CN102147359B - In-site chromogenic and quantitative method for determining free radicals of leaf tissue - Google Patents
In-site chromogenic and quantitative method for determining free radicals of leaf tissue Download PDFInfo
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Abstract
The invention discloses an in-site chromogenic and quantitative method for determination of free radicals of leaf tissue, belonging to the determination field of the free radicals of leaves. The method comprises the following steps: step one: collecting fresh the leaves of a plant and quickly rinsing the leaves; step two: chromogenic reaction of the free radicals of the leaves, (1) superoxide anion free radicals: placing the leaves in a vacuum filtration device, adding solution containing SOD enzyme and MnC12, and vacuumizing till achieving the effect that obvious air bubbles burst out of the surfaces of the leaves; and (2) hydrogen peroxide free radicals: placing the leaves in the vacuum filtration device, adding solution containing ascorbic acid and mm catalase, and vacuumizing till achieving the effect that obvious air bubbles burst out of the surfaces of the leaves; and step three: removing the chlorophyll of the leaves, washing the leaves and scanning and imaging the leaves for software analysis and quantitation. The method can visually display non-uniform distribution condition of O2.- and H2O2 in the leaf tissue, can realize more accurate semi-quantitation, has low cost and is simple to operate and easy to popularize.
Description
Technical field
The present invention relates to a kind of original position colour developing and quantitative method of leaf tissue, say more specifically a kind of original position colour developing and quantitative method of leaf tissue determining free radicals.
Background technology
The photosynthesis of plant tissue, breathing, oxidative phosphorylation, fatty acid--oxidation and many oxidasic metabolic processes all can produce active oxygen radical (being referred to as ROS).More and more many research reports, ROS is the normal part of plant metabolism, can be used as signaling molecule inducing plant body to the response of environment-stress, and the accumulation of excessive ROS can be induced oxidative modification or the degraded of membrane lipid peroxidating, protein molecular, and the oxidative damage of dna molecular etc.Therefore, the variation of the generation of research freedom base, accumulation and metabolism can reflect oxidative stress and the response level of plant under adverse environmental factor, and the early diagnosis that can be environmental contaminants provides early warning signal.Therefore, the generation of Accurate Determining plant tissue ROS and accumulating level have very important theory significance and application prospect.
There is ultra-oxygen anion free radical (O usually in ROS in the plant
2 .-
), hydrogen peroxide (H
2O
2), hydroxyl radical free radical (OH
. ), singlet oxygen (
1O
2) etc. type, wherein produce the earliest be O
2 .-
, that oxidability is the strongest is OH
. These ROS types can form the reaction network of a complexity, mutually transform by acting synergistically.For example, O
2 .-
And H
2O
2Can react by Fenton, the processes such as Haber-Weiss reaction, Winterbourn reaction and photolysis convert OH to
. The assay method of ROS generally includes the methods such as spectrophotometric method, original position development process, spin seizure-electron paramagnetic resonance method (EPR), cells were tested by flow cytometry.Wherein about plant O
2 .-
And H
2O
2Research report at most, also receive publicity most.
The half life period of free radical is very of short duration, and exists concentration very low in plant, directly catches very difficult.Therefore, people usually use indirect method and remove to detect generation O in the plant
2 .-
Speed.Wang Aiguo and Luo Guanghua
[1]The azanol reaction of appliable plant homogenate produces O
2 .-
Intermediate product, use again spectrophotometry and calculate O in the plant
2 .-
Generation speed.Able AJ
[2]Use tetrazole compound and catch the interior O of the artificial tobacco cell of cultivating
2 .-
, use spectrophotometry O
2 .-
The content of intermediate product.These two kinds of methods all are to measure plant to produce O
2 .-
The indirect method of speed is extensively quoted.
