Method for observing chromosomal morphology of dinoflagellate

Abstract

The present application belongs to the field of biotechnology, cell biology and microscopic imaging technology, and particularly relates to a method for observing the morphology of dinoflagellate chromosomes. The method comprises the following steps: performing molecular anchoring treatment on dinoflagellate cells, performing gelation treatment on the anchored dinoflagellate cells to form a cell-embedded hydrogel, performing denaturation and hydration expansion treatment on the cell-embedded hydrogel, and then performing DNA fluorescent staining to realize observation of the morphology of dinoflagellate chromosomes through optical microscopic imaging. The present application performs molecular anchoring treatment on the fixed dinoflagellate cells, so that the chromosomes can be connected with the expanded hydrogel network, and then denaturation treatment is used to eliminate the interaction between proteins, so as to realize isotropic expansion of the gel, thereby significantly enlarging the spatial scale of the chromosomes on the premise of maintaining the relative spatial configuration, and combining with the fluorescent dye, high-resolution visualization of the morphology of dinoflagellate chromosomes can be realized.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, cell biology and microscopic imaging technology, and specifically relates to a method for observing the chromosome morphology of dinoflagellates. Background Technology

[0002] Dinoflagellates are a group of single-celled eukaryotic planktonic organisms widely distributed in marine and freshwater environments, playing a vital role in marine primary production, nutrient cycling, and the formation of harmful algal blooms. Some dinoflagellates can produce paralytic and neurotoxins, posing threats to marine ecosystem safety and human health. Therefore, in-depth analysis of the genetic structure and chromosomal characteristics of dinoflagellates is crucial for understanding their physiological and ecological functions, toxin formation mechanisms, and evolutionary adaptations.

[0003] However, unlike most eukaryotes, dinoflagellates have evolved highly specialized nuclear structures. Their chromosomes lack typical histone and nucleosome structures, and their DNA exists in a highly condensed liquid crystal state, remaining condensed throughout the cell cycle. Furthermore, the nuclear membrane of dinoflagellates does not disintegrate during mitosis, and the chromosomes do not form typical metaphase plates, resulting in highly dense and overlapping chromosomes in space. Additionally, some species possess a crust-like structure. These characteristics make it difficult to directly apply traditional methods of chromosome compression, spreading, and conventional fluorescent staining in plants or animals, and traditional optical microscopy struggles to obtain clear morphological information about the chromosomes.

[0004] Currently, research on the chromosome structure of dinoflagellates mainly relies on ultrastructural observation techniques such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM). However, these techniques typically require complex sample dehydration, embedding, and ultrathin sectioning, demanding high levels of experimental conditions and operator experience. Furthermore, the equipment is expensive and has low throughput, hindering its widespread application under conventional laboratory conditions. Therefore, there is an urgent need to develop a method that does not rely on electron microscopy equipment, has a relatively simple operating procedure, and enables clear observation of dinoflagellate chromosomes under conventional optical microscopy conditions. Summary of the Invention

[0005] To address the problem that dinoflagellate chromosomes are difficult to observe clearly using conventional optical microscopy, this invention aims to provide a method for observing the morphology of dinoflagellate chromosomes. This method significantly improves the spatial separation between chromosomes by physically magnifying the dinoflagellate cells without disrupting the overall spatial structure of the chromosomes, enabling clear differentiation of dinoflagellate chromosome morphology under conventional optical microscopy or laser confocal microscopy.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for observing the chromosome morphology of dinoflagellates involves molecularly anchoring dinoflagellate cells, gelling the anchored cells to form a cell-embedded hydrogel, denaturing and hydrating the cell-embedded hydrogel, performing DNA fluorescence staining, and then observing the chromosome morphology of the dinoflagellates using optical microscopy.

[0007] Specifically, it includes the following steps: (1) Dinoflagellate cell fixation: Dinoflagellate cells were fixed to obtain fixed cells; (2) Anchoring treatment: Molecular anchoring treatment is performed on the fixed dinoflagellate cells to make the chromosomes or DNA-bound proteins stably bound to polymerizable groups; (3) Gelation treatment: The anchored dinoflagellates are perfused into an expandable hydrogel solution to initiate a polymerization reaction and form a hydrogel that embeds the cells; (4) Denaturation treatment: The hydrogel sample is denatured; (5) Hydration and swelling treatment: The denatured sample is induced to undergo isotropic swelling of the hydrogel under hydration conditions, thereby amplifying the spatial scale of the dinoflagellate chromosome; (6) DNA fluorescent staining: DNA fluorescent staining is performed on the swollen sample; (7) Microscopic imaging: The morphology of dinoflagellates chromosomes can be observed through optical microscopic imaging.

