CN104531824A - Labeling method of apple pollen tube microfilament - Google Patents

Abstract

The invention discloses a labeling method of an apple pollen tube microfilament. The labeling method comprises the following steps: cultivating apple pollen, curing the apple pollen, performing enzyme treatment on the apple pollen, treating the apple pollen with a surfactant, performing dye labeling, and performing scanning and imaging. The labeling method has the beneficial effect that the labeling method of gymnosperm pollen tube microfilament is referred and improved on the basis of the labeling method of pear pollen tube microfilament, dyes enter cells easier through curing, enzymolysis of cell walls and the surfactant, the interference of cell outer wall autofluorescence due to insufficient enzymolysis as TRITC-Phalloidin is applied are avoided when FITC-Phalloidin labeling is applied, so that relatively complete apple pollen tube microfilament can be stored; moreover, the labeling method is efficient and quick, can be used for obtaining relatively clear and bright pictures, thus filling up the blank of the apple pollen tube microfilament labeling, providing reference for labeling microfilament in rosaceae and angiosperm pollen tube, and achieving a high application value.

Description

A kind of marking method of apple flower tube cell microfilament

Technical field

The present invention relates to a kind of marking method of apple flower tube cell microfilament.

Background technology

At present both at home and abroad almost not to the report that microfilament in apple flower tube cell marks.In pollen, the technology of microfilament mark mainly contains chemical staining methods: marked by the Phalloidine with fluorescence dye, secondly cryofixation in conjunction with microinjection and utilize gene recombination technology by green fluorescence protein gene the method proceeded in live body pollen tube observe, but the method for current multiplex chemical staining is simpler and more direct, efficient.In gymnosperm Pinaceae pollen, the research of microfilament was relatively many in recent years, and the Rhodamine-Phalloidin that adopts marks more.In quilt rosaceous plant, the mark of pear pollen tube microfilament has been reported, and not carrying out the penetrating step of pollen enzymolysis tube wall, Triton decontamination in labeling process may be the reason causing marking less effective.And microfilament mark is slightly aobvious blank in apple flower tube cell.

Summary of the invention

Object of the present invention is exactly for above-mentioned defect of the prior art, provides a kind of marking method of apple flower tube cell microfilament.

To achieve these goals, technical scheme provided by the invention is: a kind of marking method of apple flower tube cell microfilament, comprises the following steps:

1) Apple Pollen is cultivated: taken out by the pollen being stored in-20 DEG C and rise again, adopt liquid culture method, take pollen 10mg, be suspended in 10ml substratum, temperature 30 DEG C, 100rpm/min vibrates light culture, and substratum consists of: 20% sucrose+0.015%CaCl ₂+ 0.01%H ₃bO ₃;

2) fixing: to wash with the paraformaldehyde containing mass percentage being 4% the cultured Apple Pollen twice that step 1) obtains, fixing 1.5h, vacuum suction 10-15min, is fixed rear pollen simultaneously, and the paraformaldehyde of described 4% configures with the 50mM PIPES damping fluid that pH value is 6.9;

3) ferment treatment: by step 2) obtain fixing after pollen with the PIPES buffer solution three times of 50mM, again the pollen after washing is placed in the 50mM PIPES damping fluid of the cellulase of polygalacturonase containing mass percentage 1% and mass percentage 1%, cultivate 15-20min for 37 DEG C, obtain pollen after ferment treatment;

4) after ferment treatment step 3) obtained, pollen is with after 50mM PIPES buffer solution three times, then is placed in the Triton X-100 solution that volumn concentration is 1%, and incubated at room 1-2h, obtains hatching pollen;

5) pollen of hatching step 4) obtained marks with 200nM Alexa Fluor 488 phalloidin, labeling process is that 200nM Alexa Fluor488 phalloidin dilutes with PBS damping fluid, pH value is 6.9, cultivate 2h under being placed in light culture environment, directly observe or observe after spending the night with 4 DEG C can obtain labeled pollen mounting with glycerine mounting again;

6) labeled pollen mounting step 5) obtained utilizes Leica TCS SP5 laser confocal scanning imaging system, FITC, Ar laser, wavelength 488nm, and 180-200 different optical section scanning got by each sample, then by all optical section stacking images.

Beneficial effect of the present invention is: the present invention carries out using for reference in conjunction with the marking method of gymnospermae pollen pipe microfilament and improves on the method basis that pear pollen microfilament marks, by fixing, enzymolysis, use FITC-Phalloidin mark, can obtain preserving relatively intact Apple Pollen microfilament, and have efficiently, feature fast, more clear, bright picture can be obtained, fill up the blank of apple flower tube cell microfilament mark, for in the Rosaceae and even Angiosperm pollen pipe, the mark of microfilament provides reference, using value is high.

Accompanying drawing explanation

Fig. 1 is in the marking method of a kind of apple flower tube cell microfilament provided by the invention, the distribution figure (bar=25 μm) of Apple Pollen microfilament.

