CN107574166B - Cell lysate for extracting soybean seed genome DNA and extraction method thereof - Google Patents
Cell lysate for extracting soybean seed genome DNA and extraction method thereof Download PDFInfo
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Abstract
The invention relates to a cell lysate for extracting soybean seed genome DNA, which is a pure water solution containing 0.015-0.025 g/ml SDS, 0.04-0.06 mol/L Tris-HCl, 0.04-0.06 mol/L EDTA and 0.14-0.16 mol/L NaCl, and has a pH value of 8; the invention also relates to a method for extracting the soybean seed genome DNA by adopting the cell lysate; the inventor finds that the cell lysate adopting the specific formula is particularly suitable for extracting the soybean seed genome DNA, and can improve the yield and the purity of the soybean seed genome DNA.
Description
Technical Field
The invention relates to a cell lysate for extracting soybean seed genome DNA and an extraction method thereof, belonging to the field of molecular biology.
Background
Soybean (Glycine max), a leguminous soybean belonging to annual herbaceous plant, originated from China, is rich in protein, fat, carbohydrate, cellulose, mineral substances, vitamins and the like, wherein the protein content is about 40 percent, the soybean is rich in 8 essential amino acids and is an ideal intake source of vegetable protein of a human body, the soybean oil content is about 20 percent, the soybean oil has high unsaturation degree and is typical health oil of linoleic acid, and the soybean also contains various nutritional factors with special effects such as phenols, vitamin E, polypeptide, saponin, oligosaccharide and the like.
Soybean is an important source for protein intake of residents in China, and China is still a net export country of soybean before 1995, but then along with the rapid increase of the demand of the soybean in China and the large-area commercial planting of transgenic soybean in other countries, the import of the soybean in China is increased year by year. In 2016, 8900 ten thousand tons of soybeans are imported in China, the yield per year is 1290 ten thousand tons, and the import quantity is about 7 times of the yield per year. The imported soybeans in China mainly come from the transgenic soybean planting countries such as the United states, Brazil, Argentina and the like, and 3387 ten thousand hectares of soybeans are planted globally in 2016, wherein the sowing area of the transgenic soybeans with herbicide resistance accounts for 94%, and the spraying of glyphosate potassium salt and glyphosate isopropylamine salt accounts for 85% of all herbicides, so that the soybeans imported to China mostly have the transgenic (glyphosate resistant) characteristics. However, the transgenic soybean imported to China is approved by the Ministry of agriculture in China only to be used as a processing raw material, and is forbidden to be planted in China.
Under the background, it is important to research, develop and master a reliable and accurate soybean transgenic detection technology based on the protection of food safety and biodiversity in China, and the successful completion of the transgenic detection technology based on qualitative or quantitative PCR requires an ideal DNA template.
At present, several main methods for extracting genomic DNA comprise an SDS method, a CTAB method, a kit method and the like, however, because high-content components such as protein, fat and the like in soybean influence the extraction of the genomic DNA to different degrees, the components cannot be effectively removed, and the concentration and purity of the DNA which directly interferes with the final product can not be effectively removed.
Therefore, the specific selection of which method and the optimization of the reagents and operation methods used in the method can be better applied to the extraction of soybean genomic DNA is a technical problem to be solved by those skilled in the art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a cell lysate for extracting soybean seed genome DNA, wherein the cell lysate is a pure water solution containing 0.015 to 0.025g/ml SDS, 0.04 to 0.06mol/L Tris-HCl, 0.04 to 0.06mol/L EDTA and 0.14 to 0.16mol/L NaCl; the pH of the cell lysate is 8.
Preferably, the cell lysate is a pure water solution containing 0.02g/ml SDS, 0.05mol/L Tris-HCl, 0.05mol/L EDTA and 0.15mol/L NaCl, and the pH value of the cell lysate is 8.
The invention also provides an extraction method of the soybean seed genome DNA, which comprises a cell lysis process and a DNA purification process, wherein the cell lysis process adopts the cell lysate.
Preferably, the cell lysis process is: and uniformly mixing the obtained soybean seed fine powder with the cell lysate, wherein 0.08-0.1 g of soybean seed fine powder is mixed in each 1ml of cell lysate, then placing the mixture in a water bath at 55-65 ℃ for at least 60 minutes, and then centrifuging the mixture to obtain a supernatant.
Preferably, the fine powder of the soybean seeds is fine powder passing through a 80-mesh sieve.
