Application of Nanos2 molecular marker system in identification and enrichment of germ stem cells in Epinephelus lanceolatus

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to the application of the Nanos2 molecular marker system in the identification and enrichment of germline stem cells from the grouper *Sinocyclocheilus saddleback*. This invention obtained the complete nanos2 mRNA sequence from *Sinocyclocheilus saddleback*, and its corresponding cDNA nucleotide sequence is shown in SEQ ID NO. 1. Based on this, this invention establishes a continuous Nanos2 molecular marker technology system suitable for *Sinocyclocheilus saddleback*, including... nanos2 RNA probes, recombinant Nanos2 protein, and anti-Nanos2 polyclonal antibodies were used. The anti-Nanos2 polyclonal antibody was applied to Percoll enriched cells for immunofluorescence identification, enabling direct, rapid, and quantitative evaluation of the distribution of Nanos2-positive cells in layers of different densities, and determining the 30%–35% Percoll layer as the optimal enrichment layer. Compared to Percoll isolation methods or RNA probe detection methods alone, this invention simultaneously supports tissue-level localization, protein-level identification, and enrichment layer determination, providing a technical foundation for subsequent research on germ cell acquisition from saddleback grouper donors, pre-transplant cell quality evaluation, and surrogate reproduction.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of the Nanos2 molecular marker system of the grouper in the identification and enrichment of reproductive stem cells. Background Technology

[0002] Grouper is an important marine aquaculture fish, mainly distributed in tropical and subtropical seas. The saddleback grouper, also known as the giant grouper, is scientifically named... Epinephelus lanceolatus The grouper is the largest species of grouper, characterized by rapid growth, large size, and high economic value in aquaculture. The saddle-tailed grouper is an important aquaculture species, often used as the paternal parent in grouper hybridization breeding to cultivate hybrid groupers with better growth performance, such as the tiger-dragon hybrid grouper, the green-dragon hybrid grouper, and the cloud-dragon hybrid grouper.

[0003] In the artificial breeding of grouper, the relative shortage of male parent fish is one of the important factors restricting fry production and hybridization breeding. Grouper often exhibit hermaphroditism and female-to-male transformation, with a long natural maturation cycle for males, making it challenging to obtain stable functional males under artificial breeding conditions. Previous studies have attempted to induce male transformation in females through androgen treatment; however, the effectiveness of induced masculinization is affected by species, age, season, treatment method, and gonadal development status. Furthermore, some induced males may suffer from unstable gonadal development or insufficient sperm production.

[0004] Fish germ cell transplantation, also known as surrogate reproduction or surrogate broodstock technology, involves transplanting primordial germ cells, spermatogonial stem cells, oogonial stem cells, or other germline cells from a donor fish into a recipient fish of the same or different species. This allows the donor-derived germ cells to colonize, proliferate, and differentiate in the recipient's gonads, ultimately producing donor-derived functional gametes. This technology can utilize fish with short reproductive cycles, easy rearing, or those that have undergone sterilization as recipients, providing a new technical pathway for the preservation and breeding of germplasm in long-cycle, large, or difficult-to-breed fish. Therefore, establishing a germ cell transplantation technology suitable for grouper holds promise for providing new insights into alleviating the problems of long male broodstock formation and difficulty in obtaining male grouper. The core of surrogate reproduction technology includes donor germ cell acquisition, sterile recipient preparation, transplantation, and donor-derived gamete formation, among which accurate identification and effective enrichment of donor germ cells are prerequisites.

[0005] Fish reproductive stem cells and primordial germ cells are important cellular materials for research on fish reproductive development, germ cell transplantation, germplasm preservation, and genetic breeding. Because these cells are present in limited numbers in gonadal tissues and are often mixed with germ cells and somatic cells at different developmental stages, they typically require molecular markers, tissue localization, immunoassay, and cell isolation techniques for identification, characterization, and enrichment.

[0006] Nanos2These are genes related to the development of reproductive stem cells in vertebrates. Previous studies have shown that reproductive stem cell populations exist in both male and female gonads of zebrafish, and these cells can express specific genes... nanos2 And be identified; relevant methods can detect nanos2 mRNA, combined with pan-germ cell markers such as Vasa, is used to locate and identify germline stem cells. Regarding fish Nanos2 antibodies, a method for preparing polyclonal antibodies that can label fish germline stem cells has been disclosed. This technique uses zebrafish Nanos2 protein as an immunogen, clones the coding region of the zebrafish Nanos2 protein into an expression vector, expresses and purifies the recombinant Nanos2 protein in E. coli, and then immunizes New Zealand white rabbits with this recombinant protein to obtain anti-Nanos2 polyclonal antibodies. This antibody can be used for immunohistochemistry and immunofluorescence detection, and can label Nanos2-positive germline stem cells in zebrafish testes and ovaries. Cross-validation was performed in the testes of Yellow River carp and crucian carp.

