CN108982192A - Rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination - Google Patents
Rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination Download PDFInfo
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- CN108982192A CN108982192A CN201710400298.9A CN201710400298A CN108982192A CN 108982192 A CN108982192 A CN 108982192A CN 201710400298 A CN201710400298 A CN 201710400298A CN 108982192 A CN108982192 A CN 108982192A
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- 239000000725 suspension Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 240000008866 Ziziphus nummularia Species 0.000 title claims description 3
- 210000000349 chromosome Anatomy 0.000 title abstract description 9
- 210000003855 cell nucleus Anatomy 0.000 title abstract 3
- 239000006166 lysate Substances 0.000 claims abstract description 20
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 12
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 10
- 239000012498 ultrapure water Substances 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 108091092562 ribozyme Proteins 0.000 claims abstract description 4
- 239000000975 dye Substances 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 235000019628 coolness Nutrition 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 8
- 230000001568 sexual effect Effects 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 238000012549 training Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 241001247821 Ziziphus Species 0.000 abstract description 27
- 238000000034 method Methods 0.000 abstract description 14
- 239000007788 liquid Substances 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000001914 filtration Methods 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 210000004940 nucleus Anatomy 0.000 description 17
- 239000000523 sample Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 5
- 244000126002 Ziziphus vulgaris Species 0.000 description 4
- 235000008529 Ziziphus vulgaris Nutrition 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001365031 Isodon japonicus Species 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 239000012805 animal sample Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000000579 hyperploidy effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N15/1434—Optical arrangements
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a rapid preparation method of a cell nucleus suspension liquid suitable for jujube chromosome ploidy determination. Collecting young and tender leaves, washing with ultrapure water for several times, placing into a precooled culture dish, and adding precooled Tris-MgCl2Quickly cutting the lysate by using a blade, adding the precooled lysate, uniformly mixing, standing for several minutes, filtering by using gauze, putting into a centrifugal tube, adding RNA enzyme and PI dye solution, and keeping out of the sun at low temperature to obtain a cell nucleus suspension; and measuring by using a flow cytometer, and calculating the fluorescence intensity of the target peak to obtain the ploidy relation. The method improves multiple links in preparation of the jujube chromosome sample and ploidy identification, is simple, rapid, accurate and low in cost, and can be used for rapid detection of ploidy of mass jujube chromosomes. The method is also suitable for Zizyphus plants such as Ziziphi Spinosae and Maoye.
Description
Technical field
The invention belongs to ploidy breeding technical fields, and in particular to a kind of nucleus suitable for the measurement of jujube ploidy is outstanding
Supernatant liquid fast preparation method.
Background technique
Jujube tree (Ziziphus jujubaIt Mill.) is that the special advantage fruit tree for originating in China, the first big dry fruit and five are big
One of advantage economic forest.But current jujube cultivars structure is reasonable not enough, and big fruit, precocity, disease-resistant variety lack, and needs to accelerate training
Educate the excellent jujube tree new varieties of Comprehensive Traits.On jujube tree, due to crossbreeding difficulty, ploidy breeding becomes that jujube tree is important educates
Kind means.With the research of jujube ploidy breeding deepen continuously and mature application, the rapidly and accurately chromosome of qualification test material
Ploidy becomes more and more important and urgently.
