Application of RBX1 gene in cow ovary granular cells

Abstract

The invention relates to the technical field of biological breeding, in particular to application of an RBX1 gene in cow ovarian granular cells. According to the invention, the RBX1 gene is specifically regulated on the ovarian granulosa cells, and the RBX1 gene is found to have the effect of promoting the proliferation of the granulosa cells; and the gene is provided for the creation of a new high-fecundity breeding material for gene editing cattle in the later period.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, and particularly relates to the application of the RBX1 gene in bovine ovarian granulosa cells. Background Art

[0002] As a monovular animal, a dairy cow generally gives birth to one calf per pregnancy. Compared with polyovular animals such as pigs and poultry, its reproductive capacity is relatively low. In addition, the reproductive traits of dairy cows are low-heritability traits controlled by multiple minor genes. Due to the limitations of traditional breeding techniques, the genetic potential of Holstein cows in economic traits such as reproductive traits and long milk production years has not been fully explored. Currently, there are still insufficient key genes that can be utilized and the mechanisms of their effects on reproductive traits clarified. Therefore, finding key functional loci that regulate the reproductive traits of dairy cows and applying them to molecular marker-assisted breeding and gene-editing biological breeding is of great significance for improving the reproductive traits of dairy cows and increasing economic benefits.

[0003] The ovary is an important reproductive organ of female cows, and its functions are to produce and discharge eggs and secrete estrogen and progesterone. The follicle is the basic structural and functional unit of the ovary and consists of an oocyte and the surrounding granulosa cells and theca cells. During the follicular development of mammals, the proliferation of granulosa cells secretes a large amount of estrogen and growth factors to support the development and maturation of oocytes. However, when the proliferation rate of granulosa cells slows down or even apoptosis occurs, the estrogen level will decrease, which in turn affects the maturation of oocytes and follicular development.

[0004] RBX1 (RING Box Protein-1) is the RING component of the E3 ubiquitin ligase complex, interacts with Cullins, activates the Cullin-RING E3 ubiquitin ligase (CRL), regulates protein ubiquitination modification, and then regulates cell functions. In Caenorhabditis elegans, specific interference with RBX1 leads to defects in germ cell meiosis and proliferation; silencing RBX1 in mouse embryos leads to embryonic death. Therefore, RBX1 may play an important regulatory role in mammalian reproductive regulation.

[0005] Currently, there is little research on RBX1 in cattle, and in addition, the RBX1 gene has not been studied in bovine ovarian granulosa cells yet. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to address the above problems and provide the application of the RBX1 gene in bovine ovarian granulosa cells.

[0007] To achieve the above invention purpose, the present invention provides the following technical solutions: In the first aspect, the present invention provides the application of regulating the expression of the RBX1 gene in vivo or in vitro in enhancing or reducing the function of bovine ovarian granulosa cells.

[0008] In some specific embodiments of the present invention, regulating the expression of the RBX1 gene includes overexpressing the RBX1 gene or inhibiting the expression of the RBX1 gene.

[0009] In some specific embodiments of the present invention, the application of overexpressing the RBX1 gene in any of the following: (I-1), enhancing the viability of the ovarian granulosa cells; (I-2), enhancing the proliferation rate of the ovarian granulosa cells; (I-3), enhancing the confluence or proliferation rate of the ovarian granulosa cells; (I-4), reducing the apoptosis rate of the ovarian granulosa cells; (I-5), enhancing the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0010] In some specific embodiments of the present invention, the application of inhibiting the expression of the RBX1 gene in any of the following: (II-1), reducing the viability of the ovarian granulosa cells; (II-2), reducing the proliferation rate of the ovarian granulosa cells; (II-3), reducing the confluence or proliferation rate of the ovarian granulosa cells; (II-4), enhancing the apoptosis rate of the ovarian granulosa cells; (II-5), reducing the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0011] In some specific embodiments of the present invention, the accession number of the RBX1 gene is: 518880; the cattle include Holstein cattle.

[0012] In some specific embodiments of the present invention, inhibiting the expression of the RBX1 gene is achieved using siRNA.

