The division is periodic, that is, the cells that divide continuously start from the completion of one division to the completion of the next division, and start from the formationDaughter cellFrom the beginning to the end of forming daughter cells again (Figure 1)cell cycle。A cell cycle consists of two stages:InterphaseandFission period。
Interphase G1, S andPhase G2The interphase is the active material preparation for the fission phaseDNA moleculeAt the same time, the cells have moderate growth (the time of these two stages is quite different, generally the interphase accounts for 90%~95% of the cell cycle, and the division period accounts for 5%~10% of the cell cycle. The time of a cell cycle is different for different cell types.)
mitosis[1]Intervals are divided into G1（DNA synthesisEarly stage), S (DNA synthesis stage), G2 (DNA synthesis late stage), of whichPhase G1RNA with G2 phase (i.eRibonucleic acid）Replication of is related toproteinSynthesis of,Phase Sconductdna replication ；Among them, Phase G1 is mainlychromosomeProtein andHelicaseThe G2 phase is mainly related to cell division related enzymes andSpindle yarnProtein synthesis.BipolarChromatinInstead of highly spiraling to form chromosomes, DNA is carried out in the form of chromatin (i.eDeoxyribonucleic acid）Single chain replication.FilamentousInterphaseIt is an important preparatory process for the whole process of mitosis and an important basic work.
(Modern medicine uses relevant drugs to stopSpindleSo as to inhibit cell mitosis and stop cell division at G0 stage (G0 stage refers to the stage when cell division is stopped due to some factors and cell division can be resumed by changing external factors). The relevant drugs using this technology have effectively curbedcancer cellMalignant proliferation and spread of
Cell mitosisThe prophase refers to the period from the beginning of division tonuclear membraneThe period until disintegration.
Prophase of mitosis
When interphase cells enter the prophase of mitosis,nucleusThe volume ofDyed threadSpiral windingAnd gradually shorten and thicken to form chromosomes.Because chromatin has replicated in interphase, each chromosome is composed of two chromatids, that is, two parallel chromatidsSister chromatidThese two chromatids have a commonCentromereconnect.nucleolusIt gradually disappeared in the second half of the early stage.At the end of prophase, the nuclear membrane breaks, so the chromosomes are scatteredcytoplasmMedium.
In the prophase of mitosis of animal cells, there are twoCentrosome。Each centrosome consists of a pairCentrioleAnd the bright areas surrounding them, called centroplasm orCentral ball。Stellar filaments (also called spindle filaments orStar ray）That is radial microtubule.The two centroids with star filaments (spindle filaments) gradually separate and move towards the opposite poles (Figure 2).This separation process is speculated to be due to the stellar filament (spindle filament) between the two centroidsmicrotubuleThe interaction continues to grow, pushing the two centrosomes (two pairs of centrioles) to the poles, and finally forming a spindle between the poles after the rupture of the nuclear membrane.
The first and middle stages are from the rupture of nuclear membrane toChromosome arrangementAt the equator.Fragments of the nuclear membrane remain in the cytoplasm, andEndoplasmic reticulumIt is not easy to distinguish them, but they can sometimes be seen around the spindle.
The main process of the prophase and metaphase is the final formation of the spindle and the orientation of the chromosomeEquatorial surfaceThe movement of.There are two types of spindles: one is a star spindle, that is, there is a star with a pair of centrioles at each pole, which is found in mostAnimal cellAnd some lowerplant cell。One is star free spindle.Two pole astral bodies are found in higher plant cells (Fig. 3).
Academics once believed that there were three kinds of spindle filaments in the star spindle, namely, three kinds of microtubules.
SecondMicrotubule, are microtubules extending from the two poles to the opposite direction, and the polar microtubules from the two poles overlap each other in the equatorial region.They believe that the polar microtubules may be formed by the elongation of stellar microtubules.
ThirdCentromereMicrotubules, microtubules associated with the centromere, also known asCentromere filamentOr traction wire.The centromere is located in the chromosomeCentromereA structure developed on both sides of the.
But there are reports that centromere hasTubulinThe function of polymerization into microtubules.(The starless spindle has only polar microtubules and centromere microtubules.)
