A method for synchronously marking germ cells and interstitial cells in the testis tissue of paralichthys olivaceus

Abstract

The application discloses a method for synchronously marking germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus, which comprises the following steps: cloning MIR17HG-a (long non-coding RNA) of Paralichthys olivaceus by using a probe primer, obtaining a target plasmid after plasmid extraction, carrying out double enzyme digestion and purification to obtain an enzyme-digested plasmid as a probe template, synthesizing Paralichthys olivaceus MIR17HG-a probe by using an SP6 / T7 RNA polymerase, preparing a tissue slice from the testis tissue of Paralichthys olivaceus, and carrying out fluorescent in situ hybridization on the tissue slice by using the Paralichthys olivaceus MIR17HG-a probe, so that the Paralichthys olivaceus MIR17HG-a probe can synchronously mark the germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus. The method solves the defects of traditional marking technology, such as the need for combined marking or multiple step-by-step marking, and fills the blank of synchronous marking of germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus.

Description

Technical Field

[0001] This invention relates to the field of reproductive biology and molecular marker technology for marine fish, specifically to a method for simultaneous labeling of germ cells and mesenchymal cells in the testes of turbot. Background Technology

[0002] The cells in fish testes mainly consist of germ cells and somatic cells. Germ cells can be further classified according to their developmental stage: spermatogonia (St), spermatocytes (Sc), spermatids (Sd), and mature sperm (Sperm, Spermatozoa). Somatic cells are diverse, primarily including Sertoli cells (Se, responsible for providing nutrients to sperm) and Leydig cells (Le, responsible for synthesizing sex hormones). Other cells include peritubular myoid cells, fibroblasts, vascular endothelial cells, and macrophages. Because somatic cells play a crucial role in spermatogenesis and the maintenance of spermatogenesis, Sertoli cells and Leydig cells are the most important target cell types besides germ cells in fish reproductive development research.

[0003] The localization of fish germ cells and somatic cells typically relies on histological immunohistochemistry / immunofluorescence and in situ hybridization techniques. For example, commonly used germ cell-specific marker genes in fish testis tissue include... vasa , dazl , sycp3 and prm1 Specific marker genes for mesenchymal cells include... cyp11a1 , cyp17a1 and cyp11b2 However, when locating different cell types, it is necessary to select their respective specific markers for localization analysis.

[0004] Current research indicates that a single marker can simultaneously label multiple cell types. For example, patent document CN108330195A discloses a method and application for labeling germ cells and somatic cells at different developmental stages of the seminiferous epithelium of turbot. By amplifying the Amh / Sox9 / Gsdf fragment from the testis cDNA of turbot for probe preparation, it is possible to simultaneously label germ cells and supporting cells. However, due to the different cell types, this probe combination cannot simultaneously label germ cells and mesenchymal cells.

[0005] The role of non-coding RNAs (such as miRNAs and lncRNAs) in fish reproductive regulation has been gradually revealed, but they have not yet been used as in situ hybridization probes for spatial localization studies of fish germ cells and mesenchymal cells. MIR17HG These are the host genes and primary transcripts of the microRNA-17-92 gene cluster. The mature microRNAs (such as miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a) produced by their splicing and processing regulate cell proliferation, apoptosis, and tumorigenesis in mammals, but their expression patterns and functions in the fish reproductive system are rarely reported.

[0006] flounder ( Paralichthys olivaceus The turbot (Scourgette flounder) is an important marine aquaculture fish in northern my country, exhibiting significant sexual dimorphism, with females typically larger than males. The turbot uses an XX / XY sex determination system; during gonadal differentiation, environmental factors such as water temperature and exogenous hormones can induce sex reversal, producing pseudo-males (XX). Previous studies have found that in the testicular tissue of the turbot... MIR17HG The processed mature miRNAs mainly target and negatively regulate germ cell-specific PIWI / piRNA pathway genes by binding to the 3'UTR region of mRNA in the cytoplasm. piwil2 , tdrd12 , tdrd3 , tdrd7a ),at the same time MIR17HG The processed mature miRNAs mainly target and positively regulate key genes involved in the synthesis of mesenchymal cell-specific steroid hormones by binding to the 5' promoter region of genes in the cell nucleus. cyp17a1 , cyp21a , hsd3b7 Although the role of non-coding RNAs (such as miRNAs and lncRNAs) in reproductive regulation is gradually being revealed, they have not yet been used as in situ hybridization probes for spatial localization studies of fish germ cells and mesenchymal cells.

[0007] Therefore, there is an urgent need to develop an innovative in situ hybridization probe primer and probe for turbot to achieve synchronous labeling of germ cells and mesenchymal cells in turbot testis tissue. This would solve the shortcomings of traditional labeling techniques that require combined labeling or multiple stepwise labeling, fill the current gap in synchronous labeling of germ cells and mesenchymal cells in turbot testis tissue, and provide an efficient detection tool for marine fish reproductive development research and genetic breeding. Summary of the Invention

[0008] The purpose of this invention is to develop a method for simultaneous labeling of germ cells and mesenchymal cells in the testis tissue of turbot. This aims to achieve simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue, overcoming the shortcomings of traditional labeling techniques that require combined labeling or multiple stepwise labeling. It fills the current gap in the simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue, providing an efficient detection tool for research on the reproductive development of marine fish and their genetic breeding.

