CN113655222A - Method for researching micro distribution of hemicellulose in plant cell wall - Google Patents
Method for researching micro distribution of hemicellulose in plant cell wall Download PDFInfo
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- CN113655222A CN113655222A CN202110929455.1A CN202110929455A CN113655222A CN 113655222 A CN113655222 A CN 113655222A CN 202110929455 A CN202110929455 A CN 202110929455A CN 113655222 A CN113655222 A CN 113655222A
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- 229920002488 Hemicellulose Polymers 0.000 title claims abstract description 74
- 210000002421 cell wall Anatomy 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000005406 washing Methods 0.000 claims abstract description 18
- 238000011534 incubation Methods 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 238000007789 sealing Methods 0.000 claims abstract description 6
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 claims abstract description 4
- 229910001863 barium hydroxide Inorganic materials 0.000 claims abstract description 4
- 241000196324 Embryophyta Species 0.000 claims description 43
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- 239000008055 phosphate buffer solution Substances 0.000 claims description 30
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 27
- 239000000706 filtrate Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 19
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 229940098773 bovine serum albumin Drugs 0.000 claims description 18
- 238000003125 immunofluorescent labeling Methods 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 229920001221 xylan Polymers 0.000 claims description 10
- 150000004823 xylans Chemical class 0.000 claims description 10
- 241001494479 Pecora Species 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 3
- 229910052788 barium Inorganic materials 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000001000 micrograph Methods 0.000 claims description 3
- UKLNMMHNWFDKNT-UHFFFAOYSA-M sodium chlorite Chemical compound [Na+].[O-]Cl=O UKLNMMHNWFDKNT-UHFFFAOYSA-M 0.000 claims description 3
- 238000011282 treatment Methods 0.000 claims description 3
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 claims description 2
- 241000283707 Capra Species 0.000 claims description 2
- 229920002324 Galactoglucomannan Polymers 0.000 claims description 2
- 229920000926 Galactomannan Polymers 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002023 wood Substances 0.000 claims 2
- 244000025254 Cannabis sativa Species 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 239000008366 buffered solution Substances 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 229920005610 lignin Polymers 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 239000002028 Biomass Substances 0.000 description 7
- 235000011609 Pinus massoniana Nutrition 0.000 description 6
- 241000018650 Pinus massoniana Species 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 229920000057 Mannan Polymers 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 229920002522 Wood fibre Polymers 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- -1 galactosyl glucose mannans Chemical class 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 239000002025 wood fiber Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000003517 fume Substances 0.000 description 2
- 239000011121 hardwood Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011122 softwood Substances 0.000 description 2
- 229920002581 Glucomannan Polymers 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003209 petroleum derivative Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
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Abstract
The invention belongs to the technical field of plant ultrastructure detection, and particularly relates to a method for researching micro-distribution of hemicellulose in plant cell walls. Firstly, carrying out wax removal, ash removal and lignin removal on a plant tissue slice, and then carrying out barium hydroxide selective fractional extraction on the plant tissue slice to separate hemicellulose; then, washing and sealing the plant tissue slices at different extraction stages and incubating the slices with a first antibody; washing the plant tissue section after the first antibody incubation and performing second antibody incubation; and finally observing the plant tissue section by using a laser confocal microscope. According to the invention, the hemicellulose is selectively extracted by barium hydroxide in a grading manner and the first antibody capable of specifically binding different types of hemicellulose is combined, so that the distribution of different types of hemicellulose in plant cell walls can be effectively and accurately identified and observed.
Description
Technical Field
The invention belongs to the technical field of plant ultrastructure detection, and particularly relates to a method for researching micro distribution of hemicellulose in plant cell walls.