[0007] Regarding the isolation and purification of fish oocytes, existing technologies disclose a method for isolating and purifying turbot oocytes. This technique uses the gonads of female turbot as material. After removing the white membrane, the tissue is minced and digested using a combined enzyme solution containing 0.20%–0.30% trypsin and 0.050%–0.060% DNase I. The cell suspension is then filtered, centrifuged, and resuspended to obtain a cell suspension. Subsequently, a Percoll density gradient of 15%–25%, 30%–40%, and 45%–55% is prepared, and the cell suspension is added to the top layer of the gradient. The mixture is centrifuged at 180G–220G for 20–40 min, and the top two cell layers are collected to obtain turbot oocytes. This method also uses Vasa protein immunofluorescence to identify the oocytes, and the purified oocyte percentage can reach over 80%. In addition, existing technologies also disclose screening schemes for reproductive cell markers in other economically important aquatic animals. For example, the large yellow croaker spermatogonium-specific marker gene lhh2a.2.2 and its screening method and application were obtained through single-cell transcriptome screening. In situ hybridization, Vasa co-localization, and PCNA co-localization demonstrated that this gene can identify spermatogonia in the proliferative state. This technology also proposes that this marker gene can be used for large yellow croaker spermatogonium transplantation, stem cell culture, and genetic breeding. Another example is a scallop spermatogonium surface marker protein and its application in genetic breeding. The scallop spermatogonium surface marker protein Fgfr and its coding sequence were disclosed, along with recombinant proteins, recombinant expression vectors, engineered strains, antibody products, and applications in detecting scallop spermatogonia through immunohistochemistry or immunofluorescence. However, due to the species specificity of aquatic animals, there is currently a lack of Nanos2-based molecular marker systems for reproductive stem cells in grouper, and its application in the identification and enrichment of reproductive stem cells in saddle-tailed grouper has not been observed. Summary of the Invention

[0008] The purpose of this invention is to provide the application of the Nanos2 molecular marker system of the saddleback grouper in the identification and enrichment of reproductive stem cells, so as to solve the problem of the lack of species-specific molecular identification tools for Nanos2 positive reproductive cells of the saddleback grouper in the prior art.

[0009] This invention provides the application of the Nanos2 molecular marker system of the grouper in the identification and enrichment of reproductive stem cells. The Nanos2 molecular marker system of the grouper is constructed using the nucleotide sequence shown in SEQ ID NO:1.

[0010] Preferably, the Nanos2 molecular marker system includes at least one of the following (1)-(3): (1) nanos2 RNA probe; (2) Recombinant Nanos2 protein; (3) Anti-Nanos2 polyclonal antibody.

[0011] Preferably, in (1), the nanos2 RNA probes include: antisense RNA probes and sense RNA probes; The template for the antisense probe was obtained by amplification using the primer pairs shown in SEQ ID NO: 11 and SEQ ID NO: 12; the template for the positive probe was obtained by amplification using the primer pairs shown in SEQ ID NO: 13 and SEQ ID NO: 14.

[0012] Preferably, in (2), the preparation method of the recombinant Nanos2 protein is as follows: the coding region of the gene shown in SEQ ID NO:1 is cloned into the expression vector PET-28a, and then the recombinant expression vector pET-28a-nanos2 is transformed into Escherichia coli for expression, and the expression product is purified to obtain the protein.

[0013] Preferably, in (3), the anti-Nanos2 polyclonal antibody is prepared using the recombinant Nanos2 protein described in (2) as an immunogen.

[0014] This invention provides a method for identifying and enriching reproductive stem cells of the grouper saddleback, comprising the following steps: S1. The gonadal tissue of the saddle-banded grouper is sequentially digested with a compound enzyme solution, filtered through a cell sieve, and centrifuged and resuspended to obtain a gonadal cell suspension; the compound enzyme solution includes trypsin, collagenase, and DNase I solution. S2. After preparing the Percoll density gradient solution, add the gonadal cell suspension from S1 to the top layer of the Percoll density gradient solution and centrifuge to obtain cell suspensions of different layers. S3. The cell suspensions at different levels were blocked in sequence, and the primary antibody was incubated with the anti-Nanos2 polyclonal antibody as described in the above technical solution. The secondary antibody was then incubated with fluorescence, stained and observed under a microscope to identify the reproductive stem cells of the saddle-banded grouper. S4. Based on the identification results in S3, collect cell suspensions containing saddle-banded grouper reproductive stem cells to enrich saddle-banded grouper reproductive stem cells.

[0015] Preferably, in step S3, when performing primary antibody incubation, the primary antibody incubation solution is obtained by mixing blocking solution and anti-Nanos2 polyclonal antibody; The blocking solution uses PBS as a solvent and contains 2% w / v BSA, 0.5% v / v Triton X-100 and 1% v / v DMSO.

[0016] Preferably, the concentration of the anti-Nanos2 polyclonal antibody is 1.64 mg / mL, and the titer is >512K; In the primary antibody incubation solution, the volume ratio of blocking solution to anti-Nanos2 polyclonal antibody is 499:1.

[0017] Preferably, in step S2, the volume concentration of the Percoll density gradient solution is 50%~60%, 40%~50%, 35%~40%, 30%~35%, 25%~30%, and 20%~25% from bottom to top.

[0018] Preferably, after collecting cell suspensions at 30%~35% and 25%~30% levels, reproductive stem cells of the grouper are enriched.

[0019] Beneficial effects: (1) This invention obtained the nucleotide sequence of the grouper saddleback as shown in SEQ ID NO:1 by conserved fragment amplification, 5'RACE, 3'RACE and open reading frame verification. nanos2 The complete mRNA sequence provides the sequence basis for establishing the Nanos2 marker system for this species. Furthermore, this invention establishes a continuous Nanos2 marker technology system suitable for the saddleback grouper, which includes the saddleback grouper. nanos2 Complete mRNA sequence nanos2 RNA probes, recombinant Nanos2 protein, and anti-Nanos2 polyclonal antibodies can be used for the identification and enrichment of reproductive stem cells in grouper.

