CN106636320A - Method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage - Google Patents
Method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage Download PDFInfo
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- 238000007901 in situ hybridization Methods 0.000 title claims abstract description 18
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- C12Q1/6841—In situ hybridisation
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Abstract
The invention belongs to the technical field of crop molecular cytogenetics, and particularly relates to a method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage. The method comprises the steps of determination of flower buds in the mature pollen period of the Chinese cabbage, section making of mature pollen cells, probe marking, in-situ hybridization, signal detection and the like. The invention is characterized in that: taking flower buds of Chinese cabbage 1-2 days before flowering, separating fresh pollen grains in 50% glacial acetic acid solution, and breaking pollen walls by adopting a vertical tabletting method to release complete vegetative cells and germ cells; after being pretreated, the cell sample is subjected to covariation with a denaturation probe for 3 min at 80 ℃, and in-situ hybridization is directly carried out, so that a fluorescent hybridization picture with clear signals is obtained, and the signal expression difference of target DNA in vegetative cells and germ cells is revealed. The invention fills the blank of the mature pollen cell fluorescence in-situ hybridization technology of brassica plants in brassicaceae, and is applied to the molecular cytogenetic research of cabbage pollen development.
Description
Technical field
The present invention relates to a kind of Chinese cabbage mature pollen cell fluorescence in-situ hybridization method, belongs to molecular and cytogenetic techniques
Field.
Background technology
Pollen（pollen）It is the amphigenetic executor of flowering plant, not only to crop sterility changing and heterotic
Using most important, and it is the excellent materials for studying development and cell differentiation mechanism.The development of pollen occurs including sporidiole
（microsporogenesis）Occur with male gametophyte（male gametogenesis）, sporidiole is in plant anther
Archesporium differentiation and development into pollen mother cell, then Jing meiosis forms monoploid sporidiole；Male gametophyte is
Sporidiole first passes through that 1 asymmetrical karyokinesis is divided into 1 big and loose vegetative cell and 1 little and the reproduction of consolidation is thin
Born of the same parents, subsequent vegetative cell no longer divides, and reproduction cell mitosis Jing 1 subnormal again forms two spermatids（Hero is matched somebody with somebody
Son）, that is, form the pollen grain of maturation（Male gametophyte）.During pollen development, meiosis makes the individual cells gene of parent 2
Group occur restructuring produce 1 it is new with subgenom, and sequencing and spontaneity are experienced during mitosis later
Change, so as to cause vegetative cell and reproduction cell nuclear genetic material different.In recent years, male gametophyte genetic mechanism
A study hotspot is had become, using different molecular genetic techniques and high flux omics technology, it has been found that some regulation and control are male
Gametophyte forms the important gene with pollen tube growth, and also the functional genome's properties study for vegetative cell and reproduction cell is carried
New opportunity is supplied.But for distinguished sequence or gene are in function and the diverse vegetative cell of destiny and reproduction cell
The molecular cytogenetic of performance difference is few.
Fluorescence in situ hybridization technique（Fluorescent in situ hybridization, FISH）It is 80 years 20th century
A kind of generation last on-radiation molecular cytogenetics technology grown up on the basis of radioactive in situ hybridization technology, extensively should
The research of chromosome or specific DNA sequence for animal and plant cells each stage of interphase cycle, is related to genetic mapping, gene fixed
Many aspects such as position, chromosome aberration detection, chromosomal rearrangement, genome structure and spore analysis.On plant, pollen
Grain is a kind of unique accessible cell type, and for monoploid, in the G1 phases, without endogenous polyploidization.It is enterprising in pollen cell
Row FISH hybridizes, and contributes to us and directly observe some special repeating or gene order or chromosome is logical whether has crossed pollen development
Stage, and determine in vegetative cell and reproduction cell with the presence or absence of difference.At present this or a new research field, there is no phase
Close research report.Additionally, carrying out pollen cell FISH, also there is a very challenging job, that is, spend
Powder expoeridium has the hard pollen wall being completely dried, and how to remove the obstruction of pollen wall, is completely spread out pollen cell
It is that can impact pollen cell FISH so that probe can penetrate into nuclear chromosome through cytoplasm on slide
It is successfully crucial.