[0009] To solve the above problems:

[0010] This patent provides a MIR17HG-a probe, which includes a MIR17HG-a positive probe and a MIR17HG-a antisense probe. The sequence of the MIR17HG-a positive probe is shown in SEQ ID NO.6, and the sequence of the MIR17HG-a antisense probe is shown in SEQ ID NO.7.

[0011] Another aspect of this patent provides the application of the MIR17HG-a probe in the synchronous labeling of germ cells and mesenchymal cells in the testes of turbot.

[0012] Furthermore, the MIR17HG-a probe includes a MIR17HG-a positive probe and a MIR17HG-a antisense probe. The sequence of the MIR17HG-a positive probe is shown in SEQ ID NO.6, and the sequence of the MIR17HG-a antisense probe is shown in SEQ ID NO.7.

[0013] Another aspect of this patent provides a primer pair for detecting the MIR17HG-a probe. The primer pair includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.2, and the sequence of the reverse primer is shown in SEQ ID NO.3.

[0014] This patent also provides a method for simultaneous labeling of germ cells and mesenchymal cells in the testes of turbot, comprising the following steps:

[0015] Step A: The turbot MIR17HG-a was cloned using probe primers to obtain recombinant DNA, and the target plasmid was obtained after plasmid extraction;

[0016] Step B: Linearize and purify the target plasmid to obtain the enzyme digestion plasmid; use the enzyme digestion plasmid as a probe template, and synthesize the MIR17HG-a sense probe and MIR17HG-a antisense probe using SP6 RNA polymerase and T7 RNA polymerase, respectively. The sequence of the MIR17HG-a sense probe is shown in SEQ ID NO.6, and the sequence of the MIR17HG-a antisense probe is shown in SEQ ID NO.7.

[0017] Step C: Tissue sections were prepared using turbot testis tissue. Fluorescence in situ hybridization was performed on the tissue sections using the MIR17HG-a sense probe and the MIR17HG-a antisense probe, respectively, so that the MIR17HG-a antisense probe could simultaneously label germ cells and mesenchymal cells in turbot testis tissue.

[0018] Furthermore, in step A, the forward primer sequence of the probe primer is shown in SEQ ID NO.2, and the reverse primer sequence of the probe primer is shown in SEQ ID NO.3.

[0019] Furthermore, in step A, the cloning step includes:

[0020] Step A-1: ​​PCR amplification of turbot MIR17HG-a was performed using SEQ ID NO.2 and SEQ ID NO.3 to obtain the first PCR amplification product;

[0021] Step A-2: The first PCR amplification product is cut into a gel for recovery and purification to obtain the gel-recovered product;

[0022] Step A-3: The gel recovery product was ligated using the pMD19-T vector to obtain recombinant DNA, which was then transformed into DH5α competent cells for plasmid extraction of the target plasmid.

[0023] Furthermore, the recombinant DNA is pMD19-T-MIR17HG-a, and the sequence of the recombinant DNA is shown in SEQ ID NO.5.

[0024] Further, in step B, the linearization process involves digesting the target plasmid with restriction endonucleases NcoI and SpeI. The digestion system is as follows: 3 μg target plasmid, 5 μL 10×Buffer, 1 μL NcoI, 1 μL SpeI, and ddH2O to a final volume of 50 µL. The digestion conditions are: digestion at 37℃ for 1 h.

[0025] Further, in step B, SP6 RNA polymerase is used to synthesize the sense probe. The reaction system for synthesizing the sense probe is as follows: 10×SP6 RNA Polymerase Buffer 2 μL; 0.1% BSA 2 μL; 100 mM DTT 2 μL; DIG RNA Labeling RNA 2 μL; 40 U / μL RNase Inhibitor 0.5 μL; enzyme digestion plasmid 1 μg; SP6 RNA Polymerase 1 μL; DEPC water is added to bring the system to 20 μL; the entire process is performed on ice.

[0026] Furthermore, in step B, T7 RNA polymerase is used to synthesize the antisense probe. The reaction system for synthesizing the antisense probe is as follows: 10×T7 RNA Polymerase Buffer 2 µL; 50 mM DTT 2 µL; DIG RNA Labeling RNA 2 µL; 40 U / μL RNase Inhibitor 0.5 µL; enzyme digestion plasmid 1 μg; T7 RNA Polymerase 1 mL; DEPC water is added to bring the system to 20 μL; the entire process is performed on ice.

[0027] Furthermore, the turbot MIR17HG-a probe is located upstream of the first mature miRNA-17 contained in MIR17HG-a.

[0028] Further, in step C, the fluorescence in situ hybridization step includes: after dewaxing and rehydrating the tissue sections, they are placed in a prehybridization solution at 65℃ for 1-2 h, then covered with MIR17HG-a sense probe and MIR17HG-a antisense probe respectively, and reacted in the dark at 62-70℃ for 24-72 h. Then, biotinylated mouse anti-digoxin antibody and FITC-labeled antibody are added, and the cell nuclei are stained with DAPI. After mounting, the slides are observed.