Background
The wood fiber biomass is wide in source, renewable, rich in resource and low in price, can realize high-value utilization of the wood fiber biomass through a certain biochemical conversion process, can be used for preparing biomass-based energy, biomass-based materials and biomass-based chemicals, can replace fossil fuels, petroleum products and the like, and provides possibility for developing high-efficiency clean new energy. The hemicellulose is the second most renewable biomass resource with the content second to that of the cellulose in the nature, is widely applied to the industries of food, pharmacy, chemistry and the like, and has wide development prospect. However, the kinds of polysaccharides constituting hemicellulose are different from raw material to raw material, and hemicellulose is closely bound or chemically linked to other components in cell walls, so that the distribution and extraction of hemicellulose in cell walls are very difficult. For example, there is a chemical linkage between hemicellulose and lignin, forming lignin and carbohydrate complexes; hydrogen bonding and van der waals force exist between the hemicellulose and the cellulose to form tight combination between the hemicellulose and the cellulose.
The traditional methods for researching the distribution of hemicellulose in plant cell walls comprise a dyeing method, a skeleton method, a chemical peeling method and the like, the distribution of the hemicellulose is mainly indirectly judged by a chemical analysis and a chromatographic method, the experimental operation is complex, and the types and the branching degrees of glycan can not be distinguished for accurate positioning.
Disclosure of Invention
Aiming at the problems, the invention provides a method for researching the micro distribution of hemicellulose in plant cell walls, and by adopting the method, the distribution of different types of glycans with different degrees of branching in the cell walls can be displayed, so that the defect of the traditional method for researching the distribution rule of the hemicellulose is overcome.
The invention relates to a method for researching micro distribution of hemicellulose in plant cell walls, which comprises the steps of sequentially carrying out dewaxing, ash removal and delignification treatment on a plant tissue slice, and carrying out barium hydroxide selective fractional extraction on the delignified plant tissue slice to separate the hemicellulose; then, washing and sealing the plant tissue slices at different extraction stages and incubating the slices with a first antibody; washing the plant tissue section after the first antibody incubation and performing second antibody incubation; and finally observing the plant tissue section by using a laser confocal microscope.
The method comprises the following specific steps:
(I) the part that interception trunk (stem) middle part is close to bark (crust) is the sample, cuts the section that gets thickness and be 5um, puts into the soxhlet extractor, adopts the volume ratio to be 2:1 toluene/ethanol was extracted at 90 ℃ for 8 hours to remove ash, wax, etc. Placing the extracted slices in a fume hood for air drying overnight, and drying in an oven at 40 ℃ for 20 hours;
(II) use of 6 w/v% NaClO at pH 3.6-4.02Delignifying the extracted plant sample, performing immunofluorescence labeling on the sliced sample, and observing the distribution of hemicellulose in cell walls by using a laser confocal microscope;
(III) Using 1.8 w/v% Ba (OH)2Treating delignified samples by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; and then treating the sample by using 10 w/v% KOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 1, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 1 in cell walls by using a laser confocal microscope;
(IV) treating the sliced sample treated in the step (3) by using 1 w/v% NaOH aqueous solution, wherein the solid-to-liquid ratio is 1: 20(g/mL), reacting at room temperature for 10 hours, adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 2, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 2 in cell walls by using a laser confocal microscope;
(V) Using 3 w/V% H3BO3And (3) treating the sliced sample treated in the step (4) by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; then using 18 w/v% NaOH waterTreating a sample with the solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 3, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 3 in cell walls by using a laser confocal microscope;
(VI) respectively carrying out nuclear magnetic analysis on the hemicellulose 1, the hemicellulose 2 and the hemicellulose 3 to obtain corresponding hemicellulose structures, and positioning the dissolved hemicellulose glycan in each step;
(VII) analyzing the result of the laser confocal microscope image of the plant section left in each step, observing the distribution and dissolution condition of the hemicellulose components, and reducing the distribution of different hemicelluloses in the cell wall.
Preferably, 6 w/v% NaClO at pH 3.6-4.0 is used in step (II)2The main steps of delignifying the extracted plant sample are as follows: sample, NaClO2And distilled water according to a solid-to-liquid ratio of 1: 20(g/mL), solid-to-solid ratio of 1: 1.2(g/g), adjusting pH to 3.6-4.0 with acetic acid, reacting at 70-80 deg.C for 1 hr, adding NaClO2And acetic acid, in mass ratio, sample: NaClO2: acetic acid 1: 0.5: 0.5, after 2 hours of reaction, the sample was repeatedly washed with distilled water and finally with ethanol.