The O of plant tissue
2 .-
Also can directly catch with quantitative.The research report is arranged, and the specificity trapping agent is O in detection of biological and the chemical system in conjunction with EPR-spin capturing technology
2 .-
The most direct and the most responsive technology of generation.O
2 .-
Specificity trapping agent phenyl-N-
t-butylnitrone (PBN) can with O
2 .-
Rapid combination also forms stable intermediate product, just can overcome O in conjunction with EPR-spin capturing technology again
2 .-
The limitation that has of short duration and difficult seizure of cycle can be to O
2 .-
Carry out qualitative and quantitative analysis
[3]Yet the applied equipment of this method is very expensive, and the same trapping agent also has its specificity to different plants, therefore, has limited popularization.
H
2O
2The mensuration of content is mainly used spectrophotometric method.1977, Brennan T and Frenkel C
[4]Set up application TiCl
4-Plants with Spectrophotometry is organized H
2O
2Method.Afterwards, the method is widely used in measuring plant tissue H under biology or abiotic stress
2O
2Level change.The somebody uses the outer H of peroxidase-Amplex Red (N-acetyl group-3,7-dihydroxy phenoxazine)-spectrophotometry tobacco cell
2O
2Variation
[5]And use dimethylaminobenzoic acid, 3-methyl-2-benzothiazolinone hydrazone hydrochloride and horseradish peroxidase mixed solution, organize H in conjunction with the spectrophotometry corn seedling
2O
2Content
[6]Although the chemical reaction reagent that is applied in the said method is different, finally all uses spectrophotometric method middle product is measured.
Plant leaf blade is organized O
2 .-
And H
2O
2The research of carrying out original position colour developing also has report, yet has after the colour developing problem of how the free radical reaction product being carried out scientific quantitative analysis, to O in the plant leaf blade
2 .-
And H
2O
2Accurate quantitative analysis become an insoluble difficult problem.
Summary of the invention
, the technical problem to be solved in the present invention
For free radical (O in the plant leaf blade
2 .-
And H
2O
2) distribution and the difficult problem of accurate quantitative analysis, the invention provides a kind of original position colour developing and quantitative method of leaf tissue determining free radicals, and application Image-Pro plus software is to O
2 .-
And H
2O
2Carry out quantitative test, improved the accuracy to the quantitative test of plant leaf blade free radical.
, technical scheme
A kind of original position colour developing and quantitative method of leaf tissue determining free radicals comprise following several step:
Step (1), the blade collection cleans up.
The fresh blade of clip plant take young leaflet tablet as good, is clamped petiole or blade base is clean with the quick rinsing of pure water with tweezers, blots with filter paper.
Step (2), blade free radical chromogenic reaction.
The blank group is set, different according to the survey Kinds of Free Radicals, before the chromogenic reaction control group blade is done following pre-service 1) and ultra-oxygen anion free radical: blade is placed in the vacuum filtration device, add and contain 10 ~ 20 U/mL SOD enzymes and 10 mM MnCl
2The pH value be 6.5 ~ 7.5 phosphate buffer solution (PBS), vacuumize 3 times, each 3 ~ 5 min, effect is advisable with the blade surface obvious bubble of emerging; 2) hydrogen peroxide free radical: blade is placed in the vacuum filtration device, and adding and containing 10 mM ascorbic acid and the catalatic pH value of 1 ~ 5 mM is 6.5 ~ 7.5 PBS solution, vacuumizes each 3 ~ 5min 3 times.
Pretreated control group blade and the blade to be measured that is untreated are placed in the vacuum filtration device, different according to the survey Kinds of Free Radicals, by following processing 1) ultra-oxygen anion free radical: use immediately the pH value contain 0.5 ~ 1.0 mg/mL nitroblue tetrazolium (NBT) and be 6.5 ~ 7.5 PBS solution and vacuumize each 5 ~ 10 min 3 times; 2) hydrogen peroxide is got free radical: use immediately the pH value contain 1 ~ 2 mg/mL diaminobenzidine (DAB) and be 6.5 ~ 7.5 PBS solution and vacuumize each 5 ~ 10 min 3 times.After vacuumizing end, blade is placed the dark place together with solution, leave standstill 5 ~ 12 h, it is fully developed the color.
Step (3) removes leaf chlorophyll, and it is quantitative that cleaning blade and scanning imagery are used for software analysis.
