CN106092864A - A kind of method of tail alpine ash flow cytometer detection - Google Patents
A kind of method of tail alpine ash flow cytometer detection Download PDFInfo
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- CN106092864A CN106092864A CN201610402098.2A CN201610402098A CN106092864A CN 106092864 A CN106092864 A CN 106092864A CN 201610402098 A CN201610402098 A CN 201610402098A CN 106092864 A CN106092864 A CN 106092864A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000006109 Eucalyptus delegatensis Species 0.000 title claims abstract description 24
- 239000006166 lysate Substances 0.000 claims abstract description 43
- 238000005336 cracking Methods 0.000 claims abstract description 24
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- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to plant Ploidy detection field, a kind of method disclosing tail alpine ash flow cytometer detection.The method comprises the following steps: (1) takes the tender leaf of tail alpine ash DH32 29 and adds in lysate, cuts tender leaf with blade, cracking, obtains cell cracking suspension；(2) being filtered by the cell cracking suspension of gained in step (1), the liquid after filtering is centrifuged, and outwells supernatant, again adds lysate, makes the nucleus Eddy diffusion in precipitation, then refilter, obtain filtrate；(3) filtrate of gained adds in step (2) RNase enzymatic solution and carry out enzymolysis, obtain enzymolysis solution；(4) enzymolysis solution of gained adds in step (3) PI dye liquor to dye, obtain dyeing liquor；(5) dyeing liquor of gained in step (4) is detected on flow cytometer.The method can realize immediately preparing sample after drawing materials in field, it is thus achieved that nuclear targeting liquid within 2 days, after (48 hours), do flow detection and analysis under the conditions of, it can be ensured that the stability of Detection results, relieve the limitation immediately carrying out detecting after flow cytometer detection to be adopted.
Description
Technical field
The invention belongs to plant Ploidy detection field, particularly to a kind of method of tail alpine ash flow cytometer detection.
Background technology
Flow cytometry (Flow cytometry, FCM) is a new technique in the twentieth century birth fifties, but directly
Just being used widely in plant science field to twentieth century this technology in the latter stage eighties, it is mainly used in nucleus
DNA content measures, and DNA ploidy qualification and cell cycle analysis etc. are studied.This technology pass that success uses in plant
Can key point be prepare unicellular (or slender karyon) suspension of vegetable material, and scientific research personnel is for this difficult problem always
Updating cell detection pretreatment technology, such as Heller (1973) utilizes the cell wall of hydrolytic enzyme digestion plant to discharge
Nucleus, but this mode is very time-consuming, and descendant seldom continues to prolong in this way；After this, Ulrich (1986,1988)
Improve method with (1988,1992) such as Bergounioux, utilize the hypotonic lysis effect of intact protoplasts to carry out release cells
Core, this improvement is the most time-consuming, and, the difficulty obtaining complete protoplast from some species or certain organization type also limits
Make this application.Additionally, Galbraith etc. (1983) develop a kind of cell organized in lysate by mulch
The method carrying out karyorhexis, this method is quick, practical, and becomes a kind of cell lysis core in plant flow cytometry
Main and most reliable method.But, because there is the problems such as difficulty when releasing the fixing of cell wall and constraint is big in plant, because of
This, although the pre-treatment having Activities of Some Plants streaming to crack obtains successfully, but, still there is a large amount of plant to there is no so far and split
Solve effect and reach the lysate of flow cytometer detection requirement.Especially for xylophyta, its degree of lignification is high, the firmest and
Thick cell wall, makes Cytoplasm support the dyeing of lysate possibly together with a large amount of secondary metabolites (such as materials such as tannic acid)
Anti-effect is big, more difficult when these factors cause cracking and attachment independent single cells.In practice, it is thus achieved that excellent effect lysate
Species are the most less.Further, even if cracking obtains successfully, there is also fragment many, nucleus is imperfect, and cytoadherence is assembled, dye
Color is uneven, yields poorly and CV value (index of reacting cells core integrity) does not reaches the problems such as requirement.It addition, blade is fresh
Testing result is also had a significant impact by degree, and the lysate having been developed over to crack in time and to detect after requiring to adopt, otherwise, and inspection
Surveying result can be greatly affected, because plant is many times field sampling, but flow cytometer is large-scale instrument, it is impossible to
Meaning is moved, and the requirement detected in the wild or nearby in time after being extremely difficult to process, therefore, if sample can be often after cracking
Keep the long period under temperature reaches stablizing of Detection results, then can solve this difficult problem.
