CN108195801A - Single molecules level observation Stomacal guard cell memebrane protein distribution and dynamic method - Google Patents
Single molecules level observation Stomacal guard cell memebrane protein distribution and dynamic method Download PDFInfo
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- CN108195801A CN108195801A CN201711146160.7A CN201711146160A CN108195801A CN 108195801 A CN108195801 A CN 108195801A CN 201711146160 A CN201711146160 A CN 201711146160A CN 108195801 A CN108195801 A CN 108195801A
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Abstract
Single molecules level observes the distribution of plant stomata guard cell memebrane protein and dynamic method in real time, is related to biotechnology.This method includes the following steps：A, the transgenic line of fluorescent marker target gene is obtained；B, the real-time imaging of guard cell's memebrane protein is obtained with utilizing total internal reflection fluorescence microscope；C, it is handled as Image J softwares time-series image by obtained by；D, individual particle follow-up analysis is carried out to destination protein by Matlab R2014a, determines the dynamic of single protein polymers；E, residence time of the Stomacal guard cell memebrane protein on cytoplasma membrane is calculated；F, Gauss curve fitting is carried out to the residence time parameter of destination protein, determines residence time and the variation of destination protein.The advantages of the method has the signal-to-noise ratio for improving imaging, can carry out high-resolution observation, and image taking speed is fast, live body visualization and high repeatability.The present invention has actual application value in plant gene function research.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of single molecules level observes plant stomata guard cell in real time
Memebrane protein is distributed and dynamic method.
Background technology
Stomata is that plant carries out gas and the portal of exchange of moisture with external environment, and photosynthesis is controlled to absorb CO2, water
Divide evaporation, play an important roll in the vital movement of plant.The guard cell of stomata is formed by perceiving environmental stimulus, is regulated and controled
The closing and folding of stomata.The albumen particularly positioned on Stomacal guard cell film is experiencing external environment and regulation and control gas
There is critical function in hole response biology and abiotic stress.However, the research to albumen in guard cell in the past, mainly sharp
Plant stomata aperture is measured in the genetic stocks such as mutant with light microscope.This method be only using stomata as
One entirety is studied, it is impossible to which guard cell's memebrane protein is directly analyzed.Particularly, it is impossible to be safeguarded after stimulating impression
The behavioral characteristics of epicyte protein are tested and analyzed in real time.
Studies have reported that, cell membrane is a heterogeneous highly dynamic biological systems, therefore plasmalemma protein
Space-time dynamic is inevitable closely related with many basal cell processes.But there are cell wall thickness, spontaneous by the guard cell of plant
Fluorescence is strong, and cell light damage and photobleaching etc. caused by interference is easily generated to final data and is irradiated for a long time all may
The measurement of molecular dynamics can be had an impact.Particularly guard cell causes to be imaged with leaf epidermal cell not in same plane
It is difficult.
Lack a kind of method of real-time observation plant stomata guard cell memebrane protein distribution in the prior art, especially lack one
It plants high-resolution and can the method observed into Mobile state quickly be distributed to Stomacal guard cell memebrane protein.
Invention content
It is distributed it is an object of the present invention to provide a kind of memebrane protein of observation plant stomata guard cell in real time and dynamic
Method is provided in particular in a kind of high-resolution and observes albumen distribution on Stomacal guard cell film, movement and residence time in real time
Method.
Specifically include following steps：
(1) transfer-gen plant with fluorescence labels is obtained；
(2) with utilizing total internal reflection fluorescence microscope, the exciting light of wavelength corresponding to selected marker fluorescin is to obtained by step (1)
Transfer-gen plant is observed, when adjusting laser incident angle, the experiences total internal reflection between cell wall and cell membrane, and obtaining
Between sequence image；
(3) time-series image obtained using 1.48 softwares of Image J to step (2) is handled, and obtains image information,
Carry out destination protein distributional analysis；
(4) fluorescence signal in the time-series image obtained to step (2) using Matlab 2014a (The Mathworks)
Individual particle tracking is carried out, analyzes albumen movement locus；
(5) time-series image obtained according to step (2), using the fluorescence intensity of 1.48 analysis of fluorescence points of Image J, note
Record occurs from fluorescence signal to completely disappearing lasting frame number, with time for exposure and progress of shooting time interval is corresponding changes
Calculate, calculate fluorescent molecular on focal plane there are duration (lifetime), so as to obtain destination protein on cytoplasma membrane
Degree that is residence time (dwell time) and being dissociated from cytoplasma membrane；
(6) Gauss curve fitting is carried out to the Stomacal guard cell memebrane protein residence time.
