CN101413033A

CN101413033A - Fluorescence labeling method for cell DNA during cotton pollen development process

Info

Publication number: CN101413033A
Application number: CNA2008102345665A
Authority: CN
Inventors: 唐灿明; 岳洁瑜; 吴李君; 吴跃进
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2008-11-21
Filing date: 2008-11-21
Publication date: 2009-04-22
Anticipated expiration: 2028-11-21
Also published as: CN101413033B

Abstract

The invention discloses a fluorescence labeling method for detecting DNA matters of a cotton pollen mother cell, and belongs to the biological technical field. The method comprises the following steps: on a sunny morning, buds are picked up, and a sample passes through ethanol with reducing concentrations to enter distilled water; pollen mother cells with a diameter of less than 7mm of the buds are placed in glycerin for infiltration for 2 to 3 hours, and, after washing, are dyed by DAPI in the dark for 1 to 2 hours to label DNA matters in the cells; mature pollen grains with the diameter of more than 7mm of the buds are added with a 10 percent sodium hypochlorite aqueous solution, and subjected to water bath treatment at a temperature of 60 DEG C for 10 minutes, washed, placed in 0.1 to 0.2 percent glycerin for infiltration for 2 to 3 hours, washed, added with DAPI solution, stood for 10 minutes and subjected to microscopic examination to label DNA matters in the cells. The method has the advantages of simple and quick operation, less damages to cells, good labeling effect and easy observation, and can play an important role in research on the development process of cotton male gametophyte, and has high application value.

Description

The fluorescence labeling method of cell DNA in a kind of cotton pollen growth course

One, technical field

The present invention relates to a kind of fluorescence labeling method of cotton pollen parent cell dna material, belong to biological technical field.

Two, background technology

The sophisticated change procedure that the sporule of plant occurs to pollen mother cell is one of important content of biology of reproduction research always.For the cotton sporule take place and the research of microgamete growth course since 20th century just, early stage research method mainly is a paraffin method, with phenodin or sarranine, fast green dyeing, develop into subsequently with ultrathin section(ing), electron microscopy observation or isolated culture, fluorescent dye.But up to now, because these method complicated operations are difficult to observe the whole process that microgametophyte is grown fast, observing effect is undesirable.This mainly contains the reason of two aspects, and the one, the limitation of experiment material self; The 2nd, the limitation of detection method.

The wall of plant mature pollen can be divided into outer wall and inwall, and outer wall mainly is made up of materials such as sporopollenin and Mierocrystalline celluloses.Wherein sporopollenin is a kind of decomposition, strong stress resistance, resistant to elevated temperatures material of being difficult to, and stability is strong, has anti-strong acid, alkaline characteristic, and it makes the outer wall of pollen granule very firm.Inwall mainly is made up of Mierocrystalline cellulose and pectin substance, is easily decomposed by enzyme material.Volume was bigger when the pollen granule of cotton was ripe, and diameter is approximately about 85-105 μ m, after discharging from tetrad, formed the very fine and close outer wall of the opaque structure of one deck, and the autofluorescence of outer wall is very strong, makes dyestuff penetration less than in the pollen.If therefore adopt the method for dyeing thin-walled pollen granule (as paddy rice, wheat pollen granule) to come cotton mature pollen grain is dyeed, then do not observe its internal structure fully.In addition, the specificity of forefathers' used dyestuff when detecting the process of cotton microgametophyte growth is not strong, and experimental arrangement is more loaded down with trivial details, and the cycle is longer.Therefore still there is not the adequate solution method.

After the exine-detached pollen grain was meant that pollen granule is sloughed outer wall, inwall that stays and protoplastis were a kind of structure units between complete pollen granule and pollen granule protoplastis, were a kind of unique material that can be used for studying POLLEN MORPHOLOGY and physiological property.The exine-detached pollen grain can be used to study many-sided contents such as synthesizing of the deposition of the surface tissue of inwall, the outer wall construction of peeling off and composition, inwall, new wall; The exine-detached pollen grain helps entering of dye molecule owing to got rid of the barrier action of outer wall to biomacromolecule.After DNA dyes to nuclear with fluorescence dye, observe with fluorescent microscope or laser scanning co-focusing microscope again, can study the nuclear structure and the Changing Pattern thereof of pollen granule inside.Present method prepares the exine-detached pollen grain by heat shock-oxidation style, use DNA fluorescence dye DAPI (4 again, 6-diamidino-2-phenylindole, 4,6-diamidine-2-phenylindone) dyes, set up a kind of easy film-making program, fluorescent microscope has been studied the constructional feature and the nuclear state thereof of cotton pollen grain.DAPI be a kind of can with the powerful bonded fluorescence dye of DNA, it can stick to the ditch district of dna double spiral.Excite and the emission wavelength of DAPI-DNA mixture is respectively 360nm and 460nm to have very high photobleaching and bears level.And it can also see through complete cytolemma, is used for the dyeing of viable cell and fixed cell.Present method has been explored the test that detects cotton male gametophyte growth course with the DAPI fluorescence dye, set up complete feasible test method, operation steps is simple, and test effect is stable, clear, therefore no matter can both well be used in scientific research, detection and student's test.

