CN112730352A - Method for observing structure, area and spatial distribution of plant cell nucleus protein in real time - Google Patents
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Abstract
本发明公开了实时观察植物细胞核蛋白的结构、面积和空间分布的方法，包括步骤一，转基因材料获得；步骤二，成像观察；步骤三，分析处理；步骤四，计算参数信息；其中在上述步骤一中，首先通过PCR技术扩增出目的基因条带，然后通过同源重组的方法将目的基因连接到真核表达载体pCM2300中；ZeissLSM880Airyscan快速超分辨激光共聚焦显微镜的Airyscan模式下成像具有超高分辨率，可以对植物细胞核内蛋白质的结构进行超高分辨率的成像观察；对细胞的光毒性伤害小，能够在活体状态下、原位观察细胞核蛋白的面积和分布等特征；成像速度快，能够真实地反映植物细胞核内蛋白分子的状态，并在单分子水平上对细胞核蛋白的面积异质性进行分析；重复性高。
The invention discloses a method for real-time observation of the structure, area and spatial distribution of plant nuclear proteins, including step 1, obtaining transgenic material; step 2, imaging observation; step 3, analyzing and processing; In the first, the target gene band was amplified by PCR technology, and then the target gene was connected to the eukaryotic expression vector pCM2300 by homologous recombination; High resolution, can carry out ultra-high-resolution imaging observation of the structure of proteins in plant nucleus; less phototoxic damage to cells, can observe the area and distribution of nuclear proteins in vivo and in situ; fast imaging speed, It can truly reflect the state of protein molecules in the plant nucleus, and analyze the area heterogeneity of nuclear proteins at the single-molecule level; the repeatability is high.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for observing the structure, area and spatial distribution of plant cell nuclear protein in real time.
Background
The nucleus is the largest and most important cell structure in eukaryotic cells, is also the main site for storage, replication and transcription of genetic information, and plays an important role in the metabolism, growth and differentiation of eukaryotic cells. In the past, the research on plant cell nuclear protein is mainly carried out by utilizing in vitro analysis methods such as heredity, biochemistry and the like, however, the method cannot truly reflect the characteristics such as the structure, distribution and the like of molecules in a living cell nucleus, so that the heterogeneity information of the protein cannot be revealed under the condition of ultrahigh space-time resolution, and further the deep understanding of the functional mechanism of the protein is hindered;
the method adopts a rapid super-resolution laser confocal microscope ZeissLSM880Airyscan to image and analyze the cell nucleus protein in a plant living body sample, and can track the fluorescence area of a single molecule through the resolution of a sub-pixel so as to obtain the information of the distribution of fluorescent particles, the precision of the information can reach the nanometer level and far exceeds the resolution of a conventional optical microscope, and research reports have been reported, the structural characteristics of the single protein in the nucleus of an animal cell can be observed by using a super-resolution technology, but the super-resolution technology is not expanded to observe the structure and the distribution characteristics of the single protein in the nucleus of the plant cell, and the problems of difficult observation and the like exist in the plant cell due to the cell wall thickness, the autofluorescence intensity of chloroplast and the limitation of the growth form of the plant cell; aiming at the defects, it is necessary to design a method for observing the structure, the area and the spatial distribution of the plant cell nuclear protein in real time.
Disclosure of Invention
The present invention aims at providing a method for observing the structure, area and spatial distribution of plant cell nuclear protein in real time, so as to solve the problems proposed in the background art.
In order to solve the technical problems, the invention provides the following technical scheme: a method for observing the structure, area and spatial distribution of plant cell nuclear protein in real time comprises the steps of obtaining transgenic material; step two, imaging observation; step three, analyzing and processing; step four, calculating parameter information;
wherein in the first step, the transgenic material obtaining comprises the following steps:
1) firstly, amplifying a target gene band by a PCR technology, and then connecting the target gene to a eukaryotic expression vector pCM2300 by a homologous recombination method;
2) finally, infecting the arabidopsis thaliana plant by an agrobacterium transformation method, and obtaining a transgenic plant by resistance screening;
in the second step, the imaging observation comprises the following steps:
1) observing the obtained transgenic plant sample by using a ZeissLSM880Airyscan fast super-resolution laser confocal microscope and selecting exciting light with proper wavelength;
2) adjusting the corresponding laser intensity, carrying out imaging observation on the fluorescent protein in the cell nucleus, and recording and backing up the observation result;
in the third step, the image obtained by the super-resolution laser confocal microscope is analyzed by means of ImageJ1.48 and originPro8 software to analyze the structural details and fluorescence intensity of the protein in the cell nucleus;
in the fourth step, the specific position information of the fluorescent molecules distributed in the plant cell nucleus is observed according to the image information imaged by the super-resolution laser confocal microscope, and the area occupied by the fluorescent molecules is further calculated, so that the parameter information such as the area distribution is obtained.