Further, in the step (1), pass through polymerase chain reaction (Polymerase chain first
Reaction, PCR) amplification testing protein gene, by double digestion, connection means the gene of destination protein is inserted into table
Up in carrier pCM2300, the Gene Fusion with coding fluorescence labelled protein infects to obtain transgenosis plant finally by Agrobacterium
Strain, so as to realize the fluorescent marker to destination protein in the transfer-gen plant.The target gene is safeguarded for plant stomata
The gene expressed on cell membrane, used carrier are plant binary expression vector.
Further, in the step (1), planting the culture dish of target gene needs upright placement, and plant roots is made to hang down completely
Straight aerial growth is observed convenient for utilizing total internal reflection fluorescence microscope.
Further, in the step (1), it is the great expression in plant stomata to obtain transgenic line, and fusion green is glimmering
Photoprotein GFP is marked and the memebrane protein with critical function.
Further, the transgenic arabidopsis seedling that transfer-gen plant is growth 3-5 days is observed in the step (2).
Further, in the step (2), 50 μ L 1/2Murashige-skoog solution is added dropwise on wave carrier piece, will grow
The transgenic line seedling of 3-5 days is placed on glass slide, by blade abaxial side close to glass slide, is then capped common coverslip；
The refractive index of the glass slide is 1.52.
Further, in the step (2), the excitation wavelength of green fluorescent protein GFP is 488nm, collects wavelength and is
525nm.Time for exposure 100ms is set, EM Gain (electron gain) are 300 times, and frequency 20Hz carries out continuous imaging to sample.
The setting of time for exposure and electron gain is the problem that the art those skilled in the art are encountered, and unreasonable setting can make
Into weaker GFP dropouts, that is, lose the fluorescence signal of single protein polymers；Or increase background signal, i.e., in image
The noise signals such as excessive plant cell wall autofluorescence can be mixed into.Using the setting in the present invention can realize cover it is single
Protein polymers avoid being mixed into excessive background noise simultaneously, can obtain unexpected technique effect.
Further, in the step (2), 100X, the mirror that numerical aperture (numerical aperture) is 1.45 are used
Mirror oil is added dropwise in head, and it is 1.325 to adjust 488nm laser incident angles numerical value, adjusts laser intensity and is to objective exit laser intensity
1mW；Time series imaging is carried out to sample, time interval is 0 between every two field pictures, obtains 200 frame of consecutive image.Laser enters
Firing angle degree and objective exit intensity are the key that realize to carry out guard cell's memebrane protein single-molecule resolution imaging, unreasonable
Setting can cause guard cell's memebrane protein to be unable to blur-free imaging and can not carry out dynamic analysis or cell by light injury.It adopts
With the setting in the present invention cell can be avoided simultaneously because of light loss to the single fluorescent grain blur-free imaging of guard cell's memebrane protein
Wound loses activity, and can realize the effect that single-molecule resolution imaging and analysis are carried out in the cell of life.
Further, in the step (3), the analysis of albumen distribution is carried out by Image J 1.48, obtains image information.
Further, in the step (3), wavelet transformation is used to choosing appropriate threshold by the obtained image of step (2)
Algorithm process carries out background removal, and the position of phosphor dot can be worth to by calculating the local maximum of 3 × 3 pixels around it
Sub-pix exact value calculates the weight center of gravity (weighted-centroid) of phosphor dot and determines.
Further, in the step (3), green channel images (File- is opened in Image J softwares>Open…->Choosing
Select picture->Open), select process->Subtract Background- settings " Rolling Ball Radius " are set
For 25Pixels；Run Image->Adjust->Brightness/Contrast adjusts the brightness and contrast of image, setting
JPEG Quality (1-100) are 100, and image is then saved as tiff format.
