CN105112402A - Rapid extraction method for plant mitochondrion DNA - Google Patents

Abstract

The invention discloses a rapid extraction method for plant mitochondrion DNA. The method includes the steps of firstly, preparing Buffer A (containing betaine and cysteine), Buffer B (containing BSA), a solution W1, a solution W2, a solution W3, buffer WT and buffer TB; secondly, adding Buffer A for grinding after preprocessing fresh and tender tissue, collecting filtered fluid, keeping the pH value at 7.0 or above, and conducting 12000g centrifugation on the supermatant after conducting 1000g centrifugation on collected fluid; thirdly, taking sediment, and adding Buffer B to sediment for resuspension, and conducting 1000g centrifugation; fourthly, taking supermatant, adding DNase I and EDTA for reaction, and then conducting 12000g centrifugation; fifthly, adding the solution W1 to the sediment for suspending sediment; sixthly, adding the solution W2 and the solution W3 for gentle perversion, taking supermatant after centrifugation, and extracting the mitochondrion DNA through an adsorption column. By means of the method, the high-quality plant mitochondrion DNA can be efficiently and rapidly obtained.

Description

A kind of rapid extracting method of plant mitochondiral-DNA

Technical field

The present invention relates to biological technical field, be specifically related to a kind of rapid extracting method of plant mitochondiral-DNA.

Background technology

Plastosome is the important organelle of vegetable cell, and it is the genome self with certain Genetic Function structure, can self-replacation, and the important enzyme composition in some respiratory metabolism processes of codified, for cellular physiological events provides energy.Plastosome, as the maincenter of cellular energy metabolism and Substance Transformation, plays an important role in cell synthesis, substance transportation and information transmission.In addition, show much higher plant cell matter male sterile research, plastosome and cytoplasmic male sterility have substantial connection, are the important carriers of the male sterile factor.The structure and fuction of research Mitochondrial DNA (mtDNA), to crop improvement, explores the vortex rooms of organoid, has important meaning especially in the research, different male sterile cytoplasmic pyruvate kinase of cytoplasmic male sterility in plants.

Extracting high-quality Mitochondrial DNA is the basic premise furtherd investigate mitochondrial inheritance system.Plant mitochondiral-DNA is compared with core DNA, very micro-at intracellular level, is very easily polluted by core and chloroplast DNA in leaching process.In addition, plant mtDNA great majority are ring molecule, and in each plastosome, DNA molecular number does not copy tens copies to not etc. from several, and different plant species mtDNA molecular size difference is very large, and from tens kb to several thousand kb not etc., corresponding mtDNA extracting method is also different.All in all, plant mtDNA extracting method, mainly contains the method such as density gradient centrifugation and differential centrifugation.The Mitochondrial DNA purity that density gradient centrifugation obtains is high, but complicated operation, consuming time, large to the demand of material, productive rate is low; Common differential centrifugation is simple to operate, but the mtDNA purity extracted is low, easily degrades, and the contaminating impurities such as core DNA, RNA and protein are serious.The restriction of these unfavorable factors causes existing plastosome extracting method can not meet follow-up study requirements of one's work.

Summary of the invention

The object of the present invention is to provide a kind of rapid extracting method of plant mitochondiral-DNA, the method can must obtain high-quality Mitochondrial DNA efficient, simple, fast, can meet pcr amplification, quantitatively and qualitative analysis, order-checking equimolecular biological experiment requirement.

Technical scheme of the present invention is: a kind of rapid extracting method of plant mitochondiral-DNA, is characterized in that, specifically comprises the steps:

(1) solution preparation

BufferA:0.4M sucrose, 5mMEDTA (ethylenediamine tetraacetic acid (EDTA)), 50mMTris-HCl, 10mM Trimethyl glycine, 8mM halfcystine, 0.1%BSA (bovine serum albumin) and 1%PVP (polyvinylpyrrolidone), pH7.8;

BufferB:0.4M sucrose, 1mMEDTA, 10mMTris-HCl, 0.1%BSA, pH7.2;

Solution W 1:1% glucose, 50mMEDTA, 25mMTris-HCl, pH8.0;

Solution W 2:0.2mMNaOH, 1%SDS (sodium lauryl sulphate);

Solution W 3:5mol/LKAC (Potassium ethanoate), pH4.8;

Rinsing liquid WT:70% alcohol;

Elution buffer TB:10mMTris-HCl, 1mMEDTA, PH=8.0;

Remarks: wherein, the percentage ratio of 70% alcohol is volume ratio, and all the other percentage ratios are mass volume ratio w/v (i.e. g/ml).