[0013] In some specific embodiments of the present invention, the siRNA has: (i), a nucleotide sequence as shown in any one of SEQ ID No.1 to 4; or (ii), a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (i), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (iii), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and having the same or similar function as the nucleotide sequence shown in (i) or (ii); or (iv) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence described in (i), (ii) or (iii).

[0014] In a second aspect, the present invention also provides a method for improving the function of bovine ovarian granulosa cells, by overexpressing the RBX1 gene to improve the function of bovine ovarian granulosa cells.

[0015] In some specific embodiments of the present invention, the improvement of the function of ovarian granulosa cells includes: (I-1) Improving the viability of the ovarian granulosa cells; (I-2) Improving the proliferation rate of the ovarian granulosa cells; (I-3) Improving the confluence or proliferation rate of the ovarian granulosa cells; (I-4) Reducing the apoptosis rate of the ovarian granulosa cells; (I-5) Increasing the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0016] In some specific embodiments of the present invention, the accession number of the RBX1 gene is: 518880; the bovine includes Holstein cattle.

[0017] The present invention first discovers that specific regulation of RBX1 has an impact on bovine ovarian granulosa cells. Specific regulation of RBX1 on ovarian granulosa cells reveals that RBX1 has the effect of promoting granulosa cell proliferation; it provides a gene for the creation of new materials for gene editing of high-fertility cattle breeding in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0019] Figure 1 Showing the results of RBX1 immunohistochemistry (IHC) of ovarian tissue; Figure 2 Showing the gene detection results of specific regulation of RBX1; wherein, a shows the effect of overexpressing RBX1 on the expression level of the RBX1 gene in bovine ovarian granulosa cells detected by qRT-PCR; b shows the effect of interfering with RBX1 on the expression level of the RBX1 gene in bovine ovarian granulosa cells detected by qRT-PCR. Figure 3Show the protein detection results of specifically regulating RBX1; among them, a shows the effect of overexpressing RBX1 on the expression of RBX1 protein in bovine ovarian granulosa cells detected by Western Blot (WB); b shows the differential analysis of the effect of overexpressing RBX1 on the expression of RBX1 protein in bovine ovarian granulosa cells detected by WB; c shows the effect of interfering with RBX1 on the expression of RBX1 protein in bovine ovarian granulosa cells detected by WB; d shows the differential analysis of the effect of interfering with RBX1 on the expression of RBX1 protein in bovine ovarian granulosa cells detected by WB; Figure 4 Show the cell viability detection results of specifically regulating RBX1; among them, a shows the effect of overexpressing RBX1 on the viability of bovine ovarian granulosa cells detected by CCK-8 method; b shows the effect of interfering with RBX1 on the viability of bovine ovarian granulosa cells detected by CCK-8 method; Figure 5 Show the cell proliferation detection results of specifically regulating RBX1; among them, a shows the effect of overexpressing RBX1 on the proliferation of bovine ovarian granulosa cells detected by EdU method; b shows the differential analysis of the effect of overexpressing RBX1 on the proliferation of bovine ovarian granulosa cells detected by EdU method; c shows the effect of interfering with RBX1 on the proliferation of bovine ovarian granulosa cells detected by EdU method; d shows the differential analysis of the effect of interfering with RBX1 on the proliferation of bovine ovarian granulosa cells detected by EdU method; Figure 6a Show the effect of overexpressing RBX1 on the proliferation of live cells in bovine ovarian granulosa cells from 0 to 48 h detected by Incucyte method; Figure 6b Show the differential analysis of the proliferation of live cells in bovine ovarian granulosa cells at 0 h, 24 h and 48 h after overexpressing RBX1 detected by Incucyte method; Figure 6c Show the images of the proliferation of live cells in bovine ovarian granulosa cells at 0 h, 12 h, 24 h and 48 h after overexpressing RBX1 detected by Incucyte method; Figure 6d Show the effect of interfering with RBX1 on the proliferation of live cells in bovine ovarian granulosa cells from 0 to 48 h detected by Incucyte method; Figure 6e Show the differential analysis of the proliferation of live cells in bovine ovarian granulosa cells at 0 h, 24 h and 48 h after interfering with RBX1 detected by Incucyte method; Figure 6f Show the images of the proliferation of live cells in bovine ovarian granulosa cells at 0 h, 12 h, 24 h and 48 h after interfering with RBX1 detected by Incucyte method; Figure 7a Show the flow cytometry results of the apoptosis of bovine ovarian granulosa cells detected by Annexin V-FITC method after overexpressing RBX1; Figure 7b Show the differential analysis results of different apoptosis types of bovine ovarian granulosa cells detected by Annexin V-FITC method after overexpressing RBX1; Figure 7cFigure showing the flow cytometry results of detecting the effect of interfering with RBX1 on the apoptosis of bovine ovarian granulosa cells by Annexin V-FITC method; Figure 7d Figure showing the differential analysis results of detecting the effect of interfering with RBX1 on different apoptosis types of bovine ovarian granulosa cells by Annexin V-FITC method; Figure 8 Figure showing the effect of specifically regulating RBX1 on the hormone secretion levels in the culture medium of GCs; among them, a shows the effect of overexpressing RBX1 on the progesterone level of bovine ovarian granulosa cells detected by radioimmunoassay; b shows the effect of overexpressing RBX1 on the estrogen level of bovine ovarian granulosa cells detected by radioimmunoassay; c shows the effect of interfering with RBX1 on the progesterone level of bovine ovarian granulosa cells detected by radioimmunoassay; d shows the effect of interfering with RBX1 on the estrogen level of bovine ovarian granulosa cells detected by radioimmunoassay. Detailed implementation manners

