CN105784573A - Method for analyzing wheat root tip cell cycle through flow cytometry - Google Patents
Method for analyzing wheat root tip cell cycle through flow cytometry Download PDFInfo
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Abstract
The invention discloses a method for analyzing the cycle G1, the cycle S and the cycle G2/M of wheat root tip cells through flow cytometry and belongs to the technical field of biology.The method comprises the steps of immersing wheat seeds and accelerating germination, obtaining a wheat root tip sample through cutting, and fixing the sampling with Carnoy fixing liquid; rinsing the sample thoroughly with a phosphate buffer solution; placing the sample in a mixed enzyme liquid of cellulose and pectinase for conducting enzymolysis for 1-2 hours; after the sample is rinsed thoroughly, conducting grinding, so that single-cell suspension is obtained; after rinsing is conducted, conducting dyeing away from light on the wheat root tip cells through DAPI dye liquor; after rinsing and filtering are conducted, differentiating and counting the cells of cycle time phases including the cycle G1, the cycle S and the cycle G2/M which are different in DNA content through a flow cytometer, and learning about the proliferation condition of the cells.The method is simple and easy to operate, separated materials are easy to obtain, the enzymolysis method is simple, the obtained single-cell suspension is high in purity, the flow cytometer is good in detection effect, and the result is stable.
Description
The present invention obtains the subsidy of National Nature fund youth's project (31501234).
Technical field
The invention belongs to biological technical field, relate to the analysis method carrying out the Wheat root tip cells cycle (G1 phase, S phase, G2/M phase) with flow cytometry.
Background technology
Flow cytometer (Flow Cytometer, FCM) is the novel high-tech instrument being integrated with laser technology, photoelectric measurement technology, computer technology, hydromechanics technology, electronics physics, cellular immunofluorescence chemical technology and monoclonal antibody technique.It can carry out qualitative and quantitative analysis and sorting on individual cell level, is mainly used in basic research and the clinical testing of the subjects such as hematology, immunology, oncology, molecular biology and cell biology.For animal and microorganism, owing to plant tissue is complicated (such as cell membrane, special cells device etc.) with eucaryotic cell structure, the single-cell suspension liquid that preparation is suitable for flow cytometry analysis is relatively difficult, and flow cytometer is the most delayed in the application in botany field.But along with the continuous renewal of streaming sample technology of preparing, flow cytometry demonstrates wide application prospect in the every subjects such as plant science field such as plant genetics and breeding, plant chromosome analysis and library construction, adversity plant, Plant Taxonomy, Plant Pathology.
During with flow cytomery sample, it is desirable to sample must be complete cell or nuclear suspension.And nuclear concentration also to reach certain requirement.Owing to plant cell has outside cell membrane, and containing a large amount of secondary metabolites, this just gives and prepares sample and add difficulty, greatly reduces the success rate of streaming experiment.And different plant structures and composition have bigger difference, so different preparing on nuclei suspension method, conventionally used nucleus preparation method has direct shearing method and enzymatic isolation method.At present in botany research, mainly carry out flow cytometry by preparing plant monocyte core suspension, thus detect plant cell nuclear dna content and ploidy.Although utilizing flow cytometry to carry out the research of nuclear DNA content on the plants such as corn, sweet potato, Kiwi berry, wheat, but when these researchs are all the nucleus extracting these plant different tissues cells by different dissociation solution, impurity and sample fragment are more, easy and nucleus is polymerized to group, and the concentration of non-ionic octoxynol detergent is difficult to hold, destroying while cell membrane also destructible nuclear membrane, the cell nuclei thus causing extraction is few.
Plant growth is in natural environment, necessarily by the spread effect of various external environments, and the change problem that always biology is concerned about of plant responding external environment.Growing of plant is exactly growth and the division of plant cell, and the change of plant cell cycle is grown closely related with it.Wheat, as one of important cereal crops, the cell cycle of research wheat, has very important realistic meaning.There are some researches show, the same with zooblast, there is complicated cell cycle regulating mechanism in plant cell, thus affect plant grow this research forefathers study on the basis of, prepared complete by improvement, form is preferable, NA wheat is unicellular, recycling fluorescent dye DAPI carries out nucleic acid staining to Wheat root tip cells, the cell cycle is determined according to nucleic acid binding capacity, use flow cytometer by the cycle phase of different DNA contents, such as the G1 phase, the S phase, the G2/M phase, it is distinguish between and counts, understand the proliferative conditions of cell, lay the foundation for doing molecular biology research further.This method establishes the most feasible test method, and operating procedure is simple, and test effect is stable, clear, the most either can well be applied in scientific research, detection and student test.