After chromogenic reaction finishes, with tweezers blade is carefully taken out from solution, flush away surface residual solution places 80% the ethanolic solution (v/v) that boils, solution adds zeolite and notes preventing bumping, and notes replenishing in whole process the ethanolic solution because of volatilization loss.Change to some extent with plant kind difference heat time heating time, generally with the complete chlorosis of leaf tissue and become transparent, and stopped heating during visible free radical reaction color spot.O in the blade
2 .-
Be blue spot, the H in the blade with the NBT reaction
2O
2Be the sepia spot with the DAB reaction.Behind the pure water rinsing blade, scanning imagery, application image process software Image-Pro plus software carries out the optical density statistical study to the free radical painted areas.Blade can place 10% glycerine (v/v), and 4 ℃ of lower long preservation are for subsequent use.
, beneficial effect
The invention discloses a kind of original position colour developing and quantitative method of leaf tissue determining free radicals, can show O at plant leaf blade
2 .-
And H
2O
2Distribute with quantitative, compared to existing technology, have following clear and definite effect:
(1) can show intuitively O in the leaf tissue
2 .-
And H
2O
2The non-uniform Distribution situation, be conducive to correct to the equally distributed wrong views of blade free radical.The method is particularly suitable for the research to plant seedlings leaf tissue oxidative stress;
(2) the pigementation district that after the decolouring free radical reaction is produced, its its optical density of appliance computer software analysis can realize comparatively accurately sxemiquantitative, is expected to that plant is oxidated is used widely when coercing degree in research;
(3) cost is low, and is simple to operate, easily promotes.The reagent that the method is related and instrument are not expensive, and operate fairly simplely, and common lab can both be carried out substantially, therefore promote easily.
Description of drawings
Fig. 1 is the broad bean seedling leaves O that is exposed to lead-contaminated soil
2 .-Original position colour developing synoptic diagram, (a) blank group wherein, (b) 500mg/kg Exogenous Pb processed group, (c) the 500mg/kg dosage group that develops the color again of SOD enzyme pre-service;
Fig. 2 is the broad bean seedling leaves O that is exposed to lead-contaminated soil
2 .-Colorimetric detection result;
Fig. 3 is the Rice Seedling Leaves O that is exposed to cadmium pollution soil
2 .-Original position colour developing (A) and densitometric analysis result (B);
Fig. 4 lead-contaminated soil is to broad bean seedling leaves H
2O
2Induce, wherein A represents that Broad Bean Leaves organizes H
2O
2DAB original position colour developing result; B represents that Broad Bean Leaves organizes H
2O
2The colorimetric determination result.
Embodiment
Below further specify of the present invention practicing by example
The broad bean seedling leaves that embodiment 1 lead-contaminated soil exposes is organized O
2 .-
Accumulating level and distribution
Select the broad bean seedling as biological subject, use NBT original position coloration method, studied the broad bean seedling leaves that is exposed in external source interpolation 0,25,125,250,500,1000, the 2000 mg/kg lead-contaminated soils and organized O
2 .-
Accumulating level and distribution situation.The Broad Bean Seeds sowing was collected the blade of different disposal group and control group plant after one month, and each group all arranges free radical colour developing contrast, processes as follows: blade is placed in the vacuum filtration device, add and contain 10 U/mL SOD enzymes and 10 mM MnCl
2The pH value be 6.5 phosphate buffer solution (PBS), vacuumize 3 times, each 3 min, the vacuum tightness size is can make the blade surface micro-bubble of emerging be advisable; Together with free radical colour developing control group blade, placing the pH value that contains 0.5 mg/mL nitroblue tetrazolium (NBT) is 6.5 PBS solution, continues to vacuumize 3 times with each group blade, each about 5 min, afterwards blade is placed the dark place together with solution, leave standstill 5 h, it is fully developed the color; After chromogenic reaction finishes, with tweezers blade is carefully taken out from solution, flush away surface residual solution, place 80% the ethanolic solution (v/v) that boils, solution adds zeolite and notes preventing bumping, boil to Broad Bean Leaves and organize complete chlorosis and become transparent, stopped heating during visible obvious blue color spot.Take out blade, clean with pure water rinsing, the scanner scanning imaging is used in the filter paper suction, and application image process software Image-Pro plus software carries out the optical density statistical study to blue color spot.
