CN114990103B - Method for extracting erigeron breviscapus chloroplast genome DNA by improved high-salt-low pH method - Google Patents
Method for extracting erigeron breviscapus chloroplast genome DNA by improved high-salt-low pH method Download PDFInfo
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Abstract
The invention discloses a method for extracting erigeron breviscapus chloroplast genome DNA by an improved high-salt-low pH method. The method of the invention comprises the following steps: (1) Adding starved erigeron breviscapus leaves into the buffer solution A for homogenization, filtering, and collecting filtrate; centrifuging the filtrate for the first time, and collecting supernatant; centrifuging the supernatant for the second time, and collecting green precipitate to obtain crude chloroplast; (2) Adding a buffer solution B into the crude chloroplast obtained in the step (1), lightly suspending, centrifuging, and collecting precipitate to obtain chloroplast; (3) Adding buffer solution C into chloroplast obtained in the step (2), then adding protease for incubation, and cooling; (4) And (3) extracting chloroplast DNA from the system after the cleavage in the step (3) to obtain chloroplast genome DNA. The method can simply and rapidly obtain complete chloroplast and chloroplast DNA, and the obtained DNA has complete structure, high quality and good purity, does not have degradation phenomenon, and can meet the standard and the requirement of subsequent sequencing work.
Description
Technical Field
The invention relates to a method for extracting plant chloroplast genome DNA, in particular to a method for extracting erigeron breviscapus chloroplast genome DNA by an improved high-salt-low pH method.
Background
Erigeron breviscapus is also called erigeron breviscapus, is a perennial herb medicinal plant of the asteraceae, is a specific species in southwest China, and has extremely high medicinal value because the extract and active compounds of erigeron breviscapus have been widely used for treating cerebral embolism, cerebral thrombosis, coronary heart disease, angina pectoris, acute renal failure and nephrotic syndrome. But the wild resources of erigeron breviscapus are greatly exhausted due to the over-digging and use of the erigeron breviscapus in successive years.
Chloroplasts are the major organelles of plants that carry out photosynthesis, and as a semi-autonomous genetic system, chloroplasts have relatively independent genetic material, i.e., chloroplast DNA (cpDNA), which plays a very broad role in studying plant phylogenetic development. In recent years, with the rapid development of high-throughput sequencing technology, sequencing work of plant chloroplast genome also makes a certain progress, and lays a foundation for researching plant genetic diversity and genetic evolution. Currently, there are many methods for extracting plant chloroplast genome, such as sucrose density gradient centrifugation, percoll density gradient centrifugation, and modified high-salt-low pH method. The traditional density gradient centrifugation method is more suitable for extracting chloroplast DNA of Gramineae and leguminous plants, but the method has the advantages of higher extraction cost, long time consumption, low DNA yield and difficult wide application. The improved high-salt-low pH method has the characteristic of simply and rapidly obtaining complete chloroplast and high-quality chloroplast DNA.
The current extraction method of the erigeron breviscapus chloroplast genome has not been reported, and the research on the erigeron breviscapus chloroplast genome at home and abroad is very little. Therefore, extracting high quality erigeron breviscapus chloroplast genome DNA has great significance in exploring genetic diversity of erigeron breviscapus, protecting, evaluating and scientifically utilizing erigeron breviscapus germplasm resource.
Disclosure of Invention
The invention aims to provide an improved high-salt-low-pH method for extracting erigeron breviscapus chloroplast genome DNA, and the DNA extracted by the method has the advantages of complete structure, high quality, good purity, no degradation phenomenon and capability of meeting the standard and the requirement of subsequent sequencing work.
The invention provides a method for extracting erigeron breviscapus chloroplast genome DNA, which comprises the following steps:
(1) Chloroplast isolation: adding starved erigeron breviscapus leaves into the buffer solution A for homogenization, filtering, and collecting filtrate; centrifuging the filtrate for the first time, and collecting supernatant; performing secondary centrifugation on the supernatant, and collecting green precipitate to obtain crude chloroplast;
(2) Chloroplast purification: adding a buffer solution B into the crude chloroplast obtained in the step (1), lightly suspending, centrifuging, and collecting precipitate to obtain chloroplast;
(3) Chloroplast lysis: adding buffer solution C into chloroplast obtained in the step (2), then adding protease for incubation, and cooling;
(4) Extraction of chloroplast DNA: and (3) extracting chloroplast DNA from the system after the cleavage in the step (3) to obtain the chloroplast genome DNA.