Summary of the invention
The present invention prepares single cell suspension, the change of recycling its cell cycle of flow cytomery using Helminthosporium sativum as experiment material, and meaning is to provide technical support for the molecular mechanism of research Growth of Wheat and wheat response environment varying effect further.
For achieving the above object, to be disclosed directly below technical scheme as follows for the present invention:
1, a kind of being suitable for the method that flow cytometry carries out Wheat root tip cells cycle analysis, it is to utilize the Helminthosporium sativum sample fixed, and prepares the single-cell suspension liquid of Helminthosporium sativum tissue, carries out fluorescent staining, flow cytomery, including:
(1) wheat seed carries out presoaking and germinating, when root length length to 3-5 cm, cut the tip of a root of about 0.2 cm length, be fixed on 30 min in freshly prepared Ka Nuoshi fixer, with proceeding in the ethanol that volume ratio is 70% after the phosphate buffer rinsed clean of pH 7-8 again, it is stored in 4 DEG C of refrigerators；Described Ka Nuoshi fixer refers to: methyl alcohol: the volume ratio of glacial acetic acid is 3:l；It is characterized in that:
(2) tip of a root sample fixed is inserted 0.1-0.2%(v/v) glycerine in permeate 2-3h, rinse 3 times with the phosphate buffer of pH 7-8, each 10 min, upper strata buffer solution is drawn with liquid-transfering gun after staticly settling, it is immersed in cellulase again and in mixed enzyme solution that pectase is made, 37 DEG C of enzymolysis 1.5-3 h, make Helminthosporium sativum soft texture；Described cellulase and the mass concentration of pectase are all 2%, prepare by deionized water；
(3) by described step 2) in Helminthosporium sativum pH 7-8 phosphate buffer rinse 3 times, each 10 min, after drawing major part upper strata buffer solution with liquid-transfering gun, single cell suspension is ground to form by pestle gentleness, filter, collect filtrate, single cell suspension can be obtained. at present, plant is typically to extract the nucleus of plant tissue cell, extract complicated component, impurity and fragment are prone to nucleus and assemble agglomerating, it is difficult to separate, the single cell suspension impurity fragment that present invention conventional reagent is made is few, it is easier to subsequent experimental operates.
(4) single cell suspension is centrifuged 4 min through 3000 r.p.m., it is the 4 of 0.5-2 μ g/ml that the precipitation obtained adds concentration, 6-diamidine-2-phenylindone dye liquor, shake up rear lucifuge to dye 10 min, intracellular DNA material is by fluorescent staining, rinse 3 times with the phosphate buffer of pH 7-8, each 10 min；
(5) after dyeing, single cell suspension flow cytometer detects, a length of 340 nm of excitation light wave.
Wheat seed running water described in step of the present invention (1) carries out presoaking and germinating, hatches 4-5d for 25 DEG C, treats that root length length is to 3-5 cm.After described step (1)-(5) rinse with the phosphate buffer of pH 7-8 every time, all it is centrifuged 4 min with 3000 r.p.m. rotating speeds.
The present invention further discloses and be suitable for the method that flow cytometry carries out Wheat root tip cells cycle analysis, can apply the carrying out in terms of understanding the proliferative conditions of cell.Dyeing therein, determines the cell cycle according to nucleic acid binding capacity, by the cycle phase of different DNA contents, such as G1 phase, S phase, G2/M phase, is distinguish between and counts, understanding the proliferative conditions of cell.The present invention by improvement prepare complete, form wheat preferable, NA is unicellular, utilize fluorescent dye DAPI that Wheat root tip cells is carried out nucleic acid staining, determine the cell cycle according to nucleic acid binding capacity, use flow cytometer by the cycle phase of different DNA contents, such as the G1 phase, the S phase, the G2/M phase, it is distinguish between and counts, understand the proliferative conditions of cell, the results show: the streaming result figure G1 visible in detail phase in Wheat root tip cells cycle, S phase, G2/M.Wherein the G1 phase refers to the off time before mitosis is accomplished to DNA replication dna, and DNA content is minimum, i.e. first peak of flow cytometer detection result；The S phase refers to the period of DNA replication dna, is the process of one times of DNA to two times of DNA, shows that period span is bigger, i.e. second peak the highest but the widest in streaming result figure；The G2 phase refers to that DNA replication dna is accomplished to a period of time before mitosis starts, and intracellular contains two times of DNA, is second peak in streaming result figure, therefore is expressed as G2/M.
