CN115624025B - Polyacrylic acid modified Mn 3 O 4 Application of nano particles in improving high temperature resistance of crops - Google Patents
Polyacrylic acid modified Mn 3 O 4 Application of nano particles in improving high temperature resistance of crops Download PDFInfo
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- CN115624025B CN115624025B CN202211202333.3A CN202211202333A CN115624025B CN 115624025 B CN115624025 B CN 115624025B CN 202211202333 A CN202211202333 A CN 202211202333A CN 115624025 B CN115624025 B CN 115624025B
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- polyacrylic acid
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- 229920002125 Sokalan® Polymers 0.000 title claims abstract description 40
- 239000004584 polyacrylic acid Substances 0.000 title claims abstract description 39
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 25
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 46
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- 230000001965 increasing effect Effects 0.000 claims abstract description 7
- 229920000742 Cotton Polymers 0.000 claims description 71
- 239000000243 solution Substances 0.000 claims description 38
- 239000011572 manganese Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
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- 238000005507 spraying Methods 0.000 description 5
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- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical group [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 4
- 101100241858 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) OAC1 gene Proteins 0.000 description 4
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- 101150036326 PMT2 gene Proteins 0.000 description 3
- 101100043108 Schizosaccharomyces pombe (strain 972 / ATCC 24843) spb1 gene Proteins 0.000 description 3
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical group [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
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- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 3
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- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
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- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- NPOSMYDPUKBGQL-UHFFFAOYSA-N HPF Chemical compound C1=CC(O)=CC=C1OC1=CC=C2C3(C4=CC=CC=C4C(=O)O3)C3=CC=C(O)C=C3OC2=C1 NPOSMYDPUKBGQL-UHFFFAOYSA-N 0.000 description 1
- 206010021033 Hypomenorrhoea Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
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- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- MIVBAHRSNUNMPP-UHFFFAOYSA-N manganese(2+);dinitrate Chemical compound [Mn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MIVBAHRSNUNMPP-UHFFFAOYSA-N 0.000 description 1
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- 230000008635 plant growth Effects 0.000 description 1
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- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
- A01N25/10—Macromolecular compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
Abstract
The invention belongs to the technical field of crop stress resistance, and in particular relates to a polyacrylic acid modified Mn 3 O 4 Use of nanoparticles for increasing the high temperature resistance of crops. Polyacrylic acid modified Mn in the present invention 3 O 4 The nano particles can remarkably reduce the content of active oxygen and iron which are increased by the high temperature stress of the crop leaves so as to maintain the steady state of the active oxygen and the steady state of the iron in the crop leaves, remarkably enhance the resistance of the crop to the heat stress (high temperature resistance) by maintaining the steady state of the active oxygen and the steady state of the iron in the crop, and the polyacrylic acid modified Mn 3 O 4 The nanoparticle has low cost, is green and safe, and is suitable for large-scale application.
Description
Technical Field
The invention belongs to the technical field of crop stress resistance. More particularly, to a polyacrylic acid modified Mn 3 O 4 Use of nanoparticles for increasing the high temperature resistance of crops.
Background
Abiotic stress refers to abiotic environmental conditions that are detrimental to the survival and growth of crops and even lead to their damage, destruction and death, including low temperature, high temperature, drought, salt-water flooding, excessive light, ultraviolet radiation, mineral nutrient deficiency, oxygen deficiency, strong wind, injury, and air, soil or water pollution such as heavy metal, pesticide, ozone and sulfur dioxide pollution, etc. In China, crops are subjected to abiotic stress, especially salt stress and heat stress (high temperature), wherein the heat stress refers to an adversity of the crops living at high temperature, the high temperature can adversely affect physiological and metabolic changes of the crops, serious damage is caused to protein, synthesis of the crops is interfered, main enzymes are inactivated and a membrane structure is damaged, and the damage can seriously limit the growth of plants, and oxidative damage is generated, so that the growth, yield and quality of the crops are affected.
The current conventional way to cope with high temperature stress is treatment with growth regulators, osmoprotectant (Kolupaev YE, akrina GE, mokrausov av. Instruction of heat tolerance in wheat coleoptiles by calcium ions and its relation to oxidative stress. Russian Journal of Plant Physiology,2005, 52:199-204.). Wherein the growth regulator increases the high temperature stress resistance of the crop by modulating enzyme activity to increase photosynthetic rate and maintain stability of cell membranes; osmoprotectant improves the high temperature stress resistance of crops by controlling various cellular processes such as enhancing antioxidant enzyme activity and reducing lipid peroxidation under heat stress to reduce damage to crops by heat stress; however, the method has the defects of poor high temperature stress resistance effect, high cost, extremely easy environmental pollution and the like.