The content of the invention
The present invention provides a kind of purpose of the method for Chinese cabbage mature pollen cell fluorescence in situ hybridization, is for Chinese cabbage pollen
Grain containing the more difficult good tiling of hard pollen wall, pollen cell on slide, probe is difficult to penetrate into Pollen Nuclear, pollen
The problems such as cell fluorescence in situ hybridization still belongs to blank, the invention can utilize the pollen cell sample for preparing to carry out FISH hybridization and obtain
Clearly hybridization signal picture, reflects target sequence in the vegetative cell crossing pattern different with reproduction cell.The present invention is
The molecular cytogenetic of Chinese cabbage pollen development is laid a good foundation.
The present invention general principle be：Pollen mother cell Jing postmeiotics form mononuclear microspore, and further Jing is twice
Mitosis is formed containing 1 big and loose vegetative cell and 2 little and the mature pollen of the reproduction cell of consolidation（Andro gamete
Body）.In this process, meiosis makes the individual cells genome of parent 2 that restructuring to occur and produces 1 new Haploid gamete gene
Group, and sequencing and spontaneous change are experienced during mitosis.By pollen FISH, mesh is can reveal that
Whether indicated weight complex sequences or specific gene have differences between vegetative cell and reproduction cell, if can be passed by reproduction cell
Pass offspring.
It is characteristic of the invention that：The bud of 1-2 d before Chinese cabbage blooms is taken, in 50% glacial acetic acid solution fresh flowers are isolated
Powder, using vertical pressed disc method pollen-breaking wall, discharges complete vegetative cell and reproduction cell；Cell specimen is preprocessed
Afterwards in situ hybridization is directly carried out in 80 DEG C of min of co-variation 3 with denatured probe, obtains signal clearly fluorescent hybridization picture,
Disclose histological difference of the target dna in vegetative cell and reproduction cell.The present invention is completed by following steps：
（1）The determination of mature pollen period bud
Different size of bud is taken, 1 piece of flower pesticide is stripped and is placed in clean glass slide, be added dropwise one and drip DAPI dyeing liquors, it is light with tweezers
Light extruding discharges pollen grain, removes anther wall, and covered sucks unnecessary dye liquor with filter paper, and thumb is gently pressed, aobvious in fluorescence
The developmental stage of micro- Microscopic observation pollen.
（2）The preparation of mature pollen cell specimen
Compressing tablet：The bud for spending front 1-2 d is taken away, 1 piece of fresh flower pesticide is stripped, is placed in clean glass slide, drip 50% ice second
Acid solution, is gently extruded with tweezers and discharges pollen grain, removes anther wall, covered, with the vertical light cap slide of thumb
Pollen wall is ruptured, vegetative cell and reproduction cell is discharged.
Remove cover plate：Specimen is placed in into 30 s in liquid nitrogen, taking-up blade sled falls cover glass.
Degradation of cell matter：The glacial acetic acid solution of a drop 50% ~ 60% is added dropwise to specimen target area, 45 DEG C of perseverances are placed in
3 min are incubated on warm platform, make cytoplasm fully degrade.
It is fixed：Specimen is inclined, fresh Cannoy fixers are added dropwise（Alcohol：Glacial acetic acid=3:1）Rinse out 50% ice
Acetic acid simultaneously fixes 3 min, natural air drying.
Microscopy：Load is observed in phase contrast microscope, by structural integrity, reproduction cell and vegetative cell clearly slide
It is placed in of short duration in 4 DEG C of refrigerators saving backup.
（3）The preparation of probe and mark
45S rDNA and 5S rDNA probes in situ hybridization are obtained by the amplification of PCR methods by template of genomic DNA
, Cy3.5-dCTP and Cy3- dUTP marks are carried out respectively using shifting method is incised.