Eucalyptus (Eucalyptus) is one of big quick growing species of trees in the world three, has very important in world's forestry industry
Status, Eucalyptus is the most also the seeds that China's artificial forest wood yield is most.There are the seeds of such critical role and value, exploitation
Comprise every biotechnology of its genetic breeding with advancing, excavate its Exploitative potential, improve further its produce, economical and
Social benefit, has very important significance.But because its nucleus cracking technique fails to develop maturation, its DNA ploidy always
Identify that the researchs such as (being mainly used in the ploidy breeding of Eucalyptus), cell cycle, apoptosis and associated metabolic fail to carry out, sternly always
Heavily constrain carrying out of the breeding techniques such as its ploidy breeding.
Summary of the invention
For overcoming above-mentioned existing shortcoming with not enough, the primary and foremost purpose of the present invention is to provide a kind of tail alpine ash flow cytometer detection
Method.
Another object of the present invention is to the method that above-mentioned tail alpine ash flow cytometer detection is provided measure at nuclear DNA content,
The application of the aspects such as DNA ploidy qualification and cell cycle analysis.
The purpose of the present invention is realized by following proposal:
A kind of method of tail alpine ash flow cytometer detection, mainly comprises the steps that
(1) cracking: take in the lysate of tender leaf addition pre-cooling of tail alpine ash DH32-29, cut tender leaf with blade, make tender leaf
Size is 0.0025～0.0225mm2, cracking, obtain cell cracking suspension；
(2) filter: being filtered by the cell cracking suspension of gained in step (1), the liquid after filtering is centrifuged, and outwells
Clear liquid, adds the lysate of pre-cooling, makes the nucleus Eddy diffusion in precipitation, then refilter, obtain filtrate；
(3) enzymolysis: add RNase enzymatic solution in step (2) in the filtrate of gained and carry out enzymolysis, obtain enzymolysis solution；
(4) dyeing: add PI dye liquor in step (3) in the enzymolysis solution of gained and dye, obtain dyeing liquor；
(5) flow cytometer detection: the dyeing liquor of gained in step (4) is detected on flow cytometer.
The formula of the lysate described in step (1) and step (2) is 0.2mol/L Tris, 4mmol/L MgCl2·
6H2O, 2mmol/L EDTA Na2·2H2O, 86mmol/L NaCl, 10mmol/L sodium pyrosulfite, 100mmol/L citric acid,
1% (v/v) PVP-10,1% (v/v) Triton X-100,0.5% (v/v) Tween 20, the solvent of lysate is distilled water,
PH=7.5.Prepare under room temperature, in 4 DEG C of Refrigerator stores.
The lysate of the pre-cooling described in step (1) and step (2) refers to that temperature is maintained at the lysate of 0～4 DEG C.
Cracking described in step (1) refers to crack 5～20min.
The consumption of lysate used in step (1) is every 40～the lysate of the tender leaf of 50mg use 0.2～1mL.
Filtration described in step (2) refers to filter with the nylon leaching film of 300 mesh, refilters the nylon referred to 400 mesh
Membrane filtration.
Centrifugal described in step (2) refers to that 1000rpm is centrifuged 3～8min at 4 DEG C.
The time of the Eddy diffusion described in step (2) is 5～20s.
The concentration of the RNase enzymatic solution described in step (3) is 1mg/mL, and solvent is distilled water, and enzymolysis is carried out at 37 DEG C,
Enzymolysis time is 30～45min.
The concentration of the PI dye liquor described in step (4) is 1mg/mL, and solvent is distilled water.
Dyeing described in step (4) is carried out at 0～4 DEG C, and dyeing time is at least 20min.
RNase enzyme used in lysate used in lysate used in step (1), step (2), step (3) is molten
The volume ratio of PI dye liquor used in liquid and step (4) is (0.2～1): (0.2～0.5): (0.002～0.006):
(0.005～0.05).
The method of above-mentioned tail alpine ash flow cytometer detection measures at nuclear DNA content, and DNA ploidy is identified and cell cycle
The application of the aspects such as analysis.The mechanism of the present invention is:
Lysate should destroy the existing structure of plant cell, releases cell wall to the constraint of cell and fixation, makes
Nucleus can crack out, meanwhile, also to ensure that nuclear stability during whole detection, protection nucleus are not dropped
Solve and can be colored；It addition, nucleus to be ensured does not sticks together.In the lysate of the present invention, each component effect is such as
Under: Tris as buffer, maintains the stability of lysate in lysate；MgCl2·6H2O be used for keep nuclear completely
Property and stability, play the effect preventing nuclear collapse；EDTA (ethylenediaminetetraacetic acid) and citric acid, as chelating agen, are used for
In conjunction with bivalent cation, serve as the coenzyme of nuclease, strengthen colouring power and the effect of DNA；NaCl be used for maintaining suitably from
Sub-intensity；Triton X-100 and Tween 20 is non-ionic detergent, is used for releasing sticky Cytoplasm to nuclear
Parcel and isolation, release cells core, reduces nucleus and the polymerization affinity of fragment；Sodium pyrosulfite and PVP-10 (polyethylene
Ketopyrrolidine) play the effect preventing aldehydes matter contained by xylophyta oxidized, and Cytoplasm can be offset to core DNA fluorescence dye
After the negative effect xylophyta aldehydes matter of color is oxidized, its nucleus can be difficult to be colored.