[0029] Furthermore, in step C, the results of the MIR17HG-a sense probe show no signal, while the results of the MIR17HG-a antisense probe show a signal that appears only in the results of the MIR17HG-a antisense probe. The spermatocytes are those that are clustered around the periphery of the seminiferous vesicles, have an irregular diffuse signal distribution in the cytoplasm, and have no signal localization in the nucleus. The interstitial cells are those that are scattered in the interspermic zone, have a diffuse signal distribution in the cytoplasm, and have a punctate signal localization in the nucleus.

[0030] Compared with existing technologies, it has the following beneficial technical effects:

[0031] This patent provides a method for simultaneous labeling of germ cells and mesenchymal cells in the testes of turbot. It involves designing in situ hybridization probe primers for the specific region of turbot MIR17HG-a, and after probe synthesis, for the first time labeling a long non-coding RNA probe (from turbot). MIR17HG-a The probe was applied to the synchronous labeling study of fish germ cells and mesenchymal cells, breaking through the limitations of traditional markers that can only label a single cell type or cannot simultaneously label fish germ cells and mesenchymal cells. It provides an important molecular marker for judging the developmental status of fish germ cells and provides a new and efficient tool for detecting developmental functions in fish genetic breeding. Attached Figure Description

[0032] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0033] Figure 1 The image shown is an agarose gel electrophoresis image of RNA from turbot testis tissue in the example (where M is the marker; 1-4 are all RNA from turbot testis tissue, with the 28 s band around 850 bp and the 18 s band around 500 bp).

[0034] Figure 2 The image shown is an agarose gel electrophoresis image of the first PCR amplification product in the example (where M is the marker; 1-6 are all the first PCR amplification products, with a bright band at 500 bp).

[0035] Figure 3 The image shown is an agarose gel electrophoresis image of the second PCR amplification product in the example (where M is the marker; 1 represents the second PCR amplification product, with a bright band at 500 bp).

[0036] Figure 4 The image shows an agarose gel electrophoresis image of the turbot MIR17HG-a probe in the example (where M is the marker; 1-2 are both turbot MIR17HG-a probes, with a bright band at 487 bp).

[0037] Figure 5 This is a schematic diagram of the structure of the turbot MIR17HG-a probe in the embodiment;

[0038] Figure 6 The results of fluorescence in situ hybridization of the turbot MIR17HG-a probe in the testis tissue of turbot are shown in the example (where St represents sperm cells; Sc represents spermatocytes; and Le represents mesenchymal cells). Detailed Implementation

[0039] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0040] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings:

[0041] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0042] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0043] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0044] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0045] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0046] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0047] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0048] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0049] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0050] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0051] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0052] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0053] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0054] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0056] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0057] (1) Source of sample materials

[0058] In this embodiment, the turbot was purchased from Xinrisheng Aquatic Products Company in Shinan District.

[0059] In this embodiment, the transcriptome sequencing was performed by Beijing Aovisen Gene Technology Co., Ltd.

[0060] In this embodiment, primer synthesis and PCR product sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0061] (2) Sources of reagents and consumables

[0062] Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment

[0063] ;

[0064] (3) Source of instruments and equipment

[0065] Table 2. Sources and Models of Instruments and Equipment Required for the Experiment

[0066] ;

[0067] Example

[0068] This patent provides a method for simultaneous labeling of germ cells and mesenchymal cells in the testes of turbot, comprising the following steps:

[0069] S1, turbot MIR17HG-a Cloning, the specific steps are as follows:

[0070] 1. Total RNA extraction from turbot testis tissue:

[0071] Total RNA was extracted from the testes of turbot using the Trizol method. All procedures were performed in a clean bench on ice. The procedure is as follows:

[0072] (1) Disinfect the turbot with alcohol (commercially available) and iodine (commercially available), and use high-temperature and high-pressure sterilized tweezers (commercially available) and scissors (commercially available) to cut the turbot testis tissue to obtain the gonad tissue;

[0073] (2) Tissue homogenization: Take 300 µL of Trizol into a 1.5 mL centrifuge tube, add gonadal tissue; homogenize using a homogenizer, then add 700 µL of Trizol, mix well and let stand for 5 min; after standing, centrifuge at 4℃ and 12000 rpm for 5 min. Place the supernatant into a 1.5 mL centrifuge tube, discard the precipitate, and retain the supernatant.

[0074] (3) RNA isolation: Add 200 µL of chloroform to the supernatant obtained in step (2) above, mix well, and let stand for 15 min; after standing, centrifuge at 4℃ and 12000 rpm for 15 min. After centrifugation, the solution is divided into three layers, with RNA present in the uppermost aqueous phase. Transfer the upper aqueous phase to a 1.5 mL centrifuge tube and discard the residual solution.

[0075] (4) RNA precipitation: Add 500 µL of isopropanol (pre-cooled to -20℃) to the upper aqueous phase obtained in step (3) above, mix well and place at -20℃ for 60 min to precipitate; then centrifuge at 4℃ and 12000 rpm for 10 min; after centrifugation, discard the supernatant and retain the precipitate.

[0076] (5) RNA elution: Take 1 mL of 75% ethanol (pre-cooled at -20℃) into the centrifuge tube of step (4) above, and gently tap the bottom of the tube to suspend the precipitate; then centrifuge at 4℃ and 8000 rpm for 5 min; after centrifugation, the RNA settles at the bottom of the tube and the supernatant is discarded.

