CN101398381A - Fluorescence labeling method for pear pollen and pollen tube fibril framework - Google Patents
Fluorescence labeling method for pear pollen and pollen tube fibril framework Download PDFInfo
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- CN101398381A CN101398381A CNA2008102349844A CN200810234984A CN101398381A CN 101398381 A CN101398381 A CN 101398381A CN A2008102349844 A CNA2008102349844 A CN A2008102349844A CN 200810234984 A CN200810234984 A CN 200810234984A CN 101398381 A CN101398381 A CN 101398381A
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Abstract
The invention provides a fluorescence marking method used for pear pollen and pollen tube microfilament framework, belonging to the field of biotechnology; the method comprises the steps as follows: mature pollen is collected; pollen is cultivated in culture mediums respectively for 0.5h and 2h; 200Mu moL/L MBS (maleic hydrazide benzoic acid-N- succinate) prepared by the fresh pollen culture medium is pre-fixed for 5-10min and subsequently fixed for 0.5h respectively by 2 percent of paraformaldehyde and 4 percent of paraformaldehyde; the pollen (tube) is washed and dyed for 0.5-1h at the temperature of 25DEG C and lucifuge by FITC-ph staining solution (phalloidin staining solution) with the final consistency of 5 Mu g/ml and containing 5 percent of DMSO (dimethyl sulfoxide); and the microfilament framework is marked by fluorescence. The fluorescence marking method has the advantages of simple and quick operation, good marking effect, little damage on the microfilament framework structure and the like, can be used for the development and utilization of rosaceae plant pollen (tube) and has high practically application value.
Description
One, technical field
The present invention is the fluorescence labeling method of pear pollen and pollen tube microfilament framework, comprise the fluorescence labeling method of rose family orchard fruit pollen and pollen tube microfilament framework and pollen and pollen tube microfilament framework are carried out contents such as microscopic examination, belong to biological technical field with this method.
Two, background technology
Fibril framework is a kind of of cytoskeleton, and it mainly is made up of the spiral fashion polymer and the actin binding protein of actin assembling.The assembling of microfilament is relevant with many functional activities of cell with depolymerization, builds up as participating in cellular morphology, the motion positions of organelle, kytoplasm flows and apical growth etc.Pollen tube is as the carrier of male unit in the Anthophyta fertilization process, has typical apical growth characteristic, thereby become in the biological technical field idealized model system of research cell polarity growth, in addition, interaction between the pollen tube pair cell and signal conduction studies are also significant.Fibril framework is one of basic structure of pollen tube, plays an important role in the apical growth of pollen tube, and transportation, the kytoplasm that comprises foundation, the excretion vesicles in pollen tube growth process Semi-polarity site flows and the structure of apical cell's wall, and and Ca
2+Closely related Deng signaling molecule, thereby pollen tube is the type material of research vegetable cell fibril framework.
Still there is dispute so far in distribution for fibril framework in the pollen tube.At present, the method of fibril framework in existing a lot of research pollen tube, but these methods need be removed wall mostly, and be mainly used in the research that the herbaceous plant pollen tube microfilament framework distributes, and almost fibril framework in arboreal pollen and the pollen tube was not carried out research, especially effective ways and the technology that fibril framework in rose family orchard fruit pollen such as pears and the pollen tube is carried out mark yet there are no report, thus cause with rose family fruit blossom tube cell growth courses such as pears in relevant biological study relatively lag behind.
Three, summary of the invention
Technical matters the purpose of this invention is to provide a kind of faster, the effective fluorescence labeling method of pear pollen and pollen tube microfilament framework, structure and the distribution in pollen (pipe) observed to fibril framework effectively.This method can be used for other rose family orchard fruits of mark (apple, peach, apricot, Lee etc.) interior fibril framework of pollen (pipe), and also fluorescence labeling and the observation for fibril framework in other histoorgans provides method.