Preferably, the process for purifying DNA comprises the following steps:
step 1, taking supernatant obtained by cell lysis, adding a mixed solution of chloroform and isoamylol with the same volume, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of the chloroform and the isoamylol is 24:1, fully mixing uniformly, and centrifuging to obtain supernatant;
step 2, adding the mixed solution of chloroform and isoamylol with the same volume into the supernatant obtained in the step 1, fully and uniformly mixing, and centrifuging to obtain the supernatant;
step 3, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 2, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 4, adding 70% ethanol into the precipitate obtained in the step 3 for washing, centrifuging, removing supernatant, taking the precipitate, repeatedly washing once, and drying in the air;
step 5, adding TE solution into the precipitate obtained in the step 4 to dissolve the precipitate, adding RNA enzyme, and uniformly mixing;
step 6, adding a mixed solution of chloroform and isoamylol in an equal volume into the mixed solution obtained in the step 5, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of chloroform and isoamylol is 24:1, fully and uniformly mixing, and centrifuging to obtain a supernatant;
step 7, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 6, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 8, adding 70% ethanol into the precipitate obtained in the step 7 for washing, centrifuging, discarding the supernatant, taking the precipitate, repeatedly washing once, and drying in the air;
and 9, adding TE solution into the precipitate obtained in the step 8 to dissolve the precipitate, and storing at the temperature of-20 ℃.
Preferably, in the step 1, the step 2 and the step 6, the centrifugation is carried out under the conditions of 12000-14000 r/min and at normal temperature for 10-20 min.
Preferably, in the step 4 and the step 8, the centrifugation condition is 10000-12000 r/min, and the centrifugation is carried out for 5-10 min at 4 ℃.
Preferably, in the step 3 and the step 7, after adding absolute ethyl alcohol, the mixture is kept stand at-20 ℃ for more than 30 minutes and then centrifuged at 12000-16000 rpm for 10-20 minutes at 4 ℃.
Preferably, in the step 5, after adding RNase and mixing, incubation at 37 ℃ for 30 minutes
The cell lysate for extracting the soybean seed genome DNA can improve the yield and the purity of the soybean seed genome DNA. Furthermore, the method for extracting the genomic DNA of the soybean seeds adopts the specific extraction and precipitation reagents and the specific sequence in the process of extracting and precipitating the DNA, namely chloroform isoamyl alcohol extraction, absolute ethyl alcohol precipitation, chloroform isoamyl alcohol extraction and absolute ethyl alcohol precipitation are sequentially carried out twice, and is particularly suitable for extracting the genomic DNA of the soybean seeds.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention.
Example embodiments will now be described more fully. However, the example embodiments can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al) or according to the product instructions.
Cell lysate for extracting soybean seed genome DNA
Regarding the extraction of soybean seed genomic DNA, because high content of components such as protein and fat in soybeans affect the extraction of genomic DNA to different degrees, the effect of extracting soybean seed genomic DNA by using conventional lysis solutions in the prior art, such as an extract solution used in an SDS method in the ministry of agriculture 1485 bulletin-4-2010 and a lysis solution (product model NEP 023-1) in the sanchi nationality novel plant genomic DNA rapid extraction kit, needs to be improved, and therefore, the inventors have made a great deal of experimental studies and research to find a formula of an optimal cell lysis solution suitable for the extraction of soybean seed genomic DNA.
In a specific embodiment of the invention, the cell lysate for extracting the soybean seed genome DNA is a pure water solution containing 0.015-0.025 g/ml SDS, 0.04-0.06 mol/L Tris-HCl, 0.04-0.06 mol/L EDTA and 0.14-0.16 mol/L NaCl, and the pH value of the cell lysate is 8.
In a preferred embodiment of the invention, the cell lysate is a pure aqueous solution containing 0.02g/ml SDS, 0.05mol/L Tris-HCl, 0.05mol/L EDTA, and 0.15mol/L NaCl, said cell lysate having a pH of 8.
The cell lysate adopting the formula can obviously improve the yield of the soybean seed genome DNA.
Example 1
The formula of the cell lysate for extracting the soybean seed genome DNA comprises the following steps:
1000ml of pure water
SDS 20g
1mol/L Tris-HCl（pH8.0） 50ml
0.5mol/L EDTA（pH8.0） 100ml
NaCl 8.76g
The preparation process comprises the following steps: adding 600ml of distilled water preheated at 65 ℃ into 20g of SDS powder, stirring uniformly, adding 8.76g of NaCl, placing in a 65 ℃ water bath, stirring, fully dissolving, adding 50ml of 1mol/L Tris-HCl (pH 8.0) into the solution, stirring, adding 100ml of 0.5mol/L EDTA (pH 8.0), stirring uniformly, placing the mixed solution at room temperature, adding NaOH or HCl to adjust the pH value to 8.0, transferring into a 1000ml volumetric flask, fixing the volume, mixing uniformly, transferring into a blue-covered flask, and sterilizing at 121 ℃ for 15 min.