[0020] The present invention discloses the application of the RBX1 gene in bovine ovarian granulosa cells. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0021] The present invention aims at the application of the RBX1 gene in bovine ovarian granulosa cells.

[0022] The following technical solutions are adopted: RBX1 gene

gene ID: 518880; NC_037332.1 (112164162..112177109)

[0023] The raw materials and reagents used in the application of the RBX1 gene provided by the present invention in bovine ovarian granulosa cells can all be purchased from the market.

[0024] The present invention will be further described below in conjunction with embodiments: Example 1 Immunohistochemistry (IHC) staining of cow ovarian tissue 1. Sampling of cow ovarian tissue: (1) Repeatedly wash the cow ovaries collected from the abattoir and brought back with a pre-warmed 0.9% sodium chloride (NaCl) solution (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) at 37°C; (2) Cut off the surrounding tissues of the ovaries with sterile surgical scissors and rinse with 75% alcohol. Then, wash the ovaries 4 - 5 times with pre-warmed NaCl (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) solution; (3) Wash the ovaries once with pre-warmed PBS. In a sterilized laminar flow hood, cut the ovaries into tissue blocks approximately 10 mm in length and width and about 3 mm in thickness, and place the tissues into embedding molds.

[0025] 2. Dehydration, clearing, and wax infiltration (1) 70% ethanol for 1 h; 80% ethanol for 1 h; 90% ethanol for 1 h; 95% ethanol I for 1 h; 95% ethanol II for 1 h; 100% ethanol I for 1 h; 100% ethanol II for 1 h; (2) Ethanol - xylene for 40 min; xylene I for 25 min; xylene II for 15 min; (3) Wax infiltration I at 65°C for 1 h; wax infiltration II at 65°C for 1 h; wax infiltration III at 65°C for 1 h.

[0026] 3. Embedding: Embed the tissues with the cut surface facing down. After the wax blocks cool, remove them from the molds and trim the blocks.

[0027] 4. Sectioning: Pre-cool the wax blocks on a freezing table for 20 minutes in advance, then perform 4-μm sectioning on a microtome. Then, place the cut tissue wax sections in a constant-temperature water bath in a spreading machine to spread the sections. After the tissue wax sections are spread flat, adhere them to glass slides.

[0028] 5. Baking the slides: Place the glass slides with adhered tissue wax sections on a baking machine to dry the residual moisture, and then put them into an oven at 65°C for baking for 1 hour.