Further, in the step (3), albumen trajectory track is carried out by Matlab 2014a (The Mathworks)
Analysis.
Further, in the step (3), picture charge pattern parameter is, in Matlab R2014a softwares, starts
" fusion " plug-in unit, File->Open ... opens intact TIFF pictures, in " Control panel " dialog box, if
It is 3, w2 to determine wavelet, clicks the region that analysis is wanted in " Select ROI " selection, clicks " Find particles " and has selected
The object to be tracked watches the selection situation of protein site, if parameter is suitable, protein site can be selected, if it is desired to be marked
Albumen without the point of label that is labeled or being done be not protein site, the parameter in " Control panel " need to be carried out
Adjustment.
Further, in the step (3), the parameter of target protein tracking is set as, is clicked " Track ", appearance
" input for process " dialog box, wherein gap values input 6, motion range is according to the motion feature of albumen, least radius
Input 1, maximum radius input 3, clicks confirming button；After Tacking finished, " the Tacking- of menu bar is clicked
>View tracking result”；In " View tracking " progress " Edit track ", save is clicked, to the knot of tracking
Fruit is preserved.
Further, in the step (4), by continuously tracking fluorescence signal, it is seen that height is presented in fluorescence signal on film
Dynamically, constantly appearing and subsiding, the time that some phosphor dots occur on film is shorter, other phosphor dots then have longer
Residence time (lifetime) takes and stops residence Time Analysis on the phosphor dot progress film more than 2 frames.
Further, in the step (4), occur on focal plane from fluorescent molecular to completely disappearing, lasting frame number,
With time for exposure and progress of shooting time interval is corresponding converts, the residence time (lifetime) of fluorescent molecular is calculated,
It can reflect protein residence time and the degree dissociated from cytoplasma membrane on cytoplasma membrane.
Further, in the step (5), the image after tracking is analyzed, the wherein residence time is set as：
“Analysis->tracking based->lifetime”；Derived Excel data are preserved, that is, when obtaining stop
Between.
Further, in the step (6), the image after tracking is analyzed, clicks menu " Analysis->
tracking based->Lifetime " then clicks " File->export->Last analysis data " export number
According to；Derived Excel data are subjected to Gauss curve fitting in Origin, right click " Frequency count " is divided into 0.5, then
“Analysis->Fit Exponential " carry out exponential fitting to it.
Further, in the step (6), fitting coefficient R is required during fitting2More than 0.99.
The present invention provides a kind of methods of real-time observation plant stomata guard cell memebrane protein distribution, especially provide a kind of
High-resolution observes albumen distribution, movement and the method for residence time on Stomacal guard cell film in real time.This method has following
Advantage：1) signal-to-noise ratio of imaging is improved by the present invention, can realize that the distribution to protein on guard cell's film carries out high score
The observation of resolution；2) it is small to cellular damage, can under condition of living organism, the distribution of home position observation Stomacal guard cell memebrane protein,
The features such as movement and residence time；3) image taking speed of the present invention is fast, can reflect that plant cell guard cell experiences outer signals
Early stage response dynamics afterwards, and the heterogeneous dynamic of memebrane protein is analyzed on single molecules level；4) present invention is repeatable
Property it is high.
Description of the drawings
Fig. 1 is aquaporin (PIP2 on Stomacal guard cell film；1) distribution figure；Figure 1A is in normal growth
Under the conditions of, the distribution map of aquaporin on guard cell's film；Figure 1B is to handle arabidopsis with 10 μM of abscisic acid ABA simulating droughts
30min, the distribution map of aquaporin on guard cell's film.
Fig. 2 is aquaporin (PIP2 on guard cell's film；1) the diffusion track signal tracked in time series
Figure；The movement locus figure of aquaporin when Fig. 2A is 10 frame, the movement locus figure of aquaporin when Fig. 2 B are 20 frame；
Fig. 3 is aquaporin on guard cell's film under normal growing conditions and abscisic acid ABA simulating drought growth conditions
(PIP2；1) the residence time schematic diagram on film；Fig. 3 A are aquaporin on guard cell's film under normal growing conditions
(PIP2；1) the residence time schematic diagram on film；Fig. 3 B is on guard cell's films under abscisic acid ABA simulating drought growth conditions
Aquaporin (PIP2；1) the residence time schematic diagram on film.