The present invention, relative to prior art, has such advantages as and beneficial effect:
This streaming lysate, for the characteristic of xylophyta, is improved: (1) xylophyta contains more tannin
Deng material, Cytoplasm viscosity is big, causes cracking and dyeing difficulty to become big, therefore, adds antioxidative composition pyrosulfurous acid
Sodium and PVP-10；(2) high for xylophyta degree of lignification, cell wall is firm and thick, set effect strong, and cell is difficult to separate
The feature gone out, have employed Triton X-100 and Tween20, reduces the Cytoplasm dyeing resistant function to lysate, significantly
Improve cell cleavage rate, it is thus achieved that can reach testing requirement (mainly fragment rate, the index such as CV value reaches testing requirement)
Lytic effect.
It addition, after using lysate of the present invention to carry out cracking process, examine after preserving 48h at room temperature 25 DEG C
Survey, find that its analysis result, without significant change, illustrates that this lysate has good stability, it is possible to achieve field draw materials after between
Do the feasibility of flow detection and analysis every 2 days (48 hours), relieve the limitation immediately carrying out detecting after flow cytometer detection to be adopted, aobvious
Write application scenario and the range of application having widened flow cytometer detection.
Accompanying drawing explanation
Fig. 1 is tail alpine ash DH32-29 blade nucleus DNA Nogata of detection after cracking poststaining 20min with lysate
Figure.
Fig. 2 is tail alpine ash DH32-29 blade at dyeing 20min the nucleus DNA Nogata that again detects after placing 48h
Figure.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
In embodiment, agents useful for same is without specified otherwise, all can buy from market routine.
Flow cytometer uses Beckman Coulter Inc. of the U.S. (Beckman Coulter, Inc.)
CytoFLEX flow cytometer, is configured to air cooled argon ion laser, and testing result carries software CyoExpert from it
Software (version 1.1.10.0.) is upper to be obtained.In detection, sample flow rate is defined to low speed 10 μ L/min, and in whole reality
Keep constant during testing.Each material arranges twice repetition, is spaced more than 24h, time interval of i.e. drawing materials between twice repetition
More than one day, averaging, nucleus amount during each duplicate detection is all more than 8000.
The parameter evaluating each material tests effect is: FS, for evaluating the relative size of granule；SS, is used for evaluating
The opposing optical complexity of granule；FL, refers to catch the nuclear fluorescence intensity of PI；CV value, evaluate nuclear integrity and
The effect of DNA dyeing；BF, evaluates sample quality；YF, compares the cell in nucleus amount and leaf tissue used in suspension
Core total quantity.The preferable standard of lytic effect is high FL value, YF value and low CV value, DF value, wherein:
The computing formula of CV (%) is:
The calculating formula of BF (%) is:
YF (the individual * second-1Milligram-1) computing formula be:
Embodiment 1
The preparation of lysate: first weigh 24.23g Tris, 0.81g MgCl with analytical balance2·6H2O、0.82g
EDTA Na2·2H2O, 5.03g NaCl, 1.90g sodium pyrosulfite, 19.21g citric acid, 10.00g PVP-10, put into wash dry
The clean beaker through distilled water rinse, adds distilled water, and Glass rod stirring is to all dissolving；Then 10mL is pipetted with liquid-transfering gun
Triton X-100 and 5mL Tween 20, with the volumetric flask constant volume of 1L after all dissolving；After finally using pH meter pH value determination,
HCl solution and 10mol/L NaOH solution adjustment pH with 1mol/L are to designated value 7.5, in 4 DEG C of Refrigerator stores.
Winning tail alpine ash DH32-29 cultivation Seedling top 2～3 tender leaf 40mg, material is placed in culture dish, and culture dish is placed on
On ice, drawing 1mL 0～the lysate submergence material of 4 DEG C with liquid-transfering gun, cut material with blade, being switched to material is 0.01mm2Left
Right size, cracks 10min；Cell cracking suspension filters to remove cell debris and big residue through 300 mesh nylon leaching films；Filter
Liquid is at 4 DEG C, and 1000rpm is centrifuged 5min；Outwelling supernatant, the lysate adding 400 μ L 0～4 DEG C makes the nucleus in precipitation
Eddy diffusion 10s (this operation is still carried out on ice)；Again filter with 400 mesh nylon leaching films, obtain filtrate；Filtrate adds 4 μ L
37 DEG C of enzymolysis 30min of 1mg/mL RNase enzymatic solution (purchased from the raw work in Shanghai)；It is eventually adding 20 μ L 1mg/mL PI dye liquor dyeing
20min；Then carrying out flow cytometer detection, its nucleus DNA rectangular histogram is as it is shown in figure 1, its cracking assessment parameter is as shown in table 1.