[0029] 6. Immunofluorescence staining: (1) Xylene (Ⅰ) for 10 min; Xylene (Ⅱ) for 10 min; Xylene: 100% ethanol = 1:1 for 2 min; 100% ethanol (Ⅰ) for 5 min; 100% ethanol (Ⅱ) for 5 min; 80% ethanol for 5 min; Wash with water for 5 min; Rinse with PBS 3 times, each for 5 min; Repair with antigen repair solution for 15 min (microwave repair method), cool to room temperature; (2) Block with 3% hydrogen peroxide and incubate at room temperature for 30 min; Rinse with PBS 2 times, each for 5 min; Spin dry and circle the tissue with an immunohistochemistry pen; Incubate with 3% BSA at room temperature for 30 min; (3) Dropwise add the primary antibody and incubate overnight at 4°C; Rinse with PBS 3 times, each for 5 min; (4) Dropwise add the secondary antibody working solution from the corresponding primary antibody source and incubate at 37°C for 30 min; Rinse with PBS 3 times, each for 5 min; (5) DAB color development (in the dark, observe under the microscope until brown) for about 3 min; Wash with water for 5 min; Stain with hematoxylin solution for 5 min; Wash with water for 5 min; Differentiate with hydrochloric acid ethanol differentiation solution for 1 s; Blue with ammonia water blueing solution for 10 s; Wash with water for 30 s (antibodies are shown in Table 1); (6) 95% ethanol (Ⅰ) for 1 min; 95% ethanol (Ⅱ) for 1 min; 100% ethanol (Ⅰ) for 3 min; 100% ethanol (Ⅱ) for 3 min; Xylene (Ⅰ) for 3 min; Xylene (Ⅱ) for 3 min; (7) Mount with neutral gum and observe under a light microscope.

[0030] Table 1. Antibody information

[0031] As Figure 1 shown, RBX1 showed positive reactions on granulosa cells and oocytes in dairy cow ovarian tissue, indicating that RBX1 was expressed in both granulosa cells and oocytes.

[0032] Example 2. Culture of ovarian granulosa cells and transfection with overexpressing / interfering RBX1 plasmids 1. Cell granule culture (1) Wash the dairy cow ovaries collected and brought back from the slaughterhouse repeatedly with a preheated 0.9% sodium chloride (NaCl) solution (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) at 37°C.

[0033] (2) Cut off the surrounding tissues of the ovaries with sterile surgical scissors and rinse with 75% alcohol. Then, wash the ovaries 4 - 5 times with the preheated NaCl (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) solution.

[0034] (3) Wash the ovaries once with pre-warmed PBS. Inside a sterilized laminar flow hood, use a 10 mL syringe to aspirate the follicular fluid from healthy yellow and transparent follicles with a diameter of 2 - 6 mm. Then use a 40 μm filter sieve to filter the collected follicular fluid into a new 15 mL centrifuge tube, wash it three times with DPBS, centrifuge at 1000 g for 5 min, and discard the supernatant.

[0035] (4) Resuspend the GCs with pre-warmed DMEM / F12 complete medium (containing 10% PBS and 1% double antibiotics) and inoculate them in a culture dish. Subsequently, culture them in an incubator at 37 °C with 5% carbon dioxide for 24 h, then replace the fresh medium, and change the new medium every 24 - 48 h.

[0036] (5) When the GCs reach 80% - 90% confluence, perform cell passage. Aspirate the medium, digest with 0.25% trypsin, and when the cells become round and detach from the bottom of the dish under the microscope, add an equal volume of medium containing serum to terminate the digestion. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 g for 5 min, and discard the supernatant. Resuspend with fresh DMEM / F12 complete medium and inoculate in a new culture dish, and continue to culture in an incubator at 37 °C with 5% carbon dioxide.

[0037] 2. Design siRNA targeting RBX1 and RBX1 overexpression plasmid (pcDNA3.1 - RBX1) according to the GenBank reference sequence number NM 001046241.1, both of which are designed and synthesized by GenePharma. In this experiment, use Lipofectamine 3000 transfection reagent to transfect siRNA - RBX1 or pcDNA3.1 - RBX1 into the prepared GCs.