Traditional methods for ploidy determination is mainly chromosome microscopic inspection and morphologic observation.Chromosome microscopic inspection
Most directly, most accurate, but there are technical steps cumbersome, heavy workload, it is at high cost, be difficult to carry out extensive Rapid identification etc. to ask
Topic.Morphologic observation is mainly according to " epimegetic " feature of polyploid, but morphologic appearance and ploidy are not in just in Hyperploidy
Correlation, and mode of appearance is easy to be influenced by environment and tree body state etc., lacks enough accuracys, it is general to be only preferably used as ploidy
The householder method or preliminary determining method of identification.In consideration of it, establishing a kind of jujube ploidy mirror quick, accurate, at low cost
Method is determined with important value.In recent years, flow cytometry is with its sample preparation simplicity, high degree of automation, quick, sensitive and can be same
The advantages that Shi Jinhang multi-parameter detects is widely used in Ploidy Identification, has become most common skill in plant Ploidy Identification at present
One of art.Its principle are as follows: the nucleus through fluorescent staining, under a certain pressure one by one by nozzle, into streaming exposure cell；
By laser irradiation, nucleus launches scattering light, and last optical signal is then converted into electric signal, inputs computer through data processing,
2 kinds of streaming maps of point diagram and peak figure are showed, peak figure ordinate is population, and abscissa is fluorescence intensity, by calculating target
The fluorescence intensity at peak is to obtain times sexual intercourse.It is following several that flow cytometry judges that the confidence level of ploidy depends primarily on
A parameter: one, peak figure, main peak is obvious, and noise peak is smaller, clear background；Two, CV value, animal sample require CV value to be lower than 3%, plant
Object sample CV value is lower than 5%.But flow cytometry requires detection sample to have complete cell or nuclei suspension, if
Cells in sample fragment impurity is more, and it is more to be directed at miscellaneous peak fragment, and miscellaneous peak is prevented to separate to influence to tie from good with signal peak
Fruit, then peak value histogram is invalid.
Nuclei suspension preparation is the key that flow cytometry.Its process is generally broadly divided into three steps: one, suitable
Sample is handled in lysate, generally Mechanical Crushing；Two, liquid obtained above is filtered, and with centrifugation phase
In conjunction with removing cell fragment；Three, to gained filtrate addition coloring agent to get nuclei suspension.But nucleus is outstanding on jujube
Supernatant liquid preparation also lacks systematic Study.
Inventor herein with ' Lizao Jujube Variety ' and its autotetraploid ' time ' for material, systematic comparison cell cracking
The influence of liquid type, blade maturity, blade holding time, dye liquor dosage to detection effect.The study found that 3 kinds of lysates
(WPB, Tris-MgCl2, GPB) and it so that ' Lizao Jujube Variety ' is generated signal peak, but WPB lysate cell fragment is more, cannot make
' time ' generates main peak, and lytic effect is worst, other 2 kinds of lysates can make ' time ' to generate clearly main peak, cell fragment
It is less, it can accurately judge a times sexual intercourse.Tris-MgCl2Compared with GPB, CV value is below 5%, but in four times of measurement
When body ' time ', Tris-MgCl is utilized2CV value be lower than 3%, hence it is evident that be better than GPB, and prepare it is convenient, it is low in cost.Therefore,
Tris-MgCl2It is chosen as the best lysate of jujube Methods of Ploidy Identification.
To select to be applicable in the Optimum Leaves state of jujube Methods of Ploidy Identification, to the tender, mature leaf of children and aging blade into
It has gone and has compared.Aging blade fails to generate apparent main peak as the result is shown, and children is tender, mature leaf can generate apparent main peak,
But the former main peak is clear, and background fragment is less, and CV value good with miscellaneous peak separating degree is substantially better than the latter.It proposes to be suitable for accordingly
The Optimum Leaves state of jujube Methods of Ploidy Identification is young leaflet tablet.
PI(propidium iodide) it is common coloring agent in flow cytometer ploidy identification, belong to non-specific dyestuff, it can be with
Base is perfectly combined, but PI price is higher and toxic.The shadow that PI dye liquor by studying different amounts generates main peak
It rings, has researched and proposed suitable PI dye liquor dosage.
On aforementioned Research foundation, the jujube nuclei suspension fast preparation method of the invention patent is formd.Utilize this
The step of method, nuclei suspension is prepared simply, eliminates centrifugation using highdensity filtered through gauze, operation is time saving, each
Sample processing time only needs 5 minutes or so (conventional method needs 20 minutes or so).Through actually detected, this method is suitable for all
The jujubes such as the kind and wild jujube of jujube and Rabdosia japonica can carry out Ploidy Identifications to more than 200 materials in 1 day, can meet pair
The needs of extensive sample Ploidy Identification.
