CN102589942A - Wheat tissue active oxygen fluorescence labeling method - Google Patents
Wheat tissue active oxygen fluorescence labeling method Download PDFInfo
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- CN102589942A CN102589942A CN2012100154797A CN201210015479A CN102589942A CN 102589942 A CN102589942 A CN 102589942A CN 2012100154797 A CN2012100154797 A CN 2012100154797A CN 201210015479 A CN201210015479 A CN 201210015479A CN 102589942 A CN102589942 A CN 102589942A
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Abstract
The invention belongs to the technical field of plant stress and physiologic response, and particularly relates to the labeling method for active oxygen generation, generation positions and relative content under biotic stress and abiotic stress during the growth and development process of wheat. The labeling method comprises the following steps of: preparing a label load buffer solution, preparing a material tissue block, rinsing the tissue block with the load buffer solution, placing the tissue block in a dark place at 25 DEG C, exciting the tissue block by using a laser excitation device, observing the tissue block and taking a photo. Compared with the traditional labeling method of DAB (Dimethylaminoazobenzene), NBT (Nitroblue Tetrazolium) and other active oxygen species, the labeling method saves time and avoids the distortion of active oxygen content and positions of a sample during long-time labeling load period. When the active oxygen is labeled by DCFH-DA (Dichlorofluorescein Diacetate), the material is fixed, and the accuracy of active oxygen labeling is ensured. The used device has simple requirements and is easy to operate.
Description
Technical field
The invention belongs to plant environment stress and physiological responses technical field; Relate to the fluorescence labeling method that a kind of plant tissue active oxygen location and relative content detect, suffer biological adverse circumstance and abiotic stress to coerce down the labeling method of oxygen production, generation position and relative content in particularly a kind of wheat growth growth course.
Background technology
Plant suffers various adverse circumstances and inevitable aging course constantly in growth and development process, thereby causes vegetable cell active oxygen (reactive oxygen species) metabolism disorder, and promptly the interior active oxygen of plant is like ultra oxygen thing negative ion (OH
-), hydrogen peroxide (H
2O
2), hydroxyl radical free radical (OH), singlet oxygen (
1O
2)Deng bursting; And the scavenger of active oxygen; Like biosynthesizing or activity level declines such as superoxide dismutase (SOD), hydrogen peroxidase (CAT) and peroxidase (POD), carotenoid, vitamin E, vitamin Cs; It is disorderly to cause active oxygen to produce with removing mobile equilibrium, the active oxygen accumulation.The active oxygen accumulation is the peroxidation that directly or indirectly starts film fat to the important mechanisms that plant produces injury; Cause cell membrane system damage, degradeds such as cell membrane, thylakoid lamellar structure, mitochondrial membrane, nuclear membrane; MDA (MDA) content increases, and chlorophyll degradation and photosynthesis enzymatic activity descend, the plant photosynthesis decrease in efficiency; Plant can not be kept normal g and D, organizes too early aging death.
Wheat is two annual crops, in the whole history of life, will suffer natural adverse circumstances such as hot evil of freeze injury in winter, spring arid, early summer, and biological adverse circumstance such as disease and pest, and spending the post-grouting process simultaneously is again an inevitable aging course.Therefore, detecting wheat tissue active oxygen generation position and content is a key index weighing wheat resistance, stable high yield, is the important measures that the breeding scholar screens good wheat breed.In the document in the past, the mark of active oxygen is generally used the ultra oxygen thing of nitro blue tetrazolium (NBT) mark negative ion (in the active oxygen a kind of), 3,3'-diaminobenzidine (DAB) mark H
2O
2A kind of in the active oxygen).Yet reactive oxygen species is various in the plant tissue, and its generation also differs greatly, and therefore, is difficult to resistance or senescence process with the variation explanation plant of 1-2 kind active o content.The more important thing is at present the comprehensive generation of active oxygen in the material of wheat tissue, particularly larger volume also there is not the detection method of science.