[0008] The dinoflagellates are both crusted and non-crusted dinoflagellates.

[0009] Further, in step (1) Dinoflagellate cell fixation: Dinoflagellate cells in the exponential growth phase are collected by low-speed centrifugation. The collected cells are washed with 1×PBS buffer and then fixed with fixative.

[0010] The fixative is one or more of formaldehyde, paraformaldehyde, glutaraldehyde, methanol, or ethanol. Preferably, it is a PBS buffer solution of formaldehyde and glutaraldehyde. The fixation treatment temperature is 25℃±1℃, and the time is 10-15 min to maintain the stability of the cell nucleus and chromosome structure.

[0011] Step (2) Anchoring treatment: The fixed dinoflagellate cells are resuspended in the anchoring reaction solution for anchoring treatment.

[0012] The anchoring reaction solution contains acrylamide and aldehydes; the anchoring treatment temperature is 37°C, and the time is 2-8 hours, preferably 2 hours. Preferably, the anchoring reaction solution contains acrylamide and formaldehyde. This step involves resuspending the fixed dinoflagellate cells in the anchoring reaction solution to introduce acrylamide, allowing chromosomes or DNA-bound proteins to stably bind to polymerizable groups.

[0013] Step (3) Gelation treatment: The expandable hydrogel is an acrylate hydrogel; the acrylate hydrogel is composed of acrylate monomers, acrylamide monomers, crosslinking agents, polymerization initiators and accelerators; The mass concentration of the acrylate monomer is 100–400 g / L; The mass concentration of the acrylamide is 50–200 g / L; The crosslinking agent is N,N′-(1,2-dihydroxyethylene)diacrylamide (DHEBA) or N-dimethylacrylamide (MBAA). The polymerization initiator is ammonium persulfate (APS) or potassium persulfate (KPS). The accelerator is N,N,N,N-tetramethylethylenediamine (TEMED) with a mass concentration of 2.5 g / L; The polymerization reaction was carried out at a temperature of 37°C for 1.5 hours.

[0014] Step (4) Denaturation treatment: The denaturation treatment is a structural decoupling and softening treatment. The denaturation treatment is carried out in a buffer system containing sodium dodecyl sulfate. After incubation at 37°C for 30 min, the temperature is raised to 76°C for 1 h to achieve full decoupling, weaken the restriction of intracellular protein cross-linking on gel expansion, and reduce the restriction of intracellular cross-linking on gel expansion.

[0015] Preferably, the buffer system containing sodium dodecyl sulfate contains approximately 200 mM sodium dodecyl sulfate, 200 mM sodium chloride, and 50 mM Tris.

[0016] Step (5) Hydration and swelling treatment: Place the denatured gel sample in ultrapure water or low ionic strength buffer for hydration and swelling until the gel volume is stable; the linear swelling ratio of the hydrogel is 2-10 times, preferably 3-5 times.

[0017] Step (6) DNA fluorescent staining: The DNA fluorescent staining uses DAPI or other nucleic acid fluorescent dyes at a concentration of 2-5 mg / L, preferably 5 mg / L, and is incubated at 25℃±1℃ in the dark for 30-60 min, preferably 30 min. After staining, the sample is washed with buffer and then placed back in ultrapure water to restore its swelling state.

[0018] Step (7) Microscopic imaging: The microscopic imaging includes wide-field fluorescence microscopy or laser confocal microscopy to achieve clear observation of the morphology of dinoflagellates chromosomes. A laser confocal microscope, such as the Zeiss LSM 900 confocal microscope, can clearly distinguish the morphology of dinoflagellates chromosomes under conventional optical microscope conditions using an objective lens of about 40×; the imaging results can be acquired, processed and analyzed by the accompanying software.

[0019] The present invention has the following advantages: (1) The operation process is stable and highly repeatable. The method of the present invention optimizes the gel reaction system and molecular anchoring conditions. In the hydrogel system composed of acrylate and acrylamide, a stable gel embedding effect can be obtained with an anchoring reaction time of about 2 hours. The cell swelling process is uniform and controllable, and the swelling ratio has good repeatability.

