in the CTAB lysate can be better matched with the extraction steps of SDS and EDTA, the complexing of protein and SDS is promoted to form a compound, and the aim of separating and removing impurities such as protein and the like is better fulfilled.

Further preferably, the addition amount of the lysis solution is such that the mass-to-volume ratio g of CTAB in the lysis system: ml is 1.8-2.2%, and the mass-volume ratio g of PVP40 is as follows: 1.8-2.2% of ml, 90-110 mM of Tris-HCl, 22.5-27.5 mM of EDTA, 315-385 mM of D-Sorbitol, 1.8-2.2M of NaCl, 90-110 mM of anhydrous sodium sulfite and 1.5-3% of beta-mercaptoethanol by volume. The CTAB lysis method can be better matched with the extraction steps of SDS and EDTA while ensuring the full lysis of plant histiocyte, and is more beneficial to the acquisition of high-quality genome DNA.

Further preferably, the CTAB cracking is carried out for 30-50 min at 60-65 ℃.

In the present invention, the precipitation is preferably performed using isopropanol.

The isopropanol precipitation is preferably carried out by adding sodium acetate and isopropanol into the extracted lower layer solution and standing at-20 ℃ for 20-30 min.

Further preferably, the addition amount of the sodium acetate is such that the concentration of the sodium acetate in a precipitation system is 150-200 mM; the addition amount of the isopropanol is 35-40% of the total volume of the precipitation system.

As a preferred embodiment of the present invention, the method for extracting plant genomic DNA comprises the following steps:

(1) fully grinding plant tissues in liquid nitrogen to obtain plant tissue powder;

(2) adding the CTAB lysate into the plant tissue powder in the step (1) until the mass-volume ratio g of CTAB in a lysis system is as follows: ml is 1.8-2%, and the mass-volume ratio g of PVP40 is as follows: 1.8-2% of ml, 90-100 mM of Tris-HCl, 22.5-25 mM of EDTA, 315-350 mM of D-Sorbitol, 1.8-2M of NaCl, 90-100 Mm of anhydrous sodium sulfite and 1.5-2% of beta-mercaptoethanol by volume, and treating the mixture at 65 ℃ for 30-50 min;

(3) standing at room temperature for 3-5 min, and centrifuging at low temperature to collect supernatant; adding the extracting agent into the collected supernatant, and standing at-20 ℃ for 5-10 min for extraction; the addition amount of the extraction agent is that the mass volume ratio g of SDS in an extraction system is: ml is 0.9-1%, and the concentration of EDTA is 4-5 mM;

(4) centrifuging at low temperature, and collecting the lower layer solution; adding sodium acetate and isopropanol into the collected lower-layer solution, and precipitating at-20 ℃ for 20-30 min; the addition amount of the sodium acetate is such that the concentration of the sodium acetate in a precipitation system is 150-200 mM; the addition amount of the isopropanol is 35-40% of the total volume of the precipitation system;

(5) centrifuging at low temperature, removing supernatant, washing precipitate with ethanol for 2 times, drying, dissolving genome DNA, and adding RNase to remove RNA.

As a more preferred embodiment of the present invention, the method for extracting plant genomic DNA comprises the steps of:

(1) fully grinding plant tissues in liquid nitrogen to obtain plant tissue powder;

(2) adding preheated CTAB lysate into the plant tissue powder in the step (1) until the mass-volume ratio g of CTAB in a lysis system is as follows: ml is 2.0%, and the mass-to-volume ratio g of PVP40 is as follows: the solution is treated at 65 ℃ for 40min, wherein ml is 2.0 percent, the concentration of Tris-HCl is 100mM, the concentration of EDTA is 25mM, the concentration of D-Sorbitol is 350mM, the concentration of NaCl is 2M, the concentration of anhydrous sodium sulfite is 100mM, and the volume percentage of beta-mercaptoethanol is 2 percent;

(3) standing at room temperature for 5min, centrifuging at 4 deg.C and 15000 Xg for 10min, and collecting supernatant; adding the extractant into the collected supernatant, and standing at-20 deg.C for 10min for extraction; the addition amount of the extractant is such that the mass volume ratio g of SDS in the extraction system is as follows: ml is 1%, and the concentration of EDTA is 5 mM;

(4) centrifuging at 15000 Xg for 10min at 4 deg.C, and collecting the lower layer solution (avoiding the upper solid protein layer); adding 1/10 volume of 3M sodium acetate and 0.7 volume of isopropanol into the collected lower layer solution, and precipitating at-20 deg.C for 30 min;

(5) centrifuging at low temperature, discarding supernatant, washing precipitate with ethanol for 2 times, drying, dissolving genomic DNA with TE solution containing 10 ng/. mu.l RNase, and incubating at 37 deg.C for 30min to obtain genomic DNA.

The invention also provides a plant genome DNA extraction kit, which comprises a reagent A, a reagent B and a reagent C; the reagent A comprises CTAB in a mass-to-volume ratio g: 1.8-2.2% of ml, and the mass-volume ratio g of PVP 40: 1.8-2.2% ml, 90-110 mM Tris-HCl, 22.5-27.5 mM EDTA, 315-385 mM D-Sorbitol, 1.8-2.2M NaCl, 90-110 mM anhydrous sodium sulfite; the reagent B comprises beta-mercaptoethanol; the reagent C comprises SDS in a mass-to-volume ratio of g: ml 9-11% and 45-55 mM EDTA.

Preferably, the solvent of the reagent a and the reagent C is water.

The invention also provides an extraction method of the plant genome DNA or an application of the plant genome DNA extraction kit in construction of a plant genome sequencing library.

The invention has the beneficial effects that:

the invention provides a method for extracting plant genome DNA, which adopts CTAB cracking reagent to crack plant tissue cells and adopts SDS and EDTA as extraction reagent to precipitate and remove impurities such as protein and the like. The plant genome DNA extraction method provided by the invention can be used for obtaining the plant genome DNA with high concentration, high purity and high integrity, and can meet the requirement of genome sequencing.

By utilizing the method for extracting the plant genome DNA, the quality of the extracted genome DNA is ensured, the use of phenol/chloroform toxic chemical reagents is avoided, and the potential harm to the body health of experimenters and the damage of experimental waste liquid to the ecological environment are effectively reduced. Meanwhile, the plant genome DNA extraction method provided by the invention has the advantages of low cost, simple operation steps, short extraction time and the like, has excellent cost and time advantages compared with genome DNA extraction methods such as a column purification method, a magnetic bead method and the like, and is suitable for extracting genome DNA for sequencing a large number of plant sample genomes.

Drawings

FIG. 1 is a schematic flow chart of a method for extracting plant genomic DNA according to example 1 of the present invention.

FIG. 2 is a diagram showing the integrity of genomic DNA detected by agarose gel electrophoresis in Experimental example 1 of the present invention; wherein M represents Marker (HindIII-digested product of lambda DNA), CK is lambda DNA as a control, lane 1 is a wheat leaf genome DNA sample extracted by the method of example 1, and lane 2 is a wheat leaf genome DNA sample extracted by the method of comparative example 1.

FIG. 3 is a diagram showing the integrity of genomic DNA detected by agarose gel electrophoresis in Experimental example 2 of the present invention; wherein M represents Marker (HindIII digest of lambda DNA), lanes 1-7 are, in order, leaf genomic DNA samples of rice, cotton, sorghum, soybean, peanut, millet, and corn extracted using the method of example 1.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.