Technical scheme is provided by the present invention carries out fluorescently-labeled method to pear pollen and pollen tube microfilament framework, comprises the following steps:
1) collection of pollen: gather the pollen that pear tree blooms the same day, seal-20 ℃ of cryopreservation after the air dry;
2) the pollen nutrient culture media prepares according to mass ratio:
Sucrose, 10%; PEG4000 (Macrogol 4000), 15%; H
3BO
3, 0.01%; Ca (NO
3)
24H
2O, 0.03%; MgSO
47H
2O, 0.02%; KNO
3, 0.01%; Solvent, water; MES damping fluid (2-(N-morpholinyl) ethyl sulfonic acid damping fluid) adjust pH to 6.5 with 30mmol/L;
3) cultivation of pollen:
Get the dried pollen of step 1), put into the pollen nutrient culture media, under 25 ℃ of dark conditions of constant temperature, cultivate 0.5h or 2h respectively, cultivate the pollen of 0.5h, be used for the fluorescence labeling of fibril framework in the pollen granule; Cultivate 2h and obtain pollen tube, be used for the fluorescence labeling and the observation of fibril framework in the pollen tube;
4) pre-fix: pollen after the cultivation or pollen tube, the 200 μ mol/L MBS (maleic acid amides benzoic acid-N-succinate) that prepare with fresh pollen nutrient culture media pre-fix 5-10min;
5) fixing: as to be fixedly pollen or pollen tube 0.5h under 6.8 0.1mol/L PBS damping fluid (phosphate buffer) room temperature condition with containing mass ratio 2% paraformaldehyde and mass ratio 4% paraformaldehyde, pH then; 0.1mol/L the preparation of PBS damping fluid (phosphate buffer): NaH
2PO
42H
2O 2.964g and Na
2HPO
412H
2O 28.998g adding distil water is settled to 1000ml, and pH 6.8;
6) washing: clean 2-3 time with 0.1mol/LPBS damping fluid (phosphate buffer), remove cleaning fluid;
7) preparation of dyeing liquor:
Dyeing liquor is prepared with the 0.1mol/L PBS damping fluid (phosphate buffer) of pH6.8, wherein add final concentration mass ratio 5% DMSO (dimethyl sulfoxide (DMSO)), final concentration 5mmol/L EGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)), final concentration mass volume ratio 10% sucrose adds final concentration 5 μ g/ml FITC-ph (phalloidine of marked by fluorescein isothiocyanate) again;
8) dyeing: add dyeing liquor, under the room temperature dark condition, hatch 0.5-1h;
9) washing: clean 2-3 time with 0.1mol/LPBS damping fluid (phosphate buffer), obtain the good sample of mark;
10) mounting: the sample drop that mark is good is added on the microslide, and with volume ratio 50% glycerine mounting, waits to observe.
Above-mentioned steps 4) time that pre-fixes with MBS (maleic acid amides benzoic acid-N-succinate) is preferably 7min, and the step 8) incubation time is preferably 1h.
Beneficial effect
1) first rose family orchard fruit pear pollen and pollen tube microfilament framework are carried out fluorescence labeling, the present invention will utilize in the process in the research and development of rose family orchard fruit pollen and play a significant role the actual application value height.
2) the present invention is simple to operate, quick, has omitted so far the step that in other labeling methods pair cell wall carries out enzymolysis;
3) pre-fix with MBS (maleic acid amides benzoic acid-N-succinate) after again with the fixing fibril framework structure that can preserve preferably in pollen and the pollen tube of paraformaldehyde;
4) use DMSO (dimethyl sulfoxide (DMSO)) as bleeding agent, increased the permeability of cell membrane, reduced the damage of pair cell, preserve fibril framework structure in pollen and the pollen tube preferably.
5) the present invention selects for use FITC-ph (phalloidine of marked by fluorescein isothiocyanate) as fluorescent marker dyes, overcome the interference of pear pollen outer wall autofluorescence, mark is effective, can clearly observe fibril framework and be pencil and distribute along the pollen tube major axis under laser confocal microscope.
6) this method can be used for other rose family orchard fruits of mark (apple, peach, apricot, Lee etc.) interior fibril framework of pollen (pipe), and also fluorescence labeling and the observation for fibril framework in other histoorgans provides method.
Four, description of drawings
The fluorescently-labeled process flow diagram of Fig. 1 pear pollen (pipe) fibril framework.
The pollen that Fig. 2 observes under laser confocal microscope (excitation wavelength is 488nm) after according to Fig. 1 process flow diagram mark with the dyeing liquor that contains FITC-ph (phalloidine of marked by fluorescein isothiocyanate) and the structure and the distribution situation of pollen tube microfilament framework.Microfilament is thread in the pollen granule, assembles at the pollen germination pore place that (Fig. 2 a), in pollen tube, microfilament presents pencil and distributes along the pollen tube major axis, and pollen tube apex zone (about 10 μ m from the tip) does not find have microfilament to have (Fig. 2 b).
