Application of RBX1 gene in bovine ovarian granulosa cells

Abstract

This invention relates to the field of biological breeding technology, and particularly to... RBX1 Application of genes in bovine ovarian granulosa cells. This invention specifically regulates genes in ovarian granulosa cells. RBX1 Genes, Discovery RBX1 The gene has the function of promoting the proliferation of granulosa cells; it provides genes for the creation of new high-fertility breeding materials for gene-edited cattle in the later stage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological breeding technology, in particular to the application of RBX1 gene in cow ovarian granulosa cells. BACKGROUND

[0002] Cows are monovular animals, generally one calf per pregnancy, and have lower reproductive capacity compared with pigs and poultry. In addition, the reproductive traits of cows are low heritability traits controlled by multiple genes with small effects. Due to the limitations of traditional breeding techniques, the genetic potential of Holstein cows in economic traits such as reproductive traits and long lactation traits has not been fully exploited. At present, there are still insufficient key genes that can be utilized and elucidated to affect the mechanism of reproductive traits. Therefore, it is of great significance to find key functional sites regulating the reproductive traits of cows and use them for molecular marker-assisted breeding and gene editing biological breeding to improve the reproductive traits of cows and improve economic benefits.

[0003] The ovary is an important reproductive organ of a cow, and its function is to produce and discharge oocytes and secrete estrogen and progesterone. The ovarian follicle is the basic structure and functional unit of the ovary, which is composed of oocytes, surrounding granulosa cells and theca cells. During the development of mammalian follicles, granulosa cells proliferate and secrete 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, the estrogen level decreases, which affects the maturation of oocytes and the development of follicles.

[0004] RBX1 (RING Box Protein-1) is a RING component of E3 ubiquitin ligase complex, which interacts with Cullins to activate Cullin-RING E3 ubiquitin ligase (CRL) and regulate protein ubiquitination modification, thereby regulating cell function. In C. elegans, specific interference of 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 the regulation of mammalian reproduction.

[0005] At present, RBX1 has been less studied in cattle, and RBX1 gene in cow ovarian granulosa cells has not been studied. SUMMARY

[0006] Therefore, the purpose of the present application is to provide the application of RBX1 gene in cow ovarian granulosa cells to solve the above problems.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides the application of in vivo or in vitro regulation of the expression of RBX1 gene in improving or reducing the function of bovine ovarian granulosa cells.

[0009] In some embodiments of the present application, the expression of the RBX1 gene is regulated by overexpressing the RBX1 gene or inhibiting the expression of the RBX1 gene.

[0010] In some embodiments of the present application, the overexpression of the RBX1 gene is used for any of the following purposes:

[0011] (I-1), improving the viability of the ovarian granulosa cells;

[0012] (I-2), improving the proliferation rate of the ovarian granulosa cells;

[0013] (I-3), improving the confluence or proliferation rate of the ovarian granulosa cells;

[0014] (I-4), reducing the apoptosis rate of the ovarian granulosa cells;

[0015] (I-5), improving the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0016] In some embodiments of the present application, the expression of the RBX1 gene is inhibited for any of the following purposes:

[0017] (II-1), reducing the viability of the ovarian granulosa cells;

[0018] (II-2), reducing the proliferation rate of the ovarian granulosa cells;

[0019] (II-3), reducing the confluence or proliferation rate of the ovarian granulosa cells;

[0020] (II-4), improving the apoptosis rate of the ovarian granulosa cells;

[0021] (II-5), reducing the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0022] In some embodiments of the present application, the RBX1 gene has the accession number: 518880; and the bovine includes Holstein.

[0023] In some embodiments of the present application, the expression of the RBX1 gene is inhibited by using siRNA.

[0024] In some embodiments of the present application, the siRNA has:

[0025] (i) the nucleotide sequence as shown in any one of SEQ ID Nos. 1-4; or

[0026] (ii) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (i) but differing from the nucleotide sequence shown in (i) due to the degeneracy of the genetic code; or

[0027] (iii) a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence shown in (i) or (ii), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (i) or (ii); or

[0028] (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).