[0020] (2) This invention is based on the saddle-banded grouper. nanos2 RNA probes were prepared by sequencing and a system was established. nanos2 A method for detecting mRNA tissue localization. In situ hybridization was used to demonstrate... nanos2The mRNA is mainly distributed in the gonads of the saddle-banded grouper, in a cell population with early germ cell or germ cell-like morphological characteristics, providing a tissue localization basis for Nanos2 as a germ cell marker in the saddle-banded grouper.

[0021] (3) This invention prepared a polyclonal antibody against Nanos2 protein in grouper, realizing the expansion from RNA-level detection to protein-level detection. Existing RNA in situ hybridization is mainly applicable to tissue section localization, while the anti-Nanos2 polyclonal antibody prepared in this invention can be used for immunofluorescence detection of free cell populations after Percoll separation, thereby identifying Nanos2-positive cells in different density layers.

[0022] (4) This invention uses anti-Nanos2 polyclonal antibodies for immunofluorescence identification of Percoll-enriched cells, which can directly, rapidly, and quantitatively evaluate the distribution of Nanos2-positive cells in different density layers after Percoll separation, and determine the 30%~35% Percoll layer as the optimal enrichment layer. Compared with the Percoll separation method alone or the RNA probe detection method alone, this invention can simultaneously support tissue-level localization, protein-level identification, and enrichment layer determination, providing a technical basis for subsequent research on the acquisition of germ cells from saddleback grouper donors, pre-transplant cell quality evaluation, and surrogate reproduction. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Picture 1 Electrophoretic images of different treatments provided for this invention (a is) nanos2 Agarose gel electrophoresis image of PCR amplification products of conserved fragments; b is nanos2 Agarose gel electrophoresis image of the second-round PCR amplification product of the 5'RACE clone; c is nanos2 Agarose gel electrophoresis image of the second-round PCR amplification products of the 3'RACE clone; d is nanos2 Agarose gel electrophoresis image of the PCR amplification product of the complete ORF sequence; M is the marker). Picture 2 The gonadal tissue of the saddle-banded grouper provided by the present invention nanos2 In situ hybridization results of mRNA (left figure shows the results of the antisense probe group; right figure shows the results of the sense probe group). Picture 3The SDS-PAGE images provided for this invention are as follows: (a) SDS-PAGE images of the induced expression of recombinant Nanos2 protein at 16℃ / 16h and 37℃ / 4h, respectively, where M is the marker and C is the uninduced strain (negative control); (b) SDS-PAGE image of purified Nanos2 protein. Picture 4 The Percoll enriched cell immunofluorescence identification based on anti-Nanos2 polyclonal antibody provided by the present invention (a is the result of Percoll discontinuous density gradient centrifugation of gonad cell suspension of grouper saddleback; b is the result of immunofluorescence of different cell layers using anti-Nanos2 protein antibody of grouper saddleback). Detailed Implementation

[0025] Unless otherwise specified, the experimental methods described in the following examples were performed in accordance with conventional methods in the art; and the materials and reagents used, unless otherwise specified, were all commercially available products.

[0026] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Saddleback Grouper nanos2 Obtaining the complete mRNA sequence Using gonadal tissue from *Siniperca saddlebrook* as material, conserved fragment amplification, 5'RACE, 3'RACE, and open reading frame verification were performed to obtain *Siniperca saddlebrook*. nanos2 The complete mRNA sequence of the gene is as follows: 1. Experimental materials and total RNA extraction A 2.5-year-old grouper with a saddleback fin was dissected, and its gonadal tissue was isolated, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. A suitable amount of gonadal tissue was collected, and total RNA was extracted using the TRIzol method. The integrity of the extracted total RNA was assessed by agarose gel electrophoresis, and the RNA concentration and purity were determined using a nucleic acid and protein analyzer. The qualified total RNA was used for subsequent cDNA synthesis and RACE amplification.

[0028] 2. nanos2 Amplification of conserved fragments According to the leopard gill spiny perch Plectropomus leopardus and oblique grouper E. coioides of nanos2 Primers were designed from conserved sequence regions to amplify the saddle-banded grouper. nanos2 Conserved fragment. The primers used are as follows: Cons_F (SEQ ID NO.3):5'-tgcttcgacatgtggca-3'; Cons_R (SEQ ID NO.4): 5'-acagatgggacaggtgtag-3'; PCR amplification was performed using cDNA obtained by reverse transcription with random primers as a template. The PCR reaction volume was 25 μL, including 12.5 μL of 2×PCR Master Mix, 1.0 μL of upstream primer, 1.0 μL of downstream primer, 1.0 μL of cDNA template, and ddH2O to a final volume of 25 μL.

[0029] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 25 s, for a total of 34 cycles; 72℃ final extension for 5 min, and storage at 12℃.

[0030] The PCR product was detected by 1.5% agarose gel electrophoresis, yielding a specific band of 555 bp. The electrophoresis results are shown below. Picture 1 a. The target band was recovered, ligated into a T-vector, transformed into competent cells, and positive clones were selected for sequencing. Sequencing results showed that the obtained fragment was 555 bp in length and belonged to the grouper *Spodoptera litura*. nanos2 Conserved gene regions sequence.