According to the technical scheme, the transgenic material obtained in the step one 2) is green fluorescent protein GFP which is expressed in a large amount in plant cell nucleuses and has an important function.
According to the technical scheme, the excitation wavelength of the GFP-labeled proteins in the step two 1) is 488nm, the collection wavelength is 525nm, the laser intensity is set to be 80%, the EMGain electronic gain is 800 times, and continuous super-resolution imaging is carried out on the sample by utilizing an Airyscan mode.
According to the technical scheme, the image obtained in the third step selects a proper threshold value and applies wavelet transformation algorithm processing to remove the background, the position of the fluorescence point can be determined by calculating the local maximum value of 3 × 3 pixels around the fluorescence point to obtain the sub-pixel accurate value and calculating the weighted center weighted-centroid of the fluorescence point.
According to the technical scheme, through continuous tracking of fluorescence signals in the fourth step, MED18-GFP is found to be in highly heterogeneous distribution in cell nuclei, some fluorescence points are in punctate distribution in cell nuclei, other fluorescence points have larger areas and are in cluster distribution, and the fluorescence points with more than 2 frames are used for analyzing fluorescent proteins in cell nuclei.
Compared with the prior art, the invention has the following beneficial effects:
the imaging under the Airyscan mode of the ZeissLSM880Airyscan fast super-resolution laser confocal microscope has ultrahigh resolution, the resolution of 120nm can be realized on an XY axis, and the ultrahigh resolution imaging observation can be carried out on the structure of the protein in the plant cell nucleus;
2. the phototoxicity damage to cells is small, and the characteristics of the area, distribution and the like of the nuclear protein can be observed in situ under the living state;
3, the ZeissLSM880Airyscan fast super-resolution laser confocal microscope has high imaging speed, can truly reflect the state of protein molecules in plant cell nuclei, and can carry out cell alignment on a single molecule level
Analyzing the area heterogeneity of the nucleoprotein;
4. the repeatability is high.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a common confocal fluorescence image of the MED18-GFP molecule of the present invention;
FIG. 3 is a super-resolution image of the present invention imaged by ZeissLSM880Airyscan fast super-resolution confocal laser microscope;
FIGS. 4-5 are enlarged views of the dotted areas 1 and 2 of FIGS. 2 and 3, respectively, in accordance with the present invention;
FIG. 6 is a graph of the spatial distribution of MED18-GFP molecules identified in FIG. 2 analyzed by ImageJ, the size of the color indicating the size of the area occupied by the molecules, in accordance with the present invention;
FIG. 7 shows the area distribution of MED18-GFP molecules of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-7, the present invention provides a technical solution:
example 1:
a method for observing the structure, area and spatial distribution of plant cell nuclear protein in real time comprises the steps of obtaining transgenic material; step two, imaging observation; step three, analyzing and processing; step four, calculating parameter information;
wherein in the first step, the transgenic material obtaining comprises the following steps:
1) firstly, amplifying a target gene band by a PCR technology, and then connecting the target gene to a eukaryotic expression vector pCM2300 by a homologous recombination method;
2) finally, the arabidopsis thaliana plant is infected by an agrobacterium transformation method, and a transgenic plant is obtained by resistance screening, and the obtained transgenic material is green fluorescent protein GFP which is expressed in a large amount in the plant nucleus and has an important function;
in the second step, the imaging observation comprises the following steps:
1) observing the obtained transgenic plant sample by using a ZeissLSM880Airyscan fast super-resolution laser confocal microscope by selecting exciting light with proper wavelength, wherein the protein exciting wavelength marked by GFP is 488nm, the collecting wavelength is 525nm, the laser intensity is set to be 80%, the EMGain electronic gain is 800 times, and carrying out continuous super-resolution imaging on the sample by using an Airyscan mode;
2) adjusting the corresponding laser intensity, carrying out imaging observation on the fluorescent protein in the cell nucleus, and recording and backing up the observation result;
in the third step, the image obtained by the super-resolution laser confocal microscope is analyzed for structural details of proteins in the cell nucleus and fluorescence intensity by means of ImageJ1.48 and originPro8 software, an appropriate threshold value is selected for the obtained image, the obtained image is processed by a wavelet transform algorithm to remove the background, the position of a fluorescence point can be determined by calculating the local maximum value of 3 pixels around the fluorescence point to obtain a sub-pixel accurate value, and the weight gravity center weighted-centroid of the fluorescence point is calculated;