In the above preparation method, in step (1), the ratio of the erigeron breviscapus leaf to the buffer solution a may be 20g: 60-100 mL. Specifically, 20g:100mL;
the pH of the buffer a may be 3.8; the buffer a may consist of the following components: 1.25mol/L NaCl,0.25mol/L ascorbic acid, 10mmol/L sodium metabisulfite, 50mmol/L Tris-HCl with pH 8.0, 7mmol/L EDTA with pH 8.0, 1% w/v PVP-40 (1% w/v: polyvinylpyrrolidone with a mass concentration of 10g/L and PVP-40: K40), 0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water.
In the above preparation method, in the step (1), the centrifugal force of the first centrifugation may be 200 to 500×g, and specifically may be 500×g; the time can be 10-20 min, and can be specifically 20min;
the centrifugal force of the second centrifugation can be 2500-3500 Xg, in particular 2500 Xg; the time can be 12-20 min, and can be specifically 12min.
In the preparation method, in the step (2), the volume of the buffer solution B may be 1/4 of the volume of the buffer solution A;
the pH of the buffer B may be 8.0; the buffer B may consist of the following components: 1.25mol/L NaCl,50mmol/L Tris-HCl with pH value of 8.0, 25mmol/L EDTA with pH value of 8.0, 1% w/v PVP-40,0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water.
In the above preparation method, in the step (2), the centrifugal force of the centrifugation may be 2500 to 3500×g, specifically 2500×g; the time can be 12-20 min, and can be specifically 12min.
In the preparation method, in the step (3), the volume of the buffer solution C may be 1/25 of the volume of the buffer solution B;
the pH of the buffer C may be 8.0; the buffer C may consist of the following components: 50mmol/L Tris-HCl,25mmol/L EDTA with a pH of 8.0, 1.25mol/L NaCl,2.0% sodium dodecyl sulfate and the balance water.
In the preparation method, in the step (3), the volume of the protease can be 0.3-0.4% of the volume of the buffer solution C; the concentration of the protease may be 20mg/mL;
the protease may be proteinase K;
the temperature of the incubation may be 50-60 ℃, in particular 55 ℃; the time can be 3-5 hours, and can be specifically 3 hours.
In the preparation method, in the step (4), the extracting step is as follows: extracting the system cracked in the step (3) by using a solvent I, uniformly mixing, centrifuging for the first time, and collecting supernatant to obtain supernatant I;
adding a solvent II into the supernatant I for secondary centrifugation, and collecting the supernatant to obtain a supernatant II;
and adding a solvent III into the supernatant II, uniformly mixing, settling, centrifuging for the third time, and collecting the sediment to obtain the chloroplast genome DNA.
In the preparation method, in the step (4), the volume of the cracked system and the volume of the solvent I can be equal; the solvent I can be prepared from the following components in percentage by volume: 24:1, phenol, chloroform and isoamyl alcohol; the centrifugal force of the first centrifugation can be 10000-12000 Xg, and can be 12000 Xg in particular; the time can be 10-20 min, and can be specifically 10min.
The volumes of the supernatant I and the solvent II can be equal; the solvent II can be prepared from the following components in percentage by volume: 1 chloroform and isoamyl alcohol;
the centrifugal force of the second centrifugation can be 10000-12000 Xg, and can be 12000 Xg in particular; the time can be 10-20 min, and can be specifically 10min.
In the preparation method, in the step (4), the volumes of the supernatant II and the solvent III can be equal; the solvent III may be isopropanol;
the sedimentation temperature can be-18 to-22 ℃, and can be specifically-20 ℃; the time can be 10-30 min, and can be specifically 10min;
the rotation speed of the third centrifugation can be 10000-12000 Xg, and can be 12000 Xg; the time can be 20-30 min, and specifically can be 20min.