The flow cytometry that is suitable for disclosed by the invention carries out the good effect that the method for Wheat root tip cells cycle analysis compared with prior art had and is:
1) the inventive method is simple to operate, fast and convenient feasible, and agents useful for same is all laboratory common agents；2) the single cell suspension purity prepared is high, and impurity fragment is few；3) before fluorescent staining, unicellular glycerine is as penetrating solvent so that follow-up staining efficiency is higher, and effect is more preferable；4) sample recall rate on flow cytometer is high and stable.5) applied range.The present invention is applicable to the tissues such as the root of various kind wheat, leaf.The inventive method and result can provide new approach and foundation for carrying out with Reproduction stage correlative study.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates that south agriculture 9918 Wheat root tip cells carries out flow cytomery；Wherein ordinate Nomalized Frequency represents the frequency shared by effective cell number counting down to；Abscissa Intensity_MC_Ch07 represents fluorescence intensity, i.e. amount of DNA；R2, R3, R4 represent G1 phase, G2 phase, S phase respectively；
Fig. 2 is the collection of illustrative plates that Jingdone district 8 Wheat root tip cells carries out flow cytomery；Wherein ordinate Nomalized Frequency represents the frequency shared by effective cell number counting down to；Abscissa Intensity_MC_Ch07 represents fluorescence intensity, i.e. amount of DNA；R2, R3, R4 represent G1 phase, G2 phase, S phase respectively；
Fig. 3 is the flow process figure being suitable for the method that flow cytometry carries out Wheat root tip cells cycle analysis.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and unrestricted the scope of the present invention, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, on the premise of without departing substantially from spirit and scope of the present invention, the various changes or the change that carry out the material component in these embodiments and consumption fall within protection scope of the present invention.
In following embodiment, method therefor is conventional method if no special instructions, and described percentage composition is volumn concentration if no special instructions.
Embodiment 1
Method with Flow cytometry south agriculture 9918 Wheat root tip cells cycle analysis
First by the method for the present invention, south agriculture 9918 Wheat root tip cells is carried out cell cycle analysis, comprises the following steps:
(1) choose well-developed wheat seed running water and carry out presoaking and germinating, treat that wheat seedling root length length is to 3 cm, the tip of a root of about 0.2 cm length is cut with blade, the tip of a root cut is continued to be cut into the least tissue block by continuation blade, it is fixed on 30 min in freshly prepared Ka Nuoshi fixer (methyl alcohol: glacial acetic acid is made into the solution of volume ratio 3:1), rinse 3 times with the phosphate buffer of pH 7.4, after standing 5 min every time, with liquid-transfering gun by upper strata buffer solution sucking-off, proceed to again, in the ethanol that volume ratio is 70%, be stored in 4 DEG C of refrigerators；
(2) tip of a root sample fixed is inserted in the glycerine of 0.1-0.2% and permeate 2.5 h, rinse 3 times with the phosphate buffer of pH 7.4, after each 10 min, staticly settle 5 min, collect Helminthosporium sativum, it is immersed in 2% cellulase again and mixed enzyme solution that 2% pectase is made, it is placed in enzymolysis in 37 DEG C of incubators, treat Helminthosporium sativum soft texture, grip a tissue pieces at random to be placed on slide with tweezers, at microscope 10 × times Microscopic observation after dissipating with tweezers folder, when in the visual field cell of about 80% be complete unicellular time, stop enzymolysis, last about 2 h.