Second, extraction method of soybean seed genome DNA
The extraction method of the soybean seed genome DNA mainly comprises a cell lysis process and a DNA purification process.
Based on the development of the optimal cell lysate suitable for extracting the soybean seed genome DNA, the inventor further searches and optimizes the operation conditions for purifying the DNA, particularly the operation conditions for extracting and precipitating the DNA suitable for extracting the soybean seed genome DNA.
In a particular embodiment of the invention, the cell lysis process is: uniformly mixing the obtained soybean seed fine powder with the cell lysate of the invention, wherein 0.08-0.1 g of soybean seed fine powder is mixed in each 1ml of cell lysate, then placing in a water bath at 55-65 ℃ for at least 60 minutes, and then centrifuging to obtain a supernatant.
In a preferred embodiment of the present invention, the soybean seed fine powder is a fine powder passing through an 80-mesh sieve.
In a preferred embodiment of the present invention, the process for purifying DNA comprises the steps of:
step 1, taking supernatant obtained by cell lysis, adding a mixed solution of chloroform and isoamylol with the same volume, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of the chloroform and the isoamylol is 24:1, fully mixing uniformly, and centrifuging to obtain supernatant;
step 2, adding the mixed solution of chloroform and isoamylol with the same volume into the supernatant obtained in the step 1, fully and uniformly mixing, and centrifuging to obtain the supernatant;
step 3, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 2, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 4, adding 70% ethanol into the precipitate obtained in the step 3, washing, centrifuging, removing supernatant, and taking the precipitate; repeatedly washing once and drying;
step 5, adding TE solution into the precipitate obtained in the step 4 to dissolve the precipitate, adding RNA enzyme, and uniformly mixing;
step 6, adding a mixed solution of chloroform and isoamylol in an equal volume into the mixed solution obtained in the step 5, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of chloroform and isoamylol is 24:1, fully and uniformly mixing, and centrifuging to obtain a supernatant;
step 7, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 6, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 8, adding 70% ethanol into the precipitate obtained in the step 7, washing, centrifuging, removing supernatant, and taking the precipitate; repeatedly washing once and drying;
and 9, adding TE solution into the precipitate obtained in the step 8 to dissolve the precipitate, and storing at the temperature of-20 ℃.
The inventors have found through a large number of experiments that the specific extraction and precipitation reagents and the specific sequence are adopted in the process of purifying the DNA in the method for extracting the genomic DNA of the soybean seeds, namely chloroform isoamyl alcohol extraction, absolute ethyl alcohol precipitation, chloroform isoamyl alcohol extraction and absolute ethyl alcohol precipitation are sequentially carried out twice, and the method is particularly suitable for extracting the genomic DNA of the soybean seeds.
Example 2
Extraction of soybean seed genome DNA
First, soybean seed fine powder is obtained: selecting soybean seeds without impurities, grinding the soybean seeds in liquid nitrogen by using a high-fat sample, sieving the crushed particles with a 80-mesh sieve, and storing the sieved soybean powder at-20 ℃ for later use;
secondly, the cell lysis process: 0.1g of fine soybean seed powder was weighed, placed in a 2ml sterile centrifuge tube, 1ml of the cell lysate obtained in example 1 (which was preheated at 65 ℃) was added thereto, mixed by manually inverting the cell lysate up and down, placed in a 65 ℃ water bath for about 60min, wherein the mixture was inverted about every 10min, and then centrifuged (12000 rpm, room temperature) to obtain a supernatant.