The invention also provides an extraction method of erigeron breviscapus chloroplast, which comprises the following steps: adding starved erigeron breviscapus leaves into the buffer solution A for homogenization, filtering, and collecting filtrate; centrifuging the filtrate for the first time, and collecting supernatant; and (3) performing secondary centrifugation on the supernatant, and collecting green precipitate to obtain chloroplasts.
In the above chloroplast extraction method, the ratio of the erigeron breviscapus leaf to the buffer solution A is 20g: 60-100 mL;
the pH value of the buffer solution A is 3.8;
the buffer solution A consists of the following components: 1.25mol/L NaCl,0.25mol/L ascorbic acid, 10mmol/L sodium metabisulfite, 50mmol/L Tris-HCl with pH 8.0, 7mmol/L EDTA with pH 8.0, 1% w/v PVP-40 (1% w/v: polyvinylpyrrolidone with a mass concentration of 10g/L and PVP-40: K value of 40), 0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water;
the centrifugal force of the first centrifugation may be 200 to 500×g, and in particular, may be 500×g; the time can be 10-20 min, and can be specifically 20min;
the centrifugal force of the second centrifugation can be 2500-3500 Xg, in particular 2500 Xg; the time can be 12-20 min, and can be specifically 12min.
In the chloroplast extraction method, the method further comprises the steps of adding a buffer solution B into the precipitate after collecting the green precipitate, suspending, centrifuging, and collecting the precipitate to obtain chloroplast;
the volume of the buffer solution B is 1/4 of that of the buffer solution A;
the pH value of the buffer solution B is 8.0;
the buffer B consists of the following components: 1.25mol/L NaCl,50mmol/L Tris-HCl with pH value of 8.0, 25mmol/L EDTA with pH value of 8.0, 1% w/v PVP-40,0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water;
the centrifugal force of the centrifugation can be 2500-3500 Xg, and can be 2500 Xg in particular; the time can be 12-20 min, and can be specifically 12min.
The invention has the following beneficial effects:
(1) The invention establishes a high-efficiency erigeron breviscapus chloroplast DNA separation and extraction method based on an improved high-salt-low pH method, and lays a foundation for deep development of sequencing and research work of erigeron breviscapus chloroplast genome.
(2) The method can simply and rapidly obtain complete chloroplast and chloroplast DNA, and the obtained DNA has complete structure, high quality and good purity, does not have degradation phenomenon, and can meet the standard and the requirement of subsequent sequencing work.
Drawings
FIG. 1 is a photograph of chloroplast precipitate isolated by a modified high salt-low pH process in the examples.
FIG. 2 is a micrograph of chloroplasts isolated by the modified high salt-low pH method of the examples (after purification).
FIG. 3 is an electropherogram of erigeron breviscapus chloroplast genomic DNA extracted in the examples.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 improved high salt-Low pH extraction of erigeron breviscapus chloroplast genomic DNA
1. Sample preparation and pretreatment
The test material is erigeron breviscapus aseptic seedling, which is planted in a Chinese medical science college traditional Chinese medicine resource center tissue culture room, each sample is about 50g, wrapped by gauze, starved in a refrigerator dark place at 4 ℃ for 48-72 hours, placed in a fresh-keeping bag, and placed in a refrigerator at-80 ℃ for standby.
(1) Preparation of buffer A (pH 3.8)
1.25mol/L NaCl,0.25mol/L ascorbic acid (Vc), 10mmol/L sodium metabisulfite, 50mmol/L Tris-HCl (pH 8.0), 7mmol/L EDTA (pH 8.0), 1% PVP-40 (w/v), 0.1% BSA (w/v), 1mmol/L DTT (dithiothreitol), BSA and DTT were added as is. PVP-40 was purchased from VWR International, LLC under the trade designation 0507-500G.