[0029] In a second aspect, the present application further provides a method for improving the function of bovine ovarian granulosa cells by overexpressing the RBX1 gene.

[0030] In some embodiments of the present application, the improvement of the function of the ovarian granulosa cells comprises:

[0031] (I-1) improving the viability of the ovarian granulosa cells;

[0032] (I-2) improving the proliferation rate of the ovarian granulosa cells;

[0033] (I-3) improving the confluence or proliferation rate of the ovarian granulosa cells;

[0034] (I-4) reducing the apoptosis rate of the ovarian granulosa cells;

[0035] (I-5) improving the level of progesterone and / or estrogen in the ovarian granulosa cells.

[0036] In some embodiments of the present application, the accession number of the RBX1 gene is: 518880; and the bovine includes Holstein.

[0037] The present application first discovers that the specific regulation of RBX1 has an effect on bovine ovarian granulosa cells. The specific regulation of RBX1 on the ovarian granulosa cells discovers that RBX1 has the effect of promoting the proliferation of granulosa cells; and provides a gene for the creation of new materials for the breeding of high-fertility bovine in the later gene editing. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the description of the embodiments or the prior art will be briefly introduced below.

[0039] Figure 1RBX1 immunohistochemistry (IHC) results of ovary tissue;

[0040] Figure 2 Detection results of genes specifically regulating RBX1; wherein a shows the influence of overexpression of RBX1 on the expression amount of RBX1 gene of cow ovary granulosa cells by qRT-PCR method; b shows the influence of interference of RBX1 on the expression amount of RBX1 gene of cow ovary granulosa cells by qRT-PCR method;

[0041] Figure 3 Detection results of proteins specifically regulating RBX1; wherein a shows the influence of overexpression of RBX1 on the expression of RBX1 protein of cow ovary granulosa cells by Western Blot (WB) method; b shows the differential analysis of the expression of RBX1 protein of cow ovary granulosa cells by overexpression of RBX1 by WB method; c shows the influence of interference of RBX1 on the expression of RBX1 protein of cow ovary granulosa cells by WB method; d shows the differential analysis of the expression of RBX1 protein of cow ovary granulosa cells by interference of RBX1 by WB method;

[0042] Figure 4 Detection results of cell viability of specific regulation of RBX1; wherein a shows the influence of overexpression of RBX1 on the viability of cow ovary granulosa cells by CCK-8 method; b shows the influence of interference of RBX1 on the viability of cow ovary granulosa cells by CCK-8 method;

[0043] Figure 5 Detection results of cell proliferation of specific regulation of RBX1; wherein a shows the influence of overexpression of RBX1 on the proliferation of cow ovary granulosa cells by EdU method; b shows the differential analysis of the proliferation of cow ovary granulosa cells by overexpression of RBX1 by EdU method; c shows the influence of interference of RBX1 on the proliferation of cow ovary granulosa cells by EdU method; d shows the differential analysis of the proliferation of cow ovary granulosa cells by interference of RBX1 by EdU method;

[0044] Figure 6a Detection results of Incucyte method for detecting the influence of overexpression of RBX1 on the proliferation of live cells of cow ovary granulosa cells within 0-48h; Figure 6b Detection results of Incucyte method for detecting the differential analysis of the proliferation of live cells of cow ovary granulosa cells within 0h, 24h and 48h after overexpression of RBX1; Figure 6c Detection results of Incucyte method for detecting the image of the proliferation of live cells of cow ovary granulosa cells within 0h, 12h, 24h and 48h after overexpression of RBX1; Figure 6d Detection results of Incucyte method for detecting the influence of interference of RBX1 on the proliferation of live cells of cow ovary granulosa cells within 0-48h; Figure 6eFigure showing the difference analysis of the proliferation of live cells in bovine ovarian granulosa cells at 0h, 24h and 48h after interference with RBX1 by the Incucyte method; Figure 6f Figure showing the image of the proliferation of live cells in bovine ovarian granulosa cells at 0h, 12h, 24h and 48h after interference with RBX1 by the Incucyte method;

