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CN-121975953-A - Method for synchronously marking germ cells and interstitial cells in spermary tissue of paralichthys olivaceus

CN121975953ACN 121975953 ACN121975953 ACN 121975953ACN-121975953-A

Abstract

The invention discloses a method for synchronously marking germ cells and interstitial cells in a flounder spermary tissue, which comprises the steps of cloning flounder MIR17HG-a (long non-coding RNA) by using a probe primer, obtaining a target plasmid after plasmid extraction, carrying out double enzyme digestion and purification to obtain an enzyme digestion plasmid serving as a probe template, synthesizing the flounder MIR17HG-a probe by using SP6/T7 RNA polymerase, preparing the flounder spermary tissue into a tissue slice, and carrying out fluorescence in situ hybridization on the tissue slice by using the flounder MIR17HG-a probe so that the flounder MIR17HG-a probe can synchronously mark germ cells and interstitial cells in the flounder spermary tissue. Solves the defect that the traditional marking technology needs to mark in combination or step by step for many times, and fills the blank of synchronous marking of germ cells and interstitial cells in the current flounder spermary tissue.

Inventors

  • CHENG JIE
  • WANG FENGCHI
  • YANG FAN
  • ZHANG QINGKE

Assignees

  • 中国海洋大学三亚海洋研究院

Dates

Publication Date
20260505
Application Date
20260402

Claims (10)

  1. 1. A method for synchronously marking germ cells and interstitial cells in a spermary tissue of a paralichthys olivaceus, which is characterized by comprising the following steps: Cloning the paralichthys olivaceus MIR17HG-a by using a probe primer to obtain recombinant DNA, and extracting plasmids to obtain target plasmids; Step B, linearizing and purifying the target plasmid to obtain an enzyme-digested plasmid, wherein the enzyme-digested plasmid is used as a probe template, an MIR17HG-a sense probe and an MIR17HG-a antisense probe are respectively synthesized by utilizing SP6 RNA polymerase and T7 RNA polymerase, 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; And C, preparing a tissue slice by utilizing the flounder spermary tissue, and respectively carrying out fluorescence in-situ hybridization on the tissue slice by utilizing the MIR17HG-a sense probe and the MIR17HG-a antisense probe so that the MIR17HG-a antisense probe can synchronously mark the germ cells and the mesenchymal cells in the flounder spermary tissue.
  2. 2. The method for synchronously labeling germ cells and interstitial cells in the testis tissue of the paralichthys olivaceus according to claim 1, wherein 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. 3. The method of synchronizing the marking of germ cells and mesenchymal cells in the testis tissue of bastard halibut according to claim 2, wherein the cloning step comprises: Step A-1, performing PCR amplification on the paralichthys olivaceus MIR17HG-a by using the SEQ ID NO.2 and the SEQ ID NO.3 to obtain a PCR amplification product; Step A-2, performing gel cutting recovery and purification on the PCR amplification product to obtain a gel recovery product; And step A-3, connecting the gel recovery products by using a pMD19-T vector to obtain the recombinant DNA, and carrying out plasmid extraction after DH5 alpha competent cell transformation to obtain the target plasmid.
  4. 4. The method for synchronously marking germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus according to claim 3, The recombinant DNA is pMD19-T-MIR17HG-a, and the sequence of the recombinant DNA is shown as SEQ ID NO. 5.
  5. 5. The method for synchronously labeling germ cells and interstitial cells in the testis tissue of the paralichthys olivaceus according to claim 1, wherein in the step B, The linearization treatment is to enzyme-cut the target plasmid by using restriction enzymes NcoI and SpeI, wherein the enzyme-cutting system is that 3 mug of the target plasmid, 10 XBuffer 5 mu L, ncoI 1 mu L, speI 1 mu L and ddH 2 O are complemented to 50 mu L, and the enzyme-cutting condition is that enzyme-cutting is 1 h under the condition of 37 ℃.
  6. 6. The method for synchronously marking germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus according to claim 5, The SP6 RNA Polymerase is used for synthesizing a sense probe, the reaction system of the sense probe is 10×SP6 RNA Polymerase Buffer mu L, 0.1% BSA 2 mu L, 100 mM DTT 2 mu L, DIG RNA Labeling RNA mu L, 40U/mu L RNase Inhibitor 0.5 mu L, 1 mu g of the digested plasmid, 1 mu L of SP6 RNA Polymerase, and 20 mu L of DEPC water are added to the system, and the whole process is operated on ice.
  7. 7. The method for synchronously marking germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus according to claim 6, The T7 RNA Polymerase is used for synthesizing an antisense probe, the reaction system of the antisense probe is 10 xT 7 RNA Polymerase Buffer mu L, 50mM DTT 2 mu L, DIG RNA Labeling RNA mu L, 40U/muL RNase Inhibitor 0.5 mu L, 1 mu g of the enzyme-digested plasmid, 1 mL of T7 RNA Polymerase and DEPC water are used for supplementing the system to 20 mu L, and the whole process is operated on ice.
  8. 8. The method for synchronously marking germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus according to claim 5, The paralichthys olivaceus MIR17HG-a probe is located upstream of the first mature body miRNA-17 contained in MIR17 HG-a.
  9. 9. The method for synchronously labeling germ cells and interstitial cells in the testis tissue of the paralichthys olivaceus according to claim 1, wherein in the step C, The fluorescence in situ hybridization step comprises the steps of placing the tissue slice in 65 ℃ prehybridization liquid for hybridization for 1-2 hours after dewaxing and rehydration operation, respectively covering the tissue slice by using the MIR17HG-a sense probe and the MIR17HG-a antisense probe, performing a dark reaction at 62-70 ℃ for 24-72 h, then dropwise adding a biotinylated mouse anti-digoxin antibody and a FITC labeled antibody, dyeing cell nuclei by using DAPI, and observing after sealing the cell nuclei.
  10. 10. The method for synchronously marking germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus according to claim 9, The MIR17HG-a sense probe has no signal, is arranged in clusters at the periphery of the seminal vesicle, is distributed in the cytoplasm in an irregular dispersion manner, and is positioned in the nucleus without signal, and is distributed in the seminal vesicle, is distributed in the cytoplasm in a dispersion manner, and is positioned in the nucleus in a punctiform manner.

