Probe for marking neuronal cells of gill tissue of macrobrachium hainanense and application of probe

Abstract

The invention discloses a probe for marking gill tissue neuronal cells of macrobrachium hainanense and application of the probe, and belongs to the technical field of biology. The nucleotide sequence of the probe is as shown in SEQ ID NO. 1. According to the method, the probe is designed by screening and utilizing the specific high-expression gene AChE of the macrobrachium hainanense gill tissue neuronal cells for the first time, so that accurate in-situ labeling of the cells can be realized, and cross reaction with non-neuronal cells is effectively avoided. Through a fluorescence in situ hybridization (FISH) technical process, neuronal cells are mainly distributed in a gill axis, only a small part of neuronal cells are distributed in gill filaments, and an AChE gene expression signal is enhanced under alkalinity stress, which indicates that the gene can be used as an alkali-resistant molecular marker. According to the invention, the blank of the technology for marking the neuronal cells of the gill tissue of the crustacean is filled, an important genetic target and a technical means are provided for breeding a new strain of the high-alkali-resistant crustacean, and the method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a probe for labeling neuronal cells in the gill tissue of the Hainan prawn and its application. Background Technology

[0002] The neuroendocrine system of crustaceans is mainly composed of the nervous and endocrine systems, which work in a coordinated manner to regulate growth, development, molting, reproduction, and osmotic pressure balance. This system is crucial for maintaining homeostasis under normal conditions and stress. The crustacean nervous system is a chain-like system, consisting of cerebral ganglia, subesophageal ganglia, and ventral nerve chains. Multiple ganglia are distributed along the nerve cords, which innervate sensory organs, muscles, and internal organs through peripheral nerves.

[0003] As vital respiratory and excretory organs in crustaceans, gills maintain a variety of important physiological functions. These functions primarily rely on hormonal regulation by the nervous system. Through the release of various neurotransmitters and hormones, such as crustacean hyperglycemic hormone (CHH), serotonin (5-HT), dopamine (DA), and octopamine (OA), gills regulate ion transport activity, osmotic pressure balance, and immune responses in gill epithelial cells. This is a key pathway for the body to rapidly respond to environmental stresses. Studies have shown that dopamine is widely distributed in the nervous system of crustaceans and can directly bind to ions in gill tissue. Na + / K + -ATPase It upregulates its activity and regulates osmotic pressure balance. Therefore, analyzing the nerve innervation patterns and neuronal cell distribution within the gill tissue is a core step in elucidating its physiological regulatory mechanisms. In Atlantic salmon, neurons are composed of... Notch1 , Occludin and Hes1 These three markers define clusters of neuroepithelial cells, while DOPA Decarboxylase It is specifically expressed in the neuroepithelial cells of crucian carp, zebrafish, and tilapia. However, in crustacean research, our understanding of gill neurons is still very limited, lacking specific molecular markers, and studies on their tissue distribution and function are still in their early stages.

[0004] In recent years, the application of single-cell transcriptomics technology has provided a new pathway for mining cell type-specific genes. Currently, research on single-cell sequencing in crustaceans is still in its early stages, lagging significantly behind the rapid development in vertebrate research in terms of both technological application and theoretical exploration. Although in Litopenaeus vannamei (… Litopenaeus vannamei ), Japanese shrimp ( Penaeus japonicus Single-cell transcriptome analysis of specific tissues has been preliminarily achieved in economically important species such as the Hainan prawn (Macrobrachium hainanense), but it is worth noting that single-cell research on crustaceans under highly alkaline environments is almost non-existent. Macrobrachium hainanense Belonging to the order Decapoda, family Palaemonidae, and genus Macrobrachium, it is also known as the Pujiang River Shrimp or the Big Claw Shrimp. At present, it is mainly distributed in the coastal areas of southern my country and has a strong adaptability to environmental stresses such as changes in salinity, alkalinity, and pH.

