研究目的
To establish a sensitive and wide-applicable approach to visualize wall polymer distribution and destruction during plant growth and biomass process using quantum dots (QDs).
研究成果
The established QDs labeling approach is applicable to in situ observe two major wall polysaccharides distribution in different types of cells in rice stem, coupled with lignin view at the same dissection. It is specifically sensitive for profiling wall polymers destruction distinctive from alkali and acid pretreatments with stem dissections.
研究不足
The study focuses on the application of QDs in visualizing wall polymer distribution and destruction in rice stem, and may not be directly applicable to other plant species or tissues without further validation.
1：Experimental Design and Method Selection
Semiconductor QDs (CdSe/ZnS) were employed to label the secondary antibody directed to the epitopes of pectin or xylan, and sorted out the optimal conditions for visualizing two polysaccharides distribution in cell walls of rice stem.
2：Sample Selection and Data Sources
Rice cultivar (Nipponbare, NPB) was collected from a previously described field trial. Rice sections (3 mm - 5 mm) were cut from the upper second internode.
3：List of Experimental Equipment and Materials
QDs (CdSe/ZnS, 610nm) and QD-labeled goat anti-mouse IgG were obtained from Beijing Najing Biotechnology co., Ltd. The synthetic process of QDs and QD-labeled goat anti-mouse IgG can refer to the methods described by Wang et al [33] and East et al [34] respectively.
4：Experimental Procedures and Operational Workflow
Paraffin sections were de-waxed with xylene and rehydrated through an ethanol series from 100% to 0%. Sections were then washed extensively in PBS, incubated with 5-fold dilution of cell wall glycan-directed monoclonal antibodies (mAbs) followed by 200-fold and 100-fold dilution of anti-mouse IgG linked to fluorescein isothiocyanate (FITC) or QDs respectively.
5：Data Analysis Methods
FITC fluorescence was observed with an Olympus BX61 microscope equipped with epifluorescence optics. QDs fluorescence was visualized by an Olympus IX71 inverted microscope equipped with DP72 camera using cellSens software.
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CdSe/ZnS
610nm
Beijing Najing Biotechnology Co., Ltd
Labeling the secondary antibody directed to the epitopes of pectin or xylan for visualizing polysaccharides distribution in cell walls.
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Olympus BX61 microscope
Olympus
Observation of FITC fluorescence.
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Olympus IX71 inverted microscope
Olympus
Visualization of QDs fluorescence.
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Hamamatsu ORCA285 camera
Hamamatsu
Capturing images of FITC fluorescence.
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DP72 camera
Olympus
Capturing images of QDs fluorescence.
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UV–VIS spectrometer
MAPADA V-1100D
MAPADA
Absorbance reading for colorimetric assay of hexoses, pentoses and uronic acids.
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Hitachi JSM-6390/LV
Hitachi
Scanning electron microscopic (SEM) observation.
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