[0045] Figure 7a Figure showing the flow cytometry result of the apoptosis of bovine ovarian granulosa cells caused by overexpression of RBX1 by the Annexin V-FITC method; Figure 7b Figure showing the difference analysis result of different apoptosis types of bovine ovarian granulosa cells caused by overexpression of RBX1 by the Annexin V-FITC method; Figure 7c Figure showing the flow cytometry result of the apoptosis of bovine ovarian granulosa cells caused by interference with RBX1 by the Annexin V-FITC method; Figure 7d Figure showing the difference analysis result of different apoptosis types of bovine ovarian granulosa cells caused by interference with RBX1 by the Annexin V-FITC method;

[0046] Figure 8 Figure showing the effect of specific regulation of RBX1 on the hormone secretion level of GCs culture solution; wherein, a shows the effect of overexpression of RBX1 on the progesterone level of bovine ovarian granulosa cells by the radioimmunoassay method; b shows the effect of overexpression of RBX1 on the estrogen level of bovine ovarian granulosa cells by the radioimmunoassay method; c shows the effect of interference with RBX1 on the progesterone level of bovine ovarian granulosa cells by the radioimmunoassay method; d shows the effect of interference with RBX1 on the estrogen level of bovine ovarian granulosa cells by the radioimmunoassay method. DETAILED DESCRIPTION

[0047] The application discloses the application of the RBX1 gene in bovine ovarian granulosa cells, and those skilled in the art can refer to the content herein and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as being included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0048] The application aims at the application of the RBX1 gene in bovine ovarian granulosa cells.

[0049] The following technical scheme is adopted:

[0050] RBX1 gene

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

[0051] The application constructs RBX1 overexpression vector and synthesizes small interfering RNA (siRNA-RBX1) to specifically regulate bovine ovarian granulosa cells to explore whether RBX1 has an effect on the function of granulosa cells; it is found that RBX1 can promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of granulosa cells; overexpression of RBX1 in ovarian granulosa cells can increase the protein expression level, promote the proliferation of granulosa cells, increase the rate of living cells and inhibit the rate of cell apoptosis, and promote the secretion of steroid hormones; interference of RBX1 can inhibit the protein expression level, cell proliferation and steroid hormone secretion, and promote the rate of cell apoptosis.

[0052] The raw materials and reagents used in the application of RBX1 gene in bovine ovarian granulosa cells can be purchased from the market.

[0053] The application is further described below in combination with examples:

[0054] Example 1: Immunohistochemical (IHC) staining of bovine ovarian tissue

[0055] 1. Sampling of bovine ovarian tissue:

[0056] (1) repeatedly wash the collected bovine ovaries from the slaughterhouse with 0.9% sodium chloride (NaCl) solution (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) preheated to 37℃;

[0057] (2) cut off the tissue around the ovary with a sterile surgical scissors, and then wash the ovary with 75% alcohol, and then wash the ovary with preheated NaCl (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) solution for 4-5 times;

[0058] (3) wash the ovary with preheated PBS once. In a sterile clean bench, cut the ovary into tissue blocks with a length of about 10 mm, a width of about 10 mm and a thickness of about 3 mm, and place the tissue into an embedding mold.

[0059] 2. Dehydration, transparency and wax immersion

[0060] (1) 70% ethanol for 1h; 80% ethanol for 1h; 90% ethanol for 1h; 95% ethanol I for 1h; 95% ethanol II for 1h; 100% ethanol I for 1h; 100% ethanol II for 1h;

[0061] (2) ethanol xylene 40min; xylene I 25min; xylene II 15min;

[0062] (3) 65℃ wax immersion I 1h; 65℃ wax immersion II 1h; 65℃ wax immersion III 1h.

[0063] 3. Embedding:

[0064] Embed the tissue section downward, and take it out of the mold after the wax block cools down, and trim the block.