（4）FISH
Pollen cell Pretreated：By step（1）The specimen preparation of preservation copies the min of piece 30 under the conditions of 65 DEG C；Then divide
Do not carry out RNase A enzymes and pepsin incubates process and formalin is fixed, after 2 × SSC rinsings dehydration of alcohol is carried out, it is natural
It is dried.
The preparation and denaturation of probe hybridization solution：The fluorescence probe of mark is hybridly prepared into 20 by proper proportion and hybridization solution
The hybridization solution of μ L, the min of denaturation 10 in boiling water bath is immediately placed on more than the min of cryostat on ice 5.
Co-variation and hybridization：20 μ L denatured probe hybridization solutions are added drop-wise into the target area of specimen preparation, close the lid glass
Piece, is placed in the min of co-variation 3 on 80 DEG C of thermostatic platforms, in being then placed in the hybridizing box of moisturizing, is placed in 37 DEG C of insulating boxs and incubates
Overnight.
Elute after hybridization：The specimen preparation after hybridization is moved after cover glass under the conditions of lucifuge is immersed in 2 × SSC solution
Rinsing, is then dehydrated in alcohol；Spontaneously dry under the conditions of lucifuge.
Redye：The DAPI dyeing liquors containing anti-quencher are added dropwise per a hybridization specimen, cover glass, room temperature lucifuge is added a cover
5 more than min of lower dyeing.
Microscopy：Specimen after in situ hybridization is carried out into hybridization signal observation loaded on Zeiss, Germany fluorescence microscope, is used
Cooled CCD devices are captured image and are taken a picture, and with Adobe Photoshop CS softwares the synthesis of row image is entered.
The technique effect and advantage that the present invention is obtained is as follows：
1）FISH is mutually carried out for target using Chinese cabbage mature pollen cell, target-probe is in vegetative cell and reproduction cell
On obtain clear and different hybridization signal, the molecular cytogenetic for Chinese cabbage pollen development provides technology
Hold.
2）With 50% glacial acetic acid solution as medium, fresh flowers powder is crushed using the mechanical means of the vertical light cap piece of thumb
Pollen wall, the pollen cell for discharging is complete, vegetative cell and 2 reproduction cell dispersions, and technology is simple, workable.
3）In the operation of existing fluorescence in situ hybridization technique method, the denaturation of target phase sample is usually by pretreated mark
This slide immerses 70% formamide solution（2 × SSC is prepared）In in 70 DEG C of min of denaturation 2.5 ~ 3, then chilled dehydration of alcohol,
Spontaneously dry, then hybridize with denatured probe.Pollen cell sample directly adds after pretreatment denatured probe at 80 DEG C in the present invention
The min of co-variation 3, that is, hybridized on thermostatic platform, simplifies process of the test, accelerates test process.
4）The fresh bud of 1-2 d before blooming is adopted for material, it is in liberal supply, draw materials conveniently；Material is not required to fixer and consolidates
Fixed, pollen grain is fresh, and pollen wall is broken.
Description of the drawings
Fig. 1 is the Chinese cabbage mature flower powder of DAPI dyeing（Containing 1 big vegetative cell and 2 little reproduction cell）.
Fig. 2 is the three core pollen cells discharged from fresh flowers powder.
Fig. 3 is the FISH results of Chinese cabbage ' B153 ' pollen cell, and redness is 45S rDNA signals, and yellow is 5S rDNA
Signal.
Fig. 4 is that with Chinese cabbage, ' the FISH results of yellow sarson ' pollen cells, redness is 45S rDNA signals to oil, yellow
Color is 5S rDNA signals.
Specific embodiment
The present invention is further illustrated below by way of example, but is not intended to limit protection scope of the present invention.
Embodiment 1 with 45S rDNA and 5S rDNA probes Chinese cabbage ' B153 ' mature pollen cell fluorescent in situ
The present invention will be described as a example by hybridization.