Fig. 1 is the broad bean seedling leaves O that is exposed to lead-contaminated soil
2 .-The synoptic diagram of original position colour developing; Fig. 2 is the broad bean seedling leaves O that is exposed to lead-contaminated soil
2 .-Colorimetric detection result.Experimental result shows, the Broad Bean Seeds sowing is after one month, blade O
2 .-
Grey matter degree conspicuousness increase, and carry out again the original position colour developing after the blade applications SOD enzyme pre-service with plumbous processed group, O
2 .-
The grey matter degree obviously reduces.The results show, lead-contaminated soil can induce the broad bean seedling leaves to organize O
2 .-
Accumulation, and O
2 .-
Distribution in blade is (see figure 1) heterogeneous.Experiment adopts colourimetry to O in the blade synchronously
2 .-Content is analyzed, and finds software statistics result and colorimetric determination result basically identical (Fig. 2).
Select rice seedling as biological subject, use NBT original position coloration method, studied and be exposed to the Rice Seedling Leaves of cadmium pollution soil after one month and organize O
2 .-
Accumulating level and distribution situation, Cadmium in Soil content adopts aas determination.Expose after one month, collect the blade of different disposal group and control group paddy rice, each group all arranges free radical colour developing contrast, processes as follows: blade is placed in the vacuum filtration device, add and contain 20 U/mL SOD enzymes and 10 mM MnCl
2The pH value be 7.0 phosphate buffer solution (PBS), vacuumize 3 times, each 5 min, the vacuum tightness size is can make the blade surface micro-bubble of emerging be advisable; Together with free radical colour developing control group blade, placing the pH value that contains 1 mg/mL nitroblue tetrazolium (NBT) is 7.0 PBS solution, continues to vacuumize 3 times with each group blade, each about 10 min, afterwards blade is placed the dark place together with solution, leave standstill 8 h, it is fully developed the color; After chromogenic reaction finishes, with tweezers blade is carefully taken out from solution, flush away surface residual solution places 80% the ethanolic solution (v/v) that boils, to the complete chlorosis of rice leaf tissue and become transparent, stopped heating during visible obvious blue color spot.Take out blade, clean with pure water rinsing, the scanner scanning imaging is used in the filter paper suction, and application image process software Image-Pro plus software carries out the optical density statistical study to blue color spot.
Fig. 3 (A) is the Rice Seedling Leaves O that is exposed to cadmium pollution soil
2 .-The synoptic diagram of original position colour developing; Fig. 3 (B) is the Rice Seedling Leaves O that is exposed to cadmium pollution soil
2 .-Optical density software analysis result.Experimental result shows, exposes after one month, and cadmium pollution soil has significantly been induced paddy rice O
2 .-
Accumulation, and its optical density value presents increase tendency along with the increase of Added Cadmium concentration.
Embodiment 3 lead-contaminated soils induce the broad bean seedling leaves to organize H
2O
2Accumulation and distribution
Select the broad bean seedling as biological subject, use DAB original position coloration method, studied the broad bean seedling leaves that is exposed in external source interpolation 0,25,125,250,500,1000, the 2000 mg/kg lead-contaminated soils and organized H
2O
2Accumulating level and distribution situation.The Broad Bean Seeds sowing is after one month, collect the blade of different disposal group and control group plant, each group all arranges free radical colour developing contrast, process as follows: blade is placed in the vacuum filtration device, adding and containing 10 mM ascorbic acid and the catalatic pH value of 1 mM is 7.5 phosphate buffer solution (PBS), vacuumize 3 times, each 5 min, the vacuum tightness size is can make the blade surface micro-bubble of emerging be advisable; Together with free radical colour developing control group blade, placing the pH value that contains 1 mg/mL diaminobenzidine (DAB) is 7.5 PBS solution, continues to vacuumize 3 times with each group blade, each about 8 min, afterwards blade is placed the dark place together with solution, leave standstill 12 h, it is fully developed the color; After chromogenic reaction finishes, with tweezers blade is carefully taken out from solution, flush away surface residual solution, place 80% the ethanolic solution (v/v) that boils, solution adds zeolite and notes preventing bumping, organize complete chlorosis and become transparent to Broad Bean Leaves, stopped heating during visible obviously sepia pigementation.Take out blade, clean with pure water rinsing, the scanner scanning imaging is used in the filter paper suction, and application image process software Image-Pro plus software carries out the optical density statistical study to blue color spot.Experiment also adopts colourimetry to H in the blade synchronously
2O
2Content detects.