[0077] (6) RNA redissolving: After centrifugation in step (5) above, let the centrifuge tube air dry for 5-10 min, take 42.5 µL DEPC water (preheated to 56℃) to dissolve the RNA precipitate, and incubate in a metal bath at 56℃ for 1-2 min.

[0078] (7) RNA purification: DNA in RNA was removed using a DNase I kit (all reagents are provided with the kit). The reaction system was as follows: 20-50 μg of RNA dissolved in step (6) above, 5 μL of 10×DNase I Buffer, 2 µL of DNase I (concentration of 10 U), 0.5 µL of RNase Inhibitor (concentration of 20 U), and DEPC water was added to bring the volume to 50 µL. The purified RNA was obtained.

[0079] (8) Quality control and preservation of RNA: The OD value and concentration of purified RNA were measured using an ultra-micro UV spectrophotometer, and the quality of RNA was tested. The qualified RNA was stored in a -80℃ freezer for later use, which is the total RNA of turbot testis tissue.

[0080] The results are as follows Figure 1 As shown, total RNA was completely extracted from the testes of turbot. The RNA bands showed that the 28 s band was around 850 bp and the 18 s band was around 500 bp.

[0081] 2. Reverse transcription

[0082] cDNA was synthesized using the All-In-One 5× RT Master Mix reverse transcription kit (all reagents described below are included in the kit). The reverse transcription system consisted of: 4 μL of All-In-One 5× RT Master Mix, 1 μg of total RNA extracted from turbot testis tissue in step 1 above, and DEPC water added to a final volume of 50 µL. The reverse transcription program was: 37℃ for 30 min, 60℃ for 10 min, and storage at 4℃ to obtain turbot testis tissue cDNA.

[0083] 3. Turbot MIR17HG-a clone:

[0084] (1) Obtaining turbot MIR17HG-aTotal RNA was extracted from the testes and ovaries of turbot (using the same method as in step 1 above) and sent to Beijing Aovisen Gene Technology Co., Ltd. for transcriptome library sequencing. After the RNA samples passed quality control, rRNA was removed, strand-specific libraries were prepared, and after quality control, sequencing was performed (Illumina HiSeq sequencing platform). The raw sequencing data were quality controlled (accuracy, GC content, adapter removal), and the filtered sequencing sequences were aligned to the turbot reference genome using Hisat2 software. Transcripts were assembled and quantified using StringTie software. The assembled transcript sequences were identified as long non-coding RNAs (lncRNAs), including: 1) longer than 200 bp and containing at least two exons; 2) not belonging to other known types of non-coding RNAs (rRNA, tRNA, snRNA, snoRNA, pseudogene, etc.) and mRNA; 3) lacking coding potential, including CPC analysis, CNCI analysis, CPAT analysis, and pfam protein structure analysis. The final selected turbot lncRNA transcript sequence was annotated using the NCBI nucleotide collection-nr / nt database, yielding the turbot MIR17HG-a transcript sequence, which is 2777 bp in length (SEQ ID NO. 1). This sequence is located on chromosome 1 of the turbot genome (ASM2471397v2) at positions 21799322-21803478 bp. Turbot MIR17HG-a contains two exons (1-579 bp and 579-2777 bp) and shares 73.82% similarity with the human MIR17HG transcript (NR_027350.2). In conclusion, those skilled in the art can obtain the turbot MIR17HG-a transcript sequence, which is 2777 bp in length (SEQ ID NO. 1), using the aforementioned transcriptome sequencing steps.

[0085] (2) According to step (1) above MIR17HG-a Probe templates were designed to amplify primers based on specific regions of the nucleic acid sequence. The primer sequences are shown in Table 3.

[0086] Table 3 MIR17HG-a Nucleic acid amplification primer sequence list

[0087] ;

[0088] (3) Using the turbot testis tissue cDNA obtained in step 2 above as a template, the first PCR amplification reaction was carried out using the amplification primers shown in Table 3. The system (25 μL system) for the first PCR amplification reaction was designed as follows:

[0089] Taq DNA polymerase 0.125 µL;

[0090] Buffer (Mg) 2+ 10×) 2.5 µL;

[0091] dNTP (2.5 mM) 2 µL;

[0092] Forward primer (MIR17HG-a-ISH-Fw) 0.5 µL;

[0093] Reverse primer (MIR17HG-a-ISH-Rv) 0.5 µL;

[0094] Template cDNA 1µL;

[0095] Add ddH2O to 25 µL.

[0096] (2) The procedure for the first PCR amplification reaction is as follows:

[0097] Pre-denaturation at 95℃ for 5 min;

[0098] Denaturation at 95℃ for 30 seconds;

[0099] Anneal at 56℃ for 30 seconds;

[0100] Extend at 72℃ for 1 min, for a total of 35 cycles from denaturation to extension;

[0101] Extend the heat at 72℃ for 5 minutes.

[0102] Store at 4°C.

[0103] After the first PCR amplification reaction, the first PCR amplification product was obtained. The first PCR amplification product was detected by 1.5% agarose gel electrophoresis (second agarose gel electrophoresis).

[0104] The results of the second agarose gel electrophoresis are as follows: Figure 2 As shown, M is the marker; 1-6 are the target sequences of the first PCR amplification product, with a bright band at 500 bp.