Three, summary of the invention

Technical problem the object of the present invention is to provide a kind of faster, the method for effective detection cotton male gametophyte growth course.The bud diameter is during greater than 7mm, and pollen mother cell has been grown and has been young tender pollen granule, needs slough extine by the hypochlorite oxidation heat shock, detects the nuclear change that cell includes dna material by DAPI dyeing again.The bud diameter is during less than 7mm, and pollen mother cell does not also develop into young tender pollen granule, and pollen wall also forms, and does not then need to handle and get final product dyeing microscopic examination through taking off outer wall.

Technical scheme

A kind of fluorescence labeling method of cotton pollen parent cell dna material may further comprise the steps:

1) win the cotton buds of different sizes between 6-7 of fine mornings at random, peel off bract, be fixed in dehydrated alcohol: Glacial acetic acid is made into the Ka Nuoshi stationary liquid of volume ratio 3:1, changes volume ratio behind 12-24h over to and is in 70% the ethanol, is stored in 4 ℃ of refrigerators;

2) sample that fixes is that 50%, 30%, 10% ethanol comes downwards to distilled water step by step through volumetric concentration;

3) diameter is less than the bud of 7mm, separates wherein pollen mother cell with dissecting needle, places the glycerine of volume ratio 0.1-0.2% to permeate 2-3h; With the phosphoric acid buffer washing pollen mother cell of pH7-8, with the DAPI lucifuge dyeing 1-2h of pollen mother cell with 0.5-1 μ g/ml, dna material is by fluorescent mark in the cell;

4) diameter is greater than the bud of 7mm, separates wherein mature pollen grain with tweezers, adds the aqueous sodium hypochlorite solution of mass volume ratio 10%, and 10min is placed in the room temperature vibration, again in 60 ℃ of water bath processing 10min; Phosphoric acid buffer washing pollen granule with pH7-8; Pollen granule places the glycerine of volume ratio 0.1-0.2% to permeate 2-3h; Phosphoric acid buffer washing pollen granule with pH7-8; Again pollen granule is moved to slide glass, adding 100-200 μ L concentration is the DAPI solution of 2 μ g/ml, cover plate, appropriateness firmly barocline to outer wall take off and the pollen granule cell not break be degree, be placed on and leave standstill microscopy behind the 10min in the magazine, dna material is by fluorescent mark in the cell.Cotton described in the above-mentioned method is a upland cotton.

Beneficial effect

The invention provides a kind of method for quick to cotton male gametophyte growth course.Mark of the present invention and mature pollen grain take off the outer wall method and have the following advantages: 1) simple to operate, fast: omitted the slicing step in the routine paraffin wax section; 2) use glycerine as osmotic agent, make that fluorescent dye efficient is higher; 3) mark is effective; 4) pollen granule was still complete after mature pollen took off outer wall.The inventive method provides new approach and foundation with the result for carrying out the biology of reproduction research and development relevant with the cotton pollen grain.

Four, Figure of description

Fig. 1 cotton male gametophyte is grown the observation of different times

A, F) zygotene stage (* 400), Bar=90 μ m; B) subtract the I later stage (* 400), Bar=100 μ m; C) subtract I latter stage (* 400), Bar=100 μ m; D) dyad period (* 400), Bar=90 μ m; E) latter stage II; F-I) tetrad period (* 400), Bar=90 μ m; J-L) mature pollen grain monokaryon, two nuclears, three nuclear phases (* 400), Bar=90 μ m

Fig. 2 detects the techniqueflow chart of the cotton 21 male gametophyte growth courses in Shandong

Five, embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and described percentage composition is volumn concentration if no special instructions.

The fluorescent mark and the microscopic (main technical flows is seen Fig. 2) thereof of embodiment 1, cotton 21 male gametophytes in Shandong

At first use method of the present invention that Shandong cotton 21 (producing with planting) microgametophyte is carried out fluorescent mark, may further comprise the steps:

1) gathers different big or small buds about 6:30 in morning, peel off bract, be fixed in freshly prepared Ka Nuoshi stationary liquid (dehydrated alcohol: Glacial acetic acid is made into the solution of volume ratio 3:1) rapidly, bleeding changes in 70% ethanol after fixing 12-24h, be stored in 4 ℃ of refrigerators, standby;

2) sample that fixes is that 50%, 30%, 10% ethanol (distilled water preparation) comes downwards to distilled water step by step through concentration, and every grade stops 2h;

3.1) diameter is less than the bud of 7mm, separates wherein pollen mother cell (removing whole impurity such as flower pesticide fragment) with dissecting needle, places 0.15% glycerine (dilution of PBS damping fluid), at room temperature leaves standstill infiltration 2h;

3.1.1) with the PBS damping fluid of pH7.4 washing 3 times, in damping fluid, stop 10-20min at every turn after, leave standstill several minutes treat the sample post precipitation again with liquid-transfering gun with upper strata damping fluid sucking-off;

3.1.2) with pollen mother cell with the DAPI of 0.5 μ g/ml (1h USA) is at room temperature secretly hatched in Sigma company, then without washing directly under fluorescent microscope, detect (observations such as Fig. 1, A-I).