[0038] The primers of siRNA - RBX1 are as follows: si RBX1 - 1 - sense(5′ - 3′): 5′ - GGAGGUUCACGAAGUUCCUTT - 3′, as shown in SEQ ID No.1; si RBX1 - 1 - antisense(5′ - 3′): 5′ - AGGAACUUCGUGAACCUCCTT - 3′, as shown in SEQ ID No.2; si RBX1 - 2 - sense(5′ - 3′): 5′ - GCUGUCUUCUGCUAAGUCATT - 3′, as shown in SEQ ID No.3; si RBX1 - 2 - antisense(5′ - 3′): 5′ - UGACUUAGCAGAAGACAGCTT - 3′, as shown in SEQ IDNo.4.

[0039] Since U cannot be presented in the sequence list production software WIPO, U in the sequence list is modified to T. Please refer to the sequence in the specification for the correct sequence.

[0040] 3. Transfection of ovarian granulosa cells: Transfect GCs with a confluence of about 70% - 80%. Replace the DMEM / F12 medium in the culture dish with Opti-MEM medium and transfect according to the instructions of the siRNA / overexpression transfection reagent Lipofectamine™ 3000; collect the cells 24 h after transfection.

[0041] Example 3 RNA Isolation and qRT-PCR Detection (1) Cell sampling Sample the GCs to be transfected 24 h after culture. Aspirate the medium, digest with 0.25% trypsin. When the cells become round and detach from the bottom of the dish under the microscope, add an equal volume of medium containing serum to terminate digestion. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 g for 5 min, and discard the supernatant.

[0042] Add 1 mL of PBS to resuspend the cells, wash twice, and discard the supernatant. Store at -80 °C in the refrigerator.

[0043] (2) Total RNA extraction: Use the Novizan RC101 kit to extract cell RNA. The specific steps are as follows: Directly add 1000 μL of TRIzol to the cell tube for sample collection, pipette the mixture, and shake for 15 s; add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 s, and let it stand in a 4 °C refrigerator for 5 min; centrifuge at 12,000 rpm (~13,400 × g) at 4 °C for 10 min; transfer the upper aqueous phase to a new RNase Free centrifuge tube; add 1.6 volumes of Buffer RL2 (anhydrous ethanol has been added) to the obtained aqueous solution and gently mix. Transfer the mixture from the previous step to RNAPureColumns (RNAPure Columns have been placed in the collection tube), centrifuge at 13,000 × g for 1 min, and discard the waste liquid; after discarding the waste liquid, place the RNAPure Columns adsorption column back into the collection tube, add all the remaining liquid to the adsorption column, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; add 500 μL of Buffer RW1 to RNAPureColumns, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; add 700 μL of Buffer RW2 (anhydrous ethanol has been added) to RNAPureColumns, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; repeat the previous step; place the RNAPure Columns adsorption column back into the collection tube, centrifuge at 13,000 × g for 2 min to completely remove the residual Buffer RW2 in RNAPureColumns; transfer the adsorption column to a new 1.5 mL RNase-free Collection Tubes centrifuge tube, and suspend and add 50 - 200 μL of RNase-free ddH2O to the central part of the adsorption column. Let it stand at room temperature for 2 min, and centrifuge at 13,000 × g for 1 min to elute the RNA; detect the RNA concentration and quality using a BioDrop-μLite ultra-micro nucleic acid analyzer and store at -80 °C.

[0044] (3)cDNA synthesis: Reverse transcribe RNA (1,000 ng) into cDNA using the PrimeScript™ IV 1st strand cDNA Synthesis Mix kit. The specific steps are as follows: Prepare the mixture according to Table 2; gently mix, and the reaction program is 30 °C for 10 min; 42 °C for 15 min; 95 °C for 5 min. Store the product obtained by reverse transcription at -20 °C.

[0045] Table 2. Preparation of cDNA synthesis reaction solution

[0046] (4)qRT-PCR detection: The gene expression levels were detected using the Thermo Fisher PowerUp™ SYBR™ Green Premix and QuantStudioTM 7 Flex System (ABI), and each sample was subjected to 3 technical replicates. In this study, RPL-19 was used as the reference gene, and the 2 −ΔΔCT -ΔΔCt method was used to calculate the expression levels of each gene. The qRT-PCR reaction system is shown in Table 3. The reaction program was as follows: 95 °C for 30 s; then 95 °C for 5 s and 60 °C for 34 s for 40 cycles; 95 °C for 15 s; 60 °C for 15 s; 60 °C for 1 min; 95 °C for 15 s. All primers used for qRT-PCR were designed online using the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ), and the primer sequences were synthesized by BGI in Beijing.