[0031] 3. nanos2 Cloning of the 5' end sequence of a gene According to the obtained nanos2 5' RACE-specific primers were designed based on conserved fragment sequences to amplify the saddle-banded grouper. nanos2 The 5' end sequence of the gene. The primers used are as follows: nanos2_5'race_GSP1 (SEQ ID NO.5):5'-tcctcccaagatgccctgaagt-3'; nanos2_5'race_GSP2 (SEQ ID NO.6):5'-tcgaagccctccatgtctgt-3'; 5'RACE cDNA was prepared according to the instructions of the 5'RACE kit (18374-058, Invitrogen, CA, USA). Using the 5'RACE cDNA as a template, the first round of PCR amplification was performed using the specific primer nanos2_5'race_GSP1 (SEQ ID NO. 5) and the universal primer AAP. The expected amplified fragment length was approximately 331 bp.

[0032] The first round of PCR reaction consisted of 50 μL of 2×PCR Master Mix, including 25.0 μL of nanos2_5'race_GSP1, 1.0 μL of AAP primer, 2.0 μL of 5'RACE cDNA template, and ddH2O to a final volume of 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 15 s, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0033] The first-round PCR product was diluted 100-fold and used as a template for the second-round PCR. The second-round PCR amplification was performed using the nested specific primer nanos2_5'race_GSP2 (SEQ ID NO. 6) and the universal primer AUAP.

[0034] The second round of PCR reaction consisted of 50 μL of 2×PCR Master Mix, including 25.0 μL of nanos2_5'race_GSP2, 1.0 μL of AUAP primers, 2.0 μL of diluted first-round PCR product, and ddH2O to bring the total volume to 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 15 s, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0035] The second round of PCR products were detected by 1.5% agarose gel electrophoresis, and clear target bands were visible. The electrophoresis results are shown below. Picture 1 b. The target band was recovered, ligated into the T vector, and then sequenced as a positive clone. Sequencing results showed that the sequence length obtained by 5'RACE was 283 bp.

[0036] 4. nanos2 Cloning of the 3' end sequence of a gene According to the obtained nanos2 Design 3' RACE-specific primers based on conserved fragment sequences for amplification of saddle-banded grouper. nanos2 The 3' end sequence of the gene. The primers used are as follows: nanos2_3'race_GSP1 (SEQ ID NO.7):5'-gcggactgctgaagatgctgag-3'; nanos2_3'race_GSP2 (SEQ ID NO.8): 5'-cagacatggagggcttcgagaca-3'; 3'RACE cDNA was prepared according to the instructions of the 3'RACE kit (18373-019, Invitrogen, CA, USA). Total RNA was obtained from the gonadal tissue of *Gymnocypris saddle-shaped grouper*, and reverse transcription was performed using universal primer AP to obtain 3'RACE cDNA. Using the 3'RACE cDNA as a template, the first round of PCR amplification was performed using the specific primer nanos2_3'race_GSP1 (SEQ ID NO. 7) and the universal primer UAP. The expected amplified fragment length was approximately 331 bp.

[0037] The first round of PCR reaction consisted of 50 μL of 2×PCR Master Mix, including 25.0 μL of nanos2_3'race_GSP1, 1.0 μL of UAP primers, 2.0 μL of 3'RACE cDNA template, and ddH2O to a final volume of 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0038] The first-round PCR product was diluted 100-fold and used as a template for the second-round PCR. The second-round PCR amplification was performed using the nested specific primer nanos2_3'race_GSP2 (SEQ ID NO. 8) and the universal primer AUAP.

[0039] The second round of PCR reaction consisted of 50 μL of 2×PCR Master Mix, including 25.0 μL of nanos2_3'race_GSP2, 1.0 μL of AUAP primers, 2.0 μL of diluted first-round PCR product, and ddH2O to bring the total volume to 50 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0040] The second round of PCR products were detected by 1.5% agarose gel electrophoresis, and clear target bands were visible. The electrophoresis results are shown below. Picture 1 c. The target band was recovered, ligated into the T vector, and then sequenced as a positive clone. Sequencing results showed that the sequence length obtained by 3'RACE was 884 bp.

[0041] 5. nanos2 Assembly and verification of complete ORF sequences Will nanos2 Conservative fragment sequences, 5' RACE sequences, and 3' RACE sequences were compared, and the sequences were spliced ​​together based on their overlapping areas to obtain the saddle-banded grouper. nanos2Complete mRNA sequence of the gene. The splicing results indicate that the saddle-banded grouper... nanos2 The complete mRNA sequence is 1148 bp in length. To verify the spliced ​​result... nanos2 The accuracy of the complete mRNA sequence, in nanos2 Primers were designed for the non-coding regions on both sides of the open reading frame (ORF) to perform PCR validation of fragments covering the ORF. The primers used are as follows: test_ORF_F (SEQ ID NO.9): 5'-acggtctgtccaacctgatgc-3'; test_ORF_R (SEQ ID NO.10): 5'-ttcaacatagttgtctcaaggtttg-3'; PCR amplification was performed using cDNA obtained by reverse transcription from the gonadal tissue of *Gymnocypris saddle-shaped* fish as a template. The PCR reaction volume was 25 μL, including 12.5 μL of 2×PCR Master Mix, 1.0 μL of test_ORF_F, 1.0 μL of test_ORF_R, 1.0 μL of cDNA template, and ddH2O to a final volume of 25 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0042] The PCR products were detected by 1.5% agarose gel electrophoresis, yielding a specific band of approximately 1063 bp. The electrophoresis results are shown below. Picture 1 d. The target band was recovered, ligated into the T vector, and then sequenced as a positive clone. Sequencing results showed that the obtained sequence was consistent with the sequence obtained from the splicing. nanos2 The complete mRNA sequence is consistent, indicating that the grouper obtained in this embodiment is a saddle-shaped grouper. nanos2 The complete mRNA sequence of the gene is accurate and reliable.

