CN102589942B - Wheat tissue active oxygen fluorescence labeling method - Google Patents
Wheat tissue active oxygen fluorescence labeling method Download PDFInfo
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- CN102589942B CN102589942B CN2012100154797A CN201210015479A CN102589942B CN 102589942 B CN102589942 B CN 102589942B CN 2012100154797 A CN2012100154797 A CN 2012100154797A CN 201210015479 A CN201210015479 A CN 201210015479A CN 102589942 B CN102589942 B CN 102589942B
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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 Adversity-stressed Plant and physiological responses technical field, relate to the fluorescence labeling method that a kind of plant tissue active oxygen location and relative content detect, particularly suffer the labeling method of generation, generation position and the relative content of active oxygen under biological adverse circumstance and abiotic stress in a kind of Growth of Wheat process.
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, i.e. active oxygen such as superoxide anion (OH in plant
-), hydrogen peroxide (H
2O
2), hydroxyl radical free radical (OH), singlet oxygen (
1O
2)Deng bursting; And the scavenger of active oxygen, as biosynthesizing or activity level declines such as superoxide dismutase (SOD), hydrogen peroxidase (CAT) and peroxidase (POD), carotenoid, vitamin E, vitamin Cs, cause Active oxygen generation and clearing mobile equilibrium disorderly, accumulated active oxygen.Accumulated active oxygen is the peroxidation that directly or indirectly starts film fat to the important mechanisms that plant produces injury, cause cell membrane system damage, the degradeds such as cell membrane, Thylakoids structure, mitochondrial membrane, nuclear membrane, MDA (MDA) content increases, 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, will suffer the natural adverse circumstances such as hot evil of winter freeze calamity, spring arid, early summer in the whole history of life, and the 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 Resistance of Wheat To Adversity, stable high yield, is the important measures that the breeding scholar screens good wheat breed.In document in the past, the mark of active oxygen is generally used nitro blue tetrazolium (NBT) mark superoxide anion (in active oxygen a kind of), 3,3'-diaminobenzidine (DAB) mark H
2O
2A kind of in active oxygen).Yet in plant tissue, reactive oxygen species is various, 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 be there is no the detection method of science.
2 ', 7 '-dichloro fluorescin diacetate (DCFH-DA) can enter in cell by cell membrane, by non-specific lipase catalysis in cell, is generated 2 ', 7 '-dihydro dichlorofluorescein (DCFH), and DCFH can not send fluorescence.When having active oxygen to exist in cell, DCFH can be oxidized to 2 ', 7 '-dichlorofluorescein (DCF), excites the green fluorescence that issues out wavelength 530 nm left and right at 488 nm wavelength lasers.DCF formation volume and cellular oxidation product level are proportional.Therefore, the DCF fluorescence intensity reflects the level of aggregation (Schopfer et al., 2001) of reactive oxygen species indirectly.At first DCFH-DA mark active oxygen is applied on zooblast, the application on plant also is only limited to as 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's application DCFH-DA fluorescence labeling, at first the detection of generation, location and the relative content of mark active oxygen in the larger organization materials such as wheat leaf blade, stem stalk, keep to greatest extent cytoactive, the active oxygen that produces in wheat plant carried out accurate mark.
Summary of the invention
, in order to solve above-mentioned technical matters, the invention provides the generation that suffers active oxygen under biological adverse circumstance and abiotic stress in a kind of Growth of Wheat process, the labeling method that produces position and relative content.
The invention provides a kind of active oxygen fluorescence mark method, adopt DCFH-DA to carry out comprehensive mark, location and relative content to the active oxygen in wheat different tissues material and detect.
The 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 by the 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 2.2-2.8%(percent by volume) glutaraldehyde.
B. use sharp cutter, as razor blade or knife blade, it is 0.5 cm tissue block that the materials such as wheat stalk stalk, blade are cut into length;
C. make tissue block with sample mark load buffer rinse step b, at least rinsing twice, then tissue block is placed in step a and fills the vessel of mark load buffer, bleeds after sealing, make the plant tissue piece all sink to the vessel bottom, impel load buffer to enter fast material internal;
D. place 20-30 minute 25 ℃ of lower lucifuges;
E. the material in steps d is placed on microslide, with the laser excitation device, excites, fluorescence microscopy Microscopic observation, ccd image acquisition system are 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 more scientific detection to the comprehensive generation of active oxygen in larger 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) specimen in use mark load buffer of the present invention to contain final concentration be the 2.2-2.8%(percent by volume) glutaraldehyde, can when DCFH-DA is to the active oxygen mark, material be fixed, guarantee the accuracy of active oxygen mark; Simultaneously, with syringe, the vessel that seal rear placement material are bled, make load buffer enter fast material internal, improve fixing and load effect;
(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 pre-treatment and the equipment used of experiment material, easy operating.