[0020] (2) Significantly improved chromosome spatial resolution. After isotropic expansion treatment, the linear expansion ratio of the whole dinoflagellate cells can be stably reached about 3-6 times, and up to about 5.1 times in non-crusted dinoflagellates. Combined with conventional DAPI fluorescence staining and optical microscopy, the effective imaging resolution can be improved to about 40-70 nm, enabling clear separation and visualization of highly condensed chromosomes that were originally difficult to distinguish under ordinary optical microscopes.

[0021] (3) It has a wide range of applications and good compatibility with different crustacean structures of dinoflagellates. The method of this invention is applicable to both non-crusted and crusted dinoflagellates.

[0022] (4) Good preservation of chromosome in situ structure. The method of this invention, through molecular anchoring and physical amplification of gel network, achieves scale magnification while maintaining no significant distortion of the relative spatial configuration of chromosomes. After expansion, the outline of the cell nucleus is clear and the chromosomes are arranged in an orderly manner, meeting the structural fidelity requirements of karyotype analysis and chromosome behavior research.

[0023] (5) Compatible with conventional experimental conditions and with low technical threshold. The method of this invention does not require an electron microscope or complex super-resolution imaging equipment. It can complete chromosome observation using only a conventional fluorescence microscope or confocal microscope. The operating cost is low, the experimental cycle is short, and it is easy to promote and apply under different laboratory conditions.

[0024] (6) It has good scalability and versatility. Based on the method of this invention, it can be further combined with multicolor fluorescent labeling, fluorescence in situ hybridization or three-dimensional reconstruction imaging technology for dinoflagellate chromosome karyotype analysis, cell cycle behavior research and chromosome localization analysis of functional genes, which has a general and reliable technical application prospect. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the microscopic imaging results of chromosome morphology in chain-like Alexandrium cells, where... Figure 1 a represents the cell nuclear imaging results after processing using the method described in Example 1 and undergoing DNA fluorescence staining. Figure 1 b shows the results of nuclear imaging after DNA fluorescence staining only.

[0026] Figure 2 This is a schematic diagram of the microscopic imaging results of chromosome morphology in Pacific Alexandrium cells, where... Figure 2 a represents the cell nuclear imaging results after processing using the method in Example 2 and undergoing DNA fluorescence staining. Figure 2 b shows the results of nuclear imaging after DNA fluorescence staining only.

[0027] Figure 3 This is a schematic diagram of the microscopic imaging results of chromosome morphology in Alexandrium microphyllum cells, where... Figure 3 a represents the cell nuclear imaging results after expansion treatment using the method described in Example 3 and subsequent DNA fluorescence staining. Figure 3 b shows the results of nuclear imaging after DNA fluorescence staining only.

[0028] Figure 4 This is a schematic diagram of the microscopic imaging results of chromosome morphology in red tide algae cells. Figure 4 a represents the cell nuclear imaging results after expansion treatment using the method described in Example 4 and subsequent DNA fluorescence staining. Figure 4 b shows the results of nuclear imaging after DNA fluorescence staining only.

[0029] Figure 5 This is a schematic diagram of the microscopic imaging results of chromosome morphology in cells of the highly toxic Alternaria leptospira. Figure 5 a represents the cell nuclear imaging results after expansion treatment using the method described in Example 5 and subsequent DNA fluorescence staining. Figure 5 b shows the results of nuclear imaging after DNA fluorescence staining only.

[0030] Figure 6 This is a schematic diagram of the microscopic imaging results of chromosome morphology in *Gymnodinium chaineri* cells, where... Figure 6 a represents the cell nuclear imaging results after processing using the method described in Example 6 and undergoing DNA fluorescence staining. Figure 6 b shows the results of nuclear imaging after DNA fluorescence staining only. Detailed Implementation

[0031] This invention involves molecularly anchoring fixed dinoflagellate cells to allow chromosomes to connect with an expanded hydrogel network. Subsequently, denaturation treatment is used to eliminate protein-protein interactions, achieving isotropic expansion of the gel. This allows the chromosomes to be significantly enlarged in spatial scale while maintaining their relative spatial configuration. Combined with fluorescent dyes, this enables high-resolution visualization of dinoflagellate chromosome morphology.

[0032] This invention provides a method for clearly observing the morphology of dinoflagellates chromosomes under conventional optical microscopy or laser confocal microscopy by physically swelling dinoflagellate cells and combining this with DNA fluorescence staining.

[0033] This invention is applicable to various dinoflagellates, both with and without crusts, and can be used in research and application scenarios such as dinoflagellate chromosome structure analysis, karyotype analysis, cell cycle research, and functional gene chromosome localization.