(3) phosphate buffer of soft Helminthosporium sativum pH 7.4 rinses 3 times, stands 10 min every time.Being ground to be formed single cell suspension, molecular sieve filtration by pestle gentleness again, collect filtrate, 3000 r.p.m. rotating speeds are centrifuged 4 min, can obtain unicellular precipitation；
(4) add, in above-mentioned precipitation, the DAPI dye liquor (phosphate buffer dilution) that appropriate concentration is 2 μ g/ml, lucifuge dyes 10 min, intracellular DNA material is by fluorescent staining, rinsing 3 times with the phosphate buffer of pH 7.4, after standing 10 min, 3000 r.p.m. rotating speeds are centrifuged 4 min every time, after the phosphate buffer of the unicellular precipitation 300 μ L pH 7.4 collected suspends, molecular sieve filtration, the single cell suspension made can be stored in 4 DEG C of refrigerators, to be ready for use on the detection of flow cytometer；
(5) detecting with flow cytometer, a length of 340 nm of excitation light wave, through exciting, the DNA molecular of dyeing fluoresces, and determinator measures its fluorescence intensity, and fluorescence intensity can be analyzed by the Computer aided analysis being connected with determinator.As it is shown in figure 1, ordinate represents the frequency shared by effective cell number counting down to；Abscissa represents fluorescence intensity, i.e. amount of DNA；R2, R3, R4 represent G1 phase, S phase, G2/M respectively.The G1 phase refers to the off time before mitosis is accomplished to DNA replication dna, and DNA content is minimum, i.e. first peak of flow cytometer detection result；The S phase refers to the period of DNA replication dna, is the process of one times of DNA to two times of DNA, shows that period span is bigger, i.e. second peak the highest but the widest in streaming result figure；The G2 phase refers to that DNA replication dna is accomplished to a period of time before mitosis starts, and intracellular contains two times of DNA, is second peak in streaming result figure, therefore is expressed as G2/M.
Embodiment 2
Method with No. 8 Wheat root tip cells cycle analyses in Flow cytometry Jingdone district
First by the method for the present invention, Jingdone district 8 Wheat root tip cells is carried out cell cycle analysis, comprises the following steps:
(1) choose well-developed wheat seed running water and carry out presoaking and germinating, treat that wheat seedling root length length is to 4cm, the tip of a root of about 0.2 cm length is cut with blade, the tip of a root cut is continued to be cut into the least tissue block by continuation blade, it is fixed on 30 min in freshly prepared Ka Nuoshi fixer (methyl alcohol: glacial acetic acid is made into the solution of volume ratio 3:1), rinse 3 times with the phosphate buffer of pH 7.4, after standing 5 min every time, with liquid-transfering gun by upper strata buffer solution sucking-off, proceed to again, in the ethanol that volume ratio is 70%, be stored in 4 DEG C of refrigerators；
(2) tip of a root sample fixed is inserted in the glycerine of 0.1-0.2% and permeate 3 h, rinse 3 times with the phosphate buffer of pH 7.4, after each 10 min, staticly settle 5 min, collect Helminthosporium sativum, it is immersed in 2% cellulase again and mixed enzyme solution that 2% pectase is made, it is placed in enzymolysis in 37 DEG C of incubators, treat Helminthosporium sativum soft texture, grip a tissue pieces at random to be placed on slide with tweezers, at microscope 10 × times Microscopic observation after dissipating with tweezers folder, when in the visual field cell of about 80% be complete unicellular time, stop enzymolysis, last about 1.5 h.