[0005] Studies have shown that a type of neuron exists in the gill tissue of the Hainan freshwater prawn, exhibiting significant changes in response to alkaline stress. These cells highly express acetylcholinesterase (AChE), suggesting its potential involvement in internal neural signal transduction and regulation. Based on these findings, a neuron-specific highly expressed gene was screened— AChE Based on this, specific molecular probes were developed. RNA fluorescence in situ hybridization (FISH) was used to locate neurons in the gill tissue of the Hainan shrimp, filling a gap in gill tissue neuronal labeling technology for this species and providing a theoretical basis for subsequent breeding of new high-alkali resistant strains. Summary of the Invention

[0006] Technical Problem Solved: To address the aforementioned technical problem, this invention provides a probe for labeling neuronal cells in the gill tissue of the Hainan freshwater shrimp and its application. This probe is targeted at... AChE Gene design can be used to detect the upregulation of gene expression under alkaline stress, providing a basis for the analysis of stress resistance mechanisms and molecular breeding.

[0007] Technical solution: In a first aspect, the present invention provides a probe for labeling neuronal cells in the gill tissue of *Macrobrachium hainanense*, the nucleotide sequence of which is shown in SEQ ID NO.1: SEQ ID NO. 1: 5'-TCCAGTTCAGAGAGGCCCACTAACA-3'.

[0008] Preferably, the probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end.

[0009] Furthermore, the fluorescent reporter group is CY3, and the quencher group is BHQ1.

[0010] Secondly, the present invention provides a kit for labeling neuronal cells in the gill tissue of Hainan shrimp, comprising the probe described in the first aspect.

[0011] Thirdly, the present invention provides the application of the probe described in the first aspect in the preparation of products for detecting neuronal cells in the gill tissue of Hainan prawns.

[0012] Preferably, the process of detecting neurons in the gill tissue of *Macrobrachium hainanense* involves using a probe to detect neurons in the gill tissue of *Macrobrachium hainanense* via RNA fluorescence in situ hybridization.

[0013] Furthermore, RNA fluorescence in situ hybridization was used to detect alkaline stress conditions. AChE Enhanced gene expression signals.

[0014] Furthermore, real-time quantitative PCR technology was used to detect alkaline stress conditions. AChE Gene expression levels increased significantly under alkaline stress conditions of 8–15 mmol / L.

[0015] Beneficial effects: This invention is the first to screen and utilize genes specifically highly expressed in neurons of gill tissue from Hainan prawns. AChE The designed probes enable precise in-situ labeling of this type of cell, effectively avoiding cross-reactivity with non-neuronal cells. Using fluorescence in situ hybridization (FISH) technology, it was found that neurons are mainly distributed in the gill axis, with only a small portion distributed in the gill filaments, and under alkaline stress... AChE Enhanced gene expression signals indicate that this gene can serve as a molecular marker for alkali tolerance. This invention fills a gap in neuronal cell marker technology for crustacean gill tissue, providing important genetic targets and technical means for breeding new strains of crustaceans resistant to high alkali conditions, and has broad application prospects. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of HE staining of gill tissue of Hainan freshwater shrimp under alkalinity stress in one embodiment of the present invention; Figure 2 This is an embodiment of the present invention of Hainan freshwater prawns under alkalinity stress. AChe Schematic diagram of fluorescent in situ hybridization of gene gill axis; Figure 3 This is an embodiment of the present invention of Hainan freshwater prawns under alkalinity stress. AChe Schematic diagram of fluorescent in situ hybridization of gene gill filaments; Figure 4 This is an embodiment of the present invention of Hainan freshwater prawns under alkalinity stress. AChe A diagram illustrating gene expression levels; Figure 5 This is an embodiment of the invention of Hainan freshwater prawns under different alkalinity stresses. AChe A diagram illustrating gene expression levels. Detailed Implementation

[0017] The present invention will be described in detail below with reference to specific embodiments: Example 1: Alkalinity stress experiment and sample collection of Hainan freshwater prawns This embodiment aims to establish a short-term high-alkalinity stress model in Hainan prawns and collect gill tissue 96 hours after stress to provide standardized samples for subsequent paraffin section preparation, HE staining observation, and fluorescence in situ hybridization (FISH) experiments, in order to study the early response of neurons under alkalinity stress. The operation steps are as follows: (1) Experimental animals: healthy, uniformly sized adult Hainan prawns, weighing 9.2±0.5g, were temporarily raised in the laboratory environment for one week.

[0018] (2) Stress conditions were set up: control group: tap water with normal aeration for 24 hours; stress group: water was adjusted to 10 mmol / L using NaHCO3. The experiment was conducted in a 300L recirculating aquaculture system, with 3 replicates in each group and 20 shrimp stocked in each replicate.