Fig. 3 contrast.Fig. 3-a handles with phalloidine (phalloidin) earlier, use the image of being observed under laser confocal microscope (excitation wavelength is 488nm) behind FITC-ph dyeing liquor (the phalloidine dyeing liquor of the marked by fluorescein isothiocyanate) mark then, the result does not observe fibril framework in pollen and the pollen tube; The image of Fig. 3-b for replacing with FITC (fluorescein isothiocynate) under laser confocal microscope (excitation wavelength is 488nm), being observed behind FITC-ph (phalloidine of the marked by fluorescein isothiocyanate) mark, hyperfluorescence is all arranged in pollen and the pollen tube, but do not observe the fibril framework structure.FITC-ph (phalloidine of marked by fluorescein isothiocyanate) labeled pollen or the interior fibril framework of pollen tube specifically are described.
The pollen that the dyeing liquor that Fig. 4 substitutes FITC-ph (phalloidine of marked by fluorescein isothiocyanate) with TRITC-ph (phalloidine of tetramethyl rhodamine B marked by fluorescein isothiocyanate) observes under laser confocal microscope (excitation wavelength is 568nm) after according to Fig. 1 process flow diagram mark and the structure and the distribution situation of pollen tube microfilament framework, in the pollen granule because the autofluorescence (shown in the triangle) of outer wall, can't determine the structural form and the distribution of the inner fibril framework in clear zone, but see dots structure (Fig. 4-b at the pollen germination pore place of not sprouting, shown in the arrow), can see that in pollen tube the microfilament fasciculation distributes along the pollen tube major axis, also can see the distribution (Fig. 4-c is shown in the arrow) of microfilament at the pollen germination pore place.Fig. 4-a is not for containing the autofluorescence of TRITC-ph dyeing liquor (the phalloidine dyeing liquor of tetramethyl rhodamine B marked by fluorescein isothiocyanate) according to the exposore that is observed behind Fig. 1 process flow diagram mark under laser confocal microscope (excitation wavelength is 568nm).
Annotate: Fig. 2, the target in 3,4 is all represented 10 μ m
Five, concrete embodiment
1) collection of plant sample: gather the pollen that pear tree blooms the same day, seal-20 ℃ of cryopreservation after the air dry.
2) preparation of pollen nutrient culture media: sucrose, 10%; PEG4000 (Macrogol 4000), 15%; H
3BO
3, 0.01%; Ca (NO
3)
24H
2O, 0.03%; MgSO
47H
2O, 0.02%; KNO
3, 0.01%; Solvent, water; MES damping fluid (2-(N-morpholinyl) ethyl sulfonic acid damping fluid) adjust pH to 6.5 with 30mmol/L;
3) cultivation of pollen: get the pollen of preserving after the step 1) drying, the 2h that thaws at normal temperatures earlier cultivates pollen on above-mentioned pollen nutrient culture media, and temperature is 25 ℃, and dark condition is cultivated 0.5h and 2h respectively.Cultivate the pollen of 0.5h, be used for the fluorescence labeling of fibril framework in the pollen granule; Cultivate 2h and obtain pollen tube, be used for the fluorescence labeling and the observation of fibril framework in the pollen tube;
4) pre-fix: pollen after the cultivation or pollen tube, the 200 μ mol/L MBS (maleic acid amides benzoic acid-N-succinate) that prepare with fresh pollen nutrient culture media pre-fix 7min;
5) fixing: as to be fixedly pollen or pollen tube 0.5h under 6.8 0.1mol/L PBS damping fluid (phosphate buffer) room temperature condition with containing mass ratio 2% paraformaldehyde and mass ratio 4% paraformaldehyde, pH then; 0.1mol/L the preparation of PBS damping fluid (phosphate buffer): NaH
2PO
42H
2O 2.964g and Na
2HPO
412H
2O 28.998g adding distil water is settled to 1000ml, pH6.8;
6) washing: clean 3 times with 0.1mol/L PBS damping fluid (phosphate buffer), remove cleaning fluid;
7) preparation of dyeing liquor: dyeing liquor is prepared with the 0.1mol/L PBS damping fluid (phosphate buffer) of pH6.8, wherein add final concentration mass ratio 5%DMSO (dimethyl sulfoxide (DMSO)), final concentration 5mmol/L EGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)), final concentration mass volume ratio 10% sucrose adds final concentration 5 μ g/ml FITC-ph (phalloidine of marked by fluorescein isothiocyanate) again;
8) dyeing: add dyeing liquor after removing cleaning fluid, under dark condition, hatch 1h for 25 ℃;
9) washing: the sample behind the mark cleans 3 times with 0.1mol/L PBS damping fluid (phosphate buffer), obtains the good sample of mark;
10) mounting and observation: the sample drop that mark is good is added on the microslide, and with volume ratio 50% glycerine mounting, at last at laser confocal microscope (Bio-Rad MRC 1024 ES, Ar/Kr laser, optical maser wavelength is 488nm) under observe, carry out continuous optical section along the Z axle, and all optical sections carried out three-dimensionalreconstruction, result such as Fig. 2 can clearly observe the distribution situation of fibril framework in pollen and pollen tube.Microfilament is thread in Fig. 2 a pollen granule, assembles at the pollen germination pore place.Fig. 2 b is in pollen tube, and microfilament presents pencil and distributes along the pollen tube major axis, and pollen tube apex zone (about 10 μ m from the tip) does not find have microfilament to exist.