[0047] Table 3. qRT-PCR reaction system

[0048] The qRT-PCR primers were as follows: qRT-PCR-RPL19 Forward: 5′-ATCGCCAATGCCAACTC-3′, as shown in SEQ ID No.5; Reverse: 5′-CCTTTCGCTTACCTATACC-3′, as shown in SEQ ID No.6; qRT-PCR-RBX1 Forward: 5′-GAATGTCAAGCCAACCAGGC-3′, as shown in SEQ ID No.7; Reverse: 5′-AAGCATGGTTACAGACGCCC-3′, as shown in SEQ ID No.8.

[0049] As Figure 2 shown, compared with the control group, the overexpression efficiency of oe-2 in the overexpression group was significantly higher than that of oe-1 (a); the interference efficiency of the si-2 group in the interference group was significantly higher than that of the si-1 group (b); according to the PCR results, the best interference fragment siRNA-RBX1-2 and the best overexpression fragment OE-RBX1-2 were selected as the primer sequences for subsequent experiments.

[0050] Example 4 Protein extraction and Western Blot (WB) detection of transfected cells (1)Cell sampling After culturing the GCs to be transfected for 24 h, sampling was carried out. The culture medium was aspirated, and 0.25% trypsin was used for digestion. When the cells became round and detached from the bottom of the dish under the microscope, an equal volume of serum-containing medium was added to terminate the digestion. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 g for 5 min, and the supernatant was discarded.

[0051] 1 mL of PBS was added to resuspend the cells, and the cells were washed twice, and the supernatant was discarded. The cells were stored in a -80 °C refrigerator.

[0052] (2)Protein extraction: (Taking the cell amount in 3 wells of a six-well plate as an example) In the 1.5 mL centrifuge tube for sampling, 200 μL of RIPA lysis buffer (1.5 μL of PMSF was added before use, and the final concentration was 1 mM) was added, and the mixture was pipetted to fully lyse the cells. The cells were lysed at 4 °C for 1 h (the mixture was shaken every 15 min); the lysed sample was placed in a centrifuge at 4 °C and centrifuged at 12,000 g for 10 min, and the supernatant was taken; then an enhanced BCA protein detection kit was used to measure the protein concentration.

[0053] (3)Protein immunoblotting detection Denaturation: After the protein sample was mixed with 5 × Loading Buffer buffer in proportion, it was boiled at 100 °C for 5 min; Electrophoresis: 20 - 40 μg of the denatured sample was separated by SDS-PAGE under the conditions of: 200 V, 30 min; Transfer: The protein was transferred to an NC membrane (HATF00010, Merck, Germany) under the conditions of: 200 mA, 2 h; Blocking: Incubated with 5% skim milk at room temperature for 2 h; Primary antibody incubation: The primary antibody (Table 4) was incubated overnight at 4 °C; Secondary antibody incubation: The secondary antibody (Table 4) was incubated at room temperature for 2 h; Development: Developed with ECL chemiluminescent solution.

[0054] Table 4. Antibody information

[0055] As Figure 3 shown, compared with the control group, the protein expression level in the RBX1 overexpression group increased (a), and differential analysis found that overexpression of RBX1 significantly promoted the expression of RBX1 protein (b); the protein expression level in the RBX1 interference group decreased (c), and differential analysis found that interference with RBX1 significantly inhibited the expression of RBX1 protein (d).

[0056] Example 5 Cell viability detection (CCK8 method) Ovarian granulosa cells (GCs) were seeded into 96-well plates. When the cell density reached over 70%, transfection was carried out. After 6 h of transfection, 100 μL of complete medium (90% DMEM / F12 + 10% FBS + 1% PS) was added and cultured for 24 h. Then, 10 μL of CCK-8 solution was added to each well and incubated in an environment of 5% CO2 and 37 °C for 1 h. CCK-8 solution was added to the culture media of the blank group, si-RNA group, and overexpression group with equal volumes as a blank control. The OD values of each well were measured using an enzyme-labeling instrument at a wavelength of 450 nm.