[0019] (3) Sampling procedure: Samples were collected 96 hours after the start of the alkalinity stress (10 mmol / L) experiment. The specific operating steps are as follows: a. Randomly select 6 individuals in good condition from each parallel group and anesthetize them in an ice-water mixture for 2-3 minutes.

[0020] b. Using sterile dissecting scissors, cut open the cephalothorax of the shrimp from the side and carefully remove the gills on both sides completely. The operation should be completed within 1 minute.

[0021] c. Immediately rinse the removed gill tissue gently with PBS water to remove blood cells and impurities.

[0022] d. Quickly immerse the cleaned gill tissue in 4% paraformaldehyde fixative and in situ hybridization fixative (4% paraformaldehyde, 0.1M phosphate buffer, DEPC water, pH 7.0-7.5) respectively, and fix at 4°C for 24-48 hours. The volume of fixative should be 10-15 times the volume of the tissue. After fixation, it can be used for subsequent paraffin embedding and sectioning. Separately, place the gill tissue directly into cryovials, flash freeze in liquid nitrogen, and store at -80°C for real-time quantitative PCR.

[0023] Example 2: Paraffin embedding, sectioning, and HE staining of gill tissue from Hainan freshwater prawns 2.1 The fixed gill tissue of the Hainan freshwater prawn obtained in Example 1 was paraffin-embedded and sectioned, and the steps are as follows: (1) Gradient dehydration: The sample is placed in an alcohol gradient for dehydration: 70% alcohol for 2 hours, 80% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol for 1 hour, and anhydrous ethanol for 1 hour.

[0024] (2) Transparency treatment: xylene for 45 min, the tissue turns a clear amber color, indicating that the transparency is complete.

[0025] (3) Wax impregnation and embedding: soft wax (52-54℃) for 1 hour, medium-hard wax for 1 hour, hard wax for 1 hour. Pour the melted paraffin wax into the mold box, then place the wax-impregnated gill tissue flat at the bottom, making sure the cut surface is facing down. Cool on a freezing table. After it has completely cooled and hardened, remove the wax block from the embedding frame and trim the wax block.

[0026] (4) Slicing: The trimmed wax block is placed stably on the paraffin microtome and sliced. The slice thickness is 5μm.

[0027] (5) Slide preparation and removal: Gently place the cut tissue sections into warm water at 42°C to flatten the tissue. Then, use a clean glass slide to remove the fully expanded tissue from the water and remove excess water. Place the slide containing the tissue in a 60°C oven for baking until the moisture in the tissue is completely dried and the paraffin is completely melted. Store at room temperature for later use.

[0028] 2.2 HE staining (1) Section preparation: Take the paraffin section of the gill tissue of the Hainan prawn prepared above, with a thickness of 5μm.

[0029] (2) Dewaxing to water: Place the sections in xylene I and xylene II for 10 minutes each to fully dewax; place them in 100% ethanol I and 100% ethanol II for 5 minutes each; place them in a gradient of 95%, 90%, 80%, and 70% ethanol for 3 minutes each; and finally rinse twice with distilled water for 1 minute each time.

[0030] (3) Hematoxylin staining: Immerse the sections completely in Harris hematoxylin staining solution and stain at room temperature for 5-8 minutes. Rinse the sections with slow-flowing tap water for 1 minute to remove excess stain.

[0031] (4) Differentiation: Immerse the slice in 1% hydrochloric acid ethanol differentiation solution, lift and insert it several times (about 2-5 seconds), and take it out immediately.

[0032] (5) Blueing: After differentiation, the slices are quickly rinsed with running water for 5 minutes, then immersed in 0.2% ammonia blueing solution for about 30 seconds, and rinsed with running water for 10 minutes.

[0033] (6) Eosin counterstaining: Immerse the blue-returned sections in eosin staining solution and stain at room temperature for 1-3 minutes.

[0034] (7) Dehydration: Place the sections into 70%, 80%, and 90% ethanol solutions in sequence and quickly lift and insert them several times (about 3-5 seconds each); place them into 95% ethanol I and 95% ethanol II for 1 minute each; place them into 100% ethanol I and 100% ethanol II for 2 minutes each; place the dehydrated sections into xylene I and xylene II for 5 minutes each to make the tissue transparent.