[0065] 4. Sectioning:

[0066] Pre-cool the wax block on the freezing table for 20 minutes in advance, then section it on the microtome at 4um, and then place the cut tissue wax section in the section expander in the constant temperature water area to expand the section, and then adhere it to the glass slide after the tissue wax section is flat.

[0067] 5. Baking:

[0068] Place the glass slide with the adhered tissue wax section on the section baking machine to bake the residual moisture, and then place it in a 65℃ oven for 1 hour.

[0069] 6. Immunofluorescence Staining:

[0070] (1) Xylene (I) 10 min; Xylene (II) 10 min; Xylene: 100% ethanol = 1:1 2 min; 100% ethanol (I) 5 min; 100% ethanol (II) 5 min; 80% ethanol 5 min; water wash 5 min; PBS rinse 3 times for 5 min each; antigen repair solution repair for 15 min (microwave repair method), cool to room temperature;

[0071] (2) 3% hydrogen peroxide blocking at room temperature for 30 min; PBS rinse 2 times for 5 min each; spin dry and draw the tissue with an immunohistochemical pen; 3% BSA incubation at room temperature for 30 min;

[0072] (3) Add the first antibody, incubate overnight at 4℃; PBS rinse 3 times for 5 min each;

[0073] (4) Add the second antibody working solution corresponding to the source of the first antibody, incubate at 37℃ for 30 min; PBS rinse 3 times for 5 min each;

[0074] (5) DAB color development (avoid light, observe under the microscope until brown) for about 3 min; water wash for 5 min; hematoxylin staining solution staining for 5 min; water wash for 5 min; hydrochloric acid ethanol differentiation solution differentiation for 1 s; ammonia blue solution blueing for 10 s; water wash for 30 s (antibodies are shown in Table 1);

[0075] (6) 95% ethanol (I) 1 min; 95% ethanol (II) 1 min; 100% ethanol (I) 3 min; 100% ethanol (II) 3 min; xylene (I) 3 min; xylene (II) 3 min;

[0076] (7) Neutral gum fixation, observe under a light microscope.

[0077] Table 1. Antibody Information

[0078]

[0079] As Figure 1 shown, RBX1 was positively reacted on granulosa cells and oocytes in the ovary tissue of dairy cows, indicating that RBX1 was expressed in both granulosa cells and oocytes.

[0080] Example 2 Culture and transfection of ovary granulosa cells to overexpress / interfere RBX1 plasmid

[0081] 1. Cell granulosa culture

[0082] (1) The ovary collected from the slaughterhouse was repeatedly washed with 0.9% sodium chloride (NaCl) solution (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) preheated at 37°C.

[0083] (2) The surrounding tissue of the ovary was cut off with a sterile surgical scissors, and washed with 75% alcohol. Then, the ovary was washed with preheated NaCl solution (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin) for 4-5 times.

[0084] (3) The ovary was washed once with preheated PBS. In a sterile clean bench, 10 mL syringe was used to extract follicular fluid in yellow and transparent healthy follicles with a diameter of 2-6 mm. Then, the collected follicular fluid was filtered into a new 15 mL centrifuge tube using a 40 μm filter screen, washed with DPBS for three times, centrifuged at 1000 g for 5 min, and the supernatant was discarded.

[0085] (4) GCs were resuspended with preheated DMEM / F12 complete medium (containing 10% PBS and 1% double antibody) and inoculated in culture dishes, and then cultured in a 37°C, 5% carbon dioxide incubator for 24 h, and the fresh medium was replaced every 24-48 h.

[0086] (5) When the GCs were confluent to 80%-90%, cell passage was performed. The medium was aspirated, and digestion was performed using 0.25% trypsin. When the cells were observed to round and fall off from the dish bottom under a microscope, an equal volume of serum-containing medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1000 g for 5 min, and the supernatant was discarded. Fresh DMEM / F12 complete medium was used to resuspend and inoculate in new culture dishes, and the culture was continued in a 37°C, 5% carbon dioxide incubator.