Specific embodiment
Understand to make the object, technical solutions and advantages of the present invention clearer, With reference to embodiment and join
According to attached drawing, the present invention is described in more detail.It should be understood that these descriptions are merely illustrative, and it is not intended to limit this hair
Bright range.In addition, in the following description, the description to known method, structure and other technologies is omitted, to avoid unnecessary
Obscure idea of the invention in ground.Therefore, it is any modification for being made in the case of without departing from present disclosure and range, equivalent
Replace, improve etc., it should all be included in the protection scope of the present invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified, in this hair
With arabidopsis thaliana aquaporin (PIP2 in bright embodiment；1) it for, is described in detail.
Embodiment 1：Aquaporin (PIP2 on Arabidopsis leaf Stomacal guard cell film；1) distribution, movement and stop
Time：
1. aquaporin base is expanded by polymerase chain reaction (Polymerase chain reaction, PCR) first
Purpose water channel protein gene is inserted into 2300 expression vectors of pCAMBIA, with volume by cause by the means of double digestion, connection
The Gene Fusion of code green fluorescent protein GFP, infects wild type Columbia arabidopsis finally by Agrobacterium and obtains transgenosis
Plant, so as to realize the green fluorescent label to aquaporin in the transfer-gen plant；
2. Green Fluorescent Protein aquaporin transfer-gen plant arabidopsis seed is placed on filter paper, by thimerosal
(85% ethyl alcohol：H2O2=3：1) it is spread across on arabidopsis seed, after seed is done, is seeded in containing 0.1% sucrose, pH 5.8
1/2Murashige-skoog solid mediums on.Culture dish is put in 4 DEG C of refrigerators and carries out vernalization, vernalization is taken out, put afterwards for 24 hours
It is cultivated in intelligent illumination box, cultivation temperature is 22 DEG C, and illumination condition was followed for 16 hours illumination/8 hour light cultures
Ring；
3. 50 μ L 1/2Murashige-skoog culture agent solutions are added dropwise on glass slide, the seedling of growth 4-5 days is taken, makes it
It is fully deployed on glass slide, and by seedling cotyledon abaxial side close to glass slide.It is capped coverslip, 1/2Murashige-skoog
The gap between coverslip and glass slide is completely filled with, but is overflowed without redundant solution, and ensures that bubble-free generates in solution；
4. above-mentioned Arabidopsis thaliana Seedlings blade is observed with TIREM microscopes.First, instrument parameter is set：Camera selection 00,
It is 1mW to adjust objective exit laser intensity according to the expression quantity of albumen, and adjustment laser incident angle is 1.325,100 × object lens,
Numerical aperture (numerical aperture) is 1.45；Secondly, acquisition parameters setting：Time for exposure is 100ms, image taking speed
For 20MHz, EM Gain are 300, and the frame number of shooting is 200；Finally, to the Arabidopsis thaliana Seedlings guard cell under the same visual field into
Row is continuously shot, and obtains consecutive image；
5. starting Image J softwares, above-mentioned gained time-series image (File- is opened in Image J softwares>Open…->
Selection picture->Open), select Process->Subtract Background- setting " Rolling Ball Radius " be
25Pixels；Run Image->Adjust->Brightness/Contrast adjusts the brightness and contrast of image, then will
Picture saves as tiff format (File->Save As->Tiff…)；
6. in Matlab R2014a softwares, tracking plug-in unit is write using multi-hypothesis algorithm.Start " fusion " plug-in unit,
File->Open…->New file->Selection picture->It opens, in " Control panel " dialog box, setting
" wavelet " is 3, w2, clicks the region that analysis is wanted in " Select ROI " selection, clicks " Find particles " and chooses institute
The object to be tracked is clicked " Process next frames ", pop-up dialog box " Enter the number of image
Frames you want to process ", it is 200 to key in parameter, clicks and determines, end of run shows Detection
It after finished, clicks " Track ", " input for process " dialog box of appearance, wherein gap values input 6, movement is most
Minor radius input 1, maximum radius input 3, clicks and determines.Individual particle tracking terminates pop-up Tacking finished, clicks dish
" the Tacking- on single column>View tracking result”；In " View tracking " progress " Edit track ", click
Save, the results are shown in Figure 2；
7. the image after tracking is analyzed, menu " Analysis- is clicked>tracking based->Lifetime ", so
" File- is clicked afterwards>export->Last analysis data " export data；
Derived data are subjected to Gauss curve fitting in Origin, right click " Frequency count " is divided into 0.5, then
“Analysis->Fit Exponential " carry out exponential fitting to it, regardless of whether with 10 μM of ABA processing, fitting coefficient
0.99 is all higher than, Gauss curve fitting image result is as shown in Figure 3.