2 ', 7 '-dichloro fluorescin diacetate (DCFH-DA) can get in the cell through cell membrane, is generated 2 ', 7 '-dihydro dichlorofluorescein (DCFH) by non-specific lipase catalysis in the cell, and DCFH can not send fluorescence.When having active oxygen to exist in the cell, DCFH can be oxidized to 2 ', 7 '-dichlorofluorescein (DCF), excites the green fluorescence that issues out about wavelength 530 nm at 488 nm wavelength lasers.DCF formation amount and cellular oxidation product level are proportional.Therefore, the DCF fluorescence intensity reflects the level of aggregation (Schopfer et al., 2001) of intracellular reactive oxygen indirectly.DCFH-DA mark active oxygen is at first used on zooblast, and the application on plant also is only limited to like individual cells or the very little materials of volume such as suspended culture cell, mushroom, bioplasts, as the blade table cortical cell of peeling off.
The present invention uses the DCFH-DA fluorescence labeling; The at first detection of mark oxygen production, location and relative content in big organization material such as wheat leaf blade, stem stalk keeps cytoactive to greatest extent, the active oxygen that produces in the wheat plant is carried out accurate mark.
Summary of the invention
In order to solve above-mentioned technical matters, the invention provides and suffer biological adverse circumstance and abiotic stress to coerce down oxygen production in a kind of wheat growth growth course, produce the labeling method of position and relative content.
The invention provides a kind of active oxygen fluorescence mark method, adopt DCFH-DA that the active oxygen in the wheat different tissues material is carried out comprehensive mark, location and relative content and detect.
This method comprises following step:
A. prepare wheat tissue sample mark load buffer, draw sample mark load buffer 5 ml and insert in the vessel of volume 6-10ml;
Above mark load buffer is prepared through following method:
Prepare 10 mM Tris-HCl or phosphate (PBS) damping fluid (pH 7.2-7.5); With DMSO, alcoholic solution preparation DCFH-DA mother liquor (can be frozen under 20 ℃ of-80 ℃ of conditions), concentration is 3-5 mM; With damping fluid preparation fluorescence labeling load liquid, contain the DCFH-DA that ultimate density is 30-50 μ M, KCl and the final concentration that final concentration is 50 mM is the glutaraldehyde of 2.2-2.8% (percent by volume).
B. use sharp cutter, like razor blade or knife blade, it is 0.5 cm piece of tissue that material cut such as wheat stalk stalk, blade are become length;
C. make piece of tissue with sample mark load buffer rinse step b; At least rinsing twice, places step a to fill the vessel of mark load buffer piece of tissue then, bleeds after the sealing; Make the plant tissue piece all sink to the vessel bottom, impel load buffer to get into material internal fast;
D. placed 20-30 minute 25 ℃ of following lucifuges;
E. the material in the steps d is placed on the microslide, excites with the laser excitation device, fluorescent microscope is observed down, the ccd image acquisition system is taken pictures, and the microscope condition of work is: excitation wavelength is 460-500 nm, and dispersing wavelength is 510-560 nm.
Above alcoholic solution is ethanol or methyl alcohol.
The present invention is applicable to that 0.5 cm is long, thickness is less than the various plant tissue materials of 1 mm, and particularly the present invention successfully uses on wheat lines.
Beneficial effect of the present invention is:
(1) traditional DAB, NBT etc. need 2-12 hour to the mark of reactive oxygen species, and the time is longer, and the present invention only needs 20-30 minute to the mark of active oxygen, have avoided the distortion of active o content and position during the long-time mark load of sample;
(2) adopt method of the present invention to carry out the more detection of science to the comprehensive generation of active oxygen in the bigger organization material; For example can detect less than the cane tissue of 1mm such as the active oxygen in the wheat cane plant leaf blade tissue, the wall thickness of thickness less than 1mm;
(3) to contain final concentration be 2.2-2.8% (percent by volume) glutaraldehyde to specimen in use mark load buffer of the present invention, can when DCFH-DA is to the active oxygen mark, material be fixed, and guarantees the accuracy of active oxygen mark; Simultaneously, the vessel that seal back placement material are bled, make load buffer get into material internal fast, improve fixing and load effect with syringe;
(4) the present invention can detect the comprehensive generation of active oxygen in the organization material of larger volume, more can precisely reflect resistance or the senescence process of plant;
(5) the present invention requires simply easy operating to the pre-treatment and the used equipment of experiment material.