Description

Method for synchronously marking germ cells and interstitial cells in spermary tissue of paralichthys olivaceus Technical Field The invention relates to the technical field of marine fish reproduction biology and molecular markers, in particular to a method for synchronously marking germ cells and interstitial cells in a spermary tissue of a paralichthys olivaceus. Background The Cells in the fish testis tissue mainly include germ Cells and somatic Cells, wherein germ Cells can be classified into spermatogonia (St, spermatogonia), spermatocyte (Sc, spermatocytes), sperm Cells (Sd, SPERMATIDS) and mature Sperm (specm, spermatozoa) according to the development period, and somatic cell types are various and mainly include supporting Cells (Se, sertoli Cells for providing nutrition to Sperm) and interstitial Cells (Le, LEYDIG CELLS for synthesizing sex hormone), and further myoid Cells (Peritubular Myoid Cells), fibroblasts (Fibroblasts), vascular endothelial Cells, macrophages, and the like. Because somatic cells play an important role in spermatogenesis and functional maintenance, support cells and mesenchymal cells are the most important target cell types in fish reproductive development studies in addition to germ cells. The positioning of fish germ cells and somatic cells usually depends on histological immunohistochemical/immunofluorescence and in situ hybridization technologies, for example, currently commonly used specific marker genes of fish testis tissue germ cells comprise vasa, dazl, sycp, prm1 and the like, specific marker genes of interstitial cells comprise cyp11a1, cyp17a1, cyp11b2 and the like, but when positioning different cell types, the positioning analysis needs to be carried out by selecting the specific markers. Currently, research has shown that a plurality of cell types can be marked simultaneously by using one mark, for example, patent document with publication number of CN108330195A discloses a marking method and application of germ cells and somatic cells in different development periods of turbot seminiferous epithelium, and the germ cells and supporting cells can be marked simultaneously by amplifying Amh/Sox9/Gsdf fragments from turbot testis cDNA to prepare probes, but the probe combination can not be marked simultaneously by germ cells and interstitial cells due to different cell types. The role of non-coding RNA (such as miRNA and lncRNA) in fish reproduction regulation is gradually revealed, but the role of non-coding RNA serving as an in-situ hybridization probe for spatial localization research of fish reproduction cells and mesenchymal cells is not seen. MIR17HG is a host gene and a primary transcript of a microRNA-17-92 gene cluster, mature microRNAs (such as miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92 a) generated by shearing processing regulate cell proliferation, apoptosis and tumorigenesis in mammals, but expression patterns and functions in fish reproductive systems are rarely reported. The paralichthys olivaceus (PARALICHTHYS OLIVACEUS) is an important seawater aquaculture fish in northern China, the growth of the fish has obvious sex two-state, and female fish is usually bigger than male fish in size. Paralichthys olivaceus is a XX/XY sex determination system, and sex-reversal pseudo-male fish (XX) can be induced to generate in the gonad differentiation period under the action of water temperature, exogenous hormone and other environmental factors. Earlier studies found that mature mirnas produced by MIR17HG processing target negative regulatory germ cell specific PIWI/piRNA pathway genes (piwil, tdrd, tdrd3, tdrd a) primarily through binding to the 3'utr region of mRNA in the cytoplasm, while mature mirnas produced by MIR17HG processing target positive regulatory germ cell specific steroid hormone synthesis key genes (cyp 17a1, cyp21a, hsd3b 7) primarily through binding to the 5' promoter region of genes in the nucleus. Although the roles of non-coding RNAs (e.g., mirnas, lncrnas) in reproductive regulation are gradually revealed, their use as in situ hybridization probes for spatial localization studies of fish germ cells and mesenchymal cells has not been seen. Therefore, innovative development of a primer and a probe for in-situ hybridization of the paralichthys olivaceus is needed to realize synchronous marking of germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus so as to solve the defect that the traditional marking technology needs to be combined for marking or multi-step marking, fill the blank of synchronous marking of germ cells and interstitial cells in the spermary tissue of the paralichthys olivaceus at present, and provide a high-efficiency detection tool for research on reproductive development of marine fishes and genetic breeding of the marine fishes. Disclosure of Invention The invention aims to develop a method for synchronously marking germ cells and interstitial cells in a spermary tissue of a par