[0035] (8) Mounting: Remove the slide from xylene, wipe away excess liquid around the tissue with a paper towel, add 1-2 drops of neutral resin to the center of the tissue, carefully cover with a coverslip to avoid air bubbles, and let it air dry at room temperature.

[0036] (9) Results Observation and Analysis: Observation was performed under an optical microscope, and the results are as follows: Figure 1 As shown: In the control group, the gill tissue structure was intact and the epithelial cells were arranged in an orderly manner; while after 96 hours of alkaline stress, the gill filaments showed obvious swelling, hemolymph invaded the intercellular spaces, and the intercellular arrangement was disordered.

[0037] Example 3

[0038] 3.1 Probe Design and Synthesis The specific detection used in this embodiment AChe The probe for the gene has the nucleotide sequence SEQ ID NO.1: 5'-TCCAGTTCAGAGAGGCCCACTAACA-3'. The 5' end of the probe is labeled with a CY3 fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.

[0039] The probes in this embodiment were synthesized and provided by Shanghai Saive Biotechnology Co., Ltd. in accordance with the above sequence and modification requirements.

[0040] 3.2 Fluorescent in situ hybridization Using the above probe to study the neuronal cells of the gill tissue of the Hainan freshwater shrimp AChe The specific steps for gene localization using fluorescence in situ hybridization are as follows: (1) The above paraffin sections were sequentially placed in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and 50% alcohol for 5 min for dewaxing.

[0041] (2) Place the dewaxed sections in a humidified chamber (35 mL of 5×SSC, 35 mL of formamide), cover the tissue with a mixture (30% H2O2 + pure methanol, volume ratio 1:9), treat at room temperature for 10 min, then wash three times with DEPC water for 1 min each time; drop 0.25% hydrochloric acid onto the tissue, treat at room temperature for 15 min, wash twice with DEPC water for 1 min each time. Cover the tissue with proteinase K, react at 37℃ for 20 min, wash for 1 min with 0.1 mol / L glycine washing solution (glycine washing solution must be prepared fresh for use) to terminate the proteinase K reaction; then wash twice with PBS for 1 min each time. Fix the tissue with 4% paraformaldehyde solution for 10 min; wash three times with PBS for 1 min each time; wash twice with acetic anhydride solution (126 μL of acetic anhydride added to every 50 mL of triethanolamine aqueous solution, prepared fresh for use) at room temperature for 5 min each time; wash five times with PBS for 1 min each time. Wash twice with 5×SSC for 1 min each time; place the sections in a humidified chamber, cover the tissue with prehybridization solution, and prehybridize at 65°C for 1 h.

[0042] (3) The 100 μM probe stock solution was diluted 1:1000, the probe was used to cover the slide, and the slide was reacted in the dark at 65℃ for 48 h.

[0043] (4) Wash once with 2×SSC at room temperature for 1 min; prepare a mixture of formamide and 4×SSC at a volume ratio of 1:1 and wash three times at 65℃ for 20 min each time; wash five times with PBS at room temperature for 1 min each time; dilute DAPI 1000 times with DEPC-treated water and stain the nuclei for 5 min; wash three times with PBS for 5 min each time. Finally, add an anti-quenching agent, cover with a coverslip; seal with nail polish and observe under a fluorescence microscope.

[0044] The results are as follows Figure 2 and Figure 3 As shown: AChe The gene is mainly expressed on the gill axis, with only a small amount expressed on the gill filaments, indicating that neurons are mainly distributed on the gill axis and that gene expression signals are enhanced under alkaline stress.

[0045] Example 4 Real-time quantitative PCR detection AChe Gene expression level 4.1 RNA extraction using the Trizol method Take the frozen gill tissue sample above into a 1.5 mL centrifuge tube, add 1 mL of Trizol and homogenize thoroughly; add 0.2 mL of chloroform, mix vigorously, place on ice for 5 min, centrifuge at 4℃ and 12000 rpm for 15 min; take 500 μL of the upper aqueous phase into another new centrifuge tube; add an equal volume of isopropanol, mix well, place on ice for 15 min, centrifuge at 4℃ and 12000 rpm for 15 min; discard the supernatant, add 1 mL of pre-chilled 75% ethanol, centrifuge at 4℃ and 12000 rpm for 10 min, repeat the above process once; discard the supernatant, dry for 10 min; add 20 μL of RNase-free water to dissolve the RNA, and store temporarily at -80℃.