(2) Preparation of buffer B (pH 8.0)
1.25mol/L NaCl，50mmol/L Tris-HCl(pH 8.0)，25mmol/L EDTA(pH 8.0)，1％PVP-40(w/v)，0.1％BSA(w/v)，1mmol/L DTT。
(3) Preparation of buffer C (pH 8.0)
50mmol/L Tris-HCl，25mmol/L EDTA(pH 8.0)，1.25mol/L NaCl，2.0％SDS。
2. Extraction of erigeron breviscapus chloroplast genome DNA
The erigeron breviscapus chloroplast genome DNA is extracted by adopting an improved high-salt-low pH method, and the specific steps are as follows:
(1) Chloroplast isolation
Taking out the leaves after starvation treatment for two days, cutting the leaves into strips with the length of 1cm, putting the strips into a refiner, adding 100mL of precooled buffer solution A into each 20g of leaves, fully homogenizing the leaves, filtering the leaves by 8 layers of gauze, collecting filtrate, filtering the filtrate by 6 layers of gauze, collecting the filtrate into 50mL centrifuge tubes, and subpackaging 25mL each tube. Chloroplasts were separated by differential centrifugation at 4 ℃,500 Xg, for 20min, and the supernatant was carefully transferred to another centrifuge tube. Centrifugation was carried out at 2500 Xg for 12min, the supernatant was removed and the green pellet was retained. The chloroplast precipitate obtained is protected from light to prevent aggregation of starch. A photograph of chloroplast precipitate is shown in FIG. 1.
(2) Chloroplast purification
The chloroplast pellet was purified by adding 25mL of pre-chilled buffer B, resuspended in sterile soft brush, centrifuged at 2 Xg for 12min and the supernatant discarded, and the procedure repeated once. The obtained precipitate is purified chloroplast, and is preserved at-80deg.C.
And sucking 10 mu L of purified chloroplast extract, dripping the purified chloroplast extract on a glass slide, and placing the glass slide under a fluorescence inversion microscope to observe chloroplast morphology. As shown in the experimental result in figure 2, the chloroplast isolated by the method has a relatively complete structure, basically has no fragmentation and pollution, has a relatively clear background, has considerable chloroplast content and can ensure that high-quality chloroplast genome DNA is obtained.
(3) Chloroplast cleavage
1mL of buffer C was added to the purified chloroplast, followed by 30. Mu.L of proteinase K (20 mg/mL by mass) and shaking vigorously, incubation was performed at 55℃for 3h, and the incubation was reversed several times every 20min and shaking was performed.
(4) Extraction of chloroplast DNA
Cooling to room temperature after water bath, extracting with equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, for preparation at present), slowly mixing, centrifuging for 10min at a speed of 12×g, adding equal volume of chloroform/isoamyl alcohol (24:1) into the supernatant, and centrifuging for 10min at a speed of 12×g; adding the supernatant into the pre-cooled isopropanol with equal volume, gently mixing, settling at-20deg.C for 10min, centrifuging for 20min, discarding supernatant, adding 70% ethanol, washing for 2 times, air drying (until no alcohol smell exists), and adding sterilized water at 50-68deg.C for dissolving to obtain chloroplast DNA.
3. Detection of chloroplast DNA
(1) Chloroplast DNA ultraviolet spectrophotometric detection
The experimental method comprises the following steps: taking 1.5 mu L of erigeron breviscapus chloroplast DNA solution, measuring the concentration by using a Thermo Nano Drop 1000 ultraviolet spectrophotometer, and determining the concentration according to OD 260nm And OD (optical axis) 280nm 、OD 260nm And OD (optical axis) 230nm Is subjected to purity analysis.
Experimental results: erigeron breviscapus chloroplast DNA sample concentration is 329 ng/. Mu.L, OD 260nm And OD (optical axis) 280nm The ratio is 1.99, which indicates that the DNA sample has no interference and pollution of protein, phenols and polysaccharide, low impurity content, high purity and OD 260nm And OD (optical axis) 230nm The ratio of (2) was 2.23, indicating that the DNA sample was low in polysaccharide carbohydrate and the like. In conclusion, chloroplasts extracted by the improved high-salt-low pH method meet the test requirements.
(2) Chloroplast DNA agarose gel electrophoresis detection
In order to further identify the size and purity of erigeron breviscapus chloroplast genomic DNA, a erigeron breviscapus chloroplast DNA solution was taken and subjected to electrophoresis detection on agarose gel with a concentration of 1%.