[0087] 2. siRNA targeting RBX1 and RBX1 overexpression plasmid (pcDNA3.1-RBX1) were designed according to GenBank reference sequence No. NM_001046241.1, and were designed and synthesized by GenePharma. siRNA-RBX1 or pcDNA3.1-RBX1 were transfected into prepared GCs using Lipofectamine 3000 transfection reagent.

[0088] siRNA-RBX1 primers are as follows:

[0089] si RBX1-1-sense (5'-3'): 5'-GGAGGUUCACGAAGUUCCUTT-3', as shown in SEQ ID No. 1;

[0090] si RBX1-1-antisense (5'-3'): 5'-AGGAACUUCGUGAACCUCCTT-3', as shown in SEQ ID No. 2;

[0091] si RBX1-2-sense (5'-3'): 5'-GCUGUCUUCUGCUAAGUCATT-3', as shown in SEQ ID No. 3;

[0092] si RBX1-2-antisense (5'-3'): 5'-UGACUUAGCAGAAGACAGCTT-3', as shown in SEQ ID No. 4.

[0093] Since U cannot be presented in the sequence listing software WIPO, U in the sequence listing is modified to T, and the correct sequence is the sequence in the specification.

[0094] 3. Ovarian granulosa cell transfection: GCs grown to 70%-80% confluence were transfected, the DMEM / F12 medium in the culture dish was replaced with Opti-MEM medium, and transfection was performed according to the siRNA / overexpression transfection reagent Lipofectamine™3000 instructions; cells were collected 24 h after transfection.

[0095] Example 3 RNA isolation and qRT-PCR detection

[0096] (1) Cell sampling

[0097] The GCs to be transfected were cultured for 24 h, then sampled. The culture medium was aspirated, and digestion was performed using 0.25% trypsin. When the cells were observed to round and fall off the dish bottom under a 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, centrifuged at 1000 g for 5 min, and the supernatant was discarded.

[0098] The cells were resuspended by adding 1 mL PBS, washed twice, and the supernatant was discarded. The cells were stored in a -80°C refrigerator.

[0099] (2) Total RNA extraction:

[0100] The cell RNA was extracted using the Novozyme RC101 kit, and the specific steps were as follows:

[0101] In the cell tube, 1000 μL TRIzol was directly added, the mixture was mixed by blowing, and oscillation was performed for 15 s. 200 μL chloroform was added, the tube cap was covered, and vigorous oscillation was performed for 15 s. The sample was placed in a 4°C refrigerator for 5 min, and then centrifuged at 4°C at 12,000 rpm (~13,400 × g) for 10 min. The upper aqueous phase was transferred to a new RNase Free centrifuge tube. In the obtained aqueous phase solution, 1.6 times the volume of Buffer RL2 (to which anhydrous ethanol had been added) was added, and the mixture was mixed gently. The mixture in the previous step was transferred to an RNAPure Column (the RNAPure Column was placed in a collection tube), and centrifuged at 13,000 × g for 1 min. The waste liquid was discarded. After the waste liquid was discarded, the RNAPure Column adsorption column was placed back into the collection tube, and the remaining liquid was added to the adsorption column. The mixture was centrifuged at 13,000 × g for 1 min, and the waste liquid was discarded. 500 μL Buffer RW1 was added to the RNAPure Column, and the mixture was centrifuged at 13,000 × g for 1 min. The waste liquid was discarded. 700 μL Buffer RW2 (to which anhydrous ethanol had been added) was added to the RNAPure Column, and the mixture was centrifuged at 13,000 × g for 1 min. The waste liquid was discarded. The previous step was repeated. The RNAPure Column adsorption column was placed back into the collection tube, and the mixture was centrifuged at 13,000 × g for 2 min to completely remove the residual Buffer RW2 from the RNAPure Column. The adsorption column was transferred to a new RNase-free Collection Tubes 1.5 mL centrifuge tube, and 50-200 μL RNase-free ddH2O was added to the center of the adsorption column. The mixture was left to stand at room temperature for 2 min, and then centrifuged at 13,000 × g for 1 min to elute the RNA. The RNA concentration and quality were detected using a BioDrop-μLite ultramicro nucleic acid analyzer, and the RNA was stored at -80°C.

