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Observation method for specifically recognizing different parts of pollen, corresponding dye and application

A specific, pollen-based technology, applied in the analysis of materials, material stimulation analysis, preparation of test samples, etc., can solve the problems of inapplicable living body observation, inapplicable living body observation, complex sample processing process, etc., and achieve excellent coloring effect , wide applicability, and the effect of expanding the application field

Active Publication Date: 2021-06-08
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, both techniques have some disadvantages. For example, although SEM is a non-destructive detection method, it is not suitable for the observation of the living body, and it can only be used for the imaging observation of the surface layer of the sample; while TEM can be used for Observation of internal structure, but it requires complex sample processing process, and it is still not suitable for in vivo observation

Method used

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  • Observation method for specifically recognizing different parts of pollen, corresponding dye and application
  • Observation method for specifically recognizing different parts of pollen, corresponding dye and application
  • Observation method for specifically recognizing different parts of pollen, corresponding dye and application

Examples

Experimental program
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Effect test

Embodiment 1

[0081] This example provides a staining method that can specifically mark the pollen (spore) outer wall (exine), pollen (spore) inner wall (intine) and pollen coat (pollen coat) structure respectively

[0082] 1. The inventors found that basic fuchsin can specifically mark the pollen (spore) exine:

[0083] Weigh 10mg of basic fuchsin dry powder into a 15mL BD tube, add 10ml of double distilled water or PBS phosphate buffered saline and other buffer reagents to prepare a 1mg / mL basic fuchsin staining liquid mother solution. The mother liquor of basic fuchsin can be stored in a refrigerator at 4°C for a long time (~1 month), and it can also be stored at room temperature for a short time (~1 week). When staining, use double distilled water or phosphate buffer solution to dilute the mother solution 100 times to prepare a working solution for basic fuchsin staining. Take an appropriate amount of pollen and place it in a 1.5mL EP tube, add 1mL of the basic fuchsin dyeing solution ...

Embodiment 2

[0091] Embodiment 2. can respectively simultaneously specifically label pollen (spore) outer wall (exine) and pollen (spore) inner wall (intine), can simultaneously specifically label pollen (spore) outer wall (exine) and pollen coat (pollen coat) structure and can Staining method for simultaneous labeling of pollen (spore) exine and pollen coat structures

[0092] 1. A staining method that can specifically mark the pollen (spore) outer wall (exine) and pollen (spore) inner wall (intine) at the same time

[0093] Referring to the dyeing method in Example 1, basic fuchsin is used to specifically stain and mark the pollen exine, and fluorescent whitening agent 220 or fluorescent whitening agent VBL is used to specifically stain and mark the pollen intine. After staining, observation and imaging were performed using a laser scanning confocal microscope. The detection channel 1 can be excited at 405 nm to collect the signal of fluorescent whitening agent 220 or fluorescent whiten...

Embodiment 3

[0099] Example 3. A dyeing method capable of simultaneously marking pollen exine and pollen coat and compatible with green fluorescent protein (GFP)

[0100] First, based on previous experimental results, the inventors found that Arabidopsis glycine-rich protein 19 (GRP19) was specifically located in the pollen coat structure. Therefore, in this example, the inventors constructed a transgenic construction in which the GRP19 genome was fused with the fluorescent reporter gene GFP. First, we introduced this construct into Agrobacterium using thermal transformation. Next, we grew wild-type Arabidopsis and bred until the plants flowered. Infection of Arabidopsis thaliana by Agrobacterium was achieved by the flower dipping method (immersing Arabidopsis flowers in the Agrobacterium solution containing the transgene construct). Using Agrobacterium to transform GRP19-GFP into Arabidopsis, a marker line containing fluorescent protein was constructed. Cultivate according to conventio...

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Abstract

The invention provides an observation method for specifically recognizing different parts of pollen, corresponding dye and application. According to the invention, a dye liquor screening method which can be shared with green fluorescent protein (GFP) / yellow fluorescent protein (YFP) is established for the first time; a dye liquor, namely basic fuchsin, capable of specifically marking pollen outer walls (sporopollen components), a dye solution capable of specifically marking the inner wall (cellulose component) of pollen, namely a fluorescent whitening agent 220 (FB-220) and a fluorescent whitening agent VBL (FB-VBL), a dye liquor capable of specifically marking pollen coating (ultra-long chain fatty acid component), namely a fluorescent whitening agent 184 (PCS-184), and a dye liquor capable of non-specifically marking pollen coating, namely a fluorescent whitening agent 52 (PCS-52), arescreened from whitening agents / coloring agents such as fluorescent whitening agents and direct dyes through large-scale screening; and a dyeing method of the dye liquor composition in plant pollen (spores) is established and optimized, and the compatibility of the dyeing method and the green fluorescent protein (GFP) is tested.

Description

technical field [0001] The invention relates to the field of biological tissue / cell staining, in particular to a dyeing of pollen exine, pollen intine and pollen coat that can be shared with green fluorescent protein (GFP) / yellow fluorescent protein (YFP) Dye composition, dyeing method and application thereof. Background technique [0002] Plant landing is an important event in the evolution of life on Earth. Plants have greatly expanded their living space from aquatic to terrestrial, but at the same time, they need to resist various adversities in the terrestrial environment, such as: drought, ultraviolet radiation, high temperature and strong light. Spores or pollen are plant reproductive organs that carry important genetic material that determines the genetic stability of offspring. Therefore, in the long-term life evolution process, they have evolved a more complex and fine pollen / spore wall structure different from the general plant cell wall. In living plants, wheth...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N21/64
CPCG01N1/30G01N21/6428G01N2021/6439
Inventor 杨仲南薛景石贾鑫磊
Owner SHANGHAI NORMAL UNIVERSITY
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