[0057] As Figure 4 shown, compared with the control group, the cell viability was significantly increased after overexpression of RBX1 in ovarian granulosa cells (GCs) (a); on the contrary, the cell viability was significantly decreased after interfering with RBX1 in GCs (b).

[0058] Example 6 Cell Proliferation Detection (EdU Method) Cell Proliferation Detection (EdU Method): The BeyoClick™ EdU-488 Cell Proliferation Detection Kit (C0071S, Beyotime, Shanghai, China) was used for cell proliferation detection. The specific steps were as follows: GCs were seeded in confocal dishes. When the confluence reached over 70%, transfection was carried out. After 6 h of transfection, the original serum-reduced medium was removed and replaced with complete medium for 24 h of culture. The 10 mM EdU stock solution was diluted with DMEM / F12 to prepare a 20 μM EdU working solution. The preheated 37 °C EdU working solution was added to the confocal dish with an equal volume, and the final working concentration was 10 μM, followed by incubation for 2 h. After the EdU labeling was completed, the mixture was aspirated, and 4% paraformaldehyde was added for fixation at room temperature for 15 min. It was washed three times with PBS solution containing 3% BSA, 5 min for each wash. PBS solution containing 0.3% Triton X-100 was added and incubated at room temperature for 15 min. It was washed twice with PBS solution containing 3% BSA, 5 min for each wash. One tube of Click Additive was dissolved in 1.3 mL of deionized water and mixed until completely dissolved. The Click reaction solution was prepared according to Table 5. 500 μL of Click reaction solution was added to each dish and incubated at room temperature in the dark for 30 min. Then, it was washed three times with PBS solution containing 3% BSA, 5 min for each wash. 1× Hoechst 33342 solution was added and incubated at room temperature in the dark for 10 min. After incubation, it was washed three times with PBS solution containing 3% BSA, 5 min for each wash. Fluorescence inverted microscopy was used for photography and Image J software for analysis.

[0059] Table 5. Preparation of Click Reaction Solution

[0060] As Figure 5 shown, after overexpressing RBX1 in GCs, the number of EdU-positive cells increased (a), and the cell proliferation rate increased significantly compared with the control group (b); after interfering with the expression of RBX1 in GCs, the number of EdU-positive cells decreased (c), and the cell proliferation rate decreased significantly compared with the control group (d).

[0061] Example 7 Incucyte Real-Time Live Cell Analysis

[0062] Bovine ovarian granulosa cells (GCs) (5×10 5 / 500ul) were seeded in six-well plates and transfected when the confluence reached more than 50%. After 6 hours of transfection, they were sent to the Incucyte Real-Time Live Cell Analysis System (Sartorius Incucyte SX5) for cell proliferation detection;

[0063] As shown in the results, in the overexpression group, after overexpressing RBX1 in GCs, the trend line of cell confluence gradually increased after 48 hours of culture compared with the control group ( Figure 6a ); Analysis of the cell confluence results at 0h, 24h, and 48h time periods found that the confluence increased significantly after overexpressing RBX1 ( Figure 6b ); According to the live cell growth state diagrams at 0h, 12h, 24h, and 48h collected by the Incucyte Real-Time Live Cell Analysis System, it was found that cell proliferation accelerated after overexpressing RBX1 compared with the control group ( Figure 6c ); In the interference group, after interfering with the expression of RBX1 in GCs, the trend line of cell confluence tended to be flat after 48 hours of culture compared with the control group ( Figure 6d ); Analysis of the cell confluence results at 0h, 24h, and 48h time periods found that the confluence decreased significantly after interfering with RBX1 ( Figure 6e ); According to the live cell growth state diagrams at 0h, 12h, 24h, and 48h collected by the Incucyte Real-Time Live Cell Analysis System, it was found that the cell proliferation rate decreased after interfering with RBX1 compared with the control group ( Figure 6f ). Example 8 Cell Apoptosis Analysis The apoptosis of cells was detected using the Annexin V-FITC Apoptosis Detection Kit (C1062M, Beyotime, Shanghai, China). The treated cells were collected by digestion with 0.25% trypsin, resuspended in 195 µL of Annexin V-FITC binding buffer, and then 5 µL of Annexin V FITC was added. The cells were incubated at room temperature for 30 min. Finally, 5 µL of propidium iodide (PI) was added and incubated at room temperature for 5 min. At the same time, unstained cells were used as negative controls, and FITC- and PI-single-stained cells were used as compensation controls. Then, the stained cells were detected using a flow cytometer (BD FACSVerse, USA). Analysis was performed using FlowJo software. As shown in the results, flow cytometry analysis data showed that compared with the control group, the number of viable cells in GCs increased and the number of early apoptotic cells decreased after overexpression of RBX1 ( Figure 7a ); differential analysis found that the proportion of viable cells in the overexpression group was significantly higher than that in the control group, and the proportion of early apoptotic cells decreased significantly, but there was no significant difference in the proportion of late apoptotic cells ( Figure 7b ); in addition, the number of early and late apoptotic cells in GCs increased after interfering with the expression of RBX1 ( Figure 7c ); differential analysis found that the proportions of early and late apoptotic cells in the interference group increased significantly, but the difference in the proportion of viable cells was not significant ( Figure 7d ).