[0046] 4.2 cDNA Synthesis Use Hifair ® III. 1st Strand cDNA Synthesis Kit (Reverse Transcription Kit): Perform reverse transcription according to the following system and procedure: (1) gDNA digestion: Remove residual genomic DNA. The system is shown in Table 1 below: Table 1 gDNA digestion system , gDNA digestion procedure: Incubate at 42℃ for 2 min.

[0047] (2) The reverse transcription reaction system (20 μL) is shown in Table 2 below: Table 2 Reverse Transcription Reaction System , (3) The reverse transcription procedure is shown in Table 3 below: Table 3 Reverse transcription reaction procedure , The obtained cDNA was stored at -20℃ for later use.

[0048] 4.3 Real-time quantitative PCR reaction The embodiment used AChe The nucleotide sequences of the gene-specific primers are AChe-RT-F (SEQ ID NO.2): CATCGTCGTTGTCTCCAT; AChe-RT-R (SEQ ID NO.3): GCAGTCCAGTAGGTAGGT.

[0049] (1) Using Hieff UNICON ® Prepare a 20 µL reaction system using Universal Blue qPCR SYBR Green Master Mix reagent according to Table 4 below: Table 4. Real-time quantitative PCR reaction system

[0050] (2) The real-time quantitative PCR reaction procedure is shown in Table 5 below: Table 5. Real-time quantitative PCR reaction procedure

[0051] (3) Use 2 -△△CT The data was analyzed, and a bar chart was drawn. The results are as follows: Figure 4 As shown: Under alkalinity stress, AChe Gene expression increased significantly ( p <0.05).

[0052] Example 5

[0053] This embodiment aims to investigate the detection of different alkalinity gradients (8 mmol / L and 15 mmol / L NaHCO3) under stress using real-time quantitative PCR (qRT-PCR) technology. AChe Changes in gene expression levels.

[0054] (1) Laboratory animals and stress treatment The experimental animals were subjected to the same stress conditions as in Example 1, with the following three groups of stress conditions: Control group: Normal water body (tap water aerated for 24 hours); Low concentration stress group: The water concentration was adjusted to 8 mmol / L using NaHCO3; High concentration stress group: The water concentration was adjusted to 15 mmol / L using NaHCO3; Each group consisted of 3 parallel studies, with the same culture and sampling procedures as in Example 1. Gill tissue was collected for testing 96 hours after the stress treatment.

[0055] (2) Real-time quantitative PCR (qRT-PCR) RNA extraction, cDNA synthesis, and qPCR experimental procedures, primers (SEQ ID NO. 2 and SEQ ID NO. 3), and reaction systems were the same as in Example 4. Gill tissue samples from the control group, low-concentration stress group, and high-concentration stress group were subjected to... AChe Gene expression level detection. Results are as follows: Figure 5 As shown: AChe The gene was significantly upregulated under both 8 mmol / L and 15 mmol / L alkaline stress, indicating that the nervous system was activated to maintain homeostasis.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probe for labeling neuronal cells in the gill tissue of *Macrobrachium hainanense*, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID NO.

1.

2. The probe for labeling neuronal cells in the gill tissue of *Macrobrachium hainanense* according to claim 1, characterized in that: The probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end.

3. The probe for labeling neuronal cells in the gill tissue of *Macrobrachium hainanense* according to claim 2, characterized in that: The fluorescent reporter group is CY3, and the quencher group is BHQ1.

4. A kit for labeling neuronal cells in the gill tissue of *Macrobrachium hainanense*, characterized in that, Includes the probe as described in any one of claims 1-3.

5. The use of the probe according to any one of claims 1-3 in the preparation of a product for detecting neuronal cells in the gill tissue of *Macrobrachium hainanense*.

6. The application according to claim 5, characterized in that: The process of detecting neurons in the gill tissue of the Hainan freshwater prawn involves using probes and RNA fluorescence in situ hybridization (RNAF) to detect neurons in the gill tissue of the Hainan freshwater prawn.

7. The application according to claim 6, characterized in that: RNA fluorescence in situ hybridization combined with real-time quantitative PCR was used to detect alkaline stress conditions. AChE Gene expression levels increased, and alkaline stress conditions were 8-15 mmol / L.

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