Chromosomes were dispersed in cytoplasm after nuclear membrane rupture.Two per chromosomeChromatidThe centromere is respectively connected with the two poles through the spindle wire.Because of the interaction between polar microtubules and centromere microtubules, chromosomes move towards equatorial plane.Finally, various forces reach equilibrium, so the chromosomes are arranged on the equatorial plane.
metaphase（metaphase）
Metaphase mitosis
Metaphase refers to the arrangement of chromosomes from the equatorial plate to theirChromatidThe period between the two poles.SometimesEarly and middle stageIt is also included in the medium term.
Metaphase chromosomeThe so-calledEquatorial plate。From one end, it can be seen that these chromosomes are radially arranged on the equatorial plate. At this time, they are not stationary, but in a state of constant swinging.metaphasechromosomeConcentrate and thicken, showing the unique number and morphology of the species.Therefore, the metaphase of mitosis is suitable for studying the morphology, structure and number of chromosomeskaryotype analysis。And the medium-term period is longer.
Anaphase refers to the period when two sister chromatids of each chromosome separate and move to two poles.
Chromosomes that are separated at a later stage are calledDaughter chromosome。The end of the anaphase is when the daughter chromosome reaches the two poles.The separation of chromatids is usually fromCentromereThe arms of the two chromatids gradually separate.When they are completely separated, they move towards opposite poles.The speed of this movement depends on
Anaphase of mitosis
The type of cells varies from 0.2 to 5 μ m/min.average velocityIt is about 1 μ m/min.Each chromosome in the same cell moves to the two poles at the same speed.Daughter chromosomeThe movement towards the poles is achieved by the activity of the spindle.
The telophase refers to the period from the arrival of daughter chromosomes at the poles to the formation of two daughter cells.The main process of this period isDaughter nucleusFormation andCell bodyThe split of.
The formation of the daughter nucleus is generally a process opposite to that of the earlier stage.The subchromosomes that reach the two poles are first uncoiled and the outline disappears, and all the subchromosomes form a largeChromatinBlocks, assembling aroundnuclear membraneThe nuclear membrane that fuses to form a daughter nucleus. With the re composition of the daughter nucleusnucleolus。Nucleolar formation andNucleolar organizer regionRelated to the activities of.
Cell division scaleCytokinesis。Cytokinesis in animals and some lower plants is accomplished by constriction or furrowing.The dynamics of constriction is generally speculated to be due toEquatorial platePericytoplasmicMicrofilamentThe result of shrinkage.The constriction of the microfilament causes the cell to form a constriction in this area, and the constriction gradually deepens so that the cell body is finally divided into two.
Cytokinesis of higher plant cells depends onCell plateFormation of.
Telophase mitosis
In telophaseSpindle yarnFirst, they disintegrate and disappear near the two poles, but the spindle fibers in the middle area remain, and the number of microtubules increases and expand around to form a barrel structure, calledFilm-forming body。
The cell plate gradually expands to the originalcell wallIt divides the cytoplasm into two parts (as shown in Figure 4).Related in cytoplasmOrganelle, such asmitochondrion，chloroplastWait is not equally distributed, but randomly enters twoDaughter cellMedium.The cell plate consists of two layers of films, which accumulate between the two layersPectin, developed intoIntercellular layerThe films on both sides accumulate cellulose, and each develops into daughter cellsPrimary wall。
[Cell mitotic memoryPithy formula】
（1） Mitosis[2]
Early stage: membranous kernel disappears and two bodies appear
Starry and Starless Spindles
Medium term: form definite number, clear equator
Later stage: increased average poles of point splitting body
End stage: separation of two elimination and two emergence
Spindle——Spindle is a special organelle produced from early to late stages of cell division.
Fission mechanism
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Condensed constitution of chromosomechromosomeIn the prophase of mitosis, the thin lines of chromosomes become shorter and thicker, and this contraction movement of chromosomes is realized through the spiralization of the dyeing lines.The process of chromatin concentration is related to some factors in the cytoplasm.If we use experimental methods to make the cell in the mitosis phase and the interphasecell fusionIt can be observed that the chromatin of interphase cells will condense into chromosomes in advance.This meansFission periodThere is something in the cytoplasm of the cell that can cause the chromosome to shrink.