[0034] Example 1: Alexandrium chainii ( Alexandrium catenella ), with carapace (1) Dinoflagellate cell fixation: Alexandrium chainii was cultured at 18±1℃ in f / 2 medium. When it was in the exponential growth phase, the culture medium was collected. About 1 mL of culture medium was taken and the algal cells were collected by low-speed centrifugation (1700×g, 4 min). The supernatant was discarded and the cells were gently washed twice with 1×PBS buffer. Then, fixation solution was added. The fixation solution was PBS buffer solution containing 3% formaldehyde and 0.1% glutaraldehyde. The cells were fixed at 25℃ for 15 min to maintain the stability of the cell nucleus and the overall structure of the chromosome.

[0035] (2) Anchoring treatment: After fixation, the algal cells were resuspended in the anchoring reaction solution containing 1% acrylamide and 0.7% formaldehyde. The solution was incubated at 37°C for 2 hours to allow the chromosome proteins to be linked to the acrylamide and covalently connected to the gel network formed subsequently.

[0036] (3) Gelation treatment: After anchoring, the cells were placed in the gel reaction chamber and a swellable hydrogel solution was added. The swellable hydrogel solution contained 188 g / L sodium acrylate, 100 g / L acrylamide, N,N′-(1,2-dihydroxyethylene)diacrylamide (DHEBA) with a mass concentration of 1.0 g / L, ammonium persulfate (APS) with a mass concentration of 2.5 g / L, and N,N,N,N-tetramethylethylenediamine (TEMED) with a mass concentration of 2.5 g / L. The gelation reaction was carried out at 37°C for 1.5 h.

[0037] (4) Denaturation treatment: After the gel is formed, it is placed in denaturation buffer, which is a buffer solution containing 200mM sodium dodecyl sulfate, 200mM sodium chloride and 50mM Tris. After incubation at 37°C for 30min, the temperature is raised to 76°C for 1h to achieve full expansion of the internal structure of the cell.

[0038] (5) Hydration and swelling treatment: The denatured gel sample was placed in ultrapure water for hydration and swelling. The ultrapure water was then replaced multiple times to make the gel swell evenly until the gel volume no longer changed significantly and the cell linear expansion factor was about 3 times.

[0039] (6) DNA fluorescence staining: DAPI staining was performed on the swollen gel sample at a concentration of 5 mg / L. The sample was incubated at 25°C in the dark for 30 min. After staining, the sample was washed with PBS buffer and then placed back into ultrapure water to restore its swollen state.

[0040] (7) Microscopic imaging: The stained and expanded samples were imaged and observed using a laser confocal microscope (Zeiss LSM 900 confocal microscope).

[0041] The results showed that the spatial separation between chromosomes of *Alexandrium chaineri* was significantly improved after swelling treatment, and the morphology of multiple chromosomes could be clearly distinguished under a 40× objective lens (see...). Figure 1 a).

[0042] Example 2: Alexandrium paciensis ( Alexandrium pacificum ), with carapace Alexandrium paclitaxum was treated using the same method as in Example 1. The expansion factor was approximately 3 times. After expansion, the cell nucleus exhibited a U-shaped structure, the chromosomes were clearly arranged, and multiple relatively long chromosome structures could be observed (see Example 1). Figure 2 a).

[0043] Example 3: Alexandrium microphyllum ( Alexandrium minutum ), with carapace The microalgae were treated using the same method as in Example 1. After swelling, the cells were smaller in volume but showed significantly improved chromosome segregation, with fewer and shorter chromosomes, making them suitable for comparing karyotype differences among different Alexandrium species (see Example 1). Figure 3 a).

[0044] Example 4: Red tide algae ( Akashiwo sanguinea ( ), without shell Red tide algae were treated using the same method as in Example 1, resulting in a linear expansion of 4–5 times. After expansion, the cell nucleus volume increased significantly, and the chromosomes exhibited a relatively uniform dispersed arrangement. Individual chromosomes could be clearly observed under a 40× objective lens (see Example 1). Figure 4 a).

[0045] Example 5: Highly toxic Calvatia ( Karlodinium veneficum ( ), without shell The highly toxic *Carya calcei* was treated using the same method as in Example 1. The cell nuclei of *Carya calcei* were clearly outlined, the chromosomes were distributed in short rod-like shapes, and the staining signals were well separated, making it suitable for qualitative analysis of chromosome number and relative length (see Example 1). Figure 5 a).