The present invention solves the quick and precisely identification problem of jujube ploidy, and to the chromosome of other xylophytas times
Property Rapid identification have reference value.
Summary of the invention
The present invention provides a kind of nuclei suspension fast preparation method suitable for the measurement of jujube ploidy, can use
In flow cytometry Rapid identification jujube ploidy, and compounding medicine is simple, at low cost, measurement effect is accurate, can be significantly
Improve the efficiency of identification jujube ploidy.
The technical solution used in the present invention is: acquiring tender blade, shreds rapidly, the Tris-MgCl of pre-cooling is added2
Lysate stands after mixing, is entered in centrifuge tube with filtered through gauze, and RNA enzyme and PI dye liquor is added, is placed in 4 DEG C of refrigerators and is protected from light 5-
30min, nuclei suspension；Then using the fluorescence intensity of flow cytometer measurement nuclei suspension, pass through calculating
The fluorescence intensity of target peak can be obtained a times sexual intercourse.
Particular content of the invention, i.e., a kind of nuclei suspension fast preparation method suitable for the measurement of jujube ploidy
It is as follows:
(1) acquisition and preservation of sample: tender blade is acquired, is saved under the conditions of being placed in -20 ~ 4 DEG C；
(2) lysate is prepared: taking 2.422g Tris-HCl, 0.0813g MgCl2·6H2O is added in 100mL volumetric flask
The Triton-100 of 0.5mL adjusts pH value to 7.0-7.5 using NaOH with ultrapure water constant volume；
(3) it the configuration of PI dye liquor: takes 1mg PI dyestuff to be melted into the ultrapure water or PBS buffer solution of 1mL, is placed in -20 DEG C of refrigerators
It saves, the used time, which takes out, thaws, and places in 4 DEG C；
(4) preparation of nuclei suspension: choosing young leaflet tablet 2 ~ 4, cleaned 1 ~ 3 time with ultrapure water, is put into the training of 4 DEG C of pre-coolings
It supports in ware, the Tris-MgCl of 4 DEG C of 1mL pre-coolings is added2Lysate adds 4 DEG C of 1mL pre-coolings with the rapid chopper blade of knife
Tris-MgCl2Lysate stands 1 ~ 10 minute after mixing, is entered in centrifuge tube with 500 mesh filtered through gauze, and it is dense that about 1 μ L is added
Degree is the RNA enzyme of 10mg/mL and the PI dye liquor of 10 ~ 30 μ L 1mg/mL, and 4 ~ 30min of dyeing is protected from light under the conditions of being placed in 4 DEG C, is obtained carefully
Karyon suspension；
(5) Ploidy Identification: nuclei suspension is measured with Germany's Partec company Cube8 type flow cytometer, by calculating mesh
The fluorescence intensity for marking peak obtains times sexual intercourse.
Beneficial effects of the present invention: one is omitted the step of nuclei suspension is centrifuged, under the premise of guaranteeing effect
Keep process simpler (the processing time 5 minutes or so), cost lower (1 yuan/sample or so)；Second is that improving lysate is added journey
Sequence；Third is that PI dye liquor suitable amounts (10 ~ 30 μ L) has been determined.The present invention overcomes chromosome microscopic inspection step operation is numerous
The problems such as trivial, cost height, speed are slowly and morphologic observation accuracy is low.
Specific embodiment
Tris-HCl lysate is configured first, takes 2.422g Tris-HCl, 0.0813g MgCl2·6H2O is in 100ml volumetric flask
In, the Triton-100 of 0.5mL is added, with ultrapure water constant volume to 100mL, adjusts pH value to 7.5, in 4 DEG C of refrigerators using NaOH
Middle pre-cooling；Secondly configuration PI dye liquor, takes 1mg PI dyestuff to be dissolved in the ultrapure water of 1mL, is placed in -20 DEG C of refrigerators and saves.Used time takes out
It thaws, is placed in 4 DEG C.