The fertilized egg of medaka splits (the chromosome is magenta fluorescence, and the spindle microtubule is green fluorescence)[5]
Spindle formation
fromTubulinPolymerize intoSpindlemicrotubuleProcess.There are two basic forms of tubulin polymerization: one is self-assembly, the other is site initiation assembly. The latter has a special site as the starting site of polymerization, while the former has no such special site.The sites where spindles are formed are collectively referred to as“Microtubule Organization Center”（MTOC）。CentrosomeandCentromereThey are MTOC, and they can all show the ability to polymerize tubulin into microtubules in vitro.The formation of spindle is obviously inseparable from the activities of these MTOCs.
Metaphase chromosome movement
Anaphase of mitosis
Medications (colchicineMercaptoethanolDestructionSpindle, the chromosome cannot be arranged toEquatorial surface, after removing the drug,spindleWhen the weight is newly formed, the chromosomes can be arranged to the equatorial plane again. It can be seen that the arrangement of chromosomes to the equatorial plane isSpindleRelated to the activities of.fromradiation damageOr other reasonsChromosome fragmentIt cannot be arranged on the equatorial plane.Therefore, the equatorial alignment of chromosomes is related to the centromere activity.With microbeamultraviolet raysWhen the bivalent is irradiated, one side of the centromere or centromere filament, the chromosome cannot be located exactly on the equatorial plane, but is closer to the pole facing the unexposed centromere.This indicates that the coordination of chromosomes on the equatorial plane must be performed by two centromeres and the centromeres on both sides connected to the two poles.According to the above facts and other observations, it is speculated that the two centromeres are respectively connected to the two poles by the centromere filament in the first and middle stagesTractive forceThe chromosome is located on the equatorial plane.In addition to this traction balance force, there may be other factors that play an auxiliary role.
Chromosome movement
At anaphase, two sets of daughter chromosomes move to the poles[3]In some cells, the poles are pushed further away.The mechanism of this movement is inconclusive.At the later stage, the centromere microtubules continuously depolymerize at the end of the poleward, and thus gradually become shorter.This may be an important reason why chromosomes are pulled to the poles.Because inIn vitro experimentWhen O is added to the model cell to prevent the depolymerization of microtubules, the movement of chromosomes to the poles stops. On the contrary, if a small amount of colchicine is added to accelerate the depolymerization of microtubuleschromosomeBipolarMovement speedAlso accelerated.In some cells, the further separation of the two poles in the anaphase of division may be caused by the following mechanism: polar microtubules from the two poles overlap each other in the equatorial region, and tubulin aggregates at their free ends to lengthen microtubules.These overlapping microtubules from the two poles slide with each other, pushing the two poles farther apart.
Organelle changes
CellularmitochondrionIt increases in mitosis, because mitosis requires mitochondria to provide a lot of energy.Unequalrandom distributionIn the daughter cells after division.
Related organelles in the cytoplasm, such asmitochondrion，chloroplastWait is not equally distributed, but randomly enters into two daughter cells.
Ribosomes, Golgi apparatus, endoplasmic reticulumvacuole、lysosomeIsoorganelles disappear before mitosis and reconstruct in daughter cells after mitosis.
Comparison of animals and plants
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Different
The process of mitosis of animal cells is basically the same as that of plant cells, with different characteristics as follows:
one
Animal cells have centrosomes. At the interval of cell division, the two centrioles of the centrosome pass throughInterphase replicationThere are two groups of centrioles in the cell.In the process of cell division, two groups of centrioles move to the two poles of the cell respectively.Numerous star rays are emitted around the two groups of centrioles, and the star rays between the two groups of centrioles form a spindle.
At the end of plant cell division, cell plates appear at the equatorial plate, and expand from the center to the periphery to form cell walls.At the end of cell division, animal cells do not form cell plates, butcell membraneIt depresses inward from the middle of the cell, and finally constricts the cell into two parts, each containing a nucleus[4]。
The significance of mitosis is to transform thechromosomeAfter replication (the key is the replication of DNA), it is evenly distributed to the two daughter cells accurately.Because there aregenetic materialDNA，Therefore, it is maintained between the biological parents and offspringGenetic traitStability.It can be seen that cell mitosis is of great significance for biological genetics.
identical
There is a very important similarity between the process of animal cell mitosis and that of plant cell division:
Both animal cell division and plant cell division have chromosomes and spindles.(Plants: star ray spindles; animals: star ray spindles).Chromosomes are evenly distributed after replication.
significance
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1、 Maintain normal growth and development of individuals (tissue and intercellularGenetic compositionConsistency);
Observe the process of plant cell mitosis and identify different stages of division.
three
Have a preliminary grasp of the method of drawing a biological map.