(2) pre-treatment

Get the fresh tender tissue of plant dark treatment 24h in 4 DEG C of environment, cleaning material [if need sterilization, being soak 5min in the clorox diluent of 0.7% (w/v) at final concentration] in the distilled water of precooling (being chilled to 2-4 DEG C in advance); Extract immediately after pretreatment, to avoid the plastosome of cell expansion pressure and acquisition maximum, following step all need be carried out at 2-4 DEG C;

(3) grind

In the mortar of precooling, add BufferA, then add pretreated material and grind; Filtered by the hospital gauze of 4-8 layer after grinding, collect filtered liquid and keep its pH (if not enough, to adjust with NaOH) more than 7.0;

(4) separate mitochondria

Liquid will be collected in 4 DEG C, the centrifugal 5 ± 1min of 1000g, collect supernatant; 4 DEG C, the centrifugal 20 ± 2min of 12000g, discards supernatant; Add BufferB, with the resuspended green precipitate particle of banister bruss; Then 4 DEG C, the centrifugal 5 ± 1min of 1000g, collects supernatant; Add DNase I, place 1-2h; Add 0.5mol/LEDTA (pH8.0), static 10 ± 2min after mixing, termination reaction; 4 DEG C, the centrifugal 20 ± 2min of 12000g, discards supernatant;

(5) Mitochondrial DNA is extracted

Solution W 1 suspension precipitation is added in precipitation; Then add solution W 2, gentleness is put upside down 6-8 time, then adds solution W 3, and gentleness puts upside down centrifuge tube, fully mixes solution; Centrifugal 10 ± 2min, draws supernatant and puts into adsorption column (the pellosil centrifugal adsorbing column with nucleic acid pellosil), more centrifugal, outwells waste liquid; Successively rinsing liquid WT is added 2 times, centrifugal rinsing adsorption column in adsorption column; Finally adsorption column is put into a clean centrifuge tube, drip elution buffer TB to adsorption film middle part, room temperature places 2-5min, centrifugal 2-3min, by solution collection in centrifuge tube, and-20 DEG C of preservations.

Further, the BufferA that described step (3) uses, if non-green tissue, then ratio is 2ml/g; If chlorenchyma, 4ml/g.

Further, the BufferB consumption of described step (4) is 40ml/g (material); Preferably, first add BufferB5-10ml/g, with the resuspended green precipitate particle of banister bruss; Re-suspension liquid is transferred in a new centrifuge tube, add BufferB to 40ml/g.The consumption of the consumption of DNase I to be final concentration be 40 μ g/ml, 0.5mol/LEDTA is 1mL/g (material).

Further, in described step (5), centrifugal centrifugal speed is 10000-15000g, is preferably 12000g.

Further, described step (5) adsorption column is put into before a clean centrifuge tube drips elution buffer TB, the more centrifugal 2min of 12000g.

Further, the every 1g material of described step (5), after adopting 2ml solution W 1 to suspend, adds 250 μ l solution W 2 and 350 μ l solution W 3.

The present invention has the following advantages:

1, in BufferA of the present invention, sucrose can maintain osmotic pressure, and EDTA provides divalent cation, and Tris-HCl provides between buffer zone, and this formula can provide stable pH value and osmotic pressure for plant tissue, at utmost ensures the integrity of vegetable cell Mitochondria; The more important thing is in BufferA and add Trimethyl glycine and halfcystine, productive rate improves, and Mitochondrial DNA is without obvious degradation;

2, keep more than PH7.0 after ground material, productive rate and quality are improved;

3, owing to adding 0.1%BSA in BufferB in step (4), there is the effect maintaining and stablize pH value and osmotic pressure, so productive rate and quality are improved;

4, because centrifugal force in step (4) is 1000g, can better cell walls residual tissue and thick line plastochondria be separated;

5, owing to using solution W 1, W2, W3 and adsorption column to extract Mitochondrial DNA in step (5), the treatment time becomes 30min from 4-5h, greatly shortens experimental period, but productive rate and quality are without considerable change.