[0064] Example 9 Determination of E2 and P4 Hormones After the cells were seeded in six-well plates and transfected, 1 mL of cell culture medium was collected and placed in a 1.5 mL centrifuge tube after 24 h of culture, and stored in a -20 °C refrigerator. The cell culture medium was sent to Beijing North Biotechnology Research Institute Co., Ltd. for the detection of progesterone (P4) and estrogen (E2).

[0065] As Figure 8 shown, compared with the control group, the levels of P4 (a) and E2 (b) increased significantly in GCs after overexpression of RBX1; the levels of P4 (c) and E2 (d) decreased significantly after interfering with the expression of RBX1.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. In vivo or in vitro regulation RBX1 Application of gene expression in increasing or decreasing bovine ovarian granulosa cell function.

2. The use according to claim 1, characterized in that The regulation RBX1 Gene expression includes overexpression RBX1 Gene or inhibition RBX1 Gene expression.

3. The use according to claim 2, characterized in that The overexpression RBX1 Use of genes in any of the following: (I-1), improving the activity of the ovarian granulosa cells; (I-2) increasing the proliferation rate of the ovarian granulosa cells; (I-3) increasing the confluence or proliferation rate of the ovarian granulosa cells; (I-4), reducing the apoptosis rate of the ovarian granulosa cells; (I-5) Increasing the level of progesterone and / or estrogen in the ovarian granulosa cells.

4. The use according to claim 2, characterized in that The inhibition RBX1 Application of gene expression in any of the following: (II-1), reducing the activity of the ovarian granulosa cells; (II-2), reducing the proliferation rate of the ovarian granulosa cells; (II-3), reducing the confluence or proliferation rate of the ovarian granulosa cells; (II-4), increasing the apoptosis rate of the ovarian granulosa cells; (II-5) reducing the level of progesterone and / or estrogen in the ovarian granulosa cells.

5. The use according to claim 4, characterized in that described RBX1 The accession number of the gene is: 518880; the cattle include Holstein cattle.

6. The use according to claim 4, characterized in that The method of inhibiting the expression of RBX1 gene adopts siRNA.

7. The use according to claim 6, characterized in that The siRNA has a nucleotide sequence as shown in any one of SEQ ID No. 1 to 4.

8. A method for improving the function of bovine ovarian granulosa cells, characterized in that: Overexpression RBX1 Gene, improves the function of bovine ovarian granulosa cells.

9. The method according to claim 8, wherein Improving the function of ovarian granulosa cells includes: (I-1), improving the activity of the ovarian granulosa cells; (I-2) increasing the proliferation rate of the ovarian granulosa cells; (I-3) increasing the confluence or proliferation rate of the ovarian granulosa cells; (I-4), reducing the apoptosis rate of the ovarian granulosa cells; (I-5) Increasing the level of progesterone and / or estrogen in the ovarian granulosa cells.

10. The method according to claim 8, wherein described RBX1 The accession number of the gene is: 518880; the cattle include Holstein cattle.

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