Experimental principle
Cell mitosis is a continuous and dynamic change process, but it can be changed through its morphological changes, especiallynucleusChromosome behavior in, artificially divided into stages, andcomparative study 。staynatural stateNext, a large group of peopleFission periodThe cells of.It is necessary to observe carefully and look for typical mitotic stagesmorphological characterTo establishcell cycleThe concept of.
Vegetalmeristem(such as root tipMeristematic region、Stem apexGrowth point, etc.) cells that can increase their number through mitosis.According to plantsCell division cycleIn cells at various stagesChromatinOr changes in the shape, number and position of chromosomes to determine the period of the cell.To see chromosomes or chromatin, usealkaline dyeDye it.
Experimental materials
Broad beanRoot tip meristematic zone of garlic and onion.
(1) When cultivating onions to take root, avoid using newly harvested onions, because they are still dormant and are not easy to take root.If it is necessary to use the newly harvested onion to cultivate and take root, try to break its dormancy.The common method is to use low concentrationgibberellinSoak the onion chassis in solution to promote its rooting.During the cultivation process, change the water at least once a day to prevent root rot.
(2) For onions harvested in the first year, the following methods can be used to promote their rooting:
① Choose onions with large chassis as rooting materials.
② Peel off the outer layer of the old skin, and cut off the old roots with a knife (from the center of the chassis to the periphery). Be careful not to cut off the "roots" around.
③ UseBeakerFill with clean water, add onions, and place in the light.Keep the water clean, and change the water 1-2 times a day.Generally, the materials needed for the experiment can be obtained within 2-3 days (root length is 5cm).
(3) Take materials at fixed time.The mitosis time of onion root tip cells is regular.Usually, it splits and becomes active from 10 a.m. to 2 p.m. every day, especially at 2 p.m. below, when materials can be obtained.The cultured onion roots can be fixed with Karol's fixative for use.Because it is not easy to cultivate onion roots once.
2. Dissociation.Cut off the root tip about 2-3 mm long with a blade, and put it into the solution containing 15% hydrochloric acid and 95% alcohol by volumeMixed liquid(1:1)Petri dishThe cells in the root tip tissue are separated from each other for 3-5 min, and the dissociation stops when the root tip is transparent and soft.If you want to accelerate the dissociation, put the culture dish on the alcohol lamp and slightly heat it (do not boil it) for 3~5min.After the root is soft, take it out with tweezers.
Sufficient dissociation is a prerequisite for the success of the experiment;The purpose of dissociation is to dissolve the drug solutionIntercellular substance, makeHistiocyteSeparate from each other.
3. Rinse.Rinse the extracted root tip with clean water for about 10 minutes, or cover the mouth of the petri dish with a layer of gauze, and rinse it with tap water for 3-5 minutes.
The purpose of rinsing is to remove the excess in the rootDissociation solution。If the excess dissociation solution is not washed away, the dyeing effect will be affected on the one hand, because the dissociation solution contains HCl solution and is used for dyeingdyestuffAlkaline;On the other hand, it will corrode the lens of the microscope.
4. Staining.Place the rinsed root tip on the slide, cut off the root tip about 3mm long with a pair of tweezers, discard the rest, and drop a drop on the root tipAcetic acid carmineDye the dye solution for 3~5min. If you want to speed up the dyeing, put the glass slide in theflameDo not boil it for several times.
5. Loading.Cover the stained material with a cover glass, and then cover it with a slide. Press it gently with your thumb to make the root tip tissue become a uniform thin layer of cells.
6. Microscopic examination
(1) Low power microscope observation
Put the made onion root tip packing piece under the low power microscope to observe, slowly move the packing piece, and find the cells in the meristematic zone. Its characteristics are: the cells are square, closely arranged, and some cells are dividing.
(2) High power microscope observation
After finding the meristem cells, remove the low power lens and replace it with the high power lensreflectorAdjust the vision clearly until you can see the cell image clearly.
(3) Observe carefully
The purpose of careful observation is to distinguishcell divisionThe characteristics of chromosome changes in different periods of.You can first find out the cells in the middle of cell division, and then find out the cells in the early, late and late stages. The cells in the interphase are the most numerous and easiest to find.