[0043] Analysis revealed that the saddle-banded grouper nanos2 The amino acid sequence corresponding to the coding region shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2, specifically as follows: MQELLRQTGLHGSLLPDGECFDMWHDYMNLGGLLKMLSGNHDLGYTDMEGFETEAAAPWSYFRASWEEDFKNYYGTSSPSGSLLGTTSPLTMSGTTSQSSSLLGTSSAFTLPQAASSRST SFSGTTSPRSTTLCGTTSPRSSSLSGPASPSSLSDSSCSEASSLYCPFCRHNGETAKVYRSHNLKSEDGKVSCPILYNYTCPICEATGDNAHTRSYCPQARRPDAAGILPWLRLRARKGN.

[0044] Example 2: Preparation and tissue localization detection of nanos2 RNA probe from saddleback grouper. To verify the saddle-banded grouper obtained in Example 1 nanos2 To determine whether the sequence (SEQ ID NO. 1) is a truly expressed endogenous transcript in gonadal tissue and to further clarify its distribution in different gonadal cell types, in situ hybridization was performed on frozen sections of gonads from the saddleback grouper using a digoxigenin-labeled RNA probe. Antisense probes were also used for detection. nanos2 mRNA expression was assessed, with a positive probe serving as a negative control.

[0045] 1. Probe Design and Fabrication The saddle-shaped grouper obtained according to Example 1 nanos2 Primers for in situ hybridization probe amplification were designed based on the sequence (SEQ ID NO. 1). Primers for antisense probe template amplification are as follows: Nanos2_ISH_F (SEQ ID NO.11): 5'-ACTACATGAACCTGGGCGGACTG-3'; Nanos2_ISH_R (SEQ ID NO.12): 5'-TAATACGACTCACTATAGGGTAGCAGAGCATGACTGGAGCACAA-3'.

[0046] The reverse primer has a 5' end linked to the T7 promoter sequence for in vitro transcription to synthesize the antisense RNA probe. The primers for the positive probe control template amplification are as follows: Nanos2_ISH_F (SEQ ID NO.13): 5'-TAATACGACTCACTATAGGGACTACATGAACCTGGGCGGACTG-3'; Nanos2_ISH_R (SEQ ID NO.14): 5'-TAGCAGAGCATGACTGGAGCACAA-3'.

[0047] The forward primer has a T7 promoter sequence attached to its 5' end for in vitro transcription to synthesize a sense RNA probe. After PCR amplification to obtain the corresponding DNA template, it was detected by agarose gel electrophoresis and purified. Using the purified PCR product as a template, in vitro transcription was performed using T7 RNA polymerase, and DIG-RNA labeling mix was added to synthesize a digoxigenin-labeled RNA probe. The transcription reaction was carried out at 37℃ for 2 hours, followed by the addition of DNase I to remove the DNA template. After purification by LiCl / ethanol precipitation, the RNA probe was dissolved in DEPC water. A small amount of probe was taken for electrophoresis to confirm probe integrity before aliquoting and storage at -80℃ for later use.

[0048] 2. In situ hybridization Gonadal tissue from grouper with saddleback saddleback was fixed overnight in 4% paraformaldehyde at 4°C. The fixed tissue was then dehydrated sequentially with 5%, 10%, 15%, and 20% sucrose solutions. Subsequently, it was embedded in a 1:1 volume mixture of 20% sucrose solution and OCT to prepare frozen sections with a thickness of 12 μm. The sections were attached to adhesive slides and dried at 37°C for 30 min.

[0049] After fixation with 4% paraformaldehyde at room temperature for 20 min, the sections were washed with RNase-free PBS. Prehybridization buffer was then added, and prehybridization was performed at 68–70 °C for 15 min. DIG-labeled RNA probes were added to the hybridization buffer to a final concentration of 0.15–0.5 ng / μL, and after denaturation at 68–70 °C for 10 min, the probes were dropped onto the surface of the sections and hybridized overnight in a humidified chamber at 70 °C.

[0050] After hybridization, the probe was recovered, and the slides were washed three times in preheated (70°C) washing buffer for 30 min each time; then washed three times in TNT buffer at room temperature for 10 min each time. After blocking with blocking buffer at room temperature for 3 h, alkaline phosphatase-conjugated anti-DIG antibody was added, and the slides were incubated overnight at 4°C in the dark. The next day, the slides were washed four times in TNT buffer for 10 min each time, followed by three times in AP buffer for 5 min each time. Then, NBT / BCIP chromogenic solution was added for chromogenic development in the dark. The reaction was terminated when a clear purplish-black positive signal was observed, and the slides were mounted and photographed under a microscope.