Disclosure of Invention
The invention aims to overcome the defects of poor high temperature stress resistance effect, high cost and extremely easy environmental pollution caused by the existing abiotic stress resistance method, and provides a polyacrylic acid modified Mn 3 O 4 Use of nanoparticles for increasing the high temperature resistance of crops.
The above object of the present invention is achieved by the following technical solutions:
polyacrylic acid modified Mn 3 O 4 Use of nanoparticles for increasing the high temperature resistance of crops.
Preferably, the polyacrylic acid modified Mn 3 O 4 The preparation method of the nano-particles comprises the following steps: dissolving manganese salt in pure water to obtain manganese salt solution, and dissolving polyacrylic acid (PAA) in the pure water to obtain polyacrylic acid solution; mixing manganese salt solution with polyacrylic acid solution to obtain solution A; dropwise adding the solution A into 30-40 wt% ammonia water, stirring for 20-30 hours, performing hydrothermal reaction at 100-130 ℃ for 20-30 hours, centrifuging, taking the supernatant, and purifying to obtain the product.
Preferably, the manganese salt is manganese sulfate, manganese nitrate or manganese chloride.
Preferably, the concentration of the manganese salt solution is 0.8-1.2 mol/L.
Preferably, the concentration of the polyacrylic acid solution is 800-1000 g/L.
Preferably, the volume ratio of the manganese salt solution to the polyacrylic acid solution is 1: (1.5-2.5).
Preferably, the volume ratio of the solution A to the ammonia water is 1 (1.5-2.5).
Preferably, the crop is cotton.
Preferably, the polyacrylic acid modified Mn 3 O 4 The nanoparticles increase the high temperature resistance of crops by maintaining active oxygen homeostasis within the crop.
Preferably, the polyacrylic acid modified Mn 3 O 4 The nanoparticles increase the high temperature resistance of crops by maintaining iron homeostasis in the crop.
More preferably, the iron in the iron steady state is ferrous or ferric.
Preferably, the method of application is: mn modified with polyacrylic acid 3 O 4 The nanometer particles and the surfactant are mixed to prepare dispersion liquid which is evenly sprayed on the leaf surfaces of crops.
Preferably, the polyacrylic acid modified Mn in the dispersion 3 O 4 The concentration of the nano particles is 80-120 mg/L.
More preferably, the polyacrylic acid modified Mn in the dispersion 3 O 4 NanoparticleThe concentration is 90-110 mg/L.
Preferably, the surfactant comprises a polyacrylic acid modified Mn 3 O 4 0.02 to 0.05 percent of nanoparticle dispersion liquid.
More preferably, the surfactant comprises a polyacrylic acid modified Mn 3 O 4 0.03 to 0.04 percent of nanoparticle dispersion liquid.
Preferably, the surfactant is a silicone surfactant.
More preferably, the silicone surfactant is Silwet L-77.
The invention has the following beneficial effects:
polyacrylic acid modified Mn in the present invention 3 O 4 The nano particles can obviously reduce the content of active oxygen and iron which are increased by the high temperature stress of the crop leaves, so as to achieve the effect of maintaining the steady state of the active oxygen and the steady state of the iron in the crop leaves, obviously enhance the resistance of the crop to the heat stress (high temperature resistance) by maintaining the steady state of the active oxygen and the steady state of the iron in the crop, and the polyacrylic acid modified Mn 3 O 4 The nanoparticle has low cost, is green and safe, and is suitable for large-scale application.
Drawings
Fig. 1 is a phenotype diagram of three groups of cotton subjected to high temperature stress for 7 days, a is a growth state diagram, B is a photosynthetic index diagram, C is a weight diagram, wherein a, B represents a significant level, B has a P value of 0.05 relative to a, P represents P <0.05, P <0.01, and P <0.001.
Fig. 2 is a graph showing the result of the study on the position of PMO in cotton leaves, wherein A is a picture of PMO entering mesophyll cell chloroplasts after being sprayed on the leaves for 3 hours, and B is a graph showing the result of co-localization rate of PMO in first and second true leaf chloroplasts of cotton.