The experimental method comprises the following steps: 1% agarose gel was used, 1. Mu.L of GelRed nucleic acid dye was added, the DNA loading was 1. Mu.L, the BioRad electrophoresis apparatus 180V,10min, and the UV gel imaging system was used to take photographs and to check the integrity of the DNA.
Experimental results: the result is shown in figure 3, and the result is shown in figure 3 that the erigeron breviscapus chloroplast DNA has a clear target band and no obvious tailing phenomenon, thus indicating that the extracted erigeron breviscapus chloroplast DNA has higher purity.
Claims (3)
1. A method for extracting erigeron breviscapus chloroplast genome DNA comprises the following steps:
(1) Chloroplast isolation: adding starved erigeron breviscapus leaves into the buffer solution A for homogenization, filtering, and collecting filtrate; centrifuging the filtrate for the first time, and collecting supernatant; performing secondary centrifugation on the supernatant, and collecting green precipitate to obtain crude chloroplast;
in the step (1), the ratio of the erigeron breviscapus leaves to the buffer solution A is 20g: 60-100 mL;
the pH value of the buffer solution A is 3.8; the buffer solution A consists of the following components: 1.25mol/L NaCl,0.25mol/L ascorbic acid, 10mmol/L sodium metabisulfite, 50mmol/L Tris-HCl with pH 8.0, 7mmol/L EDTA with pH 8.0, 1% w/v PVP-40,0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water;
in the step (1), the centrifugal force of the first centrifugation is 500 Xg, and the time is 10-20 min;
the centrifugal force of the second centrifugation is 2500 Xg, and the time is 12min;
(2) Chloroplast purification: adding a buffer solution B into the crude chloroplast obtained in the step (1), lightly suspending, centrifuging, and collecting precipitate to obtain chloroplast;
in the step (2), the pH value of the buffer solution B is 8.0; the buffer B consists of the following components: 1.25mol/L NaCl,50mmol/L Tris-HCl with pH value of 8.0, 25mmol/L EDTA with pH value of 8.0, 1% w/v PVP-40,0.1% w/v bovine serum albumin, 1mmol/L dithiothreitol and the balance water;
in the step (2), the centrifugal force of the centrifugation is 2500 Xg, and the time is 12min;
(3) Chloroplast lysis: adding buffer solution C into chloroplast obtained in the step (2), then adding protease for incubation, and cooling;
in the step (3), the pH value of the buffer solution C is 8.0; the buffer solution C consists of the following components: 50mmol/L Tris-HCl,25mmol/L EDTA with pH value of 8.0, 1.25mol/L NaCl,2.0% sodium dodecyl sulfate and the balance water;
(4) Extraction of chloroplast DNA: and (3) extracting chloroplast DNA from the system after the cleavage in the step (3) to obtain the chloroplast genome DNA.
2. The method according to claim 1, characterized in that: in the step (4), the step of extracting is as follows: extracting the system cracked in the step (3) by using a solvent I, uniformly mixing, centrifuging for the first time, and collecting supernatant to obtain supernatant I;
adding a solvent II into the supernatant I for secondary centrifugation, and collecting the supernatant to obtain a supernatant II;
and adding a solvent III into the supernatant II, uniformly mixing, settling, centrifuging for the third time, and collecting the sediment to obtain the chloroplast genome DNA.
3. The method according to claim 2, characterized in that: in the step (4), the volume of the system after cracking is equal to that of the solvent I; the solvent I is prepared from the following components in percentage by volume: 24:1, phenol, chloroform and isoamyl alcohol; the centrifugal force of the first centrifugation is 10000-12000 Xg, and the time is 10-20 min;
the volume of the supernatant I is equal to that of the solvent II; the solvent II comprises the following components in percentage by volume: 1 chloroform and isoamyl alcohol; the centrifugal force of the secondary centrifugation is 10000-12000 Xg, and the time is 10-20 min;
the volume of the supernatant II is equal to that of the solvent III; the solvent III is isopropanol; the rotation speed of the third centrifugation is 10000-12000 Xg, and the time is 20-30 min.
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