Preferably, in the step (III), the KOH concentration of the samples from different sources is different, and the KOH concentrations of the samples from the softwood plants, the hardwood plants and the gramineous plants are respectively 10 w/v%, 10 w/v% and 5 w/v%.
Preferably, the filtrate obtained by the distillation under reduced pressure in the steps (III), (IV) and (V) is added dropwise to the absolute ethanol under stirring.
Preferably, the immunofluorescent labeling in steps (II), (III), (IV) and (V) mainly comprises the following steps:
(1) washing the sample with distilled water for 2 minutes, and repeating the steps once;
(2) the samples were washed for 30 minutes with 0.01M phosphate buffered saline containing 0.1M glycine (pH 7.2-7.4, 0.01M phosphate buffered saline appearing below);
(3) washing with 0.01M phosphate buffer solution for 2 minutes, and repeating three to five times;
(4) blocking the sample with sheep serum at room temperature for 40-50 min, the sheep serum needs to be diluted with 0.01M phosphate buffer solution;
(5) incubating the sample with a first antibody; diluting the primary antibody with 0.01M phosphate buffer solution containing 1-3 w/v% bovine serum albumin, placing the sample in a sterile culture dish, covering, and incubating in a refrigerator at 4 ℃ for 48 hours;
(6) washing the sample with 0.01M phosphate buffer solution containing 1-3 w/v% bovine serum albumin for 2 minutes, and repeating the steps;
(7) the sample was washed with 0.01M phosphate buffer for 2 minutes, repeated four times;
(8) incubating the sample with a second antibody diluted with 1-3 w/v% 0.01M phosphate buffer, placing the sample in a sterile petri dish, capping, and incubating at room temperature for 2-4 hours;
(9) washing with 0.01M phosphate buffer solution for 10 min, repeated three times;
(10) the sample was washed with distilled water for 3 minutes;
(11) placing the sample in a sterile culture dish, covering the sample with a cover, and airing the sample overnight under the condition of darkness at room temperature;
(12) sealing the plate with an anti-fluorescence quencher, and observing under a laser confocal microscope.
Preferably, the first antibody is LM10 against linear or low-branching xylan or LM11 against low-branching and high-branching xylan or LM21 against linear galactoglucomannan or LM22 against high-branching galactomannan.
Preferably, the second antibody is Alexa
488 labeled goat anti-mouse antibody.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for researching the micro-distribution of hemicellulose in plant cell walls, which can display the distribution of different types and different branching degrees of glycan in the cell walls and make up for the defects of the traditional method for researching the distribution rule of the hemicellulose. The invention has positive promotion effect on the local chemical research of the hemicellulose, and provides an effective means for researching the deposition rule of the hemicellulose and the selective separation and extraction of the hemicellulose in the plant cell wall forming process.
Drawings
FIG. 1 shows the results of confocal laser microscopy of linear or low-branching xylan distribution in the cell wall of Pinus massoniana;
FIG. 2 is the result of laser confocal microscope observation of the distribution of linear galactose glucose mannan in the cell wall of masson pine.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples, which are not intended to limit the scope of the invention as defined by the claims. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a method for researching a micro distribution rule of hemicellulose in a plant cell wall, which comprises the following steps:
step 1: cutting the part of the middle part of the masson pine trunk close to the bark as a sample, cutting a slice with the thickness of 5 mu m, putting the slice into a Soxhlet extractor, and adopting a volume ratio of 2:1 toluene/ethanol was extracted at 90 ℃ for 8 hours to remove ash, wax, etc. Placing the extracted slices in a fume hood for air drying overnight, and drying in an oven at 40 ℃ for 20 hours;
step 2: using 6 w/v% NaClO at pH 3.6-4.02Delignifying the extracted plant sample, performing immunofluorescence labeling on the sliced sample, and observing the distribution of hemicellulose in cell walls by using a laser confocal microscope;
the delignification treatment step comprises: sample, NaClO2And distilled water according to a solid-to-liquid ratio of 1: 20(g/mL), solid-to-solid ratio of 1: 1.2(g/g) ratioAdding into the mixture, adjusting pH to 3.6-4.0 with acetic acid, reacting at 70-80 deg.C for 1 hr, adding NaClO2And acetic acid, in mass ratio, sample: NaClO2: acetic acid 1: 0.5: 0.5, after 2 hours of reaction, the sample was repeatedly washed with distilled water and finally with ethanol.