FIG. 3 is a dye method for determining H in PMO versus cotton leaf 2 O 2 And O 2· - Is the influence result graph of (A) is DCF dye monitoring H 2 O 2 Is the confocal imaging of (a), B is the DCF fluorescence statistical image, C is the DHE dye monitoring O 2· - D is a DHE fluorescence statistic.
FIG. 4 is a graph showing the effect of dye method on PMO on OH in cotton leaves, A is a confocal imaging of HPF dye monitoring in vivo OH, and B is an HPF fluorescence statistical graph.
FIG. 5 is a kit for detecting H in PMO versus cotton leaf 2 O 2 And. O 2 - Is the effect of PMO on H in cotton leaves 2 O 2 Effect of content on results, B is PMO vs. O in cotton leaves 2 - Effect of content results plot, wherein P is represented by<0.05 represents P<0.01 represents P<0.001。
Fig. 6 is a graph of the effect of PMO on cotton leaf cell activity, a is a confocal imaging of DAPI dye monitoring cotton leaf cell activity, B is a statistical plot of the number of dead cells in graph a, C is a confocal imaging of PI dye monitoring cotton leaf cell activity, D is a statistical plot of the number of dead cells in graph C, where P <0.05 represents P <0.01 and P <0.001.
FIG. 7 shows PMO versus Fe in cotton leaf 2+ Is shown in the influence result graph of (A) and A is Fe 2+ Distribution pattern in mesophyll cells of cotton, B is Fe 2+ Fluorescent statistical plot, wherein:. Represents P<0.01。
FIG. 8 shows PMO versus Fe in cotton leaf 3+ Is shown in the figure, wherein A is Prussian blue staining chart of cotton leaf, B is Prussian blue staining fluorescence statistical chart of A, and P is represented by<0.05。
Fig. 9 is a graph of the effect of PMO on MDA in cotton leaf, where P <0.01 is represented.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 polyacrylic acid modified Mn 3 O 4 Preparation of nanoparticles (PMO)
0.425g MnSO 4 ·H 2 O was dissolved in 2.5mL of pure water to obtain a manganese sulfate solution, and 4.5g of polyacrylic acid (PAA, weight average molecular weight: 1800) was dissolved in 5mL of pure water to obtain a polyacrylic acid solution; mixing manganese sulfate solution with polyacrylic acid solution, and mixing for 15 minutes on a vortex machine at 2500rpm to obtain solution A; dropwise adding the solution A into 15mL of 30wt% ammonia water (sigma), stirring on a magnetic stirrer at 500rpm for 24 hours, then placing into a 50mL polytetrafluoroethylene reaction kettle, carrying out hydrothermal reaction at 120 ℃ for 24 hours, then packaging into a 2mL centrifuge tube, centrifuging at 6000g at normal temperature for 1 hour, collecting the supernatant, dialyzing with a dialysis bag (MW 3500) for 24 hours, and changing water every 8 hours to obtain polyacrylic acid modified Mn 3 O 4 Nanoparticles (PMO).
Example 2PMO increases the growth status of Cotton under high temperature stress
Chlorophyll fluorescence imaging
PMO group: 100mg/L PMO dispersion containing 0.04% Silwet L-77 was used;
MnSO 4 control group (inorganic salt control group): using 13.72mg/L MnSO containing 0.04% Silwet L-77 4 Mixing the solutions;
ctrl control group: using a dispersion containing 0.04% Silwet L-77;
the test selects Xinjiang main cotton variety Xinlunzao 74 (XLZ) and uses Hogland nutrient solution for water culture, the growth temperature is 25 ℃ 22 ℃, the humidity is 60%, and the illumination intensity is 200 mu mol m -2 s -1 PAR, illumination period of 14/10h (day/night), and spraying PMO group and MnSO on leaf surface of cotton seedling when cotton seedling grows to two-leaf period 4 The treatment solutions of the control group and the Ctrl control group are sprayed, the redundant solution on the leaf surfaces is wiped off by a tissue after the spraying is finished, the treated cotton seedlings are placed at a weak light place for adaptation for 3 hours and then are subjected to high temperature treatment (45 ℃/42 ℃ and 14 hours/10 hours), after the high temperature treatment for 7 days, the 1 st real leaf (1) st leaf), leaf 2 nd true leaf (2 nd leaf) to measure various indicators. The cotton leaves after the 3 groups of treatments were measured using a chlorophyll fluorescence imaging system (WALZ, germany). The following parameters were determined: initial fluorescence (F0), maximum fluorescence yield (Fm), maximum photochemical efficiency (Fv/Fm) and non-lightChemical quenching coefficient (NPQ).