（1）The determination of mature pollen period bud
In Chinese cabbage ' B153 ' full-bloom stage, the bud not of uniform size that 1-5 d can bloom is taken, each bud strips 1 piece of flower pesticide and puts
In clean glass slide, it is added dropwise one and drips DAPI dyeing liquors, gently extruded with tweezers and discharge pollen grain, remove anther wall, covers
Cover plate, is gently pressed with thumb, the developmental stage of microscopy pollen under fluorescence microscope, it is determined that pollen in the bud of 1-2 d before blooming
Develop for three ripe core pollen grains（（Fig. 1）.
（2）The preparation of mature pollen cell specimen
Compressing tablet：The big bud for spending front 1-2 d is taken away, 1 piece of fresh flower pesticide is stripped and is placed in clean glass slide, drip 50% ice
Acetic acid solution, is gently extruded with tweezers and discharges pollen grain, removes anther wall, covered, with the vertical light cap glass of thumb
Piece ruptures pollen wall, discharges vegetative cell and reproduction cell（Fig. 2）.
Remove cover plate：Specimen is placed in into 30 s in liquid nitrogen, taking-up blade sled falls cover glass.
Degradation of cell matter：The glacial acetic acid solution of a drop 50% is added dropwise, is placed on 45 DEG C of thermostatic platforms and is incubated 3 min, make cell
Matter is fully degraded.
It is fixed：Fresh Cannoy fixers are added dropwise（Alcohol：Glacial acetic acid=3:1）Rinse out 50% glacial acetic acid and fix 3
Min, natural air drying.
Microscopy：Load is observed in phase contrast microscope, by structural integrity, reproduction cell and vegetative cell clearly slide
It is placed in of short duration in 4 DEG C of refrigerators saving backup.
（3）The preparation of probe and mark
The genomic DNA of Chinese cabbage ' B153 ' is extracted using CTAB methods, with it as template, 45S is obtained by the amplification of PCR methods
RDNA and 5S rDNA fragments, shifting method is incised in the purified employing afterwards of amplified production carries out respectively Cy3.5-dCTP and Cy3- dUTP
Fluorescein mark.
（4）FISH
Pollen cell Pretreated：By step（1）The specimen preparation of preservation copies the min of piece 30 under the conditions of 65 DEG C；Then divide
Do not carry out RNase A enzymes and pepsin incubates process and formalin is fixed, after 2 × SSC rinsings dehydration of alcohol is carried out, it is natural
It is dried.
The preparation and denaturation of probe hybridization solution：The hybridization liquid system of every slide is 20 μ L, comprising 2 × SSC, 10% sulfuric acid
The fluorescence probe of glucan, 50% deionized formamide and debita spissitudo, after mixing in boiling water bath the min of denaturation 10, then
It is immediately placed on more than the min of cryostat on ice 5.
Co-variation and hybridization：20 μ L denatured probe hybridization solutions are added drop-wise into the target area of specimen preparation, close the lid glass
Piece, is placed in the min of co-variation 3 on 80 DEG C of thermostatic platforms, in being then placed in the hybridizing box of moisturizing, is placed in 37 DEG C of insulating boxs and incubates
Overnight.
Elute after hybridization：The specimen preparation after hybridization is moved after cover glass under the conditions of lucifuge immerses 2 × SSC solution
In, rinse 3 times, 5 min every time, 3 min are then dehydrated in 70%, 95%, 100% alcohol successively；Spontaneously dry.
Redye：DAPI dyeing liquors of the 14 μ L containing anti-quencher is added dropwise per a hybridization specimen, cover glass, room temperature is added a cover
5 more than min are dyeed under lucifuge.
Microscopy：Specimen after in situ hybridization is carried out into hybridization signal observation loaded on Zeiss, Germany fluorescence microscope, is used
Cooled CCD devices are captured image and are taken a picture, and the synthesis of row image is finally entered with Adobe Photoshop CS softwares, are obtained
Image as shown in Figure 3.
In oil, with Chinese cabbage, ' yellow sarson ' mature pollens are thin with ribosomes 45S rDNA and 5S rDNA probes for embodiment 2
The present invention will be described as a example by the FISH of born of the same parents.