The immunofluorescence labeling step is as follows:
a. washing the sample with distilled water for 2 minutes, and repeating the steps once;
b. the sample was washed with 0.01M phosphate buffer solution containing 0.1M glycine (pH 7.4, which is the 0.01M phosphate buffer solution appearing below) for 30 minutes;
c. washing with 0.01M phosphate buffer solution for 2 minutes, repeated three times;
d. blocking the sample with sheep serum for 40 min at room temperature, diluting the sheep serum with 0.01M phosphate buffer;
the volume ratio of the sheep serum to the 0.01M phosphate buffer solution is 1: 20;
e. incubating the sample with a first antibody (LM10, specifically binding to linear or low-branching xylans; LM11, specifically binding to low-and high-branching xylans; LM21, specifically binding to linear galactosyl glucose mannans; LM22, specifically binding to high-branching galactosyl glucose mannans); diluting the primary antibody with 0.01M phosphate buffer solution containing 1 w/v% bovine serum albumin, placing the sample in a sterile culture dish, covering, and incubating in a refrigerator at 4 ℃ for 48 hours; the manufacturers of the primary antibodies LM10, LM11, LM21 and LM22 used in the examples of the present invention are Paul knock Cell Wall Lab at the University of seeds.
The first incubation liquid was incubated with a first antibody LM 10: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution 1: 20 (v/v); or by the first antibody LM 11: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution 1: 20 (v/v); or by the first antibody LM 21: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution 1: 50 (v/v); or by the first antibody LM 22: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution 1: 50 (v/v);
f. the sample was washed with 0.01M phosphate buffer solution containing 1 w/v% bovine serum albumin for 1 minute and repeated once;
washing the sample with 0.01M phosphate buffer solution for 2 minutes, and repeating for four times;
h. incubating the sample with a second antibody (which specifically binds to the first antibody), the second antibody being diluted with 0.01M phosphate buffer containing 1 w/v% bovine serum albumin, the sample being placed in a sterile petri dish, covered, and incubated at room temperature for 4 hours;
the second antibody incubation was performed with a second antibody: phosphate buffer solution containing 1 w/v% bovine serum albumin ═ 1: 50 (v/v);
i. washing with 0.01M phosphate buffer solution for 10 min, repeated three times;
j. the sample was washed with distilled water for 3 minutes;
k. placing the sample in a sterile culture dish, covering the sample with a cover, and airing the sample overnight under the condition of darkness at room temperature;
sealing the plate with the anti-fluorescence quencher, and observing under a laser confocal microscope. The obtained laser confocal microscope image is used for analyzing the distribution characteristics of linear or low-branching-degree xylan, low-branching-degree and high-branching-degree xylan, linear galactose glucose mannan and high-branching-degree galactose glucose mannan in the cell wall of the masson pine.
And step 3: utilizing 1.8 w/v% Ba (OH)2Treating delignified samples by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; and then treating the sample by using 10 w/v% KOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure to about 30mL, dropping the filtrate into anhydrous ethanol with the volume three times that of the filtrate, continuously stirring the filtrate in the period, standing the filtrate overnight to obtain hemicellulose 1, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 1 in cell walls by using a laser confocal microscope;
the KOH concentration varies with the source of the sample, and the KOH concentrations of samples derived from softwood plants, hardwood plants, and gramineous plants are 10 w/v%, and 5 w/v%, respectively.