' Lizao Jujube Variety ' and ' time ' (autotetraploid of ' Lizao Jujube Variety ') young leaflet tablet is chosen in field, is put into sampling
In bag, it is placed in ice chest and takes back laboratory and be stored in 4 DEG C；Young leaflet tablet 50mg is taken, with ultrapure water 3 times, is put after drying
In the culture dish for entering 4 DEG C of pre-coolings, the lysate of 4 DEG C of 1mL pre-coolings is added, is quickly shredded with sharp blade, adds 1mL
Tris-HCl lysate stands 1min after mixing；The lysate in culture dish is drawn, with 500 mesh membrane filtrations to 3mL testing tube
In, 20 μ L PI dye liquors and 1 μ L RNase is added, mixing is placed on 4 DEG C of refrigerators and is protected from light dyeing 4min；After dyeing, Germany is utilized
8 type flow cytometer of Partec company Cube is detected, and 5000 ~ 10000 particles are collected, and each sample is repeated 3 times；It is logical
The fluorescence intensity for crossing calculating target peak obtains a times sexual intercourse: the time/Lizao Jujube Variety=1.89 ± 0.15, ratio are approximately equal to 2.
Claims (1)
1. it is a kind of suitable for jujube ploidy measurement nuclei suspension fast preparation method, it is characterised in that specifically include with
Under several steps:
(1) acquisition and preservation of sample: tender blade is acquired, is saved under the conditions of being placed in -20 ~ 4 DEG C；
(2) Tris-MgCl2The preparation of lysate: 2.422g Tris-HCl, 0.0813g MgCl are taken2·6H2O is in 100mL capacity
In bottle, the Triton-100 of 0.5mL is added, with ultrapure water constant volume, adjusts pH value to 7.0-7.5 to get Tris- using NaOH
MgCl2Lysate；
(3) it the configuration of PI dye liquor: takes 1mg PI dyestuff to be melted into the ultrapure water or PBS buffer solution of 1mL, obtains PI dye liquor；
(4) preparation of nuclei suspension: choosing young leaflet tablet 2 ~ 4, cleaned 1 ~ 3 time with ultrapure water, is put into the training of 4 DEG C of pre-coolings
It supports in ware, the Tris-MgCl of 4 DEG C of 1mL pre-coolings is added2Lysate adds 4 DEG C of 1mL pre-coolings with the rapid chopper blade of knife
Tris-MgCl2Lysate stands 1 ~ 10 min, is entered in centrifuge tube with 500 mesh filtered through gauze after mixing, it is dense that about 1 μ L is added
Degree is the RNA enzyme of 10mg/mL and the PI dye liquor of 10 ~ 30 μ L 1mg/mL, and 4 ~ 30 min are protected from light under the conditions of being placed in 4 DEG C, obtain nucleus
Suspension；
(5) Ploidy Identification: nuclei suspension measures ploidy with flow cytometer, and the fluorescence intensity by calculating target peak obtains
Times sexual intercourse.
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| CN201710400298.9A CN108982192A (en) | 2017-05-31 | 2017-05-31 | Rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110146431A (en) * | 2019-06-05 | 2019-08-20 | 福建农林大学 | A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant |
| CN110749699A (en) * | 2019-10-10 | 2020-02-04 | 南京农业大学 | Method for screening triploid new germplasm from white loquat seedling |
| CN113358547A (en) * | 2021-06-08 | 2021-09-07 | 四川农业大学 | Efficient ploidy detection method for akebia trifoliata |
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2017
- 2017-05-31 CN CN201710400298.9A patent/CN108982192A/en active Pending
Patent Citations (3)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110146431A (en) * | 2019-06-05 | 2019-08-20 | 福建农林大学 | A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant |
| CN110749699A (en) * | 2019-10-10 | 2020-02-04 | 南京农业大学 | Method for screening triploid new germplasm from white loquat seedling |
| CN113358547A (en) * | 2021-06-08 | 2021-09-07 | 四川农业大学 | Efficient ploidy detection method for akebia trifoliata |
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Application publication date: 20181211 |
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