Claims (10)
1. a kind of single molecules level observes the distribution of plant stomata guard cell memebrane protein and dynamic method in real time, including following step
Suddenly：
The gene of destination protein is inserted into expression vector by step A, and the Gene Fusion with coding fluorescence labelled protein passes through
Agrobacterium infects to obtain transfer-gen plant, and the fluorescent marker to destination protein is realized in the transfer-gen plant；
Step B, using 100X, numerical aperture be 1.45 camera lens, be added dropwise mirror oil, adjust 488nm laser incident angle numerical value be
1.325, adjusting laser intensity to objective exit laser intensity is 1mW；Under this physical condition, time for exposure 100ms, electronics
Gain is 300, and time series imaging is carried out to sample with 20Hz frequencies, and time interval is 0 between every two field pictures, is obtained continuous
200 frame of time-series image；
Step C carries out Subtract Background processing to the time-series image obtained；
D steps carry out the time-series image obtained individual particle tracking, and application software Matlab R2014a are obtained each
The coordinate information and fluorescence intensity information of each phosphor dot, generalized time and phosphor dot coordinate information in the image at time point,
Calculate the movement locus of destination protein；
E steps, generalized time and fluorescence intensity information analyze the residence time dynamic of destination protein；
F-step carries out Gauss curve fitting to the Stomacal guard cell memebrane protein residence time.
2. a kind of single molecules level according to claim 1 is observed the distribution of plant stomata guard cell memebrane protein and is moved in real time
The method of state, it is characterised in that：Be placed on filter paper with transfer-gen plant arabidopsis seed in the step A, by 85% ethyl alcohol and
H2O2Match is 3：1 thimerosal is spread across on arabidopsis seed, and after the thimerosal of the surface of the seed is done, the seed is seeded in
On solid medium containing 1/2MS；Culture dish is put in 4 DEG C of refrigerators and carries out vernalization, vernalization is taken out afterwards for 24 hours, is placed in intelligent illumination training
It supports and is cultivated in case, cultivation temperature is 22 DEG C, and illumination condition is 16 hours illumination/8 hour light culture cycles；On wave carrier piece
Culture medium solution is added dropwise, Arabidopsis thaliana Seedlings are placed on glass slide, are then capped common coverslip；Between coverslip and glass slide
Full of solution but bubble-free.
3. a kind of single molecules level according to claim 1 or 2 observes the distribution of plant stomata guard cell memebrane protein in real time
With dynamic method, it is characterised in that：First by PCR amplification target gene in the step A, pass through double digestion, connection
Target gene is inserted into expression vector pCM2300 by means, and infecting to obtain finally by Agrobacterium has turning for fluorescence labels
Genetic material.
4. a kind of single molecules level according to claim 1 or 2 observes the distribution of plant stomata guard cell memebrane protein in real time
With dynamic method, it is characterised in that：Destination protein is the functional protein expressed on Plant Guard Cells film in the step A,
Used carrier is plant binary expression vector, and planting the culture dish of transfer-gen plant needs upright placement, and plant roots is made to hang down completely
Straight aerial growth is observed convenient for utilizing total internal reflection fluorescence microscope.