The immunofluorescent labeling procedure described above was similar to that in step 2, except that the first antibody incubation was performed with a first antibody LM 21: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 50 (v/v); or by the first antibody LM 22: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 50 (v/v);
and 4, step 4: the samples were treated with 1 w/v% NaOH at a solid to liquid ratio of 1: 20(g/mL), reacting at room temperature for 10 hours, adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure to about 30mL, dropping the filtrate into absolute ethyl alcohol with three times of volume, continuously stirring during the period, standing overnight to obtain hemicellulose 2, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 2 in cell walls by using a laser confocal microscope;
the immunofluorescent labeling procedure described above was similar to that in step 2, except that the first antibody incubation was performed with a first antibody LM 10: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 20 (v/v); or by the first antibody LM 11: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 20 (v/v);
and 5: using 3 w/v% H3BO3And (3) treating the sliced sample treated in the step (4) by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; and then treating the sample by using 18 w/v% NaOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure to about 30mL, dropping the filtrate into anhydrous ethanol with the volume three times that of the filtrate, continuously stirring the filtrate in the period, standing the filtrate overnight to obtain hemicellulose 3, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 3 in cell walls by using a laser confocal microscope;
the immunofluorescent labeling procedure described above was similar to that in step 2, except that the first antibody incubation was performed with a first antibody LM 10: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 20 (v/v); or by the first antibody LM 11: 1 w/v% bovine serum albumin in 0.01M phosphate buffer solution ═ 1: 20 (v/v);
the results of confocal laser microscopy of linear or low-branched xylan distribution in the cell wall of Pinus massoniana are shown in FIG. 1.
The results of confocal laser microscopy of the distribution of linear galactosyl glucomannan in the cell wall of masson pine are shown in fig. 2.
The result shows that the method not only can observe the distribution of the hemicellulose in the plant cell walls, but also can effectively distinguish the distribution conditions of different types of hemicellulose in the plant cell walls through the local chemical analysis of the hemicellulose polysaccharide obtained by each stage of separation and the corresponding slices. The method has universality for the research of other types of wood fiber biomass raw materials.
Claims (9)
1. A method for studying the micro-distribution of hemicellulose in plant cell walls comprises sequentially carrying out dewaxing, deashing and delignification treatments on plant tissue slices, and is characterized in that barium hydroxide selective fractional extraction is carried out on the delignified plant tissue slices to separate the hemicellulose; then, washing and sealing the plant tissue slices at different extraction stages and incubating the slices with a first antibody; washing the plant tissue section after the first antibody incubation and performing second antibody incubation; and finally observing the plant tissue section by using a laser confocal microscope.
2. The method for studying the micro-distribution of hemicellulose in plant cell walls according to claim 1, comprising the steps of:
(I) cutting a plant tissue sample, cutting into slices, putting into a Soxhlet extractor, and extracting by adopting a mixed solution of toluene and ethanol; air-drying and drying the extracted slices;
(II) delignifying the dried sliced sample, carrying out immunofluorescence labeling on the sliced sample, and observing the distribution of hemicellulose in cell walls by using a laser confocal microscope;
(III) Using 1.8 w/v% Ba (OH)2Treating delignified samples by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; and then treating the sample by using 10 w/v% KOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 1, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 1 in cell walls by using a laser confocal microscope;
(IV) treating the sliced sample treated in the step (3) by using 1 w/v% NaOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 10 hours, adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 2, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 2 in cell walls by using a laser confocal microscope;
(V) Using 3 w/V% H3BO3And (3) treating the sliced sample treated in the step (4) by using an aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, reacting at room temperature for 8-10 hours; and then treating the sample by using 18 w/v% NaOH aqueous solution, wherein the solid-to-liquid ratio is 1 g: 20mL, and reacting at room temperature for 10 hours; adjusting the pH value of the filtrate to 5.5-6.0, distilling under reduced pressure, dripping the filtrate into absolute ethyl alcohol, standing overnight to obtain hemicellulose 3, performing nuclear magnetic analysis, performing immunofluorescence labeling on the sliced sample, and observing the distribution condition of the hemicellulose 3 in cell walls by using a laser confocal microscope;
(VI) respectively carrying out nuclear magnetic analysis on the hemicellulose 1, the hemicellulose 2 and the hemicellulose 3 to obtain corresponding hemicellulose structures, and positioning the dissolved hemicellulose glycan in each step;
(VII) analyzing the result of the laser confocal microscope image of the plant section left in each step, observing the distribution and dissolution condition of the hemicellulose components, and reducing the structure of the hemicellulose in the cell wall.