（1）The preparation of mature pollen Cell sheet glass sample
In oil, with Chinese cabbage, ' yellow sarson ' full-bloom stages, take away the big bud for spending front 1-2d, strip 1 piece of fresh flower pesticide and are placed in
In clean glass slide, follow-up flaking method step is with embodiment 1.
（2）The preparation of probe and mark
Using CTAB methods extract oil, with Chinese cabbage, ' genomic DNA of yellow sarson ', with it as template, is expanded by PCR methods
45S rDNA and 5S rDNA fragments are obtained, PCR reaction systems composition, reaction condition, amplified production purifying and probe mark are with real
Apply example 1.
（3）FISH
1. the pretreatment of sample is with embodiment 1.
2. the preparation of probe hybridization solution and denaturation are with embodiment 1.
3. co-variation and hybridization are with embodiment 1.
4. same embodiment 1 is eluted after hybridizing.
5. DAPI redyes same embodiment 1.
6. microscopy obtains image as shown in Figure 4 with embodiment 1.
Claims (4)
1. a kind of method of Chinese cabbage mature pollen cell fluorescence in situ hybridization, it is characterised in that：
With prepare Chinese cabbage mature pollen cell as target phase, by FISH（FISH）, it is thin in vegetative cell and reproduction
Born of the same parents are it is observed that different FISH signal modes.
2. according to right 1 preparation Chinese cabbage mature pollen cell specimen method, it is characterised in that comprise the steps：
1）1 piece of flower pesticide is stripped from Chinese cabbage bud, is placed in clean glass slide, drip 50% glacial acetic acid solution, it is light with tweezers
Light extruding discharges pollen grain, removes anther wall, and covered ruptures pollen wall with the vertical light cap piece of thumb, release
Go out vegetative cell and reproduction cell；
2）Specimen is placed in into 30 s in liquid nitrogen to take out, with blade sled cover glass is fallen；
3）The glacial acetic acid solution of a drop 50% ~ 60% is added dropwise to specimen target area, is placed on 45 DEG C of thermostatic platforms and is incubated 3
Min, makes cytoplasm fully degrade；
4）Specimen is inclined, fresh Cannoy fixers are added dropwise（Alcohol：Glacial acetic acid=3:1）Rinse out 50% glacial acetic acid and consolidate
Fixed 3 min, natural air drying；
5）Load is observed in phase contrast microscope, and by structural integrity, reproduction cell and vegetative cell, clearly slide is placed in 4
It is of short duration in DEG C refrigerator to save backup.
3. according to right 2 preparation Chinese cabbage mature pollen cell specimen method, it is characterised in that：
The Chinese cabbage bud for being taken be bloom before 1-2 d big bud, sent out by the pollen in DAPI dyeing microscopic examinations now flower pesticide
Educate to mature period；Bud need not be fixed liquid and fix when preparing sample, and detached fresh pollen easily passes through vertical compressing tablet
Pollen-breaking wall, the pollen cell of release is complete.
4. the method for a kind of Chinese cabbage mature pollen cell fluorescence in situ hybridization according to right 1, it is characterised in that fluorescence is former
Position hybridization is completed by following steps：
1）65 DEG C of pollen cell specimen preparation Jing copies the pre- places such as piece, RNase A enzymes and pepsin are incubated, formalin is fixed
After reason, rinse, be dehydrated, be dried；
2）With manufacturing probe hybridization solution, the min of denaturation 10 in boiling water bath is immediately placed on more than the min of cryostat on ice 5；
3）Denatured probe hybridization solution is added drop-wise into the target area of specimen preparation, covered is placed on 80 DEG C of thermostatic platforms altogether
The min of denaturation 3, in being then placed in the hybridizing box of moisturizing, 37 DEG C of hybridization incubations are overnight；
4）Under the conditions of lucifuge the specimen preparation after hybridization is moved rinsed, be dehydrated, be dried, redying after cover glass, microscopy,
Obtain FISH hybridization signal images.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610135140.9A CN106636320B (en) | 2016-03-10 | 2016-03-10 | Method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage |
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