[0046] Example 6: Chain-like Gymnodinium ( Gymnodinium catenatum ( ), without shell Treating *Gymnodinium fasciatus* using the same method as in Example 1 resulted in a linear expansion factor of over 5 times. Given the large number of chromosomes and significant differences in their length, the expansion treatment significantly improved the spatial separation between chromosomes, making it possible to analyze complex karyotypes (see Example 1). Figure 6 a).

[0047] Comparative Example The dinoflagellates from Examples 1-6 were subjected to fluorescent staining only. Results are shown below. Figure 1 b- Figure 6 b shows that, without cell swelling treatment, the fluorescence signals of chromosomes in the nuclei of dinoflagellates are diffuse or highly overlapping, with insufficient spatial separation between individual chromosomes, making it difficult to distinguish the morphology and arrangement characteristics of individual chromosomes; however, after swelling treatment, the relatively separated chromosome morphology can be clearly observed under the same objective magnification. Figure 1 a-6a).

Claims

1. A method for observing the morphology of dinoflagellate chromosomes, characterized in that, Dinoflagellate cells were molecularly anchored, and then gelled to form a hydrogel containing the cells. The hydrogel containing the cells was denatured and hydrated to expand, and then DNA fluorescence staining was performed. The morphology of the dinoflagellates chromosomes was observed by optical microscopy.

2. The method according to claim 1, characterized in that: Includes the following steps: (1) Dinoflagellate cell fixation: Dinoflagellate cells were fixed to obtain fixed cells; (2) Anchoring treatment: Molecular anchoring treatment is performed on the fixed dinoflagellate cells to make the chromosomes or DNA-bound proteins stably bound to polymerizable groups; (3) Gelation treatment: The anchored dinoflagellates are perfused into an expandable hydrogel solution to initiate a polymerization reaction and form a hydrogel that embeds the cells; (4) Denaturation treatment: The hydrogel sample is denatured; (5) Hydration and swelling treatment: The denatured sample is induced to undergo isotropic swelling of the hydrogel under hydration conditions, thereby amplifying the spatial scale of the dinoflagellate chromosome; (6) DNA fluorescent staining: DNA fluorescent staining is performed on the swollen sample; (7) Microscopic imaging: The morphology of dinoflagellates chromosomes can be observed through optical microscopic imaging.

3. The method according to claim 1 or 2, characterized in that: The dinoflagellates are both crusted and non-crusted dinoflagellates.

4. The method according to claim 2, characterized in that: Step (1) Fix the dinoflagellate cells with a fixative solution; the fixation temperature is 25℃±1℃ and the time is 10-15min; the fixative solution is one or more of formaldehyde, paraformaldehyde, glutaraldehyde, methanol or ethanol.

5. The method according to claim 2, characterized in that: Step (2) The fixed dinoflagellate cells are resuspended in the anchoring reaction solution for anchoring treatment; the anchoring treatment temperature is 37℃ and the time is 2-8h; the anchoring reaction solution contains acrylamide and aldehydes.

6. The method according to claim 2, characterized in that: The expandable hydrogel mentioned in step (3) is an acrylate hydrogel; the acrylate hydrogel is composed of acrylate monomers, acrylamide monomers, crosslinking agents, polymerization initiators and accelerators; The crosslinking agent is N,N′-(1,2-dihydroxyethylene)diacrylamide (DHEBA) or N-dimethylacrylamide (MBAA). The polymerization initiator is ammonium persulfate (APS) or potassium persulfate (KPS); The accelerator is N,N,N,N-tetramethylethylenediamine (TEMED); The polymerization reaction was carried out at a temperature of 37°C for 1.5 hours.

7. The method according to claim 2, characterized in that: The denaturation treatment in step (4) is a structural decoupling and softening treatment; the denaturation treatment is carried out in a buffer system containing sodium dodecyl sulfate to reduce the restriction of intracellular cross-linking on gel expansion.

8. The method according to claim 2, characterized in that: Step (5) Place the denatured gel sample in ultrapure water or low ionic strength buffer for hydration and swelling until the gel volume is stable; the linear expansion ratio of the hydrogel is 2-10 times.

9. The method according to claim 2, characterized in that: The DNA fluorescent staining in step (6) uses DAPI or other nucleic acid fluorescent dyes at a concentration of 2-5 mg / L and is incubated at 25℃±1℃ in the dark for 30-60 min.

10. The method according to claim 2, characterized in that: The microscopic imaging described in step (7) includes wide-field fluorescence microscopy or laser confocal microscopy.

Images