[0102] (3) cDNA synthesis:

[0103] The RNA (1,000 ng) was reverse transcribed into cDNA by PrimeScript™ IV 1st strand cDNA Synthesis Mix kit, and the specific steps were as follows:

[0104] The mixture was prepared according to Table 2; gently mix, the reaction program was 30 ℃, 10 min; 42 ℃, 15 min; 95 ℃, 5 min. The product obtained by reverse transcription was stored at -20 ℃.

[0105] Table 2. Preparation of cDNA synthesis reaction solution

[0106]

[0107] (4) qRT-PCR detection:

[0108] The gene expression level was detected by using Thermo PowerUp™ SYBR™ Green Premix QuantStudioTM 7 Flex System (ABI), and each sample was repeated for 3 technical repeats. In this study, RPL-19 was used as a reference gene, and the expression level of each gene was calculated by 2 −ΔΔCT The reaction system of qRT-PCR is shown in Table 3, and the reaction program is: 95 ℃, 30 s; 95 ℃, 5 s and 60 ℃, 34 s cycle 40 times; 95 ℃, 15 s; 60 ℃, 15 s; 60 ℃, 1 min; 95 ℃, 15 s. All primers used for qRT-PCR were designed online by National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ), and the primer sequence was synthesized by Beijing Huada Gene.

[0109] Table 3. qRT-PCR reaction system

[0110]

[0111] The qRT-PCR primers were:

[0112] qRT-PCR-RPL19 Forward: 5'-ATCGCCAATGCCAACTC-3', as shown in SEQ ID No. 5;

[0113] Reverse: 5'-CCTTTCGCTTACCTATACC-3', as shown in SEQ ID No. 6;

[0114] qRT-PCR-RBX1 Forward: 5'-GAATGTCAAGCCAACCAGGC-3', as shown in SEQ ID No. 7;

[0115] Reverse: 5'-AAGCATGGTTACAGACGCCC-3', as shown in SEQ ID No. 8.

[0116] As shown in Figure 2 (a) The overexpression efficiency of oe-2 in the overexpression group was significantly higher than that of oe-1 compared with the control group; (b) The interference efficiency of si-2 group in the interference group was significantly higher than that of si-1 group; 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.

[0117] Example 4 Protein extraction of transfected cells and Western Blot (WB) detection

[0118] (1) Cell sampling

[0119] After the GCs to be transfected were cultured for 24 h, the medium was aspirated, and trypsin at a concentration of 0.25% was used for digestion. When the cells were observed to round and fall off the dish bottom under a microscope, an equal volume of serum-containing medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, which was centrifuged at 1000 g for 5 min, and the supernatant was discarded.

[0120] 1 mL of PBS was added to resuspend the cells, which were washed twice and the supernatant was discarded. The cells were stored in a -80°C refrigerator.

[0121] (2) Protein extraction: (taking the cell amount in 3 wells of a six-well plate as an example)

[0122] In the 1.5 mL centrifuge tube for sampling, 200 ul of RIPA lysis buffer (1.5 uL of PMSF was added before use, with a final concentration of 1 mM) was added, and the mixture was blown and mixed to fully lyse the cells. The lysed cells were incubated at 4°C for 1 h (the mixture was shaken every 15 min), and then the lysed sample was placed in a 4°C centrifuge, which was 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.

[0123] (3) Western Blot detection

[0124] Denaturation: Protein samples were mixed with 5x Loading Buffer buffer and boiled at 100 °C for 5 min; Electrophoresis: 20-40 pg of denatured samples were separated by SDS-PAGE under the following conditions: 200 V, 30 min; Membrane transfer: Proteins were transferred to NC membranes (HATF00010, Merck, Germany) under the following conditions: 200 mA, 2 h; Blocking: 5% skim milk incubated at room temperature for 2 h; Primary antibody incubation: primary antibody (Table 4) incubated at 4 °C overnight; Secondary antibody incubation: secondary antibody (Table 4) incubated at room temperature for 2 h; Development: developed with ECL chemiluminescence.

