CN106770134A - A kind of method for inducing spinach root-tip cells mitotic synchronization - Google Patents
A kind of method for inducing spinach root-tip cells mitotic synchronization Download PDFInfo
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- CN106770134A CN106770134A CN201710046079.5A CN201710046079A CN106770134A CN 106770134 A CN106770134 A CN 106770134A CN 201710046079 A CN201710046079 A CN 201710046079A CN 106770134 A CN106770134 A CN 106770134A
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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Abstract
The invention discloses a kind of method for inducing spinach root-tip cells mitotic synchronization, belong to plant cytogenetics technical field.Technical scheme main points are：A kind of method for inducing spinach root-tip cells mitotic synchronization, using hydroxycarbamide and N2The double blocked methods of O induce spinach synchronizing of cell mitosis to obtain a large amount of division phases cells.The present invention uses HU as blocking agent, and combines renewal cultivation and N2O treatment, greatly increase spinach division phases cell, medium cell can be made to account for more than the 50% of TCS using conventional compression, metaphase chromosome volume morphing simultaneously through laughing gas treatment is good, greatly increase the success rate and stability of spinach chromosome sectioning, for the application of the technologies such as spinach karyotyping, FISH is provided a great convenience, and laughing gas is nontoxic, in operation, the pollution to environment can be not only reduced, can also be reduced and the degree and risk poisoned are produced to human body.
Description
Technical field
The invention belongs to plant cytogenetics technical field, and in particular to one kind induction spinach root-tip cells mitosis
Synchronized method.
Background technology
Chromosome is most important part in nucleus, and the variation of chromosome number and structure is provided for plant breeding
Abundant genetic resources.Root-tip cells film-making mitosis metaphase is a basic cytogenetical study technology and hand
Section, the technology such as the technology and the karyotyping based on this technology and FISH is in species identification and discriminating, thing
It is widely used in the researchs such as Interspecific relationship determination, species Ploidy Identification and crossbreeding.
Spinach is Amaranthaceae lamb's-quarters subfamily spinach life in 1 year or 2 years raw herbaceous plant, its blade rich in iron, lutein, folic acid,
Vitamin, mineral matter and antioxidant, are a kind of important economic class green vegetables.Spinach is under normal circumstances for male and female are different
Strain, but monoecism also occurs under a few cases.Its genetic constitution is 2n=2x=12, and sex is determined by XY chromosomes.In recent years
Come, the research to spinach is concentrated mainly on the aspects such as nutritional ingredient, cultivation and molecular biology, it is relative to its cytological research
Delayed, this is dense mainly due to the cytoplasm of spinach, and cell membrane is harder, and chromosome is smaller, it is difficult to obtain form it is clear, point
Dissipate good split coil method.Also, although meristem zone's separatist activities of the spinach tip of a root is more vigorous, the cell of metaphase
A little part is only occupied, still in a phase, this causes very big obstacle to most cells to obtain preferable chromosome sectioning,
Generally require to carry out substantial amounts of chromosome sectioning, can just pick out a small amount of slide that preferably can be used for analysis and later experiments,
Waste substantial amounts of human and material resources and time.Induction plant cell cycle is synchronized and can make the cell proportion in metaphase
Greatly increase, greatly improve the success rate of chromosome sectioning and stability.But for spinach, or even Amaranthaceae is also without correlation
Report.Current most common method is to use hydroxycarbamide（HU）And methyl thymol blue complecon（APM）Double blocked methods induce plant cell
Mitotic synchronization, can obtain high-frequency mitosis metaphase index.But, HU and APM are toxic, and through APM at
After reason, spinach root tip cell chromosome easily trails, and form is bad, be unfavorable for carrying out the later stage deeper into research and analysis.
The content of the invention
Present invention solves the technical problem that there is provided a kind of method for inducing spinach root-tip cells mitotic synchronization.
The present invention adopts the following technical scheme that one kind induces spinach root-tip cells mitosis to solve above-mentioned technical problem
Synchronized method, it is characterised in that：Using hydroxycarbamide and N2Double blocked method induction spinach cell the synchronizing mitosis of O
To obtain a large amount of division phases cells.
The method of induction spinach root-tip cells mitotic synchronization of the present invention, it is characterised in that specific steps
For：
（1）Root tip culture：Spinach seed is soaked into 24h in 30 DEG C of water, then seed is placed on the filter of paving layer 2-3 water-soaked
In the culture dish of paper, then cultivated 4 days to a length of 0.5-1.5cm of root in 25 DEG C；
（2）Hydroxycarbamide treatment：Under the conditions of 25 DEG C a length of 0.5-1.5cm of root is processed with the hydroxycarbamide that molar concentration is 3mmol/L
Spinach seed 18h；
（3）Renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, tip of a root 6- is then cultivated in 25 DEG C of clear water
8h；
（4）N2O is pre-processed：Cut the tip of a root and be put into N21-3h is processed in O air chambers；
（5）It is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min.
Further preferably, step（1）Middle Radix spinach is long to be preferably 1cm.
