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Method for observing distribution and dynamic states of proteins on stomatal guard cell membrane at single-molecule level

A protein distribution and cell membrane technology, applied in the biological field, can solve the problems of difficult imaging, final data interference, strong autofluorescence, etc., and achieve the effects of high repeatability, fast imaging speed, and improved signal-to-noise ratio.

Active Publication Date: 2018-06-22
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

However, plant guard cells have thick cell walls and strong autofluorescence, which are easy to interfere with the final data, and cell photodamage and photobleaching caused by long-term irradiation may affect the measurement of molecular dynamics
In particular, guard cells are not in the same plane as leaf epidermal cells, making imaging difficult
[0004] In the prior art, there is a lack of a method for real-time observation of the distribution of plant stomatal guard cell membrane proteins, especially a high-resolution method that can quickly and dynamically observe the distribution of stomatal guard cell membrane proteins

Method used

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  • Method for observing distribution and dynamic states of proteins on stomatal guard cell membrane at single-molecule level
  • Method for observing distribution and dynamic states of proteins on stomatal guard cell membrane at single-molecule level
  • Method for observing distribution and dynamic states of proteins on stomatal guard cell membrane at single-molecule level

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Embodiment 1

[0037] Example 1: Distribution, movement and residence time of aquaporin (PIP2; 1) on the stomatal guard cell membrane of Arabidopsis leaves:

[0038] 1. First amplify the aquaporin gene by polymerase chain reaction (Polymerase chain reaction, PCR), insert the target aquaporin gene into the pCAMBIA 2300 expression vector by means of double enzyme digestion and ligation, and encode the green fluorescent protein GFP The gene fusion of Agrobacterium infects wild-type Columbia Arabidopsis thaliana to obtain transgenic plants, so as to realize the green fluorescent labeling of water channels in the transgenic plants;

[0039] 2. Place the Arabidopsis thaliana seeds of the green fluorescent protein-labeled aquaporin transgenic plant on the filter paper, and disinfect the solution (85% ethanol: H 2 o 2 =3:1) sprinkled on Arabidopsis seeds, and after the seeds were dry, sow them on 1 / 2 Murashige-skoog solid medium containing 0.1% sucrose and pH 5.8. Put the culture dish in a refrige...

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Abstract

The invention provides a method for observing the distribution and dynamic states of proteins on a stomatal guard cell membrane at a single-molecule level, belonging to the field of biotechnology. Themethod comprises the following steps: A, acquiring a transgenic material in which a target gene is fluorescently labeled; B, acquiring the real-time images of proteins on a guard cell membrane by using a total internal reflection fluorescence microscope; C, processing the time series images by using the Image J software; D, carrying out single-particle tracing analysis on target proteins via Matlab R2014a so as to determine the dynamic state of each individual protein polymer; E, calculating the dwell time of the proteins on the cell membrane; and F, subjecting the dwell time parameters of the target proteins to Gaussian Fitting so as to determine the dwell time and changes of the target proteins. The method has the advantages of capacity of improving the signal-to-noise ratio of imagingand realizing high-resolution observation, fast imaging speed, in-vivo visibility and high repeatability. The method has practical application value in the research of the functions of plant genes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for real-time observation of the distribution and dynamics of plant stomatal guard cell membrane proteins at the single-molecule level. Background technique [0002] Stomata are the gateways for gas and water exchange between plants and the external environment, controlling the absorption of CO by photosynthesis 2 , Water evaporation plays an important role in the life activities of plants. Guard cells that form stomata regulate the closing and opening of stomata by sensing environmental stimuli. In particular, proteins located on the stomatal guard cell membrane have important functions in sensing the external environment and regulating stomatal responses to biotic and abiotic stresses. However, previous studies on proteins in guard cells have mainly used optical microscopy to measure plant stomatal opening in genetic material such as mutants. This method only studies th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6458
Inventor 李晓娟崔亚宁林金星
Owner BEIJING FORESTRY UNIVERSITY
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