[0125] Table 4. Antibody information

[0126]

[0127] As shown in Figure 3 , compared with the control group, the protein expression of the overexpression RBX1 group increased (a), and the difference analysis found that overexpression of RBX1 significantly promoted the expression of RBX1 protein (b); the protein expression of the RBX1 interference group decreased (c), and the difference analysis found that interference of RBX1 significantly inhibited the expression of RBX1 protein (d).

[0128] Example 5 Cell viability detection (CCK8 method)

[0129] Ovarian granulosa cells (GCs) were seeded into 96-well plates, and after the cell density reached more than 70% for 6 h, 100 uL of complete culture medium (90% DMED / F12 + 10% FBS + 1% PS) was added for 24 h; 10 uL of CCK-8 solution was added to each well, and incubated at 5% CO2, 37 °C for 1 h. CCK-8 solution was added to the same volume of blank group, si-RNA group and overexpression group culture medium as blank control. The OD value of each well was measured by enzyme marker at 450 nanometer wavelength.

[0130] As shown in Figure 4 , compared with the control group, the cell viability of RBX1 overexpression in ovarian granulosa cells (GCs) was significantly increased (a); on the contrary, the cell viability of RBX1 interference in GCs was significantly decreased (b).

[0131] Example 6 Cell proliferation detection (EdU method)

[0132] Cell proliferation detection (EdU method):

[0133] BeyoClick™ EdU-488 cell proliferation detection kit (C0071S, Biyun Tian, Shanghai, China) was used for cell proliferation detection, and the specific steps were as follows:

[0134] GCs were seeded in confocal culture dishes and transfected when the confluence reached over 70%. Six hours after transfection, the original serum-depleted medium was removed and replaced with complete medium for 24 hours. A 10 mM EdU stock solution was diluted with DMEM / F12 to prepare a 20 μM EdU working solution. An equal volume of preheated 37 ℃ EdU working solution was added to the confocal dish, resulting in a final concentration of 10 μM. Incubation was performed for 2 hours. After EdU labeling, the mixture was aspirated, and 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. The mixture was washed three times with PBS containing 3% BSA for 5 minutes each time. PBS containing 0.3% Triton X-100 was added, and the mixture was incubated at room temperature for 15 minutes. The mixture was then washed twice with PBS containing 3% BSA for 5 minutes each time. One tube of Click Additive was dissolved in 1.3 mL of deionized water and mixed until completely dissolved.

[0135] Prepare the Click reaction solution according to Table 5. Add 500 μL of Click reaction solution to each dish and incubate at room temperature in the dark for 30 min. Then wash three times with PBS solution containing 3% BSA, each time for 5 min. Add 1× Hoechst 33342 solution and incubate at room temperature in the dark for 10 min. After incubation, wash three times with PBS solution containing 3% BSA, each time for 5 min. Take pictures using a fluorescence inverted microscope and analyze them using ImageJ software.

[0136] Table 5. Preparation of Click reaction solution

[0137]

[0138] like Figure 5 As shown, after overexpressing RBX1 in GCs, the number of positive cells in the EdU group increased (a), and the cell proliferation rate increased significantly compared with the control group (b); after interfering with RBX1 expression in GCs, the number of positive cells in the EdU group decreased (c), and the cell proliferation rate decreased significantly compared with the control group (d).

[0139] Example 7: Incucyte Real-Time Live Cell Analysis

[0140] Bovine ovarian granulosa cells (GCs) (5×10 5 500 μL of cells were seeded in six-well plates. When the confluence reached more than 50%, the cells were transfected. Six hours after transfection, the cells were sent to the Incucyte Real-Time Live Cell Analysis System (Sartorius Incucyte SX5) for cell proliferation detection.