The results are shown in FIG. 1: the performance of cotton with PMO coated leaves in the growing state (A in FIG. 1), photosynthetic index (B in FIG. 1) and fresh weight (C in FIG. 1) is better than that of the other two groups of treatments, and the change of Dry weight is not obvious.
EXAMPLE 3 investigation of the location of PMO in cotton leaves
DiI-PMO synthesis
Mixing 9g/L of PMO and 7.2mL of deionized water in a 20mL glass beaker, placing the beaker on a magnetic stirrer and stirring (500 r/min) to obtain a PMO solution, taking 24 mu L of fluorescent dye DiI (1, 1 '-octacosyl-3, 3' -tetramethylindocarbocyanine) (2.5 mg/mL) dissolved in 176 mu L of dimethyl sulfoxide (DMSO) to obtain a DiI dye solution, dropwise adding the DiI dye solution into the PMO solution, stirring at 1000r/min on the magnetic stirrer for 1min at room temperature to obtain a mixed solution (DiI-PMO), transferring the mixed solution (DiI-PMO) into a 15mL (MWCO 10Kd, millipore) ultrafiltration tube, adding deionized water to enable the final volume to be 15mL, centrifuging for 5min at 400 g, repeating the steps 5-7 times to finally obtain the DiI-PMO solution for fluorescent positioning, and storing the DiI-PMO solution in a refrigerator at 4 ℃ for standby.
Treatment of cotton
The grouping situation is as follows:
DiI-PMO group: 100mg/L DiI-PMO dispersion containing 0.04% Silwet L-77 was used;
ctrl control group: using a dispersion containing 0.04% Silwet L-77;
the test selects Xinjiang main cotton variety Xinlunzao 74 (XLZ) and uses Hogland nutrient solution for water culture, the growth temperature is 25 ℃ 22 ℃, the humidity is 60%, and the illumination intensity is 200 mu mol m -2 s -1 PAR, light period of 14/10h (day/night), spraying treatment solution of DiI-PMO group and Ctrl control group on leaf surface of cotton seedling when cotton seedling grows to two leaf stage, wiping off excessive solution on leaf surface with paper towel after spraying, placing the treated cotton seedling in dim light place for 3h, applying high temperature treatment (45 deg.C/42 deg.C, 14h/10 h), treating at high temperature for 7 days, collecting 1 st true leaf (1 st leaf), leaf 2 nd true leaf (2 nd leaf) determination of DiI-PMO in cotton trueCo-localization rate with chloroplasts (chlorophplasts) in mesophyll cells in leaves (Colocalization rate).
The results are shown in FIG. 2: the fluorescent image (Overlay) of DiI-PMO and chloroplast after coverage shows that after 3 hours of leaf surface spraying, the DiI-PMO enters mesophyll cells (A in figure 2), compared with a Ctrl group, the co-localization rate of DiI-PMO in the first true leaves of cotton and chloroplasts in mesophyll cells can reach 20%, and the co-localization rate of the second true leaves is about 26% (B in figure 2), so that the PMO can enter chloroplasts of the first true leaves and the second true leaves of cotton.
EXAMPLE 4 effect of PMO on the active oxygen content in cotton leaves
PMO group: 100mg/L PMO dispersion containing 0.04% Silwet L-77 was used;
ctrl control group: using a dispersion containing 0.04% Silwet L-77;
fluorescent dye quantitative method detection:
active oxygen in cotton seedling leaves was studied using a laser confocal microscope. Use of Dihydroethidium (DHE) and 2',7' -dichlorofluorescein Diacetate (DCF) and hydroxyphenyl fluorescein (HPF) as active oxygen (H) 2 O 2 、·O 2 - Fluorescent dye of OH). The cotton leaves obtained by the above two treatments were subjected to high temperature treatment (punching on the true leaves using a punch with a diameter of 5 mm) according to the method of example 1, and then immersed in 25 mu M H 2 DCFDA or 10 μm DHE or 10 μm HPF dye (diluted with 10mM TES, ph=7.5) was incubated for 30min in dark conditions. After the incubation was completed, the slides were washed three times with TES buffer and loaded with (a drop of Perfluoronaphthylamine (PFD) was previously added dropwise to the slide to enhance the fluorescence imaging effect), covered with coverslips, and ensured that there were no air bubbles. The confocal laser microscope was set as follows: a 40-fold objective lens, 488nm excitation light; PMT1:500nm to 600nm (DHE, DCF, HPF); PMT2:700nm to 800nm (chloroplasts); DCF and DHE and HPF fluorescence intensities were calculated using Image J software for 4-6 replicates.