Then, the process of purifying the DNA comprises the following specific steps:
step 1, taking the supernatant obtained after cell lysis, adding a mixed solution of chloroform and isoamylol with the same volume ratio of 24:1 into the supernatant, fully and uniformly mixing the chloroform and the isoamylol by manual inversion, and centrifuging (14000 r/min at normal temperature) for about 10 minutes to obtain the supernatant;
step 2, carefully sucking the supernatant obtained in the step 1 by using a liquid transfer device, transferring the supernatant into a new 2ml sterile centrifuge tube, adding the mixed solution of chloroform and isoamylol with the same volume, fully and uniformly mixing the mixture by manual inversion, and centrifuging the mixture (14000 r/min at normal temperature) for about 10 minutes to obtain the supernatant;
step 3, carefully sucking the supernatant obtained in the step 2 by using a pipette, transferring the supernatant into a new 2ml sterile centrifuge tube, adding 2.5 times of anhydrous ethanol with pre-cooling at the temperature of-20 ℃, manually reversing and uniformly mixing, standing at the temperature of-20 ℃ for 30 minutes, centrifuging again (15000 r/min, 4 ℃) for about 10 minutes, and pouring out the supernatant to obtain a precipitate;
step 4, adding 1ml of 70% ethanol pre-cooled at-20 ℃ into the precipitate obtained in the step 3, washing, centrifuging (12000 r/min, 4 ℃) for about 10 minutes, removing the supernatant, and taking the precipitate; washing again, and drying at room temperature;
step 5, adding 400 mul TE solution into the precipitate obtained in the step 4 to dissolve the precipitate, then adding 10 mul RNA enzyme, uniformly mixing, and carrying out water bath at 37 ℃ for about 30 minutes;
step 6, adding a mixed solution of chloroform and isoamylol with the same volume into the mixed solution obtained in the step 5, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of chloroform and isoamylol is 24:1, fully mixing the mixture by manual inversion, and centrifuging (14000 r/min at normal temperature) to obtain a supernatant;
step 7, carefully sucking the supernatant obtained in the step 6 by using a pipette, adding 2.5 times of volume of absolute ethanol precooled at the temperature of minus 20 ℃, manually reversing and uniformly mixing, standing at the temperature of minus 20 ℃ for about 30 minutes, then centrifuging (15000 r/min, 4 ℃) for about 10 minutes, and pouring out the supernatant to obtain a precipitate;
step 8, adding 1ml of 70% ethanol pre-cooled at-20 ℃ into the precipitate obtained in the step 7, washing, centrifuging (12000 r/min, 4 ℃) for about 10 minutes, removing the supernatant, and taking the precipitate; washing again, and drying at room temperature;
step 9, adding 200. mu.l of TE solution to the precipitate obtained in step 8 to dissolve the precipitate, and storing at-20 ℃.
III,
To demonstrate the effect of the cell lysate of the present invention, the inventors designed a comparative experiment of the cell lysate of example 1 with a conventional lysate of the prior art.
First, soybean seed fine powder is obtained: selecting soybean seeds without impurities, grinding the soybean seeds in liquid nitrogen by using a high-fat sample, sieving the crushed particles with a 80-mesh sieve, and storing the sieved soybean powder at-20 ℃ for later use;
secondly, the cell lysis process:
cell lysis procedure for experimental group 1: weighing 0.1g of soybean seed fine powder, placing the soybean seed fine powder into a 2ml sterile centrifuge tube, adding 1ml of the cell lysate (which is preheated at 65 ℃) obtained in the example 1, manually reversing the cell lysate from top to bottom, uniformly mixing the cell lysate and the cell lysate, placing the cell lysate into a 65 ℃ water bath for about 60min, wherein the cell lysate is reversely mixed once every 10min, and then centrifuging (12000 r/min, normal temperature) to obtain a supernatant;
cell lysis procedure for comparative group 1: the cell lysate is replaced by the lysate A.3.2.19 in the section 1485, notice-4-2010 of the Ministry of agriculture, and other operation processes are the same as the cell lysis process of the experimental group 1;
cell lysis procedure for comparative group 2: replacing the cell lysate with a novel plant genome DNA rapid extraction kit lysate of Dingguo, and performing other operation processes in the same manner as the cell lysis process of the experimental group 1;
then, the procedure for purifying the DNA: experimental group 1, comparative group 1 and comparative group 2 all employed the procedure of a3.3 in bulletin-4-2010 of Ministry of agriculture 1485.
Experimental group 1, comparative group 1 and comparative group 2, each group comprising 5 parallel tests.
Respectively detecting the mass concentration and purity of DNA obtained by 5 parallel tests in each group by using a nucleic acid protein analyzer, and taking the average value of 5 parallel results as the result of each group; see table 1 below for results:
as can be seen from table 1, on the premise of adopting the same conventional operation procedure, the DNA extracted from the soybean seeds by using the cell lysate of example 1 has the highest mass concentration and better purity, so that, compared with the existing cell lysate, the cell lysate of the specific formula of the present invention is more favorable for cracking the soybean cells, releasing the genomic DNA, can improve the yield of the genomic DNA of the soybean seeds, and is particularly suitable for extracting the genomic DNA of the soybean seeds.
Fourthly,
To prove the technical effect of the method for extracting the soybean seed genome DNA, the inventor designs a comparative experiment.
Experimental group 2: the method for extracting the soybean seed genome DNA of the embodiment 2 is adopted, wherein the lysate of the embodiment 1 of the invention is adopted;
comparative group 3: the extraction method disclosed in the ministry of agriculture 1485, bulletin-4-2010A 3.3, wherein the lysate of example 1 of the present invention was used;
comparative group 4: the novel Dingguo plant genome DNA rapid extraction kit is adopted, wherein the lysis solution of the embodiment 1 is adopted;
experimental group 2, comparative group 3 and comparative group 4, each group comprising 5 parallel tests.