Embodiment 2
Lysate after carrying out flow cytometer detection in embodiment 1 is placed in 48h in the foam box under the conditions of 25 DEG C of black outs, again
Carrying out flow cytometer detection, its nucleus DNA rectangular histogram is as in figure 2 it is shown, its cracking assessment parameter is as shown in table 1.
Result shows, the peak type that the detection of two embodiments obtains is constant, and sample 25 DEG C of black outs guarantors of room temperature after dyeing be describeds
Deposit 48h the stability of lysate is not affected, therefore, it can field sampling, detect after room temperature places 48h after dyeing.
Table 1 tail alpine ash DH32-29 cracking assessment parameter under difference detection time conditions compares
From the point of view of the cracking parametric results of flow cytomery, fluorescence average FL has raised, and BF value has declined,
The longest dyeing of explanation time may be more abundant, and the fluorescence intensity that can detect that strengthens, and also illustrate that the good stability of lysate；YF
It is declined slightly, thus it is speculated that be probably part nucleus and decompose；CV value is increased to 4.51% from 3.87%, but does not the most surpass
Cross 5%.From the point of view of the nucleus DNA rectangular histogram of tail alpine ash DH32-29 blade, the peak type obtained after room temperature placement 48h and the same day
Detection gained is basically identical.In sum, after sample dyeing, room temperature placement 48h is little to the stability influence of lysate, detection
Effect is without significant change.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, any change of being made under other spirit and principle without departing from the present invention, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (8)
1. the method for a tail alpine ash flow cytometer detection, it is characterised in that comprise the following steps:
(1) cracking: take in the lysate of tender leaf addition pre-cooling of tail alpine ash DH32-29, cut tender leaf with blade, make the size of tender leaf
It is 0.0025～0.0225mm2, cracking, obtain cell cracking suspension；
(2) filter: being filtered by the cell cracking suspension of gained in step (1), the liquid after filtering is centrifuged, and outwells supernatant
Liquid, adds the lysate of pre-cooling, makes the nucleus Eddy diffusion in precipitation, then refilter, obtain filtrate；
(3) enzymolysis: add RNase enzymatic solution in step (2) in the filtrate of gained and carry out enzymolysis, obtain enzymolysis solution；
(4) dyeing: add PI dye liquor in step (3) in the enzymolysis solution of gained and dye, obtain dyeing liquor；
(5) flow cytometer detection: the dyeing liquor of gained in step (4) is detected on flow cytometer.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
The formula of the lysate described in step (1) and step (2) is 0.2mol/L Tris HCl, 4mmol/LMgCl2·
6H2O, 2mmol/L EDTA Na2·2H2O, 86mmol/L NaCl, 10mmol/L sodium pyrosulfite, 100mmol/L citric acid,
Volume fraction is the PVP-10 of 1%, and volume fraction is the Triton X-100 of 1%, and volume fraction is the Tween 20 of 0.5%,
The solvent of lysate is distilled water, pH=7.5, prepares, 4 DEG C of Refrigerator stores under room temperature；
The lysate of the pre-cooling described in step (1) and step (2) refers to that temperature is maintained at the lysate of 0～4 DEG C.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
Cracking described in step (1) refers to crack 5～20min；
The consumption of lysate used in step (1) is every 40～the lysate of the tender leaf of 50mg use 0.2～1mL.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
Filtration described in step (2) refers to filter with the nylon leaching film of 300 mesh, refilters the nylon leaching film referred to 400 mesh
Filter；
Centrifugal described in step (2) refers at 4 DEG C, under the conditions of 1000rpm centrifugal 3～8min；
The time of the Eddy diffusion described in step (2) is 5～20s.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
The concentration of the RNase enzymatic solution described in step (3) is 1mg/mL, and solvent is distilled water, and hydrolysis temperature is 37 DEG C, enzymolysis
Time is 30～45min.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
The concentration of the PI dye liquor described in step (4) is 1mg/mL, and solvent is distilled water；
Dyeing described in step (4) is carried out at 0～4 DEG C, and dyeing time is at least 20min.
The method of tail alpine ash flow cytometer detection the most according to claim 1, it is characterised in that:
RNase enzymatic solution used in lysate used in lysate used in step (1), step (2), step (3) with
And the volume ratio of used PI dye liquor is (0.2～1) in step (4): (0.2～0.5): (0.002～0.006): (0.005～
0.05)。
8. measure at nuclear DNA content according to the method for the tail alpine ash flow cytometer detection described in any one of claim 1～7, DNA
Application in terms of Ploidy Identification and cell cycle analysis.
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