(4) In one view, it is often difficult to find cells at all stages of holomitosis.If so, you can move the loading slowly and look for it from the adjacent meristem cells.
7. Drawing.After carefully observing the cells at each stage of mitosis, draw a diagram of mitosis of onion root tip cells.
matters needing attention
1. The material for producing onion root tip is onion bulb.The bottom of the bulb is in contact with water and cannot leave the water or be flooded.Its purpose is to meet its needs for water and air at the same time.At the same time, the water should be changed frequently because of the activities of microorganisms and root cells,MetabolitesIncrease;In addition, due to the continuous production of CO by the respiration of root cellstwo, make H in watertwoCOthreeIncrease and lack of oxygen;Microbes, such as bacteria, also pollute the water quality, which is not conducive to root tip growth, so water should be changed frequently.Generally, at least once a day, and appropriate temperature shall be provided.
2. When cutting onion root tip materials, the most active time of onion splitting in a day should be around 11am.If the experiment is conducted at the above time due to the influence of temperature or not, the materials can be taken at the peak of cell mitosis and fixed in the culture dish containing the fixative, that is, soaked for half a day to one day.After fixation, flush several times with 75% ethanol, and then put in a small container containing 70% ethanolJarSave in.Note that the onion can only be cut when the root tip is 1~5cm long, and the length of the onion root tip is 2~3mm.According to the experiment, when the root grows to 1~5cm, the root grows best. At this time, the cell division is most vigorous, and it is easy to find the division images of different periods.At this time, you can take the robust root for observation, cut the onion root tip 2~3mm, and this length should be from the top of the root（root cap）From the beginning, this length has included the graduation area.If the sampling is too long, it will be difficult to find the meristematic zone under the low power microscope.
3. After the root tip is cultivated, the key step to the success of the mounting is dissociation.15% hydrochloric acid solution can dissolvecell wallThe middle layer material (intercellular substance) between the cells, which separates the cells in the tissue from each other.Otherwise, when observed under the microscope, cells will overlap, leading to experimental failure.Insufficient dissociation and cell overlap;If dissociation is excessive, cells will rot, so dissociation should be moderate.In order to achieve this goal, the concentration of hydrochloric acid prepared should be appropriate. The cut root tips should be immediately put into 15% hydrochloric acid and dissociated at room temperature for 10-15 min.The dissociation time depends on the indoor temperature. The temperature is low, the time is slightly longer, the temperature is high, and the time is short.You can also hold a pair of tweezers and gently press the root tip that is being detached to feel soft.
4. The purpose of rinsing is to wash away the excess dissociation liquid in the root.If the excess dissociation solution is not washed away, on the one hand, the dyeing effect will be affected, because the dissociation solution contains HCl, and the dye used for dyeing is alkaline;On the other hand, it will corrode the lens of the microscope.
5. When dyeing, the concentration of dyeing solution and dyeing time must be well controlled.In particular, the staining should not be too deep, otherwise it will be purple under the mirror and cannot be observed.It should not be too shallow, otherwise the morphology and number of chromatin and chromosome are not easy to distinguish.
6. When making the film, on the one hand, use tweezers to crush the onion root tip, and on the other hand, press the tablet, so that the cells can be dispersed.The purpose of adding another slide to the cover glass during compression is to avoid crushing and moving the cover glass.The second is to make the pressure moderate, so that the tissue cells are evenly dispersed.At the same time, the force must be appropriate. If it is too heavy, the tissue will be crushed. If it is too light, the cells will not disperse. Both will affect the observation.
7. Results and discussion
Most of the cells seen in one field of the microscope are mitoticinterval Because in a cell cycle, interphase takes up 90%~95% of the whole cell cycle, and the time is proportional to the number of cells.In addition, it is often difficult to find cells at all stages of holomitosis in a single field of vision. You can slowly move the slides and look for cells from adjacent meristematic regions.
Image discrimination steps
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First, look at the number of chromosomes: odd number is minus II (only one pole for sister separation);Second, see if there isHomologous chromosome: Not subtracting II (only one pole is needed for separation of sisters);Third, look at the behavior of homologous chromosomes: determine the filamentous or subtractive Ⅰ
Note: IfcytoplasmFor unequal splitOogoniaThe later period of subtracting I or subtracting II.
HOMOLOGOUS CHROMOSOME DIVERSION - anaphase of minus Ⅰ;Sister separation - minus late stage II