To sum up, the present invention efficiently (extraction efficiency is organized by existing 0.1 μ gmtDNA/g and doubled 0.2 μ gmtDNA/g plant tissue), fast (extraction time shortens to 3-4h by 6-7h) can must obtain the plant mitochondiral-DNA of high quality (detected through gel electrophoresis mtDNA is band limpid in sight), can meet pcr amplification, quantitatively and qualitative analysis, order-checking equimolecular biological experiment requirement.

Accompanying drawing explanation

Fig. 1 is 5 Mitochondrial DNA gel electrophoresis figures;

The PCR gel electrophoresis comparison diagram of Fig. 2 to be the Mitochondrial DNA extracted in two ways be template; Wherein, A1-A5 is the mitochondrial DNA amplification product that extracting method of the present invention obtains; B1-B5 is the mitochondrial DNA amplification product that traditional method (common differential centrifugation) is extracted.

Embodiment

Embodiment 1

(1) solution preparation

BufferA:0.4M sucrose, 5mMEDTA (ethylenediamine tetraacetic acid (EDTA)), 50mMTris-HCl, 10mM Trimethyl glycine, 8mM halfcystine, 0.1%BSA (bovine serum albumin) and 1%PVP (polyvinylpyrrolidone), pH7.8;

BufferB:0.4M sucrose, 1mMEDTA, 10mMTris-HCl, 0.1%BSA, pH7.2;

Solution W 1:1% glucose, 50mMEDTA, 25mMTris-HCl, pH8.0;

Solution W 2:0.2mMNaOH, 1%SDS (sodium lauryl sulphate);

Solution W 3:5mol/LKAC (Potassium ethanoate), pH4.8;

Rinsing liquid WT:70% alcohol;

Elution buffer TB:10mMTris-HCl, 1mMEDTA, PH=8.0;

(2) Antigen repairing:

Get the fresh tender tissue of 1g radish dark treatment 24h in 4 DEG C of environment, cleaning material (if need sterilization, being soak 5min in the clorox diluent of 0.7% at final concentration) in the distilled water of precooling (being chilled to 2-4 DEG C in advance);

(3) grind:

Material is once readyly need extract immediately, and to avoid cell expansion pressure and to obtain the plastosome of maximum, institute all need carry out in steps at 2-4 DEG C, be not greater than in the environment of 4 DEG C or the operating time interval extended in step.

BufferA is added, abrasive substance in the mortar of precooling; Filter homogenate by the hospital gauze of 4-8 layer, collect filtered liquid; Gauze finally by press filtration merges collects liquid in the centrifuge tube of 50ml, and the filtrate of homogenization needs to keep pH more than 7.0, if not enough, adjusts with NaOH; If the BufferA non-green tissue used, then ratio is 2ml/g; If chlorenchyma, 4ml/g;

(4) separate mitochondria:

Liquid will be collected in 4 DEG C, the centrifugal 5min of 1000g, collect supernatant; Then 4 DEG C, the centrifugal 20min of 12000g, discards supernatant; Add BufferB5-10ml, with the resuspended green precipitate particle of banister bruss; Re-suspension liquid is transferred in a new 50ml centrifuge tube, add BufferB to 40ml; Then 4 DEG C, the centrifugal 5min of 1000g, collects supernatant; Add DNase I (final concentration is 40 μ g/ml), place 1-2h; Add 1mL0.5mol/LEDTA again, pH8.0, leave standstill 10min after mixing, termination reaction; 4 DEG C, the centrifugal 20min of 12000g, discards supernatant (if desired obtaining rough mitochondrial pellet, resuspended with 2mlBufferB);

(5) Mitochondrial DNA is extracted:

In precipitation, add 2ml solution W 1, use pipettor to suspend and precipitate, add 250 μ l solution W 2, gentleness is put upside down 6-8 time, and add 350 μ l solution W 3, gentleness puts upside down centrifuge tube, fully mixes solution, the centrifugal 10min of 12000g.Draw supernatant and put into adsorption column (the pellosil centrifugal adsorbing column with nucleic acid pellosil), the centrifugal 30sec of 12000g, outwells the waste liquid in collection tube; Adsorption column is put into collection tube, adds the rinsing liquid WT of 600 μ l in adsorption column, the centrifugal 30sec of 12000g, outwells the waste liquid in collection tube; Again adsorption column is put into collection tube, add the rinsing liquid WT of 600 μ l in adsorption column, the centrifugal 30sec of 12000g, outwells the waste liquid in collection tube; Adsorption column is put into collection tube, and the centrifugal 2min of 12000g, outwells the waste liquid in collection tube; Adsorption column is put into a clean centrifuge tube, drip 50 μ l elution buffer TB to adsorption film middle part, room temperature places the centrifugal 2min of 2min, 12000g, by solution collection in centrifuge tube.The Mitochondrial DNA finally extracted is put in-20 DEG C of preservations.