Fig. 3 contrast.Fig. 3-a handles with phalloidine (phalloidin) earlier, use the image of being observed under laser confocal microscope (excitation wavelength is 488nm) behind FITC-ph (phalloidine of marked by fluorescein isothiocyanate) the dyeing liquor mark then, the result does not observe fibril framework in pollen and the pollen tube; The image of Fig. 3-b for replacing with FITC (fluorescein isothiocynate) under laser confocal microscope (excitation wavelength is 488nm), being observed behind FITC-ph (phalloidine of the marked by fluorescein isothiocyanate) mark, hyperfluorescence is all arranged in pollen and the pollen tube, but do not observe the fibril framework structure.FITC-ph (phalloidine of marked by fluorescein isothiocyanate) labeled pollen or the interior fibril framework of pollen tube specifically are described.
The pollen that the dyeing liquor that Fig. 4 substitutes FITC-ph (phalloidine of marked by fluorescein isothiocyanate) with TRITC-ph (phalloidine of tetramethyl rhodamine B marked by fluorescein isothiocyanate) observes under laser confocal microscope (excitation wavelength is 568nm) after according to Fig. 1 process flow diagram mark and the structure and the distribution situation of pollen tube microfilament framework, in the pollen granule because the autofluorescence (shown in the triangle) of outer wall, can't determine the structural form and the distribution of the inner fibril framework in clear zone, but see dots structure (Fig. 4-b at the pollen germination pore place of not sprouting, shown in the arrow), can see that in pollen tube the microfilament fasciculation distributes along the pollen tube major axis, also can see the distribution (Fig. 4-c is shown in the arrow) of microfilament at the pollen germination pore place.Fig. 4-a is the autofluorescence of the exposore that do not contain the TRITC-ph dyeing liquor and observed down at laser confocal microscope (excitation wavelength is 568nm) after according to Fig. 1 process flow diagram mark.
Claims (3)
1, the fluorescence labeling method of a kind of pear pollen and pollen tube microfilament framework comprises:
1) collection of pollen: gather the pollen of blooming the same day, gather the pollen that pear tree blooms the same day, seal-20 ℃ of cryopreservation after the air dry;
2) the pollen nutrient culture media prepares according to mass ratio:
Sucrose, 10%; Polyglycol PEG 4000,15%; H
3BO
3, 0.01%; Ca (NO
3)
24H
2O, 0.03%; MgSO
47H
2O, 0.02%; KNO
3, 0.01%; Solvent, water; 2-(N-morpholinyl) ethyl sulfonic acid MES damping fluid adjust pH to 6.5 with 30mmol/L;
3) cultivation of pollen:
Get the dried pollen of step 1), put into the pollen nutrient culture media, under 25 ℃ of dark conditions of constant temperature, cultivate 0.5h or 2h respectively, cultivate the pollen of 0.5h, be used for the fluorescence labeling of fibril framework in the pollen granule; Cultivate 2h and obtain pollen tube, be used for the fluorescence labeling and the observation of fibril framework in the pollen tube;
4) pre-fix: pollen after the cultivation or pollen tube, the 200 μ mol/L maleic acid amides benzoic acid-N-succinate MBS that prepare with fresh pollen nutrient culture media pre-fix 5-10min;
5) fixing: as to be fixedly pollen or pollen tube 0.5h under 6.8 the 0.1mol/L phosphate buffer PBS room temperature condition with containing mass ratio 2% paraformaldehyde and mass ratio 4% paraformaldehyde, pH then; 0.1mol/L the preparation of PBS damping fluid: NaH
2PO
42H
2O 2.964g and Na
2HPO
412H
2O 28.998g adding distil water is settled to 1000ml, pH6.8;
6) washing: use 0.1mol/L PBS buffer solution for cleaning 2-3 time, remove cleaning fluid;
7) preparation of dyeing liquor:
Dyeing liquor is prepared with the 0.1mol/L PBS damping fluid of pH6.8, the dimethyl sulfoxide (DMSO) DMSO that wherein adds final concentration mass ratio 5%, the ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) EGTA of final concentration 5mmol/L, the sucrose of final concentration mass volume ratio 10% adds the phalloidine FITC-ph of the marked by fluorescein isothiocyanate of final concentration 5 μ g/ml again;
8) dyeing: add dyeing liquor, under the room temperature dark condition, hatch 0.5-1h;
9) washing: use 0.1mol/L PBS buffer solution for cleaning 2-3 time, obtain the good sample of mark;
10) mounting: the sample drop that mark is good is added on the microslide, and observes with volume ratio 50% glycerine mounting.