[0105] (4) Gel recovery: The first PCR amplification product from step (3) above was purified by gel recovery using an agarose gel DNA recovery kit (all reagents mentioned below are included in the kit); the specific steps are as follows:

[0106] 1) Gel cutting: Under UV light, quickly cut the agarose gel block containing the target DNA fragment using a clean, sharp blade. Place the gel block into a clean 1.5 mL centrifuge tube of known weight, weigh it, and calculate the weight of the gel block.

[0107] 2) Sol: Add the sol solution to the centrifuge tube from step 1) above, and incubate the centrifuge tube in a water bath or metal bath at 50-60℃ for 5-10 minutes. During this time, gently invert the tube every 2-3 minutes to mix until the gel is completely dissolved. Ensure that the gel is completely dissolved and there are no visible gel lumps.

[0108] 3) DNA binding: Transfer the completely dissolved and cooled gel solution to the adsorption column (already placed in the collection tube); let stand at room temperature for 1-2 min, centrifuge at 12000 rpm for 30-60 s, and discard the waste liquid in the collection tube.

[0109] 4) Washing: Add 600-700 μL of washing buffer (already containing anhydrous ethanol) to the adsorption column; let stand at room temperature for 1 min, centrifuge at 12000 rpm for 30-60 s, and discard the waste liquid. Repeat step 4) by adding washing buffer and centrifuging once to thoroughly remove impurities; then place the adsorption column back into the empty collection tube, centrifuge at 12000 rpm with the cap open for 2 min. After centrifugation, the adsorption column can be left uncapped at room temperature to allow the ethanol to evaporate completely.

[0110] 5) Elution: Transfer the adsorption column to a new, clean 1.5 mL centrifuge tube, add 30-50 μL of elution buffer to the center of the adsorption membrane, centrifuge at 12000 rpm for 1-2 min, and the liquid collected in the centrifuge tube is the purified DNA solution, which is the gel recovery product.

[0111] (5) pMD19-T vector ligation: using pMD TM The 19-T Vector cloning kit was used to ligate the gel recovery product in step (4) above (all reagents mentioned below are provided in the kit); 0.5 µL of pMD19-T vector, 4.5 µL of gel recovery product and 5 μL of Solution I were added to a microcentrifuge tube and reacted at 16 °C for 1 h to obtain the ligation product.

[0112] (6) DH5α competent cell transformation: Remove DH5α competent cells from the -80℃ freezer and quickly place them on ice to thaw. Add the ligation product prepared in step (5) above to 100 µL of DH5α competent cells, gently tap the tube wall to mix, and let stand on ice for 30 min. After heat shock in a 42℃ water bath for 45 s, quickly place them on ice for 5 min. Then add 750 μL of LB liquid medium (without antibiotics) to the centrifuge tube, mix well, and place it in a shaker at 37℃ and 200 rpm for 40 min to recover and obtain the recovered bacterial solution. Take 50 μL of the recovered bacterial solution and spread it evenly on a medium containing Amp + Spread the resistant LB solid medium plates and invert them in a 37°C incubator overnight to obtain the spread plates.

[0113] (7) After removing the plate obtained in step (6) above, pick a single clone for shaking culture, prepare an 8× system mix according to the ordinary PCR system, and dispense it into 200 µL PCR tubes. Place 8 1.5 mL centrifuge tubes on a plate rack in a clean bench, and add 800 µL of ampoule to each 1.5 mL centrifuge tube. + For liquid LB medium, use a toothpick to pick up a single colony from the plate, dip it 4-5 times into a 200 µL PCR tube, then dip it 4-5 times into a 1.5 mL centrifuge tube, and then discard it. The 200 µL PCR tube and the 1.5 mL centrifuge tube should be labeled accordingly.

[0114] A 200 µL PCR tube was placed in a PCR instrument for a second PCR amplification (colony PCR). The reaction program for the second PCR amplification was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, for a total of 25 cycles from denaturation to extension; 72℃ for a final extension for 5 min; and storage at 4℃. After the second PCR amplification reaction, the second PCR amplification product was obtained and detected by 1.5% agarose gel electrophoresis (third agarose gel electrophoresis).

[0115] The results of the third agarose gel electrophoresis are as follows: Figure 3 As shown, M is the marker; 1 is the target sequence, which has a bright band at 500 bp.

[0116] Then, place the 1.5 mL centrifuge tube in a shaker at 37°C for incubation. Send the tube with the correct third agarose gel electrophoresis result for sequencing. The sequencing primer is the universal primer M13 for the pMD19-T vector (SEQ ID NO.4: 5'-TGTAAAACGACGGCCAGT-3').

[0117] Sequencing results indicate that the cultivar has been successfully cloned from the testis tissue of turbot. MIR17HG-a probe, among which MIR17HG- a The complete nucleotide sequence of the probe is shown in SEQ ID NO.5 (pMD19-T- MIR17HG-a ).

[0118] (8) Plasmid extraction: The strain containing the pMD19-T-MIR17HG-a sequence was inoculated into 20 mL of a solution containing 100 µg / mL plasmid. + After culturing in resistant LB liquid medium at 37℃ and 200 rpm for 12-16 h, the plasmid was extracted using the Tiangen endotoxin-free plasmid mini-extraction kit and stored at -80℃ to obtain the target plasmid.