(6) according to the method described above, the Mitochondrial DNA of 5 radish samples is extracted altogether, the quality of detected through gel electrophoresis Mitochondrial DNA.As shown in Figure 1, the Mitochondrial DNA of 5 samples amplifies band that is complete, clear, bright, nothing hangover at >2kb place, show that the Mitochondrial DNA genome that the method is extracted is complete, concentration is high, almost without degraded.

Method described in the present invention and traditional method (common differential centrifugation) is utilized to extract the Mitochondrial DNA of 5 radish samples further respectively, as pcr template, utilize a pair radish special primer, carry out pcr amplification to 10 samples respectively, Gel electrophoresis results as shown in Figure 2.As shown in Figure 2, the Mitochondrial DNA that extracting method of the present invention obtains, as masterplate, is clear, bright target stripe at the amplified band at 500bp place; The Mitochondrial DNA of traditional method for extracting, as template, does not obtain target stripe at 500bp place.

To sum up, adopt this kind to extract mitochondrial method, every 1g tissue extraction is to 0.2 μ gmtDNA, and total used time is 3-4h, the Mitochondrial DNA purity extracted is high, quality good, meet completely pcr amplification, quantitatively and qualitative analysis, order-checking equimolecular biological experiment requirement.

Comparing embodiment 1:

BufferA does not add 10mM Trimethyl glycine, pH value step-down in step (3), and ultimate yield obtains 0.2 μ g Mitochondrial DNA from every gram of sample and becomes every gram of sample and obtain 0.1 μ g Mitochondrial DNA.

Comparing embodiment 2:

In step (3), BufferA adds 0.1% beta-mercaptoethanol, and productive rate and quality are without considerable change.

Comparing embodiment 3:

In step (3), BufferA does not add halfcystine, and productive rate reduces, and Mitochondrial DNA degraded obviously.

Comparing embodiment 4:

Step (3) is if the rear PH less than 7.0 of middle grinding, and productive rate reduces.

Comparing embodiment 5:

In step (4), BufferA, B do not add 0.1%BSA, and productive rate reduces.

Comparing embodiment 6:

In step (4), centrifugal force 1000g changes 2000g into, and productive rate reduces.

Comparing embodiment 7:

Centrifugal force 12000g in step (4) changes 20000g into, improve, but productive rate and quality is without considerable change to the requirement of whizzer.

Comparing embodiment 8:

Step (4) middle 2mlBufferB is resuspended obtain rough plastosome after, add damping fluid C5-10ml, damping fluid C formula be: sucrose 0.6mol/L, Tris-Cl10mmol/L, EDTA20mmol/L, pH=7.2, experimental procedure increases, but productive rate and quality are without considerable change.

Comparing embodiment 9:

Further, after adding damping fluid C in step (4), 4 DEG C, the centrifugal 5min of 1000g, collects supernatant; 4 DEG C, the centrifugal 20min of 12000g, experimental procedure increases, but productive rate and quality are without considerable change.

Comparing embodiment 10:

Step (5) changes into and add 2ml preheating CTAB solution, the centrifugal 10min of 65 DEG C of cracking 1h, 12000g, Aspirate supernatant in precipitation; Add isopyknic phenol: chloroform, mixing shakes up, 12000g, centrifugal 10min, draws supernatant; Add isopyknic chloroform: primary isoamyl alcohol (24:1), mixing shakes up, 12000g, centrifugal 10min, draws supernatant; Add the pre-cold isopropanol of 2/3 volume, ice bath 30min; 12000g, centrifugal 1min, with 70% ethanol wash precipitation several; MtDNA is air-dry, and add 30ulTE and dissolve mtDNA ,-20 DEG C of preservations, for subsequent use, productive rate and quality there is no considerable change, but the treatment time becomes 4-5h from 30min.