Respectively detecting the mass concentration and purity of DNA obtained by 5 parallel tests in each group by using a nucleic acid protein analyzer, and taking the average value of 5 parallel results as the result of each group; see table 2 below for results:
as can be seen from Table 2, the concentration of the DNA obtained by the experiment group 2 is the highest under the condition of ensuring good DNA purity by adopting the same cell lysate and different DNA extraction methods, so that compared with the conventional DNA extraction method, the reagent and the steps adopted by the DNA extraction method are better in applicability, the yield of the soybean seed DNA can be improved, and the method is particularly suitable for extracting the soybean seed genome DNA.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (5)
1. A method for extracting soybean seed genome DNA, which comprises a cell lysis process and a DNA purification process, and is characterized in that:
the cell lysis process is as follows: uniformly mixing the obtained soybean seed fine powder with cell lysate, wherein 0.08-0.1 g of soybean seed fine powder is mixed in each 1ml of cell lysate, then placing the mixture in a water bath at 55-65 ℃ for at least 60 minutes, and then centrifuging the mixture to obtain supernatant;
the cell lysate is 0.02g/ml SDS, 0.05mol/L Tris-HCl, 0.05mol/L EDTA and 0.15mol/L NaCl pure water solution; the pH value of the cell lysate is 8;
the process for purifying the DNA comprises the following steps:
step 1, taking supernatant obtained by cell lysis, adding a mixed solution of chloroform and isoamylol with the same volume, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of the chloroform and the isoamylol is 24:1, fully mixing uniformly, and centrifuging to obtain supernatant;
step 2, adding the mixed solution of chloroform and isoamylol with the same volume into the supernatant obtained in the step 1, fully and uniformly mixing, and centrifuging to obtain the supernatant;
step 3, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 2, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 4, adding 70% ethanol into the precipitate obtained in the step 3 for washing, centrifuging, removing supernatant, taking the precipitate, repeatedly washing once, and drying in the air;
step 5, adding TE solution into the precipitate obtained in the step 4 to dissolve the precipitate, adding RNA enzyme, uniformly mixing, and culturing at 37 ℃ for 30 minutes;
step 6, adding a mixed solution of chloroform and isoamylol in an equal volume into the mixed solution obtained in the step 5, wherein the volume ratio of the chloroform to the isoamylol in the mixed solution of chloroform and isoamylol is 24:1, fully and uniformly mixing, and centrifuging to obtain a supernatant;
step 7, adding 2.5 times of volume of absolute ethyl alcohol into the supernatant obtained in the step 6, standing at-20 ℃, centrifuging, removing the supernatant, and taking a precipitate;
step 8, adding 70% ethanol into the precipitate obtained in the step 7 for washing, centrifuging, discarding the supernatant, taking the precipitate, repeatedly washing once, and drying in the air;
and 9, adding TE solution into the precipitate obtained in the step 8 to dissolve the precipitate, and storing at the temperature of-20 ℃.
2. The extraction method according to claim 1, characterized in that:
the soybean seed fine powder is fine powder passing through a 80-mesh sieve.
3. The extraction method according to claim 1, characterized in that:
in the step 1, the step 2 and the step 6, the centrifugation is carried out for 10-20 minutes at normal temperature under the condition of 12000-14000 r/min.
4. The extraction method according to claim 1, characterized in that:
in the step 4 and the step 8, the centrifugation condition is 10000-12000 r/min, and the centrifugation is carried out for 5-10 min at 4 ℃.
5. The extraction method according to claim 1, characterized in that:
in the steps 3 and 7, after adding absolute ethyl alcohol, standing at-20 ℃ for more than 30 minutes, and then centrifuging at 12000-16000 rpm for 10-20 minutes at 4 ℃.
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| US20090083883A1 (en) * | 2006-03-17 | 2009-03-26 | Basf Plant Science Gmbh | D-Amino Acid Selection For Soybean |
| CN104450677A (en) * | 2014-11-04 | 2015-03-25 | 青岛康大分析检测有限公司 | Method for extracting soybean DNA |
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| US20090083883A1 (en) * | 2006-03-17 | 2009-03-26 | Basf Plant Science Gmbh | D-Amino Acid Selection For Soybean |
| CN104450677A (en) * | 2014-11-04 | 2015-03-25 | 青岛康大分析检测有限公司 | Method for extracting soybean DNA |
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