[0119] S2. Linearization of plasmids and quantification, the specific steps are as follows:

[0120] 1. The target plasmid obtained in step S1 above was subjected to double digestion with restriction endonucleases (NcoI, SpeI). The double digestion reaction system was as follows: MIR17HG-a 3 μg, 10× Buffer 5 μL, NcoI 1 µL, SpeI 1 µL, ddH2O added to 50 µL, digested at 37℃ for 1 h to obtain the digestion product.

[0121] 2. The enzyme digestion products were then subjected to gel digestion. The enzyme digestion products were recovered and purified using an agarose gel DNA recovery kit, dissolved in DEPC-treated water, and quantified using a nucleic acid quantification instrument to obtain the enzyme digestion plasmid.

[0122] S3. Synthesize the in situ hybridization probe, the specific steps of which are shown below:

[0123] The enzyme digestion plasmid obtained in step S2 above was used as a probe template to synthesize the in situ hybridization probe using the SP6 / T7 RNA polymerase kit. All reagents mentioned below are included in the kit.

[0124] 1. The T7 antisense probe synthesis reaction system was as follows: 10×T7 RNA Polymerase Buffer 2 µL; 50 mMDTT 2 µL; DIG RNA Labeling RNA 2 µL; 40 U / μL RNase Inhibitor 0.5 µL; probe template 1 μg; T7 RNA Polymerase 1 mL; DEPC water was added to bring the system to 20 μL; the entire process was performed on ice.

[0125] 2. The reaction system for synthesizing the SP6 positive probe was as follows: 2 μL of 10×SP6 RNA Polymerase Buffer; 2 μL of 0.1% BSA; 2 μL of 100 mM DTT; 2 μL of DIG RNA Labeling RNA; 0.5 μL of 40 U / μL RNase Inhibitor; 1 μg of probe template; 1 μL of SP6 RNA Polymerase; and DEPC water to bring the system to a final volume of 20 μL. The entire process was performed on ice.

[0126] 3. Gently mix the above reaction system, briefly centrifuge, and then react overnight in a metal bath at 37°C. Add 2 μL of DNase I and then place it in 37°C for 30 min to remove DNA.

[0127] 4. Add 2 μL EDTA (0.5 M, pH 8.0) and 3 μL LiCl (4 M), and mix well.

[0128] 5. Pre-cool the anhydrous ethanol, add 75 μL of pre-cooled anhydrous ethanol, mix well, and then place at -20℃ for 2-3 h to precipitate.

[0129] 6. Centrifuge at 2000 rpm and 4℃ for 30 min.

[0130] 7. Discard the supernatant, add 100 μL of pre-cooled 70% ethanol, gently shake to suspend the precipitate, centrifuge at 12000 rpm and 4℃ for 20 min, and repeat twice.

[0131] 8. Discard the supernatant as much as possible and place it on ice for 5-10 minutes to dry the precipitate.

[0132] 9. Preheat DEPC water to 68℃, add 30 μL of preheated DEPC water to dissolve the precipitate, and incubate in a 58℃ metal bath for 7 min to obtain the probe, which is the turbot. MIR17HG-a Probe.

[0133] 10. The bands of the digoxigenin-labeled probe were detected by 1.5% agarose gel electrophoresis. The results are as follows: Figure 4 As shown, M is the marker; 1-2 are the target sequences, which have a bright band at 487 bp. The prepared probe samples are stored in a -80℃ freezer for later use.

[0134] flounder MIR17HG-a The location of the probe is as follows Figure 5 As shown, upstream of the first mature miRNA-17 contained in MIR17HG-a, turbot MIR17HG-a The positive probe sequence is shown in SEQ ID NO.6, turbot. MIR17HG-a The antisense probe sequence is shown in SEQ ID NO.7.

[0135] S4. Fluorescence in situ hybridization (FISH) of turbot testis tissue sections, the specific steps of which are as follows:

[0136] 1. Preparation of tissue sections before the experiment:

[0137] (1) Turbot testis tissue was fixed overnight in 4% paraformaldehyde. The next day, it was sequentially subjected to a methanol gradient of 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%, with each gradient lasting 2 hours. Finally, it was stored in 100% methanol at 4°C. Approximately 3 mm in volume was cut out. 3The small sample pieces were sequentially dehydrated (methanol-anhydrous ethanol for 15 min, anhydrous ethanol for 15 min), cleared (anhydrous ethanol:xylene = 1:1 for 15 min, xylene for 10 min, xylene for 5 min), and impregnated with paraffin (paraffin:xylene = 1:1 for 1.5 h, paraffin for 1.5 h, paraffin for 1.5 h). Then, the paraffin and the sample were poured together into a wax box and solidified overnight to obtain the embedded wax block.

[0138] (2) Dilute the DEPC stock solution at a ratio of 1:1000, stir overnight, soak the pipette tips, 1.5 mL centrifuge tubes and 24-well plates in the stirred DEPC water overnight, and then sterilize them with clean DEPC water at 121°C for 40 min.

[0139] (3) Soak the round glass slide in heavy acid overnight, then rinse it with triple-distilled water. Place the soaked round glass slide, glass culture dish and tweezers in an oven at 180℃ for 6 h. Then add 1× polylysine under the round glass slide and place it in an oven at 60℃ for 2 h.