Fig. 4 A is the broad bean seedling leaves H that is exposed to lead-contaminated soil
2O
2The synoptic diagram of original position colour developing; Fig. 4 B is the broad bean seedling leaves H that is exposed to lead-contaminated soil
2O
2Colorimetric detection result.Experimental result shows that the Broad Bean Seeds sowing is after one month, and lead-contaminated soil can induce the broad bean seedling leaves to organize H
2O
2Accumulation, and its distribution is heteropical, the variation of its optical density and spectrophotometry result are basically identical.
Claims (2)
1. the colour developing of the original position of a leaf tissue determining free radicals and quantitative method the steps include:
The first step: the fresh blade of herborization, rinsing is clean fast; Control group is set synchronously, and different according to the survey Kinds of Free Radicals, control group blade pre-treating method is respectively 1) ultra-oxygen anion free radical: blade is placed in the vacuum filtration device, add and contain 10 ~ 20 U/mL SOD enzymes and 10 mM MnCl
2Solution, vacuumize 3 times, each 3 ~ 5 min, effect is with the blade surface obvious bubble of emerging; 2) hydrogen peroxide free radical: blade is placed in the vacuum filtration device, add and to contain 10 mM ascorbic acid and the catalatic solution of 1 ~ 2 mM, vacuumize 3 times, each 3 ~ 5min, effect is with the blade surface obvious bubble of emerging;
Second step: blade free radical chromogenic reaction: pretreated control group blade and the blade to be measured that is untreated are placed in the vacuum filtration device, different according to the survey Kinds of Free Radicals, by following processing 1) ultra-oxygen anion free radical: use 0.5 ~ 1.0 mg/mL nitroblue tetrazolium NBT solution and vacuumize each 5 ~ 10 min 3 times; 2) hydrogen peroxide is got free radical: use immediately 1 ~ 2 mg/mL diaminobenzidine DAB solution and vacuumize each 5 ~ 10 min 3 times; Suction filtration places the dark place with blade together with solution after finishing, and leaves standstill 5 ~ 12 h, and it is fully developed the color;
The 3rd step: remove leaf chlorophyll, it is quantitative that cleaning blade and scanning imagery are used for software analysis, be specially: after chromogenic reaction finishes, with tweezers blade is carefully taken out from solution, flush away surface residual solution, place the 80(v/v that boils) ethanolic solution of %, solution adds zeolite and notes preventing bumping, and notes replenishing in whole process the ethanolic solution because of volatilization loss; With the complete chlorosis of leaf tissue and become transparent, stopped heating during visible free radical reaction color spot; Blade ultra-oxygen anion free radical and NBT reaction are blue spot, and hydrogen peroxide free radical and DAB reaction are the sepia spot; Behind the pure water rinsing blade, scanning imagery, application image process software Image-Pro plus software carries out the optical density statistical study to the free radical painted areas; Blade places the glycerine of 10 (v/v) %, and 4 ℃ of lower long preservation are for subsequent use.
2. the original position of a kind of leaf tissue determining free radicals according to claim 1 develops the color and quantitative method, it is characterized in that NBT that solution that the control group pre-service is used and chromogenic reaction are used and DAB solution all with 10 mM phosphate buffer solution PBS preparation, the pH value remains on 6.5 ~ 7.5.
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