Embodiment

reagent source:

Paraformaldehyde: lot number: 20080512, Beijing Tuo Yingfang Science and Technology Ltd.;

PIPES damping fluid: SIGMA, 80635-50G, 50mM PIPES takes 7.55925g and is dissolved in distilled water, adjusts sheet pH to be 6.9, is settled to 500ml.Sigma-Aldrich (Shanghai) trade Co., Ltd;

Polygalacturonase: Pectolase Y-23, MFCD00131809, Beijing North unit high-tech agricultural development center;

Cellulase: Cellulase R-10, lot number 201097,105-0004, Beijing North unit high-tech agricultural development center;

Triton X-100:SIGMA, T9284-500ML, Sigma-Aldrich (Shanghai) trade Co., Ltd;

Alexa Fluor 488 phalloidin:SIGMA, A12379, Sigma-Aldrich (Shanghai) trade Co., Ltd;

PBS damping fluid: 100mM PBS: take 8.0 g NaCl respectively, 0.2g KCl, 2.9 g Na ₂hPO ₄12H ₂o, 0.2g KH ₂pO ₄, be dissolved in 800 ml distilled waters successively in order, adjust pH 7.2 with 1 N HCl, be then settled to 1000 ml.

embodiment 1:

A marking method for apple flower tube cell microfilament, comprises the following steps:

1) Apple Pollen is cultivated: taken out by the pollen being stored in-20 DEG C and rise again, adopt liquid culture method, take pollen 10mg, be suspended in 10ml substratum, temperature 30 DEG C, 100rpm/min vibrates light culture, and substratum consists of: 20% sucrose+0.015%CaCl ₂+ 0.01%H ₃bO ₃;

2) fixing: with containing mass percentage being the cultured Apple Pollen twice that 4% paraformaldehyde (being the 50mM PIPES damping fluid configuration of 6.9 by pH value) is washed step 1) and obtained, fixing 1.5h, while vacuum suction 10-15min, be fixed rear pollen;

3) ferment treatment: by step 2) obtain fixing after pollen with the PIPES buffer solution three times of 50mM, again the pollen after washing is placed in the 50mM PIPES damping fluid of the cellulase of polygalacturonase containing mass percentage 1% and mass percentage 1%, cultivate 15-20min for 37 DEG C, obtain pollen after ferment treatment;

4) after ferment treatment step 3) obtained, pollen is with after 50mM PIPES buffer solution three times, then is placed in the Triton X-100 solution that volumn concentration is 1%, and incubated at room 1-2h, obtains hatching pollen;

5) pollen of hatching step 4) obtained marks with 200nM Alexa Fluor 488 phalloidin, labeling process is that 200nM Alexa Fluor488 phalloidin dilutes with PBS damping fluid, pH value is 6.9, cultivate 2h under being placed in light culture environment, directly observe or observe after spending the night with 4 DEG C can obtain labeled pollen mounting with glycerine mounting again;

6) labeled pollen mounting step 5) obtained utilizes Leica TCS SP5 laser confocal scanning imaging system, FITC, Ar laser, wavelength 488nm, and 180-200 different optical section scanning got by each sample, then by all optical section stacking images.

Apple Pollen microfilament distribution (bar=25 μm) as shown in Figure 1.

The marking method of a kind of apple flower tube cell microfilament provided by the invention compared with prior art, has following effect:

1, improve on pear pollen cultivation basis, stationary liquid directly adopts 4% paraformaldehyde to fix, but not use the paraformaldehyde of 2% and 4% respectively, save time for lysed cells wall, the proportioning of 1% cellulase and 1% polygalacturonase makes cell walls enzymolysis and does not affect the form of microfilament in pollen, uses 1% non-ionic detergent Triton X-100, increase the permeability of film, make dyestuff more easily enter born of the same parents' internal labeling microfilament, in picture, microfilament clearly, and maintain the integrity of microfilament.

2, the Phalloidine not adopting gymnosperm TRITC to mark in the application of dyestuff, adopt the Phalloidine of the FITC mark of suitable concentration 200nM, when the pollen cell outer wall of TRITC mark exists, outer wall autofluorescence is stronger, impact mark effect, select FITC-ph that may be far away with outer wall excitation wavelength to mark, the mark of microfilament can not be subject to not having the cell walls autofluorescence of abundant enzymolysis to affect, and can observe microfilament form more clearly.

Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (1)

1. a marking method for apple flower tube cell microfilament, is characterized in that, comprises the following steps:

1) Apple Pollen is cultivated: taken out by the pollen being stored in-20 DEG C and rise again, adopt liquid culture method, take pollen 10mg, be suspended in 10ml substratum, temperature 30 DEG C, 100rpm/min vibrates light culture, and substratum consists of: 20% sucrose+0.015%CaCl ₂+ 0.01%H ₃bO ₃;

2) fixing: to wash with the paraformaldehyde containing mass percentage being 4% the cultured Apple Pollen twice that step 1) obtains, fixing 1.5h, vacuum suction 10-15min, is fixed rear pollen simultaneously, and the paraformaldehyde of described 4% configures with the 50mM PIPES damping fluid that pH value is 6.9;

3) ferment treatment: by step 2) obtain fixing after pollen with the PIPES buffer solution three times of 50mM, again the pollen after washing is placed in the 50mM PIPES damping fluid of the cellulase of polygalacturonase containing mass percentage 1% and mass percentage 1%, cultivate 15-20min for 37 DEG C, obtain pollen after ferment treatment;

4) after ferment treatment step 3) obtained, pollen is with after 50mM PIPES buffer solution three times, then is placed in the Triton X-100 solution that volumn concentration is 1%, and incubated at room 1-2h, obtains hatching pollen;

5) pollen of hatching step 4) obtained marks with 200nM Alexa Fluor 488 phalloidin, labeling process is that 200nM Alexa Fluor488 phalloidin dilutes with PBS damping fluid, pH value is 6.9, cultivate 2h under being placed in light culture environment, directly observe or observe after spending the night with 4 DEG C can obtain labeled pollen mounting with glycerine mounting again;

6) labeled pollen mounting step 5) obtained utilizes Leica TCS SP5 laser confocal scanning imaging system, FITC, Ar laser, wavelength 488nm, and 180-200 different optical section scanning got by each sample, then by all optical section stacking images.