[0051] 3. Results like Picture 2As shown, the antisense probe group exhibited clear purple positive signals in gonad sections of *Gymnocypris saddle-shaped*. Positive cells were mainly small in size, with a high nucleoplasm-to-cytoplasm ratio, and were round or oval in shape, distributed singly or in small groups. The signal was mostly located in the cytoplasm and perinuclear region. The morphological characteristics of these cells are consistent with germline stem cells or early germ cells. The sense probe control group showed no corresponding strong positive signals, only a small amount of background staining. These results indicate that... nanos2 mRNA is mainly enriched in cell populations with early germ cell or germ cell-like morphological characteristics, and can serve as one of the molecular markers for Nanos2-positive germ cells in saddle-banded grouper.

[0052] Example 3: Recombinant Nanos2 protein expression and preparation of anti-Nanos2 polyclonal antibody 1. Construction of prokaryotic expression vectors cDNA sequence of the Nanos2 protein coding region extracted from the saddle-banded grouper was introduced. Nde I and Xho The target fragment was subcloned into the prokaryotic expression vector pET-28a after I restriction site I extraction, constructing the pET-28a-nanos2 recombinant plasmid. Positive clones were selected for PCR identification and sequencing verification to ensure the target gene sequence was correct and the reading frame was accurate.

[0053] 2. Induced expression of recombinant proteins The correctly sequenced recombinant plasmid pET-28a-Nanos2 was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. The plates were incubated overnight at 37°C with the plates inverted. Single colonies were picked and inoculated into 3 mL of LB liquid medium containing kanamycin, and cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 ≈0.6-0.8. The bacterial culture was divided into two portions. One portion was incubated with 1 mM IPTG at 37°C for 4 h; the other portion was incubated with the same concentration of IPTG at 16°C overnight. A negative control (not induced) was also included. After induction, the bacterial cells were collected by centrifugation at 10,000 rpm for 1 min, 25 μL of 5× reducing loading buffer was added, and the mixture was boiled for 10 min before 12% SDS-PAGE electrophoresis. Results are shown below. Picture 3 α, Nanos2 is expressed in a precipitate at 37°C, and the protein size is approximately 30 kDa.

[0054] 3. Purification of recombinant proteins The optimal expression conditions for amplification culture were selected as 37℃ and 1mM IPTG induction for 5h. After cell collection, the cells were resuspended in lysis buffer (PBS pH 7.5 + 10% glycerol + 1mM PMSF) and sonicated (300W, 3 s operation time, 5 s interval, total time 20 min). The cells were centrifuged at 12000g for 10 min, and the supernatant and precipitate were collected separately. Since the target protein mainly exists in the form of inclusion bodies, the precipitate was resuspended in PBS (pH 7.5) containing 8M urea, sonicated again for 20 min, centrifuged at 12000g for 10 min, and the supernatant was collected. The supernatant was mixed with pretreated Ni-NTA resin and incubated at 4℃ for 30 min. After resin collection, contaminating proteins were washed sequentially with PBS (pH 7.5) containing 8M urea, and the target protein was finally eluted with PBS (pH 7.5) containing 300mM imidazole and 8M urea. The eluted fractions were collected and analyzed by SDS-PAGE. Results are shown below. Picture 3 b. The protein size is approximately 30 kDa, which is in line with expectations.

[0055] The eluents containing the target protein were combined and dialyzed into 1×PBS + 6M urea buffer. The protein concentration was determined by the BCA method. The final product, recombinant Nanos2 protein, had a concentration of 5.65 mg / mL, and its purity met the immunoassay requirements. After aliquoting, it was stored at -80℃ for later use.

[0056] 4. Animal immunization Four healthy female New Zealand white rabbits, aged 4 months and weighing approximately 2.1 kg, were selected as immunization animals. For the initial immunization, 400 μg of purified Nanos2 protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Three booster immunizations were administered on days 21, 35, and 49, with 300 μg of Nanos2 protein emulsified with an equal volume of Freund's incomplete adjuvant for each booster immunization. During the immunization period, 1 mL of blood was collected via the ear vein on day 42 (after the third immunization) and day 56 (after the fourth immunization) for titer monitoring. After confirming that the antiserum titer reached the target on day 57, whole blood was collected via the carotid artery, and the antiserum was separated and collected, incubated overnight at 4°C, separated, aliquoted, and stored at -20°C.

[0057] 5. Indirect ELISA detection of antiserum titer Nanos2 antigen was diluted to 2 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6), and 50 μL was used to coat each well of an ELISA plate. The plate was incubated overnight at 4°C. After washing three times with PBST, 250 μL of 1% BSA blocking buffer was added to each well, and the plate was blocked at 37°C for 120 min. After washing, serially diluted rabbit antiserum (starting from 1:500) was added, and the plate was incubated at 37°C for 1 h. After washing, HRP-labeled goat anti-rabbit IgG (1:5000 dilution) was added, and the plate was incubated at 37°C for 1 h. After washing again, TMB substrate chromogenic solution (100 μL / well) was added, and the reaction was incubated at 37°C for 5 min. Finally, 50 μL of 2 mol / L sulfuric acid was added to terminate the reaction. The absorbance (OD value) was measured at 450 nm using an ELISA reader. The results showed that when the antiserum was diluted to 1:512K, the OD value was still significantly higher than that of the negative control by 4 times, indicating that the titer of the antiserum at the final bloodletting was >512K.