Further preferably, step（3）The time of the middle clear water culture tip of a root is preferably 7h.
Further preferably, step（4）Middle N2The treatment pressure of O is 1.01MPa, N2The process time of O is preferably 2h.
Further preferably, by step（5）After treatment, it is placed in isolation liquid after the tip of a root washing that will be fixed and is isolated
1.5min, the isolation liquid is that volume ratio is 1:1 concentrated hydrochloric acid and the mixed liquor of absolute ethyl alcohol, then cut after washing removal isolation liquid
Tip of a root growing point part 2mm long is placed on wave carrier piece, and the pinkish red dye liquor dyeing 15min of improvement is fully added dropwise after chopping, adds lid
Slide carries out Conventional compression, and slide then is placed in into basis of microscopic observation, takes pictures and calculate mitosis metaphase division Phase Proportion.
The present invention has the advantages that compared with prior art：
1st, cells blocks can be made in the interkinesis using HU, renewal cultivation is carried out after removal HU, inducible synchronizing of cell,
By the renewal cultivation of 6-8h, a large amount of cells is located at fissional mid-term, after preprocessed, fixed and compressing tablet, there is silk point
The ratio for splitting medium cell is greatly increased；
2nd, the present invention uses N2O pre-processes the tip of a root, can help to remove cytoplasm background, makes chromosome morphology good, and method
Simply, in addition, during film-making, the pollution to environment and human body can be substantially reduced and risk is poisoned；
3rd, substantial amounts of spinach division phases can be obtained using the present invention, the success rate for preparing spinach chromosome specimen is carried significantly
Height, saves substantial amounts of human and material resources and time；
4th, using the method for the present invention can provide facility for the preparation of spinach chromosome specimen, for spinach physical chromosomal map
Spectrum structures, genetic breeding, evolution of sex chromosomes study and spinach platymiscium Study on Evolution etc. with important theoretical and real
Trample value.
Brief description of the drawings
Fig. 1 is the cytological map observed by Conventional compression after the spinach tip of a root is synchronized；
Fig. 2 is the cytological map observed by Conventional compression after the spinach tip of a root is not synchronized；
Fig. 3 drips the chromosome map that piece method is obtained after being synchronized by the spinach tip of a root；
Fig. 4 is positioning figures of the 45S rDNA on spinach chromosome（The chromosome that A is redyed for DAPI, B is Alexa Fluor-
The signal site of 488 fluorescein-labeled 45S rDNA, C is composite diagram）.
Specific embodiment
The above of the invention is described in further details by the following examples, but this should not be interpreted as this
The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair
Bright scope.
Embodiment 1
A kind of method for inducing spinach root-tip cells mitotic synchronization, comprises the following steps：
1st, root tip culture：Spinach seed is soaked into 24h in 30 DEG C of water, then seed is placed on the filter of paving layer 2-3 water-soaked
In the culture dish of paper, then cultivated 4 days to a length of 1cm of root in 25 DEG C；
2nd, hydroxycarbamide treatment：The spinach kind of a length of 1cm of root is processed with the hydroxycarbamide that molar concentration is 3mmol/L under the conditions of 25 DEG C
Sub- 18h；
3rd, renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, seed 7h is then cultivated in 25 DEG C of clear water；
4、N2O is pre-processed：Cut the tip of a root and be put into N22h is processed in O air chambers, the N2Pressure in O air chambers is 10atm（1.01MPa）；
5th, it is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min；
6th, compressing tablet：Isolation liquid is put into after the tip of a root washing that will be fixed（Volume ratio is 1:1 concentrated hydrochloric acid and absolute ethyl alcohol mixing
Liquid）Middle isolation 1.5min, cuts 2mm tip of a root growing point parts long and is placed on slide after washing removal isolation liquid, fully cuts
The pinkish red dye liquor dyeing 15min of improvement is added dropwise after broken, adding cover glass carries out Conventional compression；
7th, microscopy：Slide is placed in basis of microscopic observation, mitosis metaphase division Phase Proportion is taken pictures and calculate.
After using the method, as shown in figure 1, spinach metacinesis Phase Proportion can reach more than 50%, and it is not synchronized
Change, the spinach metacinesis Phase Proportion obtained by conventional treatment is about 1%（Fig. 2）.
Embodiment 2
Drop piece method prepares spinach metaphase chromosome sample
The spinach tip of a root after synchronization process is cut into Root apical meristem area, the fruit that 20 μ L volume fractions containing quality are 1% is placed in
In glue enzyme and the mixed liquor that quality volume fraction is 2% cellulase, 2h is digested in 37 DEG C.It is with volume fraction after the completion of enzymolysis
70% ethanol washes the tip of a root twice, then smashs the tip of a root to pieces with dissecting needle in 40 μ L ethanol, and vortex suspension cell, centrifugation retains heavy
30 μ L anhydrous acetic acids are formed sediment and added, 5-8 μ L drop pieces are inhaled after mixing, room temperature can microscopy after placing 5min.The good slice, thin piece of microscopy
Put in UV-crosslinked instrument by 120-125mJ/cm2It is standby that treatment 2min fixes chromosome.Microscopy result is as shown in figure 3, spinach
Chromosome spread is good, and form is clear, and cytoplasm background is relatively low.