The results are shown in FIG. 3: DCF and DHE dyes monitor H in the blade, respectively 2 O 2 And. O 2 - PMO treated cotton first leaf and second leafThe control group of che She Junbi Ctrl had less H 2 O 2 (A and B in FIG. 3), in the first true leaf of PMO-treated cotton 2 - The content of (2) is obviously reduced compared with that of Ctrl control group, and the first part is in true leaf 2 - The content of (C and D) in the cotton has no obvious change (FIG. 3), and the PMO has the function of eliminating H in the first true leaf and the second true leaf of the cotton 2 O 2 And O in the first leaf 2 - Is effective in (1).
As shown in fig. 4: the Hydroxyl Phenyl Fluorescein (HPF) is taken as a fluorescent dye of active Oxygen (OH), cotton leaves treated by PMO have lower OH content than that of a control group, and the second true She Duibi first true leaves of the Ctrl control group show higher OH content, so that the PMO has the effect of removing the cotton first true leaves and the second true She Zhong OH, and the removing effect of the second true leaves is better.
The kit detects the content of active oxygen in cotton leaves:
hydrogen peroxide (H) 2 O 2 ) The content was measured using a "hydrogen peroxide content measuring kit" (A064-1-1, nanjing built biosystems Co., ltd.) and the procedure was performed according to the specification. Accurately weighing 0.1g of two groups of treated cotton leaf samples, adding 1mL of physiological saline, grinding for 120sec (65 Hz) by using a grinder, centrifuging for 20min at 4 ℃, and taking the supernatant for later use. Adding samples according to the instruction sample adding flow, uniformly mixing, and measuring the absorbance value at the wavelength of 405nm and the optical path of 1 cm.
Superoxide anion (.o) 2 - ) The content was measured using a "superoxide anion content measuring kit" (Beijing Soy Bao technology Co., ltd.) according to the specification. Accurately weighing 0.1g of two groups of treated cotton leaf samples, and adding 1 mL.O 2 - The Lysis Buffer was milled for 180 seconds (65 Hz) using a milling machine, 10000g was centrifuged at 4℃for 10 minutes, and the supernatant was taken for use. And (3) adding samples according to a sample adding flow of the specification, zeroing by using a blank, and measuring absorbance values at 530nm of the samples by using a spectrophotometer.
The results are shown in FIG. 5: PMO treated cotton leaves had less H than Ctrl control 2 O 2 Content (in FIG. 5)A)、·O 2 - The content also decreased significantly in the first leaf (B in fig. 5), demonstrating that PMO treatment helped to remove H from both the first and second leaves of cotton 2 O 2 And O in the first leaf 2 - 。
The high-temperature stress can cause more reactive oxygen species to generate more reactive oxygen species to cause oxidative damage, and the result shows that the PMO can obviously reduce the ROS content in cotton leaves, and is consistent with the result of fluorescent dye detection, so that the PMO can maintain the ROS steady state in the leaves, and the high-temperature stress resistance of the leaves is improved.
Example 5 effect of PMO on cellular Activity in cotton leaf
DAPI and PI dyes can bind to DNA in the nucleus to fluoresce and after cell activity is reduced, cell membrane permeability changes to allow the dye to readily enter the nucleus, thus reducing cell activity bound by DAPI and PI.
50. Mu.g/L Propidium Iodide (PI)/4', 6-diamidino-2-phenylindole (DAPI) dye was prepared using 0.05M sodium phosphate buffer (PBS, pH=7.4). After 7 days of high temperature stress, two sets of cotton leaves in example 4 were punched out of the true leaves using a punch (diameter 5 mm) to remove leaf discs as in example 1. Leaf discs were immersed in PI dye and incubated for 1h at 4 ℃. Leaf discs were immersed in DAPI dye and incubated for 5min at ambient temperature. After completion, the leaf disk was rinsed three times with deionized water and loaded into the slide (a drop of Perfluorodecalin (PFD) was previously added dropwise to the slide to prevent fluorescence quenching), the coverslip was covered, and no air bubbles were ensured between the slides. The parameters of the confocal laser microscope are set as follows: 40 times of objective lens, PI 514nm excitation light, intensity 30%; PMT1:610nm-630nm (PI fluorescence); PMT2:700nm-800nm (chloroplast fluorescence); DAPI, 405nm excitation light, intensity 30%; PMT1:435nm-500nm (DAPI fluorescence); PMT2: repeating for 3-6 times at 700-800 nm (chloroplast fluorescence), and counting the number of dead cells in a single visual field.