[0140] (4) Trim the embedded wax block in step (1) with a knife, cut the wax block into small pieces with a thickness of 5 μm using a microtome, spread the pieces in DEPC water at 45℃, pick up the pieces with the round glass slides prepared in step (3) above, and place them in an incubator at 37℃ overnight to dry.

[0141] 2. Fluorescence in situ hybridization:

[0142] (1) Prepare a humidification chamber: Place 35 mL each of 5×SSC (pH 7.5) and formamide into the humidification chamber.

[0143] (2) Mix 30% H2O2 with pure methanol at a volume ratio of 1:9 to obtain the first mixture. Use the mixture to treat the wet box at room temperature for 10 min, and then wash it with DEPC water 3 times, 1 min each time.

[0144] (3) Place the slide in a humidified chamber, drop 0.25% hydrochloric acid onto the tissue, leave at room temperature for 15 min, and wash twice with DEPC water for 1 min each time (hydrochloric acid can neutralize charged basic proteins and reduce background).

[0145] (4) By covering the tissue with proteinase K and in a molecular hybridization instrument at 37°C for 20 min, proteinase K can digest and expose the masked target nucleic acid, thereby increasing the binding efficiency of the probe.

[0146] (5) Wash with 0.2% or 0.1 mol / L glycine solution for 1 min (prepare fresh before use) to stop proteinase K, and wash twice with PBS for 1 min each time.

[0147] (6) Fix the tissue with 4% PFA paraformaldehyde for 10 min, and wash it with PBS 3 times for 1 min each time.

[0148] (7) Wash twice at room temperature for 5 min each time with acetic anhydride (pH 8.0, acetic anhydride solution preparation: add 126 μL of acetic anhydride to every 50 mL of triethanolamine aqueous solution, prepare fresh for use, prepare according to the ratio, acetylation reduces background) and then wash five times with PBS for 1 min each time.

[0149] (8) Wash twice with 5×SSC (pH 7.5), 1 min each time.

[0150] (9) Place the slides in a humidified box, cover the tissue with prehybridization solution, and prehybridize at 65°C for 1 h.

[0151] (10) Using 500 ng / mL turbot MIR17HG-a Cover sections with probes (sense probes and antisense probes are covered separately), and react in the dark at 62-70℃ for 24-72 h in a hybridization apparatus. The reaction temperature and reaction time depend on the different tissues and probes.

[0152] (11) Wash once with 5×SSC (pH 7.5) at room temperature for 1 min.

[0153] (12) Mix formamide with 4×SSC (pH 4.5) at a ratio of 1:1 to obtain a second mixture. Add the second mixture and wash at 60℃ or 65℃ for 20 min each time. Then wash with PBS at room temperature for 1 min each time.

[0154] (13) Place the slices in a humidified box, cover the slices with a sealing solution, and react at room temperature for at least 30 min.

[0155] (14) Add biotinylated mouse anti-digoxin antibody, react at 37°C for 60 min, and wash three times with PBS for 5 min each time.

[0156] (15) Add FITC-labeled antibody, react at 37°C in the dark for 30 min, and wash three times with PBS for 5 min each time.

[0157] (16) Use DAPI to stain cell nuclei and wash with PBS 3 times for 5 min each time.

[0158] (17) Add antiquenching agent, cover with a coverslip, seal with nail polish, and observe under a fluorescence microscope or laser confocal microscope.

[0159] The results are as follows Figure 6As shown, spermatocytes and mesenchymal cells only appeared in the antisense probe results. Spermatocytes were clustered around the periphery of the seminiferous vesicles, exhibiting an irregular diffuse signal distribution in the cytoplasm but no signal localization in the nucleus. Mesenchymal cells were scattered in the interspermial region, exhibiting a diffuse signal distribution in the cytoplasm and a punctate signal localization in the nucleus. This indicates that the turbot prepared by the method of this embodiment... MIR17HG-a The probe can simultaneously label germ cells and mesenchymal cells in the testis tissue of turbot. Furthermore, this labeling method can also be applied to the simultaneous labeling of germ cells and mesenchymal cells in other marine fish, filling the current gap in the simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue and providing an efficient detection tool for research on the reproductive development and genetic breeding of marine fish.

[0160] Comparative Example

[0161] The difference between this comparative example and the embodiment is that this comparative example uses the Amh / Sox9 / Gsdf mark combination described in Chinese patent document CN108330195A for positioning.

[0162] The results showed that the Amh / Sox9 / Gsdf marker combination could not achieve synchronous labeling of germ cells and mesenchymal cells in turbot testes.

[0163] Therefore, it can be concluded that this patent provides a method for simultaneous labeling of germ cells and mesenchymal cells in the testes of turbot. By designing in situ hybridization probe primers for the specific region of turbot MIR17HG-a, and after probe synthesis, a long non-coding RNA probe (from turbot) is used for the first time. MIR17HG-a The probe was applied to the synchronous labeling study of fish germ cells and mesenchymal cells, which broke through the limitation of traditional markers that could only label a single cell type. It provides an important molecular marker for judging the developmental status of fish germ cells and provides a new and efficient tool for detecting developmental functions in fish genetic breeding.