Embodiment 3
Spinach metaphase chromosome FISH
45S probes are marked：By following component in centrifuge tube is added on ice：Corn 45S rDNA 2 μ g, 10 × nick
Translation buffer 2 μ L, Labeled-dNTP（Texas red-dCTP or Alexa Fluor-488-dUTP）0.5µ
2 μ L, DNA polymerase I of L, Non-labeled-dNTPs 5,0.5 μ L of μ L, DNaseI, are mended to 20 μ L with deionized water.
2h is processed in 15 DEG C in PCR instrument after mixing, plus 5 × the μ L of TAE+140ng/ μ L salmon sperm DNAs 175, addition volume fraction 90% ethanol-
The μ L of 10% sodium acetate of volume fraction 500, mix precipitation more than probe 2h, are subsequently placed in centrifuge the centrifugation speed in 13000rpm
Rate is centrifuged 30min, and absolute ethyl alcohol is washed twice, and lucifuge room temperature preservation 30min plus 2 × the μ L dissolution precipitations of 1 × TE of SSC 20.
FISH：Prepare probe solution：The probe that 0.5 μ L are marked adds 2 × SSC, the 1 × TE of 5.5 μ L, will
Probe solution is added dropwise the place for having cell on slide, gently puts plastic coverslip.Slice, thin piece is placed in and is covered with moist tissue
In canister, then be put into slice, thin piece in hybridizing box by boiling water bath 5min, 55 DEG C of hybridization more than 3h.Hybridize slice, thin piece after terminating
Take out, be put into 5min in 2 × SSC, remove cover plate.
Signal detection：Add DAPI fluorescence dye liquors of the 20 μ L containing anti-quencher, lucifuge dyeing on chromosome slide sample
10min, covers cover plate long（22cm×40cm）Microscopy can be carried out under fluorescence microscope afterwards.Microscopy uses Olympus BX63
Fluorescence microscope, CCD carries out chromosome analysis with Metamorph softwares after taking pictures, and Photoshop carries out image procossing.As a result
As shown in figure 4, being observed that bright hybridization signal by fluorescence microscope.
Embodiment above describes general principle of the invention, principal character and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, simply original of the invention is illustrated described in above-described embodiment and specification
Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements each fall within
In the scope of protection of the invention.
Claims (6)
1. it is a kind of induce spinach root-tip cells mitotic synchronization method, it is characterised in that：Using hydroxycarbamide and N2The double resistances of O
Disconnected method induces spinach synchronizing of cell mitosis to obtain a large amount of division phases cells.
2. the method for induction spinach root-tip cells mitotic synchronization according to claim 1, it is characterised in that specific
Step is：
（1）Root tip culture：Spinach seed is soaked into 24h in 30 DEG C of water, then seed is placed on the filter of paving layer 2-3 water-soaked
In the culture dish of paper, then cultivated 4 days to a length of 0.5-1.5cm of root in 25 DEG C；
（2）Hydroxycarbamide treatment：Under the conditions of 25 DEG C a length of 0.5-1.5cm of root is processed with the hydroxycarbamide that molar concentration is 3mmol/L
Spinach seed 18h；
（3）Renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, tip of a root 6- is then cultivated in 25 DEG C of clear water
8h；
（4）N2O is pre-processed：Cut the tip of a root and be put into N21-3h is processed in O air chambers；
（5）It is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min.
3. it is according to claim 2 induction spinach root-tip cells mitotic synchronization method, it is characterised in that：Step
（1）Middle Radix spinach is long to be preferably 1cm.
4. it is according to claim 2 induction spinach root-tip cells mitotic synchronization method, it is characterised in that：Step
（3）The time of the middle clear water culture tip of a root is preferably 7h.
5. it is according to claim 2 induction spinach root-tip cells mitotic synchronization method, it is characterised in that：Step
（4）Middle N2The treatment pressure of O is 1.01MPa, N2The process time of O is preferably 2h.
6. it is according to claim 2 induction spinach root-tip cells mitotic synchronization method, it is characterised in that：By
Step（5）After treatment, it is placed in isolation liquid after the tip of a root washing that will be fixed and isolates 1.5min, the isolation liquid is that volume ratio is 1:
1 concentrated hydrochloric acid and the mixed liquor of absolute ethyl alcohol, then wash after removal isolation liquid and cut 2mm tip of a root growing point parts long and be placed on load
On wave plate, the pinkish red dye liquor dyeing 15min of improvement is fully added dropwise after chopping, adding cover glass carries out Conventional compression, then by slide
Basis of microscopic observation is placed in, mitosis metaphase division Phase Proportion is taken pictures and calculate.
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| CN109856330A (en) * | 2019-01-29 | 2019-06-07 | 南京农业大学 | A method of chrysanthemum tip of a root Mitotic phase is improved by artificial regulatory |
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