As shown in fig. 6: after dyeing with DAPI (a and B in fig. 6) and PI (C and D in fig. 6) dyes, PMO-treated cotton leaves had stronger cell activity than Ctrl control and the first true leaves were more active than the second true leaves, demonstrating that after PMO treatment, cotton leaf cells grew better under high temperature conditions, PMO could significantly improve cotton high temperature resistance.
EXAMPLE 6 Effect of PMO on ferrous ions in cotton leaf
FerroOrange (Dojindo Japan) is used as iron ion (Fe 2+ ) Is a fluorescent dye of (a); FM 4-64 (Thermo Fisher Scientifc) as a plasma membrane dye can help differentiate between vacuoles and cytoplasmic locations. The cotton leaves treated in example 4 were subjected to the high temperature treatment in the method of example 1, and then were punched out of the cotton true leaves with a punch (diameter: 5 mm) to remove the leaf discs. Ferroorange was diluted to 1. Mu.M with TES buffer (10 mM, pH=7.5) and FM 4-64 probe was diluted to 20. Mu.M. Soaking leaf discs in mixed dye solution of Ferroorange and FM 4-64 for Fe 2+ Staining, incubation for 30min in dark conditions. After incubation, the leaf disk was rinsed 3-5 times with TES buffer and loaded into the slide (one drop of Perfluorodecalin (PFD) was previously added dropwise to the slide to prevent fluorescence quenching), covered with a cover slip, and ensured that there were no air bubbles. The confocal laser microscope was set as follows: a 40-fold objective lens, 514nm excitation light; PMT1:550nm to 585nm (Fe) 2+ Fluorescence); 610nm to 660nm (plasma membrane fluorescence); fe was calculated using Image J software with 4-6 replicates 2+ Fluorescence intensity.
The results are shown in FIG. 7: ferroorange as Fe 2+ The fluorescent probe (green fluorescent part in the figure) monitors its distribution in the leaf, magnifies the field of view 6 times (zoom=6), and the result shows Fe 2+ Mainly distributed in the cytoplasm (red arrow), while Fe in PMO-treated mesophyll cell cytoplasm 2+ Significantly reduced, while Fe in the vacuoles (yellow arrow) 2+ There was no increase (fig. 7 a), and fig. 7B also shows a significant decrease in ferrous ion content in the true leaves of PMO group treated cotton compared to Ctrl control. This suggests that the PMO treatment group was not prepared by mixing Fe in the cytoplasm 2+ Transport into vacuoles but by reducing cell-to-Fe 2+ The way in which this is taken up is to maintain intracellular iron homeostasis.
EXAMPLE 7 effect of PMO on ferric ion in cotton leaf
The reaction of high-valence iron in plant tissues with potassium ferrocyanide can produce insoluble blue precipitates (Prussian blue). Therefore, prussian blue staining can detect Fe in cotton leaves 3+ Distribution.
Preparing a solution:
4% hydrochloric acid (solution a): dissolving 2ml of concentrated hydrochloric acid in 50ml of deionized water;
4% potassium ferrocyanide (Coolaber) (solution B): 2g of potassium ferrocyanide was dissolved in 50ml of deionized water.
Dyeing liquid: equal amounts of solution A and solution B were mixed and allowed to stand for 15 minutes before being used for staining.
Taking two groups of cotton leaves treated in example 4, carrying out high-temperature treatment according to the method in example 1, immersing the whole cotton leaves in the dyeing liquid, vacuumizing for 10min to help the dyeing liquid to immerse the cotton leaves, washing the cotton leaves three times by deionized water, and using absolute ethyl alcohol: glycerol = 9:1 decolorization solution was boiled to leaf decolorization via absolute ethanol: glycerol = 6:4 mix for leaf softening and observation using a camera.