Description of drawings
The result that Fig. 1 excites the back under fluorescent microscope, to observe for embodiment 1 wheat leaf blade histofluorescence;
The result that Fig. 2 excites the back under fluorescent microscope, to observe for embodiment 2 wheat leaf blade histofluorescences;
The result that Fig. 3 excites the back under fluorescent microscope, to observe for embodiment 3 wheat leaf blade histofluorescences.
Embodiment
Come the present invention is done explanation further below in conjunction with accompanying drawing and embodiment,, but do not limit the present invention with this so that those skilled in the art more understands the present invention.
Embodiment 1
A kind of plant tissue active oxygen fluorescence labeling method is coerced the application in wheat (Jimai 22) the stem stalk tissue under (nitrogen shortage) in nutrition, concrete operations are following:
A. prepare wheat tissue sample mark load buffer, draw sample mark load buffer 5 ml and insert in the vessel that volume is 10 ml;
Prepare 10 mM Tris-HCl damping fluids (pH 7.2); With DMSO preparation DCFH-DA mother liquor, concentration is 5 mM; With damping fluid preparation fluorescence labeling load liquid, contain the DCFH-DA that ultimate density is 50 μ M, KCl and the final concentration that final concentration is 50 mM is the glutaraldehyde of 2.5% (percent by volume).
B. the wheat stalk stalk is cut into the piece of tissue that length is 0.5 cm, and on average cuts, be divided into two along the longitudinal axis;
C. make piece of tissue twice with sample mark load buffer rinse step b, place step a to fill the vessel of mark load buffer then, bleed after the sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 30 minutes 25 ℃ of following lucifuges;
E. be placed on the stem stalk piece of tissue outside on the microslide up; Take pictures with fluorescent microscope (Leica DM 2500) observation and with ccd image acquisition system (Leica DFC 420); The microscope condition of work is: excitation wavelength is 495 nm, and dispersing wavelength is 518 nm.
The wheat stalk stalk organization material of finally making is excited with the fluorescence excitation device; Under fluorescent microscope, observe; Observations is shown in accompanying drawing 1, and the arrow indication produces position and relative content for property oxygen among the figure, and high brightness position explanation active o content is higher; Low-light level position active o content is relatively low, scale=200 μ m).
Embodiment 2
A kind of plant tissue active oxygen fluorescence labeling method, the application in grouting wheat declining period (Jimai 22) leaf tissue, concrete operations are following:
A. prepare wheat tissue sample mark load buffer, draw sample mark load buffer 5 ml and insert in the vessel that volume is 10 ml;
The compound method of above mark load buffer is: prepare 10 mM PBS damping fluids (pH 7.4); With ethanol preparation DCFH-DA mother liquor, concentration is 3 mM; With damping fluid preparation fluorescence labeling load liquid, contain the DCFH-DA that ultimate density is 30 μ M, KCl and the final concentration that final concentration is 50 mM is the glutaraldehyde of 2.5% (percent by volume);
B. wheat leaf blade is cut into the piece of tissue that length is 0.5 cm;
C. use sample mark load buffer rinse step (2) to make piece of tissue twice, insert then in the vessel of step (1) preparation, bleed after the sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 20 minutes 25 ℃ of following lucifuges;
E. blade is placed on the microslide, takes pictures with fluorescent microscope (Leica DM 2500) observation and with ccd image acquisition system (Leica DFC 420), the microscope condition of work is: excitation wavelength is 495 nm, and dispersing wavelength is 518 nm.
The wheat leaf blade organization material of finally making is excited with the fluorescence excitation device, under fluorescent microscope, observe, the arrow indication produces position and relative content, scale=200 μ m for property oxygen in 2 diagrammatic sketch of observations such as accompanying drawing.
Embodiment 3
A kind of plant tissue active oxygen fluorescence labeling method, the application of wheat under drought stress (Jimai 22) leaf tissue, concrete operations are following:
A. prepare wheat tissue sample mark load buffer, draw sample mark load buffer 5 ml and insert in the vessel that volume is 10 ml;
Prepare 10 mM PBS damping fluids (pH 7.2); With methyl alcohol preparation DCFH-DA mother liquor, concentration is 3 mM; With damping fluid preparation fluorescence labeling load liquid, contain the DCFH-DA that ultimate density is 30 μ M, KCl and the final concentration that final concentration is 50 mM is the glutaraldehyde of 2.2-2.8% (percent by volume).