Description of drawings
Fig. 1 is that embodiment 1 wheat leaf blade histofluorescence excites rear result at the fluorescence microscopy Microscopic observation;
Fig. 2 is that embodiment 2 wheat leaf blade histofluorescences excite rear result at the fluorescence microscopy Microscopic observation;
Fig. 3 is that embodiment 3 wheat leaf blade histofluorescences excite rear result at the fluorescence microscopy Microscopic observation.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art more understands the present invention, but with this, does not limit the present invention.
Embodiment 1
A kind of plant tissue active oxygen fluorescence labeling method, the application in lower wheat (Jimai 22) stem tissue of Nutrient Stress (nitrogen shortage), concrete operations are as follows:
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 2.5%(percent by volume) glutaraldehyde.
B. the wheat stalk stalk is cut into the tissue block that length is 0.5 cm, and along the longitudinal axis, on average cuts, be divided into two;
C. make tissue block twice with sample mark load buffer rinse step b, then be placed in step a and fill the vessel of mark load buffer, bleed after sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 30 minutes 25 ℃ of lower lucifuges;
E. be placed on the stem tissue piece outside on microslide up, observe and use ccd image acquisition system (Leica DFC 420) to take pictures with fluorescent microscope (Leica DM 2500), 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, at the fluorescence microscopy Microscopic observation, observations as shown in Figure 1, in figure, the arrow indication is that property oxygen produces position and relative content, 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 as follows:
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: preparation 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 2.5%(percent by volume) glutaraldehyde;
B. wheat leaf blade is cut into the tissue block that length is 0.5 cm;
C. use sample mark load buffer rinse step (2) to make tissue block twice, then insert in the vessel of step (1) preparation, bleed after sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 20 minutes 25 ℃ of lower lucifuges;
E. blade is placed on microslide, with fluorescent microscope (Leica DM 2500), observes and use ccd image acquisition system (Leica DFC 420) to take pictures, 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, at the fluorescence microscopy Microscopic observation, observations as shown in Figure 2 in figure the arrow indication be that property oxygen produces position and relative content, scale=200 μ m.
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 as follows:
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 2.2-2.8%(percent by volume) glutaraldehyde.
B. wheat leaf blade is cut into the tissue block that length is 0.5 cm;
C. use sample mark load buffer rinse step (2) to make tissue block twice, then insert in the vessel of step (1) preparation, bleed after sealing, make the plant tissue piece all sink to the vessel bottom;
D. placed 20 minutes 25 ℃ of lower lucifuges;
E. then blade is placed on microslide, observe and use ccd image acquisition system (Leica DFC 420) to take pictures with fluorescent microscope (Leica DM 2500), 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, and at the fluorescence microscopy Microscopic observation, as shown in Figure 3, in figure, the arrow indication is that property oxygen produces position and relative content, scale=200 μ m to observations.
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 be placed in vessel;
Above sample mark load buffer is prepared by the following method:
Prepare 10 mM Tris-HCl or phosphate PBS damping fluid, its pH 7.2-7.5;
Be the mother liquor of 3-5 mM DCFH-DA with DMSO, alcoholic solution compound concentration;
Fluorescence labeling load liquid with damping fluid preparation, 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%, and wherein the concentration of glutaraldehyde is percent by volume;
B. with blade, wheat stalk stalk, blade material being cut into length is 0.4-0.6cm tissue block;
C. use the tissue block in sample mark load buffer rinse step b, then be placed in step a and fill the vessel of sample mark load buffer, bleed after sealing;
D. place 20-30 minute 25 ℃ of lower lucifuges;
E. the material in steps d is placed on microslide, with the laser excitation device, excites, in fluorescence microscopy Microscopic observation ccd image acquisition system, 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 step b is any in methyl alcohol or ethanol.
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| US7378282B2 (en) * | 2003-05-14 | 2008-05-27 | Tetsuo Nagano | Method for measuring hypochlorite ion |
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