The results are shown in FIG. 8: after 7 days of high temperature stress, compared with a Ctrl control group, the Prussian blue color development of the first true leaf of the PMO group has no obvious change, the Prussian blue color development of the second true leaf is obviously reduced (A in FIG. 8), and the PMO treatment group obviously reduces Fe in the second true leaf 3+ Content (B in fig. 8), indicating that cotton after PMO use generally reduced ferric ion content in the leaves, thereby improving the high temperature resistance of the cotton leaves.
H in the blade after the blade is subjected to high temperature stress 2 O 2 With Fe 2+ The Fenton reaction generates hydroxyl free radicals to cause lipid peroxidation, and the iron steady state can be effectively maintained by reducing the concentration of Fe ions, so that the contents of ferrous ions and ferric ions are obviously reduced after PMO treatment, and the iron steady state in cotton leaves can be effectively maintained.
Example 8 effect of PMO on MDA in cotton leaf
Plant organs are damaged under adverse conditions, membrane lipid peroxidation often occurs, and Malondialdehyde (MDA) is a final decomposition product of membrane lipid peroxidation, and the content of Malondialdehyde (MDA) can be used as one of indexes for examining the severity of stress on cells.
0.15g of the two cotton leaf samples of example 4 were weighed, treated at high temperature in accordance with the method of example 1, 1.5mL of 5% trichloroacetic acid (TCA) was added, and the mixture was ground for 180 seconds (65 Hz) using a grinder, 12000g and centrifuged at 4℃for 20 minutes, and the supernatant was collected for use. 1mL of the supernatant was taken, 1mL of 0.67% thiobarbituric acid (TBA) was added, and after mixing, the mixture was subjected to a boiling water bath for 30 minutes, and after cooling, the mixture was centrifuged. The absorbance of the supernatant at 450nm, 532nm and 600nm was measured using deionized water instead of the mixture of the extracts as a control, and repeated 4 times, and the Malondialdehyde (MDA) content was calculated according to the following formula:
MDA content (mmol/g FW) = [6.452 × (A) 532 -A 600 )-0.559×A 450 ]×[2×V×W]
W is cotton leaf mass (g); v is the volume of the supernatant (mL) taken during the measurement
The results are shown in FIG. 9: the MDA content of PMO treated group was significantly less than Ctrl control group, demonstrating that the degree of high temperature stress to cotton was significantly reduced after PMO treatment.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. Polyacrylic acid modified Mn 3 O 4 Use of nanoparticles for increasing the high temperature resistance of crops;
the crop is cotton;
mn modified by polyacrylic acid 3 O 4 The nanoparticles increase the height Wen Kangxing of the crop by maintaining active oxygen homeostasis within the crop;
mn modified by polyacrylic acid 3 O 4 The nanoparticles increase the high temperature resistance of crops by maintaining iron homeostasis in the crop.
2. The application according to claim 1Characterized by the fact that said polyacrylic acid modified Mn 3 O 4 The preparation method of the nano-particles comprises the following steps: dissolving manganese salt in pure water to obtain manganese salt solution, and dissolving polyacrylic acid in pure water to obtain polyacrylic acid solution; mixing manganese salt solution with polyacrylic acid solution to obtain solution A; dropwise adding the solution A into 30-40 wt% ammonia water, stirring for 20-30 hours, performing hydrothermal reaction at 100-130 ℃ for 20-30 hours, centrifuging, taking the supernatant, and purifying to obtain the product.
3. The application according to claim 1, characterized in that the method of application is: mn modified with polyacrylic acid 3 O 4 The nanometer particles and the surfactant are mixed to prepare dispersion liquid, and the dispersion liquid is uniformly sprayed on the leaf surfaces of crops.
4. Use according to claim 3, characterized in that the polyacrylic acid modified Mn in the dispersion 3 O 4 The concentration of the nano particles is 80-120 mg/L.
5. The use according to claim 4, wherein the dispersion comprises polyacrylic acid modified Mn 3 O 4 The concentration of the nano particles is 90-110 mg/L.
6. Use according to claim 3, characterized in that the surfactant comprises a polyacrylic acid modified Mn 3 O 4 0.02 to 0.05 percent of nanoparticle dispersion liquid.
7. Use according to claim 3, wherein the surfactant is a silicone surfactant.
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| CN113735175A (en) * | 2021-08-17 | 2021-12-03 | 华中农业大学 | Nano-mimetic enzyme, preparation method and seed soaking agent containing nano-mimetic enzyme |
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