[0164] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0165] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0166] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0167] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A method for synchronously labeling germ cells and somatic cells in the spermary tissue of Paralichthys olivaceus, characterized by, It comprises the following steps: Step A: cloning of MIR17HG-a of Paralichthys olivaceus by using probe primers, preparing recombinant DNA connected with a vector, and obtaining a target plasmid by plasmid extraction after transformation of competent cells; Step B: linearization treatment and purification of the target plasmid to obtain a restriction enzyme digestion plasmid; using the restriction enzyme digestion plasmid as a probe template, using SP6 RNA polymerase and T7 RNA polymerase to synthesize MIR17HG-a sense probes and MIR17HG-a antisense probes respectively, the sequence of the MIR17HG-a sense probe is shown as SEQ ID NO. 6, and the sequence of the MIR17HG-a antisense probe is shown as SEQ ID NO. 7; Step C: preparing a tissue section by using testis tissue of Paralichthys olivaceus, and performing fluorescent in situ hybridization on the tissue section by using the MIR17HG-a sense probe and the MIR17HG-a antisense probe respectively, so that the MIR17HG-a antisense probe can simultaneously label the germ cells and the interstitial cells in the testis tissue of Paralichthys olivaceus; The MIR17HG-a sense probe has no signal, and only appears in the MIR17HG-a antisense probe result, is located in the periphery of the small sperm sac and is arranged in clusters, is irregularly dispersed in the cytoplasm and has no signal positioning in the nucleus, and is a spermatocyte; is scattered in the interstitial area of the small sperm sac, is dispersed in the cytoplasm and has a point-like signal positioning in the nucleus, and is an interstitial cell.

2. The method for synchronous labeling of germ cells and interstitial cells in the testis tissue of Pseudosciaena crocea according to claim 1, characterized in that, In the step A, the forward primer sequence of the probe primer is shown as SEQ ID NO. 2, the reverse primer sequence of the probe primer is shown as SEQ ID NO.

3.

3. The method for synchronous labeling of germ cells and interstitial cells in the testis tissue of Pseudosciaena crocea according to claim 2, characterized in that, The cloning step comprises: Step A-1: PCR amplification of MIR17HG-a of Paralichthys olivaceus by using the SEQ ID NO. 2 and the SEQ ID NO. 3 to obtain a PCR amplification product; Step A-2: gel recovery and purification of the PCR amplification product to obtain a gel recovery product; Step A-3: connecting the gel recovery product by using a pMD19-T vector to obtain the recombinant DNA, and obtaining the target plasmid by plasmid extraction after transformation of DH5α competent cells.

4. The method for simultaneously labeling germ cells and interstitial cells in testis tissue of Paralichthys olivaceus according to claim 3, wherein the recombinant DNA is pMD19-T-MIR17HG-a, and the sequence of the recombinant DNA is shown as SEQ ID NO.

5.

5. The method for synchronous labeling of germ cells and interstitial cells in the testis tissue of Pseudosciaena crocea according to claim 1, characterized in that, In the step B, the linearization treatment is enzyme digestion of the target plasmid by using restriction enzymes NcoI and SpeI; the enzyme digestion system is: 3 μg of the target plasmid, 5 μL of 10×Buffer, 1 μL of NcoI, 1 μL of SpeI, and ddH2O supplemented to 50 µL; and the enzyme digestion conditions are: enzyme digestion at 37°C for 1 h.

6. The method for simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue according to claim 5, characterized in that, The SP6 RNA polymerase was used to synthesize the sense probe. The reaction system for synthesizing the sense probe was as follows: 2 μL of 10×SP6RNA Polymerase Buffer; 2 μL of 0.1% BSA; 2 μL of 100 mM DTT; 2 μL of DIG RNA Labeling RNA; 0.5 μL of 40 U / μL RNase Inhibitor; 1 μg of the enzyme digestion plasmid; 1 μL of SP6 RNA Polymerase; and DEPC water to bring the system to 20 μL. The entire process was performed on ice.

7. The method for simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue according to claim 6, characterized in that, The T7 RNA polymerase was used to synthesize the antisense probe. The reaction system for synthesizing the antisense probe was as follows: 2 µL of 10×T7 RNAPolymerase Buffer; 2 µL of 50 mM DTT; 2 µL of DIG RNA Labeling RNA; 0.5 µL of 40 U / μL RNase Inhibitor; 1 μg of the enzyme-digested plasmid; 1 mL of T7 RNA Polymerase; and DEPC water to bring the system to a final volume of 20 μL. The entire process was performed on ice.

8. The method for simultaneous labeling of germ cells and mesenchymal cells in turbot testis tissue according to claim 5, characterized in that, The turbot MIR17HG-a probe is located upstream of the first mature miRNA-17 contained in MIR17HG-a.

9. The method for synchronous labeling of germ cells and interstitial cells in the testis tissue of Pseudosciaena crocea according to claim 1, characterized in that, In step C The fluorescence in situ hybridization step includes: after dewaxing and rehydration, the tissue sections are placed in a prehybridization solution at 65°C for 1-2 h, then the tissue sections are covered with the MIR17HG-a sense probe and the MIR17HG-a antisense probe, respectively, and reacted in the dark at 62-70°C for 24-72 h. Biotinylated mouse anti-digoxin antibody and FITC-labeled antibody are then added, and the cell nuclei are stained with DAPI. After mounting, the sections are observed.

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