B. wheat leaf blade is cut into the piece of tissue that length is 0.5 cm;
C. use sample mark load buffer rinse step (2) to make piece of tissue twice, insert then in the vessel of step (1) preparation, bleed after the sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 20 minutes 25 ℃ of following lucifuges;
E. then blade is placed on the microslide; Take pictures with fluorescent microscope (Leica DM 2500) observation and with ccd image acquisition system (Leica DFC 420); The microscope condition of work is: excitation wavelength is 495 nm, and dispersing wavelength is 518 nm.
The wheat leaf blade organization material of finally making is excited with the fluorescence excitation device, under fluorescent microscope, observe, observations is shown in accompanying drawing 3, and the arrow indication produces position and relative content, scale=200 μ m for property oxygen among the figure.
Claims (2)
1. wheat tissue active oxygen fluorescence labeling method comprises following step:
A. prepare wheat tissue sample mark load buffer, draw wheat tissue sample mark load buffer 5 ml and place vessel;
Above sample mark load buffer is prepared through following method:
Prepare 10 mM Tris-HCl or phosphate PBS damping fluid, its pH 7.2-7.5;
Use DMSO, alcoholic solution compound concentration mother liquor as 3-5 mM DCFH-DA;
Fluorescence labeling load liquid with damping fluid preparation contains the DCFH-DA that ultimate density is 30-50 μ M, KCl and the final concentration that final concentration is 50 mM is the glutaraldehyde of 2.2-2.8%, and wherein the concentration of glutaraldehyde is percent by volume;
B. using blade that wheat stalk stalk, blade material are cut into length is 0.4-0.6cm piece of tissue;
C. use the piece of tissue among the sample mark load buffer rinse step b, place step a to fill the vessel of sample mark load buffer then, bleed after the sealing;
D. placed 20-30 minute 25 ℃ of following lucifuges;
E. the material in the steps d is placed on the microslide, excites with the laser excitation device, under fluorescent microscope, observe the ccd image acquisition system and take pictures, the microscope condition of work is: excitation wavelength is 460-500 nm, and dispersing wavelength is 510-560 nm.
2. wheat tissue active oxygen fluorescence labeling method as claimed in claim 1 is characterized in that the alcoholic solution described in the step b is any in methyl alcohol or the ethanol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149189A (en) * | 2013-03-11 | 2013-06-12 | 山东省农业科学院作物研究所 | Method for rapidly detecting wheat tissue callose |
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| CN1737539A (en) * | 2005-08-04 | 2006-02-22 | 浙江大学 | Rapid trace amount method for detecting tissue active oxygen |
| CN1818620A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improved method for determinating intracellular active oxygen |
| CN1818621A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improvement for intracellular ROS inspecting sensitivity |
| WO2009036120A1 (en) * | 2007-09-11 | 2009-03-19 | Cornell Research Foundation, Inc. | Cellular antioxidant activity (caa) assay |
| CN101671722A (en) * | 2009-09-28 | 2010-03-17 | 华东理工大学 | Evaluation method of cell biology safety of silicon dioxide nanoparticle |
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2012
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Patent Citations (9)
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| WO2002061422A1 (en) * | 2001-01-29 | 2002-08-08 | Young-Mee Park | Method for measuring antioxidative activity of cell |
| US20040229371A1 (en) * | 2003-05-14 | 2004-11-18 | Tetsuo Nagano | Method for measuring hypochlorite ion |
| CN1737539A (en) * | 2005-08-04 | 2006-02-22 | 浙江大学 | Rapid trace amount method for detecting tissue active oxygen |
| CN1818620A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improved method for determinating intracellular active oxygen |
| CN1818621A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improvement for intracellular ROS inspecting sensitivity |
| WO2009036120A1 (en) * | 2007-09-11 | 2009-03-19 | Cornell Research Foundation, Inc. | Cellular antioxidant activity (caa) assay |
| CN101855538A (en) * | 2007-11-13 | 2010-10-06 | 蒂埃里·帕特里斯 | Method for measuring the ability of a sample to withstand reactive oxygen species (ROS) |
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Non-Patent Citations (6)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103149189A (en) * | 2013-03-11 | 2013-06-12 | 山东省农业科学院作物研究所 | Method for rapidly detecting wheat tissue callose |
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