in the fourth step, according to the image information imaged by the super-resolution confocal laser microscope, observing the specific position information of the fluorescent molecules distributed in the plant cell nucleus, further calculating the area occupied by the fluorescent molecules, further obtaining the parameter information such as the area distribution and the like, and finding out that MED18-GFP presents highly heterogeneous distribution in the cell nucleus, some fluorescent points present point-shaped distribution in the cell nucleus, other fluorescent points have larger area and present cluster-shaped distribution, and the fluorescent points with more than 2 frames are used as the fluorescent protein in the cell nucleus for analysis by continuously tracking the fluorescent signals;
example 2:
a method for observing the structure, area and spatial distribution of plant cell nuclear protein in real time comprises the steps of obtaining transgenic material; step two, imaging observation; step three, analyzing and processing; step four, calculating parameter information;
wherein in the first step, the transgenic material obtaining comprises the following steps:
1) seeds of Arabidopsis thaliana with MED18-GFP marker were placed on filter paper, sterile water 85% ethanol: 30% H2O23: 1, spraying the arabidopsis thaliana seeds, after the seeds are dried, sowing the sterilized seeds on 1/2MS solid culture medium containing 0.1% of cane sugar and having the pH value of 5.8 by using sterile toothpicks, placing the flat plates with the seeds in a refrigerator at 4 ℃ for vernalization for 24-48h, placing the flat plates in an incubator for culture under the culture condition of 22 ℃, and carrying out 16h/8h light-dark circulation, and taking seedlings growing for 4-5d for confocal microscopic imaging observation;
2) the process of flaking the plant arabidopsis sample comprises the following steps: firstly, placing the whole plant seedling on a glass slide, enabling the paraxial surface of a leaf to be upward, laying and placing the root of arabidopsis thaliana, dripping about 30 mu L of 1/2MS liquid culture medium, and slightly covering the glass slide, wherein the thickness of the glass slide is about 0.17 mm;
in the second step, observing the arabidopsis seedling sample by using a ZeissLSM880Airyscan fast super-resolution laser confocal microscope, wherein 63-fold oil lens is needed by an objective lens, a plant sample is excited by 488nm laser, the collection wavelength is 525nm, and the instrument parameters are set as follows: setting the laser intensity to be 60% and the Gain of Gain to be 600 times in a confocal mode; setting the laser intensity to be 80% and the Gain to be 800 times in an Airyscan mode, and finally continuously shooting the cell nucleus of the Arabidopsis under the same visual field to obtain images under different modes, wherein FIG. 2 is a common confocal fluorescence image of MED18-GFP molecules, and FIG. 3 is a super-resolution image imaged by a Zeiss LSM880Airyscan fast super-resolution laser confocal microscope;
wherein in the third step, the analysis process comprises the following steps:
1) utilizing ImageJ software to perform background removal analysis on an Image, firstly, starting the ImageJ software, opening the obtained green channel Image File- > Open … - > selecting an Image picture- > opening, running the Image- > Adjust- > Brightness/Contrast in the ImageJ software, adjusting the Brightness and Contrast of the Image, and then storing the Image in a TIFF format File- > SaveAs- > TIFF …;
2) performing post-analysis on the super-resolution Image by using ImageJ software, importing the saved Image into the ImageJ software again, setting the Image Type, selecting Image- > Type- >32-bit, selecting Image- > Lookuptables- > Fire, selecting Image- > Adjust- > Size, selecting the Image Type to be enlarged by 10 times, selecting Plugins- > MeanShift-to set the Color Distance to be 25.0, then selecting Process- > Subtractickground-to set the Rolling Ball iRad to be 20Pixels, checking Preview, and storing the Image again as TIFF format File- > SaveAs- > TIFF …;
3) utilizing ImageJ software to carry out later data analysis on the processed super-resolution Image, firstly, selecting Image- > Adjust- > Threshod-, properly adjusting the size of fluorescent particles on the Image, then selecting Analyze- > AnalyzeParticiles-, setting Show to be of an overlayMasks type, namely obtaining a picture of the marked fluorescent particles, storing the picture again and exporting data;
in the fourth step, the derived area data is analyzed in OriginPro8 software, Frequency count is selected after right-click, the interval is set to be 500, and then data of FreqsY column is selected and analyzed to obtain a histogram of area distribution.
Based on the above, the invention has the advantages that the imaging of ZeissLSM880Airyscan rapid super-resolution laser confocal microscope in the Airyscan mode has ultrahigh resolution, the resolution of 120nm can be realized on the XY axis, and the ultrahigh resolution imaging observation can be carried out on the structure of the protein in the plant cell nucleus; the phototoxicity damage to cells is small, and the characteristics of the area, distribution and the like of the nuclear protein can be observed in situ under the living state; the ZeissLSM880Airyscan fast super-resolution laser confocal microscope has high imaging speed, can truly reflect the state of protein molecules in plant cell nuclei, and analyzes the area heterogeneity of the cell nuclear protein on a single molecule level; the repeatability is high.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
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| CN115165827A (en) * | 2022-06-28 | 2022-10-11 | 北京林业大学 | Method for observing cell wall cellulose microfibrils by using SIM ultrahigh-resolution imaging technology |
| CN115439400A (en) * | 2021-06-02 | 2022-12-06 | 卡尔蔡司显微镜有限责任公司 | Synthetic fluorescence images with sample-specific references |
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