5. a kind of single molecules level according to claim 2 is observed the distribution of plant stomata guard cell memebrane protein and is moved in real time
The method of state, it is characterised in that：It is molten that 50 μ L 1/2Murashige-skoog culture mediums are added dropwise on wave carrier piece in the step A
Liquid will grow the transgenic line seedling of 3-5 days and be placed on glass slide, by blade abaxial side close to glass slide；The glass slide
Refractive index be 1.52.
6. a kind of single molecules level according to claim 1 or 2 observes the distribution of plant stomata guard cell memebrane protein in real time
With dynamic method, it is characterised in that：In the step B, the excitation wavelength of exciting light is 488nm, and collection wavelength is 525nm；
Time for exposure 100ms is set, EM Gain are 300 times, and frequency 20Hz carries out continuous imaging to sample.
7. a kind of single molecules level according to claim 1,2 or 5 observes plant stomata guard cell memebrane protein point in real time
Cloth and dynamic method, it is characterised in that：In the step C, to being calculated by the obtained image of the step B with wavelet transformation
Method processing carries out background removal, and sub-pix exact value is worth to by the local maximum for calculating 3 × 3 pixels around phosphor dot,
Calculate the weight center of gravity of phosphor dot and the position of determining phosphor dot；The analysis of albumen distribution is carried out by Image J 1.48；
Green channel images are opened in ImageJ softwares, select process->Subtract Background- set " Rolling
Ball Radius " are set as 25Pixels；Run Image->Adjust->Brightness/Contrast adjusts image
Brightness and contrast, setting JPEG Quality are 100, and image is then saved as tiff format.
8. a kind of single molecules level according to claim 1,2 or 5 observes plant stomata guard cell memebrane protein point in real time
Cloth and dynamic method, it is characterised in that：In the D steps, in Matlab R2014a softwares, start " fusion " plug-in unit,
File->Open ... opens intact TIFF pictures, in " Control panel " dialog box, sets wavelet as 3,
W2 clicks the region that analysis is wanted in " Select ROI " selection, clicks " Find particles " and has selected pair to be tracked
As；The parameter of target protein tracking is set as, is clicked " Track ", " input for process " dialog box of appearance,
Middle gap values input 6, motion range are clicked and are determined according to the motion feature of albumen, least radius input 1, maximum radius input 3
Button；After Tacking finished, " the Tacking- of " fusion " plug-in unit in Matlab R2014a is clicked>View
tracking result”；In " View tracking " progress " Edit track ", save is clicked, the result of tracking is carried out
It preserves.
9. a kind of single molecules level according to claim 1,2 or 5 observes plant stomata guard cell memebrane protein point in real time
Cloth and dynamic method, it is characterised in that：By continuously tracking fluorescence signal in the D steps, guard cell's memebrane protein is in film
Highly dynamic, the constantly appearing and subsiding of upper presentation takes stop to divide more than the phosphor dot of 2 frames for carrying out the residence time on film
Analysis；In the E steps, occur on focal plane from fluorescent molecular to completely disappearing, lasting frame number, with time for exposure and bat
It takes the photograph time interval to be converted accordingly, calculates the residence time of fluorescent molecular.
10. a kind of single molecules level according to claim 1,2 or 5 observes plant stomata guard cell memebrane protein point in real time
Cloth and dynamic method, it is characterised in that：In the F-step, the image after tracking is analyzed, and clicks Matlab R2014a
In " fusion " plug-in unit " Analysis->tracking based->Lifetime " then clicks " File->export->
Last analysis data " export data；Derived Excel data are subjected to Gauss curve fitting, right click in Origin
" Frequency count ", is divided into 0.5, then " Analysis->Fit Exponential " carry out exponential fitting to it,
Fitting coefficient R is required during fitting2More than 0.99.
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| CN112760340A (en) * | 2021-01-21 | 2021-05-07 | 北京林业大学 | Method for tracking protein movement in living plant cell nucleus in real time by using ultrahigh resolution imaging technology |
| CN113466194A (en) * | 2021-06-24 | 2021-10-01 | 北京林业大学 | Method for observing protein distribution dynamics of stigma mastoid cell membrane at single molecule level |
| CN114317593A (en) * | 2021-12-31 | 2022-04-12 | 北京林业大学 | Method for observing plant protoplast membrane protein dynamics by using super-resolution microscopy |
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