[0058] 6. Affinity purification of polyclonal antibodies Polyclonal antibodies were purified using antigen affinity chromatography. 1 mL of affinity chromatography packing material was loaded onto a column and equilibrated with coupling buffer. 2 mg of purified Nanos2 antigen was mixed with the packing material and incubated at room temperature for 2 h to couple the antigen to the resin. After washing the packing material, blocking buffer was added and incubated at room temperature for 2 h to block free sites. After washing with coupling buffer, 10 mL of rabbit antiserum was added and incubated at room temperature for 2 h to allow specific antibodies to fully bind to the antigen. The flow-through was collected, and non-specific binding proteins were eluted with 2 column volumes of washing buffer. Then, 2 mL of elution buffer was added to suspend the column material, and after standing for 2 min, the elution was collected into a centrifuge tube pre-filled with 10% neutralization buffer. This elution step was repeated 3 times. The collected polyclonal antibody was dialyzed against PBS and concentrated to approximately 1 mL to obtain the final specific antibody. ELISA confirmed that the purified antibody titer was >512 K, and the measured concentration was 1.64 mg / mL. An equal volume of glycerol (final concentration 50%) was added, and the solution was aliquoted and stored at -20℃ for later use.

[0059] Example 4: Percoll enrichment cell immunofluorescence identification based on anti-Nanos2 polyclonal antibody This embodiment is used to establish a Percoll density gradient enrichment method for gonadal germ cells of the saddle-banded grouper, and to use the anti-Nanos2 polyclonal antibody (concentration of 1.64 mg / mL) prepared in Example 3 to perform immunofluorescence identification on cells in different Percoll layers to determine the main distribution layer of germ cells.

[0060] 1. Preparation of donor cell suspension The gonads of donor grouper were anesthetized with MS-222 and dissected. The gonads were placed in L-15 medium containing 1% penicillin and antibiotics, and the surface adipose tissue and black membrane were removed. The gonads were then minced and enzymatically digested. Each tube contained 1 mL of trypsin, 30 μL of collagenase, and 50 μL of DNase I solution (DNase I concentration: 10 mg / mL), with a trypsin to collagenase volume ratio of 30:1. The gonadal tissue was digested and dissociated using a cell preparation instrument until flocculent dispersion and transparent tissue blocks were observed, at which point digestion was terminated. The digested cell suspension was filtered through a 300-mesh sieve into a new centrifuge tube. 1 / 10 volume of FBS was added to terminate the reaction, and the tube was centrifuged at 800g for 5 min, discarding the supernatant. The precipitate was resuspended in 1% FBS-L15 medium, centrifuged at 800g for 3 min, and the supernatant was discarded. The washing process was repeated three times. Finally, add 1 mL of 1% FBS-L15, gently pipette to resuspend the cells, and obtain a gonadal cell suspension.

[0061] 2. Percoll discontinuous density gradient separation Take 1 mL of the above cell suspension and slowly add the top layer of the pre-prepared Percoll density gradient separation solution along the tube wall using a low-absorption pipette tip. The Percoll density gradient, from bottom to top, is 50%~60%, 40%~50%, 35%~40%, 30%~35%, 25%~30%, and 20%~25%. After adding the sample, seal the tube and centrifuge using a slow acceleration method at 700g for 30 minutes, with acceleration set to 0 and braking set to 3. Picture 4 As shown in Figure a, after centrifugation, approximately 1 mL of cell layers from each Percoll gradient interface was aspirated using a low-absorption pipette tip and transferred to separate 2 mL low-absorption centrifuge tubes. An equal volume of 1% FBS-L15 medium was added to each gradient cell layer, and the mixture was centrifuged at 800g for 5 min, discarding the supernatant. Subsequently, 500 μL of 2% FBS-L15 was added for resuspending, and the mixture was filtered through a 500-mesh nylon mesh into a 1.5 mL centrifuge tube, centrifuged at 800g for 5 min, and the supernatant was discarded. Finally, 10 μL of 2% FBS-L15 was added for resuspending, yielding cell suspensions from different Percoll gradient layers.

[0062] 3. Immunofluorescence staining of Nanos2 cells Centrifuge the cell suspensions from each layer at 800g for 5 min, discard the supernatant, add 1 mL of fixative (PBS containing 4% PFA, 0.1% Triton X-100, pH 7.4), and fix at room temperature for 20 min. Centrifuge and discard the supernatant. Then wash three times with PBST (PBS containing 0.1% Triton X-100, pH 7.4), 5 min each time. After washing, permeabilize with PBST containing 0.5% Triton X-100 at room temperature for 30 min, centrifuge and discard the supernatant. Add 300 μL of blocking buffer (PBS containing 0.5% Triton X-100, 2% BSA, and 1% DMSO, pH 7.4), and block at 4°C for 30 min. After blocking, 300 μL of anti-Nanos2 primary antibody (prepared from the anti-Nanos2 polyclonal antibody prepared in Example 3) incubation solution was added. The primary antibody incubation solution was prepared by mixing blocking buffer and the anti-Nanos2 polyclonal antibody prepared in Example 3 at a volume ratio of 499:1. Cells were incubated overnight at 4°C for more than 16 hours. After incubation, the cells were centrifuged at 2500g, the supernatant was discarded, and the cells were washed three times with blocking buffer, vortexing each time and then allowing to stand for 5 minutes. Then, 300 μL of secondary antibody incubation solution was added. This secondary antibody incubation solution was prepared by mixing blocking buffer and fluorescent secondary antibody (Alexa Fluor™ 488-labeled goat anti-rabbit IgG secondary antibody, purchased from Invitrogen, catalog number A-11008) at a volume ratio of 999:1, and incubated at room temperature in the dark for 1 hour. After incubation, the cells were centrifuged at 2500g, the supernatant was discarded, and the cells were washed once with PBST containing 0.1% Triton X-100 for 6 minutes each time. Subsequently, 300 μL of DAPI staining solution was added for nuclear staining. The DAPI staining solution was prepared by mixing PBST and DAPI at a ratio of 499:1, and staining was performed at room temperature for 20 min. After staining, the cells were washed three times with PBST containing 0.1% Triton X-100, 5 min each time. After washing, 50 μL of PBST was added to resuspend the cells. 20 μL of the cell suspension was dropped onto the area circled by the immunohistochemical pen on a glass slide, dried at room temperature for 5 min, and then an anti-fluorescence quenching mounting medium was added. The slide was then covered with a coverslip and observed under a confocal microscope or an inverted fluorescence microscope.