Claims (9)

1. a rapid extracting method for plant mitochondiral-DNA, is characterized in that, specifically comprises the steps:

(1) solution preparation

BufferA:0.4M sucrose, 5mMEDTA, 50mMTris-HCl, 10mM Trimethyl glycine, 8mM halfcystine, 0.1%BSA and 1%PVP, pH7.8;

BufferB:0.4M sucrose, 1mMEDTA, 10mMTris-HCl, 0.1%BSA, pH7.2;

Solution W 1:1% glucose, 50mMEDTA, 25mMTris-HCl, pH8.0;

Solution W 2:0.2mMNaOH, 1% sodium lauryl sulphate;

Solution W 3:5mol/L Potassium ethanoate, pH4.8;

Rinsing liquid WT:70% alcohol;

Elution buffer TB:10mMTris-HCl, 1mMEDTA, PH=8.0;

Wherein, the percentage ratio of 70% alcohol is volume ratio, and all the other percentage ratios are mass volume ratio;

(2) pre-treatment

Get the fresh tender tissue of plant dark treatment 24h in 4 DEG C of environment, then be chilled to cleaning material in the distilled water of 2-4 DEG C in advance or at final concentration be 0.7% (w/v) sodium hypochlorite solution in soak and carry out disinfection;

(3) grind

In the mortar of precooling, add BufferA, then add pretreated material and grind; Filtered by the hospital gauze of 4-8 layer after grinding, collect filtered liquid and keep its pH more than 7.0;

(4) separate mitochondria

By the filtered liquid of collection in 4 DEG C, the centrifugal 5 ± 1min of 1000g, collects supernatant; 4 DEG C, the centrifugal 20 ± 2min of 12000g, discards supernatant; Add BufferB, with the resuspended green precipitate particle of banister bruss; Then 4 DEG C, the centrifugal 5 ± 1min of 1000g, collects supernatant; Add DNase I, place 1-2h; Add 0.5mol/LEDTA again, static 10 ± 2min after mixing, termination reaction; 4 DEG C, the centrifugal 20 ± 2min of 12000g, discards supernatant;

(5) Mitochondrial DNA is extracted

Solution W 1 suspension precipitation is added in precipitation; Then add solution W 2, gentleness is put upside down 6-8 time, then adds solution W 3, and gentleness puts upside down centrifuge tube, fully mixes solution; Centrifugal 10 ± 2min, absorption supernatant puts into the pellosil centrifugal adsorbing column with nucleic acid pellosil, more centrifugal, outwells waste liquid; Successively rinsing liquid WT is added 2 times, centrifugal rinsing adsorption column in adsorption column; Finally adsorption column is put into a clean centrifuge tube, drip elution buffer TB to nucleic acid pellosil middle part, room temperature places 2-5min, centrifugal 2-3min, by solution collection in centrifuge tube, and-20 DEG C of preservations;

Above-mentioned steps (2)-(4) all need to operate at 2-4 DEG C.

2. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 1, is characterized in that, the material of employing is non-green tissue, and BufferA consumption is 2ml/g, in the total mass of material.

3. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 1, is characterized in that, the material of employing is chlorenchyma, and BufferA consumption is 4ml/g, in the total mass of material.

4. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 1, is characterized in that, the BufferB consumption of described step (4) is 40ml/g, in the total mass of material.

5. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 4, is characterized in that, first add BufferB5-10ml/g, with the resuspended green precipitate particle of banister bruss; Re-suspension liquid is transferred in a new centrifuge tube, then add BufferB to 40ml/g.

6. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 1, is characterized in that, in described step (5), centrifugal centrifugal speed is 10000-15000g.

7. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 6, is characterized in that, in described step (5), centrifugal centrifugal speed is 12000g.

8. the rapid extracting method of a kind of plant mitochondiral-DNA as claimed in claim 1, is characterized in that, described step (5) adsorption column is put into before a clean centrifuge tube drips elution buffer TB, the more centrifugal 2min of 12000g.

9. as the rapid extracting method of a kind of plant mitochondiral-DNA in claim 1-8 as described in any one, it is characterized in that, the every 1g material of described step (5), after adopting 2ml solution W 1 to suspend, adds 250 μ l solution W 2 and 350 μ l solution W 3.