2, the fluorescence labeling method of a kind of pear pollen according to claim 1 and pollen tube microfilament framework is characterized in that: step 4) is 7min with the time that maleic acid amides benzoic acid-N-succinate MBS pre-fixes.
3, method according to claim 1 and 2 is characterized in that: the step 8) incubation time is 1h.
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| CN102192901A (en) * | 2011-03-10 | 2011-09-21 | 山东理工大学 | Method for imaging pollen surface ornamentation based on laser scanning cofocal microscope |
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| CN103451147A (en) * | 2013-09-17 | 2013-12-18 | 武汉市蔬菜科学研究所 | Lactuca sativa L. pollen in vitro germination culture medium and method for measuring pollen activity |
| CN104531824A (en) * | 2014-12-16 | 2015-04-22 | 北京农学院 | Labeling method of apple pollen tube microfilament |
| CN105486565A (en) * | 2015-12-16 | 2016-04-13 | 青岛农业大学 | Method for loading fluorescent dye into pollen tube |
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| CN102192901A (en) * | 2011-03-10 | 2011-09-21 | 山东理工大学 | Method for imaging pollen surface ornamentation based on laser scanning cofocal microscope |
| CN103109737A (en) * | 2013-03-10 | 2013-05-22 | 通化师范学院 | Pollen liquid for high yield of hazelnut fruit and cultivation and pollination of hybrid hazel variety, and preparation method thereof |
| CN103451147A (en) * | 2013-09-17 | 2013-12-18 | 武汉市蔬菜科学研究所 | Lactuca sativa L. pollen in vitro germination culture medium and method for measuring pollen activity |
| CN104531824B (en) * | 2014-12-16 | 2017-04-19 | 北京农学院 | Labeling method of apple pollen tube microfilament |
| CN104531824A (en) * | 2014-12-16 | 2015-04-22 | 北京农学院 | Labeling method of apple pollen tube microfilament |
| CN105486565B (en) * | 2015-12-16 | 2018-07-03 | 青岛农业大学 | A kind of method that pollen tube loads fluorescent dye |
| CN105486565A (en) * | 2015-12-16 | 2016-04-13 | 青岛农业大学 | Method for loading fluorescent dye into pollen tube |
| CN108424876A (en) * | 2017-02-15 | 2018-08-21 | 中国农业大学 | A kind of method and its kit of labeled plant pollen microfilament microtubules cytoskeleton |
| CN108424876B (en) * | 2017-02-15 | 2020-06-16 | 中国农业大学 | Method for marking plant pollen microfilament microtubule cytoskeleton and kit thereof |
| CN108680418A (en) * | 2018-06-01 | 2018-10-19 | 广东金作农业科技有限公司 | A kind of rapid fluorescence colouring method of crop in cruciferae pollen |
| CN108680418B (en) * | 2018-06-01 | 2021-03-23 | 广东金作农业科技有限公司 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
| CN115220132A (en) * | 2022-07-04 | 2022-10-21 | 山东浪潮智慧医疗科技有限公司 | Method for forecasting pollen concentration in atmosphere |
| CN115615785A (en) * | 2022-09-05 | 2023-01-17 | 南京农业大学 | Method for improving pear pollen tube cell wall methyl esterification pectin dyeing effect by using pre-liquid changing technology |
| CN115615785B (en) * | 2022-09-05 | 2024-06-14 | 南京农业大学 | Method for improving pear pollen tube cell wall methyl esterification pectin dyeing effect by using pre-liquid exchange technology |
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