3. The method for researching micro-distribution of hemicellulose in plant cell walls according to claim 2, wherein the volume ratio of toluene to ethanol in step (I) is 2: 1; the extraction temperature is 90 ℃, and the extraction time is 8 h; the drying temperature is 40 ℃, and the drying time is 20 hours.
4. The method for researching the micro distribution of the hemicellulose in the plant cell wall as claimed in claim 2, wherein the step of delignifying the sliced sample in the step (II) is as follows: sample, NaClO2And distilled water according to a solid-to-liquid ratio of 1 g: 20mL, solid-solid ratio 1 g: adding 1.2g of the above mixture, adjusting pH to 3.6-4.0 with acetic acid, reacting at 70-80 deg.C for 1 hr, adding NaClO2And acetic acid, in mass ratio, sample: NaClO2: acetic acid 1: 0.5: 0.5, after 2 hours of reaction, the sample was repeatedly washed with distilled water and finally with ethanol.
5. The method for researching micro-distribution of hemicellulose in plant cell walls according to claim 2, wherein in the step (III), KOH concentration of samples from needle-leaved wood plants, broad-leaved wood plants and grass plants is 10 w/v%, 10 w/v% and 5 w/v% respectively according to different KOH concentration of samples from different sources.
6. The method for researching micro-distribution of hemicellulose in plant cell walls according to claim 2, wherein in the steps (III), (IV) and (V), the filtrate after reduced pressure distillation is dripped into absolute ethyl alcohol without continuous stirring; the volume of the absolute ethyl alcohol is 3 times of the volume of the filtrate after reduced pressure distillation.
7. The method for studying the micro-distribution of hemicellulose in plant cell walls according to claim 2, wherein the immunofluorescent labeling in steps (II), (III), (IV), (V) comprises the following steps:
(1) washing the sample with distilled water for 2 minutes, and repeating the steps once;
(2) wash the sample with 0.01M phosphate buffer solution containing 0.1M glycine for 30 minutes;
(3) washing with 0.01M phosphate buffer solution for 2 minutes, and repeating three to five times;
(4) blocking the sample with sheep serum at room temperature for 40-50 min, the sheep serum needs to be diluted with 0.01M phosphate buffer solution;
(5) incubating the sample with a first antibody; diluting the primary antibody with 0.01M phosphate buffer solution containing 1-3 w/v% bovine serum albumin, placing the sample in a sterile culture dish, covering, and incubating in a refrigerator at 4 ℃ for 48 hours;
(6) washing the sample with 0.01M phosphate buffer solution containing 1-3 w/v% bovine serum albumin for 2 minutes, and repeating the steps;
(7) the sample was washed with 0.01M phosphate buffer for 2 minutes, repeated four times;
(8) incubating the sample with a second antibody diluted with 1-3 w/v% 0.01M phosphate buffer, placing the sample in a sterile petri dish, capping, and incubating at room temperature for 2-4 hours;
(9) washing with 0.01M phosphate buffer solution for 10 min, repeated three times;
(10) the sample was washed with distilled water for 3 minutes;
(11) placing the sample in a sterile culture dish, covering the sample with a cover, and airing the sample overnight under the condition of darkness at room temperature;
(12) sealing the sheet by using an anti-fluorescence quencher, and observing under a laser confocal microscope;
the pH of the 0.01M phosphate buffered solution was 7.2-7.4.
8. The method of studying the micro-distribution of hemicellulose in plant cell walls according to claim 1, wherein the first antibody is LM10 resistant to linear or low-branching xylan or LM11 resistant to low-branching and high-branching xylan or LM21 resistant to linear galactoglucomannan or LM22 resistant to high-branching galactomannan.
9. The method of claim 1, wherein the second antibody is Alexa
488 labeled goat anti-mouse antibody.
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