By operations such as fixing, infiltration, dyeing and microscopies, observed the different development stage of cotton microgametophyte.Maiotic zygotene stage: the karyomit(e) more open (Fig. 1-A) that distributes; Later stage I: homologous chromosomes is separated by spindle fibre traction, evenly pulls to two-stage (Fig. 1-B); Latter stage I: the karyomit(e) evenly distribute, arrive respectively the two poles of the earth (Fig. 1-C), cell walls does not form, tenuigenin divides, and forms dyad, illustrates that it is not simultaneous type division of cytoplasm (Fig. 1-D); Latter stage II: karyomit(e) progresses into uncoiling (Fig. 1-E); Tetrad period: the quadrantal tetrahedral structure that is arranged as, 4 sporules be by common callose bag quilt, also cut apart by callose between the sporule (Fig. 1-F-I).

In to the observation of cotton microgametophyte growth course, also find, the speed of the pollen mother cells of the different Xiao Hua of different inflorescences of different plants, same plant and same bud is also asynchronous, even the cell fission speed in the same flower pesticide also there are differences.Therefore, in same film-making, can observe division phase more than 1 different times (Fig. 1-F) in the experiment.

3.2) diameter is greater than the bud of 7mm, separates wherein mature pollen grain with tweezers, adds 10% aqueous sodium hypochlorite solution, 10min is placed in the room temperature vibration, again in 60 ℃ of water bath processing 10min;

3.2.1) with the PBS damping fluid of pH7.4 washing 3 times, in damping fluid, stop 10-20min at every turn after, leave standstill several minutes treat the sample post precipitation again with liquid-transfering gun with upper strata damping fluid sucking-off;

3.2.2) sample after the washing places 0.15% glycerine (dilution of PBS damping fluid) to permeate 2h again;

3.2.3) with the PBS damping fluid of pH7.4 washing 3 times, in damping fluid, stop 10-20min at every turn after, leave standstill several minutes treat the sample post precipitation again with liquid-transfering gun with upper strata damping fluid sucking-off;

3.2.4) with liquid-transfering gun the pollen granule cell is moved to slide glass again, add DAPI (the Sigma company that 100 μ L concentration are 2 μ g/ml, USA) solution, rapid cover plate, appropriateness is firmly baroclined to outer wall and is taken off and the pollen granule cell does not break and is degree, be placed on leave standstill in the magazine behind the 10min again microscopy under fluorescent microscope (observations such as Fig. 1, J-L).

Under fluorescence microscope, can clearly see the inner core structure through the exine-detached pollen grain after the DAPI dyeing, (((Fig. 1-L) waits three kinds of dissimilar pollen granules (the arrow indication is all represented to examine among the figure) for Fig. 1-K) and three nuclear pollen granules for Fig. 1-J), double-core pollen granule can clearly to tell the monokaryon pollen granule.Nucleus in the monokaryon pollen granule does not also carry out mitotic division, has perhaps carried out a mitotic division, and still, vegetative nucleus and germ nucleus still flock together.The double-core pollen granule has carried out a mitotic division, form a vegetative nucleus and a germ nucleus, and both separates obviously.Three nuclear pollen granules had carried out twice successive mitotic division, comprise a vegetative nucleus and two spermatid nucleuses, and the three separated obviously.

Claims

1, the fluorescence labeling method of cell DNA in a kind of cotton pollen growth course may further comprise the steps:

1) win the cotton bud between 6-7 of fine mornings, peel off bract, be fixed in dehydrated alcohol: Glacial acetic acid is made into the Ka Nuoshi stationary liquid of volume ratio 3:1, changes volume ratio behind 12-24h over to and is in 70% the ethanol, is stored in 4 ℃ of refrigerators;

4) diameter is greater than the bud of 7mm, separates wherein mature pollen grain with tweezers, adds the aqueous sodium hypochlorite solution of mass volume ratio 10%, and 10min is placed in the room temperature vibration, again in 60 ℃ of water bath processing 10min; Phosphoric acid buffer washing pollen granule with pH7-8; Pollen granule places the glycerine of volume ratio 0.1-0.2% to permeate 2-3h; Phosphoric acid buffer washing pollen granule with pH7-8; Again pollen granule is moved to slide glass, adding 100-200 μ L concentration is the DAPI solution of 2 μ g/ml, cover plate, firmly barocline to outer wall take off and the pollen granule cell not break be degree, be placed on and leave standstill microscopy behind the 10min in the magazine, dna material is by fluorescent mark in the cell.

2, method according to claim 1 is characterized in that: described cotton is a upland cotton.