[0141] As the results show, in the overexpression group, after overexpression of RBX1 in GCs, the cell confluence gradually increased compared to the control group after 48 hours of culture (using a broken-line trend).Figure 6a ) and the confluence degree of cells at 0h, 24h and 48h time periods was analyzed. It was found that the confluence degree of cells was significantly increased after overexpression of RBX1 ( Figure 6b ) and the growth state of live cells at 0h, 12h, 24h and 48h was found to be accelerated compared with the control group after overexpression of RBX1 ( Figure 6c ) and the growth state of live cells at 0h, 12h, 24h and 48h was found to be accelerated compared with the control group after overexpression of RBX1 ( Figure 6d ) and the growth state of live cells at 0h, 12h, 24h and 48h was found to be accelerated compared with the control group after overexpression of RBX1 ( Figure 6e ) and the growth state of live cells at 0h, 12h, 24h and 48h was found to be accelerated compared with the control group after overexpression of RBX1 ( Figure 6f ).

[0142] Example 8. Apoptosis analysis

[0143] Apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit (C1062M, Biyun Tian, Shanghai, China). The treated cells were collected by 0.25% trypsin digestion, resuspended with 195 µL Annexin V-FITC binding solution, then 5 µL Annexin V FITC was added, and incubated at room temperature for 30 min. Finally, 5 µL 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). FlowJo software was used for analysis.

[0144] As shown in the results, flow cytometry data showed that the number of live cells in GCs increased and the number of early apoptotic cells decreased after overexpression of RBX1 compared with the control group ( Figure 7a ) and the difference analysis found that the proportion of live cells in the overexpression group was significantly higher than that in the control group, and the proportion of early apoptotic cells was significantly decreased, but there was no significant difference in the proportion of late apoptotic cells ( Figure 7b ) and the number of early and late apoptotic cells in GCs increased after RBX1 expression was interfered ( Figure 7c ) and the difference analysis found that the proportion of early and late apoptotic cells in the interference group was significantly increased, but the proportion of live cells was not significantly different ( Figure 7d ).

[0145] Example 9. E2 and P4 hormone determination

[0146] Cells were seeded in six-well plates and transfected. After 24 hours of culture, 1 mL of cell culture medium was collected in a 1.5 mL centrifuge tube and stored at -20°C. The cell culture medium was then sent to Beijing Northern Biotechnology Research Institute Co., Ltd. for the detection of progesterone (P4) and estrogen (E2).

[0147] like Figure 8 As shown, compared with the control group, the levels of P4(a) and E2(b) were significantly increased after overexpression of RBX1 in GCs; and the levels of P4(c) and E2(d) were significantly decreased after interference with RBX1 expression.

[0148] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of RBX1 gene overexpression in improving bovine ovarian granulosa cell function; The application is shown in any of the following: (I-1) Enhance the vitality of the ovarian granulosa cells; (I-2) Increase the proliferation rate of the ovarian granulosa cells; (I-3) Increase the confluence or proliferation rate of the ovarian granulosa cells; (I-4) Reduce the apoptosis rate of the ovarian granulosa cells; (I-5) Increase the levels of progesterone and / or estrogen in the ovarian granulosa cells.

2. The application as described in claim 1, characterized in that, The gene ID of the RBX1 gene is 518880; NC_037332.1 (112164162..112177109); the cattle include Holstein cattle.

3. A method for improving the function of bovine ovarian granulosa cells, characterized in that, Overexpression of the RBX1 gene enhances the function of bovine ovarian granulosa cells; The improvement of ovarian granulosa cell function includes: (I-1) Enhance the vitality of the ovarian granulosa cells; (I-2) Increase the proliferation rate of the ovarian granulosa cells; (I-3) Increase the confluence or proliferation rate of the ovarian granulosa cells; (I-4) Reduce the apoptosis rate of the ovarian granulosa cells; (I-5) Increase the levels of progesterone and / or estrogen in the ovarian granulosa cells.

4. The method as described in claim 3, characterized in that, The gene ID of the RBX1 gene is 518880; NC_037332.1 (112164162..112177109); the cattle include Holstein cattle.

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