(3) phosphate buffer of soft Helminthosporium sativum pH 7.4 rinses 3 times, stands 10 min every time.Being ground to be formed single cell suspension, molecular sieve filtration by pestle gentleness again, collect filtrate, 3000 r.p.m. rotating speeds are centrifuged 4 min, can obtain unicellular precipitation；
(4) add, in above-mentioned precipitation, the DAPI dye liquor (phosphate buffer dilution) that appropriate concentration is 2 μ g/ml, lucifuge dyes 10 min, intracellular DNA material is by fluorescent staining, rinsing 3 times with the phosphate buffer of pH 7.4, after standing 10 min, 3000 r.p.m. rotating speeds are centrifuged 4 min every time, after the phosphate buffer of the unicellular precipitation 300 μ L pH 7.4 collected suspends, molecular sieve filtration, the single cell suspension made can be stored in 4 DEG C of refrigerators, to be ready for use on the detection of flow cytometer；
(5) detecting with flow cytometer, a length of 340 nm of excitation light wave, through exciting, the DNA molecular of dyeing fluoresces, and determinator measures its fluorescence intensity, and fluorescence intensity can be analyzed by the Computer aided analysis being connected with determinator.As in figure 2 it is shown, ordinate represents the frequency shared by effective cell number counting down to；Abscissa represents fluorescence intensity, i.e. amount of DNA；R2, R3, R4 represent G1 phase, S phase, G2/M respectively.The G1 phase refers to the off time before mitosis is accomplished to DNA replication dna, and DNA content is minimum, i.e. first peak of flow cytometer detection result；The S phase refers to the period of DNA replication dna, is the process of one times of DNA to two times of DNA, shows that period span is bigger, i.e. second peak the highest but the widest in streaming result figure；The G2 phase refers to that DNA replication dna is accomplished to a period of time before mitosis starts, and intracellular contains two times of DNA, is second peak in streaming result figure, therefore is expressed as G2/M.
Comparative test: in summary, the inventive method be applicable to different cultivars Wheat root tip cells cell cycle detection, all can stable detection to G1 phase, S phase, the cell distribution of G2/M；With traditional experiment by separating nucleus compared with the FCM analysis, during separation wheat organization of root tips single cell suspension of the present invention, medicine used is only cellulase and pectase, has quick, easy, low cost and the feature of effect stability.
Claims (4)
1. being suitable for the method that flow cytometry carries out Wheat root tip cells cycle analysis, it is to utilize the Helminthosporium sativum sample that fixes, prepares the single-cell suspension liquid of Helminthosporium sativum tissue, carries out fluorescent staining, flow cytomery, including:
(1) wheat seed carries out presoaking and germinating, when root length length to 3-5 cm, cut the tip of a root of about 0.2 cm length, be fixed on 30 min in freshly prepared Ka Nuoshi fixer, with proceeding in the ethanol that volume ratio is 70% after the phosphate buffer rinsed clean of pH 7-8 again, it is stored in 4 DEG C of refrigerators；Described Ka Nuoshi fixer refers to: methyl alcohol: the volume ratio of glacial acetic acid is 3:l；It is characterized in that:
(2) tip of a root sample fixed is inserted 0.1-0.2%(v/v) glycerine in permeate 2-3h, rinse 3 times with the phosphate buffer of pH 7-8, each 10 min, upper strata buffer solution is drawn with liquid-transfering gun after staticly settling, it is immersed in cellulase again and in mixed enzyme solution that pectase is made, 37 DEG C of enzymolysis 1.5-3 h, make Helminthosporium sativum soft texture；Described cellulase and the mass concentration of pectase are all 2%, prepare by deionized water；
(3) by described step 2) in the phosphate buffer of Helminthosporium sativum pH 7-8 rinse 3 times, each 10 min, after drawing major part upper strata buffer solution with liquid-transfering gun, grind to form single cell suspension by pestle gentleness, filter, collect filtrate, single cell suspension can be obtained；
(4) single cell suspension is centrifuged 4 min through 3000 r.p.m., it is the 4 of 0.5-2 μ g/ml that the precipitation obtained adds concentration, 6-diamidine-2-phenylindone dye liquor, shake up rear lucifuge to dye 10 min, intracellular DNA material is by fluorescent staining, rinse 3 times with the phosphate buffer of pH 7-8, each 10 min；
(5) after dyeing, single cell suspension flow cytometer detects, a length of 340 nm of excitation light wave.
Method the most according to claim 1, it is characterised in that: described in described step (1), wheat seed running water carries out presoaking and germinating, hatches 4-5d for 25 DEG C, treats that root length length is to 3-5 cm.
Method the most according to claim 1, it is characterised in that: after described step (1)-(5) rinse with the phosphate buffer of pH 7-8 every time, all it is centrifuged 4 min with 3000 r.p.m. rotating speeds.
4. it is suitable for flow cytometry described in claim 1 and carries out the application in terms of understanding the proliferative conditions of cell of the method for Wheat root tip cells cycle analysis.
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