[0063] 4. Results like Picture 4As shown in b, Nanos2 immunofluorescence identification (i.e., fluorescence indicates Nanos2-positive cells) revealed differences in the distribution of Nanos2-positive cells (i.e., germline stem cells) across different Percoll density gradient layers. The 30%–35% Percoll layer had the highest number of Nanos2-positive cells, representing the primary enrichment layer for Nanos2-positive germ cells in the grouper; the 25%–30% Percoll layer had the second highest number, representing a secondary enrichment layer; and the remaining gradient layers had relatively fewer Nanos2-positive cells. These results indicate that anti-Nanos2 polyclonal antibodies can be used for protein level identification of cell populations after Percoll separation and can be used to determine the enrichment layers of Nanos2-positive germ cells in the grouper.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the Nanos2 molecular marker system of *Siniperca saddleback* in the identification and enrichment of reproductive stem cells, characterized in that... A Nanos2 molecular marker system for saddle-banded grouper was constructed using the nucleotide sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The Nanos2 molecular marker system includes at least one of the following (1)-(3): (1) nanos2 RNA probe; (2) Recombinant Nanos2 protein; (3) Anti-Nanos2 polyclonal antibody.

3. The application according to claim 2, characterized in that, In (1), the stated nanos2 RNA probes include: antisense RNA probes and sense RNA probes; The template for the antisense probe was obtained by amplification using the primer pairs shown in SEQ ID NO: 11 and SEQ ID NO: 12; the template for the positive probe was obtained by amplification using the primer pairs shown in SEQ ID NO: 13 and SEQ ID NO:

14.

4. The application according to claim 2, characterized in that, In (2), the preparation method of the recombinant Nanos2 protein is as follows: the coding region of the gene shown in SEQ ID NO:1 is cloned into the expression vector PET-28a, and then the recombinant expression vector pET-28a-nanos2 is transformed into Escherichia coli for expression, and the expression product is purified to obtain the protein.

5. The application according to claim 2, characterized in that, In (3), the anti-Nanos2 polyclonal antibody is prepared using the recombinant Nanos2 protein described in (2) as an immunogen.

6. A method for identifying and enriching reproductive stem cells of the saddleback grouper, characterized in that, Includes the following steps: S1. The gonadal tissue of the saddle-banded grouper is sequentially digested with a compound enzyme solution, filtered through a cell sieve, and centrifuged and resuspended to obtain a gonadal cell suspension; the compound enzyme solution includes trypsin, collagenase, and DNase I solution. S2. After preparing the Percoll density gradient solution, add the gonadal cell suspension from S1 to the top layer of the Percoll density gradient solution and centrifuge to obtain cell suspensions of different layers. S3. The cell suspensions at different levels are blocked in sequence, and the primary antibody is incubated with the anti-Nanos2 polyclonal antibody as described in claim 2 or 4, followed by secondary antibody fluorescence incubation, staining, and microscopic observation to identify the reproductive stem cells of the saddle-banded grouper. S4. Based on the identification results in S3, collect cell suspensions containing saddle-banded grouper reproductive stem cells to enrich saddle-banded grouper reproductive stem cells.

7. The method according to claim 6, characterized in that, In step S3, the primary antibody incubation solution is prepared by mixing blocking solution and anti-Nanos2 polyclonal antibody during primary antibody incubation. The blocking solution uses PBS as a solvent and contains 2% w / v BSA, 0.5% v / v Triton X-100 and 1% v / v DMSO.

8. The method according to claim 7, characterized in that, The concentration of the anti-Nanos2 polyclonal antibody was 1.64 mg / mL, and the titer was >512K. In the primary antibody incubation solution, the volume ratio of blocking solution to anti-Nanos2 polyclonal antibody is 499:

1.

9. The method according to claim 6, characterized in that, In step S2, the volume concentrations of the Percoll density gradient solution are 50%~60%, 40%~50%, 35%~40%, 30%~35%, 25%~30%, and 20%~25% from bottom to top.

10. The method according to claim 9, characterized in that, After collecting cell suspensions at 30%–35% and 25%–30% levels, reproductive stem cells of the grouper were enriched.

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