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Mitochondria fluorescence labeling method for tobacco leaves and protoplasts

A technology of protoplasts and mitochondria, applied in fluorescence/phosphorescence, preparation of test samples, material analysis through optical means, etc., can solve the problems of limitation, loss and washing out, etc., to achieve clear concentration and improve dyeing effect , to avoid the effect of interference

Inactive Publication Date: 2020-06-23
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

[0004] Traditional mitochondrial fluorescent probes such as TMR and rhodamine 123 can also stain mitochondria well, but they will be washed away once the mitochondrial membrane potential is lost, so they are limited in some special experiments

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  • Mitochondria fluorescence labeling method for tobacco leaves and protoplasts
  • Mitochondria fluorescence labeling method for tobacco leaves and protoplasts

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Embodiment Construction

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments It is a part of embodiments of the present invention, but not all embodiments.

[0028] Take Ben's Tobacco as an implementation case

[0029] Mitochondrial staining of tobacco leaves:

[0030] Step 1, first we connect the CDS region (removing the stop codon) of the AtHXK1 gene to the pCAMBIA2300-GFP expression vector by homologous recombination;

[0031] Step 2, transferring the recombinant vector into GV3101 Agrobacterium by heat shock method;

[0032] Step 3. Select the single clone that is positive in PCR verification and shake it in YEB medium until the OD value is about 1.0, collect the bacteria by centrifugation, resuspe...

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Abstract

The invention relates to a mitochondria fluorescence labeling method for tobacco leaves and protoplasts. The method comprises the following steps: 1, preparing a working solution containing a Mitotracker Red CMXRos dye; 2, separating tobacco leaves and protoplast, and dyeing the tobacco leaves and the protoplast in a working solution containing a Mitotracker Red CMXRos dye; 3, cleaning the dyed tobacco leaves and protoplast by adopting a buffer solution; and 4, observing and photographing the tobacco leaves and protoplast cleaned in the step 3 by adopting a laser confocal microscope, wherein the excitation wavelength is 559 nm, and the emission wavelength is 600-630 nm. The mitochondria fluorescence labeling method for tobacco leaves and protoplasts is simple to operate, time-saving and good in dyeing effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mitochondrial fluorescent labeling method for tobacco leaves and protoplasts. Background technique [0002] Tobacco is a good material for transient expression system in plants because of its simple structure, short growth cycle, and easy injection. Agrobacterium-mediated transient transformation of tobacco is an important means to rapidly study the subcellular localization of proteins and the interaction between genes. Therefore, it is widely used in molecular biology research, including the expression of foreign genes, the interaction between transcription factors and cis-acting elements, the interaction of proteins, the functional study of promoters, the subcellular localization of proteins, and so on. [0003] Mitochondria are organelles covered by two membranes in eukaryotic cells. Their diameter is generally 0.5-1.0 μm and their length is about 3.0 μm. The number of mitocho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/34G01N21/64
CPCG01N1/30G01N1/34G01N21/6402G01N2001/302
Inventor 林金星郑文清张原杜亮冯圣年
Owner BEIJING FORESTRY UNIVERSITY
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